REGULATION OF THE MAP KINASE PATHWAY

IN Y 1 ADRENAL CELLS

Zana Todorovic

A thesis submitted in the conformity with the requirements

for the degree of Master of Science

Department of Phamiacology in the

University of Toronto

O Copyright by Zana Todorovic 1997

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The growth inhiibitory &kt of adrenocorticotropic hormone (ACTH) on &enal cclls in vibo

is weii doaunented. ACTH-induad inhibition of ceii protiferation has ban obsavcd Li the Y1

mouse adrenocortical tumot ceil line, as well as in normdadrenocorticai celis isolated f?om a

variety of species hcluding rat, cow and human. Adrenal ceii mutants defective in CAMP-

dependent protein kinase are resistant to the growth inhibitory effect of A C m indicating that

the inhibitory eff- of the hormone on adrend cell proliferation is mediated by a CAMP-

dependent siphmg pathway.

The rnitogen activateci protein kinase (MAP kinase) cascade is an important regulatory

pathway in ceIl cycle progression. In fibroblast ceU lines, inhibition of the MAP kinase cascade by

CAMP has been iinked to CAMP-dependent inhibition of ceU proliferation. In this study I

exmineci the regulation of MAP kinase in Y1 mouse adrenocortical tumor ceUs in order to

determine if the growth inhibiting effect of ACTH in vitro correlated with the inhibition of MAP

kinase. Contrary to my expectations, 1 found that ACTH activates the MAP kinase pathway in

Y1 ceiis. ACTH also activates the MAP kinase pathway in the CAMP-dependent protein kinase

defective mutant, Kin 8. These results indicate that the activation of MAP kinase by ACTH is

CAMP-independent. These hdings prompted me to search for an underlying growth promothg

effect of the hormone. I found that ACTH promotes the transition of Y1 ceUs fiom G1 to S in a

CAMP-independent manner and stimulates ce1 division when administered to Y 1 celis as a short

pulse early in the G1 phase of the ce1 cyde. These latter hdings thus unmasked an underlying

growth promothg eff- of ACTH on adrenal cells that w u not previously appreciated. Our

findïings help to reconcile the observations that ACTH acts as a trophic hormone in viw.

i

1 wish to extend my gratitude to my supe~sor Dr. B. P. Schimmer for his guidance, support and

advice &ai to me over the course of my graduate training and in the process ofwriting this thesis.

1 thank my advisor Dr J. Heersche for suggestions aven to me during experimental work.

1 thank Jennivine Tsao for her technical assistance.

1 also thank Valdi, Wai King, Tina, Sandy and Claudia for being nice and supportive hiends.

1 thank to my husband for his amazing love, patience and support throughout my academic pursuit.

LIST OF CONTENTS

ABSTRACT

ACKNOWtEDGMENTS

TABLE OF CONTENTS

LIST OF SCIEMES AM> FIGURES

ABBREVIATIONS

1 INTRODUCTION

1. Overview of adrenal cortex

a) Structure, finctions and regulation

b) Mechanism of ACTH action on adrenal cortex

2. Growth regulation in the adrenal cortex

a) in vivo

i) Proliferative response of adrenal gland: Zona glomerulosa vs. Zona

fasciculatdreticuIaris

ii) Role of ACTH in proliferative response of adrenal glands

iii) The role of other factors in adrenal growth

iv) Origin of the dividing cells

b) Growth regulation of the adrend cortex in vifro

3. MAP kinase cascade

a) Regulation of M M kinase cascade

b) Mitogen activated protein kinase (MM kinase)

c) Cyclic-AMP regulation of the MAP kinase cascade

... lll

vii

i) Inhibitory effect of CAMP on ceil proliferation

ii) Stimulatory effect of CAMP on cell proliferation

4. Other signahg pathways

a) Signal transduction through cAMP-dependent protein kinase

b) W S A P K signding pathway

C) Janus kinase (JAK) signaling pathway

5. "Cross-talk" among signaling pathways

6. Activation of transcriptional factors and protooncogenes

II RESEARCH OBJECTIVE AND RATIONALE

III MATERIAL AND METHODS

1. Cells and ce11 culture

2. Incorporation of [3HJthymidine into the DNA

3. Protein determination

4. MAP kinase phosphorylation

a) Ceiî lysate preparation

b) Western Blot analysis

5 . MAP kinase activity

a) CeIl lysate preparation

b) Immunoprecipitation

c) MAP kinase assay

d) hunoblot t ing

6. Statistical analysis

N RESULTS

1. Kinetics of ['H'Jthymidine incorporation in Y 1 ceils 52

2. Inhibition of growth by long term treatment with ACTH, 8Br CAMP and PMA in Y1

ceiis 54

3. EfFect of FGF and semm treatment on MAP base phosphoryiation in Y c l s 57

4. T i e course of serum and FGF induced MAP kinase phosphorylation in Y1 ceiis 57

5. Time course of ACTH, 8Br CAMP and PMA on MAP kinase phosphorylation in Y1

cefls 61

6. Dose dependent relationships of ACTHar and ACT',, on MAP kinase

phosphorylation in Y 1 cells 63

7. Dose dependent relationships of ACTHar and ACTH,,, on MAP kinase

phosphorylation in Kin 8 cells 63

8. MAP kinase activity assay in Y 1 : pretreatment with ACTH and 8Br CAMP and treatment

with ACTH, 8Br CAMP, S'AMP, PMA and fonkoli 66

9. Time course of ACTH, 8Br CAMP and PMA stirnulated MAP kinase phosphorylation in

Kin 8 68

10. Effect of short treatment with ACTH, 8Br CAMP and PMA on [H]thymidine

incorporation in Y 1 cells 70

1 1. ACTH concentration dependent growth induction in Y 1 cells; cornparison to synthetic

AcTHi-39 70

12. Growth stimulation with short pulse treatment with ACTH, 8Br CAMP and PMA in

Kin 8 74

V DISCUSSION 76

VI REFERENCES 86

LET OF SCHEMES AND FIGURES

Scheme 1. Graphic representation of the CAMP signaling pathway

Scherne 2. MAP kinase cascade

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure S.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Time course of [3H]thyrnidine incorporation k Y1 cells 53

Effect of ACTH, 8Br CAMP or PMA on serum-stimulated ceU cycle progression

in Y1 ceIls 55

Effect of A C m 8Br CAMP or PMA on serum-stirnulated ceil cycle progression

in Kin 8 cells

EfFect of FGF and serum treatment on MAP kinase phosphoiylation

Effects of FGF, serum, 8Br CAMP and ACTH on MAP kinase

phosphorylation

Time course of ACTH, 8Br CAMP and PMA induced MAP kinase

phosphorylation in Y 1 cells

Concentration dependent MAP kinase phosphorylation in Y 1 cells

Concentration dependent MAP kinase phosphoiylation in Kin 8 cells

MAP kinase activity assay in Y 1

Figure 10. Time course of ACTH, 8Br CAMP and PMA induced MAI? kinase

phosphorylation in Kin 8 cells

Figure 11. Growth stimulation with short pulse treatment in Y1 ceils

Figure 12. ACTH concentration dependent stimulation of [3HJthymidUie incorporation

in Y1

Figure 13. Growth stimulation with short pulse treatment in Kin 8 cells

vi

LIST OF ABBREVLATIONS

8Br CAMP - 8-bromo cyclic adenosine 3', 5' monophosphate

ACTH - adrenocorticotropic hormone

AP-I - advator protein 1

CAMP - adenosine 3', 5' monophosphate

cAMPdPK - cyclic AMP-dependent protein h a s e

CRE - CAMP response element

CREB - CAMP response element binding protein

EGF - epidermal growth factor

ERK - extracellular regulated protein kinase

FGF - fibroblast growth factor

LPA - lysophosphatidic acid

MAPK - rnitogen activated protein kinase

MEK - MM kinase kinase

PDGF - platelet derived growth factor

PKC - protein kinase C

PLC - phospholipase C

PMA - phorbol myristyi acetate ester

HS - horse serum

FCS - fetal caifserum

PVDF - polyvinilidene ditluoride

PBS - Phosphate buffered saline

TBS - Tris buffered saline

vii

1 INTRODUCTION

1 INTRODUCTION

1. Overvicw of adrend corter

a) Structure, functions and regdation

The adrenal cortex is of mesoded ongin Pad is identifiable a9 a separate organ in the 2-

month-old fetus. The adrenal medulia is of sympathetic-neurai origin and in humans occupies a

central position in the widest part of the gland. The aduit adrend glands, with a combined weight

of 8-10 g, iie in the retro pentoneum above or medial to the upper poles of kidneys. A fibrous

capsule surrounds the gland; the yellowish outer cortex comprises 90 % of the adrend weight, the

inner medulla about 10 %. Histologically the adult cortex is composed of 3 zones: an outer zona

glomerulosa, a zona fkiculata, and an inner zona reticularis. The inner two zones appear to

fùnction as a unit. The zona glomerulosa, which produces aldosterone, is deficient in 17a-steroid

hydroxylase activity and thus cannot produce cortisol or androgens. The zona giomemlosa lacks a

weli defined structure, and the small lipid-poor cels are scattered beneath the adrend capsule.

The zona fasciculata is the thickest layer of the adrenal cortex and produces cortisol and

androgens. The cells in zona fasciculata are larger and extend in colums. The inner zona

reticularis surrounds the meduiia and alw produces cortisol and androgens. The c d s in this zone

are compact and are organited in a relatively narrow zone (reviewed in, Biglien and Kater, 1994;

Tyrreli and Forsham, 1994).

The adrenal cortex is responsible for the synthesis of several types of steroid hormones,

most importantly glucocorticoids and mineraiocorticoids (Miller, 1988). The major hormones

secreted by the adrenal cortex are cortisol, the adrend androgens and aldosterone. Steroid

production is divided arnong the zones and dierent mechanisms regulate their biosynthesis: the

outer, zona glomemlosa produces aldosterone and its synthesis is primariiy regulated by the r e h -

angiotensin system and by potassium. The i ~ e r zona fasciculata and zona reticularis produce

prVnarily cortisol but in some species, including humans, androgens and srnail amounts of

estrogens are also produced. These zones are primariiy r&ulated by anterior pituitary homone - adrenocorticotropic hormone (ACTH). In addition, angiotensin II, sodium and potassium,

dopamine, norepinephrine and epinephrine, hsuün, prostaglandins as well as additional f ~ o r s ali

contribute to the regulation of adrenocortical functions. Mineraiocorticoids affect the maintenance

of normal sodium and potassium concentrations, while glucocorticoids afZect glucose metabolism,

mood and behavior, immune nsponse and other endocrine functions. M e n characterized

biochemicaily, steroid 1 lp-hydroxylase was able to cataiyze the terminal steps in both a

glucocorticoid and rnineralocorticoid biosynthesiq thus forming both aldosterone and cortisol

(Yanagibashi et al., 1986). Recently, it has been demonstrated by Parker et al. (1 99 1) that actually

two isozymes of mouse steroid 1 1 j3-hydroxylase exist, designated as 1 1 p-OHase and aldosterone

synthase that are responsible for biosynthesis of either giucocorticoids or mineralocorticoids and

are expressed selectively. In situ hybridizations of adrenal sections with gene-specific probes

showed that the 1 1p-OHase gene was expressed in the zona fasciculata, whereas aidosterone

synthase denved transcripts were detected only in the outer zona glomerulosa. The authon also

examined Y 1 adrenocortical tumor cells regarding expression of the two transcripts and found

that Y1 cells resemble zona fasciculata cells from normal adrenai coriex in that they expressed

1 lp-OHase at high levels, while transcripts encoded by the aldosterone synthase gene were

present at approximately 10-fold lower levels.

The normal mouse adrenal cortex produces the glucocorticoid corticosterone and the

3

rnineralococticoid aldosterone. The primary point of control in this biosynthetic pathway is

conversion of cholesterol to pregnenolone (Churchiil and Kimura, 1979). This conversion of

cholesterol to pregnenolone by the side chah cleavage cytochrome P-450 (P-450&, an enzyme

localized in inner mitochondrial membrane, is hormonally regulated in aU steroidogenic tissues,

and in particular, by ACTH in the adrenal cortex (Hali, 1984). The adrenal cortex rnanifests two

discrete responses to ACTH which can be separated on temporal basis. The acute response to

A C T ' occurs rapidly, within seconds or minutes, and results in increased steroidogenesis

(Simpson and Watennan, 1983). This action of ACTH is mediated by CAMP and involves the

mobilization of cholesterol fiom its storage sites (Iipid droplets) to the imer rnitochondria

membrane in the vicinity of cholesterol side-chain cleavage cytochrorne P-45 0 (P-45OsCa

(Simpson and Waterman, 1983). The transport of the cholesterol fiom intracellular stores to the

inner mitochondrial membrane in which steroid production appears to be regulated, can be divided

into two steps: first is transport of cholesterol to rnitochondna and the second step is the delivery

of cholesterol fiom the outer to the i ~ e r membrane of mitochondria. The first step is considered

as a rate limiting step in the acute response to ACTH action (Crivello and Jefcoate, 1980). It has

been reported that this process rnight be regulated by Ca*-calmodulin cornplex, since this

complex has been found to specifically stimulate transport of cholesterol to mitochondria

(Papadopoulos et al., 1990). The second step, delivery of cholesterol from the outer to the imer

mitochondnai membrane, is a process sensitive to inhibitors of protein synthesis (Pedersen and

Brownie? 1983). When cholesterol cornes into the vicinity of cytochrorne P-450 in the inner

membrane of mitochondria, this enqme eatalyzes the cleavage of isocaproaldehyde from

cholesterol with the formation of pregnenolone. The conversion of pregnenolone to progesterone

and the 17- and 21-hydroxylations occur in the soluble-microsomal fractions of the cell, and the

hnalll-8-hydroxylation is an intramitochondrial event (Haii, 1984). The precisc mechanisms of

cholesterol mobilization and transport are not cîear. nor is the manner in which A C W acting via

CAMP, regdates these pmcesses, but it has been proposed that cholesterol transport occurs via

sterol carrier proteins (Lefevre et al., 1978). Clark et ai. (1 994) reported isolation of a cDNA

nom MA40 mouse Leydig tumor cells that encodes a protein that is synthesised in response to

Iuteininng hormone Ui a time and dose-responsive manner that correlates with stimulation of

steroidogenesis. They named the protein Steroidogenic Acute Regulatory protein (StAR).

Prmirsor of this protein is rapidly synthesized in response to hormone stimulation and targeted to

the mitochondria. It is translocated across the outer and i ~ e r membrane and undergoes two

cleavage events to the final mature form. It is hypothesized that translocation of this protein

across the mitochondrial membranes generates contact sites between the inner and outer

membrane aiiowing translocation of the cholesterol to the inner mitochondrial membrane and the

P450,. Lin et al. (1995) reponed isolation of two point mutations of StAR fiom two patients

with congenital lipoid hyperplasia, an autosomal recessive disorder that is characterized by a

deficiency of adrend and gonadal steroid hormones. These single point mutations rendered

formation of truncated, completely inactive StAR proteins. Contrary to these inactive StAR

proteins, coexpression of wild type StAR with cholesterol side chah cleavage system in COS-1

ceiîs increased pregnenolone production with the cholesterol as a substrate 8-fold. These results

provided genetic evidence for the hypothesis that the StAR is the molecule that mediates the acute

trophic regulation of steroid hormone synthesis. Another interest hg hypothesis about the

existence of carrier molecules that mediate cholesterol transfer fiom the outer to the inner

mitochondrial membrane came nom the studies of Knieger and Papadopoulos (1990). They

reported that in Y1 mouse adrenocortical celi he intermembrane cholesterol transpofl was

5

coupled to benzodiazepine receptors (PBR). They found that compound PK 11 195 (a PBR

ligand) markedly stirnulated steroidogenesis when Y 1 cells were pretreated with ACTH and

cyclohexhide (which by itself blocks steroidogenesis).

The trophic action of ACTH occurs over a long time fhme and is required for the

maintenance of adrenocortical integrity and optimal steroidogenic capacity Li the adrenai cortex.

