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Wayne State University Dissertations

1-1-2012

Molecular mechanisms of strain-stimulatedintestinal epithelial cell differentiation andmigrationLisi YuanWayne State University,

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Recommended CitationYuan, Lisi, "Molecular mechanisms of strain-stimulated intestinal epithelial cell differentiation and migration" (2012). Wayne StateUniversity Dissertations. Paper 489.

MOLECULAR MECHANISMS OF STRAIN-STIMULATED INTESTINAL EPITHELIAL CELL DIFFERENTIATION AND MIGRATION

by

LISI YUAN

DISSERTATION

Submitted to the Graduate School

of Wayne State University,

Detroit, Michigan

in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

2012

MAJOR: Anatomy and Cell Biology

Approved by:

_________________________________ Advisor Date

_________________________________

_________________________________

_________________________________

_________________________________

ii

ACKNOWLEDGEMENTS

I would like to thank Dr. Marc Basson for his ongoing mentorship and

support. His professional example, guidance and support have been key to my

success as a graduate student and development as a research scientist. I would

like to thank my committee members, Drs. Adhip Majumdar, Fu-shin Yu, and

Gabriel Sosne for their thoughtful insights that have been an asset to the

development and progression of my dissertation work.

I also owe a great deal of gratitude to my fellow lab members (both past

and present), Drs. Matt Sanders, Cheri Owen, Shouye Wang, Pavlo Kovalenko,

Rebecca Hermann, Christina Downey, Hiroe Shiratsuchi, Mary Walsh, and

Lakshmi Chaturvedi, for their friendship and technical assistance through the

years.

The Department of Anatomy and Cell Biology deserve thanks as well. Drs.

Linda Hazlett and Roberta Pourcho gave me this valuable chance to study in the

U.S., and certainly without them I would not be presenting this dissertation. I also

would like to thank Dr Walker for his help and patience with me during this

process.

I would also like to acknowledge all of our collaborators, Dr. Guangxiang

Yu at Department of Anesthesiology, Wayne State University, Dr. Yingjie Yu at

Department of Internal Medicine, Wayne State University, Dr. Andrea Amalfitano

and Dr. Sergey Seregin at Department of Microbiology & Molecular Genetics,

Michigan State University, Dr. Gavin Reid at Department of Chemistry, Michigan

iii

State, and finally Dr. Melinda Frame at Michigan State University Core Facilities

for her ongoing confocal microscopy expertise. Thank you.

Finally, I would like to thank my family and friends for all of their love,

support and patience that they have given me through the difficult times that I’ve

endured during my graduate training. Thank you.

iv

TABLE OF CONTENTS

Acknowledgements ii

List of Tables ___________________________________________________ iii

Chapter 1 – Introduction ___________________________________________1

Chapter 2 – Materials and Methods __________________________________ 9

Chapter 3 – Schlafen 3 induction by cyclic strain regulates intestinal epithelial

differentiation 18

Chapter 4 – ILK mediates the effects of strain on intestinal epithelial wound

closure 33

Chapter 5 – Discussion __________________________________________ 52

References 72

Abstract ______________________________________________________ 97

Autobiographical Statement 100

v

LIST OF TABLES

Table 1 – Antibodies____________________________________________ 17

Table 2 – Inhibitors_____________________________________________ 18

1

CHAPTER 1

Introduction

Mucosal healing is central to problems as diverse as peptic ulcers, Crohn’s

disease, GERD esophagitis, gastroenteritis or ulcerative colitis, and surgical

anastomotic healing (16, 82, 131, 143, 155). During these conditions, rapid

resealing of the gut mucosal is therefore essential following injuries, or intestinal

homeostasis will not be restored and diseases deteriorates. Gut mucosal healing

is an orderly and regulated process in which gut mucosal epithelial cells adopt a

migratory phenotype and the superficial interruptions in the gastrointestinal

mucosa are repaired by the flattening and spreading of epithelial cells

surrounding the damage (143). Understanding the signals and mechanisms that

regulate epithelial migration may permit us to define targets for pharmacologic

intervention to promote mucosal healing and preserve the gut mucosa in septic

patients or those with ileus, which are unfortunately common, and cause

substantial morbidity and mortality.

The gut mucosa repetitively experiences mechanical forces causing strain

and pressure, including villus motility (164), contact with luminal contents (105),

and peristalsis (144). Increasing in vitro and in vivo evidence suggests that such

forces may substantially influence intestinal mucosal cell biology by flexing the

matrix, altering integrin binding, and initiating matrix-dependent signals that

regulate enterocyte proliferation, migration, differentiation etc.(8, 13, 21, 48, 54-

2

55, 94-95, 168-169, 172-174). Thus, mucosal healing can only be fully

understood in the context of the effects of physical forces.

Tonic (resting) strain in the rat intestine approximates 10% and can reach

200% with contraction (160). Villous motility and shear stress further strains villi

and adherent enterocytes. The intestinal villi spontaneously contracts up to 15

times per minute at frequencies depending upon the specific location within the

bowel (frequency distribution gradient decreases progressively from duodenum

to terminal ileum (164) and tightly regulated by many factors such as luminal

nutrients and neurohumoral influences (164-165). Luminal contents are generally

non-compressible or minimally compressible; thus, interaction with relatively

noncompressible endoluminal chyme results in shear stress and pressure (7).

Peristalsis contractions occur at frequencies of 7–20 per minute, with more rapid

frequencies tending to be toward the duodenum and slower frequencies toward

the distal ileum and colon (51, 76, 115). Peristaltic contractility of the intestinal

tract can also induce strain and pressure on the intestinal mucosa.

In states of total parenteral nutrition (TPN), prolonged fasting, inflammatory

bowel disease (IBD), ileus, or sepsis, normal gut forces are aberrant, sometimes

with no peristaltic contractions at all (72). Eventually, mucosa atrophies and

barrier function fails (30, 70, 103). However, mucosal atrophy during ileus and

starvation is not due to malnutrition since defunctionalized mucosa atrophies

despite TPN (70). Interestingly, this condition can be reversed by luminal water

(24), suggesting that distention supports the mucosa. Thus, the biology of

3

intestinal epithelial cells can only be fully understood in the context of the effects

of physical forces.

Previous in vitro work from our laboratory suggests that cyclic strain

stimulates Caco-2 and IEC-6 intestinal epithelial cell migration across a

fibronectin matrix (174). This process requires the activation of Src,

phosphatidylinositol 3-kinase (PI3K) and Akt, as well as a novel Src-independent

phosphorylation of focal adhesion kinase (FAK) at Tyr925 (19, 45). How these

signal proteins interact to mediate the effects of strain, however, remains poorly

understood. Integrin linked kinase (ILK) is an integrin β1 and β3 subunit binding

protein (57). Ectopic expression of active ILK in mammary epithelial cells

promotes rapid cell spreading on fibronectin (41). In contrast, ILK-deficient

cultured keratinocytes fail to spread efficiently, with impaired ability to form stable

lamellipodia upon induction of cell migration in scraped keratinocyte monolayers

(109). Overexpression of ILK increases chicken embryo fibroblast cell migration

in a PI3K dependent manner, corresponding with the activation of Akt (122).

Moreover, ILK can directly phosphorylate myosin light chain (MLC) to stimulate

cell motility by activating the cellular contractile machinery in smooth muscle cells

(33-34). Thus, ILK is of particular interest as a candidate to mediate strain-

stimulated intestinal epithelial migration across fibronectin because it is required

for migration in some other cell types and because of its interaction with PI3K,

Akt and MLC. Interestingly, Gange et.al. recently reported that the siRNA

knockdown of ILK in human intestinal cells severely affected cell migration, which

was rescued with exogenously deposited fibronectin (44).

4

However, ILK kinase activity, or the lack thereof, has been a contentious

issue. Some studies suggest that ILK is a pseudokinase and an essential

scaffold protein (87, 163). It has been argued that although mutational analysis

has been used to gain some insight into the catalytic activity of ILK, these

mutations have also been shown to disrupt the interaction of ILK with essential

binding partners (5, 166). In addition, genetic studies in Caenorhabditis elegans

and Drosophila melanogaster fail to confirm the kinase function of ILK in vivo

since the reported kinase dead ILK mutants were capable of fully rescuing the

severe phenotypes caused by ILK deletion in both species (100, 171). Moreover,

a knock-in mouse strain carrying mutations in the autophosphorylation site

(S343A or S343D) is normal and does not show changes in Akt or Gsk-3β

phosphorylation or actin organization downstream of integrins. Mice carrying

point mutations in the potential ATP-binding site (K220A/M) die shortly after birth

because of kidney agenesis, although the authors argued that this did not result

from impaired kinase activity since the mutations did not alter the

phosphorylation levels of reported ILK substrates in vivo and no evidence of

kinase activity was detected in vitro (87). Despite all of the above evidence, ILK

has been shown to act as a serine/threonine kinase and to directly activate

several signalling pathways downstream of integrins (32, 41, 57, 119-120). It has

also been reported that although ILK-deficient chondrocytes, or keratinocytes, do

not show changes in Akt or Gsk-3β phosphorylation (50, 97), ablation of ILK in

the heart, kidney, nervous system, or skeletal muscle, abrogates phosphorylation

5

of Ser473 of Akt (118, 149, 156, 162), raising the possibility of tissue-specific

kinase function.

As delineated above, many different intracellular signals may influence the

processes that we will study. However, ILK is of particular interest. Integrin linked

kinase (ILK) is an integrin β1 and β3 subunit binding protein (57). Ectopic

expression of active ILK in mammary epithelial cells promotes rapid cell

spreading on fibronectin, which is associated with constitutive activation of both

Rac and Cdc42, but not Rho (41). The use of ILK siRNA or small molecule

inhibitors to inhibit ILK expression and kinase activity, respectively, results in

diminished cell spreading. This is concomitant with a reduction in Rac and Cdc42

activation (41). Similarly, ILK-deficient cultured keratinocytes fail to spread

efficiently, with impaired ability to form stable lamellipodia and reduced activation

of Rac1 upon induction of cell migration in scraped keratinocyte monolayers

(109). Overexpression of ILK increases chicken embryo fibroblast cell migration

in a PI3K-dependent manner, corresponding with the activation of Akt and Rac1

(122). Inhibition of Rac1 inhibited the effects of ILK on cell migration, suggesting

a regulatory role of the PI3K/Akt/Rac1 signaling pathway in response to ILK

signaling (122). Moreover, both β-parvin and paxillin can interact with αPIX (130),

a guanine nucleotide exchange factor for Rac and Cdc42, indicating another link

between ILK and these small GTPases. In addition, ILK can directly

phosphorylate MLC and two protein inhibitors of myosin phosphatase (PHI-1 and

CPI-17) to stimulate cell motility by activating the cellular contractile machinery in

smooth muscle cells (33-34).

6

The differentiation of the intestinal epithelium is a complex phenomenon

during which the epithelial cells acquire the functional properties of mature

enterocytes, which are known to be regulated by growth factors (68, 81, 154),

neuro- humoral peptides (27, 107, 137), luminal nutrients (9, 80, 108, 150-151),

and the extracellular matrix (6, 14, 96).

The intestinal epithelium is subjected to repetitive deformation from diverse

physical forces including peristalsis and villous motility during normal gut function

(61, 164).Increasing in vitro and in vivo evidence suggests that such forces may

substantially influence intestinal mucosal cell biology by flexing the matrix,

altering integrin binding, and initiating matrix-dependent signals (21, 46, 94). In

vitro, such repetitive deformation stimulates intestinal epithelial proliferation and

induces an absorptive phenotype characterized by increased dipeptidyl

dipeptidase (DPPIV) expression in an amplitude-dependent fashion, changes

opposite to those observed during prolonged fasting when peristaltic contractions

and villous motility are diminished (11). In addition, different models of repetitive

mechanical deformation also promote differentiation in other cell types including

fetal type II epithelial cells (157), osteoblast-like cells (114), and embryonic stem

cell-derived cardiomyocytes (52). However, the mechanisms of how strain

modulates cell differentiation and particularly intestinal epithelial differentiation, a

complex phenomenon during which the epithelial cells acquire the functional

properties of mature enterocytes, are largely unknown.

