BIOLOGY OF REPRODUCTION 52, 124130 (1995)
Cellular and Molecular Mechanisms That Mediate Insulin-Dependent
Rat Granulosa Cell Mitosis'
JJ. PELUSO,~>*
A.M. LUCIANO,~,*A. PAPPALARDO, and B.A.
WHITE^
Departments of Obstetrics and ~ynecology*and ~ n a t o m yUniversity
,~
of Connecticut Health Center
Farmington, Connecticut 06030
ABSTRACT
Rat ovarian follicles are composed of small and large granulosa cells (GC). The present studies demonstrate that small GCs
undergo insulin- or phorbol ester-dependent mitosis in vitro. In order to examine the cellular and molecular events that account
for insulin's mitogenic action, small GCs were cultured with either insulin, phorbol ester (TPA), or both insulin and TPA. Insulin
and TPA increased GC numbers by 21
3% and 20 f 2% over control values, respectively ( p < 0.05). Simultaneous addition
of insulin and TPA increased GC numbers by 20 f 3% ( p < 0.05). In a second experiment, small GCs were exposed to control
medium, insulin, staurosporine (a protein kinase C [PKC] inhibitor), or both insulin and staurosporine. These studies revealed
that insulin induced a 21 f 5% increase in GC numbers and that staurosporine blocked insulin's mitogenic action. These observations suggest that insulin mediates its mitogenic action through a PKC-dependent mechanism. Since the proto-oncogenes,
c-fos and c-jun, are expressed during GC mitosis, studies were undertaken to determine whether or not the expression of these
two proto-oncogenes products was enhanced by insulin. The expression of c-jos and c-jun proteins was assessed by immunocytochemistry. These studies showed that after 5 h, insulin increased the percentage of cells that stained for c-fos and c-jun by
15 2% and 19 f 4, respectively ( p < 0.05). The expression of these proto-oncogenes was blocked by staurosporine. Both
progesterone and 8-br-CAMP,which block insulin-dependent GC mitosis, also inhibited the expression of c-jos and c-jun. Finally,
small GCs were cultured with insulin in the presence of sense and antisense oligonucleotides specific for c-fos and c-jun. Under
these conditions, insulin induced a 32 f 10% and 24 2 5% increase in cell numbers in the presence of c-fos and c j u n sense
oligonucleotides, respectively. Insulin-dependent GC proliferation was completely prevented in the presence of either c-fos or
c-jun antisense oligonucleotide. Taken together, these experiments support the concept that insulin stimulates small GC mitosis
by activating PKC, which induces the expression of both c-fos and c-jun. Both of these genes play essential roles in GC mitosis.
*
*
INTRODUCTION
The pituitary gonadotropins, FSH and LH, control ovarian follicular growth and steroidogenesis through very
complex and ill-defined mechanisms. This is illustrated by
the observation that rat granulosa cells (GC) temporarily
lose their ability to synthesize steroids in response to gonadotropin stimulation. This transient loss of gonadotropin
responsiveness coincides with an increase in GC mitosis
[I]. Further, gonadotropins do not stimulate rat GC proliferation in vitro [ 2 ] . In fact, FSH suppresses GC DNA synthesis, while the combined treatment of FSH and insulinlike growth factor-l (IGF-I) restores the rate of DNA synthesis
to control values [3]. These observations suggest that gonadotropins may not mediate their mitogenic action directly but rather stimulate the production of various intraovarian growth factors that in turn induce GC mitosis
[4,51.
Several gonadotropin and/or growth factor treatments
have been shown to promote GC DNA synthesis in vitro.
However, whether a specific growth factor functions as a
GC mitogen is very species-dependent [4-61. With regard
to rat GCs, Anderson and Lee [7] have shown that GC proAccepted September 5, 1994.
Received May 31, 1994.
'This work was supported in part by NIH Grant I-R55-HD27578-01A2.
'correspondence. FAX: (203) 679-1436.
3Current address: lnstituto Sperimentale Italiano, Lazzaro Spallanzani, Universita
degli Studi di Milano, Via Capolago, 2, 20133 Milano, Italy.
liferation can be induced in medium supplemented with
FSH, platelet-derived growth factor, transforming growth
factor p (TGFP), transforming growth factor a , and growth
hormone. The importance of FSH and TGFP has also been
demonstrated by Dorrington and associates [8]. Dorrington's studies have shown that the combined action of FSH
and TGFP is required to stimulate 3~-thymidineincorporation within rat GCs. The synergistic effect of FSH on GC
DNA synthesis can be replicated by epidermal growth factor (EGF) [9] or steroidogenesis-inducing protein [lo]. Insulin and IGF-1 also promote rat GC DNA synthesis and
mitosis [ l l ,121. Although several gonadotropin/growth factor combinations stimulate rat GC proliferation,'the cellular
mechanisms through which these factors mediate their mitogenic action have not been elucidated.