The long terni actions of ACTH are also mediated by an increase in the intracellular

concentrations of CAMP (Simpson and Waterman, 1983). Evidence for the chronic action of

ACTH was provided by Punis et al. (1979), utilking hypophysectomized rats. Folîowing

hypophysectomy, adrenal weight and the number of mitochondna decreased, as did the content of

mitochondrial and rnicrosomd cytochrome P-450~ including cholesterol side chah cleavage

enzyme, steroid 2 1 -hydroxylase and 1 1 p-hydroxylase. The content of reducing equivalents

required for the electron transport chah in the case of both mitochondrial and microsomal

hydroxylases also decreased, nameiy: NADPH: adrenodoxin reduaase, iron-sulfur protein

adrenodoxin, and NADPH: cytochrome P-450 reductase. Prolonged treatment of

adrenalectomized rats with ACTH resulted in an hcrease of ail these activities and components,

towards the levels found in normal rats. From these studies it was apparent that ACTH is not only

capable of increasing the amount of enzymes responsible for certain steps within the

steroidogenic pathway but also of electron transport carriers that finction in the adrenal cortex

for steroid hydroqdation reactions. The increases in amounts of cytochromes P-450, P45Ol1,

and adrenodoxin in adrenocortical cells are clearly due, in a part, to increased rates of synthesis;

the same was shown for the microsomal components of the steroidogenic pathway (Simpson and

Waterman, 1988).

Under normal physiological conditions the adrenai cortex experiences ACTH pulses that

6

occw unifody throughout a 24 h penod. The mean interpulse interval is approximately 52 min

with the major increase in ACTH secretion in the morning (iranrnanesh et al., 1990), leading to a

reguiar diumal pattern of production of glucocorticoids and adrenai androgens. Fluctuations in

the levels of ACTH lead to fluctuations in the levels of these steroid products due to changes in

the amount of cholesterol e n t e ~ g the pathway. In addition, the constant stimulation by ACTH

leads to optimal levels of the enzymes in this pathway always being present.

b) Mechanism of ACTH action on adrenal cortex

ACTH binds to cell surface receptors thereby activating an effector that produces an

intracellular signal. Considerable evidence has been gathered suppotting the role of adenosine 3',

5'-monophosphate (CAMP) as an obligatory mediator of ACTH steroidogenic d e c t (Schimmer

and Zimrnerman, 1976; Saez et al., 1981). Part of the actions of ACTH on adrenal cells is

described in Scheme 1. The interaction between hormone and the receptor stimulates the activity

of adeny lyl cyclase and the production of CAMP via the aimulatory guanine nucleotide binding

protein G-protein (reviewed in Bockaert, 199 1). A G-protein is a heterovimer composed of three

subunits: a, f3 and y. The G-protein required for the stimulation of adenylyl cyclase (Gs) is a

member of a large family of G proteins, each composed of a combination of an a subunit and f3y

dimer ( Linder and Gilman, 1992). Binding of the hormone to the specific receptor leads to

codonnational changes that promote the exchange of GDP on the a subunit for GTP and

subsequent dissociation of a-GTP f?om the f3y dimer (Gian, 1987). RendeU et al. (1977)

provided evidence for participation of GTP in the activation of adenylyl cyclase in the response to

hormones. They suggested, using analysis of the steady state kinetics of the hepatic adenylyi

cyclase, the existence of a three-state modei. They suggested that adenylyl cyclase in its response

7

CAMP

ATP

Scheme 1. Graphic representation of the CAMP signaiing pathway in a cell

to the hormones, is a system that osallates between States of low and high activity depending on

the rate of tum over of GTP at the GTPase site and the availability of the GTP to the system. The

a-GTP generaily acts by interacting with and modulating the activity of an efféctor, adenylyl

cyclase ( Tang and Güman, 1992). Schimmer (1 982) invekgat ed optimal requirements for guanyl

mcleotides of adenylyl cyclase system in Y1 d s . He suggested that in the absence of guanyl

nucleotides, adenylyl cyclase activity increased marginally in response to synthetic ACTH,,. In

the presence of guanyl nucleotides response of adenylyl cyclase increased up to 10-fold upon

treatment with ACTH,,. Begeot et ai. (1 99 1) provided further evidences for hvolvemuit of Gs

in ACTH mediated action on adrenocortical cell, by direct measurements of modulation of Gs

Ievels in these cells upon various factors including ACTH itself They reported that in bovine

adrenai zona fasciculata cells, pretreatment with ACTH for 24 h enhanced CAMP accumulation in

response to ACTH and increased amount of a subunit of Gs evaluated by cholera toxin-

stimulated ADP ribosylation and by direct immunoblotting.

The intracellular second messenger generated by the receptorleffector cornpleq CAMP,

activates CAMP-dependent protein kinase (cAMPdPK) (Taylor et ai., 1990). Holoenzymes of

cAMPdPK are composed of a regulatory subunit (R) dimer and two catalytic subunits (C) that

dissociate and become catalytically active when the regulatory subunit diier binds four molecules

of CAMP (Scheme 1). Activation of cAMPdPK leads to the phosphorylation of Ser and some Thr

residues in various cellular proteins and lead to the evenhial end-responses of the cell. It has been

shown that c AMPdPK phosp ho rylates and activates substrates such as choiesterol ester hydrolase

(Beckett and Boyd, 1977).

Another important signal transduction pathway that is used by some receptors occupied

9

by agonists is the activation of hydrolysis of pho~phatidylinositol4~5-biphosphate by the enzyme

phospholipase C (PX) which results in production of two second messenger molecules: inositol

1.4,s-triphosphate a) and diacylgiycerol (DG) (Majerus, 1992). lP3 is a smaii water soluble

molecule that can increase intracellular concentrations of Ca". Diacylglycerol is a lipid molecule

that in concert with Ca* ions and phosphatydilse~e can ktivate protein kinasc C (PKC) which is

another second messenger. Honnones which primarily utilize CAMP as theû intraceiiular "second

messengef' are known to activate the IP3-Ca" signaling system, as well. Farese and colieagues

(1986) showed in primary cultures of rat adrenal cells that ACTH at certain concentrations can

activate both CAMP and IP,-Ca* intraceilular signaling systems. Kimura et al. (1993) showed that

in Y 1 mouse adrenocortical ce11 line, specific ACTH receptors were capable of activating in

parallel both the classical adenylyl cyclase-CAMP-cAMPdPK pathway and the PKC route.

Despite the eady studies that stated CAMP as the second messenger for ACTH, the

relative importance of CAMP in ACTH- mediated steroidogenesis has been in doubt in a number

of studies. For instance: the dose-response relationships for ACTH-stimulated steroidogenesis and

CAMP accumulation were parallel but the ED, value for ACTH action on CAMP accumulation

was more than one order of magnitude greater than the ED, value for ACTH-stimulated

steroidogenesis (Bed and Sayers, 1972); secondly, a concentration of ACTH that maximally

stimulated steroidogenesis increased CAMP levels only 20% of maximum (Sayers et al., 1974); on

the other hand, low ACTH concentrations stimulated steroidogenesis c50 % of maximum activity

without changing the levels of intracellular CAMP (Beail and Sayers, 1972); studies on the

influence of ACTH aaalogs and denvatives on CAMP accumulation and steroidogenesis have

added to the uncertainties regarding the participation of CAMP in ACTH action. Some ACTH-

Ue compounds (for example: O-nitrophenylsutfenyl-ACTH) stimulated steroidogenews without

10

Kowai et ai. (1974) emphasiÿed the importance of C." in ACTH binding to the receptors and

steroid production. Neverthefess, Schimmer and Zimmerrnan (1976) have reported thot ACTH

concentrations of 1 O-'' M stimdated steroidogenesis in Y 1 adrenocorticaf tumor celis up to 4-fold

over the basal activity, and was the maximal stimulation obtained. Mavimaüy effective

concentrations of ACTH i n c r d extfaceilular concentraitions of CAMP almost 50-fold. On the

other han& much Iowa concentrations of ACTH that stimulated steroidogenesis minimalîy (7.5

pM and 15 PM) Uicreased extracellular accumulation of CAMP 1.4-fold and 2.3-fold respedvely.

These observations confirmed that ACTH increases CAMP accumulation at al1 steroidogenic

concentrations and supported the hypothesis that CAMP is an essential component of ACTH-

stimulated steroidogenesis. Moreover, studies fiom Schimmer and colleagues, added more light to

the controversy regarding CAMP as an obligatory component of ACTH-stimulated adrenal

steroidogenesis and provided genetic support for the obligatory roles of CAMP and cAMPdPK in

the actions of ACTH. Rae et al. (1979) isolated two groups of mutant clones Erom Y1

adrenocortical tumor cens. Kin group of mutants exhibited altered cytosolic cAMPdPK activity.

In those mutants, steroidogenic response to 8-bromo CAMP (8Br CAMP) was highly reduced,

compared to parent Y 1 cells. In the other group of isolated mutants (Cyc), steroidogenic

responses to 8Br CAMP exceeded those obtained with the parent Y1 cells, whereas steroidogenic

responses to ACTH were depressed. This group of mutants had diminished corticotropin-

responsive adenylyl cyclase activity. These findings strongly suggested that CAMP and cAMPdPK

were obligatory components of ACTH action on adrenai steroidogenesis. In order to examine

relationships between the mutations and the resistance to ACTH and CAMP Wong et d. (1992)

transfected Kin 8 mutants with expression vector encodimg wiid type subunits of cAWdPK. By

this experirnental approach they recovered CAMP-responsive protein kinase activity and the

11

steroidogenic and morphologid responses to ACTH and CAMP. Olson et al. (1993) showed thPt

those cAMEkesistant phenotypes of mutant Kin clones were associated with singie basa changes

causing substitutions, respectively, of Glu for Gly, Trp for A r g , and Asp for Gly, in the

type 1 reguiatory subunit (RI protein). By expressing the mutant f o m of RI in the Y1 ceiîs they

studied the relationships between those mutations and impairment of CAMP-stimulated

adrenocorticai responses. Expression of the mutant RI fonns decreased CAMP stimulated d

rounding, steroid production and growth inkîition. AU together these data established the

importance of cAMPdPK as an essential regulator of adrenocodcal function.

2. Growth regulation in the adrend cortex

a) in vivo

In vivo the growth rate of the adrenal glands is under complex and multifactorial control

which, on the whole, is poorly understood. Under normal conditions, the glands bear a predictabie

relationship to body weight, growing as the animai grows. The mechanisms of this growth are

largely unknown.

i) Proiiferative response of adrenal gland: Zona glomerulosa vs. Zona tasciculata/reticularia

There are certain experimental conditions under which cells in the adrenal cortex

prolienite, thus allowing investigations into the mechanisms controliing adrenal growth. These

conditions are: when functional adrenal mess is experimentally decreased by either enucleation or

by unilateral adrenaiectomy. Bilateral adrenal enucleation is a process in which each adrenal glsnd

is carefuly litted and the body of the gland is extrudeci fiom the capsule. After biateral adrenai

enucleation, the adrenal regenerates fiom the capsule and the thio rirn of glomedosa cells thaî are

left. Three days afta enucleation, the admial capsule is markedly edernatous and there is evidence

12

for proliferation of the remaining parenchyd celis. Ten days later, tmbeeulae of celis are fomed,

with histological evidence of organlled growth and fùrther promeration of parenchymai cells.

Regeneration appears to be completed 3 weeks afker enucleation (Holzwarth et ai., 1980).

Engeland a al. (1996) investigated adrenal regeneration d e r enucleation in rats on nonnal and

low Na+ diet, using immunocytochemistry to monitor P~s'O, and P450,,, expression, and the

presmce of nuclear marker Ki67 a celi-cyde sssociated antigen in the proüferating ceiis.

They showed that switching rats fiom n o r d to low Na' diet causeâ zona glomerulosa

cells underlying the capsule to expand several fold. Zona fasciculata ceils stained with P45OllP are

displaced inward and the zona intemedia, defined by absence of staining, was placed between

those zones but aiso shified inward. In response to enucleation 5 to 7 days afîer, in rats on n o d

Na' diet cells adjacent to the capsule were without staining, suggesting that zona glornedosa was

missing and that cells present belonged to intermedia zone. Below that zone were cells stained

with P45Ol1, and the Ki47 labelling was associateci within that zone, reaecting that the

proliferating ceUs were associated with fasciculata type cells. However, 5 to 7 days aAer

enucleation perfonned on rats placed for 3 weeks on low Na+ diet, areas adjacent to the capsule

were mostly stained with P450,,, a d with markedly reduced P450, staining. Ki-67 labehg was

associated with both, P-45011p and P-450, areas. In conclusion, foliowing enucleation zona

fàsciculata type of cells proliferate and the zona glomerulosa ceU phenotype is reduced during the

first week after enucleation. When the phenotypic configuration of adrenal cortex was altered

placing rats on low Na' diet, proliferation was observed in both zona fasciculata and zona

glomerulosa ceUs, but the mechanism responsibie for dserentiation fiom glomenilosa to a

fasucdata ce1 phenotype remains unclear.

Unilaterai adrenalectomy results in a rapid (12-24 h) increase in proüferative response in

the runainhg gland fiom the rata compareâ to the tc9ponse iffer sham opemion. The respotlse

can be detected by ce in wet wught, DNA RNA and protein content in the remaining

adrend (DPllmPa et al., 1980). Two days Iita the munben of thyrnidinatabeiied nuda are

increased in cells in the capsule, glorneruiosa and outer fosadata of the remaining giand (Reiter

and Pizzareilo, 1966) sugeesting that prolifkration of thoh ceiis occurred. Holzwarth a al. (19%)

monitored proMeration of the adrend cdls between 24 and 96 h after unilateral h d e c t o m y

and &es unilaterd adrenalectomy whm the phenotypic organization of adrenal gland was aitered

by p l h g rats on low Na+ diet. They used irnrn~~ocytochemistry to monitor P450, and P450,,,

expression, and presence of nuclear marker -7, a cell-cycle associated antigen. Compared to

n o r d rats, in the rats rnaintained on low Na' diet, zona gfomerulosa expanded more than 5-fold.

The abundance of proliferating d s throughout the expanded giomedosa cells suggested that

much of the expansion is due to proliferation. Mer unilateral adrenalectomy, compensatory

growth was associated within outer zona fàsciculata. The proliferative response to adrenelectomy

of anhais placed on low Na' diet was as weil, associated within zona fascicuiata. These results

suggested that the distribution of and phenotype of proüferating ceus is deterrnined by the

proiiferative stimulus and that this is maintained even when the phenotypic organization of the

adrenal is fiinctionaiîy aitered.

ii) Role of ACTH in proliferative mponse of adrend giandr

Fnquent injections to intact animais or sustained U o n s of ACTH r d t in ccllulpr

hypertrophy (increase RNA and protein content ocairring within kst hours or days). This

proCuUr is later foliowed by hyperpk G n ~ d DNA and ceii number) ( F i i et J., 1956;

DPllmM et al, 1980). The mitotic index fouowing treatment with ACTH for 48 h was found to

increase in glomedosrl zone (Payet et ai., 1980). Both purineci natural and synthetic ACT'&

preparations produced both of these effects (Payet et al., 1980), thus excludmg the possibility

that another peptide from the anterior pituitary which copurifies with ACTH acts as a mitogenic

in v i w . Therefore it is clear tiom these shidies that in addition to stimulating differentiated

fiinctions of the adrenal cortex such as steroidogenesis, ACTH exerts profound trophic effects. In

addition to the ACTH efféct in viw that resulted in ce1 praliferation, lack of ACTH in the

cucdation caused by surgical rernoval of the pituitary gland, results in a fairly rapid decrease in

adrenal weight and atrophy of the adrenal glands (Purvis et al., 1979). Atrophy of adrenal glands

that occun upon hypophysectomy is particularly due to atrophy of inner adrenocorticd zones

(Paimore and Mulrow, 1967). Similarly, treatment with high doses of dexamethasone, sufficient

to totally inhibit ACTH secretion in rats, resulted in rapid decreases in adrenal weight

accompanied by decreases in RNA and protein content but no changes in DNA levels for 7 days

@ a l h m et ai., 1980). This atrophy Ulcluded a decrease in the activity of e q m e s associated with

steroidogenesis and growth (Ramachandran et al., 1977). Uitrastmcturaiiy, within this period,

adrenal fàsciculata ceil volume decreased 40-60%, nuclear volume decreased by 10-30%,

mitochondrial volume dso decreased by 5040% and volume of cytoplasmic mat& decreased by

4040% (Nussdorfer and Marzochi, 1972). These decreases were reversed by administration of

ACTH to hypophysectomized animalo. Treatment of hypophysectomized animais with ACTH for

15

48 h restored the structure and the îunction of the aârenal giand (Nudorfer and Marzochi,

1972). The role of cydic nucleotides in trophic actions of ACTH on adrenal M d 8 wpr

examined. Ney (1%9) obsared that CAMP anaiog &%utyiyl CAMP, when administered to

hypophysectomizcd rats, pprtiaiiy mauitained adrenal weight and the contait of RNA and protein.