Brush border digestive enzymes such as dipeptidyl peptidase are canonical

markers for enterocytic differentiation (10, 26, 124, 126, 167). DPPIV cleaves

7

NH2-terminal dipeptides from polypeptides with either L-proline or L-alanine at

the penultimate position, including chemokines and neuropeptides, leading to

their inactivation and/or degradation (142). DPPIV specific enzyme activity is also

used in many other cell types as a marker of differentiation (3, 69, 152). Indeed,

the loss or alteration of DPPIV expression is linked to the development of many

cancers, including breast, prostate, lung, ovarian, hepatocellular cancer and

melanomas, and plays a key role in tumorigenesis and metastasis (3). In addition

to brush border digestive enzyme activity, intestinal differentiation is also

frequently assessed by villin expression (53, 64, 77, 88, 112). Villin is a key

Ca2+-regulated actin binding protein in the microvillus core of the brush border (2,

53, 64, 77, 88, 101, 106, 112). Intestinal epithelial cells and kidney proximal

tubule cells are notable examples of cells which have these highly specialized

brush border microvilli and villin accumulates at their apex. Villin content in

differentiated HT29-18 cells, a clone derived from the HT-29 human colonic

adenocarcinoma cell line, is 10 times higher than that in undifferentiated HT29-18

cells but close to that seen in normal human colonic cells (38).

We have previously observed that cyclic strain of a physiologically relevant

frequency and magnitude modulates the differentiation of well-differentiated

human intestinal epithelial Caco-2 cells, increasing in the specific activity of

DPPIV (11). Schlafen 3 belongs to a family of growth regulatory genes that was

first discovered in mice (136) and currently contains 10 intracellular protein

members (117). A unique domain named “Schlafen box” along with the adjacent

ATP/GTP binding AAA domain is common to all the members in the family (47).

8

This family has been divided into three subgroups based on their overall exon

homology and size of the encoded proteins (47, 136). Schlafen3, along with

Schlafen 4, Schlafen 6, and Schlafen 7, belongs to intermediate subgroup.

Schlafen 3 has recently been associated with cellular growth during aging and

differentiation (116-117). Other Schlafens, such as Slfn-1 or -8 plays an

important role in modulation of T cell development (89, 136). Schlafen expression

has been reported to increase during cellular differentiation in haematopoietic cell

lines (17), and Slfn-2 induction has been shown to be critical during the process

of differentiation of monocytes/macrophages to osteoclasts induced by receptor

activator of NF- кB ligand (RANKL) (89).

Based on these previous observations we hypothesize that intracellular

signals initiated by repetitively applied physical forces such as strain and

pressure can initiate intracellular signals that alter intestinal epithelial migration

and phenotype. We assessed the role of ILK and subsequent interaction with

FAK and Src modulates Caco-2 cell migration in response to strain in vitro. We

also showed that stain induction of Schlafen 3 plays a key role in the modulation

of intestinal epithelial differentiation. This work will elucidate signals that

represent important targets for medical intervention to promote mucosal healing

and reverse atrophy, thus perhaps would provide new targets for many diseases

such as ileus and sepsis treatment. In addition, these studies are also important

in other systems as diverse as endothelium, lung, and bone where cells also

experience mechanical forces.

9

CHAPTER 2

Materials and Methods

Cell culture Human intestinal epithelial Caco-2BBE cells, an established

highly differentiated human colonocyte cell line (74, 121), and non-transformed

rat intestinal IEC-6 epithelial cells (American Type Culture Collection, Manassas,

VA) were maintained at 370 C 5% CO2 as previously described (174).

Strain Application Cells were plated on Flexwell plates and maintained in a

37 °C humidified incubator with 5%CO2. Once the cell monolayers were

confluent, they were subjected to mechanical deformation using the Flexcell

Strain Unit (FX-3000; Flexcell, McKeesport, PA) as described previously (172).

Briefly, cells were subjected to cyclic deformation and relaxation at a magnitude

of 10% strain and a frequency of 10/cycles/minute by a computer-controlled 20-

kPa vacuum. Control plates were not attached to the Flexcell Unit though placed

in the same incubator. Placing a Plexiglas ring in the center addressed the

nonuniformity of strain in the center of the flexible wells as cells were plated only

around the periphery of the ring where strain is more uniform. Previous studies

have demonstrated that the cells remain adherent during deformation and

experience parallel elongation and relaxation with the repetitive deforming

membrane (11).

Matrix and inhibitors Flexwell amino plates were precoated with 12.5μg/ml

tissue fibronectin (Sigma Chemical Co, St Louis, MO) at saturating

concentrations (12).

10

Cells were seeded 300,000/well and grown to confluence. PD98059, PP2,

LY294002, SB203580 (Calbiochem, La Jolla, CA) were each dissolved in

dimethyl sulfoxide (DMSO), diluted immediately prior to use, and added to the

cells one hour before exposure to strain. Control cells in these studies were

similarly treated with 0.1% DMSO.

Migration To measure motility, confluent cell monolayers on deformable

membranes precoated with fibronectin were subjected to 0–24hours of repetitive

deformation after induction of circular wounds using a 1000-mL pipet tip (1.5-mm

diameter) as previously described (11). Wound areas were photographed,

measured on a Kodak Image Station (Perkin Elmer, Boston, MA), and the

percentage of wound closure was calculated.

In vitro kinase assay Lysates of cells maintained under static conditions or

subjected to strain for 5 minutes will be immunoprecipitated with anti-ILK and

protein A/G sepharose. The ILK protein will be incubated with GSK-3 α/βfusion

protein and ATP prior to SDS-PAGE and probing with anti-GSK-3

(phosphoserine 2/19) antibody.

Co- immunopreciptation Confluent cells serum-starved for 24 hours were

lysed with immunoprecipitation buffer and measured protein by BCA assay. 25μl

(per reaction) of antibody (ILK) and Protein A/G-Agarose (25 μl/reaction) was

added to 600 μg of protein-matched samples diluted to equal volumes (1ml) with

overnight rotation. After 3 washings with immunoprecipitation buffer, pellet was

resuspended in loading buffer. The immunocomplex suspension was then boiled

for 4 min and resolved by Western blot for FAK.

11

P85 Translocation Assays The static control cells or cells exposed to cyclic

strain were lysed in lysis buffer (50 mM HEPES, pH 7.4, 50 mM NaCl, 1 mM

MgCl2, 2 mM EDTA, 5 mM NaF, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml

aprotinin, 1mM Na3VO4, and 1 mM dithiothreitol). The cell lysates were

sonicated at 20% duty cycles 10 times to disrupt the cell membranes and release

cytosolic proteins (Sonifier 250, Branson Ultrasonic Corp., Danbury, CT). The

lysates were ultracentrifuged at 105 X g for 1 h at 4 °C. The supernatant was

collected as the “cytosolic fraction.” The resulting pellet “membrane fraction” was

washed twice with PBS and resuspended in the above lysis buffer. The pellet

was sonicated at 20% duty cycles 10 times and incubated 30 min on ice. Protein

concentration were measured by the BCA method. Equal amounts of protein

were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis,

transferred to nitrocellulose membrane, and immunoblotted with monoclonal anti-

p85 antibody to assess the amounts of p85 in each fraction. The blots were also

probed with Na-K ATPase and Rho-GDI as protein loading controls for

membrane and cytosolic fractions, respectively.

Western Blot Analysis Following strain, Caco-2 cells were lysed on ice in

modified radioimmunoprecipitation buffer (50 mmol/L Tris [pH 7.4], 150 mmol/L

NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate,

1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, 50

mmol/L NaF, 10 mmol/L sodium pyrophosphate, 2 μg/mL aprotinin, and 2 μg/mL

leupeptin [pH 7.4]) and lysates were centrifuged at 12,000g for 10 minutes at 4°C.

Supernatant protein concentrations were determined by bicinchoninic acid

12

analysis (Pierce Chemical, Rockford, IL). Equal amounts of protein were resolved

by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and

electrophoretically transferred to Hybond ECL nitrocellulose membrane

(Amersham Pharmacia Biotech, Piscataway, NJ). Nonspecific binding sites were

blocked with 5% bovine serum albumin in Tris-buffered saline (20 mM Tris-HCl,

137 mM NaCl, pH 7.6) with 0.1% Tween 20 for 1 h at room temperature.

Membranes were probed with appropriate primary and secondary antibodies.

Bands were visualized using enhanced chemiluminescence (Amersham

Pharmacia Biotech) and analyzed with a Kodak Image Station 440CF.

Membranes were then stripped and reprobed with appropriate primary and

secondary antibodies specific for total protein. All exposures used for

densitometric analysis were within the linear range.

siRNA transfection 30-40% confluent Caco-2 cells were transfected with

non-targeting siRNA (NT1), or siRNA to ILK, FAK, or Src (Dharmacon, Lafayette,

CO) asdescribed previously (46). Protein reduction (routinely 75-90%) will be

confirmed by parallel Western blots. 30-40% confluent IEC-6 cells were

transfected with non-targeting siRNA (NT1), or siRNA to Schlafen 3 (Dharmacon,

Lafayette, CO) as described previously (21). Protein reduction (routinely 70-90%)

was confirmed by parallel Western blots.

FAK Y925 mutant transfection 6 μg of HA-tagged FAK-wild type or HA-

tagged FAK mutant Phe 925 DNA or empty vector will be mixed with 60 μl of

Plus reagent in 6 ml of Opti-MEM for 15 min. Then, Lipofectamine (30.0 μl in 1 ml

of Opti-MEM) will be added. This mixture will be incubated at room temperature

13

for 20 min, and added at 1.0 ml per well to cells at 70–80% confluence for 6 h.

Cells will be rinsed twice with Opti-MEM prior to addition of the transfection

mixture. Following transfection, cells will be incubated with normal medium for 20

–24 h and then incubated with medium containing no FBS for an additional 18 –

24 h prior to exposure to cyclic strain. 20–25% of cells are transfected using this

procedure.

Brush border enzyme activity assay DPDD activities were quantitated by

synthetic substrate digestion of p-nitrophenyl phosphate hexahydrate and

glycylprolyl-p-nitroanilide tosylate (Sigma), respectively, in protein-matched

cellular lysates as quantitated by BCA (Pierce as previously described (151).

Isolation of RNA from mucosal cells Total RNA was isolated from the IEC

cells using RNA-STAT solution (Tel Test, Friendswood, TX) according to the

manufacurer’s instructions. The total RNA was treated with DNase I (Invitrogen,

Gaithersburg, MD) to remove contaminating genomic DNA. DNase I-treated

RNA was purified using RNeasy Mini Kit (Qiagen, Valencia, CA). RNA

concentration was measured spectrophotometrically at optical density (OD) 260.

Reverse Transcription-PCR The two-step RT-PCR was performed by

using the GeneAmp Gold RNA PCR Kit (Applied Biosystems, Foster City, CA).

Briefly, 1 ug of purified RNA was reverse transcribed in the presence of 2.5 mM

MgCl2, 1X RT-PCR Buffer, 1 mM dNTPs, 10 mM dithiothreitol, 10 units RNase

inhibitor, 1.25 μM random hexamers and 15 units Multiscribe Reverse

Transcriptase in a final reaction volume of 20 μl. The components were mixed,

briefly spun down and incubated at 25°C, 10min for hybridization; then reactions

14

were carried out at 42°C for 15 minutes in a Gene Amp PCR system 9600

(Perkin-Elmer) and cooled to 4°C. The RT reactions were subjected to

polymerase chain reaction (PCR) amplification. 5μl of cDNA products were

amplified with 2.5 units of Ampli Taq Gold Polymerase (Applied Biosystems), 1X

RT-PCR Buffer, 1.75 mM MgCl2, 0.8 mM dNTPs, 0.15 uM upstream primers,

and 0.15 uM downstream primers in final concentration. Reactions were carried

out in the Gene Amp PCR system 9600. Reactions were performed for 10

minutes at 95°C for activated AmpliTaq Gold DNA Polymerase, then for 20

seconds at 94°C, and then for 60 seconds at 62°C for 40 cycles for amplification

of the target gene. The rat schlafen 3 primers used were 5’-

ATTCTGCTGTGCAGTGTTCG-3’ (upstream) and 5’-

TTGCTTGGAGAAACATGCTG-3 (downstream). The beta actin primers used

were 5’-CCCAGCACAATGAAGATCAA-3’ (upstream) and 5’-

ACATCTGCTGGAAGGTGGAC-3’ (downstream).