Recent studies have shown that the phorbol ester 12-0tetradradecanoyl phorbol 13-acetate (TPA) stimulates rat GC
proliferation in vitro [13].TPA is thought to directly activate
PKC [14], thereby implicating PKC activation as part of a
mitogenic pathway of rat GCs. In rat GCs, TPA promotes
the expression of the proto-oncogenes, c-fos [15,16]and cj u n [16]. Since these proto-oncogenes are induced during
rat GC mitosis [17],it has been proposed that GC mitogens
activate PKC, which in turn promote the expression of c-fos
and c-jun products. These proto-oncogene proteins would
then dimerize to form AP-I transcription complexes and
subsequently activate a gene cascade that ultimately results
in mitosis [MI.
MECHANISM OF INSULIN-DEPENDENT GRANULOSA CELL MITOSIS
The first series of experiments described herein was designed to determine whether or not insulin mediates its
mitogenic action via a PKC-dependent pathway that subsequently promotes the expression of c-fos and c-jun. Further experiments were conducted to determine whether
regulatory agents that prevent GC mitosis affect c-fos and
c-jun expression. Finally, GCs were exposed to sense and
antisense oligonucleotides to c-fos and cjun, respectively,
in order to determine whether either of these proto-oncogene products was essential for insulin-dependent GC
mitosis.
MATERIALS AND METHODS
Animals
Immature female Wistar rats (22 days of age) were obtained from Charles River Breeding Labs. (Wilmington, MA)
and housed under controlled conditions of temperature,
humidity, and photoperiod (12L:12D; lights-on at 0700 h).
Rats were anesthetized with CO 2 and then cervically dislocated between 0930 and 1000 h when they were between
25 and 29 days of age. This protocol was approved by the
Animal Care Committee of the University of Connecticut
Health Center.
Preparationof Culture Medium
RPMI 1640 without phenol red (Gibco Labs., Grand Island, NY) was used in all culture experiments. It was supplemented with penicillin (0.14 g/L), streptomycin
(0.27 g/L), HEPES (4.76 g/L), BSA fraction V (2 g/L), sodium selenite (5 ng/ml), transferrin (5 V.g/ml), and sodium bicarbonate (2.2 g/L). The pH was adjusted to 7.4 and
the medium was filtered through a 0.2-Rm filter. Depending on the experimental design, insulin (1 M), TPA (2 nM),
staurosporine (0.7 nM; Kamiya Biomedical, Thousand Oaks,
CA), 8-br-cAMP (0.2 mM), and progesterone (640 nM) were
added to the cultures. All chemicals were purchased from
Sigma Chemical Co. (St. Louis, MO) unless indicated otherwise. Progesterone was dissolved in ethanol and then diluted in RPMI 1640 to the desired final concentration.
GC Isolation and Culture
GCs were isolated according to the procedure of Rao et
al. [19] with slight modifications. Briefly, ovaries were placed
in Medium 199 containing 0.2% BSA, 9.1 mM EGTA at pH
7.4. The follicles within the ovary were punctured with 20gauge needles and then incubated for 5 min at 37°C in 5%
CO2:95% air. The ovaries were transferred to Medium 199
containing 0.2% BSA, 2.1 mM EGTA, and 0.5 M sucrose at
pH 7.4 and then incubated at 37 0C in 5% C0 2:95% air for
10 min. The ovaries were washed, resuspended in fresh
Medium 199 containing 0.2% BSA, and then pressed to release the GCs. The cells were loaded onto the top of a 1545% Percoll gradient (8 ml total volume). The gradient was
125
centrifuged at 200 x g for 5 min and, starting from the top,
12 fractions of 666 Il1 were collected. The cells in each fraction were washed and resuspended in RPMI 1640. Small
GCs were typically collected in fractions 3-4, while larger
GCs were isolated from fractions 5-7. Cells were plated in
35-mm dishes or on 8-chamber Lab-tek slides (Nunc Inc.,
Naperville, IL) and cultured for up to 24 h in a 5% CO 2 /
air atmosphere.
