Regulation of Placental Autophagy by the Bcl-2 Family Proteins Myeloid Cell Leukemia Factor 1 (Mcl-1) and

Matador/Bcl-2 Related Ovarian Killer (Mtd/Bok)

by

Manpreet Kalkat

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Physiology University of Toronto

© Copyright by Manpreet Kalkat, 2010

ii

Regulation of Placental Autophagy by the Bcl-2 Family Proteins

Myeloid Cell Leukemia Factor 1 (Mcl-1) and Matador/Bcl-2

Related Ovarian Killer (Mtd/Bok)

Manpreet Kalkat

Master of Science

Department of Physiology

University of Toronto

2010

Abstract

The process of autophagy is defined as the degradation of cellular cytoplasmic constituents via a

lysosomal pathway. Herein I sought to examine the regulation of autophagy in the placental

pathologies preeclampsia (PE) and intrauterine growth restriction (IUGR). I hypothesized that

the Bcl-2 family proteins Mcl-1L and MtdL regulate placental autophagy and contribute towards

dysregulated autophagy in PE. My results demonstrate that Mcl-1L acts to repress autophagy via

a Beclin 1 interaction, while MtdL induces autophagy when it interacts with Mcl-1L. My data

indicate that while autophagy is elevated in PE, a pathology characterized by oxidative stress, it

is decreased in IUGR, a hypoxic pathology. Treatment with sodium nitroprusside to mimic PE

caused a decrease in Mcl-1L and an increase in MtdL levels in response to oxidative stress,

thereby inducing autophagy. Overall, my data provide insight into the molecular mechanisms

contributing to the pathogenesis of preeclampsia.

iii

Acknowledgments

I would like to acknowledge the support my supervisor, Dr. Isabella Caniggia, who has provided

me with many valuable lessons that have been instrumental in both my professional and personal

growth in this early stage of my scientific career.

I would also like to express my gratitude for the guidance, feedback and support provided to me

by the members of my student supervisory committee: Dr. Mingyao Liu, Dr. Jim Woodgett and

Dr. Stephen Lye.

I'd like to extend my heartfelt thanks to all the members of the Caniggia Lab and the other labs of

the sixth floor of TCP for their encouragement and moral support throughout the ups and downs

of research. In particular, I would like to thank Julia Garcia for her scientific feedback,

suggestions and excellent advice. I'd also like to thank Livia Deda, Tharini Sivasubramaniyam,

Antonella Racano and Jocelyn Ray for their consistent scientific and emotional support, and

importantly, for all the of the laughter and humour they have all brought to the lab every day.

Finally, I'd like to thank my family, who have not seen much of me in the last two years (for

which I apologize) but without whom I would not have had the luxury to explore my intellectual

pursuits.

iv

Contributions

I would like to acknowledge the contributions of the following people in the generation of the

data described in this thesis:

Julia Garcia, for the generation of the HEK293-GFP and HEK293-GFPMtdL stably transfected

cell lines and for the construction of the FlagMtdL, Mcl-1L and RFP-Mcl-1L vectors.

Doug Holmyard, for processing of placental tissue and HEK293 stably transfected cell lines for

electron microscopy.

Mount Sinai Hospital Biobank (Toronto, Canada) and Dr. Tulia Todros (University of Turin,

Italy) for supplying human placental tissue for immunoblot analysis and immunofluorescence

staining.

v

Table of Contents

Acknowledgments .......................................................................................................................... iii

Table of Contents ............................................................................................................................ v

List of Figures ................................................................................................................................. x

Chapter 1 ......................................................................................................................................... 1

1 Introduction ................................................................................................................................ 1

1.1 The Placenta ........................................................................................................................ 1

1.1.1 Early Placental Development .................................................................................. 1

1.1.2 Development of the Villous Structure .................................................................... 4

1.1.3 Onset of placental perfusion and oxidative stress ................................................... 8

1.1.4 Preeclampsia ........................................................................................................... 8

1.1.5 Cell Death and Placentation .................................................................................... 9

1.1.6 Intrauterine growth restriction (IUGR) ................................................................. 10

1.2 Autophagy ......................................................................................................................... 11

1.2.1 Autophagic Induction ............................................................................................ 12

1.2.2 Autophagic Nucleation and Expansion ................................................................. 17

1.2.3 Autophagosome Fusion ........................................................................................ 19

1.2.4 Mitophagy ............................................................................................................. 21

1.3 Bcl-2 family members ....................................................................................................... 21

1.3.1 Bcl-2 family regulation of apoptosis ..................................................................... 21

1.3.2 Bcl-2 Proteins and Mitochondrial Morphogenesis ............................................... 25

1.3.3 Bcl-2 proteins and non-apoptotic cell death ......................................................... 25

1.3.4 Bcl-2 family members and autophagy .................................................................. 26

1.3.5 Bcl-2 family proteins and the placenta ................................................................. 27

1.4 Myeloid Cell Leukemia Factor 1 ...................................................................................... 28

vi

1.5 Matador/Bcl-2 related ovarian killer (Mtd/Bok) ............................................................... 30

1.6 Human Placenta and Mtd/Mcl-1 ....................................................................................... 32

1.7 Rationale and Hypothesis ................................................................................................. 33

Chapter 2 ....................................................................................................................................... 34

2 Materials and Methods ............................................................................................................. 34

2.1 In Vitro Studies ................................................................................................................. 34

2.1.1 Cell Culture ........................................................................................................... 34

2.1.2 Protein Extraction ................................................................................................. 35

2.1.3 Bradford Protein Assay ......................................................................................... 35

2.1.4 Western Blot Analysis .......................................................................................... 35

2.1.5 Immunoprecipitation studies ................................................................................. 36

2.1.6 Immunofluorescence staining ............................................................................... 37

2.1.7 Electron Microscopy ............................................................................................. 39

2.1.8 Transient Transfection Experiments ..................................................................... 39

2.1.9 SNP Treatment ...................................................................................................... 40

2.1.10 Etoposide Treatment ............................................................................................. 41

2.1.11 Rapamycin Treatment ........................................................................................... 41

2.1.12 Glucose Starvation ................................................................................................ 41

2.1.13 Oxygen Experiments ............................................................................................. 41

2.2 Human Placental Tissue Experiments .............................................................................. 42

2.2.1 Collection and Sampling ....................................................................................... 42

2.2.2 Protein Extraction ................................................................................................. 44

2.2.3 Western Blotting ................................................................................................... 44

2.2.4 Immunofluorescence Staining .............................................................................. 45

2.2.5 Electron Micrographs ............................................................................................ 46

2.3 Statistical Analysis ............................................................................................................ 46

vii

Chapter 3 ....................................................................................................................................... 47

3 Results ...................................................................................................................................... 47

3.1 Mcl-1L is a repressor of autophagy .................................................................................. 47

3.2 Mcl-1L and MtdL interact ................................................................................................ 54

3.3 MtdL is a novel inducer of autophagy .............................................................................. 58

3.4 MtdL decreases the endogenous interaction of Mcl-1L and Beclin 1 .............................. 66

3.5 Autophagy is elevated in preeclampsia and decreased in IUGR ...................................... 69

3.6 Electron micrographs indicate increased presence of autophagosomes in PE.................. 73

3.7 A model of oxidative stress induces autophagy via alterations in Mcl-1L/MtdL

expression ......................................................................................................................... 80

Chapter 4 ....................................................................................................................................... 87

4 Discussion ................................................................................................................................ 87

4.1 Mcl-1 and MtdL are regulators of placental autophagy .................................................... 87

4.2 Oxidative stress induces autophagy in preeclampsia ........................................................ 92

Chapter 5 ....................................................................................................................................... 97

5 Future Directions ...................................................................................................................... 97

5.1 Is oxidative stress in trophoblast also sensed by Atg4? .................................................... 97

5.2 Are Vps34/PI3KIII complexes involved in MtdL induced autophagy? ........................... 97

5.3 Is MtdL contributing to mitophagy? ............................................................................... 100

5.4 What is the functional effect of autophagy in trophoblast? ............................................ 100

References ................................................................................................................................... 103

Appendices .................................................................................................................................. 118

6 Appendix ................................................................................................................................ 118

6.1 Rapamycin Treatment ..................................................................................................... 118

6.2 Low oxygen environment and autophagy ....................................................................... 120

6.3 Glucose Deprivation in JEG3 cells ................................................................................. 123

viii

6.4 Autophagy in Placental Development ............................................................................ 125

ix

List of Tables

Table 2.2.1 Clinical parameters of control, preeclamptic and IUGR patients. ............................ 43

x

List of Figures

Figure 1.1.1 The blastocyst. ............................................................................................................ 3

Figure 1.1.2. Diagrammatic representation of floating and anchoring placental villi. ................... 5

Figure 1.1.3 EVT cells remodel maternal spiral arteries. .............................................................. 7

Figure 1.2.1 Overview of the process of autophagy. ................................................................... 14

Figure 1.2.2. Nutrient sensing and regulation of autophagy by mTOR. ...................................... 15

Figure 1.2.3 Regulation of autophagy by mTOR. ....................................................................... 16

Figure 1.2.4 Autophagosome formation. ..................................................................................... 18

Figure 1.2.5 Process of autophagosome formation and fusion. .................................................... 20

Figure 1.3.1 Extrinsic and intrinsic pathways of apoptosis. ......................................................... 23

Figure 1.3.2. Diagrammatic representation of protein domains in Bcl-2 family proteins. .......... 24

Figure 1.4.1. Diagrammatic representation of Mcl-1 isoforms. .................................................. 29

Figure 1.5.1. Schematic diagram of Mtd-L, Mtd-P and Mtd-S. .................................................. 31

Figure 3.1.1 Identification of LC3B-II via immunoblot analysis in JEG3 cells. .......................... 48

Figure 3.1.2 Overexpression of Mcl-1L in JEG3 cells. ............................................................... 50

Figure 3.1.3 Silencing of Mcl-1L in JEG3 cells. .......................................................................... 51

Figure 3.1.4 Effect of Mcl-1L silencing on lysosomal presence in JEG3 cells. .......................... 52

Figure 3.1.5 Rapamycin treatment in HEK293 cells. .................................................................. 53

Figure 3.2.1 Immunoprecipitation of Mcl-1L in JEG3 cells transfected with FlagMtdL. ............ 55

Figure 3.2.2 Colocalization of MtdL and Mcl-1L in HEK293 cells. ........................................... 57

xi

Figure 3.3.1 Time and dose-course for doxycycline-mediated induction of MtdL. .................... 60

Figure 3.3.2 Immunoblot analysis of HEK293 cells stably expressing MtdL. ............................ 62

Figure 3.3.3 Lysotracker® Red staining in HEK293 cells. ......................................................... 63

Figure 3.3.4 Intracellular localization of MtdL in HEK293 cells. ............................................... 64

Figure 3.3.5 Electron micrographs of HEK293 cells stably expressing MtdL. ........................... 65

Figure 3.4.1 Immunoprecipitation of Beclin 1 in HEK293 cells stably expressing MtdL. ......... 68

Figure 3.5.1 Expression of LC3B-II in normal and preeclamptic placentae. .............................. 70

Figure 3.5.2 Expression of LC3B-II in normal and IUGR placentae. ......................................... 71

Figure 3.5.3 Spatial localization of Beclin 1 and Mcl-1 in normal and preeclamptic placentae. 72

Figure 3.6.1 Electron micrographs of the syncytium of normal and pathological placentae. ..... 75

Figure 3.6.2 Electron micropraphs of CT cells in normal and pathological placentae. ............... 77

Figure 3.6.3 Electron micrographs of the endothelium of normal and pathological placentae. .. 79

Figure 3.7.1 Expression of Mcl-1L and LC3B-II across time and dosage with SNP treatment in

JEG3 cells. .................................................................................................................................... 82

Figure 3.7.2 Expression of Mcl-1L, MtdL and LC3B-II in SNP treated JEG3 cells. .................. 84

Figure 3.7.3 Lysotracker® Red staining in SNP treated JEG3 cells. ........................................... 85

Figure 3.7.4 Spatial localization of Mtd in SNP treated JEG3 cells. ........................................... 86

Figure 4.1.1 Putative model of MtdL induced autophagy. ........................................................... 90

Figure 4.2.1 Proposed model of oxidative stress induced autophagy in trophoblast cells. .......... 94

Figure 5.2.1 Co-immunoprecipitation for the association of Beclin 1 with Vps34/PI3KIII in

HEK293-GFP and HEK293-GFPMtdL cells. ............................................................................... 99

xii

List of Appendices

Appendix 6.1.1 Expression of p70S6K phosphorylated at Threonine 389 in JEG3, HEK293 and

human placental lysate and rapamycin treatment in JEG3 cells. ................................................ 119

Appendix 6.2.1 Expression of Mcl-1L and LC3B-II in response to 20% and 3% oxygenation.122

Appendix 6.3.1 Expression of LC3B-II in JEG3 cells in response to glucose starvation. ......... 124

Appendix 6.4.1 Expression of LC3B-II in human placental development. ................................ 126

xiii

List of Abbreviations

3-MA 3-Methyladenine

ACOG American College of Obstetrics and Gynaecology

ALT alanine aminotransferase

AMC age-matched control

AMP adenosine monophosphate

AMPK adenosine monophosphate kinase

ATP adenosine triphosphate

BOK Bcl-2 related ovarian killer

CO2 carbon dioxide

CS caesarean section delivery

CT cytotrophoblast

D1 siRNA duplex 1

D2 siRNA duplex 2

DAPI 4',6-diamino-2-phenylindole

DME Dulbecco's modified Eagle's medium

ECL enhanced chemiluminescence

EMEM Eagle's minimum essential medium

EVT extravillous trophoblast

FBS fetal bovine serum

GFP green fluorescent protein

HBSS Hank's buffered saline solution

hCG human chorionic gonadotropin

HELLP hemolysis, elevated liver enzymes, low platelets

HIF-1 Hypoxia inducible factor 1

HRS hours

ICM inner cell mass

IP immunoprecipitation

IUGR intra-uterine growth restriction

LC3B microtubule associated light chain 3

Mcl-1 myeloid cell leukemia factor 1

mins minutes

MOMP mitochondrial outer membrane permeabilization

Mtd Matador

mTOR mammalian Target of Rapamycin

mvm microvillous membrane

O2 oxygen

OMM outer mitochondrial membrane

PBS phosphate-buffered saline

xiv

PE preeclampsia

PlGF placental growth factor

PVDF polyvinylidene fluoride

RFP red fluorescent protein

RIPA radioimmunoprecipitation assay buffer

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEM standard error of the mean

sFlt-1 soluble fms-like tyrosine kinase 1 (VEGFR-1)

siRNA small-interfering ribonucleic acid

SS siRNA scramble sequence control

ST syncytiotrophoblast

TBST tris-buffered saline with Tween-20

TC term control

TE trophectoderm

TGFβ transforming growth factor β

TNFα tumour necrosis factor α

Tsc tuberous sclerosis complex

Thr threonine

TM transmembrane

VEGF vascular endothelial growth factor

VEGFR-1 vascular endothelial growth factor receptor 1

VD vaginal delivery

WB western blot

XIAP X-linked inhibitor of apoptosis

1

Chapter 1

1 Introduction

1.1 The Placenta

The human placenta is a transient organ that serves as a conduit for the transfer of

nutrients and oxygen from the mother to fetus, while acting as a protective barrier for the fetus

from infectious agents. The placenta is an important source of hormones during pregnancy

critical for maintaining pregnancy and proper embryo development, including human chorionic

gonadotropin (hCG). hCG is a glycoprotein that maintains the corpus luteum thereby preventing

the onset of menstruation (Muyan & Boime 1997). The placenta also produces progesterone and

estrogen, hormones that stimulate uterine growth. Proper human placental development is a

prerequisite for the growth of a normal and healthy fetus, and heavily relies upon the presence of

an optimal intrauterine environment. The uterine environment changes dramatically from the

first trimester to term due to a switch from a histiotrophic to a haemotrophic nutrition, as the

result of the establishment of fetal-maternal blood flow (Burton et al., 2010). Up until 10 weeks

of gestation, nutrition to the fetus is supported by endometrial glands, while the onset of fetal-

maternal circulation occurs at about 10-12 weeks of gestation (Burton et al., 2002).

1.1.1 Early Placental Development

The fertilized zygote arrives in the uterine cavity within four to five days post conception.

During this time the zygote undergoes a series of mitotic divisions and forms the blastocyst

(Moore & Persaud 1998). At the blastocyst stage of development, the outer cell mass, termed

the trophectoderm (TE), forms and surrounds a fluid filled interior, the blastocoel, and inner cell

mass (ICM) (Figure 1.1.1). The trophectoderm forms the majority of the placental tissues, while

the inner cell mass forms the embryo.

At the end of the first week of conception, the trophectoderm attaches to the uterine

epithelial surface, the endometrium, forming a connection between the two apical surfaces of the

trophectoderm and endometrium (Pijnenborg 1998). In humans, decidualization of the

endometrium occurs under the influence of progesterone, resulting in uterine endometrial stromal

2

cells differentiating into decidual cells (Dimitriadis et al., 2010). Decidualization involves

remodelling of the extracellular matrix in order to provide a receptive substrate for the invasion

of trophoblasts. Trophoblasts secrete extracellular matrix molecules including laminins, and

their receptor integrins, as well as matrix-remodeling metalloproteinases (Moore & Persaud

1998). At the site of attachment the trophoblast cells proliferate and differentiate into two cell

types, namely the mitotically active cytotrophoblast (CT) cell layer and the multinucleated

syncytiotrophoblast (ST) layer, which form as the result of cytotrophoblast fusion. As fusion

proceeds, fluid-filled spaces form in the syncytium, termed lacunae, that perforate the syncytium

and fill with maternal blood and secretions from endometrial capillaries and uterine glands,

supporting placental histiotrophic nutrition (Moore & Persaud 1998; Burton et al., 2002).

3

Figure 1.1.1 The blastocyst.

At 4-5 days post-fertilization the blastocyst arrives in the uterus. The blastocyst is composed of

trophectoderm, which will give rise to the placenta, and the inner cell mass that will become the

embryo proper. At 8-9 days post-fertilization the blastocyst will implant into the uterine

endometrium.

4

1.1.2 Development of the Villous Structure

1.1.2.1 Floating Villi

Development of the functional unit of the placenta, the chorionic villi, initiates

immediately after implantation. Chorionic floating villi are the primary site for the exchange of

gas and nutrients between the fetal and maternal circulations (Moore & Persaud 1998).

Cytotrophoblast cells proliferate and extend into the syncytial mass forming sprouts that are

termed primary villi. At about 5 weeks of gestation, mesoderm cells extend into the primary villi

and transform them into secondary villi that contain a stromal core. The mesenchymal cells then

differentiate and undergo the process of de novo vasculogenesis, marking the creation of tertiary

villi (Demir et al., 2006). The tertiary villi undergo extensive branching which is characterized

by expansion of the stroma and increased angiogenesis, which is mediated both by vascular

endothelial growth factor-A (VEGF-A), an important factor that promotes the differentiation of

mesenchymal cells into hemangioblastic stem cells, and placental growth factor (PlGF), a factor

highly expressed by trophoblast cells (Demir et al., 2004; Burton et al., 2009).

Floating villi account for 90% of all chorionic villi and are suspended in the intervillous

space where they are bathed in maternal blood. In their mature form, they are composed of an

overlying syncytiotrophoblast layer that is maintained by the continual renewal by

cytotrophoblast cells which divide and fuse with the overlying syncytium (Figure 1.1.2). In the

second and third trimesters of gestation, the syncytium begins to aggregate and form syncytial

knots. These knots are comprised of aging nuclei and are extruded into the maternal circulation,

contributing to a pro-inflammatory response that is characteristic of normal pregnancy, and

presumed to be cleared in the maternal lung by macrophages (Johansen et al., 1999; Burton &

Jones 2009). The process by which cytotrophoblasts fuse to form syncytium and are extruded as

knots is termed trophoblast turnover.