Proliferation assay Proliferation assays were performed as previously

described (21). Briefly, IEC-6 cells were plated on collagen-coated flex-well

plates at 10% confluence, cultured overnight, and then transfected with non-

targeting siRNA (NT1), or siRNA to Schlafen 3. On the following day, cells were

serum-deprived for 6 h with 1 plate reserved for a time 0 measurement. The

remaining serum-starved cells were switched back to normal growth medium with

10% FBS under static or repetitive strain conditions, or incubate without or with

EGF for 24 h. Cell were then fixed and stained with crystal violet for dye elution

assay. Using 10% acetic acid, dye was extracted, and absorbance at 550nmwas

15

measured using a Thermomax microplate reader (Molecular Devices, Ramsey,

MN). Each experiment contained six observations.

Adenovirus infection Once 50-60% confluent, the cells were washed twice

in PBS and infected with Ad-GFP (gift from Dr. Andrea Amalfitano), or Ad-SRC

(Vector Biolabs, Philadelphia, PA). After 48 hours infection, the cells were

stimulated with or without strain. The infection efficiency of GFP adenovirus was

estimated with a fluorescent microscope.

Statistics All data are expressed as mean ±SEM of at least three

independent similar experiments. Statistical analysis was performed using paired

or unpaired t tests or analysis of variance as appropriate. P< 0.05 was

considered statistically significant.

16

Table 1. Inhibitors

Inhibitor Target Concentration

Manufacture Catalog Number

PP2 Src 10 μM Calbiochem 529573

SB203580 P38 10 μM Calbiochem 559389

LY294002 PI3K 20 μM Calbiochem 440202

PD98059 ERK 20 μM Calbiochem 513000

Akt Inhibitor IV

Akt 500 nM Calbiochem 124015

FAK FAK 2 µM Calbiochem 324877

ML-7 MLC 10 µM Calbiochem 475880

17

Table 2. Antibodies

Antibody Source Manufacture Catalog Number

FAK (phospho-Y576)

Rabbit Biosource 44-652G

ERK (phosphor-Y42/44)

Rabbit Cell Signaling 9101

Akt (phospho-S473)

Mouse Cell Signaling 4051

ERK Rabbit Cell Signaling 9102

Akt Rabbit Cell Signaling 9272

GAPDH Mouse Biodesign International

H86045M

FAK Mouse Upstate 05-537

Schlafen 3 Goat Santa Cruz sc-8902

villin Goat Santa Cruz sc-7672

,Hsp 27 Mouse Santa Cruz sc-51596

FAK (phospho-Y397)

Rabbit Biosource 44-624G

FAK -Y925)

Rabbit Cell Signaling 3284

Src (phospho-Y476)

Rabbit Cell Signaling 2101

ILK Rabbit Cell Signaling 3862

Na-K ATPase Rabbit Cell Signaling 3010

p85 Mouse Upstate 06-497

MLC (phospho-T18/S19)

Rabbit Cell Signaling 3674

MLC Rabbit Santa Cruz Biotechnology

sc-15370

GST (phospho-3α/β)

Rabbit Cell Signaling 9331

18

CHAPTER 3

SCHLAFEN 3 INDUCTION BY CYCLIC STRAIN REGULATES INTESTINAL EPITHELIAL DIFFERENTIATION

We evaluated whether Schlafen 3 mediates the effects of strain on the

differentiation of non-transformed rat IEC-6 small intestinal epithelial cells

cultured on collagen-coated membranes in the absence or presence of strain.

Strain increased both Schlafen 3 protein and mRNA.

To determine whether strain increases Schlafen 3 protein and mRNA in IEC-

6 intestinal epithelial cells, we subjected confluent serum-deprived cells to cyclic

strain for 24 hours and assayed Schlafen 3 protein levels by Western blotting and

gene expression by RT-PCR. Cyclic strain increased Schlafen 3 protein and

mRNA levels to 1.73±0.20 (n=4, p<0.05) and 1.92±0.11 (n=7, p<0.05)

respectively (Fig. 1A and B). Furthermore, Schlafen 3 mRNA increased in a time-

dependent fashion in response to strain. Cyclic strain for 0–48 hours increased

Schlafen 3 expression, with a maximal observed increase in expression of

2.46±0.31 at 12 hours time point (Fig. 1C, n=4, p<0.05). We chose to study 24

hours of an average 10% deformation at 10 cycles per minute as our primary

stimulus because we have previously demonstrated that this stimulus modulates

brush-border enzyme activity in human intestinal Caco-2 cell monolayers

(11). These parameters are similar in magnitude and frequency to those

observed during normal intestinal peristalsis (43) and villous motility (164).

19

Fig. 1

A

B

C

20

Fig.1 (A) IEC-6 monolayers were subjected to cyclic strain for 24 hours after

confluence. Cells were lysed in buffer and equal protein aliquots resolved by

SDS-PAGE, followed by Western analysis. Strain increased Schlafen 3 protein to

1.92±0.11 (n=7, *p<0.05). Bars represent densitometric analysis; representative

Western blots are shown above the graph. (B) Confluent IEC-6 monolayers were

strained for 24 hours. Total RNA was extracted followed by real-time RT-PCR

analysis. Strain increased Schlafen 3 mRNA to 1.73±0.20 (n=4, *p<0.05). (C)

Schlafen 3 mRNA increases in a time-dependent fashion in response to strain

(n=4, *p<0.05).

Reducing Schlafen 3 by siRNA inhibited the stimulation of DPPIV activity

and villin expression by strain.

We reduced Schlafen 3 protein by a specific siRNA prior to strain and

examined DPPIV activity by DPPIV activity assay. Schlafen 3 levels were

reduced by >70% 72 hours after treatment prior to application of strain (Fig. 2A,

n=3). DPPIV enzymatic specific activity was increased in lysate from IEC-6 cells

exposed to cyclic strain for 24 hours to 1.63±0.09 compared with cells not

exposed to strain for this period (Fig. 2B, n=6, p<0.05), similar in magnitude to

previously reported effects of other differentiating stimuli in IEC-6 cells (81) as

well as to the effects of cyclic strain in Caco-2 cells (11). Schlafen 3 reduction

by >70% using a specific siRNA decreased basal DPPIV to 0.35±0.05 (Fig. 2B,

n=6, p<0.05) and prevented any stimulation of DPPIV activity by strain. Exposure

to strain also increased villin protein levels to 1.34 ±0.10, and this effect was also

21

eliminated by Schlafen 3 reduction (Fig. 2C, n=4, p<0.05). Other differentiating

stimuli in IEC-6 cells have similar magnitude effects on villin (140, 158).

Fig. 2

22

Fig. 2 Reducing Schlafen 3 by siRNA inhibited the stimulation of DPPIV activity

and villin expression by strain. Schlafen 3 protein abundance was substantially

reduced 72 hours after transfection with siRNA to Schlafen 3. A typical

immunoblot showing Schlafen 3 protein level reduction in cells transiently

transfected with siRNA targeted to Schlafen 3 in comparison with cells

transfected with a nontargeting sequence (NT1) (A; 1 of 3 similar experiments).

(B) Cells were plated on collagen I and transiently transfected with siRNA

targeted to Schlafen 3 or with non-targeting NT1 sequences prior to lysis and

DPPIV specific activity assay. Strain increased DPPIV activity to 1.63±0.09 (B;

n=6, *p<0.05). Reducing Schlafen 3 prevented any stimulation of DPPIV activity

by strain in comparison with unstretched cells, while basal activity lowered to

0.35±0.05 (B; n=6, *p<0.05), strongly suggesting that Schlafen 3 mediates the

strain effect on DPPIV stimulation. (C) To determine the role of Schlafen 3 in

villin expression following strain, we transiently transfected cells plated on

collagen I with siRNA targeted to Schlafen 3 or with non-targeting NT1

sequences for 72 hours before strain and assessed villin expression by Western

blot. Schlafen 3 siRNA moderately reduced villin levels and eliminated the strain

effect (n=4, *p<0.05).

Schlafen-3 reduction by siRNA also inhibited the stimulation of DPPIV

activity by sodium butyrate (1mM) or TGF-β.

Reducing Schlafen 3 by siRNA also prevented DPPIV induction by sodium

butyrate (1mM) or TGF-β (0.1ng/ml), two unrelated differentiating stimuli.

Induction of DPPIV activity by sodium butyrate and TGF-β was significant in IEC-

23

6 cells (n=4, p<0.01, Fig. 3A and D). DPPIV activity induced by strain can be

further increased by sodium butyrate, suggesting that sodium butyrate induces

differentiation by a different mechanism from strain (n=3, p<0.05, Fig.

3B). These results further suggest the potential importance of Schlafen 3 in the

regulation of intestinal epithelial differentiation since different differentiating

stimuli each require Schlafen 3 for activity. We then treated IEC-6 cells with

sodium butyrate (1mM) or TGF-β (0.1ng/ml) after transiently transfected cells

with Schlafen 3 specific siRNA. DPPIV activity was increased by sodium butyrate

(1mM) in NT1-treated cells to 1.82±0.09 (n=5, p<0.05), but this effect was

completely blocked by transfection with Schlafen 3 siRNA, as shown in Fig. 3C.

DPPIV activity was increased by TGF-β (0.1ng/ml) in NT1-treated cells to

1.41±0.04 (n=3, p<0.05); however, the effect of TGF-β on DPPIV activity was

eliminated in cells transfected with siRNA to Schlafen 3 (n=3, p<0.05 Fig. 3E).

24

Fig. 3

25

Fig. 3 Schlafen-3 reduction by siRNA also inhibited the stimulation of DPPIV activity by 24

hours treatment with sodium butyrate (1mM) or TGF-β (0.1ng/ml). (A) Sodium butyrate, a

differentiating agent, stimulated DPPIV activity in IEC-6 cells plated on collagen I (n=4,

*p<0.01). (B) DPPIV activity was increased by strain and sodium butyrate (1mM), indicating

that sodium butyrate induces differentiation by a different mechanism from strain (n=3;

*p<0.05). (C) Cells plated on collagen I and transiently transfected with siRNA targeted to

Schlafen 3 or nontargeting NT1 sequences were treated with sodium butyrate (1mM) prior to

lysis and assay for DPPIV specific activity. DPPIV activity was increased by sodium butyrate

(1mM) to 1.82±0.09 in NT1-treated cells, but this effect was completely blocked by

transfection with Schlafen 3 siRNA (n=5, *p<0.05). (D) TGF-β, another well-characterized

differentiating agent, stimulated DPPIV activity in IEC-6 cells plated on collagen I (n=4,

*p<0.01). (E) In IEC-6 cells plated on collagen I, addition of TGF-β stimulated DPPIV to

1.41±0.04, and this effect was similarly prevented by reducing Schlafen 3 with siRNA (n=3;

*p<0.05).

Schlafen-3 reduction by siRNA did not inhibit strain or EGF-induced

proliferation in IEC-6 cells.

We have previously shown that cyclic strain also stimulates IEC-6 cell

proliferation. However, Shlafen-3 reduction by siRNA did not prevent the

mitogenic effect of strain or that of EGF (n=3, p<0.05 for each), as shown in Fig.

4.

26

Fig.4 Schlafen 3 reduction did not modulate intestinal epithelial proliferation

induced by strain or EGF. (A) IEC-6 cells transiently transfected with siRNA

targeted to Schlafen 3 or with a non-targeting NT1 sequence were maintained

under static conditions or cyclic strain for 24 h prior to crystal violet staining.