GC ProliferationAssessed by In Situ Cell Counting
To assess the capacity of GCs to undergo mitosis, 2 to 3
x 105 GCs from the different fractions were added to 35mm dishes in 1 ml of RPMI 1640 with or without insulin
as indicated. After 2 h, any unattached GCs were removed
and the medium was replaced. An aliquot of these unattached GCs was stained with trypan blue, and cell number
and viability were assessed. The remaining unattached GCs
were replated and cultured for an additional 22 h in the
presence of insulin. After culture, cells were harvested by
gently scraping the dish with a diSPO cell scraper (Scientific Products, McGraw Park, IL), and cell number and viability were reassessed.
To determine the proliferative capacity of those GCs that
attached to the culture dish within the first 2 h of culture,
the number of GCs, observed within five different large (160ptm 2) grids within the original 35-mm dish, was recorded.
Each of these large grids was divided into 100 smaller grids,
allowing for the precise location of each GC. The large grids
were located at the ends of the horizontal and vertical axes
and at the center of the dish. At this plating density, 20-30
cells per large grid were generally observed. After 24 h of
culture, the GCs in each large grid were recounted. Cell
proliferation was expressed as the percentage increase in
cell number over the 2-h control value. For each experiment, 100-150 GCs were counted. Each of these experiments was replicated two to three times.
The results obtained by counting cells in situ were similar to those obtained by harvesting the cells and counting
them in a hemacytometer [13]. For the present experiments
the method of counting cells was chosen to eliminate cell
harvesting.
Immunocytochemical Detection of Proto-Oncogene
Proteins
The localization of the c-jun and c-fos proteins was carried out as described previously [17]. All antibodies and reagents for the immunocytochemical detection of c-fos and
c-jun proteins were supplied by Oncogene Science
(Uniondale, NY). The c-fos antibody was a rabbit affinitypurified polyclonal antibody that was raised against the
peptide SGFNADYEASSSRC, which corresponds to residues
4 to 17 of human c-fos. The c-jun antibody was a rabbit
affinity-purified polyclonal antibody raised against the peptide TPTPTQFLCPKNVTD, amino acids 73 to 87, in the
N-terminal region of the c-jun.
126
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PELUSO ET AL.
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FIG. 1. The effect of insulin and TPA on the proliferation of GCs isolated from Percoll gradient fractions 3 through 7. GCs were collected from
immature rat ovaries and separated by Percoll gradient centrifugation (for
details see Materials and Methods). The cells were then plated in 35-mm
culture dishes in the presence of either insulin or TPA. After 2 h, the medium was removed and the number of cells present within five large grids
was counted. Twenty-four hours later, the number of cells within these five
grids was again counted and the percentage change in cell number calculated. This experiment was repeated three times. In this and subsequent
graphs, GC mitosis is expressed as a mean percent change + SE.
Small GCs were plated in Lab-tek chamber slides and
exposed to various regulatory factors (see Results and figure legends for experimental details). After 5 h of culture,
GCs were fixed in 10% phosphate buffered formalin for 5
min at room temperature, washed, incubated with 1% Triton X for 5 min, and stained for c-fos and c-jun, respectively, according to instructions provided with Oncogene
Science's immunohistochemistry system (Uniondale, NY).
This detection system is an avidin-biotin peroxidase-based
system that uses diaminobenzidine tetrahydrochloride as a
substrate. Cells that expressed c-fos and c-jun were detected by the presence of a reddish-brown precipitate. To
assess the specificity and number of false positives, the primary antibody was replaced with normal preimmune rabbit serum and GCs stained by means of the avidin-biotin
peroxidase-based system. In the absence of the primary antibodies, 3 ± 1% (n = 36) of the GCs stained. For each
treatment, c-fos and c-jun expression was presented as the
net percentage change from control treatment (i.e., % stained
in a given treatment minus % stained in the control). These
experiments were replicated four to six times on separate
days, and mean ± SE for each treatment was calculated.
Effect of Antisense Oligonucleotides on GC Mitosis
Oligonucleotides specific to the 5' end of c-fos and c-jun
were synthesized in both the sense and antisense orientation as outlined by McDonnell et al. [20]. The following oligonucleotides were used:
c-fos Sense 5'-ATG ATG TTC TCG GGC TTG-3'
.
0
-
Control
Insulin
TPA
Insulin
+
TPA
FIG. 2. The effect of insulin and TPA on proliferation of GCs isolated
from fractions 3 and 4 (i.e., small GCs). The asterisk indicates a value significantly different from the control value (p < 0.05).
c-fos Antisense 5'-GAA GCC CGA GAA CAT CAT-3'
c-jun Sense 5'-ATG ACT GCA AAG ATG GAA-3'
c-jun Antisense 5'-TC CAT CTT TGC AGT CAT-3'
The oligonucleotides were synthesized and reverse-phase
cartridge-purified by Integrated DNA Technologies, Inc.