5

Figure 1.1.2. Diagrammatic representation of floating and anchoring placental villi.

Floating villi are comprised of an overlying syncytial layer and underlying cytotrophoblast

progenitor cells. Syncytial knots are extruded nuclei produced by the syncytium. Anchoring

villous columns connect the placenta to the uterus. They are composed of a proximal and distal

region composed of migrating extravillous trophoblast cells that invade the maternal decidua and

remodel spiral arteries. ST: syncytiotrophoblast layer; CT: cytotrophoblast, EVT: extravillous

trophoblast.

6

1.1.2.2 Anchoring Villi

Anchoring villi comprise 10% of the chorionic villi, physically connect the placenta and

embryo to the uterine wall, and are created as the result of trophoblast invasion into the maternal

decidua (Moore & Persaud 1998). Cytotrophoblasts that escape the confines of the syncytial

layer and form columns of nonpolarized cells are termed extravillous trophoblast cells (EVT)

(Damsky et al., 1992). Villous cytotrophoblasts that are anchored to the basement membrane

express α6β4 integrin subunits and laminins, while nonpolarized cells in the anchoring column

along the invasive pathway express α5β1 integrin and a fibronectin matrix (Damsky et al., 1992).

Growth factors and cytokines regulate trophoblast invasion, in particular interleukin-1β and

epidermal growth factor have been demonstrated to stimulate invasion of trophoblast cells (Bass

et al., 1994; Librach et al., 1994). In contrast, transforming growth factor β (TGFβ) signalling in

response to hypoxia inducible factor 1 (HIF-1) inhibits the invasive capacities of trophoblast

cells (Graham et al., 1992; Caniggia et al., 1997; Caniggia et al., 2000).

Trophoblast invasion follows two different pathways, the interstitial and the endovascular

pathway (Figure 1.1.3). The interstitial pathway is controlled by EVT cells that migrate through

the decidua and enter the inner myometrium at about 8 weeks of gestation and replace smooth

muscle cells with a fibrinoid material. The endovascular pathway begins with the invasion of

arterioles and arteries by the EVTs, and relining of the vessel endothelium. Invasion of the

uterine wall occurs up to the first third of the myometrium and associated spiral arteries, and is

maximal during the first trimester of gestation, peaking at about 10-12 weeks of gestation

(Caniggia et al., 2000).

Conversion of the vessels and the replacement of the muscular wall by the EVTs results

in the spiral arteries no longer being under the control of maternal neurovascular control

mechanisms, resulting in a low resistance uterine network that efficiently increases the volume of

maternal blood being delivered to the placenta (Pijnenborg 1998; Jauniaux et al., 2006). While

fetal vessels appear in the developing villi at about 11-15 days following conception, a maternal-

fetal circulation does not commence as endovascular plugs of EVT inhibit maternal blood flow

until they regress at about 10-12 weeks of gestation. This restricted blood flow protects the fetus

from teratogenesis mediated by reactive oxygen species and maintains a low level of metabolism

in early blastocyst development (Jauniaux et al., 2000; Burton et al., 2010).

7

Figure 1.1.3 EVT cells remodel maternal spiral arteries.

In the interstitial pathway of remodeling, EVT cells degrade the smooth muscle wall of the spiral

arteries and deposit fibrinoid material. In the endovascular pathway EVT cells re-line the

endothelium and occlude the vessel until 10-12 weeks of gestation. EVT: extravillous

trophoblast.

8

1.1.3 Onset of placental perfusion and oxidative stress

An early environment of low oxygen is recognized as an important factor in proper

placental development, as low oxygen tension maintains trophoblast cells in a proliferative and

non-invasive phenotype that is essential for early placentation events (Genbacev et al., 1996;

Genbacev et al., 1997; Caniggia et al., 2000). Experiments using placental villous explants have

demonstrated that maintaining the villous in low oxygen conditions of about 3% oxygen has a

positive effect on the outgrowth capability of EVT cells, but nonetheless maintains these cells in

a noninvasive phenotype (Genbacev et al., 1997; MacPhee et al., 2001). At 10-12 weeks of

gestation, perfusion of the intervillous space results in a highly invasive EVT cell phenotype

(Rodesch et al., 1992). The onset of maternal circulation results in a three-fold increase in

intraplacental oxygen concentration. This was measured by oxygen electrode experiments that

demonstrated an increase in oxygen tension from 15-20mmHg (2-3%) at 8 weeks of gestation to

55mmHg (8%) at 12 weeks of gestation, and a subsequent decrease to 6% O2 at term due to

increased oxygen extraction by the fetus (Rodesch et al., 1992; Jauniaux et al., 2000). The

syncytial layer of the placenta is especially exposed to damaging reactive oxygen species as it is

directly bathed in maternal blood and contains low concentrations of antioxidant enzymes in the

first trimester of pregnancy (Watson et al., 1997; Watson et al., 1998). From 8 weeks of

gestation, a peak of markers of oxidative stress is observed in placental tissue, and is

hypothesized to drive trophoblast differentiation, invasion and maturation (Genbacev et al.,

1997; Caniggia et al., 2000; Jauniaux et al., 2000). Importantly, alterations in invasive properties

have been associated with trophoblast pathology.

1.1.4 Preeclampsia

Preeclampsia (PE) is a devastating disease of placental origin, affecting 5-8% of

pregnancies, and is a leading cause of fetal morbidity and mortality (2002). While many theories

exist on the cause of PE, it is recognized that the placenta is central to this disorder, as the only

symptomatic cure is the removal of the placenta. Hydatidiform moles, which develop in the

absence of a fetus, can also lead to the development of PE, supporting the important role of the

placenta in this pathology (Chun et al., 1964). Risk factors for PE include multiple gestations,

first pregnancies, chronic hypertension, diabetes, and vascular disease. Early onset PE is

characterized by onset of clinical symptoms of pregnancy-induced hypertension of greater than

140/90mmHg and proteinurea of ≥ 0.3g per 24 hrs after 20 weeks of gestation in a woman with

9

previously normal blood pressure and urine (2002). PE can also be characterized by other

symptoms including edema, headaches, epigastric pains, and visual disturbances, and in severe

cases, by hemolysis, elevated liver enzymes and low platelets (HELLP syndrome) (2002).

The etiology of preeclampsia is unknown; however some consensus exists on the origin

of this disease of pregnancy. A general agreement has emerged that preeclampsia is the result of

defects in early placental development and trophoblast invasion, resulting in shallow invasion,

insufficient conversion of spiral arteries and reduced or abnormal placental perfusion (Robertson

et al., 1985; Jauniaux et al., 2006). Histological examination of the placental bed from

preeclamptic women has revealed that there is limited migration into the decidua and reduced

invasion into the myometrial portions of the maternal spiral arteries as well as incomplete

integrin switching as the trophoblast compartment remains α5 positive (Redline & Patterson

1995; Zhou et al., 1997). Ultrasound Doppler analysis of preeclamptic women demonstrated that

they have reduced placental blood flow and increased uterine vascular resistance (Harrington et

al., 1996). This altered perfusion leads to both a status of decreased oxygenation in preeclampsia

and intermittent blood flow, leading to transient hypoxia/reoxygenation stress and nitrosative

stress to the placenta (Myatt et al., 1996; Hubel 1999; Hung et al., 2004; Myatt & Cui 2004;

Takagi et al., 2004; Soleymanlou et al., 2005). Preeclamptic placentae have been demonstrated

to have a phenotype of immature cytotrophoblast cells with increased rates of proliferation and

decreased invasive capabilities, as well as the presence of increased apoptotic cell death, all of

which have been hypothesized to be the result of decreased oxygenation of the trophoblast

(Genbacev et al., 1996; Soleymanlou et al., 2005; Soleymanlou et al., 2007; Ray et al., 2009).

1.1.5 Cell Death and Placentation

Apoptosis, a form of programmed cell death, has been recognized as an important

regulator of normal placentation in order to maintain tissue homeostasis. Normal placentation is

associated with some degree of trophoblast cell apoptosis. Caspase-8 has been determined to be

an important factor in cytotrophoblast-syncytial fusion, as knockdown of caspase-8 activity in

placental villous explants prevented fusion (Black et al., 2004). As well, nuclei of the syncytium

that display chromatin condensation shed into the maternal circulation as syncytial knots, in a

process that shares similarities with apoptosis (Jones & Fox 1991; Huppertz et al., 1998;

Johansen et al., 1999; Heazell & Crocker 2008). As stated previously, this is thought to

10

contribute to a status of increased inflammatory response that is characteristic of normal

pregnancy. However, in preeclampsia, apoptosis and shedding is increased, and this has been

hypothesized to contribute to the excessive inflammatory response typical of this disease

(Johansen et al., 1999; Goswami et al., 2006). This increased trophoblastic shedding may be the

result of oxidative injury, as hypoxia and hypoxia-reoxygenation injury has been demonstrated to

increase apoptosis in the syncytium of preeclamptic placentae (Burton & Jones 2009). The

systemic inflammatory response and endothelial dysfunction in preeclampsia is thought to arise

from factors in the maternal circulation as a result of excessive placental shedding. One factor of

interest in preeclampsia is the soluble receptor for vascular endothelial growth factor, sVEGFR-

1, also termed soluble Fms-like tyrosine kinase 1 (sFlt-1), which has been reported to be secreted

in response to hypoxia (Maynard et al., 2003; Nevo et al., 2008). When sFlt-1 is present in

excess, it binds and inactivates VEGF-A, a key factor for endothelial survival and

vasculogenesis, and is thought to induce systemic endothelial dysfunction, as evidenced by

engorged glomerular capillary endothelial cells of the kidney (Maynard et al., 2003; Khankin et

al., 2010).

1.1.6 Intrauterine growth restriction (IUGR)

The growth of a fetus is determined by both the genetic potential of the fetus and the

nutritional and hormonal environment of the conceptus. Intrauterine growth restriction (IUGR)

is defined as when the fetus fails to reach its potential growth and is generally accepted to be

when the fetus falls below the 5th percentile for its gestational age (2001; Cetin & Alvino 2009).

IUGR affects 7-15% of pregnancies and is a leading cause of perinatal mortality and morbidity

(Alexander et al., 2003; Baschat 2004; Cetin & Alvino 2009). Many factors have been shown to

contribute to low birth weight and can be grouped into maternal, fetal or placental factors (2001).

Maternal contributors include smoking, extremes of reproductive age, malnutrition, diabetes,

lung disease, and multiple gestation (2001). Placental factors include the presence of

preeclampsia; however, IUGR can occur in the absence of maternal pregnancy induced

hypertension. When IUGR is suspected, the diagnosis can be confirmed with ultrasound and

Doppler analysis, with two sets of examinations 2 weeks apart (2001). However, the cause of

most IUGR cases remains indeterminate, and share the common placental phenotype of placental

insufficiency, the notion that the placenta is unable to sustain fetal growth (Sibley et al., 2005).

While both preeclampsia and IUGR share the feature of decreased migration of the EVT cells

11

into the decidua, why some patients develop the symptoms of both preeclampsia and IUGR,

while others are not affected by preeclampsia, remains to be established.

Very low birth weight from mid to late gestation has been correlated with an increased

propensity towards adult disease such as coronary heart disease, obesity and diabetes later in life

(the metabolic syndrome) (Barker 1995; Barker et al., 2010). In IUGR, both the fetus and

placenta are small in size, however the relative ratio of placental mass to fetal mass is increased

(Pardi et al., 2002). Many groups have characterized defects in placental metabolism and

transport in IUGR, consistently demonstrating that there is decreased transport of amino acids to

the IUGR fetus, and a decreased surface for the exchange of nutrients across the placenta

(Jansson et al., 1993; Challis et al., 2000; Huppertz et al., 2006). Along with increased thickness

of the villous structure, this reduces the diffusing capacity of the placenta by up to 60%

(Huppertz et al., 2006). Severe IUGR pregnancies have been reported to exhibit reduced fetal-

placental blood flow, and also to have a malformed and hypovascularized phenotype, suggesting

IUGR placentae are hypoxic, and possibly contribute towards decreased nutrient exchange

(Kingdom & Kaufmann 1999; Kingdom et al., 2000; Chen et al., 2002; Arroyo & Winn 2008;

Nevo et al., 2008).

1.2 Autophagy

Autophagy is a cellular process that results in the degradation of cytoplasmic constituents

via a lysosomal pathway by the engulfment of this material into double-membraned vacuoles

termed autophagosomes (Deter et al., 1967; Melendez & Neufeld 2008). While there are three

different kinds of autophagy (macroautophagy, microautophagy and chaperone-mediated

autophagy), the term autophagy generally refers to macroautophagy. It is distinguishable from

proteasome-mediated degradation processes in that autophagy is the only known cellular process

that degrades entire organelles, including mitochondria, portions of the endoplasmic reticulum

and ribosomes (Klionsky & Emr 2000; Kim et al., 2007).

Autophagy is a ubiquitous cellular process that has been characterized in many organisms

from yeast to humans (Klionsky et al., 2007). While the process was identified more than 50

years ago, recent interest in this field of research has increased due to both the discovery of genes

that control this process, the autophagy-related genes (Atg), and the understanding that

autophagy has a large impact on human health and disease (Thumm et al., 1994; Harding et al.,

12

1995; Mizushima & Hara 2006; Melendez & Neufeld 2008; Mizushima et al., 2008). The best

understood function of autophagy remains as an internal nutrient source for starved cells,

however autophagy also has many other complex cellular functions. Overall, the process of

autophagy can be divided into four distinct steps: induction, nucleation, expansion and fusion

(Klionsky et al., 2007; Mizushima 2007).

1.2.1 Autophagic Induction

Autophagic induction is the upstream signaling event that results in autophagosome

nucleation and formation (Figure 1.2.1). While autophagy is active at a basal level in most cells

for protein turnover and organelle recycling, it can be rapidly activated in response to cellular

stressors (Melendez & Neufeld 2008). The most potent activator of autophagy is nutrient

deprivation of glucose and amino acids, which partially act by inactivation of the mammalian

Target of Rapamycin, mTOR (Wullschleger et al., 2006; Melendez & Neufeld 2008). mTOR is

a major nutrient sensor of the cell, and controls cell growth in response to nutrient availability,

and is an important negative regulator of autophagy (Kim et al., 2002; Yu et al., 2010). It

consists of two different complexes, entitled mTORC1 and mTORC2, of which only mTORC1 is

sensitive to inhibitory rapamycin treatment and acts as the predominant nutrient sensor, while

mTORC2 controls actin organization and possibly can also respond to the nutrient status of a cell

(Wullschleger et al., 2006). The mTOR pathway can respond to growth factors via the PI3K

class I pathway in response to insulin or insulin-like growth factors (IGFs) binding to their

receptors, leading to the recruitment and activation of PI3K class I (Wullschleger et al., 2006).

The tuberous sclerosis proteins Tsc1 and Tsc2 form a heterodimer that negatively transduces

PI3K class I signaling to mTOR (Figure 1.2.2). Amino acid starvation, especially with leucine,

results in rapid dephosphorylation of the downstream targets of mTOR, p70 ribosomal protein S6

kinase (p70S6K) and elongation factor 4 binding protein 1 (4E-BP1) (Kanazawa et al., 2004).

While the exact mechanism of amino acid sensing is not known, it has been proposed that amino

acid deprivation can by sensed via Tsc1/Tsc2 or via the Rheb GTPase (Gao et al., 2002; Garami

et al., 2003).

mTORC1 senses the energy of a cell via AMP-activated protein kinase (AMPK), which

is activated in response to low cellular energy levels, which is indicated by a high AMP to ATP

ratio (Wullschleger et al., 2006; Luo et al., 2010). Activation of AMPK downregulates energy-

13

costly cellular events such as protein synthesis and results in increased ATP-generating processes

including fatty acid oxidation and autophagic activation. Activated AMPK exerts its function by

directly phosphorylating Tsc2 and leading to the inhibition of mTORC1 signaling (Inoki et al.,

2003). mTOR also responds to cellular stress events, such as hypoxia, which results in decreased

mTOR signaling in a mechanism that involves activation of Hypoxia Inducible Factor 1 (HIF-1)

(Wullschleger et al., 2006). Prolonged hypoxia leads to ATP depletion and subsequent

activation of AMPK.

The mechanism by which mTOR negatively regulates autophagy in mammalian cells is

not entirely understood. However, in yeast and mammalian systems it is known that TOR

inhibits the protein kinase Atg1, which is an early step in the activation of autophagy (Kamada et

al., 2000; Scott et al., 2007). As well, in yeast TOR can hyperphosphorylate Atg13, reducing its

binding affinity and activation of Atg1, and this event can be reversed by rapamycin treatment

(Figure 1.2.3). Rapamycin inhibits mTOR kinase activity, resulting in downstream

dephosphorylation and activation Atg13 (Peterson et al., 1999; Kamada et al., 2000).

14

Figure 1.2.1 Overview of the process of autophagy.

An isolation membrane forms from the endoplasmic reticulum and encircles cytoplasmic

constituents forming the autophagosome. Subsequent fusion events with lysosomes result in the

breakdown and recycling of the engulfed components.

15

Figure 1.2.2. Nutrient sensing and regulation of autophagy by mTOR.

Conditions of low ATP or hypoxia can be sensed by AMPK leading to mTOR inactivation. Low

amino acid levels could inactivate mTOR either by sensing by Rheb or Tsc1/Tsc2. IGF and

IGF-like signaling is sensed through PI3K class I stimulating the protein synthesis and cell

growth. P: phosphorylation. Note: Arrows do not imply direct effects, pathways have been

collapsed for clarity.

16

Figure 1.2.3 Regulation of autophagy by mTOR.

In conditions of activated mTOR, Atg13 is hyperphosphorylated thereby reducing its affinity for

Atg1. When mTOR is inactivated by a variety of upstream signalling mechanisms, Atg13 is

dephosphorylated and binds with high affinity to Atg1, thereby activating Atg1 and leading to

the formation of the nucleation complex. P: phosphorylation.

17

1.2.2 Autophagic Nucleation and Expansion

The genes that are necessary for autophagy were first discovered in screens using yeast

mutants that were defective in vacuole formation, and homologues for these genes have been

discovered in mammalian systems (Harding et al., 1995). Autophagy genes control diverse

aspects of autophagosomal formation, including expansion and fusion events.

A functional complex involved in vesicle nucleation consists of Beclin 1/Atg6/Vps30,

Vps15/p150 and Atg14, and is termed Complex I (Itakura et al., 2008). This complex is present

at the level of the isolation membrane, which is thought to be located at the endoplasmic

reticulum or trans-Golgi network (Simonsen & Tooze 2009; Yen et al., 2010). Autophagosome

formation has been suggested to result from the accumulation of Complex I and activity of the

class III PI3K/Vps34 to produce phosphatidylinositol 3-phosphate (PI3P) (Hamasaki &

Yoshimori 2010). PI3P production is thought to locally change the ER membrane curvature to

create a site of autophagosome formation termed the autophagosome cradle (Figure 1.2.4). The

autophagosome cradle develops to form the precursor structure to the autophagosome, the

omegasome, which is still physically connected to the ER (Hamasaki & Yoshimori 2010).

Two conjugation systems that resemble ubiquitin-like protein conjugation are essential

for the expansion of autophagosomes. In order for vesicle completion, Atg12 is activated by

binding to Atg7 by a thioester bond. It is then transferred by the E2-like molecule Atg10 to Atg5

(Mizushima et al., 1998). A second ubiquitin-like system is used to anchor Atg8 (also known as

microtubule-associated light chain 3, LC3) to the autophagosomal membrane, where it is present

until autophagosome degradation (Melendez & Neufeld 2008). LC3 has two isoforms, one

which is cytoplasmic and another form that is processed and associated with the autophagosomal

membrane, termed LC3-II (Kabeya et al., 2000). Two LC3 bands, LC3-I and LC3-II can be

detected via immunoblotting, at 18 and 16 kDa respectively. Expression levels of LC3-II are

frequently used as a marker for autophagy, while LC3-I is particularly abundant in brain tissue

(Mann & Hammarback 1996; Kabeya et al., 2000). LC3-II is conjugated to

phosphatidylethanolamine by a series of reactions catalyzed by Atg4, Atg7 and Atg3, resulting

an autophagosome dotted with LC3-II (Ichimura et al., 2000).