Strain stimulated proliferation in both NT1-treated cells and cells in which

Fig. 4

A

B

27

Schlafen 3 was reduced (n=3, *p<0.05). IEC-6 cell monolayers exposed to cyclic

strain for 24hours display significantly increased cell numbers after strain

initiation compared with static control cell monolayers at the same time point, as

assessed by a colorimetric assay using crystal violet absorbance assay over the

linear range of the assay, with interpolation against a standard curve. However,

reducing Schlafen 3 by siRNA could not block this strain effect. (B) Incubation

IEC-6 cells with 10 pM EGF for 24 hours resulted in a 1.68±0.02-fold (n=3,

*p<0.05) increase in proliferation. However, reduction of Schlafen 3 by transient

transfection with siRNA targeted to Schlafen 3 also did not block EGF-induced

IEC-6 proliferation in comparison with static control.

The induction of Schlafen 3 protein by strain was prevented by blocking

Src, p38, or PI-3-K, but not ERK.

Although the upstream mediators by which strain influences differentiation

are not known, we have previously described activation of Src, ERK, p38, and PI-

3-kinase by strain and shown that Src, ERK, and PI-3-kinase mediate the

mitogenic effect of strain, which is independent of p38 activation (21, 46). We

therefore examined strain stimulation of Schlafen 3 protein levels in the absence

or presence of PP2 (10μM), PD98059 (20μM), SB203580 (10μM), and LY294002

(20μM), which block Src, ERK, p38, and PI-3-kinase respectively. To confirm that

the compounds were actually inhibiting Src, p38, and PI-3-kinase activity

respectively, we took advantage of previous studies demonstrating that

phosphorylation of FAK Y576, Hsp27 accumulation, and phosphorylation of Akt

are downstream consequences of Src (23, 138), p38 (62, 71, 111) and PI3K(60,

28

99) activation respectively in a variety of cell types. PD98059 is a specific MEK

inhibitor that prevents downstream activation of ERK1 and ERK2 in response to

various stimuli in diverse cell types (128, 170), so ERK phosphorylation served

as a marker for efficacy for the PD98059 compound. We confirmed that all the

agents used actually inhibited the activity of their target kinases (Fig. 5A, B, C, D,

lower two blots for each, 1 of 3 similar experiments for each). Blocking Src or PI-

3-kinase prevented strain induction of Schlafen 3, similar to previous

observations that Src and PI-3-kinase are required for the mitogenic effects of

strain in intestinal epithelial cell lines (21, 46). However, Schlafen 3 induction

required activation of p38 but not ERK, in contrast to the mitogenic effects of

strain which requires ERK but not p38 (20-21) (Fig. 5A, B, C, D, n=4, p<0.05 for

each). Parallel RT-PCR studies confirmed that strain stimulation of Schlafen 3

gene expression was also prevented by Src blockade (Fig. 5E, n=7, p<0.01).

These results suggest that cyclic strain modulates intestinal epithelial Schlafen 3

expression via Src, p38, and PI-3-kinase activation, and in particular identify for

the first time a role for p38 induction by strain in intestinal epithelial cells on a

collagen substrate.

29

Fig. 5

30

Fig.5 Cyclic strain (24 hours) induction of Schlafen 3 protein was blocked by (A)

PP2(10 M), (B) SB203580(10 M) or (C) LY294002 (20 M), which block Src,

p38, and PI-3-K, but not by (D) PD98059 (20 M), which blocks ERK(n=4,

*p<0.05). Each inhibitor significantly blocked basal as well as strain–stimulated

phosphorylation or accumulation of the specific downstream target of each

kinase (n=3, lower two blots for each, *p<0.05). (E) Strain stimulation of Schlafen

3 expression was prevented by Src blockade with PP2 (10 M) vs actin mRNA

(n=7, *p<0.01).

Strain did not induce Schlafen 3 expression or DPPIV activity in IEC-6 cells

cultured on fibronectin.

We have previously reported that strain effect on cell biology is matrix

dependent. Strain stimulates intestinal epithelial proliferation on collagen

substrates, but stimulates migration across fibronectin (21, 46, 174). The matrix-

dependence of the differentiating effects of strain on intestinal epithelial cells has

not previously been investigated. Therefore, we sought to determine whether

E

31

strain effect on Schlafen 3 is similarly affected by different matrix substrates. We

exposed normal small intestinal epithelial IEC-6 cells cultured on collagen or

fibronectin to strain for 24 hours and measured Schlafen 3 expression by

Western blotting. Induction of Schlafen 3 expression across collagen by strain

was statistically significant. In contrast, no stimulation of Schlafen 3 expression

by strain was observed in cells cultured on fibronectin (n=4, p<0.05, Fig. 6A). In

further studies, IEC-6 cells grown to confluence on either collagen or fibronectin

were subjected to 24 hours of strain, and DPPIV specific activity was measured.

We observed that fibronectin slightly decreased basal DPPIV activity and

prevented any stimulation of DPPIV activity by strain in contrast to collagen (n=5,

p<0.01, Fig. 6B), and in parallel with our observations of the matrix-dependence

of the effects of strain on Schlafen 3.

Fig. 6

A

32

Fig.6 Strain increased Schlafen 3 protein and DPPIV activity on collagen but not

on fibronectin. (A) IEC-6 cells cultured on membranes coated with collagen or

fibronectin were maintained under static conditions or repetitive deformation for

24 h prior to lysis and Western blot for Schlafen 3 protein. Schlafen 3 expression

of IEC-6 cells was different in response to strain on collagen compared with cells

on fibronectin. Induction of Schlafen 3 expression across collagen by strain was

statistically significant. In contrast, little stimulation of Schlafen 3 expression by

strain was observed in cells cultured on fibronectin (n=4; *p<0.05). (B) DPPIV

activity was determined by DPPIV activity assay in IEC-6 cells subjected to 24

hours of strain after grown to confluence on either collagen or fibronectin.

Fibronectin slightly decreased basal DPPIV activity and prevented any

stimulation of DPPIV activity by strain in contrast to collagen (n=5; *p<0.01).

B

33

CHAPTER 4

ILK mediates the effects of strain on intestinal epithelial wound closure

We hypothesized that ILK mediates the effects of strain on intestinal epithelial

wound closure. To test this hypothesis, we used the Flexcell apparatus to

rhythmically deform human Caco-2 intestinal epithelial cells cultured on a tissue

fibronectin substrate. Rat non-transformed IEC-6 cells were used to confirm key

results. We studied a frequency of 10 cycles per minute and an average 10%

strain, similar in order of magnitude to the frequency by which the intestinal

mucosa might be deformed by peristalsis or villous motility in vivo, and

conservative with regard to the amplitude of deformation (11, 115). We

characterized ILK activation by cyclic strain and determined the role of Src and

PI3K in this process. Additionally, we used siRNA targeting ILK to examine its

role in strain stimulated migration and characterized ILK association with FAK

and Src through coimmunoprecipitation studies. Finally, we used ILK siRNA to

characterize its role in FAK, Akt, and MLC phosphorylation, which have

previously been implicated in this process (45).

Cyclic strain stimulates ILK kinase activity.

To determine whether cyclic strain stimulates ILK kinase activity in Caco-2

intestinal epithelial cells, we subjected confluent serum-deprived cells to cyclic

strain for 0–60 minutes and ILK kinase activity was assayed by in vitro kinase

assay using GSK-3-α/β as the substrate. We tried to confirm there was little or if

any contamination of other kinases in our IP complex by adding a control sample

from cells pretreated with siRNA to ILK. If the IP complex were contaminated with

34

other kinases that can also phosphorylate GSK-3-α/β protein, then we would

have expected the phosphorylation level of GSK fusion protein substrate in ILK

siRNA sample to still be high from such other contaminating kinases. However,

performing the ILK kinase assay with lysate from cells treated with siRNA to

reduce ILK yielded only a minimal level of phosphorylation of the GSK fusion

protein, which might be attributable to the small amount of residual ILK in these

ILK-reduced but not ILK-null cells. This suggests that even if the

immunoprecipitated complex had been contaminated by other kinases, the bias

that might have resulted from this would have been minimal. We observed

increased ILK kinase activity at 5 minutes after initiation of cyclic strain (275 ±

17% of control, n=7, p<0.05; Fig. 1A). ILK kinase activity then decreased

gradually but remained higher than baseline after one hour of cyclic strain. Thus,

cyclic strain stimulated ILK kinase activity in a rapid and sustained manner.

Although Caco-2 cells are commonly used to model diverse aspects of

nonmalignant intestinal epithelial biology and signal transduction (84, 147), they

are nevertheless a transformed cell line originally derived from a colon cancer

(29, 42). We therefore confirmed our results in non-transformed small intestinal

epithelial IEC-6 cells (Fig. 1B). Similarly to Caco-2 cells, strain significantly

stimulated ILK kinase activity at 5 minutes (255 ± 14% of control, n = 7, p< 0.05),

and ILK kinase activity remained significantly higher than baseline after one hour.

Additionally, ILK protein levels are not increased in response to strain. Cyclic

strain for 0-48 hours did not increase total ILK protein levels in either Caco-2

cells or IEC-6 cells (Fig. 1B, 1 of 3 similar experiments for each).

35

Figure 1. ILK is activated in response to strain. A and B, The effect of cyclic

strain on ILK kinase activity was assessed by in vitro kinase assay in cells

subjected to cyclic strain for 0–60 minutes. Cells transfected with siRNA to ILK

served as a negative control. ILK was activated by strain in a time-dependent

fashion in both Caco-2 cells (A) and IEC-6 cells (B) (n=7, *p < 0.05 for each).

A B

p-GSK

ILK

Strain 0 5 15 60

p-GSK

ILK

Strain 0 5 15 60

Fig.1

C

36

Total ILK served as a loading control. Representative Western blots are shown

above each graph. (C) Total ILK protein levels do not increase in response to

strain (n=3, p>0.05).

ILK is required for strain-induced migration.

We next determined whether ILK was required for strain to stimulate wound

closure. We transfected Caco-2 or IEC-6 cells with either siRNA targeting ILK or

a control nontargeting siRNA sequence (NT1). Then, 72 hours after transfection,

we assessed migration with or without repetitive strain over 0-48 hours. Wounds

in NT1-transfected Caco-2 monolayers closed more rapidly in response to strain

than NT1-transfected static controls at all time points (n=17, p<0.05; Fig. 2B).

siRNA targeted to ILK reduced ILK protein by 70±10% (n=3, p< 0.05; Fig. 2A).

This reduction of ILK reversed the motogenic effect of strain at all time points.

The maximal effect of ILK reduction on strain-induced wound closure was

observed at 12 hours (n=17, p<0.05; Fig. 2B). ILK reduction by siRNA also

prevented the stimulation of migration by strain in IEC-6 cells (Fig. 2C). These

data indicate a role for ILK in the strain-induced motogenic response in intestinal

epithelial cells and suggest a role for ILK in the intestinal epithelial restitution

mechanism.

37

Fig.2

A

B

C

ILK

GAPDH

NT siILK1 NT siILK2IEC-6

ILK

GAPDH

NT siILK1 NT siILK2IEC-6

GAPDH

ILK

NT siILKCaco-2

GAPDH

ILK

NT siILKCaco-2

24 hours

NT

0 hour

staticsiILK

strain static strain

24 hours

NT

0 hour

staticsiILK

strain static strain

38

Figure 2. ILK is required for strain-induced migration. (A) Cells were transfected

with SMARTpool ILK siRNA targeted to human ILK IN Caco-2 cells, siILK1 or

siILK2 targeted to rat ILK in IEC-6 cells, or with control NT1 non-targeting siRNA

prior to lysis and Western blot for ILK protein. Transfection with the ILK siRNA

sequence achieved 60-80% reduction in ILK protein levels in both Caco-2 cells

and IEC-6 cells as shown by immunoblot. (B) ILK reduction by siRNA inhibited

strain-stimulated migration of Caco-2 cells. Circular wounds were made in

monolayers of cells transfected with either NT1 or siRNA targeted to ILK, and

these wounds were photographed. Cells were then cultured under static

conditions or conditions of strain for 0-48 hours before the holes were measured

again and wound closure was calculated. Reduction of ILK reversed the

motogenic effect of strain at all time points with maximum inhibition observed at

12 hours (n = 17, *P < 0.05). (C) A parallel study in IEC-6 cells confirmed that

strain stimulated cell migration in cells transfected with control NT1 non-targeting

siRNA but not in cells in which ILK was reduced by siRNA (n = 16, *P < 0.05).

