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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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