18

Figure 1.2.4 Autophagosome formation.

The process of autophagosome formation is thought to occur at the ER. PI3K class III activity

produces PI(3)P which is thought to lead to deformation of the ER leading to the creation of the

autophagosome cradle. The pre-autophagosomal structure is referred to as the omegasome.

19

1.2.3 Autophagosome Fusion

Autophagosomes can fuse with endosomes forming a cellular compartment termed

amphisomes, and the maturation of autophagosomes involves many sequential fusion events with

vesicles from the endosomal and lysosomal cellular compartments, during which the autophagic

compartment becomes more acidic (Figure 1.2.5) (Dunn 1990; Berg et al., 1998). The final

stages of autophagy involve the transport of autophagosomes to lysosomes in a dynein-

dependent manner, and membrane fusion of the two cellular compartments (Melendez &

Neufeld 2008). The fusion products are then termed autophagolysosomes, and the contents are

degraded by lysosomal hydrolases (Eskelinen 2005). Finally, the degradation products are

returned to the cytoplasm and reused for cellular metabolism.

20

Figure 1.2.5 Process of autophagosome formation and fusion.

Early events in the autophagic process include nucleation and bending of the membrane to form

the initiat autophagic vacuole. Fusion events with endosomes and lysosomes leads to maturation

of the autophagosome and increased acidity of the organelle, thus creating the mature late stage

degradative autophagolysosome.

21

1.2.4 Mitophagy

Mitochondria, via mitochondrial oxidative phosphorylation provide a large amount of

energy to the cell, but this organelle also is the source of cellular reactive oxygen species (ROS)

that can cause damage to lipids, DNA and proteins. Autophagy that is specific to the turnover of

mitochondria has been termed mitophagy, and is the only known pathway that results in the

clearance of mitochondria from the cytoplasm of a cell (Kim et al., 2007; Melendez & Neufeld

2008). Conditions that lead to mitochondrial stress have been reported to lead to mitophagic

activation; however, the molecular mechanisms behind mitochondrial targeting are not well

understood (Kanki et al., 2009). Studies on reticulocyte maturation have pointed to Nix (a Bcl-2

family member) and Parkin (a ubiquitin ligase), as proteins that are required for mitophagy in

mammalian cells (Narendra et al., 2008; Ding et al., 2010). In yeast, mitophagy was found to be

induced in response to nitrogen starvation, rapamycin treatment, and disturbances in

mitochondrial membrane potential (Nice et al., 2002). Upon induction of mitophagy, Atg32

binds to Atg11, recruiting mitochondria into the pre-autophagosomal structure. However, in

mammalian systems, the homologues to yeast Atg32 and Atg11 remain to be established (Nice et

al., 2002).

1.3 Bcl-2 family members

The B cell lymphoma proteins (Bcl-2) are a group of promiscuous proteins with the

capacity to hetero- and homodimerize with one another. The Bcl-2 family are important

regulators of apoptotic cell death, but have also been characterized in the regulation of other

cellular events, including cellular proliferation, mitochondrial morphogenesis, and autophagy.

Bcl-2 family proteins have conserved Bcl-2 Homology (BH) domains that mediate the direct

interactions between different members and govern how the proteins function. There are three

subcategories for Bcl-2 proteins: BH3-only proapoptotic molecules, BH-multidomain

proapoptotic molecules, and prosurvival molecules.

1.3.1 Bcl-2 family regulation of apoptosis

Apoptosis is the process of controlled cell death and is characterized by the

morphological characteristics of nuclear and cytoplasmic condensation and cellular

fragmentation (Kerr et al., 1972). Apoptotic cell death is mediated by a group of proteins termed

22

cysteine-aspartic acid proteases (caspases) that specifically cleave proteins after Asp residues

(Salvesen & Dixit 1997). Caspases exist as inactive zymogens or proenzymes which are cleaved

to generate two subunits that form a heterotetramer, the active form of the protein (Wang &

Youle 2009).

There are two pathways through which apoptosis can occur, the extrinsic and intrinsic

apoptotic pathways. The extrinsic pathway is also termed the death-receptor pathway, in which

cell death is activated from outside the cell by ligand binding to cell death receptors such as Fas,

TNF, TRAIL and DR3-6 (Figure 1.3.1.) (Wang & Youle 2009). Once the receptors are activated

each receptor can form the death-inducing signaling complex (DISC) via the recruitment of Fas-

associated death domain (FADD) and procaspase-8 and -10. Once caspase-8 and caspase-10 are

activated, they directly cleave and activate caspase-3 and 7.

The intrinsic pathway is also called the mitochondrial pathway and involves regulation at

the level of the mitochondria by the Bcl-2 antiapoptotic and proapoptic proteins. Bcl-2

antiapoptotic proteins include Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1/Bfl-1. Bcl-2 proapoptotic

multidomain proteins include Mtd/Bok, Bax and Bak. These proteins regulate the cellular

commitment to apoptosis by regulating the mitochondrial outer membrane potential (MOMP)

(Wei et al., 2001). Once the MOMP is disturbed the cell is committed to death, and cytochrome

c is released from the mitochondria. Cytochrome c binds apoptotic protease-activating factor 1

(APAF-1) which then undergoes oligomerization into the apoptosome. The apoptosome leads to

caspase-9 activation, followed by caspase 3/7 which then dismantle the cell (Liu et al., 1996;

Autret & Martin 2009; Wang & Youle 2009).

The BH3-only family members include Bid, Bad, Bim, Puma, Noxa, Hrk, Bmf, and

Nbk/Bik (Figure 1.3.2). These proteins contain only one BH3 homology domain and are

activated by cell death stimuli. Their BH3 domain is an amphipathic α-helix that can bind with

the hydrophobic grooves formed by BH1 and BH2 domains of anti-apoptotic and proapoptotic

Bcl-2 family proteins. BH3-only proteins operate upstream of the multidomain proteins, and

when the multidomain proteins are deficient, BH3-only proteins cannot induce apoptosis (Wei et

al., 2001; Zong et al., 2001). While Bcl-2 proteins share features with α-helical pore-forming

proteins, a consensus has not been reached for the molecular mechanism of how MOMP actually

is achieved.

23

Figure 1.3.1 Extrinsic and intrinsic pathways of apoptosis.

The extrinsic pathway of apoptosis is activated in response to death ligands. The intrinsic

mitochondrial pathway relies on MOMP and cytochrome c release leading to caspase activation.

24

Figure 1.3.2. Diagrammatic representation of protein domains in Bcl-2 family proteins.

There are three classes of Bcl-2 family proteins, antiapoptotic, proapoptotic multidomain and

proapoptotic BH3-only proteins. TM= transmembrane domain, BH= Bcl-2 homology domain.

25

There are three hypotheses regarding the activation of MOMP by proapoptotic Bcl-2

family proteins: direct activation, indirect activation and embedded together models (Leber et al.,

2007; Lovell et al., 2008). In the direct activation model, BH3-only proteins are required to

activate multidomain proteins by direct interaction, leading to MOMP. Evidence supports that

with regards to Bax, MOMP relies upon activation of Bax by BH3-only proteins (Yin et al.,

1994; Lovell et al., 2008). In the indirect activation model, proapoptotic proteins are stably

bound to antiapoptotic proteins. Upon the induction of apoptotic stimuli, BH3-only proteins

bind to the antiapoptotic proteins, releasing the proapoptotic molecules and leading to MOMP.

The embedded together model proposes that MOMP does not occur until the multidomain

proapoptotic molecules insert into the outer mitochondrial membrane (OMM) and undergo

conformational changes prior to assuming the conformation needed to generate MOMP (Leber et

al., 2007).

1.3.2 Bcl-2 Proteins and Mitochondrial Morphogenesis

Cells contain many long tubular mitochondria that can interconnect and form networks.

These mitochondrial networks are dynamic and undergo remodeling through cycles of

mitochondrial fission and fusion events (Autret & Martin 2009). Importantly, these networks

can undergo remodeling in response to stress such as changes in energy demand and changing

calcium levels. During apoptosis, mitochondria fragment via mitochondrial fission, which may

promote cytochrome c release, or alternatively may be a consequence of apoptosis (Frank et al.,

2001). Intriguingly, cells that lack Bax and Bak have mitochondria that are shorter with less

extensive network formation and decreased rates of fusion, implying that besides their role in

apoptosis, multidomain proapoptotic Bcl-2 family proteins play an essential role in regulating the

morphology of mitochondria in healthy cells (Karbowski et al., 2006). Conversely, Bax and Bak

have also been implicated in mitochondrial fission prior to cytochrome c release (Desagher &

Martinou 2000).

1.3.3 Bcl-2 proteins and non-apoptotic cell death

Several studies have identified a form of non-apoptotic cell death. In mouse L929

fibroblast cells, the use of the pan-caspase inhibitor benzyloxycarbonyl-valyl-alanyl-aspartic acid

(O-methyl)-fluoro-methylketone (zVAD) directly induced cell death (Yu et al., 2004). Electron

26

micrographs of these cells demonstrated the presence of autophagic vacuoles. A similar

phenomenon was observed in Bax/Bak-/-

mouse embryonic fibroblasts (MEFs). This cell line

was resistant to apoptosis induced by etoposide and staurosporine, but still underwent cell death

(Shimizu et al., 2004). Electron micrographs of these cells revealed the formation of

autophagosomes/autophagolysosomes, thus implicating autophagy in this alternative death

scenario (Shimizu et al., 2004). Treatment of the cells with 3-methyladenine, an inhibitor of the

class III PI3K complex resulted in improved cell viability, indicating that autophagy led to

cellular demise.

1.3.4 Bcl-2 family members and autophagy

The connection between regulators of autophagy and Bcl-2 family members was first

demonstrated with the discovery of Beclin 1 as a novel Bcl-2 interacting protein, and subsequent

analyses demonstrated that Beclin 1 contained a BH3 domain (Liang et al., 1998; Oberstein et

al., 2007). Beclin 1 is the homologue of yeast Atg6, and is an essential protein for the process of

autophagy to occur. Knockout/knockdown experiments of Beclin 1 have demonstrated that

Beclin 1 is required for autophagy and that decreased Beclin 1 protein is associated with an

increased likelihood of tumorigenesis (Liang et al., 1999; He & Levine 2010). Unlike the other

BH3-only Bcl-2 family proteins, Beclin 1 does not exhibit any proapoptotic activity; instead,

overexpression of Beclin 1 induces autophagy (Pattingre et al., 2005; Maiuri et al., 2007).

Subsequent studies have determined that Beclin 1 can interact not only with Bcl-2, but

also with Bcl-xL, Bcl-w and to a lesser extent with Mcl-1 via GST pull-down assays, structural

analysis and fluorescence resonance energy transfer (FRET) assays (Pattingre et al., 2005; Erlich

et al., 2007; Maiuri et al., 2007). However, Beclin 1 does not bind to the proapoptotic Bax, Bak,

Bad, or tBid (Erlich et al., 2007). Importantly, the interaction of Bcl-2 and Beclin 1 was shown

to be highly dependent on the relative abundance of each protein, and overexpression of Bcl-2

could inhibit Beclin 1-induced autophagy in mouse cardiac muscle (Pattingre et al., 2005).

Subsequent experiments demonstrated that the interaction of Beclin 1 and Bcl-2 was mediated

via the BH1 and BH2 domains of Bcl-2 and that overexpression of Bcl-2 resulted in decreased

autophagy and inhibited the formation of the Beclin 1 and Vps34 complex (Pattingre et al.,

2005). Intriguingly, only Bcl-2 that was targeted to the ER had this result on autophagic

inhibition, while mitochondrial targeted Bcl-2 did not demonstrate the same effect. An

27

inhibitory brake needs to be retained on Beclin 1 to prevent complex formation with Vps34, as

Beclin 1 mutants that were unable to bind Bcl-2 had a phenotype of constitutive autophagy and

induced cell death (Pattingre et al., 2005).

Subsequently, the effects of BH3-only proteins in autophagic activation were studied, and

it was discovered that BH3-only proteins could have an inductive effect on autophagic activation

by disrupting the interaction of Bcl-2/Bcl-xL from Beclin (Erlich et al., 2007). Specifically, the

interaction of Beclin 1 with the anti-apoptotic protein Bcl-xL was inhibited by coexpression with

the BH3-only proteins tBid and Bad, but not by the multidomain proteins Bax and Bak (Erlich et

al., 2007). Taken together, this implies that the activation of autophagy requires, to some extent,

the release of Beclin 1 from its inhibitory complexes. Maiuri et al. demonstrated that under

starvation conditions, continuously more Bcl-xL immunoprecipitated with Bad, indicating that

Beclin 1 was complexing with Bcl-xL to inhibit autophagy, providing a greater functional

understanding of autophagic regulation by Bcl-2 family members (Maiuri et al., 2007).

1.3.5 Bcl-2 family proteins and the placenta

In human placentation, a balance needs to be reached between trophoblast proliferation,

differentiation and death to produce a functioning placenta and a healthy baby, while placental

pathologies are characterized with alterations in these processes. In normal placentation, an

increase in trophoblast apoptosis is observed in the third trimester; however, preeclamptic

placentae are characterized by excessive trophoblast apoptosis. The human placenta expresses

many factors of both the extrinsic and intrinsic apoptotic pathways. In terms of the extrinsic

pathway, the placenta has been reported to express tumour necrosis factor (TNF) receptors, X-

linked inhibitor of apoptosis protein (XIAP), Fas, Fas-ligand and caspase-8 (Gruslin et al., 2001;

Kharfi et al., 2006). However, the cell damage present in placental diseases suggests that the

intrinsic pathway may be more significant than the extrinsic (Pongcharoen et al., 2004; Kharfi et

al., 2006; Heazell & Crocker 2008).

The intrinsic pathway of apoptosis is well studied in the placenta. Bcl-2 is present

throughout gestation in the syncytium, and is absent in endothelial cells (Toki et al., 1999; Soni

et al., 2010). Bcl-xL has been detected at the mRNA and protein level but its cellular

localization has not been described. Bax/Bak have been described in third trimester, where they

were found to be localized to the syncytium, and frequently observed in areas with damage,

28

likely contributing to the increased apoptosis observed in term placentae (Ratts et al., 2000;

Cobellis et al., 2007). However, in studies using placental explants to model

hypoxia/reoxygenation stress or altered oxygen conditions, no alterations in Bax or Bcl-2

expression were observed (Heazell et al., 2008). Both Mcl-1 and Mtd/Bok are highly expressed

in reproductive tissues and appear to have very distinct and important roles in trophoblast

homeostasis, and will be discussed in further detail (Hsu et al., 1997; Soleymanlou et al., 2005;

Soleymanlou et al., 2007; Ray et al., 2009).

1.4 Myeloid Cell Leukemia Factor 1

Mcl-1L is a 37 kDa prosurvival member of the Bcl-2 family. It was first cloned as an

early response gene in myeloid cells in response to cytokine treatment, but has been

demonstrated to be expressed in a variety of cell types (Kozopas et al., 1993). Mcl-1 expression

is important during development as Mcl-1 knockout blastocysts fail to undergo implant, and is

also important for differentiation along the monocyte/macrophage pathway in ML-1 human

myeloid leukemia cells (Kozopas et al., 1993; Rinkenberger et al., 2000). Mcl-1 has significant

homology to Bcl-2 and in particular the C-terminus of Mcl-1 is similar to that of Bcl-2; however,

it is unique in its N-terminal domain having PEST (proline, glutamic acid, serine, threonine)

sequences, that are known to target proteins for rapid proteasomal turnover (Figure 1.4.1)

(Kozopas et al., 1993; Day et al., 2005). The protein contains BH1-3 domains as well as a

carboxy-terminal transmembrane (TM) domain (Yang et al., 1995). The N-terminal region of

Mcl-1 plays an important role in both the turnover of Mcl-1 as well as its localization. Deletion

of the first 79 amino acids of Mcl-1 impairs both its localization to the mitochondria and its anti-

apoptotic abilities (Germain & Duronio 2007).

Mcl-1 has several different isoforms. Mcl-1S is a splice variant lacking exon 2, and

caspase-cleavage at Asp127 and Asp157 results in two different Mcl-1c products (Bae et al.,

2000; Bingle et al., 2000; Herrant et al., 2004). Contrary to the prosurvival function of Mcl-1L,

Mcl-1S, Mcl-1c127 and Mcl-1c157 are proapoptotic molecules (Bingle et al., 2000; Herrant et

al., 2004). Mcl-1 does not bind to all BH3-only proteins with equal affinity; it can tightly bind to

Bax, Bak, Mtd/Bok, Bim, Puma and Noxa and phosphorylated Bad, but its binding to Bik, Bmf

and Hrk is weaker and it does not interact with antiapoptotic proteins (Leo et al., 1999; Day et

al., 2005).

29

Figure 1.4.1. Diagrammatic representation of Mcl-1 isoforms.

Mcl-1L contains BH1-3 domains and a transmembrane domain as well as N-terminally located

PEST sequences that promotes its rapid turnover. Caspase-cleavage sites at Asp127 and Asp157

results in two caspase cleavage products. Mcl-1S is the result of exon II skipping, resulting in a

proapoptotic protein product that closely resembles BH3-only proteins. TM: transmembrane

region; BH: Bcl-2 homology domain.

30

1.5 Matador/Bcl-2 related ovarian killer (Mtd/Bok)

Matador/Bcl-2 related ovarian killer (Mtd/Bok) is a multidomain proapoptotic Bcl-2

family member with BH1-3 domains and a C-terminal transmembrane domain (Hsu et al., 1997;

Inohara et al., 1998). It was first characterized in a yeast two-hybrid screen using Mcl-1 as a bait

in an ovarian fusion cDNA library (Hsu et al., 1997). However, unlike Bax and Bak, Mtd/Bok

cannot bind to Bcl-2 or Bcl-xL, and also does not interact with Bax, Bak or Hrk (Inohara et al.,

1998). To date, Mtd/Bok has only been reported to interact with Mcl-1, Bfl-1 and viral BHRF1

(Hsu et al., 1997). Several theories exist on the mechanism by which Mtd induces apoptosis

independently of Bax and Bak. Firstly, it has been proposed that Mtd could bind and antagonize

other prosurvival factors, namely Mcl-1 and Bfl-1. However, a mutant Mtd without a BH3

domain retains apoptotic activity. Alternatively, Mtd could induce apoptosis by directly forming

pores in the OMM.

Bok/Mtd has 5 exons, and splicing of exon 3 results in a splicing variant termed

Bok/Mtd-S (Figure 1.5.1) (Hsu & Hsueh 1998). Bok-S retains apoptotic activity, but is unable to

dimerize with antiapoptotic proteins (Hsu & Hsueh 1998). Bok/Mtd also has a placental specific

splice variant lacking exon 2, termed Bok/Mtd-P, that induces apoptotic death under conditions

of reduced oxygenation and oxidative stress (Soleymanlou et al., 2005).

31

Figure 1.5.1. Schematic diagram of Mtd-L, Mtd-P and Mtd-S.

Full-length Mtd-L has all four BH domains and a transmembrane domain. Channel formation

occurs in the region between BH1 and BH2 domains. Mtd-P forms as the result of exon II

skipping, resulting in a truncated BH3 domain. Mtd-S results from exon III skipping resulting in

a fused BH3 and BH1 domain. BH: Bcl-2 homology, TM: transmembrane domain.