Cyclic AMP and its dibutyqd d o g increamd the v o l h s of subceüular organelles including the

smooth endoplasmic reticulum and mitochondrial mptrix in the fàscidata f?om the adrenals of

intact rats. Cycbc AMP and its anaiog only p d y reproduced the trophic actions of ACTH

since increases in various morphomeric parameters w u smaller in the case of CAMP treatment

compared to ACTH treatrnent (Nussdorfer and Marrochi, 1972). Nevertheless, the authors

concluded that CAMP fundons as an intracellular mediator of the trophic effects of ACTH on

adrenal cortex. The dinerences in increases of various morphomeric parameters upon ACTH and

CAMP treatrnent, were explained by the fàct that the concentration of CAMP (25 mgkg of body

weight) used in this study was smaller in cornparison to ACTH (IO IWkg). On the other hand,

cGMP was not a good substitute for CAMP: this compound did not induce changes in the

mitochondnai cornpartment (Nussdorfer and Maaochi, 1973). When administered together,

however, CAMP and cGMP maintained the physiological integrity of both zona glomerulosa and

fiisçiculata in the adrenals and completely reversed the stnictural consequences of

hypophysectomy (Mauochi et al., 1974). Therefore it is not clear whether proüferative response

of adrenai glands to ACTH is solely due to the CAMP-mechanism of action.

It has been clear from the above that ACTH has broad trophic actions on adrenal glands

since excess of ACTH k vivo increased adrenal weight, wtiich is adiieved by sequentiai cellular

hypertrophy followed by hyperplasia whiie, depletion of AC= levels in the blood decreaseâ

adrenal weight and caused atrophy of adrenai glands. Cyclic AMP acts as an intracellular mediator

of the trophic fundons of ACTH although, it is not sure whether CAMP is exclusive mediator of

trophic actions of ACTH on adrend cortex.

iii) The role of otber facîon in adrend gmwth

Numerous examples, taken mainiy ceil culture models, support the notion that

peptide powth fhctors are, in fact, multifàctorial regdaton of d growth and dinerentiation.

Serum contains -ors thai are rquvcd for optimal growth, since omission of saum fiom growth

medium results in growth cessation (Amelin et al., 1977). Cnide extracts fiorn pituitary gland

stirnulated DNA synthesis in ceU cycle arrested Y1 ceiis (Armelin et al., 1977). Gospodarowicz

and Handly (1975) described the mitogenic effect of Fibroblast Growth Factor (FGF) isolated

from bovine pituitary gland on Y1 mouse tumour adrenal cell line. The minimal FGF

concentration that was growth stimulatory was as low as 1rL2 M. Coulter et al. (1996) showed by

acamlliing fetuses of rhesus modceys from 109 days of gestation until term that the ontogenic

adrenal growth is regulated by locdy synthesùed insulin Iüce growth factor 1 (IGF-1) and that

cessation of fetal adrenal growth which occurs by the fist week der delivery may be mediated by

the decrease in IGF-1 recepton. Very similas redts were obtstined in the studies of JafEe and

Messiano (1992) where they found that epidemd growth Wor (EGF), fibtoblast growth fmor

FGF) and IGF-II were mitogens in fetal adrenal cortical celis and that their expression in fetai

zone cells was up-regulated with ACTH. In cultures of bovine adrenocortical cells, angiotensin II

was found to stimulate ce11 proliferation and [3Hlthymidine incorporation (Gill et al., 1977). I11

and Gospodarowicz (1982) investigated the growth requirements for bovine aârenai cortex ceUs

in culture, and found that senim, insulin and FGF wae rnitogenic stimuli for those cells.

The central nervous system was also found ta play a role in the growth of adrenal cds.

VIP-üke immunoreactive fibres were found in abundance in the capsuk and zona glomedosa md

some olso were found in the meduiia The fibres appcsrrd to innecvate parenchymsi d s rather

than blood vessds (Holzwarth a al., 1980). There is Jso evidence for the presence of severai

neuronal plexuses in adrenal cortex and the effect of neurotransmitters and neuropeptides strongly

suggested that the regdation of adrenocorticai growth is wntrolled by the autonomic nerwnis

system as weli as by humoral faaors. Neuropeptides and neuotransmitters that take part in

adrenal growth are: catech01amines, VIP ad NPY (Hblzwarth et ai., 1987). Haiasz and

Szentagothai (1959) reported that &er treatments that taud adrend growth (stress, ACTH)

nuclear shrinkage occurred in ceils prunarily in the ventromedial nadei (VMN). They found that

eAer uniiateral adrenalectomy nuclear Sue in VMN d s contralateral to the removed adrenal

were larger, whereas those in VMN ceîis ipdateral to the removed gland were smaller than nuclei

in VMN cells sham-operated rats. This was very suggestive evidence that adrenal size Uiformation

was transmitted neurally to the cells in VMN.

iv) Origin of dividing cellr

When adrenal glands grow, proliferation is essentially iimited to the cortex. It seems clear

that the source of new ceUs is in the corticpl periphery, derived fiom either subcapsular stem d s

or glomerulosa and outer fasciculata cells (HortlSby, 1984-85). There are two existing hypotheses

regarding proliferation of adred cortex. The "migration" hypothesis States that proliferative

adrenaî ceUs arise ftom a population of undinerentiated stem d s that expand and dfirentiate as

they move through the distinct zones of the cortex (Mitani et al., 1996). The second hypothesk

which is called the bbzonal" hypothesis argues that growth in each adrenocorticd zone U regulated

independently (Holzwarth et al., 1996). The migration theory has been supported by ni vivo

experiments which have tracked labelleâ cens inwards d e r mitosis. Labelied ceiis m i m e

c e n t r i p d y Born @ornedosa to ret.i& over 8 period of approxhateiy 3 weeh in rats

injected with [)Hlthymidhe on Qy 6 of age (Zajicek a ai., 1986). These latta studiw suaest

that aidosterone producing zona glomedosa cells 6rst becorne cortisd-producing SOM

hcicuiata ceils and then anârogen peuethg zona rdculaiis celis.

Proponents of the z o d hypothesis point to experiments in which steroidogenesis and

zone widths are often reguiated independent of changes in the other zones. FolIowing

hypophysectomy, as it was mentioned eariier, the inner adrenocortical zones atrophy and cortisol

production is much reduced leaving the zona glomerulosa with aldosterone synthesis relatively

unchanged (Palmore and Mulrow, 1967). Conversely, dietary sodium restrictions specifically

causes zona glomerulosa hypertrophy (Engeland a ai., 1996). Mitani et al. (1994) in a recent

publication argued that the zona intermedia which comprises a thin layer of ceUs between the

giomexulosa and fasciculata cells represents stem ceiis fiom which both fasciculata and

glomerulosa ceUs originate. McEwan et al. (1 995) reported that DNA synthesis within the zones

of adrenal cortex appeared to be independently regulated, excluded the possibility that the zona

intemedia could be the origin of undinerentiated hiculata and glomerulosa cells. Accordhg to

their re4ts it is not likely that adrenocortical cells migrate between zones, although migration

within zones is ükely to occur.

b) Growth regdation in adrenal cortex Ui *ibo

The fact that ceil proliferation is induced in vivo by ACTH is bUrly âiflicult to reconde

with the opposite findings that are observeci in vitro ifta treatment of adrenocortical cells Mth

ACTH. In contrast to the n o r d adrenrl in which ACTH and CAMP appear to induce DNA

synthesis, in a W o n a l adrenal hunour ceii line or in norinal adrenai d i s in culture, ACTH and

CAMP have been shown to inhibit DNA synthesis and ceii replication aithough the dserentiated

fûnction, narnely steroidogenesis, was stimulated (Masui and Garren, 1970; Gospodarowicz and

Handley, 1975; Gili and Weidman, 1977; A m e h et al., 1977; Gospodarowicz et ai., 1977).

Weidman and Gili (1977) detennined the length of dinerent phases of the celi cycle of Y1 mouse

adrenocortical tumor ceiis Y1 and examined the biological state of cells whose growth was

arrested by serum deprivation, ACTH or CAMP treatrnent. By using flow microfiuorimetry, they

detennined that the length of average doubling time of Y1 ceils was approximately 24 houn.

They also indicated that although ACTH treatment and serum deprivation arrested ceus in the G1

phase of the ceU cycle, the biochemicd States of arrested c d s diaered: ceils arrested by treatment

with high ACTH doses (0.4 U/d) or with 8Br CAMP in the presence of serum had increased ceii

size, protein and RNA content. Additionaily, while senim deprivation and ACTH treatment

arrested ceiis in G1 phase, 8Br CAMP treatment arrested ceUs in both Gl and Ui G2. GilI and

Weidman (1977) ewmined the effect of senim, ACTH and 8Br CAMP given at different times to

serum-starved Y 1 celis. They found that when senam was added to ceUs arrested by serum

removai, a characteristic lag of 8 to 10 hours before initiation of DNA synthesis occurred. After

the initial lag, G1 population of cells progressed exponentially into S phase. Addition of ACTH or

8Br CAMP four hours following semm naddition completely inhibited the onset of DNA

synthesis. On the contrary, ACTH or 8Br CAMP added 8 hours &er serum addition only panidy

20

inbibited DNA synthesis, suggesting that by 8 hom after s e m addition, substantial cornmitment

to the progression thro~gh the c d cycle occurred. According to these r d t s , they concluded that

ACTH or 8Br CAMP opposed semm induceci initiation of DNA synthesis only when added pnor

to S. Once cornmitment to DNA synthesis occurca ACTH or 8Br CAMP did not inhibit DNA

synthesis. GospodPmwia and Handley (1975) invdgatd whether ACTH can block DNA

synthesis in Y 1 ceils if added together with FGF. Indeeâ, addition d ACTH (0.75 U/rnl) togeth«

with FGF to Y 1 d i s blocked initiation of DNA synthesis as well as increase in ceU number

observed d e r addition of FGF done. Annelin et ai. (1977) hvestigated regulation of DNA

synthesis in Y1 cells upon addition of ACTH and pituitary growth factors. They suggested that

upon stimulation with piniitary factors, cells are not irreversibly committed to the DNA synthesis

in the first 6 hours and are still susceptible to the inhibitory effect of ACTH. However, d e r this

time ells be rne irreversibly committed to DNA synthesis and completely resistant to ACTH

action. Accordhg ta them, first 6 hours can be designated as Go phase and another 5 hours (which

is the rest of 1 1 h lag penod required for the first restimulated cell to enter S phase, according to

their findings) G1 phase. They indicated that control of proliferation and differentiation probably

operates at the transition of Go to G1 and through reactions taking place in Go. In bovine

adrenocortical ceUs in primary culture, Gospodarowicz et al. (1977) found that FGF was a potent

stimulator of growth, whiie ACTH stimulated steroid production and at the same tirne inhibited

the incorporation of [%l]thymidine into DNA Identical dose- response curves for stimulation of

steroidogenesis and inhibition of DNA synthesis were observed with a 50 % effhve dose of

approxhately looll M ACTH. Homsby et ai. (1 974) showed that in zona glomemlosa cells

isolated fiom adult rat adrenal cortex ACTH treatment (100 mU/ml) also inhibited growth.

Interesthg was their notion that those cuitured glomerulosa celis after treatment with ACTH

21

evenîuaily resembled fhiculata cells; the same observation was reported âom Hobarth a al.

(1996) in vivo, afta adrenal enucleation when rots were placed on a low Na+ dia. In primary

cultures of adult bovine adrenowrtid ceus, ACTH stimdated steroidogenesis mPnmally md

inbibited DNA synthesis completely at a hamaximal effiective concentration of 0.08 &f, which

is approximately l Q I O M (Hornsby and Gdl, 1978). HoweMr, stimulation of only a small M o n

of CAMP production was necessary for either stimulation of steroidogenesis or

inhibition of DNA synthesis in primary cuituns. Rainey et ai. (1983) investigated effect of ACTH

treatment on bovine and human adrenal ceUs. niey reporteci that in primary bovine adrenocortical

celî cultures and prirnary human definitive zone adrenocortical cell cultures, treatment with ACTH

for 24 to 48 h inhibited ce11 proliferation with the daerences in respect to cell morphology. Whiie

in human adrenocortical cells treatment with ACTH caused cells to round up, no such behaviour

was observed in bovine cells. Since both cell types responded similarly to ACTH in respect to

increaseà steroidogenesis and growth inhibition they concluded that steroidogenic and

morphologid responses must be triggered with ACTH caused separable events. Contrary to the

abundant data and general nile that ACTH has an inhibitory effect on adrenal ceil proliferation in

cuiture, an exception to the rule is the saidy fiom h n a t o and Andreis (1973) and Armato et al.

(1974). They reported that in fieshly isolated rat and human adrenocortical cells (from zona

Easciculata and reticularis) ACTH promoted proliferation of those cells. The percentage of

adrenocorticd celis that were labelled with [3H]thymidine was approximately 15 %, while the

percentage of dividing cells was only 4 %. These obpervations have opened the possibility that in

adrenocortical cells in culture the growth-promoting effect of ACTH is masked by o metabolic

sequence that lads to growth inhibition. Whitfield et ai. (1976) raised a sirnilar suggestion on

their studies on BALB/3T3 fetal mouse endothehl c d s and in rat liver cells. They suggested that

" any agent which in cantinuous exposure stops prolifarton, mi@ actuaiiy be a kcy positive

reguiator that under normal physiologid conditions appean only briefiy h the ceiî to tngger a

spedic phase of proüfedve development without blocking other phases". Morera and S p a

(1980) reported while native porche ACTH,, and synthetic ACTH,, i n c r d steroid

production and production of CAMP and inhibited DNA sjmthesis in Y1 cells, terminai

sequence of ACTH - ACTH,, ACT& and a-MSH in addition to causing d changes in

CAMP levels, stirnuiated steroidogenesis and increased DNA synthesis.

These studies supporteci the argument that ACTH has an inhibitory effect on adred celis

in vitro, but suggested that ACTH may have some rnitogenic action which may be underlying this

inhibitory effect .

3. MAP kinase cascade

a) Regulation of MAP kinase cascade

The intracellular transmission of growth factor signals is presumed to be mediated by

sequentidy activated protein kinases integrated into a network. Many extraceIIular mitogens,

such as platelet-derived growth factor (PDGF), EGF and nerve growth factor induce

autophosphotyiation on their respective receptors on tyrosine residues by activation of intrinsic

catalytic domains. This is ultimately tmslated hto phosphorylation of serine and threonine

residues in proteins throughout the cell (reviewed by Davis, 1993).

Autophosphorylation of the receptor acts as a signai for binding of other intracellular

proteins which are thereby activated. Recently a large number of proteins that bind to activated

growth factor receptors have been identifiecl. These proteins share two higbly conserved domains

known as SH2 and SH3 narned by their virtue of considerable sequence simüarity to the non-

23

catalytic region of the src family of protein tyrosine kinases. Broad investigations into the

interactions between growth faaor rrcepton and SHZ/SH3-domain-contallllng proteins

uncuvered pathways downstream of these initial signaiing events. One such cascade characterized

in this way, highly consexved among a Mliety of difterent species, is the M N kinase cascade.

Among those proteins that recognize phosphotyrosi~es o i the growth factor receptors through

SEI3 containing domaias and is important with respect to the MAP kinase pathway, is a

multiprotein complex that activates Ras.