PI3K is required for strain-induced ILK activation.

We next studied the involvement of PI3K in the activation response of ILK to

cyclic strain using LY294002 to inhibit PI3K. Subconfluent Caco-2 cells were

pretreated with 10 μM LY294002 for 1 hour, then treated and untreated control

cells were exposed to cyclic strain for 24 hours or cultured without repetitive

strain. Control untreated Caco-2 cells subjected to strain exhibited increased ILK

kinase activity compared with static untreated Caco-2 cells (n=3, p<0.05; Fig. 3A).

In LY294002 treated Caco-2 cells subjected to strain, however, ILK activation

39

was blocked, and LY294002 reduced basal ILK kinase activity in static cells

compared with untreated control static cells (n=3, p<0.05; Fig. 3A).

Strain Induces translocation of the p85 subunit of PI3K independent of ILK.

Translocation of the p85 subunit of PI3K from cytosol to the cell membranes

has been widely used as an indirect measure of activation (18, 133). To

determine whether PI3K activation in response to strain requires ILK, serum-

starved Caco-2 cells were subjected to static or cyclic strain conditions after ILK

siRNA transfection. Repetitive deformation stimulated translocation of the p85

subunit of PI3K from the cytosol to cell membranes in an ILK independent

manner (n=3, p<0.05; Fig 3B). Blots were also probed for the abundant

membrane protein Na-K ATPase and the abundant cytosolic protein Rho-GDI to

confirm fraction enrichment and equal loading (Fig. 3B lower panel).

ILK is required for strain-induced Akt activation.

Since ILK and Akt are both known downstream targets of PI3K and are each

required for the motogenic effect of strain (45), we determined whether ILK acts

upstream of Akt. After 5 minutes of repetitive strain, Akt phosphorylation

increased in cells transfected with NT1 control siRNA (n=6, p<0.01, Fig 3C). In

contrast, cells transfected with ILK siRNA displayed decreased basal Akt

phosphorylation and no further increase in Akt phosphorylation in response to

strain (Fig. 3C, n=6, p<0.05), suggesting Akt may be downstream of ILK in this

pathway.

40

A

B

p-GSK

ILK

p-GSK

ILK

Na/K ATPase

siRNA

P85

-GDI

cytosol membrane

NT ILKstatic strain static strain

NT ILKstatic strain static strain

Na/K ATPase

siRNA

P85

-GDI

cytosol membrane

NT ILKstatic strain static strain

NT ILKstatic strain static strain

Fig. 3

41

Figure 3. PI3K is upstream of ILK, whereas Akt is downstream. (A) Blockade of

PI3K with LY294002 prevents strain-induced ILK activation at 5 minutes (n = 3,

*p < 0.05). (B) Strain stimulated a shift in the p85 subunit of PI-3K from a cytosol-

enriched fraction to a fraction enriched for cell membranes in an ILK-independent

manner, which indicates that ILK does not modulate PI3K activation (n = 3, *p <

0.05). The membrane protein Na-K ATPase (lower panel) and the cytosolic

protein Rho-GDI (lower panel) were used to confirm fraction enrichment and

equal loading. (C) ILK reduction by siRNA inhibited strain-induced Akt

phosphorylation. Typical blots are presented at the top of the figure, and the

bottom graph summarizes densitometric analysis.

Association of ILK with FAK is reduced by FAK Tyr925Phe mutation.

Since reducing FAK by siRNA or transfection with the phosphorylation

mutant FAK Tyr925Phe blocks the strain effect on migration (19), and both ILK

and FAK are localized to focal adhesions, this suggests that ILK may interact

with FAK. Total lysates of cells exposed to 5 minutes of cyclic strain or

maintained under static conditions were immunoprecipitated with ILK antibody.

C

42

Immunoblots of immunoprecipitates were probed sequentially with FAK and ILK

antibodies. We observed that FAK co-immunoprecipitated with ILK from lysates

of both static and strained cells, although there was no significant difference in

ILK-FAK association between static and strained cells (n=3, p>0.05; Fig. 4A). An

opposite co-immunoprecipitation of ILK using anti-FAK antibody was also

performed, showing a similar result. We then examined ILK association with HA-

tagged FAK Tyr397Phe and FAK Tyr925Phe mutants. Association of HA-tagged

FAK Tyr925Phe with ILK, but not association of HA-tagged FAK Tyr397Phe with

ILK, was reduced compared to association of HA-tagged wild type FAK with ILK

(n=6, p<0.05; Fig. 4B), indicating that FAK Tyr925 phosphorylation is necessary

for ILK-FAK association.

ILK reduction selectively modulates strain-induced FAK phosphorylation.

Cyclic strain stimulated FAK Tyr397, FAK Tyr576 and FAK Tyr925

phosphorylation in comparison with static controls at 5 minutes (n=7, p<0.01, Fig.

4C). ILK reduction by siRNA did not affect basal FAK Tyr397 or FAK Tyr576

phosphorylation, and did not prevent further FAK Tyr397 or FAK Tyr576

phosphorylation in response to repetitive strain (Fig. 4C, n = 7, p < 0.01). In

contrast, ILK reduction completely blocked strain-induced FAK Tyr925

phosphorylation without affecting basal FAK Tyr925 phosphorylation (Fig. 4C,

n=7, p<0.01).

43

Figure 4. ILK associates with FAK and mediates FAKY925 phosphorylation. (A)

FAK co-immunoprecipitates with ILK independently of strain (n = 3, p>0.05).

Total Caco-2 cell lysate served as a control for the FAK band. An opposite co-

immunoprecipitation of ILK using anti-FAK antibody was also performed, showing

Fig. 4

44

ILK co-immunoprecipitates with FAK independently of strain (n=3, p>0.05). Total

Caco-2 cell lysate served as a control for the ILK band. (B) Caco-2 cells were

transfected with HA-tagged wild type or FAK point mutation constructs, and

subjected to static or cyclic strain conditions before immunoprecipitation of ILK

and immunoblotting for HA. The FAK Tyr397Phe mutation did not alter ILK-FAK

association (n=3, *p<0.05), but the FAK Tyr925Phe mutation reduced ILK-FAK

association compared to association of ILK with wild type FAK (n = 6, *p < 0.05).

Equal transfection efficiency was confirmed by immunoblotting of total cell

lysates for FAK and HA tag. Total lysates from cells transfected with KH3 control

vector served as a control. Total lysates immunoprecipitated with normal mouse

IgG also served as a control. (C) ILK reduction selectively modulates strain-

induced FAK phosphorylation. NT1 control siRNA or ILK siRNA transfected cells

were subjected to static versus strain conditions. Lysates were immunoblotted for

FAK phosphorylated at Tyr397, Tyr576 orTyr925. ILK reduction by siRNA

prevented strain-induced FAK Tyr925 phosphorylation, whereas strain-induced

FAK Tyr397 and FAK Tyr576 phosphorylation were not prevented (n = 7, *p <

0.01). Typical blots are presented at the top of each figure, whereas the bottom

graphs summarize densitometric analysis.

Src is required for ILK activation in response to strain, but strain-induced

Src phosphorylation is independent of ILK.

We examined whether Src was required for ILK activation using Src siRNA

and the Src inhibitor PP2. siRNA targeted to Src reduced Src protein by 70 ±

10% (n=3, p< 0.05; Fig. 5A, lower two blots, 1 of 3 similar experiments). To

45

confirm that the compound PP2 was actually inhibiting Src, we took advantage of

previous studies demonstrating that phosphorylation of FAK Y576 is a

downstream consequence of Src activation in a variety of cell types (23, 138).

We confirmed that PP2 at the dose used here actually inhibited the activity of its

target kinase Src because it inhibited FAK Y576 phosphorylation (Fig. 5B, lower

two blots, 1 of 3 similar experiments). Both Src siRNA (Fig. 5A) and PP2 (Fig. 5B)

prevented strain-induced ILK activation, indicating that Src is required for this

process. Furthermore, Caco-2 cells were infected with either Ad-GFP or Ad-Src.

After 48 hours infection, they were subjected to static or strain conditions, and

harvested for ILK in vitro kinase assay. Src activation by Ad-Src increased ILK

activity by 435 ± 31% in static cells, and no further increase of ILK activity was

observed in cells subjected to strain but infected with the Ad-Src virus (n=3, p<

0.05; Fig. 5D). Reduction of ILK by siRNA, however, did not inhibit strain

stimulation of Src phosphorylation (Fig. 5C). Together, this data suggests that

Src is upstream of ILK in the signaling pathway by which strain stimulates

migration.

ILK co-immunoprecipitates with Src, and ILK-Src association is increased

by strain, but this effect of strain is prevented by PP2.

Confluent serum-starved Caco-2 cells were pretreated with PP2 and then

cultured with or without cyclic strain for 5 minutes. Immunoblots of ILK

immunoprecipitates of whole cell lysates were probed sequentially with Src and

ILK antibodies. Strain increased the amount of Src that co-immunoprecipitated

with ILK compared to the same measurement in static control cells (Fig. 6, left

46

two bars, n = 3, p<0.05). Furthermore, PP2 inhibition of Src prevented the strain

effect on ILK-Src association (Fig. 6, right two bars, n = 3, p<0.05), indicating that

activated Src is necessary for the association of ILK and Src.

Fig. 5

47

Figure 5. Src associates with and is required for ILK activation by strain. ILK

activation was assessed in the presence of Src reduction by siRNA (A) or Src

inhibition by PP2 inhibitor (B). siRNA targeted to Src reduced Src protein by

70±10% (n=3, p< 0.05; Fig. 5A, lower two blots, 1 of 3 similar experiments). PP2

inhibitor significantly blocked basal as well as strain–stimulated phosphorylation

or accumulation of the specific FAK 576 downstream target of the kinase (n=3,

lower two blots for each, *p<0.05). Both siRNA to Src and the Src inhibitor PP2

blocked the strain-induced activation of ILK (n = 5, *p < 0.01). (C) Cells were

transfected with siRNA to ILK and immunoblotted for phosphorylated Src after

being subjected to static or cyclic strain conditions. Reducing ILK did not alter

strain-induced Src phosphorylation (n = 5, *p < 0.05) Typical blots are presented

at the top of each figure, and the bottom graphs summarize densitometric

analysis. (D) Caco-2 cells were infected with either Ad-GFP or Ad-GFP,

subjected to static or strain conditions, and harvested for ILK in vitro kinase

assay. Src activation by Ad-Src increased ILK activity by 435 ± 31% and 408 ±

24% in static cells and strained cells respectively (n=3, p< 0.05; Fig. 5D). Ad-

GFP infection served as control. Equal infection efficiency was confirmed by

immunoblotting of total cell lysates for Src and GFP. GAPDH served as a control

for equal loading.

48

Figure 6. Strain increased ILK-Src coimmunoprecipitation. However, the strain

effect on ILK-Src association is blocked by PP2. Serum-starved cells were

pretreated with PP2 or DMSO vehicle control and subjected to static or cyclic

strain conditions. An equal amount of whole cell lysate for each condition was

immunoprecipitated with anti-ILK antibody before immunoblotting for Src and for

ILK. ILK coimmunoprecipitated more Src in strained cells in comparison with

static control cells (left two bars, n = 3, *p<0.05). PP2 inhibition of Src abolished

the strain effect on ILK-Src association (right two bars, n = 3, *p<0.05). Total

lysates from cells transfected with siRNA targeted to Src or with non-targeting

NT1 sequences served as controls (12 μg protein/lane). Total lysates

immunoprecipitated with normal mouse IgG also served as a control. Typical

blots are presented at the top of each figure, whereas the bottom graphs

summarize densitometric analysis.