32

1.6 Human Placenta and Mtd/Mcl-1

Mtd expression in the placenta has previously been reported to induce apoptosis of the

trophoblast layers (Soleymanlou et al., 2005; Soleymanlou et al., 2007). Analysis of Mtd

expression across placental development determined that MtdL and Mtd-S expression remained

constant across the first trimester of gestation, while Mtd-P transcript levels increased in early

first-trimester compared to later gestational periods (Soleymanlou et al., 2005). The expression

of Mtd isoforms in early first trimester was predominantly localized to proliferating

cytotrophoblast cells (Ray et al., 2009). Analysis of later first trimester (after 12 weeks of

gestation) tissue revealed low Mtd immunoreactivity in cytotrophoblast cells and increased Mtd

expression was observed in the apical membrane of the syncytiotrophoblast layers (Soleymanlou

et al., 2005; Ray et al., 2009). Analysis of Mtd expression in preeclamptic placentae revealed

increased expression of MtdL and Mtd-P in preeclampsia compared to age-matched control

placentae and IUGR placentae (Soleymanlou et al., 2005).

Analysis of Mcl-1 expression in preeclampsia compared to age-matched controls

revealed increased expression of Mcl-1S and Mcl-1c and decreased expression of Mcl-1L in

preeclampsia, contributing to increased trophoblast apoptosis (Soleymanlou et al., 2007). Mtd-L

and Mtd-P overexpression in chinese hamster ovarian (CHO) and human choriocarcinoma BeWo

cells resulted in the formation of apoptotic cells, expression of cleaved caspase-3 and increased

cell death (Soleymanlou et al., 2005). Mtd-P expression was induced by hypoxia, and hypoxia-

reoxygenation experiments in explants revealed increased expression of MtdL and Mtd-P and

increased trophoblast cell death compared to control. The effect of increased cell death was

reversed by the overexpression of Mcl-1 (Soleymanlou et al., 2005; Soleymanlou et al., 2007).

Additionally, MtdL was recently demonstrated to have an effect on trophoblast

proliferation. MtdL specifically localizes to cells that are mitotically active in placental tissue,

including the cytotrophoblast cells early in first trimester gestation (Ray et al., 2009). siRNA-

mediated knockdown of MtdL expression resulted in decreased expression of cyclin E, a marker

of the G1 phase of the cell cycle, and inducing increased amounts of MtdL using a stably

transfected cell line resulted in increased cyclin E expression and BrdU incorporation, indicating

enhanced cell cycling in trophoblast cells expressing MtdL (Ray et al., 2009). In preeclampsia,

Mtd was localized to progenitor cytotrophoblast cells that coexpressed either cyclin E or the

33

proliferative marker Ki67, indicating that MtdL was also playing a role in the increased

proliferation of cytotrophoblast cells present in preeclampsia and this was correlated to the

hypoxic environment in preeclampsia (Ray et al., 2009).

1.7 Rationale and Hypothesis

Mtd and Mcl-1 play an important role in controlling trophoblast cell fate by regulating

trophoblast apoptotic cell death and proliferation. Autophagy has recently been recognized as

another important cellular response to stress, and in some cases has been reported to contribute to

accelerated cell death. As well, autophagy is an important component of normal cellular

metabolism and has been reported to be defective or altered in many human disease states and in

response to stressors such as hypoxia, nutrient deprivation and oxidative stress.

Many reports have implicated oxidative stress and hypoxia in the pathogenesis of

trophoblast-related disorders of pregnancy including PE and IUGR, and studies from our lab

have demonstrated that Mcl-1 and Mtd are important mediators of trophoblast cell fate in

response to oxidative stress. Therefore, I sought to evaluate whether autophagy is present in the

human placenta, and if levels of autophagy are altered in preeclampsia and IUGR. Given the

important role Bcl-2 has in regulating autophagy, I sought to examine if Mcl-1, a protein that

shares homology with Bcl-2 and also interacts with Beclin 1, exerted an effect on placental

autophagy, and whether its binding partner Mtd-L could also contribute to aberrant levels of

autophagy in placental pathology. Overall my hypothesis was that Mcl-1 and Mtd are two Bcl-2

family proteins that regulate aberrant autophagy in placental pathology.

34

Chapter 2

2 Materials and Methods

2.1 In Vitro Studies

2.1.1 Cell Culture

JEG3 choriocarcinoma cells (ATCC, Manassas, VA, USA) were maintained at 20%

oxygen tension in Eagle's Minimum Essential Media (EMEM) (ATCC, Manassas, VA, USA)

with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA). JEG3 cells were passaged

by washing with 10 mL of PBS without Ca2+/

Mg2+

and trypsinizing with 0.25% trypsin

(Invitrogen, Carlsbad, CA, USA). Cells were counted using trypan blue dye to exclude dead

cells (Invitrogen, Carlsbad, CA, USA) and a hematocytometer and seeded overnight into 35mm

x 6-well plate for experimentation at a concentration of 2x105 cells for 50% confluence or 4x10

5

cells for 100% confluence. In order to determine the optimal confluency for future experiments,

cells were seeded at 2x105, 4x10

5 and 6x10

5 and collected for protein analysis as JEG3 cells have

the capacity to proliferate beyond a monolayer.

Flp-In T-Rex human embryonic kidney HEK293 cells (gift of Dr. Gingras, Samuel

Lunenfeld Research Institute, Toronto, Canada) were maintained at 20% oxygen tension in

Dulbecco's Modified Eagle's (DME) high glucose medium with 10% tetracycline-free FBS

(Invitrogen, Carlsbad, CA, USA) and 0.01% Blasticidin (Invitrogen, Carlsbad, CA, USA). The

human MtdL gene was amplified from full-length cDNA hMtdL (Open Biosystems) by PCR

using the forward primer 5'-GGCGCGCCAGAGGTGCTGCGGCGCTCCTCG-3' and the

reverse primer 5'-CAGAGAGATGACCCGGATCCCG-3', as previously described (Ray et al.,

2009). The PCR was digested with AscI/BamHI and cloned into pcDNA5/FRT/TO/GFP (gift of

Dr. Gingras, Samuel Lunenfeld Research Institute, Toronto, Canada). HEK293-GFPMtdL and

HEK293-GFP cell lines were generated by co-transfection of host HEK293-Flp Recombinase

Target (FRT) cells with pOG44 vector, containing Flp recombinase gene, and

pcDNA5FRT/TO/GFPMtdL vector or pcDNA5FRT/TO/GFP vector under the control of a

hybrid human cytomegalovirus (CMV)/tetracycline promoter, and clones were selected for

hygromycin resistance as previously described (Ray et al., 2009). HEK293-GFPMtdL and

HEK293-GFP cells were maintained in DME high glucose media with 10% FBS, 0.01%

35

blasticidin and 0.4% hygromycin (Invitrogen, Carlsbad, CA, USA). Cells were washed with

Ca2+

/Mg2+

free PBS and trypsinized with 0.05% trypsin (Invitrogen, Carlsbad, CA, USA) and

were seeded at a concentration of 4x105 cells/mL into 35mm 6-well plates or in 100mm plates

for immunoprecipitation experiments. Expression of GFPMtdL or GFP was induced with

doxycycline treatment (2.5ng/mL and 5ng/mL, Sigma-Aldrich Inc., St. Louis, MO, USA).

2.1.2 Protein Extraction

Cells were collected on ice in RIPA buffer (ddH2O, 3% 5M NaCl, 1.66% 3M Tris-HCl

pH 7.5, 1% NP-40) with protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA). The

lysate was vortexed and incubated on ice for 10 mins, then centrifuged for 10 mins at 14000xg at

4oC. For immunoprecipitation studies, HEK293-GFPMtdL and HEK293-GFP cells were cross-

linked using 1% formaldehyde at room temperature for 10 mins, then rinsed in cold PBS and

collected with 1% triton-x buffer (#9803, Cell Signaling Technology Inc., Danvers, MA, USA)

containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein concentration

was measured in duplicates using Bradford Protein Assay.

2.1.3 Bradford Protein Assay

Standards were created in duplicate in PBS at the following concentrations using a stock

solution of 25µg/µL of BSA in PBS: 0, 1.25, 2.5, 5, 7.5, 10, 12.5 µg/µL protein. 1µL of each

sample was added to 800µL of PBS and 200µL of Bradford Dye (Bio-Rad Laboratories,

Hercules, CA, USA) was added to all standards and samples. The mixture was vortexed and

transferred to plastic cuvettes and the protein concentration was measured using an absorbance

spectrophotometer at 595nm.

2.1.4 Western Blot Analysis

Following Bradford protein analysis, 15 µg of protein from each sample was diluted with

15 µg of 2x sample buffer (10 mL of 1M tris pH6.8, 3.2g SDS, 16mL glycerol, 8mL β-

mercaptoethanol, 4mL 0.1% Bromophenol blue, 1mL of ddH2O, diluted in a 1:1 ratio with

ddH2O). The samples were seperated by SDS-PAGE. The SDS-PAGE gels were run at 100V

with the following buffer:14.4g Tris base, 3.03g glycine, 0.1% sodium dodecylsulfate (SDS) in

double-distilled (dd)H2O for a total volume of 1L. Proteins were transferred onto polyvinylidene

fluoride (PVDF) membranes by hydrating the membranes in 100% methanol and transferred at

36

100V using the following transfer buffer: 14.4g Tris base, 3.03g glycine, 20% methanol in

ddH2O for a total volume of 1L, for 1 hr at room temperature with an ice pack to prevent

overheating. Anti-rabbit polyclonal Mcl-1L (12% gel, dilution 1:1000, S-19, Santa Cruz

Biotechnology, Santa Cruz, CA, USA), anti-rabbit polyclonal LC3B-II (15% gel, dilution

1:2500, ab48394, Abcam, San Francisco, CA, USA), anti-rabbit monoclonal PI3KIII/Vps34 (6%

gel, dilution 1:1000, #3358, Cell Signaling Technology Inc., Danvers, MA, USA), anti-mouse

monoclonal Beclin (10% gel, dilution 1:500, am 1818a Abgent Inc., San Diego, CA, USA), anti-

goat polyclonal β-actin (dilution 1:1000, I-19 Santa Cruz Biotechnology, Santa Cruz, CA, USA),

anti-rabbit polyclonal Mtd/Bok (15% gel, dilution 1:200, AP1310a, Abgent Inc., San Diego, CA,

USA) and anti-mouse monoclonal GFP (12% gel, dilution 1:500, B-2, Santa Cruz

Biotechnology, Santa Cruz, CA, USA) membranes were blocked in 5% non-fat milk in Tris-

buffered saline Tween-20 (TBST) for 1 hr at room temperature, with incubation of primary

antibody in 5% non-fat milk at 4oC overnight. 3x10 min washes in TBST were performed and

membranes were then incubated with horse-radish peroxidase (HRP) conjugated anti-rabbit, anti-

mouse and anti-goat secondary antibodies (dilution 1:5000, Santa Cruz Biotechnology, Santa

Cruz, CA, USA) for 1 hr at room temperature in 5% non-fat milk diluted in TBST. Anti-rabbit

monoclonal PI3KIII/Vps34 (6% gel, dilution 1:1000, #4263, Cell Signaling Technology Inc.,

Danvers, MA, USA), anti-rabbit monoclonal Phospho(Thr389)-p70S6K (8% gel, dilution

1:1000, #9234, Cell Signaling Technology Inc., Danvers, MA, USA) and anti-rabbit monoclonal

Beclin (10% gel, dilution 1:1000, #3495, Cell Signaling Technology Inc., Danvers, MA, USA)

membranes were blocked in 5% bovine serum albumin (BSA) in TBST for 1 hr, and the primary

antibody was diluted in 5% BSA overnight at 4oC. The blots were subjected to 3x10 min washes

in TBST, and incubated with HRP-conjugated secondary antibody (1:5000, Santa Cruz) at room

temperature in 5% BSA for 1 hr. The membranes were then washed using 3x10 min washes in

TBST. Detection of HRP-conjugated secondaries was performed using ECL plus

chemiluminescent reagent (PerkinElmer Inc., Waltham, MA, USA) and imaged on x-ray film

(GE Healthcare Limited, Pollards Wood, Buckinghamshire, UK) or using a VersaDoc gel-

documentation system (Bio-Rad Laboratories, Hercules, CA, USA).

2.1.5 Immunoprecipitation studies

Following treatment, HEK293-GFPMtdL or HEK293-GFP cells were cross-linked using

1% formaldehyde diluted in PBS for 10 min at room temperature, then rinsed twice in cold PBS

37

and collected with 1% triton-x buffer (#9803, Cell Signaling Technology Inc., Danvers, MA,

USA) with a protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA). The lysate was

briefly sonicated on ice for 3x10 second pulses at medium speed and protein concentration was

obtained using Bradford protein assay. Two hundred micrograms of cell lysate at a final

concentration of 1 µg/µL was pre-cleared by rotating at 4oC for 3 hrs using 30 µL of protein A-

agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The beads were removed

from the sample by centrifugation of the sample at 4oC 3000xg for 5 min. The supernatant was

transferred to a new Eppendorf tube. The lysate was incubated rotating at 4oC overnight with

anti-rabbit monoclonal PI3KIII/Vps34 antibody (dilution 1:50, #3358 Cell Signaling Technology

Inc., Danvers, MA, USA), anti-rabbit monoclonal Beclin (dilution 1:100, #3495, Cell Signaling

Technology Inc., Danvers, MA, USA), anti-rabbit polyclonal Mcl-1 (dilution 1:20, S-19, Santa

Cruz Biotechnology, Santa Cruz, CA, USA) and anti-rabbit normal IgG was used as a negative

control (Santa Cruz Biotechnology, Santa Cruz, CA, USA). JEG3 cells were collected with

RIPA buffer with protease inhibitor cocktail and precleared with 30µL of protein A-agarose

beads, then incubated with anti-rabbit polyclonal Mcl-1 (dilution 1:20, S-19, Santa Cruz

Biotechnology, Santa Cruz, CA, USA) primary antibody overnight at 4oC.

After incubation with the primary antibodies, 30 µL of protein A-agarose beads were

added to each sample and incubated at 4oC for 3 hrs slowly rotating. Samples were centrifuged

at 40C at 3000xg and washed with 500 µL of buffer, and 2 washes of 500 µL of PBS. Following

the washes, 25 µL of 3x sample buffer were added to the beads and the mixture was boiled and

centrifuged at 14000xg. The immunoprecipitated lysates were subsequently immunoblotted with

20% of the sample loaded to immunoblot for the input, and 80% was loaded for the interaction of

interest.

2.1.6 Immunofluorescence staining

For experiments involving HEK293 cells, glass coverslips were first coated with poly-L-

lysine prior to plating of cells to enhance cell adherence. JEG3 cells were plated directly onto

glass coverslips.

38

2.1.6.1 Formaldehyde Fixation

Cells were fixed using a 3.7% formaldehyde fixative. 37% formaldehyde was mixed

with media to create a 3.7% fixative solution that was incubated with the cells for 15 mins at

37oC. Cells were then washed twice with PBS and refrigerated at 4

oC prior to staining.

2.1.6.2 Lysotracker® Red Staining

Lysotracker® Red (Invitrogen, Carlsbad, CA, USA) was used to monitor lysosomal

production as a surrogate marker for autophagy. Lysotracker® Red dye at a concentration of

1mM in DMSO was mixed with cell media to create a mixture at a final concentration of 50nM.

This mixture was incubated with the cells for 1 hr at 37oC. Formaldehyde fixation was

subsequently performed. The cells were washed 3x5 mins in PBS with gentle rotation, and

nuclei were subsequently counterstained with 4',6-diamidino-2-phenylindole for 20 min (DAPI,

Invitrogen, Carlsbad, CA, USA). Coverslips were mounted onto microscope slides using a drop

of 50% glycerol in PBS as mounting medium, and adhered to the coverslip with nailpolish.

2.1.6.3 Co-immunofluorescence Staining Experiments

In experiments utilizing GFP- or RFP- tagged proteins, endogenous fluorescence was

imaged by deconvolution microscopy. In order to visualize non-fluorescent proteins, following

fixation cells were permeabilized with 0.2% Triton-X solution dissolved in PBS for 5 min with

gentle rotation, and washed 2x5 min in PBS. Blocking was performed for 1 hr at room

temperature with 5% normal donkey serum (NDS). The following primary antibodies were used

overnight at 4oC: anti-rabbit polyclonal Mcl-1 (dilution 1:200, S-19 Santa Cruz), anti-rabbit

polyclonal Mtd/Bok antibody (dilution 1:400, H-151 Santa Cruz), and anti-mouse monoclonal

calreticulin (dilution 1:2000, ab22683 Abcam). Normal rabbit IgG and normal mouse IgG were

used at the same concentration as the primary antibodies as negative controls (Santa Cruz

Biotechnology, Santa Cruz, CA, USA). The primary antibodies were diluted in a solution of

antibody diluent (0.4% sodium azide, 0.625% gelatin in PBS filtered with 0.22mm syringe-

driven filter) mixed at a 1:1 ratio with 5% NDS. Following incubation with the primary

antibody, cells were washed 3x5 min with PBS on a rotating platform. Alexa Fluor® 594-

conjugated and 488-conjugated donkey secondary antibodies were diluted into antibody diluent

and used for 1 hr at room temperature (dilution 1:200, Invitrogen, Carlsbad, CA, USA). The

cells were then washed with 3x5 min PBS washes and counterstained with DAPI diluted in PBS

39

for 20 mins. A drop of 50% glycerol in PBS was placed on the section and coverslips were

adhered to the slide using nail polish. Images were subsequently captured using DeltaVision

deconvolution microscopy with z-stacking (Applied Precision, LLC, Issaquah, WA, USA).

2.1.7 Electron Microscopy

HEK293-GFP and HEK293-GFPMtdL cells were plated at 70% confluence onto

Thermanox™ plastic coverslips (Thermo Fisher Scientific, Roskilde, Denmark). The cells were

treated with 2.5µg/mL of doxycycline for 24 hrs. The cells were rinsed with PBS and room

temperature EM fixative (2% gluteraldehyde, 0.1M sodium cacodylate) was pipetted over the

cells and incubated for 1 hr. The fixed cells were then stored at 4oC for 24 hrs prior to

processing into semi-thin silver and thin gold sections by the Mount Sinai Hospital Electron

Microscopy facility. Images were captured using a FEI Tecnai 20 Transmission Electron

Microscope.

2.1.8 Transient Transfection Experiments

2.1.8.1 Overexpression of MtdL and Mcl-1L

pcDNAFlagMtdL vector was generated as previously described (Soleymanlou et al.,

2005). The forward and reverse primers encoded a Kpnl and BamHI restriction site respectively

for cloning into pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). Forward primer: 5'-

CCCGGTACCACCATGATCCGGCCCAGCGTCTAC-3', reverse primer: 5'-

CCCGGATCCGGGTCATCTCTCTGGCAACAACAGGAA-3'.

Human choriocarcinoma JEG-3 cells at 70% confluence were transfected with

3µg/35mm dish of either pcDNA3.1 vector, pcDNA3xFlag vector, pcDNAFlagMtdL or

pcDNA3Mcl-1L vector (gift of Dr. Ruth Craig, Dartmouth Medical School, Hanover, NH) with

8 µL of Lipofectamine 2000® transfection reagent (Invitrogen, Carlsbad, CA, USA). Three

micrograms of DNA were mixed with 250µL of OPTI-MEM low serum media (Invitrogen,

Carlsbad, CA, USA) and 8µL of Lipofectamine 2000® were mixed with 250µL of OPTI-MEM

and incubated at room temperature for 5 min. Subsequently, the DNA and Lipofectamine 2000®

tubes were mixed and incubated for 20 mins and pipetted over the cells, then 1.5mL of EMEM

with 10% FBS were added to each well. The medium was changed 4 hrs after transfection to

40

remove the lipofectamine, and protein extraction was performed 24 hrs and 48 hrs post-

transfection.