Schematic represmtation of the MAP kinase crucade is presented on the Scheme 2. The

initial step, that triggers propagation of the reactions through this cascade, after receptor

autophosphorylation is recniitment of the Grb2-SOS complex to the receptor and to the plasma

membrane. Grb2 binds with its SH3 domains guanine nucleotide exchange factor, SOS (son of

sevenless) via proline rich regions in the c-terminal region of SOS and the GrbZSos complex is

recruited to the receptor and to the piasma membrane by the SH2 domain of Grb2 (Egan d al.,

1993). Localization of SOS with its membrane bound target Ras catalyses formation of the active

form of p2 1 Ras by accelerating the exchange of GDP for GTP on the Ras molecule. The GTP-

bound fonn of Ras is active and is beiieved to play a centrai role in signal transduction by a variety

of growth factors. In the context of the MAP kinase pathway, activated Ras in tum activates

1 by a mechanism that appears to involve direct contact between Ras and (Moodie et al.,

1993; Kyriakis et al., 1992; Chuang a al., 1994; Vojtek a al., 1993). AcUvated Raf-1 which is a

dual, serine/threonine kinase phosphorylates MEK (hW? kinase kinase) on Ser,,, and Ser,

thereby activating it (Zhang et al., 1993; Cook et al., 1993). MM is duai specificity protein

kinase that phosphorylates substrates on both tyrosine and se~dthreonine residues including

MAP kinase (Kyriakis et al., 199 1). Following activation MAP Linase is subject to

EGF FGF PDGÇ

Plasma membrane

cAMPdPK

Transcription factors (fos & jun)

Cell proliferation

Scheme 2. MAP kinase cascade

redistribution within the ce1 and can activate target proteins such as phospholipase AZ, p90rsk

and p62TCF in the plasma membrane, cytoplasm, and nucleus, respectively. In the nucleus MAP

kinase initiates activation of nuciear protooncogenes (aiso d e d imrnediate early genes) incIuding

c-jun (Mverer et al., 1991), c-myc (Gupta et al., 1993) and c-fos (Gille et al., 1992). These

processes uitixnately l d to ce1 differentiation and proHekition. It shouid be emphasized that in

the MAP kinase pathway, it is Wrely that each t r d u a r has many substrates to act on, the resdt

being a complex signahg network rather than a linear pathway.

b) Mitogen activated protein kinase (MAP kinase)

MAP kinases also cailed extracellular regulated kinases (ERKs), have been proposed to

play an important role in ceU growth and dserentiation stimulated by growth factors. MAP kinase

was first identified by Ray and SturgU(1987) as a se~e/threonllie-directed kinase that utilized

microtubule associated protein 2 as a substrate following stimulation of adipocytes with insulin.

Cloning studies indicated presence of at least two MAP îcinases, ERK 1 and ERK 2 (Boulton et

al., 1991). Other members include two ERIO isozymes having approxVnately 50 % sequence

similady with its counterparts (Meloche d al., 1996), ERK 4 (Boulton and Cobb, 1991), ERK 5

(Zhou et al., 1995), Jun N-terminai kinaseslstress-activated protein kinases (MWSAPK; Derijard

et al., 1994), two p38 MAP kinases (Han et al., 1993; Jiang et ai., 1996) and p57 MAP kinases

(Lee et al., 1993) . Phosphorylation of ERK 2 occurs upon Thr,, and Tyr,, residues in the

mammalian species (Anderson et ai., 1990). It seems that phosphorylation on both residues is

essential for enzyme activation since single point mutations produce inactive mutants: Th,, to

Ala and Tyr,,, to Phe (Robbins et ai., 1993). Recently the three dimensional atomic structure of

MAP kinases has been resolved in its unphosphoqlated, low-activity confocmation ( Z b g et d.,

26

1994). It appears tbat Th,, is on the surface of the molecule, while Tyr,, is buried in large

hydrophobie pocket and blocks the peptide binàing site, suggesting that activation is likely to

involve both global and local conformation changes. KUietic anaiysis of the rate of incorporation

of phosphate into Tyr and Thr residues of ERK 2 by the MEK suggested that the enzyme is

phosphorylated first on tyrosine residue (Zhang et al., 1993).

c) Cyclie-AMP regulrtion of the MAP kinase cascade

Cyclic AMP was the first second messmget to be identifieci, and its role in regulating

physiological processes is well established (Sutherland, 1972). Despite the fact that there are data

in the literature about CAMP dating back 25 years, the precise role of CAMP regulating cell

growth and proliferation remains a matter of considerable debate. In some cells it has been found

that the rising concentration of CAMP is associated with inhibition of ce1 proliferation, while in

other ceil types, the effkct of CAMP on cell proliferation is quite opposite: rising concentrations of

CAMP were found to be growth stimulatory.

i) Inhibitory effect of CAMP on ceU proliferatioa

In fibroblast celis (Rat 1) two different classes of growth hctors, lysophosphatidic acid

@PA) and EGF, are necessary to fÛUy stimulate DNA synthesis and activate ERK (Cook a al.,

1993). Cook and McConnick (1993) demonstrated that dibutyryl CAMP added 10 min before

LPA, into quiescent Ratl cells, completely inhibited the formation of aaivated phosphorylated

foms of MAP kinase and inhibited ceil growth. Similarly, Sturgiii et ai. (1 993) found that

forsicolin and isobutylmethyhthine, agents that stimulate CAMP accumulation by difEerent

mechanisrns, blocked activation of Raf- 1, MAP kinase kinase and MAP kinase in Ratl hER

27

fibroblasts. In another fibroblast celi he (hurnan foresicin fibroblast ceiî line AG 1523) indudion

of CAMP synthesis with forskolin treatment was foiiowed by reduction in the expression of c-myc

messenger RNA and inhibition of (3HJthymidine incorporation in human fibroblasts (Heldin et d.,

1 989).

Inhibition of cell proliferation by CAMP was dso fiported in smooth muscle d s (Ndsson

and Olsson l984), where treatment of these d s with prostagiandins raised intracellular

concentrations of CAMP and inhibited DNA synthesis. High CAMP concentrations have been

implicated in suppression of proliferation in normal end neoplastic B cells (Blomhoff a ai., 1987).

The authors found that treatment of the human B preausor of ceil line Reh (denved fiom acute

lymphatic leukemia ceiis) and human B lymphocytes with forskolin induced a rapid increase in

CAMP level, which was folowed by an accumulation of cdls in the GdGl phase of the cell cycle.

Inhibition of ceil growth with CAMP treatment was also reported in myeloid cell line HLdO

(Bang et ai., 1994)

In ail ceil types where CAMP is found to be growth inhibitory, the mechanism underlying

CAMP-mediated growth inhibition is associated with phosphorylation of Ser, of the Raf-

regdatory domain (Sturgiil et al., 1993; Hordijk et al., 1994). This results in the reûuced binding

of c - M l to p21Ras (Sturgill et ai., 1993; Cook et al., 1993) and prevention of signal

transmission through the MAP kinase cascade.

ii) Stimulatorg effect of CAMP on ceil proliferatioii

Contrary to the d types in which CAMP wrs found to inhibit c e U proliferaton in some

ce1 types elevated concentrations of htracelluiar CAMP are mciated with stimulation of c d

proliferation. For awmple, in Swiss 3T3 ceUr accumulation of CAMP, which predominantly

activates CAMP-dependent kinase waa associatecl with ATP- stimulated proliferation of those

cdls. Huang et al. (1994) transfied Swiss 3T3 celis with a gene d e f ' v e for the regdatory

subunit of the cAMP4ependent protein kinsse or with a plasmid that caused overexprcssion of

CAMP phosphodiesterase. Response of those transfécted celis to ATP was markedly reduced,

thereby demonstrating a role of CAMP and c-ependent protein kinase in mitogenesis in

those cells. In primary cultured human thyroid celis, thyrotropin (TSH) stimulated both growth

and -ion. These effects of TSH are most probabiy mediated by an elevation in intracellulu

levels of CAMP since TSH stimulates adenylyl cyclase, and forskolin was able to fbly reproduce

the inhibitory TSH efEect (Saunier et al., 1995). In rat pheochromocytoma PC12 cells it was

demonstrated by Frodin et al. (1994) that elevation of CAMP by cholera toxin,

isobutylmethylxanthine, forskolin or by CAMP analogues stimulated the M . kinase isozyme,

ERK 1. The stimulatory role of CAMP on growth in rat ovarian granulosa cells was demonstrated

by Das et al. (1996). They showed that pretreatment with FSH for 10 min promoted a 2 to 5-fold

inaease in mitogen-activated protein kinase (MAPK) activity. The effects of FSH were mimicked

by forskolin and inhibited by the inhibitor of CAMP-dependent protein kinase, H89, but not

inhibited by the tyrosine b a s e inhibitor, Ag- 18.

In an attempt to explain this tissue specitïc lack of CAMP-mediated inhibition of

proliferation, it has been postulated that it may have resulted fiom differential sensitivity of Raf-1

to CAMP-mediated inhibition and suggest the existence of Raf-l independent, CAMP sensitive

29

pathway in these ceils. For example, it has been suggested that in PCl2 cellg CAMP-dependent

protein kinase phosphorylates the smali G-protein Rap 1. Rap- 1 is selective activator of &Ra€

and inhibitor of 1, thus providing cdls that express B-Raf a mechanism for regulation of

growth via MAP kinase (Vossler et al., 1997).

4. Other signailing pathways

a) Signai transduction through CAMP-dependent protein kinase

One of the first describd signal transduction pathways which lads to the activation of

transcriptional factors was the signalhg pathway from serpentine receptors coupled to Gs

proteins to production of CAMP. The nuclear transcription factor which has been charactensed as

a CAMP response element binding protein (CREB), is a sequence specific activator that interacts

with the CAMP response element (CRE; G o d e s and Montminy, 1989) and is located in the

vicirÜ,ty of c-fos promoter (Edwards, 1994). Transcriptional control of c-fos involves several cis-

acting regulatory DNA eiements that lie upstrearn of the promoter. One of them is CRE

(Edwids, 1994). Upon stimulation of serpentine celi-surface receptors that positively regulate

adenylyl cyclase, intracellular CAMP rises and activation of protein kinase (cAMPdPK) ensues.

The cataiytic subunit of cAMPdPK then trandocates to the nucleus where it phosphorylates CRE-

bound CREB on Ser,,, located in the critical position within the activation domain. This results in

a large increase in CREB transcriptionai advity, leadiig to activation of CRE-containhg

promoters such as the c-fos promoter (Gonzales and Montrniny, 1989; Edwards, 1994).

Following dissociation of ligand Eom the receptor CAMP Ievels drop and cAMPdPK activity wps

inhibited (Hagiwara et al., 1992). This drop of cAMPâPK activity results in net increase in

phosphatase activity, leading to dephosphorylation of CREB and its inactivation (Hagiwara et al.,

1992). It is interesting to mention that, besides having specific phosphorylation site for

CAMP@& CREB poses a specific phosphorylation site for PKC, as weU (Godes et ai., 1989).

b) JNK/SAPK signaîiing pathway

JNK (c-jun N-terminal kinase) is a member of o nive1 M y of kinases stnicturaüy related

to ER&, aithough it appears to be dienntiy regulateâ. JMC is activated in response to meral

agonists for G-protein coupled receptors, induding carbachol (Coso et al., 1995; Minden et al.,

1994), piatelet activahg factor (Squito et al., 1989) and angiotensin II (Zohn et ai., 1995).

Regdation of JNK is associated with the activation of the srnail moiecular weight G proteins,

Rac, cdc42 and Rho (Minden et al., 1995; Coso et ai., 1995; Chnanowska-Wodnicka and

Bumdge, 1992). Rho, Rac and cdc42 regulate the formation of stress fibers (adn

polymerization), lamellipodia (membrane rufbg) and filopodia, respectively (Nobes and Hall,

1995). Coilectively, Rac, Rho, and cdc42 fhction to activate TM( through the activation of a

MEK kinase (SAPK kinase kinase) (Minden et ai., 1995) whose function is analogous to Rafand

MEK in the ERK pathway, leading to the phosphorylation of c-jun (Minden et al., 1994; Coso et

ai., 1995; Squito, 1989). PDGF, insulin, bombesin and phorbol ester (PMA) activate Rac,

resulting in actin polymerization at the membrane and the formation of membrane ruffles (Nobes

et al., 1995). How G-protein coupled nceptors and heterotrhneric G proteins activate

RaJRho/cdc42 pathways is unclear. To Uiwstigate how lysophosphatidic acid and bombesin

stimulate the formation of focal adhesions and acth stress fibers in Swiss 3T3 cells, Ridley and

Haü (1994) tested the roles of three intraceliular signaling pathways known to be induced by

LPA: PKC-pathway, Ca* mobilization and decreased CAMP levels. They reported that Rh09

mediated stress fibre formation in response to LPA or bombesin is unafkted by changes in the

31

levels of CAMP, intracellular calcium or PKC activity but is significantly anenuated by receptor

tyrosine IcÀnase inhibitor, genistein, suggesting that novei Rho-mediated signaling mechanisrtu

simiiar to those reguloting the pz lras/ERK pathway exists.

c) Janus kinase (JAK) rignaiing pathway

A novel class of tyrosine kinase signahg cascades, comprising tyrosine base (TykZ) and

Janus base (JAK) which are responsible for tyrosine phosphorylation of cytoplasmic proteins

d e d signal transducers and activators of transcription (STATs) was the one most recentiy

identified.

This signaling pathway is primarüy activated in response to the a and y interferons (IFNa

and y) (Schindler et al., 1992; Shuai a al., 1992). Cytokine stimulation of the receptor lads to

association of JAKITyk2 kinases with the receptor and theù subsequent phosphorylation and

activation (Watiing et al., 1993; Sihtemnoinen et al., 1993). Activation of the JAK kinase results

in the formation of DNA binding complexes. This is mediated in part by the tyrosine

phosphorylation and activation of STAT proteins. Different cytokines activate different STATs or

possibly other unknown intermediates and initiate a unique pattern of transcriptional events

(Lamer et al., 1993).

5. 'Cross- WL" among signaling pathways

Signal transduction is, however, more cornplicated than the simple linear sequence of

raptor, transduw, Cffector. Due to this phenornenon, a tigand may simultaneousiy affect more

than one signahg ~athw~y. By linking a number of s i p i h g pathways, the ceIl is capable of

perceiving and adapting to multiple environmental fhctors:

Recent stuâies assessing agonist activation of the MAP klliase pathway have provided a

biochemicai rationaie for an opposing role of CAMP in ce11 growth and differentiation in specific

ce11 types. Its been shown by studies from Shirgill et al. (1993) that CAMP-dependent protein

kinase inhibits the MAP kinase pathway by direct phosphorylation ofHaf-1 within its regulatory

domain. Phosphorylation of Raf-1 by cAMPdPK prevents binding of Ras to Raf-1, thus

preventing its activation. Another example of cross-talk between MAP kinase pathway and

CAMP-dependent protein kinase pathway is cAMPdPK stimulation of MAP kinase cascade in

PC 12 cells. Vossler and coleagues (1 997) reported that in PC 12 cells transfected with the

catalytic subunit of CAMP-dependent proteh kinase or treated with either CAMP analog 844-

ch1orphenyithio)-cyclic AMP or nerve growth factor were able to differentiate and develop

neuntes. This c d type specific action of CAMP requires the expression of B-Raf and activation of

a smaii G protein Rap 1. Rap 1 activated by the cAMPdPK is a sdective activator of B-Raf (and

inhibitor of R&l), thereby allowing signal to progress through the MAP kinase cascade leadmg

to the activation of transcriptional factors and induction of neuronal differentiation. Tan et al.

(1994) demonstrated that fibroblast growth factor through activation of MAP kinase cascade and

CAMP through activation of cAMPdPK, synergistically activated proenkephalin gene expression

in a human neuroblastorna c d line through the activation of CRE.

An interesting example of merging signais h m tyrosine kinase recepton and G-proth

coupled recepton to activate MAP kinase pathway is through activation of Rheb (smd GTP-

binding protein, simüar to Rap or Ras). Yee and Worley (1997) reported that in PC 12 ceils md

NWT3 cells, activation of Rheb is potentiated by a combination of growth fàctors and agents

that increase CAMP leveis. Protein Irlliase A dependent phosphorylation of the regdatory do&

of Raf-1 inhibits subsequent down~fream kinase activation (Cook et al., 1993). However,

phosphorylation of =l potentiates its in tedon with Rheb and decreases its interaction with

Ras thereby allowhg signal to progress downstream through the MAP kinase pathway (Yee and

Worley, 1 997).

The phorbol ester PMA is a potent tumor promoter that aEects celi morphology,

metabolism and ceU division. It is also hown that PMA rniinics many of the effects of ACTH in

adrenal celis, namely stimulation of steroidogenesis, inhibition of celi growth and changing in ceIl

shape (Kimura and Armelin, 1990). Marquardt et al. (1994) showed that in insect cells a signal

from PMA through a PKC-dependent mechanism activates the MAP base cascade. Rapp et al.

(1993) hvestigated the mechanism by which PKC activates MAP kinase. They showed that in

NIH3T3 fibroblasts, PKCa directly phosphorylates Rafboth in vitro and in vivo, thereby

activating the MAP kinase pathway. They suggested that Raf may serve as a convergence point

integrating signals f h m tyrosine kinasedrus on one hand and fiom P W K C on the other hand.