Fig. 6

49

ILK is also required for strain-induced MLC phosphorylation.

We have previously described increased phosphorylation of MLC in

response to strain and have shown that this activation of MLC is necessary for

strain stimulation of migration (174). Since ILK has been shown to directly

phosphorylate MLC to stimulate smooth muscle cell motility (33-34), we next

asked whether ILK reduction alters strain-induced MLC phosphorylation in Caco-

2 cells. Reduction of ILK by siRNA inhibited strain stimulation of MLC

phosphorylation (n=6, p<0.05; Fig. 7), suggesting that cyclic strain modulates

MLC phosphorylation via ILK activation.

Figure 7. Effect of ILK siRNA on MLC phosphorylation. Stain-stimulated MLC

phosphorylation was prevented by siRNA reduction of ILK in Caco-2 cells (n=6,

*p<0.05). Typical blots are presented at the top of each figure, whereas the

bottom graphs summarize densitometric analysis.

AKT, FAK, and MLC are required for strain stimulation of migration.

p-MLC Thr 18/Ser 19

t-MLC

p-MLC Thr 18/Ser 19

t-MLC

p-MLC Thr 18/Ser 19

t-MLC

Fig. 7

50

Akt and MLC have been previously reported to inhibit strain-induced

migration in Caco-2 cells across fibronectin (45, 174), so we next investigated the

contribution of Akt, FAK and MLC to the ability of strain to promote IEC-6 cell

migration. For these 24 hour studies, 1 μM AKT inhibitor IV proved toxic, so we

used 500 nM, which still prevented strain-induced AKT phosphorylation (not

shown). We assessed migration after pretreatment with the Akt inhibitor Akt IV

(500 nM), FAK inhibitor (2 μM), MLC inhibitor ML-7 (10 μM), or DMSO vehicle

controls. Blocking FAK and MLC prevented the motogenic effect of strain, while

blocking AKT reversed it (n≥25, p<0.05 for each, Figure 8).

Figure 8. Inhibiting Akt, FAK and MLC prevents strain stimulation of IEC-6

migration across fibronectin. Inhibiting these signals in IEC-6 cells with the Akt

inhibitor Akt IV (500 nM), FAK inhibitor (2 μM), or MLC inhibitor ML-7 (10 μM)

blocked the motogenic effect of strain compared with wound closure in static

Fig.8

51

cells. Data summarizes results of 25-36 holes per experiment from at least three

independent experiments (*p<0.05).

Figure 9. Schematic diagram outlines a signaling pathway that may mediate

strain-induced migration in intestinal epithelial cells consistent with our present

and previous observations. Cyclic mechanical strain activates PI3K and Src,

which leads to the activation of ILK. ILK is then required for the activation of Akt,

FAK, and MLC, which results in increased proliferation of intestinal epithelial cells.

PI3K, phosphatidylinositol 3-kinase; ILK, integrin-linked kinase; FAK, focal

adhesion kinase; MLC, myosine light chain.

Fig.9

52

CHAPTER 5

Discussion

Schlafen 3 induction by cyclic strain regulates intestinal epithelial

differentiation

The gut mucosa repetitively experiences mechanical forces causing strain

and pressure, including villus motility, contact with luminal contents, and

peristalsis (61, 164). Although intestinal epithelial deformation in vivo is complex,

we chose to study the effects on cultured non-transformed IEC-6 cells of a

regular and rhythmic deformation pattern of physiologically relevant amplitude

and frequency to facilitate reproducible analysis at acute time points. We found

that repetitive strain induced an absorptive phenotype characterized by increased

DPPIV activity and increased villin expression via induction of Schlafen 3 in rat

intestinal epithelial IEC-6 cells on collagen. However, strain failed to induce

Schlafen 3 or increase DPPIV activity on a fibronectin substrate, indicating the

strain effect on differentiation is somehow matrix-dependent. Cyclic strain

modulated intestinal epithelial Schlafen 3 expression via Src, p38, and PI-3-

kinase activation. Furthermore, Schlafen 3 might also be a key factor in the

induction of intestinal epithelial differentiation by other stimuli such as sodium

butyrate or TGF-β. However, these effects are independent of the mitogenic

effect of strain since Schlafen-3 reduction by siRNA did not inhibit strain-induced

proliferation in IEC-6 cells.

Considerable evidence indicates that cyclic mechanical deformation

provides physical signals for differentiation in many cell types that experience

53

strain normally in vivo (58, 65, 67, 90, 139, 157). Although cell differentiation is

difficult to study in vivo, intestinal lines which reproducibly modulate brush border

enzyme expression permit the modeling of this process in culture. The

expression of active brush border enzymes and microvilli specific structural

protein has proven useful markers of differentiation of intestinal epithelial cells.

We have previously reported that brush border enzyme DPPIV is amplitude-

dependently stimulated by strain in Caco-2 cells (11) , and here confirm this

observation in non-malignant IEC-6 cells. Although we measured only DPPIV

specific activity in this study, we have previously reported that changes in DPPIV

protein levels in response to cyclic strain correlate with DPPIV specific activity

(11). In addition, we demonstrate here that strain affects the expression another

differentiation marker, the microvilli specific protein villin, similarly.

Although the mechanism of strain-induced differentiation is poorly

understood, our data suggest that Schlafen 3 expression stimulated by strain

plays a critical role in regulating this strain effect, and Schlafen 3 might even be a

common effector of differentiating agents such as butyrate (83, 86, 125) and

TGF-beta (59, 127, 159). Stimulation of Schlafen 3 expression was associated

with increased activity of DPPIV and increased expression of villin, while

transfection of intestinal epithelial cells with siRNA to Schlafen 3 markedly

attenuated those two, indicating a relationship between Schlafen 3 and intestinal

epithelial differentiation. In contrast to the effects of strain on differentiation,

Schlafen 3 was not required for the mitogenic effect of either strain or EGF,

despite many reports that Schlafen family members serve as negative regulators

54

of growth (15, 116, 136). Indeed, Schlafen1 and Schlafen2 were also found to

not regulate proliferative activities in vitro in murine fibroblasts or myeloid cell

lines (175).

Although we have studied collagen I here, previous studies demonstrated that

strain affects intestinal epithelial cells similarly on type I and type IV collagen

substrates (6). Interestingly, although the effects of strain on the differentiation of

the intestinal epithelial cells cultured on collagen substrates had previously been

studied (11), the effects of strain on the differentiation of intestinal epithelial cells

cultured on fibronectin had not previously been investigated. Strain was unable to

induce Schlafen 3 or stimulate DPPIV activity on fibronectin, consistent with the

model that Schlafen 3 induction is required for strain to induce IEC-6 intestinal

epithelial differentiation and suggesting that strain-induced differentiation is

matrix-dependent similarly to strain-induced proliferation (which also occurs on

collagen or laminin but not fibronectin) but inversely to strain-induced migration

(which occurs on fibronectin but not collagen) (19, 21-22, 45-46, 172, 174). This

may be important because fibronectin deposition is characteristic of gut

inflammatory states characterized by chronic mucosal injury (153) in which the

sealing of breaks in the mucosal barrier by restitution may be more critical than

enterocytic differentiation.

We have previously reported that PI3K, Src, ERK, and P38 are activated in

intestinal epithelial cells subjected to repetitive mechanical strain on collagen

substrates (20-21, 46, 94). However, PI3K, Src and ERK had previously been

implicated in the regulation of intestinal epithelial proliferation by strain (21, 46),

55

while the role of p38 in mediating physical force effects in intestinal epithelial

cells is not yet well understood. Our current results suggest that strain-associated

p38 activation plays an important role in mediating the effects of cyclic strain on

the expression of differentiation genes in IEC-6 cells independently of mitogenic

ERK signaling. Activation of specific mitogen-activated protein kinases (MAPKs)

by mechanical strain has also been shown to be involved in phenotypic

modulation in some other mechano-sensitive cell types including vascular

smooth muscle cells (123, 148) and osteoblasts (49, 75). However, the role of

p38 in mediating strain effects seems likely to be cell-type and matrix-specific. In

human bone marrow stromal cells, ERK and p38 are each activated in response

to strain, but neither seems required for strain induced enhanced differentiation

(35). In contrast, p38 activation in response to strain in embryonic stem cells is

required for strain-stimulated differentiation (135). Moreover, p38 stimulates

differentiation in intestinal cells subjected to some other stimuli as well, such as

sodium butyrate (37) and oligosaccharides (85). Our observation that p38

inhibitor SB203580 blocks strain-induced Schlafen 3 expression, suggests a

possible role for p38 in intestinal differentiation via Schlafen 3, while the different

roles of p38 in mediating strain effects in other cell types likely reflects the

different regulatory machinery of these other cells.

We additionally demonstrated that blocking PI3K by LY294002 or blocking

Src by PP2 each prevented induction of Schlafen 3 by strain. This observation is

consistent with previous suggestions that PI3K activation is required for the

stimulation of p38 MAPK during chondrogenesis of mesenchymal cells (113),

56

and PP2 regulates human trophoblast cells differentiation by activating p38 (25).

Thus, it would appear then that repetitive deformation is an important trophic

factor for intestinal epithelial cells that promote a differentiated phenotype.

In conclusion, cyclic mechanical strain applied to cells plated on collagen

activated cell signals necessary for cell differentiation in a matrix-dependent

fashion. The induction of Schlafen 3 or its human homologues by strain may

modulate intestinal epithelial differentiation and preserve the gut mucosa during

normal gut function.

57

ILK mediates the effects of strain on intestinal epithelial wound closure

The intestinal mucosa is subjected to constant injury during normal function

and in intestinal disease states such as Crohn’s disease. Mechanical deformation

generated under normal conditions of digestion by peristalsis (61, 76, 115),

contact with luminal contents, and villous motility (103) may affect cell signaling,

and regulates migration as well as other biological properties in intestinal

epithelial cells (11, 19, 21, 45-46, 174). Although the effects of cyclic strain on

signal activation and wound closure that we report here may appear relatively

small, they are highly statistically significant. We (9-11, 39, 50) and others (51-53)

have previously described changes in cell migration and signaling in intestinal

epithelial cells of similar magnitudes in response to various stimuli. Interestingly,

Gange and colleague recently observed that knockdown of ILK expression

inhibits HIEC cell spreading and migration and Caco-2/15 monolayer restitution

(44), and the magnitude of their result in Caco-2 cells is comparable to ours.

Finally, such small changes in the rate of cell migration may well be biologically

important in determining whether a mucosal wound heals or fails to heal in vivo,

since the tightly regulated intestinal epithelium is constantly injured and repaired,

and a small change in the equilibrium between injury and repair may make the

difference between an intact mucosa and a failed barrier (54-58).

In this study, we delineate a role for ILK in the strain-induced motogenic

effect. ILK activation by strain was fast and sustained and required for the strain

effect on promotion of wound closure across fibronectin. Blocking PI3K

prevented strain activation of ILK, while blocking ILK prevented strain activation

58

of Akt, indicating that PI3K and Akt are respectively upstream and downstream of

ILK in this pathway. The reduction of FAK Tyr925 phosphorylation after ILK

siRNA transfection suggests that ILK regulates some functions of FAK. Since

FAK Tyr397 and Tyr576 phosphorylation are unaffected, however, this suggests

that ILK is not an upstream regulator of FAK activation. ILK co-

immunoprecipitated with FAK. ILK-FAK association was decreased by the FAK

Tyr925Phe mutation, but not point mutation of FAK Tyr397Phe. Src is an

upstream regulator of ILK in our system. Strain increased ILK-Src association,

but this strain effect was blocked by PP2. Finally, ILK activation by strain may be

required for strain-induced phosphorylation of MLC. Although our results do not

conclusively establish that all these effects of ILK require its kinase activity, our in

vitro kinase assay certainly suggests ILK activation by strain in parallel with the

requirement for ILK in the strain-mediated signaling pathway that we describe

here.