2.1.8.2 Mcl-1L silencing experiments

Silencing of Mcl-1L protein in JEG3 cells was performed using 60 nM of 2 different

siRNA Silencer® duplexes targeted against mRNA of Mcl-1L (Ambion Inc., Austin, TX, USA).

Silencer® scramble sequence control siRNA (SS), which does not correspond to any known

gene product was used as a control. Sixty nanomolars of siRNA and 6 µL of Lipofectamine

2000® were each mixed with 250 µL of OPTI-MEM, and incubated for 5 mins. The two tubes

were subsequently mixed and incubated at room temperature for 20 mins and the mixture was

pipetted over the cells and 1.5 mL of EMEM with 10% FBS was added to each well. Media was

changed at 24 hrs post-transfection and protein was extracted 48 hrs post-transfection.

2.1.8.3 Transient Expression of RFP-Mcl1L

Mcl-1L was cloned into a pdsRed2 vector (gift of Dr. Andrea Jurisicova, Samuel

Lunenfeld Research Institute, Toronto, ON, Canada) by the use of HindIII/BamHI restriction

sites. Forward primer: 5'-CCCAAGCTTATGTTTGGCCTCAAAAGAAACGCGG-3', reverse

primer:5'-CGCGGATCCCTTATTAGATATGCCAAACCAGCTCC-3'.

HEK293-GFP or HEK293-GFPMtdL were transfected with pdsRed vector or

pdsRedMcl-1L using 6 µL of Lipofectamine 2000® for 24 hrs. Three micrograms of DNA and

6 µL of Lipofectamine 2000® were each mixed with 250 µL OPTI-MEM, incubated at room

temperature for 5 mins, and mixed together for 20 mins prior to cell treatment. 1.5mL of DME

media with 10% FBS were then added to each well. Media was changed 4 hrs after transfection

and the cells were treated with 2.5ng/mL of doxycycline to induce either GFP or GFPMtdL

expression. Formaldehyde fixation was performed 24 hrs post doxycycline treatment.

2.1.9 SNP Treatment

Sodium nitroprusside (SNP, Sigma-Aldrich Inc., St. Louis, MO, USA) crystals were

diluted into EMEM media to create a 100mM stock that was subsequently aliquotted and frozen

at -20oC. JEG3 cells at 70% confluence were treated with SNP at a concentration of 2.5 and 5

41

mM for 6, 12 and 24 hrs. After SNP treatment, either protein extraction, co-immunofluorescence

staining or lysotracker red staining was performed.

2.1.10 Etoposide Treatment

JEG3 cells were treated with etoposide, a topoisomerase II inhibitor. Twenty-five, 50

and 100 uM of etoposide dissolved in EMEM medium with 10% FBS were pippetted over the

cells and incubated for either 24 or 48 hrs. The cells were subsequently collected for protein

analysis. Two microlitres of mouse brain extract (sc-2253, Santa Cruz Biotechnology, Santa

Cruz, CA, USA) were used as a positive control for the unlipidated LC3-I band.

2.1.11 Rapamycin Treatment

Rapamycin is an inhibitor of mTOR and rapidly induces autophagy (Peterson et al., 1999;

Klionsky et al., 2007). Stock rapamycin (Sigma-Aldrich Inc., St. Louis, MO, USA) at a

concentration of 2.5 mg/mL in dimethyl sulfoxide (DMSO) was diluted in medium to perform

time-course and dosage experiments (0.1, 0.2, 0.3, 0.4 µM rapamycin) in JEG3 cells. JEG3 cells

at 70% confluence were incubated either with 10% FBS or were starved of serum for 3 hrs prior

to treatment with varying concentrations of rapamycin. DMSO was used as the vehicle control.

Protein extraction was performed at 24 and 48 hrs after treatment.

As a positive control, HEK293 cells were treated with 1µM rapamycin and collected for protein

or treated with lysotracker red and fixed 3 hrs after treatment.

2.1.12 Glucose Starvation

JEG3 cells were starved of glucose by washing cells with PBS and then providing cells at

70% confluence in 6-well 35mm plates with 2 mL Hank's Buffered Saline Solution (HBSS+).

Cells were collected for protein extraction at several different timepoints.

2.1.13 Oxygen Experiments

The medium of JEG3 cells at 70% confluence in 35mm x 6-well plates was changed prior

to the beginning of the experiment. Cells were transferred to a 3% oxygen incubator (3% O2, 5%

CO2, 92% N2) whereas control cells were maintained at 20% oxygenation (20% O2, 5% CO2,

75% N2). Cells were collected for protein extraction at 24 and 48 hr time-points.

42

2.2 Human Placental Tissue Experiments

2.2.1 Collection and Sampling

Tissue was collected with informed consent in accordance with the guidelines of

participating institutional ethics guidelines (Faculty of Medicine, University of Toronto and

Mount Sinai Hospital, Toronto, Canada). First-trimester human placental tissue were collected

from elective pregnancy terminations by dilatation and curettage (5-9 weeks of gestation:

n=18;10-15 weeks of gestation: n=21).

Placentae from pregnancies characterized by preeclampsia (PE) and intrauterine growth

restriction (IUGR) were collected based on the American College of Obstetricians and

Gynaecology (ACOG) criteria (2002). Preeclampsia (n=21) was defined using the criteria of

blood pressure ≥ 140/90mmHg after 20 weeks of gestation in women with previously normal

blood pressure and urinary excretion of ≥ 0.3g protein per 24 hrs (2002). Intra-uterine growth

restriction without signs of preeclampsia (n=16) was defined using the criteria of normotensive

blood pressure and fetal weight below the 5th percentile for gestational age. PE, IUGR, age-

matched control (AMC, n=25), and term control (TC, n=20) samples were collected from

random central and peripheral placental regions, rinsed in PBS and snap frozen in liquid nitrogen

immediately after delivery. Patients with diabetes, infection, or kidney disease were excluded.

Preterm and term control groups had no signs of placental pathology.. The clinical information

for placental samples is summarized in Table 2.2.1.

43

Table 2.2.1 Clinical parameters of control, preeclamptic and IUGR patients.

Term (TC)

(n=20)

Preterm/Age-

matched controls

(AMC) (n=25)

PE (n=21) IUGR (n=16)

Mean gestational age

at delivery (weeks)

38.20±0.21 30.97± 1.02 29.61±

0.6298

32.88± 0.7922

Blood

pressure

(mmHg)

Systolic 129.6±7.886 118.8± 4.239 162.5± 3.658 137.0± 12.38

Diastolic 74.71±6.664 75.17± 2.812 99.48± 2.182 85.83± 10.92

Proteinurea (g/24hr) Absent Absent 4.408± 2.079 Absent

Platelets (per L) 214.8± 16.23 244.8± 27.27 119.1± 11.41 218.0± 23.21

AST (U/L) 23.00± 3.464 14.33± 2.028 115.4± 30.19 21.67± 4.807

ALT (U/L) 11.50± 3.175 15.33± 4.333 108.3± 21.89 28.00± 15.10

Fetal sex M: 56%

F: 44%

M: 31%

F: 69%

M: 52%

F: 48%

M: 44%

F: 56%

Fetal weight 3177± 169.9 1822± 254.1 1725± 570.2 1246 ± 42.02

Mode of delivery CS: 100% CS: 62%

VD: 38%

CS: 81%

VD: 19%

CS: 100%

Data are represented as mean ± SEM

AST: aspartate aminotransferase

ALT: alanine aminotransferase

CS: Caesarean section delivery

VD: vaginal delivery

44

2.2.2 Protein Extraction

Protein extraction of human placental tissue was performed using a mortar and pestle

cleaned with 100% methanol and on dry ice. Snap frozen placental chunks were placed in the

mortar and ground with the addition of liquid nitrogen to create a powder. Tissue powder was

transferred to an Eppendorf tube on dry ice. 1 mL of RIPA buffer (ddH2O, 3% 5M NaCl, 1.66%

3M Tris-HCl pH 7.5, 1% NP-40) with protease inhibitor cocktail (Roche, Indianapolis, IN, USA)

was added to each tube containing powdered placental tissue and a homogenizer was used in

2x45 second bursts followed by a 30 second waiting period. Samples were left standing at 4oC

for 1 hr and then transferred to Eppendorf tubes. The lysate was vortexed and incubated on ice

for 10 mins, then cold centrifuged for 10 mins at 14000xg at 4oC. Protein concentration was

measured in duplicates using Bradford protein assay as described above.

2.2.3 Western Blotting

Thirty micrograms of human placental tissue was diluted with 2x sample buffer and was

loaded on SDS-PAGE gels running at 100V with running buffer. Proteins were transferred onto

PVDF membranes by hydrating the membranes in 100% methanol and transferred at 100V using

transfer buffer for 1 hr at room temperature with an ice pack. Anti-rabbit polyclonal LC3B-II

(ab48394 Abcam, 15% gel, dilution 1:2500) and anti-goat polyclonal β-actin (dilution 1:1000, I-

19 Santa Cruz) were blocked in 5% non-fat milk in Tris-buffered saline Tween-20 (TBST) for 1

hr at room temperature, with incubation of primary antibody at 4oC overnight. 3x10 min washes

in TBST were performed and membranes were then incubated with horse-radish peroxidase

(HRP) conjugated secondary antibody diluted in 5% non-fat milk (dilution 1:5000, Santa Cruz

Biotechnology, Santa Cruz, CA, USA) for 1 hr at room temperature. Anti-rabbit monoclonal

Phospho(Thr389)-p70S6K (8% gel, dilution 1:1000, #9234, Cell Signaling Technology Inc.,

Danvers, MA, USA) was blocked in 5% bovine serum albumin (BSA) in TBST for 1 hr, and the

primary antibody was diluted in 5% BSA overnight at 4oC. The blots were washed in TBST

3x10 min washes, and incubated with HRP-conjugated secondary antibody (dilution 1:5000,

Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature in 5% BSA for 1 hr. The

membranes were washed using 3x10 min washes in TBST. Detection of HRP-conjugated

secondaries was performed using ECL plus chemiluminescent reagent (PerkinElmer Inc.,

45

Waltham, MA, USA) and imaged on x-ray film (GE Healthcare Limited, Pollards Wood,

Buckinghamshire, UK) or using the gel-documentation system VersaDoc (Bio-Rad Laboratories,

Hercules, CA, USA).

2.2.4 Immunofluorescence Staining

Co-immunofluorescence staining in human placental sections was performed using

sections cut from paraffin embedded blocks provided by the Mount Sinai Hospital Biobank

(Toronto, Ontario, Canada). The sections were deparaffinized and hydrated by placement into

xylenes for 3x5 min, 100% ethanol for 3x2 min, followed by 2 min in 95%, 90%, 85%, 80%,

70%, 50% ethanol in ddH20, then 2 min in ddH20. Sections were then placed in PBS gently

rotating for 5 mins. In order to expose antigens for antibody binding, a solution was prepared by

mixing 9 mL of citric acid (4.2g dissolved in 200 mL ddH20) and 41 mL of sodium citrate

(14.78g of crystal dissolved in 500mL ddH20) at pH6.0. The sections were placed into a plastic

container filled with the solution and microwaved at power setting 4 for 5 mins, followed by a 15

min cool down with the lid on. The sections were then heated at power setting 4 for 3 mins

followed by a 20 min cool down with the lid off. The sections were then placed in a glass

container of PBS for 3x5 min washes gently rotating. Autofluorescence was quenched by

placing the sections into 0.1% sudan black in 70% ethanol for 15 mins. Remaining sudan black

was removed by 2x5 min PBS washes. The sections were then circled around with an immuno-

pen (Invitrogen, Carlsbad, CA, USA) and subsequently blocked using 5% NDS for 1 hr at room

temperature. Primary antibody was then used at a concentration of 1:100 for anti-mouse

monoclonal Beclin (AM1818a, Abgent Inc., San Diego, CA, USA) and 1:200 for anti-rabbit

polyclonal Mcl-1 (S-19, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in a 1:1

solution of 5% NDS and antibody diluent at 4oC overnight. Normal mouse and rabbit IgG were

used at the same dilution as the primary antibody as a negative control. The sections were then

washed 3x5mins in PBS with gentle rotation, and incubated with Alexa Fluor® 594-conjugated

and 488-conjugated donkey secondary antibodies (dilution 1:200, Invitrogen, Carlsbad, CA,

USA). The fluorophore-conjugated secondary antibodies were each used for 1 hr at room

temperature and then washed 3x5 mins in PBS, and the sections were subsequently

counterstained with DAPI for 15 mins. Coverslips were adhered to the slides using 50% glycerol

in PBS as mounting media and sealed with nailpolish. Slides were refrigerated and imaged with

46

Deltavision deconvolution microscopy with z-stacking (Applied Precision, LLC, Issaquah, WA,

USA).

2.2.5 Electron Micrographs

Placentae from consented patients were diced into 2mm pieces and rinsed with PBS to

remove remaining blood within 10 mins of delivery. The chunks were then placed into room

temperature EM fixative (2% gluteraldehyde, 0.1M sodium cacodylate). After 1 hr at room

temperature, the tissue was maintained at 4oC for 24 hrs and processed into semi-thin silver and

thin gold sections by the Mount Sinai Hospital Electron Microscopy facility. Images were

captured using a FEI Tecnai 20 Transmission Electron Microscope.

2.3 Statistical Analysis

Densitometry for quantitating immunoblot experiments was performed using Image

Quant 5.0 software (Molecular Dynamics, Piscataway, NJ, USA) and Quantity One software

(Bio-Rad Laboratories, Hercules, CA, USA). Expression of the protein of interest was

normalized to β-actin. The coefficient of correlation (R) was calculated using Volocity software

(PerkinElmer Inc., Waltham, MA, USA) to determine the degree of overlap between two

different fluorophores by tracing around the outline of an individual cell.

Statistical analyses were performed using Graphpad Prism 5 software (San Diego, CA

California). For comparisons with multiple groups, a one-way ANOVA with a Newman-Keuls

post-hoc test or Kruskall Wallis test was used, and a t-test or Mann-Whitney U-test was used for

comparisons between 2 groups. Significance was defined as p<0.05 and all data are represented

as mean ± SEM. All experiments were performed with a minimum of 3 technical replicates.

47

Chapter 3

3 Results

3.1 Mcl-1L is a repressor of autophagy

Previous studies from our lab have addressed the importance of Mcl-1L as a key

determinant of cell fate in trophoblast cells. It has been reported that Mcl-1L prosurvival protein

levels are decreased in preeclampsia, contributing to increased trophoblast cell death in this

pathology (Soleymanlou et al., 2007). Intriguingly, Mcl-1L has been reported to interact with

Beclin 1, an essential protein for autophagic initiation (Erlich et al., 2007); however, the role of

Mcl-1L in autophagic regulation has not been investigated.

To determine levels of autophagy, LC3B-II was used as a specific marker of

autophagosomes. LC3B-II is present on the membrane of autophagosomes and is commonly

used to detect autophagic induction by immunoblotting (Klionsky et al., 2007). To first

determine if autophagy could be induced in JEG3 choriocarcinoma cells, a placental cell line of

trophoblast origin, etoposide was employed. Etoposide is a topoisomerase type II inhibitor and

has previously been reported to induce autophagy in a variety of cell lines including human

glioma and cervical cancer cell lines (Katayama et al., 2006; Lee et al., 2007). A dose and time

course for etoposide treatment was performed in JEG3 cells, for 24 and 48 hrs with either 0, 25,

50, or 100 µM of etoposide dissolved into EMEM media. Mouse brain extract was used as a

positive control for the 18 kDa unlipidated LC3B-I, while etoposide mediated induction of

LC3B-II was used to identify the lipidated 16 kDa band corresponding to LC3B-II (Figure

3.1.1a). In order to determine the effect of confluency on autophagic marker expression, cells

were seeded to 50% confluence (2x105 cells per well), 100% confluence (4x10

5 cells per well)

and 6x105 cells per well, as JEG3 cells have the capacity to proliferate beyond a monolayer. The

cells were collected and immunoblotted for LC3B-II expression, demonstrating that confluency

does not affect basal levels of autophagy in JEG3 cells (Figure 3.1.1b). β-actin immunoblotting

was used as a loading control.

In order to determine if Mcl-1L is involved in autophagic regulation, loss and gain of

function studies were performed in JEG3 cells. Mcl-1L was overexpressed by a transient

transfection of pcDNA3.1Mcl-1L (+) or empty vector (control) for 24 and 48 hrs. While

48

Figure 3.1.1 Identification of LC3B-II via immunoblot analysis in JEG3 cells.

A. Representative immunoblot of JEG3 cells treated with etoposide and immunoblotted for

LC3B. LC3B-1 was identified using a positive control (B), while LC3B-II was identified by

using a known inducer of autophagy, etoposide. B = mouse brain extract, a positive control for

LC3B-I. B. Representative immunoblot of JEG3 cells plated with 50% confluence, 100%

confluence and 6x105 cells per well (150%) and immunoblotted for LC3B-II. β-actin was used

as a loading control.

49

overexpression of Mcl-1L at 24 hrs did not have an effect on LC3B-II protein expression,

overexpression of Mcl-1L for 48 hrs resulted in a more than 30% reduction in expression of

LC3B-II (Figure 3.1.2). β-actin was used as a loading control in order to perform quantification

using densitometric analysis, demonstrating that the 31.3±11.4% fold-decrease in LC3B-II

expression was significant compared to control pcDNA3.1 transfected cells (Mann Whitney U-

test, p<0.05, n=3).

To perform loss of function experiments, Mcl-1L expression was suppressed using two

different siRNA sequences targeted against Mcl-1 mRNA in JEG3 cells (Figure 3.1.3, upper

panel). Mcl-1L protein expression was quantified using densitometric analysis (Figure 3.1.3, left

lower panel). Mcl-1L protein expression was significantly decreased at 48 hrs post-treatment by

72.7± 3.9% for duplex 1 (D1), and 61.2±8.0% for duplex 2 (D2) relative to scramble sequence

(SS) control (one-way ANOVA with Newman-Keuls post-hoc test, p<0.01, n=4). LC3B-II

protein levels were then analyzed by immunoblotting and densitometric analysis (Figure 3.1.3,

lower right panel). LC3B-II protein levels were found to be significantly increased with both

sequences (D1: relative fold increase of 1.79±0.32; D2: relative fold increase of 1.64± 0.19) in

response to Mcl-1L silencing as compared to SS control (one-way ANOVA with Newman-Keuls

post hoc test, p<0.05, n=4).

Lysosomal number is another marker of autophagy and is increased in end-stage

autophagy, prior to lysosomal fusion with autophagosomes (Klionsky et al., 2007). Lysosomes

were monitored using Lysotracker® Red, a tracer dye specific to the lysosomal compartment.

To further verify the effect of Mcl-1L on autophagy, JEG3 cells were transfected with two

siRNA sequences targeted against Mcl-1L mRNA (D1 and D2) and SS control siRNA, and

lysosomal presence was monitored using Lysotracker® Red (Figure 3.1.4). Silencing of Mcl-1L

with D1 and D2 resulted in increased lysosomal presence as compared to SS control, indicating

that this increase in LC3B-II expression was associated with lysosomal activation, further

indicating a role for Mcl-1L as an inhibitor of autophagy.

Autophagy is controlled by several upstream signaling pathways, many of which impinge

upon the mammalian Target Of Rapamycin (mTOR) (Codogno & Meijer 2005). Under

conditions of mTOR activation, autophagy is inhibited and can be monitored by the

phosphorylation of p70S6K, a protein kinase downstream of mTOR. In response to mTOR

50

Figure 3.1.2 Overexpression of Mcl-1L in JEG3 cells.

Upper panel: Representative immunoblot of Mcl-1L overexpression in JEG3 cells C: control

pcDNA3.1 transfected cells, +: pcDNA3.1Mcl-1L transfected cells. Lower panel: Densitometric

analysis of the protein expression levels of LC3B-II normalized to β-actin and expressed as a

fold change compared to control pcDNA3.1 transfected cells. Overexpression of Mcl-1L

significantly decreased the expression of the autophagy marker LC3B-II at 48 hrs post-

transfection (n=3, Mann-Whitney U-test, *p<0.05).