When a membrane bound receptor acts through a G-protein, the GTP binding Ga subunit

dissociates from the Gpy dimer (Bockaert, 1991). Until recently it was thought that ody the G a

subunit was responsible for signal transmission. Evidence that G-protein bsubunits are capable

of transmit@ mitogenic signals utiliring the MAP kinase pathway, initially came fkom studies of

Crespo (1994) and Koch (1994). Crespo et ai. (1994) found that in COS-7 cells ERKl/ERK/2

activation via both pemissis toxlli-sensitive and G-protein coupled muscarhic MZ cholinergie

34

receptors QM2AchR) and pertussis toxin-insensitive G-protein coupleâ muscarinic Ml cholinergie

reseptors (MlAchR) wu attenuated by coapression of the a-subunit of transducin, which acts to

sequester G&ubunits released upon stimulation fkom endogenous G proteins. Koch et d.

(1994) showed that activation of Gi-cwpleâ receptors leads to the activation of mitogen-

activated protein klliose. In two ceIl types, Rab1 and COS7 cells, it appeared that LPA which

signais through Gi-coupled receptor activates MAP kinase pathway through the py subunit,

through a stüi unhiown mediator. A convergence point for growth factor receptors such as

receptor for EGF and Gi-coupled surface receptor signalhg pathways seemed to be Ras

activation (Koch et al., 1994). Activation of Ras leads to a sequential activation of 1 and the

rest of the MAP kinase cascade. Some clue what cwld be possible candidate for activation of Ras

came fiom studies of Langhans-Rajaselcaran et ai. (1995). They reported that plekstrin homology

domain (PH) containhg proteins Btk and Tsk are activated in vitro by the addition of Gpy-

subunits. PH domain is a comrnon structural motif found in more than 90 proteinq including Sosl

and the GTP-ase activating protein Ras-GAP (Gibmn et al., 1994).

UnWte pertussis tod-sensitive G-protein coupled receptors, pertussis toxin-insensitive

Gq/l 1 receptors a h , stimulated the MAP kinase pathway but through PLC and PKC-dependent

mechanisms, rather than through $y-subunits mwes a al., 1995).

The ability of recepton that activate a,- subunit of G-proteins to activate MAP Linase

pathway was also reported. Crespo et al. (1995), reported that in COS-7 ceils trdected with o

$-aârenergic receptor, isoproterenol raised intraceliular levels of CAMP, and effectively

stimulateci cAMPdPK and increased epitope-tagged MAP kinase activity. Activation of MAP

Linase wasn't abolished by depletion of PKC, but it was abolished upon tratlsfection of a chimenc

molecule consisting of the carboxy teminus of $-adrenergic receptor kinase, including the By-

binding domain. The pretreatrnent of ceils with 8Br CAMP, markedly decreased MAP kinase

activation and the ssimulation of MAP kinase w a d t mimickeù by cAMPdPK-stimulating agents.

Moreover, since the activation of MAP kinase was completely abolished by expression of Ras-

inhibithg molecules, they coacluded that signaling fiom &adrenergic receptors to MAP kinase

involves j3y-subunits of G-protehs.

6. Activation of transcriptional facton and protooncogener

Much attention has been directed towards improving the understanding of how growth

facton and hormones activate imrnediate eafly genes c-fos, c-myc and c-jun. Ceil-surface

stimulation results in increased synthesis of polypeptides Fos, Myc and Jun. They are members of

specific transcriptional activator complexes on specific target genes (Curan and Franca Ir., 1988).

Inhibition of Fos and Jun protein expression, or activity by, respectively, antisense RNA or

antibodies, blocks G1 phase progression of growth factor stimulated cells (Holt et ai., 1986;

Mshüaira and Murray, 1987; Kovary and Bravo, 199 1). Recent evidence indicated that MAP

kinases translocate fiom the cytoplasm to the nucleus upon senim growth factor stimulation

initiating transcriptionai responses that ultimately lead to celi proliferation and differentiation

(Chen et al., 1992).

The serum response element (SRE) Ui many Unmediate early gene promoters mediates

transcriptional activation in response to semm growth factors. A temary complex of serum

response factor (Sm) dimer and Temary complex mot (TCF)/Ekl binds to the SRE on the c-

fos protooncogene promoter (Epskind et d., 1991). Recently, TCF/EUE-I was found to be

phosphorylated and activated by MAP kinase (Marais et al., 1993). The AP-1 (Activator Protein

36

1) is a transaiptional regulator consisting of homo and heterodimers of nuclear proteins of the fos

and jun &milies and is hown to bind to AP-1 sites in DNA ( a h caiied TRE; C m md F m

Jr., 1988). C-jun appeared to be a component of the dimenc sequence-specific actiwtor of AP-1

(Angel and Karin, 1991). T t d p t i o n a l activation of c-jun by growth fimors, cytokine a d

oncogenes is mediated through the JNK pathway ud through activation of PKC (ïWq 1994).

The c-myc protooncogeae product fiinctions as a tronsaiption fâctor that bin& as hete rod i i

with Max to the DNA sequence CACGTG (Alvarez et al., 1991). Chuang and Ng (1994) showed

that difEerent MAP kinase cascades diverge with at least one specific target for each MAP kinase

isoform. They found that in N W T 3 fibroblasts, over expression of ERIC 1 c-DNA resulted in

activation of the semm response factor accessory protein, Ek-1, while over expression of ERK 2

dva ted Myc, but not Elk- 1.

Recent studies Born severai laboratories indicated that the binding of ACTH to the ACTH

receptor sites on the adrenocortical cells involved induction of expression of nuclear

protooncogenes. Imai et al. (1990) showed that treatment of hypophysectomized rats with ACTH

for 3 days increased mRNA encoding c-fos and $-adn in adrenal glands. Kimura et ai. (1993)

reported that ACTH is capable of regdating fos and jun proteins in Y 1 adrenocortical celis. The

induction of these protooncogenes was blocked by actinomycin D, but not by cycloheximide,

suggesting that ACTH regulates these genes at the transcriptional level. They also reported that

PMA closely mimicked these inductive effêcts of ACTH, while on the contrary, CAMP denvatives

were not very effective in induction of fos and jun genes. Kimura et al. (1993) reported that

ACTH induced c-fos expression with a 0.5-1 h peak. PMA induced c-fos with similar kinetics

compared to ACTH but reached only 60 % of m x h a l ACTH induction, while on the contrary,

CAMP was a weak inducer and caused c-fos to increase only to 15 % of the maximal level

37

achieved with ACTH.

1t &as been reported that inbition of fos ami jun protein expression or activity by

antisense RNA or dbodies blocks the Go to G1 to S transition of growth- tactor stimulated ceils

(Holt et pl., 1986; NiShilaira and Murray, 1987; Riabowol r ai., 1988). Thus, fos and jun

pmteins mediate activation of the transcriptionel program requited for cells to enter the ceii cycle

and uitimately proliferatc.

II RESEARCH OaTECTNE AND RATIONALE

II RESEARCH OBJECTIVE AND RATIONALE

The growth inhibitory Hect of adrenocorticotropic hormone (ACTH) on adrenal cells in

vitro is weli documented. ACTH- induced inhibition of ceii proliferation in vitro bas been

observed in Y 1 mouse adrenocortid tumor ceiis, as weii as in cultured nonnal adrenal ceUs

isolated fiom mouse, bovine, hwnan and rat adrenals. ACTX arrests dividing adred ceils by

interfiering with progression through the G1 phase ofthe cell cycle and inhibits the initiation of

DNA synthesis in G1-arrested cells following addition of serum or growth factors. Despite

existing controversies about the mechanism of action of ACTH on adrenal cells, it is widely

accepted that the mechanism of action of ACTH includes CAMP and the cAMPdPK pathway.

Experiments on Y 1 adrenal cells harboring dominant inhibitory mutations in cAMPdPK that

specifically disrupt the CAMP signahg pathway showed that these mutants were resistant to the

growth inhibitory actions of ACTH and CAMP analogs. These data indicated that CAMP and

cAMPdPK are obligatory components of the inhibitory effect of ACTH on ce1 proliferation.

There is abundant data in the literature indicating that CAMP inhibits ceU proüferation in

many c d types including fibroblastq smooth muscle cellq neuronal cells and T ce1s by inhibithg

the MAP kinase pathway. This cascade of protein kinase redons serves as an activator of

transcriptional factors such as c-fos and c-jun, leading to the transition of ceils fkom the G1 to S

phase of the celî cycle. There is increasing evidence in the literature that a cornmon point of

convergence of many growth fàcton and hormones is the MAP kinase pathway. Extracellular

mitogens, such as PDGF, EGF, and newe growth f a o r through the activation of tyrosine kiaase

a CAMP-independent, growth promoting &ect of ACTH.

III MATERIAL AND METHODS

III MATERIAL AND METHODS

1. Celis and ce1 cuItun

The cels used in this study are Y 1 BS 1 and Kin 8. Y 1 BS 1 (Schimmer, 198 1) is a

functionai mouse adrenal tumor ceii he, stable suûcione originaliy isolated by Yasumura a ai.

(Yasumura et al., 1966). Kin 8 cells are Y1 adrenocorticai -or celi mutants that hahot point

mutation in the regulatory subunit of the type 1 cAMPdPK, that render the enzyme resistant to

activation by CAMP (Rae et al., 1979). Cells were routinely grown in F 10 growth medium

supplemented with 15 % heat inactivated horse serum (HS) and 2.5 % heat inactivated fetal calf

semm (FCS), 200 U/ml penicillin G and 270 mghl streptomycin sulfate. Ce1 cultures were

maintained in tissue culture flasks (Falcon) at 37 C in a humidified atmosphen of 5 % CO2 - 95

% air. Growth medium was changed every 3 to 4 days; ceUs were subcultured into the new

bottles every 7 to 10 days using Viokase as a proteotytic solution.

To replicate plate ceUs for experimentai analysis monolayers were dispersed using 0.1 %

trypsin, 0.02 % EDTA in phosp hate-busered saline (PBS) containhg 137 mM NaCl, 2.7 m M

KCl, 8 mM Na&PO, and IS mM K H2 PO,. Cella were counted under the microscope using a

hemocytometer and transferred at appropriate dilution to a stede Erlenmyer fiask with magnetic

stir bar. Cells were maintained in unifonn suspension by gentle stirring and the equal amounts of

the ce1 suspension were distnbuted among the culture dishes.

2. Iacorpontion of [SHltbymidine into the DNA

Cells were repliate plated in 60 mm tissue culture dishes at a density 0.8 x 10' celld plate,

and grown for two days in F10 medium supplemented with serum to assure that they were in the

logarithmic phase of growth. Ceils then were piaced in a saumfiee medium (Alpha Minimal

Essentiai Medium - aMEM) to arrest d s early in G1, treatments were applied and cultures

were left for an additional 14 h to reach the peak of DNA synthesis (S phase). During the last 2

h of incubation, 2 pCilml of [3H-methyl]thyMdine (New England Nuclear, 20 Cilmrnole), was

added to the culture medium to monitor DNA synthesis (Armelin et al., 1977).

At the end of labeling period the radioactive medium was removed fkom the cells, and 0.1

% uypsin with 0.02 % EDTA in PBS was added for 10 min at 37 C to detach cells nom the

plate su~ace. Floating celis were transfered with Pasteur pipet and fltered through glas fiber

filter disks (2.4 cm, Ahlstrom Filtration). Plates were then washed twice with 2 ml of PBS and

washes were passed through the tilters, too. Filters then were , washed once with 10 mi of PBS,

twice with 10 ml of 5 % TCA and once with 10 mi of 95 % ethanol. Fiiters were air dned, and

transferred to scintillation vials and counted in a iiquid scintillation counter (12 17 Rackbeta,

LKB) using PCS (Amersham) as a scintillation fluid.

3. Protein determination

Protein concentrations wen daennineci uwig the BioRad Protein Assay Kit

(Bradford, 1976). Accordhg to the protocoi, Dye Reagent was prepared by diluthg 1 part of

Dye Reagent Concentrate with 4 parts of distillecl wota. Diluted Dye Reagent wu fltered

through Whatman #1 Fiher pqer to remove puriCulates. Four concentrations of bovine serum

aibumin ( h m 5 to 20 pg in 100 pi of distillai water) were prepared to constnict protein

standard curve for each experiment. Samples of each ceil lysate (IO pi) were assayed in dupiicate

for protein content. Standards and unknowns were brought to 100 pl with distilied water and

mked with 5 ml of diluted Dye Reagent. Samples were vortexed and dowed to stand for 5 min

at room temperature, protein absorbante was measured at 595 nrn wavelength, using Spectronic

20 spectrophotometer (Bausch and Lomb).

4. MAP kinase phosphorylation

a) Cell lysate preparation. Cells were washed twice with PBS at room temperature and

scraped in ice cold RIPA bufTer containing 150 mM NaCl 50 mM Tris-HCl, (pH 8.0), 5 rnM

EDTb 1% (voV vol) Nonidet P40,O.S % (wtlvol) sodium deoxycholate, 0.1 % SDS, 10 mM

sodium fluonde, 10 m . disodium pyrophosphate, 1mM sodium orthovanadate, O. 1 mM

phenylmethanesuironyi fluoride, 10 pg/rnl benzamid'ie, 10 pg/d leupeptin, 10 pglml soybean

trypsin inhibitor and 5 pg/rnl aprotinin (Koch a al., 1994).

CeUs were passed several times through a 21 gauge needle to break the cells and shear the

DNA Cells then were, incubated for 30 to 60 min on ice and centrifiged for 20 min at 4 C in a

microfuge (Beckman 12). The supernatant, referred to as the total ceil lysate, was trarisfemed to a

new microfbge tube and stored at -70 C.

b) Western Biot andysis. Equaî amount of di lysates (10 pg) and 2x- concentrated

SDS sample buffet (20 % glycerol 10 % B-mercaptoethanoi, 200 mM TrisWC1 @H 6.7), 4 %

SDS and 0.02 % brornphenol blue) were mixed and Wied for 3-5 min at 100 C. Protek were

electmphoresed on 10 % polyacrylamide gels (29 : 1) (Laemmli, 1970) and bioned on

microporous, polyvinyiidene difluoride (PVDF) membranes using a BioRad transbiot apparatus.

MAP kinase phosphorylation was determineci with an antibody s p d c for the tyrosine-

phosphorylated forms of MAP kinase (ERK 1 and ERK 2) (New England BioLabs.) and also by

mobiüty shift assay using an ERK 2 antibody (Transduction Labs.).

For detection of phosphorylated MAP kinase using phospho-specific antibodies, PVDF

membranes containhg transferred proteins were incubated for 1 hour at room temperature with

gentle agitation in 25 ml of blocking buffer (O. 1 % Tween 20 in PBS plus (w/v) 5 % nonfat skim

rnilk powder) to prevent nonspecific binding of antibody to the blot. Membranes then were

incubated for 1 hour at room temperature with PhosphoPIus MAP kinase antibody (1 : 1000),

(New England BioLabs.) in a buffer of 0.1 % Tween 20 in PBS with 5 % bovine senim albumin.

Blots were then washed three times in 25 ml of blocking buffer for 5 min each, and incubated 1

hour at room temperature with alkaline phosphatase-conjugated antirabbit secondary antibody

(dilution 1 : 1000) in blocking buffer. Mer incubation with secondary antibody, membranes were

washed 3 tîmes 5 min each in blocking buffer.

Detedon of secondary antibody was done using CDP-Star reagent (Lumigen-PPD).

Membranes were washed twice for 5 min with an assay buffer containing 0.1 M diethanolamine

and 1.0 mM MgCl, (pH 9.5). Blots were agitated 5 minutes at room temperature, in assay b d e r

containing the CDP-Star reagent (1:500), wrapped in Saran Wrap and exposed to Du Pont

47

Refiection radiography film 0.

A sirnüar immunoblotthg technique was wd to detect MAP kinase phosphorylation by

mobüity shiA on polyacryIamide gels. Blocking b d e r was 0.2 % Tween 20 in Tris b&ed saiine

(TBS) contauiing: 20 mM Tris-HCl and 137 m M NaCl (pH 7.6) with 5 % skim mik powdec the .

primary antibody was monoclonal ERK 2 antibody raisecl agaimt C-terminai part of the protein

(Transduction Labs.), diluted 1: 250 in blocking b&a. Washings were done bdore and a f k

incubations with primsry and secondary antibody. Each tirne blots were washed once 15 min and

twice 5 min in 0.2 % Tween 20-TBS. Secondary antibody useci was anti-mouse horse radish

peroxidase (Amersham), diluted 1 : 10000 in blocking buffer. Incubation t h e with pnmary and

secondary antibodies were the same as described above for Western blotting using PhosphoPlus

antibody kit.