PI3K plays a major role in many cellular activities and is a key signaling

molecule in regulating cell migration in response to strain (45). Our observations

suggest that ILK activation regulates intestinal epithelial cell migration in a PI3K-

dependent manner, similarly to that seen in breast cancer cells (73) and chicken

fibroblast cells (122). In contrast, Tetreault et al recently reported that treatment

of gastric epithelial cells with the PI3K inhibitor LY294002 strongly reduced basal

and TGF-α-stimulated cell spreading, while a specific siRNA targeting ILK

sequence did not affect the kinetic rates of wound closure (145). We believe that

these apparently contrary results could reflect differences in response to different

59

stimulating factors in different cell types or differences in migration across

different substrates. Tetreault et al studied migration across plastic, while we

studied migration across tissue fibronectin. We have previously shown that

although repetitive deformation stimulates motility across tissue fibronectin,

which is deposited into tissues in settings of injury, deformation actually inhibits

motility on pure collagen or laminin substrates (174). We have also observed that

cell signaling during cell motility is very different on plastic than on matrix proteins

(168-169).

Downstream of PI3K, Akt signaling also contributes to the motogenic effect

of strain (45). Our results here suggest that Akt is also downstream of ILK,

consistent with recent observations that ILK-mediated activation of Akt is

required for endothelial cell motility (40). However, silencing ILK did not affect the

wound-induced phosphorylation of Akt in smooth muscle cells (63), indicating

that ILK may regulate smooth muscle cell migration by phosphorylating other

substrates, or that the scaffolding functions of ILK in focal adhesions may be

more important in this cell type. Indeed, phosphorylation of Akt is normal in ILK-

deficient chondrocytes (50) and fibroblasts (132), and ILK-induced Akt

phosphorylation is not observed in hypertrophic human hearts exhibiting elevated

ILK expression or mouse hearts transgenically engineered to overexpress ILK

(98), suggesting that ILK and Akt can be uncoupled in some settings.

Strain effects in intestinal epithelial cells on fibronectin involve activation of

FAK within the dynamic multi-protein focal adhesion complex that links the

transmembrane matrix-binding integrins with the cytoskeleton. Previous results

60

from our laboratory indicate that, although FAK is essential for the stimulation of

migration by cyclic strain, strain stimulates FAK Tyr925 phosphorylation by a

different time course than phosphorylation of FAK Tyr397 and FAK Tyr576 (19).

Furthermore, FAK phosphorylation at Tyr925 in response to strain is independent

of Src or FAK Tyr397/576/577 phosphorylation, since Tyr925 phosphorylation

was not prevented by either PP2 or Src reduction by siRNA and since the

tyrosine phosphorylation-deficient FAK triple mutant

Tyr925Phe/Tyr576Phe/Tyr577Phe still displayed increased Tyr925

phosphorylation in response to strain (19). Thus, traditional FAK activation by Src

may be less important for transducing strain effects on fibronectin than FAK’s

role in focal adhesion assembly. ILK localizes to integrin adhesions (92, 110,

141), and deletion of the gene encoding ILK blocks maturation of focal adhesions

by destabilizing α5β1 integrin-cytoskeleton linkages (141).

The proximity of ILK and FAK to integrins raised the possibility that they

might associate with each other within focal adhesions. Indeed, our current

experiments demonstrated that ILK immunoprecipitated with FAK, and that their

association was abolished by the tyrosine phosphorylation-deficient FAK mutant

Tyr925Phe but not by Tyr397Phe. Moreover, ILK reduction by siRNA selectively

blocked strain-induced FAK phosphorylation at Tyr925 but not at Tyr397 or

Tyr576. Tyr397 is in the NH2-terminal domain of FAK, but Tyr925 is within the C-

terminal focal adhesion targeting (FAT) domain of FAK (1). The selective

requirement for the phosphorylation of FAK Tyr925 for ILK-FAK association may

suggest that focal adhesion assembly may be important in mediating their

61

association. Similarly, Li et al. reported that ILK associates with FAK in vascular

smooth muscle cells (93). The selective blockade of strain-induced FAK Tyr925

phosphorylation by siRNA to ILK may suggest that ILK-FAK association is

essential for maintaining phosphorylated FAK Tyr925 by masking this

phosphorylation site upon binding or critical for bringing other kinases to the

proximity of FAK Tyr925. White and colleagues recently reported that the loss of

ILK impacts β1-integrin/FAK signaling (162), consistent with our observations in

intestinal epithelial cells.

Absence of Src kinases causes deficiencies in intestinal epithelial cell

migration and interferes with regulation of the actin cytoskeleton (102). Our data

suggests that Src is upstream of ILK in the pathway we describe, similarly to

signaling in an integrin α5-expressing normal rat intestinal epithelial cell line after

integrin-mediated cell adhesion (79). Our present results suggest that ILK binds

to active Src, since PP2 blocked the increase of association between ILK and Src

in response to strain. This is consistent with a recent study done by another

laboratory, in which in vitro pulldown assays demonstrated that ILK associated

with a GST-c-Src fusion protein through the SH3 domain of Src and the kinase

domain of ILK, and that this association was abolished by PP2 treatment (79).

The relationship between the PI3K/ILK/AKT signaling pathway and the

FAK/Src signaling pathway is not well defined and is likely to vary with the cell

type and stimulus studied. In vascular smooth muscle cells, the association

between FAK and ILK increased significantly after the cells were pretreated with

PP2 to inhibit Src. However, the level of FAK phosphorylation was not influenced

62

by ILK siRNA (93). Previous data from our laboratory demonstrated that in

SW620 cells, the PI3K inhibitor LY294002 blocked FAK Tyr397 phosphorylation

but not Src Tyr416 phosphorylation, and FAK siRNA blocked Akt phosphorylation

at Ser473 in response to pressure, indicating that PI3K is downstream of Src, but

upstream of FAK, and that FAK is upstream of Akt in SW620 cells in response to

pressure (146). Similarly, Del Re and colleagues reported that activation of

endogenous cardiomyocyte FAK by stretch leads to concomitant activation of Akt

(31).

Although ILK has been implicated in the regulation of spreading and

migration (56, 104), its role in these processes in intestinal epithelial cells has

remained unclear. For instance, it has only recently been reported that ILK is

implicated in the formation/assembly of mature focal adhesion points in human

intestinal epithelial cells (44). Our observations here demonstrate that the

silencing ILK in Caco-2 cells blocked the promotion of migration in response to

strain. Although previous work has reported that ILK can contribute to the

activation of signaling pathways that are implicated in the regulation of cell

migration, such as Akt (91), other migration-regulating pathways that are not

susceptible to being activated by ILK following strain certainly exist, such as

extracellular signal-regulated kinase (ERK). ERK is phosphorylated in response

to strain, and blocking ERK phosphorylation inhibited the motogenic effect of

strain on fibronectin (19, 174). This may explain why the loss of ILK significantly

reduced strain-stimulated migration in Caco-2 cells without completely abrogating

basal migration. Although Akt and MLC are critical mediators of intestinal

63

epithelial migration in response to strain, they are not the only signaling

molecules to promote migration (20, 45, 91, 174).

Tétreault et al observed that treatment of confluent HGE-17 cells with the

phosphatidylinositol 3-kinase inhibitor LY294002 strongly reduced basal and

TGFalpha-stimulated cell spreading, while addition of a specific siRNA targeting

the ILK sequence did not affect the kinetic rates of wound closure (145). The

differences between the results of Tétreault et al in gastric epithelial cells and our

current observations may reflect fundamental differences between the regulation

of gastric and intestinal epithelial cell migration. In addition, the intracellular

signals mediating TGF-alpha effects are likely to be fundamentally different from

the mediation of strain effects.

The increased basal activation of MLC in the presence of ILK inhibition in

our system may represent an attempt by the cell to overcome the blockade of

one pathway by stimulating another pathway. Although basal phosphorylation of

MLC was increased when ILK was reduced, the activation of MLC by strain was

reversed in ILK siRNA treated cells. Interestingly, it has been previously reported

that ILK can act as a calcium-independent MLCK that can phosphorylate the

MLC phosphatase inhibitor, CPI-17, at Thr38 (66). Hence, it is possible that

strain can activate MLC phosphatase in ILK-reduced cells since the MLC

phosphatase inhibitor CPI-17 is no longer being activated by ILK in response to

strain when ILK is reduced.

In conclusion, this study provides evidence that ILK is a critical mediator in

the stimulation of intestinal epithelial migration across fibronectin by repetitive

64

deformation. ILK has previously been implicated in the regulation of intestinal

epithelial proliferation (4, 44), while certain bacteria modulate intestinal epithelial

ILK to prevent infected epithelial cell detachment (78). Furthermore, ILK

knockdown in vivo inhibits fibronectin deposition (44), further emphasizing the

importance of the fibronectin-dependent and ILK-dependent promotion of

migration by strain studied here. ILK could prove an important target to modulate

the maintenance, renewal, and healing of the intestinal epithelium under

pathophysiologic conditions that alter natural patterns of deformation.

We hypothesized that cancer cells are activated by the increased pressures and

shear forces of the circulation, leading to increased adhesion and metastasis,

and that blocking this activation can reduce metastasis.

It has been estimated that nearly 1 X 106 tumor cells per gram of tumor

tissue are shed into the bloodstream each day [1]. In addition, viable tumor cells

can be recovered from the peripheral blood of more than 40% of patients

immediately following “curative” colon tumor resection surgery, indicating a poor

prognosis [2-3]. Such circulating cancer cells encounter many host responses,

including immune system interference, but some surmount these hurdles and

progress to metastatic disease.

0.8-1.0% of patients may experience wound implantation following tumor

resection surgery, while laboratory studies suggest that CT-26 murine colon

cancer cells exposed to 30 minutes of 15mmHg increased pressure adhere to

surgical wounds significantly greater than those cells left in ambient pressure

conditions [4-5]. However, although local surgical site recurrence can be an

65

important adverse event, most metastasis occurs via distant spread through the

lymphatic, venous, and arterial circulation [6]. Such metastatic progression

depends upon tumor cell adhesion at a distant site, but the factors that influence

cancer cell adhesion are incompletely understood. Some studies suggest that

adhesion at a distant site is a completely random process, while others have

implicated the overexpression of adhesion molecules such as ICAM, E-Cadherin,

or integrins [7-8]. Increased extracellular pressure promotes cancer cell adhesion

in vitro by FAK and Src activation at the focal adhesion complex, Akt activation,

and ultimately increased β1 integrin binding affinity [9]. Shear stress similarly

promotes adhesion in vitro [10]. These findings seem potentially clinically

relevant, as cancer cells experience pressure and shear stresses as they

circulate.

To test our hypothesis, CT-26 murine colon cancer cells were labeled with

carboxyfluorescein succinimidyl ester (CFSE) and 1 million cells were injected

into the tail vein of Balb/c mice. After 30 minutes, the cells were recovered by

cardiac puncture and analyzed by flow cytometry. The flow cytometer gated only

fluorescent CFSE labeled tumor cells that were further evaluated for FAK, Akt

and 9EG7 phosphorylation or activation. Passage through the circulation

increased phosphorylation of FAK at tyrosine 397 and Akt at serine 473, similar

to that observed in vitro in response to increased extracellular pressure.

Activation of the 9EG7 epitope, which specifically indicates activation of β1

heterodimers, was also increased in cells recovered from the circulation

66

(n=6;p<0.05, Figure 37). Pretreatment with pharmacologic inhibitors to FAK or

Akt prevented the increased phosphorylation of these molecules (not shown).

Pressures of the portal circulation are much lower (5-10mmHg) than those

of the peripheral circulation (120-125mmHg) [11-12]. We first sought to

determine whether pre-treating cells with pressure before injection into the portal

circulation could increase adhesion and metastatic capability from within blood

vessels. We exposed luciferase tagged CT-26 cells to ambient or 15 mmHg

increased pressure and injected 750,000 cells into the spleen of Balb/c mice.