51

Figure 3.1.3 Silencing of Mcl-1L in JEG3 cells.

Upper panel: Representative immunoblot of Mcl-1L and LC3B-II protein levels in siRNA

targeted silencing of Mcl-1L in JEG3 cells. D1: duplex 1; D2: duplex 2; SS: scramble sequence

control. Lower left panel: Densitometric analysis of Mcl-1L protein expression normalized to β-

actin and expressed as a fold change compared to SS control. Right panel: Densitometric

analysis of LC3B-II protein expression levels normalized to β-actin and expressed as a fold

change relative to scramble sequence control. Silencing of Mcl-1L resulted in significantly

increased expression of LC3B-II at 48 hrs (n=4, one-way ANOVA with post-hoc Newman-Keuls

multiple comparisons test, *p<0.05, **p<0.01).

52

Figure 3.1.4 Effect of Mcl-1L silencing on lysosomal presence in JEG3 cells.

JEG3 cells were silenced with two different siRNA sequences, D1 and D2, targeted against Mcl-

1L mRNA. Forty-eight hours post-treatment the cells were stained with Lysotracker® Red to

visualize lysosomes, demonstrating increased lysosomal presence in Mcl-1L silenced cells

compared to SS. Chromatin is stained with DAPI (blue). SS: scramble sequence control; D1:

Duplex 1; D2: Duplex 2 (n=3).

53

Figure 3.1.5 Rapamycin treatment in HEK293 cells.

A. HEK293 cells were treated with 1uM rapamycin for 3 hrs and immunoblotted for the

expression of p70S6K phosphorylated at Threonine 389. Rapamycin treatment

dephosphorylated p70S6K in conditions of 3 hrs of serum starvation and in nonstarved cells. st:

starved; nst: non-starved; R: rapamycin treated B. As a positive control for lysosomal induction,

HEK293 cells were treated with 1uM rapamycin for 3 hrs resulting in increased lysosomal

induction, as visualized by the use of Lysotracker® Red dye. Nuclei are counterstained in DAPI

(blue), (n=3).

54

activation, p70S6K is phosphorylated at Thr389 (Peterson et al., 1999). Human embryonic

kidney cells (HEK293) were either starved of serum for 3 hrs (st), not-starved (nst) or treated

with rapamycin (R). Rapamycin treatment resulted in dephosphorylation of p70S6K (Figure

3.1.5a). In order to determine the specificity of Lysotracker® Red dye in monitoring lysosomal

activation, HEK293 cells were treated with 1uM rapamycin for 3 hrs and were stained with

Lysotracker® Red dye. Compared to DMSO control treated cells, lysosomes were induced in

response to rapamycin treatment in HEK293 cells (Figure 3.1.5b).

3.2 Mcl-1L and MtdL interact

Bcl-2 family proteins exert their effects on a variety of different cellular processes

dependent upon their specific interaction partners. While Mcl-1L and MtdL have been reported

to interact in a yeast two-hybrid system, I sought to examine if these proteins interact in JEG3

choriocarcinoma cells and HEK293 cells (Hsu et al., 1997).

To demonstrate that Mcl-1L and the multidomain proapoptotic molecule MtdL interact in

trophoblast cells, co-immunoprecipitation studies were conducted in JEG3 cells. JEG3 cells

were transfected with pcDNA3.1Flag vector and pcDNA3.1FlagMtdL and immunoblotted to

confirm transfection (Figure 3.2.1a). Immunoprecipitation of Mcl-1L specifically pulled-down

associated FlagMtdL in cells transfected with FlagMtdL (Figure 3.2.1b). To further confirm this

association in a HEK293 cells, co-immunofluorescence staining was performed. HEK293 cells

stably expressing GFP (HEK293-GFP) and GFPMtdL (HEK293-GFPMtdL) under the regulation

of a hybrid human cytomegalovirus (CMV)/tetracycline promoter, which express GFP and

GFPMtdL in response to doxycyline treatment were utilized. Expression of GFP or GFPMtdL in

HEK293-GFP and HEK293-GFPMtdL cells were induced by treatment of 2.5 ng/mL

doxycycline for 24 hrs, and immunostained using an antibody for endogenous Mcl-1. In

HEK293-GFPMtdL cells, MtdL depicted in green, and Mcl-1 in red, colocalized in apoptotic

cells with condensed nuclei, as indicated by yellow. Nuclei are shown in blue and normal rabbit

IgG was used as a negative control (Figure 3.2.2a).

To confirm the association of specifically the long isoform of Mcl-1 with MtdL, cells

were transfected with Mcl-1L tagged with red fluorescent protein (RFP). Expression of GFP or

GFPMtdL was induced in HEK293-GFP and HEK293-GFPMtdL cells with 2.5 ng/mL

55

Figure 3.2.1 Immunoprecipitation of Mcl-1L in JEG3 cells transfected with FlagMtdL.

A. Representative immunoblot analysis of JEG3 cells transiently transfected with either control

pcDNA3.1Flag or pcDNA3.1FlagMtdL vector and immunoblotted 24 hrs after treatment. B.

Representative immunoprecipitation of endogenous Mcl-1L in JEG3 cells transfected with

FlagMtdL for 24 hrs. Immunoprecipitation revealed an association of Mcl-1L with FlagMtdL 24

hrs post-transfection. Neg: Negative IgG control, (n=3).

56

57

Figure 3.2.2 Colocalization of MtdL and Mcl-1L in HEK293 cells.

A. HEK293-GFP and HEK293-GFPMtdL cells treated with 2.5ng/ml doxycycline for 24 hrs

were stained for endogenous Mcl-1 (red) revealing association between MtdL and Mcl-1

(yellow). Nuclei were counterstained with DAPI (blue). B. Immunofluorescence staining of

HEK293-GFP and HEK293-GFPMtdL cells treated with 2.5 ng/mL doxycycline for 24 hrs and

transfected with pdsRed or pdsRedMcl-1L vector. Nuclei were counterstained with DAPI (blue).

58

doxycycline for 24 hrs and the cells were transfected with either pdsRed vector (control) or

pdsRedMcl-1L (Figure 3.2.2b). Cells transfected with pdsRedMcl-1L and expressing GFPMtdL

demonstrated colocalization of MtdL and Mcl-1L in a perinuclear cellular location. Chromatin

was counterstained with DAPI (blue).

3.3 MtdL is a novel inducer of autophagy

To determine if MtdL was affecting autophagy, gain of function studies with MtdL were

performed in stably transfected HEK293 cells. Stably transfected HEK293 cells were used due

to the highly cytotoxic properties of MtdL, a protein that rapidly induces apoptosis when present

at high concentrations (Hsu et al., 1997). In order to examine the effects of the expression of

MtdL at low-levels, the stably transfected cells were used and treated with low concentrations of

doxycycline to induce low-level GFPMtdL expression. Initially, to elucidate the appropriate

concentration of doxycycline and timepoint for further experiments a timecourse for induction of

GFPMtdL was performed for 12, 24 and 48 hrs using 0, 2.5 and 5 ng/mL of doxycycline (Figure

3.3.1a). Densitometric analysis of GFPMtdL expression in response to doxycycline treatment

demonstrated that MtdL was significantly elevated in response to 2.5 and 5 ng/mL doxycycline

treatment at 24 hrs of treatment, and the two dose levels did not differ significantly (Figure

3.3.1b, one-way ANOVA with Newman-Keuls post-test, p<0.001, n=3). Densitometric analysis

of LC3B-II expression following treatment with 2.5 and 5 ng/mL doxycycline for 24 hrs

demonstrated that both concentrations of doxycycline elevated LC3B-II expression (2.5ng/mL:

4.47± 1.02; 5ng/mL: 6.15± 1.11 fold increase, one-way ANOVA with Newman-Keuls post-test,

p<0.05, n=3) (Figure 3.3.1c). However, the two treatments did not produce significantly

different levels of LC3B-II protein expression. Therefore, further experiments were performed

with 2.5ng/mL of doxycycline for 24 hrs of treatment.

To determine the specific effect of GFPMtdL compared to control GFP expressing cells,

HEK293-GFP and HEK293-GFPMtdL cells were induced with 2.5 ng/mL of doxycycline and

immunoblotted for LC3B-II and GFP to confirm expression (Figure 3.3.2, upper panel).

Expression of GFPMtdL resulted in significantly increased levels of LC3B-II expression with a

fold increase of 4.59± 1.03 compared GFP-expressing cells, as quantified by densitometric

analysis (Figure 3.3.2, lower panel; one-way ANOVA with Neuman-Keuls post-hoc test,

p<0.001, n=3). HEK293-GFP and HEK293-GFPMtdL cells were subsequently stained with

59

Lysotracker® Red, revealing increased lysosomal formation in cells expressing GFPMtdL as

compared to GFP control cells and cells not treated with doxycycline (Figure 3.3.3). Together,

these findings demonstrate that MtdL exerts an opposing effect to that of Mcl-1L in autophagic

induction.

60

Figure 3.3.1 Time and dose-course for doxycycline-mediated induction of MtdL.

A. Immunoblot analysis for the expression of GFPMtdL and LC3B-II protein in response to 0,

2.5 and 5 ng/mL doxycycline treatment at 12, 24 and 48 hrs post-treatment. B. Densitometric

analysis of GFPMtdL expression at 24 hrs post-treatment, normalized to β-actin relative to 0

doxycycline treatment. C. Densitometric analysis of LC3B-II expression at 24 hrs post-

treatment, normalized to β-actin relative to 0 doxycycline treatment. (n=3, one-way ANOVA

with Newman-Keuls post-hoc test, *p<0.05, ***p<0.001).

61

62

Figure 3.3.2 Immunoblot analysis of HEK293 cells stably expressing MtdL.

Upper panel: Representative immunoblot for GFP, GFPMtdL and LC3B-II expression in stably

transfected HEK293 cells in response to treatment of 2.5 ng/mL of doxycycline. Lower panel:

Densitometric analysis of the expression of LC3B-II in response to doxycyline treatment

normalized to β-actin and expressed as a fold change relative to 0 doxycycline treatment. LC3B-

II expression was significantly elevated 4.47±1.02-fold with GFPMtdL expression compared to

GFP induced cells (n=3, one-way ANOVA with Newman-Keuls Multiple comparison test,

***p<0.001).

63

Figure 3.3.3 Lysotracker® Red staining in HEK293 cells.

HEK293-GFP and HEK293-GFPMtdL cells were induced to express either GFP or GFPMtdL

using 2.5 ng/mL doxycycline. Twenty-four hrs post-induction, Lysotracker® Red staining was

performed to visualize lysosomes. Nuclei are counterstained with DAPI (blue). Increased

lysosomal formation was observed in cells expressing GFPMtdL (n=4).

64

Figure 3.3.4 Intracellular localization of MtdL in HEK293 cells.

HEK293-GFP and HEK293-GFPMtdL cells were induced with 2.5 ng/mL of doxycycline to

express GFP and GFPMtdL. Twenty-four hrs after induction, the cells were immunostained for

calreticulin, a marker of the ER. GFPMtdL localizes to the ER membrane in cells with

condensed nuclei. Correlation of overlap (R) = 0.93 ± 0.01 for apoptotic cells and 0.59 ± 0.03

for healthy cells. Green: GFP; red: calreticulin; blue: nuclei; (n=6).

65

Figure 3.3.5 Electron micrographs of HEK293 cells stably expressing MtdL.

HEK293-GFP and HEK293-GFPMtdL cells were induced to express GFP and GFPMtdL using

2.5 ng/mL doxycycline for 24 hrs. Autophagosome formation was observed in cells expressing

GFPMtdL, as indicated by the black arrows. *: mitochondria, n: nucleus.

66

To determine if MtdL exerts a stimulatory effect on autophagy at the level of the

endoplasmic reticulum (ER), where other Bcl-2 proteins have been implicated in autophagic

induction (Wada et al., 1995; Pattingre et al., 2005), staining for the ER was performed using

calreticulin (shown in red) as a specific organelle marker (Figure 3.3.4). GFP and GFPMtdL was

induced with 2.5 ng/mL of doxycycline for 24 hrs and subsequently immunostained for

calreticulin and the nuclei were counterstained with DAPI (blue). Normal mouse IgG was used

as a negative control. GFPMtdL (green) localized to the ER and the coefficient of correlation

was measured using Volocity software. The correlation coefficient (R) for MtdL and calreticulin

was calculated to be 0.93±0.01 in cells with condensed nuclei, indicating that MtdL does

translocate to the ER (n=3). Interestingly, in cells that were not condensed, the correlation factor

was determined to be 0.59 ± 0.03 (n=3). This indicates that in cells that are not rounded, a subset

of MtdL translocates to the ER membrane, however in cells that are apoptotic, as identified by

condensed nuclei and cytoplasm, MtdL extensively colocalizes with a marker of the ER.

Consequently, HEK293-GFP and HEK293-GFPMtdL cells that were treated with 2.5

ng/mL of doxycycline for 24 hrs were processed for electron micrograph images to visualize the

presence of autophagosomes. Autophagosomes were identified by characteristic features of a

double-membraned structure present in the cytoplasm of the cell, containing cellular constituents

(Klionsky et al., 2007). The presence of autophagosomes was confirmed for cells expressing

MtdL, as indicated by the black arrow (Figure 3.3.5).

3.4 MtdL decreases the endogenous interaction of Mcl-1L and Beclin 1

Beclin 1 is an important component in upstream autophagic signaling, at the level of

autophagosome nucleation, that has been reported to interact with several prosurvival Bcl-2

family members (Erlich et al., 2007). To determine if Mcl-1L associates with Beclin 1, co-

immunoprecipitation studies for the association of Beclin 1 and Mcl-1L were performed in stably

transfected HEK293 cells (Figure 3.4.1a). Co-immunoprecipitation of Beclin 1 and Mcl-1L

demonstrated that in baseline conditions, Mcl-1L does interact with Beclin 1, leading to low

levels of autophagy, as previously shown in Figure 3.3.2. However, in conditions of MtdL

induction with 2.5 ng/mL doxycycline for 24 hrs, the interaction between Mcl-1L and Beclin 1 is

reduced, as demonstrated by co-immunoprecipitation for Beclin 1 and immunoblotting for

67

68

Figure 3.4.1 Immunoprecipitation of Beclin 1 in HEK293 cells stably expressing MtdL.

A. HEK293-GFP and HEK293-GFPMtdL cells were induced to express GFP and GFPMtdL

using 2.5 ng/mL doxycycline. Immunoprecipitation for endogenous Beclin in HEK293-GFP and

HEK293-GFPMtdL cells revealed decreased association with Mcl-1L with Beclin following 24

hrs of MtdL induction. B. Left panel: Representative immunoblot for Mcl-1L expression in the

lysates (input) immunoprecipitated for Beclin 1. Right panel: Densitometric analysis of the

input normalized to β-actin. C. Left panel: Representative immunoblot from the supernatant of

lysates immunoprecipitated for Beclin 1. Right panel: Densitometric analysis of the supernatant

normalized to resulted in increased levels of Mcl-1L in cells expressing GFPMtdL. D. Relative

abundance of Mcl-1L in the supernatant of lysates immunoprecipitated for Beclin 1 normalized

to the expression of Mcl-1L in the input lysate. GFPMtdL expression resulted in a 1.25± 0.02 -

fold increase in Mcl-1L presence in the supernatant relative to cells expressing GFP alone (n=3,

paired t-test, **p<0.01).

69

associated Mcl-1L (Figure 3.4.1a). In order to quantify this change in association, the lysate was

probed for the relative amounts of Mcl-1L before (input) and after (supernatant)

immunoprecipitation of Beclin 1. HEK293 input lysates expressing GFP and GFPMtdL were

probed prior to immunoprecipitation for the expression of Mcl-1L (Figure 3.4.1b, left panel).

Densitometric analysis and normalization to β-actin revealed a trend towards increased Mcl-1L

expression in response to GFPMtdL expression (Figure 3.4.1b, right panel). The supernatant

remaining after immunoprecipitating for Beclin 1 was subsequently probed for Mcl-1L

expression (Figure 3.4.1c, left panel). Densitometric analysis and normalization to β-actin

revealed a trend towards increased Mcl-1L present in the supernatant of cells

immunoprecipitated for Beclin 1 and expressing MtdL (Figure 3.4.1c, right panel). In order to

determine the relative amount of Mcl-1L remaining in the supernatant after immunoprecipitation

of Beclin 1, the quantity of Mcl-1L in the supernatant was normalized to the amount of Mcl-1L

present prior to immunoprecipitation of Beclin 1 (Figure 3.4.1d). A significantly increased

amount of Mcl-1L protein was determined to be present in the supernatant of cells expressing

MtdL compared to cells expressing GFP, as determined by a paired t-test (n=3, p<0.01). This

indicates that MtdL expression is leading to a decreased Mcl-1L and Beclin interaction,

contributing to increased autophagy.

3.5 Autophagy is elevated in preeclampsia and decreased in IUGR

Preeclampsia is characterized by increased oxidative stress, leading to the activation of

cellular stress responses. Additionally, our lab has reported that there are decreased levels of

Mcl-1L and increased expression of the pro-death MtdL protein in preeclampsia contributing

towards increased trophoblast cell death (Soleymanlou et al., 2007). As autophagy is an

important stress defense mechanism and is regulated by MtdL expression, I sought to examine

the status of autophagy in preeclamptic tissue.

To ascertain the levels of autophagy in human placental tissue, immunoblotting was

performed using LC3B-II as a marker of autophagy (Figure 3.5.1). Immunoblotting for LC3B-II

expression in severe PE placentae compared to AMC and TC placentae revealed significantly

increased levels of autophagy in PE (n=14), with a fold increase of 2.05±0.47

70

Figure 3.5.1 Expression of LC3B-II in normal and preeclamptic placentae.

Immunoblot analysis for LC3B-II in PE, AMC and TC placentae reveals increased expression in

severe PE vs AMC and TC (PE n=14, AMC n=8, TC n=4, One-way ANOVA with Newman-

Keuls post-test, **p<0.01, ***p<0.001).

71

Figure 3.5.2 Expression of LC3B-II in normal and IUGR placentae.

Representative Immunoblot of IUGR, AMC and TC placentae probed for LC3B-II expression

(Kruskall Wallis Test, IUGR n=12, AMC n=12, TC n=7, *p<0.05).

72

Figure 3.5.3 Spatial localization of Beclin 1 and Mcl-1 in normal and preeclamptic

placentae.

Human PE and AMC placentae were immunostained for the localization of Mcl-1 (red) and

Beclin (green), revealing increased coexpression in AMC placentae. CT= cytotrophoblast,

ST=syncytiotrophoblast. Nuclei are counterstained in DAPI (blue), (n=3).

73

compared to AMC (p<0.01, n=8), and a fold increase of 7.54±0.82 compared to TC (n=4,

p<0.001, one-way ANOVA with Newman-Keuls post-test). Conversely, immunoblotting for

LC3B-II expression in IUGR placentae (n=12) demonstrated a fold decrease of 70.8±13.5%

compared to AMC (n=12) and a fold decrease of 58.6±5.7% compared to TC placentae (n=7),

(p<0.05, Kruskall Wallis test, Figure 3.5.2).

To determine the spatial localization of Mcl-1 and Beclin 1 in vivo, co-

immunofluorescence staining experiments were performed using human placental tissue (Figure

3.5.3). In PE placentae, Mcl-1, shown in red, was primarily localized to the nuclei of

cytotrophoblast cells, with minimal cytoplasmic localization. Beclin 1, depicted in green, is

widely expressed in the syncytial layer and cytotrophoblastic cells, as well as in the stroma.