For protein visualization, ECL detection kit (Amersham) w u used: 1 ml of reagent 1 and

the same volume of reagent 2 were Mxed with 8 ml of distiiied water and incubated with

membrane for exactly 1 min. Blots were then, wrapped in the Saran Wrap and expose to the

radiography film.

5. MAP kinase activity

MAP kinase activity waa assayed using a MAP kinase assay kit (New England BioLabs).

The p~ciples of the procedure are as follows: mouse MAP kinase (ERIC 2) phosphorylated at

Tyr,, was selectively immunoprecipitated with a crossreachg antibody r a i d against the Tyr,

phosphoiylated form of human MAP kinase. The resulting imrnunoprecipitate is then incubated

with an EUc 1 fbsion protein in the presence of ATP and kinase bufEer; this aliows

immunoprecipitated MAP kinase to phosphorylate Ek 1 (Marais et al., 1993). Phosphorylation

48

of Ek 1 at Ser, is detecteâ using a phospho-specific Ek 1 antibody.

a) Ce1 Iysate preparatioa. Celis were hsrvested with a celi scraper unda nondenaturing

conditions, in ice cold ceil lysis b s e r (20 mM TrW-HCI (pH 7.9, 150 mM NaCi, 1mM EDTA,

1 rnM EGTA, 1% Triton X-100,2.5 mM sodium pyrophosphate, 1 m . P-glycaophosphate,

NaJO,, 1mM phenylmethanesulfonyl fluoride and Img/ml leupeptin.

Celis were b r o b by sonication 4 times for 5 sec eadi Tubes were kept on ice al the

the. Lysates were clarified by centdùgation for 10 min at 4 C in a microcentrifbge (Beckman

12).

b) Irnmunoprecipitation. Ce11 lysates (200 d) were mixed with Phospho-specific MAP

kinase antibody (2pl) and incubated with gentle rocking ovemight at 4 C. Then protein A

sepharose beads (20 pi) were added into the ceii Iysates and incubated for 4 h to pellet antibody

antigen complex.

c) MAP kinase assay. The protein A sepharose beads with MAP kinase were

resuspended in 50 pl of kinase bufFer (25 m M Tris (pH 7.9,s mM P-glycerophosphate, 2 mM

dithiothreitol, O. 1 mM Na3V0,, 10 mM MgCl, supplemented with 100 mM ATP and 1 kg of Ek

1 fusion protein as substrate). To allow the kinase reaction to proceed, sarnples were hcubated

for 30 minutes at 30 C. The reaction was teRninated with 25 pl of 3x9 SDS sample bde r (62.5

mM Tris-HCI, (pH 6.8), 2 % w/v SDS, 10 % glyceroi, 50 mM dithiothreitol, 0.1 % (wh)

bromphenol blue). Sarnples were boiled for 5 min, vortexed, microcentrifuged for 2 min prior to

loading ont0 a 10 % SDS polyacrylamide gel. Electrophoresed proteins were transferred fiom the

gel to a PVDF membrane ushg a Bio Rad mini-transblot apparatus.

d) Immunoblotting. Blots were incubated in 25 ml of blocking buffer (0.1 % Tween 20

with 5 % w/v skim mik powder) for 3 houn at room temperature. Mots were then incubated

overnight at 4 i: 4 t h an antiody specific for phosphocylated Ek 1 (1: 1000) in 0.05 % Tween 20

-TBS witb 5 % bovine serum albumin. Blots were then washed 3 Gmes for 5 min each in O. 1 %

Tween 20-TBS. Mer washllig, blots were incubateci with an hors radish peroxidase conjugated

anti-rabbit secondary antibody (1:2000) in the blocking bufEer with sentie agitation 1 hour et

room temperature. Membranes were washed 3 timea for 5 min each in O. 1 % Twem 20-TBS.

For secondary antibody deteaion, membranes were incubated with LurniGLO reagent and

peroxide reagent diluted in water (l:20) with gentle rockhg for exacffy 1 min at room

temperature. Membranes were drained fkom the excess of developing solution, wrapped in Saran

Wrap and exposed to Du Pont radiography film.

6. Statistical analysis

Unless otheMrise stated, n values represent the numbers of experiments fiom which

means and standard deviations were caiculated. Data were analysed using the Pentz's F test, a

multiple cornparison test for statistical analysis of ail diierences among group means ( Harper,

1984).

IV RESULTS

1. Kinetics of [3mthymidine incorporation in Y1

Purpose of this experiment was to esthate the tirne course of ceîl cycle progression in Y1

celis. Y 1 cels were unifody plated and ailowed to grow for 2 days. CeUs then were divided into

three groups. The nrst group of ceUs were maintained in growth medium plus serurn for the next

72 h. As can be seen fiom Figure 1, ['Hlthymidine incorporation into DNA increased with t h e

and reached a maximal level (192700 * 5700 cpm ) after 72 h. At this point ['HJthymidine

incorporation plateaued as cells approached saturation density and stopped dividing. The second

group of ceils were rnaintained in serum-fhe medium for 72 h and in these cells [3HJthymidhe

incorporation slowly deciind over the 72 h penod reaching a low value of 9100 * 1600 cpm.

The deciiie in [3H]thymidie incorporation seen upon serum-starvation likely reflects an arrest of

cells early in the G1 stage of the ce11 cycle due to the absence of serum-denved growth factors. in

the third group, serum starved celis were treated with senim and monitored for their ability to

progress through S phase. As Figure 1 shows, [3Hlthymidine incorporation in restimulated ceUs

rose d e r a lag penod of at least 4 h, reached a peak (138200 * 13300 cpm ) at 14 h, and then

rapidly declined to a low value (40122 * 2500) at 18 h . The levets of ['Wthymidine incorporation

at the 18 h and 24 h time points were not significantly dinerent @ = 0.5). These results are

consistent with previous reports describing the lengths of Gl (14 h), S (7 h), G2+M (3 h)

(Weidman and Gill, 1977).

Figure 1. Time course of [3~]th~midine incorporation in Y1 ceUs

Cells were plated in 60 mm tissue culture dishes at a density 0.8 x 105 cellslplate

and grown for two days. One group of cells was treated with aMEM + semm

for another 72 h ( + ), while a second group was maintained in aMEM without

senim (r-=-3. S e m was added into serum starved ceils at the end of 72 h

period ( -t.) and ceils were sampled at different intervals for [3~]thymidine

incorporation. Results are means t SEM, n=3.

2. Inhibition o f gmwth by long tmtment with ACT& (Br CAMP and PMA in Y1 ceh

In this experiment we examineci the dèct of a long t h e treatment with ACTH, 8Br

CAMP and PMA on [%XJthymidim incorporation in Y 1 d s . S e m starved cells were inabated

for 14 h in the presence of serum, ACTX, 8Br CAMP and PMA As figure 2 showq saum

treatrnent significantly i n c r d [%Jthymidine incorporation approximately 1 5-fold ova

untreated, m m starved celis p < 0.001. ACTH, 8Br CAMP and PMA each given together with

serum for 14 h significafltly inhibited growth compared to serum as a control with p values for

ACTH: p < 0.001,8Br CAMP: p < 0.0001 and PMA: p < 0.001. ACTH given alone for a period

of 14 h, further inhibited [3H)thyrnidine incorporation @ < 0.00 9, compared to serurn starved

ceils (controls).

To investigate whether inhibition of ceU growth obtained with long tenn incubation with

ACTH and 8Br CAMP in Y1 ceus is mediated through cAMPdPK mechanism, we examined the

effect of those agents on [%Jthymidine incorporation in mutant Kin 8 cells. Semm starved Kin 8

cells were incubated for 14 h with ACTH, 8Br CAMP and PMA. As Figure 3 shows, semm

treatment for 14 h si@cantIy increased [%Jthymidine incorporation in Kin 8 ceh,

approximately 17-fold over control with p < 0.001. ACTH given together with serum for 14

hours stimulated growth approximately 15-fold over control: p < 0.001 and not significantly

dierent from the efEect of senun alone. 8Br CAMP kept together with senun for 14 h also

increased [3Hlthymidine incorporation in KUi 8 cellq approximately 12 times over control, with p

< 0.001 compared to control, and not signifiwitiy difFerent fiom effect of senun alone. These

observations thus c o h that ACTH-mediated inhibition of ce1 cycle progression occurs by a

cAMPâPK mechanism. PMA given together with sem for 14 h, decreased ['Wthymidine

incorporation approbtely 3-fold compand to senun effect alone: p=0.0002. These hdlligs

+ serum

Figure 2. Effect of ACTH, 8Br CAMP or PMA on serum-stimulated cell cycle

progression in Y1 cells. Cells were plated on 60 mm dishes at a density of 0.8 x

105 ceildplate and grown for 2 days. AAer 2 days in culture, semm was removed fiom medium

for the next 72 h to amest cell growth . Cells were then incubated with senun (15 % HS and

2.5 % FCS), ACTH (25 mU/ml), or a mixture of serum plus ACTH, serum plus 8Br CAMP

(3 mM) and senun plus PMA (100 nM) for 14 h. [3~]th~midine was added (2 pCi/rnl) during

the last 2 h of incubation and the level of [3~]thymidine incorporation into DNA was

measured as described in Methods. Values represent the fold stimulation of [3~]thymidine

incorporation relative to senun stanred controls and are presented as means f SEM of n

experiments camied out in triplicates.

Figure 3. Effect of ACTH, 8Br CAMP or PMA on serum-stimulated ceil cycle

progression in Kin 8 ceils. Kin 8 ce11 were assayd for [3KJthymidine incorporation into

the DNA as described in the legend of Fig. 2. Results are expressed as means of 6 individuai

deteminations in two separate experiments i SEM.

are in agreement with previously reported data about PMA action via PKC medianism. A C m

treatment done for 14 h siBnificantly stirnuiated ['Hithymidine incorporation in Kin 8 ceUs over

two fold cornparrd to control (p4.002).

3. Effcet of FGF and suum treatnent on M W kinase phosphorylation i i Y1

In order to examine FGF and semm effect on MAP kinase activation in YI, d i s were

plated at the density of2 x 10' ceIldl00 mm tissue culture dish and allowed to grow for 4 days.

Cells then were semm starved for another 3 days to mest cd growth. Confluent, serum starveû

ceiis, were incubated with serum and another known rnitogen for Y 1 cells - FGF, for difEerent

tirne intervals. As shown in Figure 4, compared to nonstirnulated, serum stawed ceiis, FGF

treatrnent for different times gradudy induced appearance of a slow migrating - phosphorylated

fonn of ERIC 2, with the best resolution of doublets af€er 15 and 20 min treatment. Semm

treatment was more potent, causing appearance of the phosphorylated, shifted form of ERK 2

isozyme after only 3 min of serum incubation with persistence of doublets for 5 and 10 min.

4. Tirne course of serum and FGF induced MAP kinase phosphorylation in Y1 cells

In order to examine time course of appearance and disappearance of MAP kinase

phosphorylated isofom, cells were treated with short pulses of FGF and serum. T a h g into

consideration that the shifted-phosphoryIated fom of ERK 2 isozyme (Figure 4), doesn't always

resolve nicely fiom the nonphosphorylated fom (iower band), to r d the presence of activated

MAP kinase isofom, 1 decided to use phospho-specinc MAP kinase antibody, that specifically

recognizes tyrosine-phosphorylated form of both ERK 1 and ERK 2.

Quiescent and m m starved ceUs were treated for 1,3,5, 10 and 15 min 6th FGF and

k 3 5 10 15 20 3 5 I O tinw (min) s"

FGF serum

Figure 4. Effect of FGF and serum treatment on MAP kinase phosphorylation.

Y 1 cells were plated on 100 mm tissue culture dishes at a density of 2 x 105 cells/plate.

Cells were grown in FI0 medium with senun for four days and another three days in

medium without serum (aMEM) to arrest ce11 growth. S e m -starved cells were then incubated

with FGF (100 ng/ml) or serurn (15 % HS and 2.5 % FCS) for indicated times.

Western Blot analysis was done and membrane blotted with ERK 2 antibody (Transduction Labs.).

Control sample was incubated 5 min in aMEM and washed the same way as other treated

samples. The same results were obtained in several repeated experiments.

for the same time (including additional 20 min treatment), with s e m (Figure 5 a md b).

Cornparrd to untreaîed d s (serum starved), where are no detected bands, FGF treatment

induced appearance of two bands at the arpected molecular weight sizes, indicating induction of

phosphorylated ERK 1 and ERIC 2 isofomu of the MAP kinase. The amount of induceâ

phosphorylated isoforms seemed to increase offer 3 min treatrnent with FGF. On the other han4

serum tnrtment rapidly and trpnsiently stimulateci appearance of phosphorylated isofonns (Fi-

5 b). Incubation of cdls with serum for l min induccd appearance of phosphorylated isofom,

white the same incubation time with FGF induced appearance only of faint bands. This effect is

probably due to the fact that serum is a combination of growth factors. Semm stirnulated MAP

kinase phosphorylation reached maximal intensity within 5 min treatment and slowly declined

towards control level by 20 min tteatment.

In order to test the hypothesis that ACTH inhibits ceil cycle progression in Y 1 cells by

inhibiting MAP kinase, 1 treated cells for 10 min with ACTH and 8Br CAMP. Cells were washed

twice with medium without serum (to assure washing away of ACTH and 8Br CAMP) and then

incubated for 2, 5, and 10 min with medium containing serum (Figure 5 c and d). Compared to

convol (semm-starved cells) in a i i semm treated samples we observed the appearance of

phosphorylated f o w of ERK 1 and ERK 2 with the maximal induction afler 5 min treatment

(Figure 5 c and d), which is in agreement with my previous data. As figure 5 c and d show, 10 min

pretreatment with either 8Br CAMP or ACTH didn't inhibit semm stimulated MAP kinase

phosphorylation. Aithough Figures 5 c and d show slight differences in intensity of

phosphorylated MAP kinase isofonns in pretreated samples compared to senun treated samples,

statistical analysis of quantitated densities of the bands on the blotc fiom three separate

experiments showed that pretreatment with ACTH and 8Br CAMP does not have a signihcaat

time (min)

k 1 3 5 10 15 20

&* time (min)

- ERK-1 - ERK-2

- ERK-1 - ERK-2

ti me (min)

pmtmatment

dP' 2 5 10 2 5 10 time (min)

de none ACTH pretmatment

Figure S. Effects of FGF, serum, 8Br CAMP and ACTH on MAP kinase

phosphorylation. Confluent, senun starved cells were treated with FGF (100 ng/ml) (a)

or serum (1 5 % HS and 2.5 % FCS ) (b), for indicated times, then solubilized

in RlPA buffer and Western Blot analysis was done using phospho-specific

MAP kinase antiôody (New England BioLabs.). Cells were pretreated with

8Br CAMP (3 mM) (c) and ACTH (25 rnU/d) (d) for 10 min, prior to serum treatment

for 2,s and 10 min (c and d). Control samples were maintained for 5 min in aMEM and

then washed the sarne way as treated samples. Al1 experiments were repeated at least

twice with similar results. 60

&ect on senimstimulated ERKl or ERK 2 phosphorylation.

S. Timt coum of ACTE, $Br CAMP and PMA on MAP kinase pbospborylntioi in Y1

In this acperiaient 1 wanted to test the timc course of signai appearance upon treatment

with ACTH, 8Br CAMP and PMA Compared to control (nonstimulated, serum-starved cells)

ACTH treatment (Acthar), which is a purifid peptide âom porcine pituitary, rapidly stimuiated

phosphorylation of ERIC 1 and ERK 2 (Figure 6 a). The signal wes clear and strong after only 2

min ACTH treatment, reached maximum Pfter 5 min treatment and slightly weakened PAer 10 min

treatment. On the contrary, 8Br CAMP was a weak inducer of MAP kinase phosphorylated

isoforms. H~wever, 5'-AMP treatment induced a significant amount of phosphorylated ERK 1

and ERK 2, suggesting low specificity of signal obtained with cyclic nucleotide in the case of 8Br

CAMP treatment. On the other hand phorbol ester very rapidly induced MAP kinase

phosphorylation for ail indicated times compared to inactive PMA isomer - 4a PMA .