Seven days later, we injected the mice with luciferin and imaged them using a

Kodak IS4000MM small animal imaging device. Exposure conditions were held

constant for all measurements and all bioluminescence intensity of the images

were pseudocolored (pink-low intensity; red-highest intensity). Quantitation using

MetaMorph software indicated a 73±6% increase in luminescence in the spleen

and liver of mice injected with pressure pre-treated cells relative to ambient

controls (n=16; p<0.05, Figure 38). Splenic weight increased 62±9% and hepatic

weight 41±11% in mice receiving pressure pre-treated cells (n=16;p<0.05, Figure

39), and long term survival was substantially decreased in mice injected with

pressure pre-treated cells compared with mice injected with cells not first

exposed to increased pressure (n=20; p<0.05, Figure 40). Prior treatment of the

cells with inhibitors of FAK or Akt before pressure treatment and splenic injection

resulted in reduced luminescence in mice receiving cells not exposed to

increased pressure and blocked the increase in luminescence associated with

injection with pressure-treated cells (Figure 41). Furthermore, long term survival

67

increased substantially in mice receiving inhibitor-treated, with or without

pressure pre-treatment (n=20 for each inhibitor; p<0.05, Figure 40). To

distinguish alterations in adhesion from any effect of this single dose of each

inhibitor on cellular proliferation, we performed MTT assays on cells treated with

the FAK or Akt inhibitor. We observed an initial decrease in cell proliferation with

each inhibitor, but a full recovery in proliferation by 24 hours compared with

untreated cells (n=5;p<0.05, Figure 42). Since we imaged the mice seven days

after surgery, our findings are thus more likely to reflect modulation of initial

tumor cell adhesion than interference with cellular proliferation.

To further confirm that cancer cells become activated during transit

through the circulation, we exposed luciferase tagged CT-26 cells to ambient or

15 mmHg increased pressure for 30 minutes and injected 1 million into the tail

vein of Balb/c mice. Seven days after surgery the mice were injected with

luciferin and bioluminescent imaging was performed. In contrast to our results

with portal injection, we observed no increase in lung metastasis in mice

receiving pressure pre-treated cells relative to those receiving control cells

preincubated at ambient (n=20, Figure 43) and we found no difference in long

term survival (n=20, Figure 44), consistent with our observation (Figure 37) that

the force-activated pathway was already activated by passage through the

circulation. When we pre-treated the cells with FAK or Akt inhibitors to block this

pathway, lung metastasis was reduced (n=12, Figure 45) and long term survival

increased (n=12, Figure 44).

68

Cancer metastasis is a complex process. Increased expression of cell

surface receptors such as VEGF, loss of function of tumor suppressor genes

such as p53 and over-expression of oncogenes all contribute [13]. Our results

suggest that the extracellular forces experienced by cancer cells during

circulation activate an adhesiogenic pathway that promotes adhesion and

subsequent metastasis. Cells pretreated with increased pressure displayed

increased adhesion and tumor burden locally, as illustrated by our splenic

injection model. However, because pressure activation is already maximized as

cancer cells travel through the circulation, pretreatment with pressure did not

further increase lung metastasis in our tail vein model.

Previous studies have confirmed the importance of FAK and Akt activation

in pressure induced signaling in vitro, as well the necessity of a dynamically

functional cytoskeleton [9]. FAK is a critical regulator in the signaling by which

pressure stimulates adhesion, proliferation and migration of cancer cells. In vitro

studies suggest that increased pressure results in FAK phosphorylation and

activation and downstream signaling. Furthermore, FAK and Akt may participate

in bi-directional signaling where FAK is necessary for Akt activation and Akt is

necessary for FAK activation. Upon activation, FAK and Akt translocate to the

cell membrane where FAK associates with 1 integrin. This association

increases in response to elevated pressures leading to increased integrin affinity

[5, 9]. Although this novel mechanism has been previously elucidated in vitro, this

is the first demonstration that physical forces promote metastasis in vivo, and

69

that interruption of the force-activated signal pathway has biological

consequences for metastasis.

In the present study, pretreatment of the cancer cells with a single dose of

an inhibitor of FAK or Akt, critical kinases activated by physical forces, decreased

metastasis and increased long term survival. FAK or Akt expression is increased

in diverse tumors, including those of colon, breast, prostate and lung [14-15].

Increased FAK expression correlates with increased cancer cell migration,

invasion, and metastasis [15-16]. Akt stimulates aerobic respiration in cancer

cells [17]. Akt activation and localization correlates with tumor invasion,

oncogene expression and an overall poor prognosis in several cancers, including

thyroid, lung and endometrial [18]. Early stage clinical trials of chronic therapy

with FAK or Akt inhibitors have shown promise. TAE226 is a FAK inhibitor that

inhibits cell proliferation and migration, induces apoptosis and inhibits cancer cell

growth through inhibition of FAK tyrosine 397 phosphorylation. Another FAK

inhibitor PF-562,271 inhibits FAK autophosphorylation and has lead to tumor

regression or stable disease in ovarian cancer patients when taken daily [19].

Similarly, the Akt inhibitors KP372-1 and GSK690693 inhibit tumor cell growth

and proliferation and induce apoptosis in a variety of cancers with daily treatment

[20]. Although such trials imply a potential benefit of these new chemotherapeutic

agents as part of a daily, long term regimen, our current results suggest that

even a single perioperative dose of these inhibitors may interfere with

perioperative tumor dissemination and metastasis when tumor cells are shed

during surgery.

70

In conclusion, our results indicate that in vivo pressures modulate cancer

cell adhesion and metastasis. Cancer cells traveling through the circulation

experience phosphorylation of FAK and Akt leading to activation of signaling that

induces adhesion. Increased adhesion at a distant site perpetuates the

metastatic process and indicates a poor prognosis for patients. These findings

may be particularly relevant, to patients with unresectable tumors or patients

undergoing surgery in whom millions of cancer cells may be shed from a primary

tumor prior to and during its removal.

In summary, this thesis is important for the assessment of intestinal cell

migration and differentiation. The intestinal mucosa is constantly subjected to

injuries that it must heal to maintain normal function. Intestinal mucosal healing is

a complex process involving lamellipodial extension of epithelial cells, migration,

and proliferation (36). Physical forces due to intestinal contraction as in

peristalsis, villous motility, and interactions with luminal contents likely interact

with the effects of cell-matrix interactions and soluble factor signaling to influence

these processes (7). When the normal homeostasis of the small bowel is

disturbed by disease as in ileus or IBD, the normal physical forces of the gut are

altered and the response to these forces also changes. Defects in cell migration

may lead to failure of barrier function of gut and bacterial translocation ensues

(129), with death being a possible outcome. A more complete understanding of

the pathways governing the complex processes of migration is required in order

to attempt therapeutic manipulation and potentially improve such pathologic

conditions.

71

The results of these studies also emphasized that multiple and potentially

redundant mechanisms are at work to achieve a differentiated phenotype, with

Schlafen 3 a common downstream mediator. The elucidation of these

mechanisms would be useful not only in delineating normal cell processes

leading to the differentiated phenotype but also in providing information regarding

abnormal processes, such as malignant transformation, that can occur when

these mechanisms go awry (28, 134). Future efforts could also be made to

design drugs that manipulate this pathway to promote mucosal differentiation,

reverse mucosal atrophy and promote intestinal adaptation in patients who have

short gut syndromes (39, 161). In addition, this work will have implications for

differentiation and development in other closely related epithelial tissues such as

the lung.

72

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ABSTRACT

MOLECULAR MECHANISMS OF STRAIN-STIMULATED INTESTINAL EPITHELIAL CELL DIFFERENTIATION AND MIGRATION

by

LISI YUAN

May 2012

Advisor: Dr. Marc Basson

Major: Anatomy and Cell Biology

Degree: Doctor of Philosophy

The intestinal epithelium is subjected to repetitive deformation during normal

gut function by peristalsis and villous motility. In vitro, cyclic strain promotes

intestinal epithelial proliferation and induces an absorptive phenotype

characterized by increased dipeptidyl dipeptidase (DPPIV) expression. Schlafen

3 is a novel gene recently associated with cellular differentiation. We sought to

evaluate whether Schlafen 3 mediates the effects of strain on the differentiation

of IEC-6 intestinal epithelial cells in the absence or presence of cyclic strain.

Strain increased Schlafen 3 mRNA and protein. In cells transfected with a control

non-targeting siRNA, strain increased DPPIV specific activity. However, Schlafen

3 reduction by siRNA decreased basal DPPIV and prevented any stimulation of

DPPIV activity by strain. Schlafen 3 reduction also prevented DPPIV induction by

sodium butyrate (1mM) or TGF-β (0.1ng/ml), two unrelated differentiating stimuli.

However, Shlafen-3 reduction by siRNA did not prevent the mitogenic effect of

strain or that of EGF. Blocking Src and PI-3-kinase prevented strain induction of

Schlafen 3, but Schlafen 3 induction required activation of p38 but not ERK.

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These results suggest that cyclic strain induces an absorptive phenotype

characterized by increased DPPIV activity via Src-, p38-, and PI-3-kinase-

dependent induction of Schlafen 3 in rat intestinal epithelial IEC-6 cells on

collagen, while Schlafen 3 may also be a key factor in the induction of intestinal

epithelial differentiation by other stimuli such as sodium butyrate or TGF-β. The

induction of Schlafen 3 or its human homologues may modulate intestinal

epithelial differentiation and preserve the gut mucosa during normal gut function.

Repetitive strain promotes intestinal epithelial migration across fibronectin in

vitro, but signaling mediators for this are poorly understood. We hypothesized

that integrin linked kinase (ILK) mediates strain-stimulated migration in intestinal

epithelial cells cultured on fibronectin. ILK kinase activity increased rapidly five

minutes after strain induction in both Caco-2 and IEC-6 cells. Wound closure in

response to strain was reduced in ILK siRNA transfected Caco-2 cell monolayers

compared to control siRNA transfected Caco-2 cells. Pharmacologic blockade of

PI3K or Src or reducing Src by siRNA prevented strain activation of ILK. ILK

coimmunoprecipitated with FAK, and this association was decreased by mutation

of FAK Tyr925 but not FAK Tyr397. Strain induction of FAK Tyr925

phosphorylation but not FAK Tyr397 or FAK Tyr576 phosphorylation was blocked

in ILK siRNA transfected cells. ILK-Src association was stimulated by strain and

was blocked by the Src inhibitor PP2. Finally, ILK reduction by siRNA inhibited

strain-induced phosphorylation of myosin light chain and Akt. These results

suggest a strain-dependent signaling pathway in which ILK association with FAK

and Src mediates the subsequent downstream strain-induced motogenic

99

response, and suggest that ILK induction by repetitive deformation may

contribute to recovery from mucosal injury and restoration of the mucosal barrier

in patients with prolonged ileus. ILK may therefore be an important target for

intervention to maintain the mucosa in such patients.

100

AUTOBIOGRAPHICAL STATEMENT

PERSONAL

Born May 25th, 1981, China

EDUCATION

2005 China Medical University, Shenyang, China; MD

PUBLICATIONS

Yu G, Dymond M, Yuan L, Chaturvedi LS, Shiratsuchi H, Durairaj S, Marsh HM, Basson MD. Propofol's effects on phagocytosis, proliferation, nitrate production, and cytokine secretion in pressure-stimulated microglial cells. Surgery. 2011 Jun 14 Yuan L, Sanders MA, Basson MD. ILK mediates the effects of strain on intestinal epithelial wound closure. Am J Physiol Cell Physiol. 2011 Feb;300(2):C356-67. Yuan L, Yu Y, Sanders MA, Majumdar AP, Basson MD. Schlafen 3 induction by cyclic strain regulates intestinal epithelial differentiation. Am J Physiol Gastrointest Liver Physiol. 2010 Jun;298(6):G994-G1003. Owen CR, Yuan L, Basson MD. Smad3 knockout mice exhibit impaired intestinal mucosal healing. Lab Invest. 2008 Oct;88(10):1101-9