Nuclei are shown in blue (DAPI). In contrast in AMC the expression of Mcl-1 is less restricted

to cytotrophoblast nuclei and is more diffuse and cytoplasmic in the trophoblastic layers. The

spatial localization of these proteins suggests that in PE, Beclin 1 is not coexpressed with Mcl-1,

while there is a higher degree of coexpression in the same cell layers in AMC placentae.

3.6 Electron micrographs indicate increased presence of autophagosomes in PE

To further confirm immunoblotting data regarding increased autophagy in PE, electron

micrographs (EM) of severe PE placentae and control placentae were imaged for the presence of

autophagosomes. Autophagosomes were identified by their characteristic double-membraned

appearance and cytoplasmic contents contained within a vacuole (Klionsky et al., 2007). In

Figure 3.6.1, the syncytial layer of placentae was found to be highly vacuolated in TC, AMC and

PE placentae, while it had a condensed appearance in IUGR placentae. TC and AMC placentae

retained an organized syncytial appearance with a well-retained microvillous membrane (mvm).

In contrast, in PE, the syncytial layer was observed to be disrupted and the mvm was highly

disorganized in appearance. Evidence for increased autophagy was found in PE placentae.

Autophagosomal structures are indicated with a black arrow while mitochondria are indicated

with an asterisk (*).

Electron micrographs for the cytotrophoblast layer revealed that the CT cells of TC,

AMC and IUGR placentae appeared similar, with minimal presence of vacuoles (Figure 3.6.2).

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Syncytial Layer

75

Figure 3.6.1 Electron micrographs of the syncytium of normal and pathological placentae.

Electron micrographs of the syncytium of PE, IUGR, AMC and TC placentae (TC n=3, AMC

n=3, PE n=3, IUGR n=4. Black arrows denote autophagosomes, n:nuclei, *:mitochondria, mvm:

microvillous membrane.

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Cytotrophoblast Cells

77

Figure 3.6.2 Electron micropraphs of CT cells in normal and pathological placentae.

Electron micrographs of cytotrophoblast of PE, IUGR, AMC and TC placentae (TC n=3, AMC

n=3, PE n=3, IUGR n=4. Black arrows denote autophagosomes, CT: cytotrphoblast cell ST:

syncytium, n:nuclei, *:mitochondria, mvm:microvillous membrane, ECM: extracellular matrix.

78

Endothelial Cells

79

Figure 3.6.3 Electron micrographs of the endothelium of normal and pathological

placentae.

Electron micrographs of the endothelium of PE, IUGR, AMC and TC placentae (TC n=3, AMC

n=3, PE n=3, IUGR n=4. Black arrows denote autophagosomes, ST: syncytium, n:nuclei,

*:mitochondria, ECM: extracellular matrix, L: vessel lumen.

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In PE placentae, however, evidence for mitophagy was found in the majority of CT cells, as

indicated by the black arrows and was not observed in AMC, TC or IUGR placentae.

Electron micrographs of the endothelial layer of placental blood vessels demonstrated

that this cell layer is very thin and elongated (Figure 3.6.3). In PE placentae however, this layer

appears disorganized in appearance and has the unique appearance of autophagosomes

encapsulating mitochondria, as indicated by the black arrows. Mitophagic vacuoles were

frequently identified in PE in all cell layers examined, indicating that the increase in autophagy

in PE in part results in increased turnover of mitochondria. This phenomenon was not observed

in IUGR, AMC or PE placentae.

3.7 A model of oxidative stress induces autophagy via alterations in Mcl-1L/MtdL expression

As PE placentae are characterized by a status of increased oxidative stress, I evaluated

the response of Mcl-1L, MtdL and autophagy in response to adverse oxidative conditions (Hubel

1999; Hung & Burton 2006). Sodium nitroprusside (SNP) is a compound that results in the

release of reactive NO species and OH- radicals leading to a status of intracellular nitrile and

oxidative stress. To determine if autophagy in PE may be the result of increased oxidative stress

in trophoblast cells, SNP was used to generate reactive oxygen species in JEG3 choriocarcinoma

cells. Time-course experiments were performed to determine the concentration of SNP (0, 2.5

and 5mM) and time-point (6, 12 and 24 hrs) for autophagic activation (Figure 3.7.1). Following

time-course experiments, 6 hrs of treatment with 2.5 and 5 mM of SNP were chosen for further

experiments.

Subsequent treatment of JEG3 cells with 2.5 and 5 mM of SNP for 6 hrs, resulted in

significantly decreased Mcl-1L expression levels (2.5mM fold decrease: 17.7±4.3%, 5mM fold

decrease: 30.5±7.2% ; p<0.05, one-way ANOVA, n=6), as depicted in Figure 3.7.2a.

Conversely, MtdL levels increased with 2.5 mM of SNP (Figure 3.7.2b; fold increase:

21.0±7.0%; p<0.01, n=4, 1-way ANOVA), but not with 5mM of SNP. These changes in Mcl-1L

and MtdL expression in response to SNP were correlated with significantly increased LC3B-II

expression in response to both 2.5 (Figure 3.7.2c; fold increase: 2.43±0.33) and 5mM (fold

increase: 2.68±0.35) of SNP (one-way ANOVA, p<0.001, n=6). Autophagic induction in

response to SNP treatment was further confirmed with Lysotracker® Red dye, revealing

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increased lysosomal formation in cells treated with 2.5 and 5mM SNP for 6 hrs (Figure 3.7.3).

Nuclei were counterstained in DAPI (blue).

To determine if SNP induced oxidative stress was causing a change in MtdL localization

along with autophagic induction, co-immunofluorescence staining was performed for MtdL and

calreticulin, an ER marker, in JEG3 cells. Staining for the localization of MtdL, depicted in

green, and calreticulin (red) demonstrated a change in MtdL intracellular distribution in response

to SNP treatment (Figure 3.7.4). In control untreated JEG3 cells, MtdL is distributed throughout

the cytoplasm of the cell, and also has strong nuclear staining, as previously reported (Ray et al.,

2009). SNP treatment altered the morphology of JEG3 cells, which gained the appearance of

condensed and abnormally shaped nuclei. SNP treatment resulted in MtdL localization into

aggregates surrounding a peri-ER region, while the ER reflected a vacuolated appearance.

Normal mouse IgG was used as a negative control. Taken together, the presented data indicates

that Mcl-1L and MtdL exert opposing functions on autophagic activation following oxidative

stress in a trophoblast cell model.

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Figure 3.7.1 Expression of Mcl-1L and LC3B-II across time and dosage with SNP

treatment in JEG3 cells.

Representative immunoblot of JEG3 cells treated with 2.5 and 5 mM of SNP for 6, 12 and 24 hrs

and immunoblotted for Mcl-1L and LC3B-II protein expression.

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Figure 3.7.2 Expression of Mcl-1L, MtdL and LC3B-II in SNP treated JEG3 cells.

Representative immunoblot of JEG3 cells treated with 2.5 and 5mM of SNP for 6 hrs. A. SNP

treatment resulted in decreased Mcl-1L expression with both 2.5 and 5 mM of SNP (n=6, one-

way ANOVA with Newman-Keuls Multiple Comparisons test,*p<0.05, **p<0.01. B. MtdL

levels increased with treatment of 2.5 mM of SNP for 6 hrs (n=4, one-way ANOVA with

Newman-Keuls Multiple Comparisons test,**p<0.01. C. LC3B-II expression increased with

both 2.5 and 5 mM of SNP. (n=6, one-way ANOVA with Newman-Keuls Multiple Comparisons

test,***p<0.001).

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Figure 3.7.3 Lysotracker® Red staining in SNP treated JEG3 cells.

JEG3 cells were treated with 2.5 and 5 mM SNP for 6 hrs and stained for lysosomes using

Lysotracker® Red, demonstrating increased lysosomal formation in response to SNP treatment

compared to control. Chromatin are stained with DAPI (blue).

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Figure 3.7.4 Spatial localization of Mtd in SNP treated JEG3 cells.

JEG3 cells treated with 2.5 mM SNP for 6 hrs were stained for MtdL (green) and calreticulin

(red). MtdL forms aggregates and localizes to peri-endoplasmic location in response to SNP

treatment. Chromatin are stained with DAPI (blue).

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Chapter 4

4 Discussion

Preeclampsia is a devastating disorder of placental origin affecting 5-7% of all human

pregnancies, and is diagnosed by the onset of the clinical symptoms of maternal pregnancy-

induced hypertension and proteinurea, with the only known symptomatic intervention being

delivery of the fetus and associated placenta (2002; Hung & Burton 2006). While the cause of

preeclampsia remains unknown, the placenta plays a central role in its genesis and it is

established that the placenta of preeclamptic pregnancies are characterized by a status of

oxidative stress, leading to a hyperproliferative phenotype of the progenitor cytotrophoblast

cells, and increased death of the trophoblastic cell layers, which extrude and are shed in excess as

debris into the maternal circulation (Hubel 1999; Soleymanlou et al., 2005; Ray et al., 2009).

This debris in the maternal circulation is believed to contribute to a status of generalized

endothelial dysfunction, leading to the clinical symptoms of preeclampsia (Roberts 1998;

Roberts & Cooper 2001). While it has been recognized that in preeclampsia, apoptosis is a

major component of trophoblastic cell death, the contribution of autophagy to this placental

pathology has yet to be determined.

4.1 Mcl-1 and MtdL are regulators of placental autophagy

My results indicate that autophagy is highly elevated in all cell layers of preeclamptic

placentae, and I present novel findings that in a trophoblast cell model, Mcl-1L is a repressor of

autophagy, while its binding partner MtdL is an inducer of this lysosomal degradation pathway.

Moreover, I present evidence to support that this regulation of autophagy is at the level of the

ER, where several key players in autophagic regulation have previously been shown to reside

(Heath-Engel et al., 2008), and mediated by changes in the interaction of Mcl-1L with Beclin 1.

Recent publications have demonstrated that crosstalk exists between regulators of

apoptosis and autophagy, which is mediated by the haploinsufficient tumour suppressor protein

Beclin 1. Beclin 1 mobilizes from the trans Golgi network to ER membranes when autophagy is

induced and forms a complex involving the lipid kinase Vps34/PI3KIII leading to autophagic

membrane nucleation (Heath-Engel et al., 2008; Levine et al., 2008; He & Levine 2010). Beclin

1 is a Bcl-2 family protein containing only a BH3 domain, exerts no apoptotic effects, and has

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been reported to interact with Bcl-2, Bcl-xL, Bcl-w and to a lesser extent with Mcl-1 (Oberstein

et al., 2007; Levine et al., 2008). Specifically, the interaction with Bcl-2 was shown to inhibit

starvation-induced autophagy via the sequestration of Beclin 1 from its autophagy activating

complex with Vps34/PI3KIII (Pattingre et al., 2005). Conversely, pro-apoptotic BH3-only

proteins of the Bcl-2 family including Noxa, Puma, BimEL and Bad have been shown to induce

autophagy by binding to and sequestering the prosurvival protein Bcl-2 from interacting with

Beclin 1 (Levine et al., 2008).

While Bcl-2 has been extensively characterized as an inhibitor of autophagy, the

involvement of Mcl-1 has remained neglected in the literature due to its relatively weaker

interaction with Beclin 1. However, in the human placenta, Mcl-1 and Mtd are the key Bcl-2

proteins mediating trophoblast cell survival and proliferation, necessitating the question of

whether they are also involved in the regulation of placental autophagy. Previous reports have

indicated that the expression levels of prosurvival Bcl-2 and proapoptotic Bax are unaltered in

response to oxidative stress in human placental explants, likely not contributing to or accounting

for the increased trophoblastic cell death present in preeclamptic placentae (Heazell & Crocker

2008; Heazell et al., 2008). Importantly, studies from our laboratory have previously described

that the proapoptotic Bcl-2 family protein MtdL and its placental specific isoform MtdP are

elevated in preeclamptic placenta in response to oxidative stress, contributing to increased

trophoblastic proliferation and apoptosis typical of preeclamptic placentae. As well, the

prosurvival Mcl-1L protein has decreased expression in preeclampsia, further promoting cell

death (Soleymanlou et al., 2005; Soleymanlou et al., 2007; Ray et al., 2009). Taken together,

this information suggests that Bcl-2 family proteins exert their functions in a tissue specific

context.

Previous reports have indicated that prosurvival Bcl-2 and Bcl-xL inhibit Beclin 1

dependent autophagy at the level of autophagosomal nucleation (Pattingre et al., 2005). My

results indicate, by the use of loss and gain of function experiments in a trophoblast cell lineage,

that Mcl-1L also has a function in regulating autophagy, and specifically inhibits the process via

an interaction with Beclin 1. Silencing Mcl-1L resulted in the rapid induction of markers of

autophagy, indicating that in baseline cellular conditions, Mcl-1L maintains autophagy at low

basal levels. I also demonstrated that the autophagosomal formation caused by MtdL induction

is due to a sequestration of Mcl-1L from its interaction with Beclin 1, as indicated by my

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immunoprecipitation and overexpression studies. I propose that this event releases the repressive

brake by Mcl-1L on Beclin 1. This allows for autophagic induction, presumably via increased

Beclin 1 interaction with Vps34/PI3KIII as indicated by other studies involving Bcl-2 (Pattingre

et al., 2005) (Figure 4.1.1). Along with MtdL localization to the ER, this provides evidence for

an important role of MtdL in autophagic regulation, and further supports the notion that Bcl-2

family proteins mediate their functional effects as the result of their specific interaction partners

and localization (Pattingre et al., 2005; Heath-Engel et al., 2008; Ray et al., 2009). Intriguingly,

Mtd/Bok is a multidomain BH3 family member, and is the first multidomain proapoptotic Bcl-2

family protein to be directly implicated in autophagic induction and this is the first report to

demonstrate MtdL localization to the ER.

Bcl-2 family proteins have been reported to localize to a variety of cellular compartments

and thereby influence different cellular processes. The most common and well understood

localization is the mitochondria. The Bcl-2 family proteins constitutively localize to the

mitochondria, and the mobilization of Bax from the cytoplasm to the mitochondria is a key event

in the induction of apoptotis (Desagher & Martinou 2000; Soleymanlou et al., 2005; Bhatt et al.,

2008; Ray et al., 2009). MtdL and MtdP mitochondrial mobilization has been reported in

response to oxidative stress in human preeclamptic placentae (Soleymanlou et al., 2005).

Conversely, nuclear localization of MtdL in the human placenta has been reported to induce

progression through S-phase of the cell cycle (Ray et al., 2009). A nuclear localization for Mcl-

1L has been reported to play a role in inhibiting cell cycle progression through the S-phase, via

an interaction with proliferating cell nuclear antigen (PCNA) and at G2 phase via an interaction

with cyclin dependent kinase 1 (Cdk1) (Fujise et al., 2000; Jamil et al., 2005). Bcl-2 family

proteins including Bax,, Bak, Bcl-2, and Mcl-1 have been reported to localize to the ER, where

they have been reported to play a role in regulating cellular Ca2+

homeostasis and signalling

(Yang et al., 1995; Demaurex & Distelhorst 2003; Lao & Chang 2007). An ER localization has

been implicated in autophagic regulation in the early steps of autophagosome membrane

formation (Pattingre et al., 2005; Heath-Engel et al., 2008; Hamasaki & Yoshimori 2010).

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Figure 4.1.1 Putative model of MtdL induced autophagy.

In baseline conditions, Mcl-1L directly interacts with Beclin 1 to inhibit autophagosome

formation. When MtdL expression is induced, MtdL complexes with Mcl-1L thereby allowing

Beclin 1 to interact with Vps34/PI3KIII at the level of the ER thereby induction autophagosome

formation.

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Studies on cell death in preeclamptic placentae have reported on apoptosis and necrosis

as mechanisms for trophoblastic shedding into the maternal circulation, but have not yet

examined the relative contribution of autophagy towards trophoblastic cell death. In this study, I

provide evidence that autophagy is significantly elevated in severely preeclamptic placentae,

corroborating findings from a previous study reporting the presence of autophagosomes in the

trophoblast cells layers of preeclamptic placentae (Oh et al., 2008). Herein, I provide evidence

for the presence of autophagy in the trophoblast cell layers and in the endothelium of placental

blood vessels via electron microscopy. The syncytiotrophoblast layer is the first layer exposed to

oxidative damage as it is directly bathed in maternal blood. In preeclampsia, the placenta

experiences aberrant blood flow, and consequently is exposed to hypoxic injury and oxidative

stress (Jauniaux et al., 2000; Soleymanlou et al., 2005; Jauniaux et al., 2006; Heazell et al., 2008;

Hung et al., 2008). The highly vacuolated and disorganized appearance of the

syncytiotrophoblast layer of PE placentae indicates that autophagy is exacerbated in this cell

layer, likely in response to oxidative stress, and may be contributing to the observed excessive

cell death in the syncytium. In stark contrast, the trophoblastic layer in AMC and TC placentae

retains an organized appearance with a well-defined syncytium and microvillous membrane.

Placentae from fetuses with IUGR had an entirely distinguishable phenotype from PE

placentae, supporting the notion that IUGR and PE are two different disorders of pregnancy

stemming from different placental insults (Burton et al., 2009). Via electron microscopy, IUGR

placentae were easily identifiable by a compact syncytial phenotype with minimal vacuolization.

Little evidence was found supporting autophagic activation in IUGR, via both western blot

analysis for LC3B-II and electron micrographs. While IUGR placentae have been reported to be

in a condition of general hypoxia, the status of oxidative stress in IUGR has not been extensively

examined (Nevo et al., 2008; Zamudio et al., 2010). Several reports indicate that IUGR

placentae are not under oxidative stress, while in contrast, placentae from patients with PE and

IUGR have elevated expression of markers for placental oxidative stress and decreased

antioxidant defense mechanisms, possibly indicating that PE placentae uniquely experience

oxidative stress while IUGR placentae are simply hypoxic (Sahlin et al., 2000; Takagi et al.,

2004; Zamudio et al., 2010).

The IUGR fetus has been reported to be nutrient deprived due to both decreased placental

to fetal amino acid transport via System A activity, and a decrease in villous surface area for

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nutrient exchange to occur (Jansson et al., 2002; Pardi et al., 2002; Regnault et al., 2005). While

amino acid transport is decreased from the placenta to the fetus, glucose transport remains

unaltered, despite many reports of the fetus being in a state of hypoglycemia in utero (Jansson et

al., 1993; Jansson et al., 2002). Although there is no change in the abundance of placental

glucose transporters, it has been reported that IUGR placentae have increased glycolytic

metabolism compared to control placentae, thus contributing to fetal hypoglycemia (Challis et

al., 2000). Recently, it has been proposed that in response to decreased oxygen availability in

IUGR placentae, the placenta undergoes reprogramming, switching it from oxidative to

anaerobic metabolism, leading to increased glucose metabolism and decreased ROS formation

via mitochondrial respiration (Illsley et al., 2010). This indicates that IUGR placentae are not in

a state of nutrient deprivation but contribute to fetal undernutrition and subsequent fetal growth

reductions via abnormal metabolism in response to a low oxygen environment (Illsley et al.,

2010). The build-up of placental amino acids due to decreased transport to the fetus would also

have a powerful inhibitory effect on autophagy, via mTOR signaling (Codogno & Meijer 2005).

These findings are corroborated by my data that the level of autophagy, as assessed by the

marker LC3B-II and electron microscopy, is significantly decreased in IUGR placentae relative

to controls, supporting the notion that IUGR placentae are not in a state of starvation or oxidative

stress.