2 5 10 time (min) CI

ACTHar

2 5 1 0 5 2 5 1 0 5 tirne (min)

8Br CAMP AMP PMA 4a PMA

Figure 6. Time course of ACTH, 8Br CAMP and PMA induced MAP kinase

phosphorylation in Y1 cells. Confluent, serum starved cells were treated with

ACTHar (25 mUIml) for indicated tirne (a). Cells were harvested in RIPA buffer and

Western Blot analysis was done using phospho-specific MAP kinase antibody

(New England BioLabs.). Cells were treated with 8Br CAMP (3 mM) for various times and

5'- AMP (3 mM) as indicated on figure. Cells were also treated with PMA (100 nM)

as indicated and with the same concentration of inactive isomer 4a P M A @) .

Similar results were obtained in three independent experiments.

6. Dort dependent relationships of ACTHar and ACTH,, on MAP kinase phosphoylation

inY1 ctHr

In this expriment Y1 celis were t r d with difli'erent concentrations of AC- and

human synthetic ACT& and monitored for dose dependent effects on MAP kinase .

phosphorylation. As Figure 7 a) and b) show, the ACTHar effkct on MAP kinase phosphoryiation

was mimicked with ACTH, treatment, mggesting that the e d f k t of ACTHar was specific and

not due to the contamlliation of ACTHPr with pituitary growth Eactors. Additionaliy, AC= and

ACTH,,-induced MAP kinase phosphorylation were effdve with concentrations as low as

0.0 1 pU/ml. MAP kinase phosphorylation caused with the highest applied concentrations of both

agents approached the level of MAP h a s e phosphorylation obtained with serum treatment.

7. Dose dependent relationships of ACTHar and ACTHI4, on MAP kinase phosphorylation

in Kin 8 cells

In previous experiment, I tested dose dependent relatonships of ACTHar and synthetic

ACTH on MAP kinase phosp horylation in Y 1 cells. In the present experiment 1 tested the same

e E i in Kin 8 mutant cells.

As Figure 8 a) and b) show, ACTHar and ACTH,, ha9 very similar effects on MAP

kinase phosphoMation in fi 8 d s as was sem before in Y1 cells, suggesting that this efEect is

probably not due to the cAMPdPK mechanism. ACTHar and ACTH,., ais0 stimulated MAP

kinase phosphorylation in fi 8 celis over the same concentration range as they did in Y1 ceiis.

MAP kinase phosphorylation wised by treatment with highest apptied concentrations of both

agents approached the levei of MAP kinase phosphorylation obtained with m m treatment.

1000 100 10 1 0.1 1000 100 pUlml None ACTH ACTH149 Serum

Figure 7. Concentration dependent MAP kinaae phosphorylation in Y1 cells.

Quiescent, senun starved cells were treated for 5 min with ACTHar and synthetic

ACTHIJ9 as indicated. Cells were harvested in RIPA buffer and Western Blot analysis

was done using phospho-specific MAP kinase antibody (New England BioLabs.).

Similar results were obtained in two independent experhents.

Figure 8. Concentration dependent MAP kinase phosphorylation in Kin 8 ceUs.

Kin 8 cells were treated and collected at the same way as it was mentioned for the Y 1 cells

(Figure 7). This experiment was done twice with similar results.

8. MAP kinase activity assay in YI: pretreatment with ACTE and 8Br CAMP and

treatment with ACTE, 8Br CAMP, 5'-AMP, PMA and Conkolin

To coda te phosphorylation of MAP kinase isofonns with increase in enzyme activity 1

treated d i s with ACTH, 8Br CAMP and PMA anâ w y e d for MAP kinase activity. As shown

on Figure 9 a), 10 min pretreatment with ACTH or 8Br CAMP foliowed by serum addition

stimulated MAP kinase activity at least as weli as ifnot even mon than senun by itself. After

getting those resuitq 1 was prompted to hvestigate whether those agents given done wouid be

capable of stimulating MAP kinase activity. Figure 9 b) shows that both higher and lower ACTH

concentrations stimulated intensively enzyme activity, compared to signal obtained in control

sarnple, although not as much as serum treatment alone. 8Br CAMP stimulated MAP h a s e

activity, but AMP was also effective suggesting low contribution of cyclic nucleotides in this

process. PMA treatment strongiy stimulated MAP kinase activity, as much as semm treatment

did. However, forskolin, an agent known as a direct stimulator of adenylyl cyclase activity and

production of CAMP, concentration of 100 pM, wasn't very effective in the stimulation of MAP

kinase activity cornpared to control and other treatments and in two separate experirnents didn't

cause any increase in MAP kinase activity (data not presented), thus confinning Our previous

observation that ACTH stimulated MAP kinase activation is not mediated via CAMP and

cAMPdPK mechanism.

The upper band present in al1 simples, includig controls represents IgG protein with

which Phospho Elk 1 antibody cross reacts (Figure 9). On the panel c) there are two important

controls: wntrol A is control sample with absence of Elk 1 fusion protein in the reaction mixture

(substrate for MAP kinase). Control B is semm treated sample with, omission of Elk 1 fusion

protein fiom reaction mixture.

pretreatment 5 min

f IgG

40 KDa + f phosph0 E I ~ - 1

A 6 Figure 9. MAP kinase activity assay in Y1

Y 1 cells were replicate plated in 100 mm tissue culture dishes at a density 2 x 105 cellslplate.

Cells were grown for four days and serum starved for another 3 days. Cells were then exposed

to variety of stimuli, hawested and MAP kinase activity assay was done, using MAP kinase

assay kit (New England BioLabs.). Senun starved cells were pretreated with Acthar (ACTH,

25 mU/ml) and 8Br CAMP (3 mM) and then treated with senun for 5 min (a). Senun starved cells

were treated with serum (1 5% HS and 2.5 % FCS), ACTH as indicated on the figure, 8Br CAMP

(3 mM), 5'-AMP (3 mM), PMA (1 00 nM) or forskolh (1 00 mM) @). Kiaase reaction was performed

on the control extract without Elk-l fusion protein which is substrate for MAP kinase (A). Kinase

reaction was performed on serum treated extract without Elk-1 fusion protein in the reaction

mixture (B) (c). This expriment was repeated twice with the same results.

67

9. Time course of ACTH, 8Br CAMP and P M . stimulated MAP kinase phosphoylatioci in

Kin 8

As was statsd More Kin 8 cells are Y 1 adrenocurtical tumor ceIl mutants that harbor a

point mutation in the regdatory subunit of the type 1 cAMPâPK that rendus the mzyme resistant

to activation by CAMP. In this expehent 1 wanted to examine t h e course of ACTH, 8Br CAMP

and PMA treatment on MAP kinase phosphorylation and compare the the ability of those agents

to activate MAP lanaSc phosphorylation in KUi 8 and Y 1 cells.

As shown on Figun 10 a), ACTHar treatment hduced MAP kinase phosphorylation at all

indicated times compared to control sample. ACTHar induced MAP kinase phosphorylation

seerned to be vexy prompt and strong. Mer only 2 min of stimulation, ACT& treatment

promptly induced appearance of phosphorylated MAP kinase isoforms. Signal seemed to be the

most potent d e r 2 min of stimulation and gradiialiy decreased towards 10 min stimulation.

8Br CAMP wasn't veiy effective and signal seem to be vew weak. However, AMP signal

seemed to lose intensity compared to the signal obtained in Y1 ceils, suggesting that the

cAMPdPK mutation affects the AMP response in Kin 8 cells (Figure 10 b). PMA was very

effective in stimulating MAP kinase phosphorylation at al1 indicated times, and far more potent

than it's control- 4a PMA Taken together these results suggest that ACTHar and PMA induced

MAP kinase phosphorylation is mediated via cAMPdPK-independent mechanism.

2 5 10 4.' I ACTHar

- ERK-1 - ERK-2

time (min)

Figure 10. Time course of ACTR, 8Br CAMP and PMA induced MAP kinase

phosphorylation in Kin 8 cells. Quiescent, serurn starved cells were treated with ACTHar

(25 mU/ml) at indicated times. Cells were harvested in RIPA buffer and Western Blot

analysis was done using phospho-specific MAP kinase antibody (New England BioLabs.) (a) . Cells were treated with 8Br CAMP (3 mM) as indicated on the figw, with 5'- AMP (3 mM),

PMA (100 nM) or with the same concentration of inactive isomer 4a PMA (b).

Experiment was done twice with similar resuits.

10. Effect of short treatment with ACTE, 8Br CAMP and PMA on rwthymidine

incorporation in Y1 eJlr

Since 1 knew fiom my p r h s orpeximaits that MAP kiwe activation is tninsient and

1ast.s for few minutes (Figure 5). in this a<periment I attempted to correlate MAP kinase activation

with the ability of arrested of ceiis to progress f?om G1 to' s phase of the cd cycle. Therefon

here, 1 tested the &ect of short pulse treatment with ACTH, 8Br CAMP and PMA on

[3H]thymidiie incorporation in Y 1 ceiis. As Figutt 12 shows, semm and FGF significantly

incread ['Wthymidine incorporation 6 and 4-fold ove control (serum starved ds), with p =

0.0004 for serum and p = 0.007 for FGF. 8Br CAMP also, significantly increased [3mthymidine

incorporation approximately Cfold over control (p = 0.01) and PMA was very effective and

increased [3athymidine incorporation 2-fold over control (p = 0.01). On the contrary, ACTH

given as a short pulse treatment and in concentration of 25 mU/d didn't increase ['Hlthymidine

incorporation in Y 1 ceiis.

11. ACTE concentration dependent growth induction in Y1 cells; cornparison to synthetic

A-,,

The ability of ACTH to stimulate MAP kinase activation (Figures 6 and 9) and at the sarne

time its inability to promote growth (Figure I l ) were in the sharp contrast. My previous data

(Figure 7) showed that treatments with ACTH induced MAP kinase phosphorylation at

concentrations that were lower than concentrations requireâ to produce sigpuficant changes in the

CAMP pool @eall and Sayers, 1972). Therefore it seand possible that higher ACTH

concentrations inhibited growth through a CAMP-dependent pathway. Therefore 1 set up an

experiment to test the hypothesis that lower concentrations of ACTH would activate MAP kinase

Figure 11. Growth stimulation with short pulse treatment in Y1 ceils

Cells were plated on 60 mm dishes at a density 0.8 x 105 cells/plate and grown

for 2 &YS. After 2 days in culture, senim was removed from medium for next 72 h

to arrest ce11 growth. Cells were then incubated for 5 min with senun (15 HS and 2.5 % FCS)

FGF (100 ngiml), ACTH (25 mU/ml), 8Br CAMP (3 mM) or PMA (100 nM) .

After treatrnents, cells were carefully washed twice with plane aMEM, and left for 14 h in

the plane M M . [3~]~hymidine was added (2 pCi/ml) during the last 2 h of incubation and

the level of [3~]th~midine incorporation was measured as described in Methods.

Values represent the fold stimulation of [3~]thymidine iacorporation relative to serum starved

controls and are presented as means I: SEM of n experiments carried out in triplkates. 71

without raising CAMP, thus promoting ceil cycle progression.

As show in Figure 12 a), ACT& over tbc concentration range fiom 0.01 mUlml to 25

m U / d produced opposite &kct on g r 0 6 in Y 1 d i s . Lower ACTHar concentrations up to 1

mU/rni, stimulated [mthymidine incorporation in Y1 d s whereas higher, up to 25 mulnd, .

were growth inhibitory. Maximai stimulation wrs obtained with the lowest concentration applied:

0.01 mU/d (approxktefy 10" M), which sisnificantly increased ['wthymidine incorporation

approximately 4-fold ove control, p = 0.00 1. Low concentration of ACTH,, (O. 1 mU1ml) also

stimulated [3H]thymidie incorporation in Y 1 d s (Figure 12 b). Synthetic ACTH,,

signtficantly increased ['Hjthymidine incorporation with a 3 -5-fold increase compared to contro1

@ = 0.00 l), that compared favorably with the 4-fold increase seen with ACTHar treatment.

To further explore the observation that ACTH aven as a short pulse in a low

concentration stimulated ce11 cycle progression in Y1 cells and to address the question whether

ACTH is a partial or full rnitogen, semm starved cells were treated with ACTHar (0.00 1 mU/rnl)

for 5 min. Cells were washed and &er 36 h the ability of ACTH to stimulate growth was assessed

through direct cell counting. ACTHar at concentration 0.001 mU1 ml given for a short period of

tirne significantiy increased c e U number (approximately 60 %) fiom (8.5 * 1 .O8 cells) x 10' celld 2

dishes to (14.5 0.8) x 10' ceild 2 dishes, p=û.002.

ACTHar, mutml

O O. 1 25

control ACTH1 -39, mutml

Figure 12. ACTH concentration dependent stimulation of P ~ t h ~ r n i d i n e incorporation in Y1

a) Cells were plated at density 0.8 x 1 6 cellslplate and grown for 2 days. After 2 days in culture

senim was removed from medium for 72 h. Cells were then incubated 5 min with decreashg

ACTHar concentrations as indicated on figure. Afier treatments cells were washed twice

with M M and left for 14 h in aMEM. [3HIThymidine was added (2 pCi/ml) during the 1st 2 h of

incubation and the level of [3HJthymidine incorporation was measured as described in Methods.

Values are means fiom single experiment done in triplicates, except hvo values with emor bars, which

are meam f SEM of indicated number of experiments done in triplicates. b) Experiment was done at

the same way as described previously, except celis were treated for 5 min with synthetic ACTHI-39 as

indicated on the figure. Values represent means fiom one experiment done in triplkates.

73

12. Gronth stimulation with short treatment with ACTE, (Br CAMP and PMA in Kin 8

To detenine mechanism by wbich ACTH stimulates MAP lrinsJe activity md

['H]thymidine incorporation in Y1 ceus, I monitord [%Jthymidine incorporation in mutant Kin 8

cdls upon high and low ACTH conentntiona. Figure 13 shows results of this experiment.

Semm treatment signincantly increased [mthymidine incorporation approximately 6-foM

over control in Kin 8 celis @ < 0.001). Another mitogen FGF was also effective, with an increase

in [3H]thyMdine hcorporation more than 4 fold over control @ < 0.001). Both high and low

ACTH concentrations (25 mU/ml and 0.1 mU/rnl) increased [3HJthymidine incorporation

approximately 3-fold over control, with p = 0.005 for 25 mU/mi of ACTH and p < 0.001 for 0.1

mU/ml of ACTH. These results further support my previously stated hypothesis that the ACTH

stimulatory effect on cell growth is cAMPdPK-independent. Both high and low 8Br CAMP

concentrations also increased [3Hlthymidine incorporation: 3-fold compared to control with p =

0.0002 for 3 mM and p = 0.0006 for 0.3 rnM 8Br CAMP. These findings are in agreement with

my hypothesis that 8Br CAMP effect on MAP kinase phosphorylation and thymidine

incorporation is not ükely due to the CAMP and cAMPdPK-dependent mechanism of action.

Incubation of Kin 8 cells with PMA significantly increased [3H]thymidine incorporation in Kin 8

cells with a 4-fold increase compared to control@ = 0.0006), which is a 2-fold increase compared

to the observed PMA effect in Y 1 cels. The ability of PMA to stimulate [3Hlthymidine

incorporation in KU1 8 cells suggested a PKC-dependent pathway in the regdation of this process.

Additionally, growth stimulation obtained with PMA treatment in fi 8 cells was 2-fold higher

compared to the same effect of P M . in Y1 celiq suggesting a possible inhibitory contribution of

cAMPdPK in PMA-stimuiated [3HJthymidine incorporation in Y1 cells.

Figure 13. Growth stimulation with short pulse treatment in Kin 8 cells.

Cells wen plated on 60 mm dishes at a density 0.8 x105 cells/plate and grown

for 2 days. After 2 days in culture, semm was removed from medium for next 72 b

to arrest ce11 growth. Cells were then incubated for 5 min with serum (1 5 % HS and 2.5 % FCS),

FGF (1 00 ng/mi), ACTH (either 25 or 0.1 mU/ml), 8Br CAMP (either 3 mM or 0.3 mM) or PMA

(100 mM). Cells were then washed twice with M M and left in aMEM for 14'h.