4.2 Oxidative stress induces autophagy in preeclampsia

It is widely accepted that preeclampsia is associated with an increased status of both

oxidative and nitrile stress in the placenta(Hubel 1999; Burton 2004; Myatt & Cui 2004; Burton

et al., 2009). In my study, SNP treatment was used to generate oxidative stress to model PE in

JEG3 cells. SNP has the ability to release NO, resulting in a status of nitrile stress, and donates

hydroxyl radicals via Fenton reaction, resulting in downstream Bcl-2 family activation (Cardaci

et al., 2008). Our lab has previously utilized SNP treatment in JEG3 cells and found that it

induced trophoblast cell death via mobilization of Mtd/Bok from the cytoplasm to the

mitochondria, and death could be reversed by the overexpression of Mcl-1L (Soleymanlou et al.,

2007; Ray et al., 2009). In this study, I report that SNP treatment in JEG3 cells resulted in

increased autophagy and this effect is correlated with decreased Mcl-1L expression, increased

MtdL expression and altered MtdL localization (Figure 4.2.1).

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While my results enhance the understanding of autophagic regulation in the human

placenta, the functional role of autophagy in pathological and normal human placentae has yet to

be definitively established. Autophagy has been demonstrated to be a cell death mechanism

independently of apoptosis. The extensive vacuolization and lysosomal activation that some

dying cells exhibit has necessitated the classification of Type II (autophagic) cell death, in which

the dying cell exhibits features of autophagy, while the exact mechanism of cellular demise is not

entirely characterized (Gozuacik & Kimchi ; Erlich et al., 2007). In mouse L929 fibroblast cells,

oxidants, ceramide and radiation have been reported to induce caspase-independent cell death

(Fiers et al., 1999). In L929 cells, caspase inhibition by use of the pan-caspase inhibitor

benzyloxycarbonyl-valyl-alanyl-aspartic acid (O-methyl) fluoro-methylketone (zVAD) also

directly induced cell death, which could be inhibited by RNAi directed against Atg7 and Beclin 1

(Yu et al., 2004). Additional data in cancer cell lines reveals that the knockdown of autophagy

machinery can lead to tumour progression, and that Beclin 1 is a haploinsufficient tumour

suppressor protein, further supporting a role for autophagy in cell death (Liang et al., 1999). In

Drosophila, autophagy has been identified as a cell death inducer in salivary glands, and in

contrast, as a cell survival mechanism in the larval fat body (Scott et al., 2004; Berry &

Baehrecke 2007). Remarkably, an engulfment receptor termed Draper (Drpr) has been

discovered to specifically regulate death-inducing autophagy in Drosophila salivary glands, but

not starvation-induced autophagy in the larval fat body, suggesting that distinct factors regulate

autophagy in different cellular contexts (McPhee et al., 2010).

It has also been proposed that autophagy is a cytoprotective response in stressed cells in

order to prolong cell survival. This is supported by studies that have demonstrated increased cell

death in cells that lack the genes essential for autophagy to occur (Levine & Yuan 2005). The

prosurvival capacity of autophagy is most frequently observed in response to nutrient

insufficiency. In autophagy deficient yeast and amoeba, starvation conditions rapidly induce cell

death, and mice that lack Atg5 die during the neonatal period due to starvation (Otto et al., 2003;

Kuma et al., 2004; Levine & Yuan 2005). In human HeLa cells, inhibition of autophagy has

been demonstrated to induce apoptosis, further supporting the cytoprotective role of autophagy

(Boya et al., 2005). Indeed, the autophagy observed in preeclamptic placentae may be defensive,

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Figure 4.2.1 Proposed model of oxidative stress induced autophagy in trophoblast cells.

In response to oxidative stress, cellular ROS formation leads to decreased levels of Mcl1-L and

increased levels of MtdL. Both decreased Mcl-1L and increased levels of MtdL contribute to

freeing Beclin 1 protein, which can then induce autophagy at the level of the ER.

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in order to turnover damaged organelles in response to hypoxia reoxygenation injury in the

trophoblast layer. This notion is supported by my finding of mitophagy in all trophoblastic

layers, suggesting autophagy is recycling damaged mitochondria.

Other reports have indicated that autophagic activation precedes apoptotic cell death and

may predispose cells towards a death phenotype (Yousefi et al., 2006). Autophagy is frequently

discussed as an independent event distinct from apoptosis with the absence of apoptotic features

(Klionsky et al., 2007). However recent reports have identified that autophagic regulation is

intricately connected to that of apoptosis, via the factors Atg5 and Beclin 1, and likely many

other proteins (Luo & Rubinsztein 2007). High levels of autophagy in trophoblast cells that are

dying, and the absence of concomitant cell survival in PE indicates that in preeclampsia, some of

the observed trophoblast cell death may be the result of increased autophagic levels, which in

trophoblast are regulated by the Mcl/Mtd system.

The data presented in this thesis supports that in response to MtdL, both cellular

processes of autophagy and apoptosis are occuring in the same cell. This observation is

consistent with my data regarding increased autophagic activation in PE placental tissue and

previous reports of elevated trophoblast apoptosis in PE (Myatt & Cui 2004; Soleymanlou et al.,

2005). In the data presented herein, extensive colocalization was seen between MtdL and the ER

membrane in apoptotic cells, which were identified by condensed nuclei, suggesting that

autophagy and apoptosis are occuring in the same cell, possibly at the same time. As well,

extensive Mcl-1L and MtdL colocalization was also observed in apoptotic cells, which may be

related to their apoptotic functions as well as their autophagic regulation.

In summary, alterations in the balance between Mtd and its prosurvival binding partner

Mcl-1L is responsible for many of the characteristics of PE placentae, including elevated

autophagy, excessive proliferation of cytotrophoblast progenitor cells and apoptosis of the

trophoblast layers. Mtd induction in PE is likely the result of increased oxidative insult to the

placenta. Many studies have attempted to ameliorate the symptoms of PE by increasing the

dosage of antioxidants consumed by women early in pregnancy, including vitamins C and E.

While antioxidants have profound effects on reversing oxidative damage in vitro, success in

large-scale clinical trials has been very limited (Tannetta et al., 2008; McCance et al., 2010;

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Roberts et al., 2010; Sibai 2010). Therefore, targeting downstream effectors of oxidative stress,

such as Mtd, might provide more promising future interventions for this disease of pregnancy.

Understanding the precise mechanisms regulating autophagic induction in PE and the

functional response in trophoblast tissue to autophagy is essential in order to understand future

therapeutic targets for PE. Therefore future studies in regards to autophagy in the human

placenta should be targeted to elucidating these concepts more fully.

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Chapter 5

5 Future Directions

5.1 Is oxidative stress in trophoblast also sensed by Atg4?

In the data presented herein, a status of oxidative stress was shown to activate autophagy

through changes in Mcl-1L and MtdL expression levels, thus resulting in increased levels of

autophagy. However, reactive oxygen species (ROS) have also been reported to activate

autophagy independently through oxidation of the protein product of autophagy-related gene 4

(Atg4) (Scherz-Shouval et al., 2007). Atg4 is an essential part of the autophagy cascade after

Atg5 conjugation to Atg12, and must become inactive following the initial cleavage of LC3B-I

into LC3B-II to ensure LC3B-II conjugation to the autophagosomal membrane (Hemelaar et al.,

2003). ROS serve as signalling molecules for many cellular processes, and not surprisingly have

also been implicated in autophagic activation in response to nutrient deprivation. However, it

remains to be established whether a cellular insult of ROS could also independently activate

autophagy via an Atg4 response. To evaluate in JEG3 choriocarcinoma cells whether SNP

treatment can inhibit Atg4, the following approach can be undertaken. The activity of Atg4 can

be measured by assessing the relative cleavage of LC3B-I to LC3B-II in lysates treated with

SNP, with and without the reducing agent dithiotheitrol (DTT). As previously reported, DTT

would recover Atg4 activity to baseline conditions if it had been oxidized by ROS in response to

SNP (Scherz-Shouval et al., 2007). This would provide valuable insight into other possible

mechanisms by which adverse oxidative stress conditions are sensed and relayed in trophoblast

cells, and the relative importance different response mechanisms independent of Bcl-2 family

activation have upon autophagic transduction.

5.2 Are Vps34/PI3KIII complexes involved in MtdL induced autophagy?

The class III phosphatidylinositol 3-kinase (PI3KIII) is a regulator of intracellular

membrane trafficking, and is the orthologue to yeast Vacuolar Protein Sorting 34 (Vps34) (Schu

et al., 1993). Vps34/PI3KIII forms a complex involving several other proteins including the

protein kinase hVps15, leading to subsequent Vps34 activation (Yan et al., 2009). In

mammalian cells, Beclin 1 forms two different PI3KIII complexes, one with Atg14, termed

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complex I, and another with UV irradiation resistance-associated gene (UVRAG), termed

complex II, providing evidence that PI3KIII and Beclin 1 can form multiple complexes that are

both involved in autophagic activation (Itakura et al., 2008). In the present study, the association

of Beclin 1 with Vps34/PI3KIII was examined via co-immunoprecipitation analysis, providing

inconclusive results pending further investigation (Figure 5.2.1). Alternatively if the interaction

between Vps34/PI3KIII and Beclin 1 is not altered due to MtdL, the activity of Vps34/PI3KIII

could be altered. Future experiments could confirm whether Vps34/PI3KIII complexes have

enhanced activity in response to MtdL expression, thus leading to autophagosome nucleation and

formation.

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Figure 5.2.1 Co-immunoprecipitation for the association of Beclin 1 with Vps34/PI3KIII in

HEK293-GFP and HEK293-GFPMtdL cells.

Preliminary co-immunoprecipitation for the association of Beclin 1 with Vps34/PI3KIII in

HEK293-GFP and HEK293-GFPMtdL cells indicates no change in assocation. Cells were

treated with 0 or 2.5 ng/mL doxycycline for 24 hr, immunoprecipitated for Vps34/PI3KIII and

immunoblotted for Beclin 1 association (n=3). Neg: negative IgG control; Pos: positive control.

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5.3 Is MtdL contributing to mitophagy?

Bax-interacting factor 1 (Bif-1) has been reported to be an essential protein for

autophagy, and interacts with complex II, which consists of Beclin 1, UVRAG and

Vps34/PI3KIII, and has been hypothesized to deform membranes leading to membrane curvature

due to its N-BAR domain (Frost et al., 2009; Simonsen & Tooze 2009). Additionally, Bif-1 is

localized to mitochondria where it has been reported to directly interact with Bax and has been

demonstrated to activate both Bax and Bak leading to apoptotic induction (Takahashi et al.,

2005). As Bax activation has been demonstrated to lead to mitochondrial fragmentation, Bif-1

has been postulated to be a candidate protein in targeting mitochondria for mitophagic events

(Takahashi et al., 2009). It remains an intriguing possibility whether, if due to the homology and

functional similarities between MtdL and Bax, if MtdL can undergo an interaction with Bif-1.

This is a putative secondary mechanism by which MtdL could be contributing to increased the

mitophagy evidenced in PE.

5.4 What is the functional effect of autophagy in trophoblast?

While the work presented in this thesis has examined the regulatory mechanisms behind

enhanced autophagic activation in response to oxidative stress, the functional outcome of

increased autophagy in trophoblast has not been fully answered in this study. It is apparent from

both the data herein, and from reports of other groups, that PE is characterized by excessive

autophagy and apoptosis within the syncytium (Soleymanlou et al., 2007; Oh et al., 2008; Burton

& Jones 2009). My data supports the proposition that in trophoblast, autophagy may be priming

the cells for subsequent apoptotic events. Several experiments could shed further insight into the

possible roles of MtdL induced-autophagy and the functional effect of autophagy in trophoblast

tissue. Firstly, live-cell imaging experiments should be performed in HEK293-GFPMtdL cells to

determine when autophagy is induced and when signs of apoptosis can be detected within a

single cell. This would shed further light on whether MtdL is inducing apoptosis and autophagy

concurrently or whether autophagy precedes apoptosis in this cell line.

Autophagy-related protein 5 (Atg5) was first discovered in yeast and is involved in the

conjugation steps of autophagosome formation (Mizushima et al., 1998). While full-length Atg5

is required for autophagosome formation, it has also been reported that a cleavage product of

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Atg5 can prime cells towards apoptosis (Yousefi et al., 2006). In response to death stimuli,

calpain-mediated cleavage of Atg5 results in a pro-apoptotic 24kDa isoform of Atg5 that is

mitochondrial targeted and complexes with Bcl-xL, leading a transition from autophagy to

apoptotic cell death via Bax displacement and oligimerization (Yousefi et al., 2006).

In this study, it was observed that MtdL was having an inductive effect on both apoptosis,

as previously reported (Inohara et al., 1998; Soleymanlou et al., 2005), and an independent effect

on autophagic stimulation via an interaction with Mcl-1L. However, whether MtdL is directly

triggering both of these events, or if the apoptotic transition is enhanced by Atg5 cleavage was

not addressed. The status of Atg5 in these cells should be determined in order to address if Atg5

cleavage is also partially responsible for the apoptotic phenotype present in the HEK293-

GFPMtdL cell line in response to low levels of MtdL induction. Alternatively, if Atg5 cleavage

is indeed occurring in these cells, it would be interesting to determine whether MtdL could

possibly be encouraging or exacerbating calpain-mediated cleavage of Atg5. As Atg5-cleaved

toxicity can be rescued by Bcl-2 overexpression, whether Mcl-1L overexpression could as well

rescue the apoptotic switch also remains to be addressed. As preeclamptic placentae are

characterized by a phenotype of both excessive apoptosis and autophagy, it is likely that these

cellular processes are activated concurrently by a variety of cell signalling pathways, including

MtdL functions and possibly via Atg5 cleavage.

Once the dynamics of Mcl-1L/MtdL regulated apoptosis and autophagy are better

understood, functional experiments in placental explants can be carried out to better understand

the effect of oxidative stress on placental autophagy. While cell lines provide a model for

trophoblast cells, human villous placental explants provide a model of the functional chorionic

unit, including the two trophoblast layers and vasculature. Placental explants from healthy

placentae can be exposed to SNP treatment as well as hypoxia/reoxygenation experiments.

Electron micrographs can be taken of the tissue to see if it develops the preeclamptic phenotype

of a vacuolated syncytium with increased evidence for mitophagy.

Finally, explants of PE placentae can be treated with 3-methyladenine (3-MA), an

inhibitor of PI3K with greater affinity for class III proteins. The tissue and media can be

subsequently monitored to detect if more or less shedding of syncytium occurs, indicating

whether cell death has increased or decreased in response to inhibited autophagy (Seglen &

102

Gordon 1962). These experiments would more fully answer the question of the role of

autophagy in placenta and if the elevated autophagy seen in PE is aiding the tissue to cope with a

deleterious environment or further accelerating cell death in response to oxidative stress.

103

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Appendices

6 Appendix

6.1 Rapamycin Treatment

When mTOR is activated, autophagy is inhibited and can be monitored by

phosphorylation status of p70S6K, a protein kinase downstream of mTOR. In response to

mTOR activation, p70S6K is phosphorylated at Thr389. P(Thr398)p70S6K was used as an

additional marker of the autophagy status in JEG3 cells, HEK293 cells and placental lysates

from three different gestational ages. JEG3 cells were either starved for 3 hrs of FBS or not

starved, and HEK293 cells were treated with 1µM rapamycin for 3 hrs and cultured in the

presence of either serum (nst) or serum starved (st). Placental lysate from 7 and 12 weeks of

gestation and term were also used. The above lysates were immunoblotted for

P(Thr389)p70S6K expression, and expression was detectable in placental tissue and in HEK293

cells but not in JEG3 cells. In HEK293 cells, P(Thr389)p70S6K expression was abrogated in

response to rapamycin treatment (Appendix 6.1.1a). Therefore, P(Thr389)p70S6K was not

further used as marker of autophagic levels in JEG3 cells. In order to determine if autophagy

could be activated by rapamycin treatment in JEG3 cells a dose and time course for rapamycin

treatment was performed. No significant changes in LC3B-II expression were found at either 24

or 48 hrs of treatment with differing concentrations of rapamycin compared to vehicle DMSO

control (Appendix 6.1.1b). Rapamycin was not found to induce autophagy in JEG3

choriocarcinoma cells.

119

Appendix 6.1.1 Expression of p70S6K phosphorylated at Threonine 389 in JEG3,

HEK293 and human placental lysate and rapamycin treatment in JEG3 cells.

A. Immunoblot for P-Thr 389 p70S6K reveals expression in HEK293 cells and placental lysate

and was diminished in response to 1 µM rapamycin treatment for 3 hrs. St: FBS starved for 3

hrs, nst: nonstarved, R: rapamycin treated, w: weeks of gestation. B. JEG3 cells were treated for

24 and 48 hrs with 0.1-0.8 µM rapamycin and immunoblotted for LC3B-II, C: control, V:

vehicle.

120

6.2 Low oxygen environment and autophagy

As trophoblast pathologies are characterized by a hypoxic environment, experiments

were performed in JEG3 cells to determine if a relatively hypoxic environment of 3% oxygen

could induce LC3B-II expression. A timecourse was performed for 24, 48 and 72 hrs in both

20% and 3% oxygen tensions and protein expression levels for Mcl-1L and LC3B-II were

monitored. In response to low oxygen, Mcl-1L levels decreased after 24 hrs, with more variable

results at 48 and 72 hrs of treatment, despite reports in the literature that Mcl-1 is a HIF-1

responsive gene (Appendix 6.2.1a) (Piret et al., 2005). LC3B-II expression levels consistently

decreased in response to hypoxic stimulus at all timepoints (Appendix 6.2.1b).

121

122

Appendix 6.2.1 Expression of Mcl-1L and LC3B-II in response to 20% and 3%

oxygenation.

Timecourse of JEG3 cells cultured in 3% and 20% oxygen conditions for 24, 48 and 72 hrs. A.

Immunoblot analysis for Mcl-1L expression at 3% and 20% oxygenation. B. Immunoblot

analysis for LC3B-II expression at 3% and 20% oxygenation (n=3).

123

6.3 Glucose Deprivation in JEG3 cells

The most powerful inducer of autophagy is glucose starvation which activates autophagy

independently of apoptosis (Kuma et al., 2004; Codogno & Meijer 2005). The previous

interventions presented, including etoposide and SNP treatment have the disadvantage of also

inducing apoptosis concurrently with autophagic activation. In order to determine the effect of

glucose deprivation upon a trophoblast cell model, JEG3 cells were starved of glucose by using

Hank's Buffered Saline Solution (HBSS+) for 3, 6, and 24 hrs and then immunoblotted for

LC3B-II expression (Appendix 6.3.1). However, no significant changes in LC3B-II expression

were observed in response to glucose starvation in JEG3 cells, indicating that JEG3 cells do not

respond to glucose starvation within the first 24 hrs of treatment (n=3).

124

Appendix 6.3.1 Expression of LC3B-II in JEG3 cells in response to glucose starvation.

JEG3 cells were starved of glucose using HBSS+ for 3, 6 and 24 hrs and immunoblotted for

LC3B-II expression (n=3).

125

6.4 Autophagy in Placental Development

Early placental development occurs at a low oxygen environment of about 3% O2 which

is essential for CT proliferation and to stimulate EVT growth at the distal ends of the villous tips

(Genbacev et al., 1996; Genbacev et al., 1997; Caniggia et al., 2000). A low oxygen

environment is maintained until the maternal circulation opens to the developing placenta as the

result of EVT spiral artery remodeling, at about 10-12 weeks of gestation, resulting in a rise in

oxygen tension to about 8% O2. In order to understand the effect of oxygen upon placental

autophagy, the levels of LC3B-II expression were immunoblotted in first trimester human

placental samples (Appendix 6.4.1). While there is significantly more LC3B-II expression in

first trimester development compared to term placentae, no significant difference was observed

in autophagic activation from placentae of 5-9 weeks of gestation compared to placentae from

10-15 weeks of gestation.

126

Appendix 6.4.1 Expression of LC3B-II in human placental development.

Upper panel: Representative immunoblot of LC3B-II expression in human placental lysate.

Lower panel: Densitometric analysis of LC3B-II protein expression normalized to β-actin

relative to TC. (5-9 weeks of gestation n=18, 10-15 weeks of gestation n=21, TC n=5).

Kruskall-Wallis test with Dunn's multiple comparisons, *p<0.05, **p<0.01.