[3HIThymidine was added (2 pCi/ml) during the last 2 h of incubation and the level of

[f HJthymidine incorporation was rneasured as described in Methods. Values represent

the fold stimulation of [3~]thymidine incorporation relative to semm starved controls and

are presented as means f SEM of n experiments canied out in triplicates.

V DISCUSSION

V DISCUSSION

It hns been weii documented that ACTH inbibits the growth of cultured n o d adrend

celis (Homsby et al., 1974; Rainey et IL, 1983) as weii as the growth of the Y 1 mouse

adrenocortical -or d line (GU and Weidman, 1977; Annelin et ai., 1977). This growth

inhibitory effect of ACTH seen in uibo was paradoxid since ACTH has a trophic action on

adrenal glands in vivo (Fiaia et al., 1956; Dailman, 1980; Paya et al., 1980). In the present shidy,

I demonstrated that ACTH activates MAP kinase and promotes transition of ceils fiom the G1 to

S phase of the ceii cycle when adrninistered to Y1 cells as a short pulse early in the G1 phase of

the ce1 cycle.

The MAP Luiase pathway has been a subject of broad investigation in the recent past. It

has been established that this cascade of enymatic reactions is conserveci among diverse species

and serves as a signal transduction pathway in proliferafve response of the cells to mitotic signais

from the variety of growth factors, as detailed above (Section I, 3a). It has been reported that

inhibition of the M N kinase cascade acwrnpanies the growth inhibitory effects of CAMP

observed in fibroblast and other celi types (Section I, 3c). Therefore, 1 hypothesized that the

mechanisrn of ACTH- mediated inhibition of growth in vitro might involve inhibition of the MAP

kinase pathway in Y1 ceils. To test my hypothesis, 1 examineci the e f f ' s of ACTH and 8Br

CAMP on MAP kinase activation. 1 employed the approach used by Cook and McCormick (1993)

in fibroblasts, in which they showed that pretreatment of quiescent Rat1 cells for 10 min with

CAMP analogues before stimulation of cells with growth factors (either EGF and LPA)

77

completely inhibited the LPA- or EGF-induced phosphorylation of MAP kinase. Con- to my

expectations, neither pretreatment with 8Br CAMP or ACTH inhibited saum Uuluced

phosphorylation of ERK 1 and ERK 2 in Y1 4 s (Figure 5). Mer these resuits, it sewd logid

to investigate the &kt of ACTH alone on MAP kinase activation.

It bas b e n documentai in the l i t m e tbot the activation of MAP kinase is transient; thpt

the enzyme is phosphorylated and activateci by MEK and persists in the active fonn for short

perioâs of time (up to 20 or 30 min), with the peak in e q m e activity between 5 and 10 min of

stimulation (Ray and Sturgill, 1987). M y results with serum treatment of Y 1 cells confimed those

findhgs with the activation peak occurring within 5 min of stimulation (Figure 5). ACTH,

administered for a short period of time (up to 10 Mn) to G1 arrested Y 1 cellq stirnulated

phosphorylation and activation of ERK 1 and ERIC 2 (Figure 6). These results, a little bit

unexpected, prompted me to formulate another hypothesis that despite having a growth inhibitory

effect in vitro, documented by others and by my experhents (Figure 2), ACTH has an underlying

growth promoting &ect which would be more consistent with the ACTH trophic e f k t seen in

vivo. Therefore, 1 fbrther examined the effect of ACTH, 8Br CAMP and PMA on MAP kinase.

Results with ACTH- induced MAP kinase phosphorylation correlated weii with increased enzyme

activity in Y1 ceils upon same treatments (Figure 9). The &e* of 8Br CAMP on MAP Linase

activation was weak compared to ACTH and AMP itselfhad a stronger effect, indicating low

specificity of signal obtained with cyclic nucleotides (Figures 6 and 9 b). However, treatment with

PMA produced a marked activation of MAP kinase, suggesting that the stimulatory effect of

ACTH may involve a PKC-dependent pathway. In KU1 8 mutant cells, ACTH treatment for a

shoa penod of t h e also stimulated phosphorylation of MAP kiwe isofom (Figure 8). PMA

treatment for a short period of tirne in Kin 8 cels also stimuîated the MAP kinase cascade (Figure

10). To the contrary, forskoh weakly stimulated MAP kinase activity cornpared to other

treatments and control (Figure 9 b) and in two other separate experiments, forskoün didn't show

my stimuletion of MAP kinase activity (daîa aot prtsuited). These data, taken together, indicsted

a CAMP-independent mecbanism of ACTH-induced MAP kinase stimulation. In the raidies of

other authors, PKC was proposed to mediate part of ACTH action on the adrenal cortex.

Lehoux et ai. (1991) showed that in rat zona glomerufosa cellg PKC content increased upon

ACTH treatment. Moreover, ACTH wu shown to activate PKC in Y 1 cdle (Widmaier and Hall,

198S), lending M e r support for the involvment of PKC in ACTH action. Supporting data for

ACTH activation of a PKC pathway came from studies by Lefkowitz et al. (1970) and Widmaer

and Hal1 (1985). Widmaer and Hall (1985) found that PKC exista in Y1 adrenai tumor cells and in

rat fasciculata cells. They showed aiso that the activity of this enzyme was stimulated by Ca* and

phosphatidylserine. Dose response curves of ACTH action showed that the hormone was

effective in stirnulating protein h a s e C at lower concentrations than those requùed to increase

steroid synthesis. Lefkowitz et al. (1970) reported that in broken cell preparations increasing Ca*

levels produced a progressive inhibition of adenylate cyclase activity. Crespo et al. (1995)

suggested that Gs-coupled receptors shultaneously mediate opposing effects. They suggested

that the Gpy-subunits denved fkom Gs mediate ERIC 1 and ERK 2 activation, whiie CAMP

generated by Gsa-mediated activation of adenylyl cyclasq opposed this effect. The ability to

activate two distinct transduction pathways was reported for some members of the G-protein

wupled receptor fàmily. For example, Chabre et pl. (1992) showed that the Gs-coupled calcitonin

receptor activated phospholipase C second messagers and stimulated accumulation of

cytoplasmic Ca* and at the same time stimulated pathwoys mediated by CAMP when slimulated

wÂth a ligand. The same was shown for the human thyrotropin receptor expressed in CHO cels

79

(VanSande et al., 1990). nierdore, the contribution of the PICC pathway to the ACTH-mediated

activation of MAP kinase d e w u aiggested, but has yet to be established.

There are seved poteuthi pathways through whidi ACTH may activate the MAP kinase

cascade. ûtha hormones acting thmugh G-protein wupled receptors have been show to

activate MAP Linsse via pathway involving activation of Dy subunits through the activation of

either phosphoinositide 3-khase andor src-Ue tyrosine kinase (van Biesen et ai., 1996; Koch et

ai., 1994; Chpham and Neer 1993; Gamowskaya et al., 1996; Lapez-ilasaca et ai., 1997).

Since ACTH given for a short penod of tirne stirnulated transiently and rapidly MAP

kinase (Figures 6, 7 and 9), we explored the possibility that ACTH Mght stimulate cell cycle

progression, as weil. Low concentrations of ACTH, up to 1 mU/mi, shulated [3Hlthymidine

incorporation 3 to Cfold compared to control whereas higher concentrations of ACTH (up to 25

mU/ml) were inhibitory Figure 12). Moreover, both high and low ACTH concentrations

stimuiated transition of G1 to S phase in Kin 8 ceils (Figure 13).Whereas ACTH stimulated MAP

kinase activation over a broad concentration range from 0.01 pU/ml to 25 mU/ml, only low

concentrations of ACTH were growth stimulatory Therefore, 1 suggest that low concentrations

of ACTH that were growth promoting, utilized a CAMP-independent pathway. When the

concentrations of ACTH was raised it produced more CAMP which caused inhibition of growth

via CAMP-dependent pathway. 8Br CAMP given for a short penod of time stimulated ce1 cycle

t r d o n fkom G1 to S phase in Y 1 and Kin 8 cells. Since 1 showed that 8Br CAMP-induced

M A , kinase phosphorylation bears low specificity in respect to cyclic nucleotide action (Figures 6

and 9 b), 1 awum that growth prornoting effect of8Br CAMP could be either the consequence of

some metabolic &ect mediated by adenine nucleotides or the consequence of adenine nucleotides

acting via pwinergic receptors (Cusain and Planka, 1979). PMA treatment aiso stllnulated

80

transition fiorn G1 to S phase of the cell cycle in Kin 8 cds (Figure 13). Since the growth

promoting &ect ofPMA was eiicited in Y1 celis snd pasisted in the cAMPdPKdefdve mumt

cd iine, 1 suggest that PMA e f k t is solely due to the activation of PKC-signahg pathway.

Further support@ my hypothesis that the meclrsnism of ACTH growth promothg &ed involves

a CAMP-independent signalhg pathway arc r d t a hrom arc se and coiieagues (1986). niy

investigated the mechanian of action of ACTH on fieshly dispersed rat adrend cells and found

that lW1' M ACTH concomitantiy actimed phospholipase C second messenger system, inaeased

concentrations of inositol-phosphates and subsequently concentration of Ca* ions. This activation

was transient and reached maximum after 5 min of stimulation. Activation of phospholipase C

a h can lead to the activation of the PKC signaling pathway. On the other hanci, higher

concentrations of ACTH (1 O4 and 1 OJ M) increased CAMP concentrations, suggesting an

involvement of CAMP as second messenger in ACTH actions upon those concentrations.

Therefore, in the mechanism of ACTH action, activation oftwo distinct pathways can be

elucidated. One is the CAMP and cAMPdPK pathway and another one is possibly PKC the

pathway through the activation of phospholipase C. Sustained production of CAMP with high

ACTH concentrations and prolongeci treatment inhibits growth in Y 1 cels, while low ACTH

concentrations, that induce modest changes in CAMP levels (Schlmmer and Zimmennan, 1976) in

a treatment of fnv min predominantly activates the PKC pathway and promotes growth through

the transition of cells ftorn G 1 to S phase. One possible exphnation why the ACTH growth

promoting effect was difficult to be registered in vàtro in the past is that activation of one of two

possible ACTH-mediated pathways through the accumdation of CAMP and activation of

cAMPdPKq tngger3 growth inhibition. The mitogenic dect of ACTH rnay have been blunted by

CAMP accumulation during hormonal stimulation.

The ability of ACTH to induce FOS and JüN protein expression in adreaal ceUs *i

vim and in vibo wu reported (Imai et ai., 1990). but seemed paradoxicd since the activation of

these so d e d eady response genes ultimateEy leads to the initiation of ceIl proliferation (Rimm

et al., 1993). This ACTH efE& is Iürely to be mediateci through the activation of the MAP h

cascade (Ofu a al., 1990; Pulverer et ai., 1991). Thesc &d9ings weîl correhted with my results of

ACTH mediated activation of MAP kinase ad stimulation of transition of G1 resting cells to S

phase of the cd cycle. Fidings of Annelin et ai. (1996) weil supported my hypothesis ad

provided a rationale for the growth promoting effed of ACTH in Y 1 ceus. They reported that

administration of ACTH induced protooncogenes c-fos and c-jun in Y 1 ceiis. They aiso noted that

while PMA mimicked this ACTH effect, 8Br CAMP was very weak inducer of c-fos and c-jun

protooncogenes. These findings supported my hypothesis that the growth promoting effect of

ACTH seen in my experiments is a PKC-dependent and cAMPdPK-independent effect. Kimun et

al. (1993) examined the degree of c-fos induction in response to ACTH, PMA and dibutyryl

CAMP. They reported that PMA induces c-fos with a sMar kinetics compared to ACTH, but

reached only 60 % of the maximal ACTH induction, while dibutyiyl CAMP was a weak c-fos

inducer and reached only 15 % of ACTH induction.

Although Y1 celîs are of tumor origin, the cell &ne behaves in many aspects like

normal adrenocortical ceiis and has long been used as a mode1 adrenocortical celi system

(Schimmer, 198 1). Thus the finding that ACTH under appropriate conditions can stimulate

transition of Gl ceîî cycle arrested Y 1 celi to S phase may be physiologically relevant. My results

provide a rationale for the trophic effèct of ACTH seen in vivo and inductive effkcta of ACTH on

genes essociated with ceil proliferation nich u fos and jun protooncogenes and ornithine

decarboxylase, that were previously considered to be paradorricd dects of the hormone.

Ornithine decarboxylase activity increaseà in respoase to ACTH treatment which is of particular

interest because thc enymt ia rate ümiting in the -s of polyamines, which are impîicaud in

the control of tissue culture growth (Kudlow et ai., 1980). My results demoMtrating a

prolifuntve &ect of ACTH on a differentirirerl ceil line that originated fkom tumors of zoni

fàsciculata cells may also help to reso1ve a controversy &out the origin of proliferating cells in the

a d r d cortex My results are more consistent with the hypothesis that the proMerathg celis arise

fkom differentiated zones of gland (Hobarth et ai., 1996) rather than fkom an undifferentiated

stem ce11 population (Mitani et al., 1996).

Progression of eucaryotic ceUs through the cell cycle is regulated by the sequentiai

formation and activation at the specific stages of the ceU cycle, and subsequent inactivation of

series of stnicturally related serindthreonine protein kinases. They wnsist of a catalytic subu&, a

cyclin dependent kinase and the regdatory subunit, a cych (Sherr, 1993). The temporal

activation of the holoenymes is primariiy dependent on the synthesis and accumulation of specific

regdatory subunits. the cyciins (Grana and Reddy. 1995). Cyclins D 1, D2, and D3, in wnjunaion

with their cataiytic partners cdk4 and cdk6, appear to regulate the initial phases of G1 progression

(Jiang a al., 1993; Quelle et al., 1993; Resnitsky et al., 1994; Lucas et al., 1995). In n o d

untransfonned cells, the growth factor-dependent accumulation of cyclin Dl has been show to

be required to dow ceUs to p a s the G1 restriction point (Queue et al., 1993). It has been show

that early appearance of cyclin D1 upon growth fkctor stimulation of resting fibroblast cellq plays

a central role in regdating the O,-G1 transition of the ceil cycle. Lavoie et ai. (1996) showed that

cych Dl expression is positively controlieâ by MAP kinase cascade in Chinese hamster fibroblast

cell line. Therefore this example semes as a potentiai mode1 bow ce11 cycle progression upon

ACTH treatment. However this relationship in Y1 d i s has yet to be established.

My fin'ngs aiso confinned previousfy reported data that prolonged treatment with ACTH

inhibits progression of the Y 1 cds throug& the ceii cycle (Figure 2). The growth inhibitory & I I

was mimicked with 8Br CAMP treatment (Figure 2). It WU suggested that the mechanism of

ACTH action involves CAMP-dependent pathway (Schirnmer and Zimme~n~n, 1976; Soez a d.,

198 1). Armelin et al. (1996) and Kimura et al. (1993) reportecf interesthg hdings which wdd

explah inhibition of G1 to S transition in YI d s a f k prolonged treatment with ACTH seen in

vitro by other authors and in my studits (Figure 2). My results indicate that thW growth inhibitory

effect of ACTH does not result fiom inhibition of the M N kinase isoforrns ERIK 1 and ERK 2

(Figures 6 and 7). Amelin et al. (1996) suggested that besides inducing expression of c-fos and c-

jun, ACTH caused down regulation of c-rnyc by posttranscriptional modification. They reported a

similar finding on c-myc down regulation caused by 8Br CAMP. Since ceU growth response is

characterized by coordinate induction of the early response genes, the uncouphg c-myc f5om c-

fos and c-jun induction may account for the growth inhibition induced by ACTH. Although in my

experiments, CAMP analog and PMA both mimicked the growth Uihibitory efEect of ACTH

(Figure 2), the hormone cleariy acts through cAMP-dependent pathway, since the inhibitory

effects of ACTH and CAMP d o g s were aôolished in cAMPdPK-defective Kin 8 mutants,

whereas the growth inhibitory e f f ' of PMA persisted (Figure 3).

The results presented here demonstrate that ACTH activates MAP kinase, promotes the

transition of cells fiom G1 to S phase of the cell cycle and stimulates ce11 division in a CAMP-

independent manner when administered to Y1 cells as a short pulse. These ACTH eEects may

involve a PKC-dependent pathway since these actions of ACTH are mimicked by a pulse of P m

although other possible pathways may a h exists. On the contrary, the prolonged treatment 4 t h

ACTH inhibits G1 ta S progression in Y 1 cells and my r d t s suggest that this ACTH d e c t does

not result fiom inhibition of MAP kinaae pathway.

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