(Coralville, IA).
In these experiments, small GCs were isolated, plated in
35-mm dishes, and exposed to insulin for 2 h. The medium
was removed and replaced with medium that was supplemented with insulin and either a sense or an antisense oligonucleotide. Both sense and antisense oligonucleotides
were used at a concentration of 40 ptg/ml. The number of
2
GCs observed within five different large (160-[Lm ) grids
within the 35-mm dish was recorded after 2 and 24 h of
culture. These experiments were replicated three times and
the data were pooled and analyzed by a Student's t-test.
StatisticalAnalysis
When appropriate and unless otherwise stated, the data
were analyzed by either a two-way or one-way ANOVA followed by Student-Newman-Keuls multiple range test. Regardless of the statistical test, only p values - 0.05 were
considered to be significant.
RESULTS
Regardless of cell size or fraction number, about 20%
(i.e., 0.5-0.6 x 105 cells/ml) of the GCs failed to attach
within the first 2 h of culture. The viability of these nonattached GCs was poor. Less than 5% of the small nonattached GCs collected from fractions 3-4 were viable. Since
MECHANISM OF INSULIN-DEPENDENT GRANULOSA CELL MITOSIS
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Insulin
0=_ - Mm Stauro.
Insulin
Stauro.
FIG. 3. The effect of the protein kinase C inhibitor, staurosporine, on
insulin-dependent GC mitosis. For this study, GCs were separated by Percoil gradient centrifugation and cells within fractions 3 and 4 were pooled.
virtually all of these cells were dead, their ability to undergo
insulin-dependent mitosis was not studied. In contrast, 42
+ 1% of the large nonattached GCs within fractions 5-7
were viable. Insulin did not produce an increase in the
number of nonattached large GCs but rather a decline in
cell viability to 19
4% (n = 8;p < 0.05).
GCs that were isolated in fractions 3 to 7 after Percoll
gradient centrifugation and attached to the culture dish within
2 h were exposed to either insulin or TPA in order to assess
their mitogenic capacity. Both insulin and TPA increased
the number of GCs present within fraction 3 by 30-40%.
127
This percentage gradually decreased with increasing fraction number, with less than 5% of the GCs within fraction
7 dividing (Fig. 1). On the basis of these observations, only
GCs collected from fractions 3 and 4 were used in subsequent studies. Since these GCs were smaller (- 50 pLm 2 in
area; 8 ILm in diameter) than those collected in fractions 5
to 7 (- 75 pum2; 11 }xm in diameter), these GCs were referred to as small GCs. These findings were similar to those
obtained from eCG-primed immature rats [19, 21].
Both insulin and TPA increased small GC mitosis by 20%.
The combined treatment of insulin and TPA did not increase the percentage of GCs undergoing mitosis compared to treatment with either mitogen alone (Fig. 2). Further, staurosporine blocked insulin-dependent GC
proliferation (Fig. 3).
Since mRNA and protein levels of c-fos and c-jun are
increased during gonadotropin-induced GC proliferation [17],
studies were undertaken to determine whether or not insulin promoted the expression of these proto-oncogene
products in vitro. A pilot study indicated that maximum
expression of c-fos and c-jun proteins occurred 5 h after
insulin exposure. Both proto-oncogene products were localized within the nucleus of many small GCs after insulin
treatment (compare Fig. 4, A or B, with Fig. 4C, the negative
control). Insulin induced a 15-20% increase in the number
of small GCs that express c-fos and c-jun products. This
induction was prevented by staurosporine (Fig. 5). Similarly, two regulatory factors that block insulin/TPA-dependent GC mitosis (i.e., 8-br-cAMP [13] and progesterone [unpublished observation]), also prevented insulin from
stimulating c-fos and c-jun protein expression (Fig. 6).
To determine whether c-fos and c-jun were necessary in
order for insulin to induce mitosis, small GCs were treated
FIG. 4. Immunocytochemical detection of c-fos and c-jun expression within small GCs exposed for 5 h to insulin.
The presence of these proto-oncogenes was revealed by a reddish-brown precipitate. Note the intense staining within
the nuclei of cells stained for c-fos (A) and c-jun (B). When the primary antibody is replaced with normal rabbit serum
(i.e., negative control), the reddish-brown precipitate is rarely observed (- 3% of the GCs) (C). All photographs are
shown at x229.
128
PELUSO ET AL.
34 +/- 8% for C-Fos
Control Values:
o
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Control Values:
34 +/- 8%
for C-Fos
21 +/- 4%
for C-Jun
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C-Jun
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Insulin
Stauro
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FIG. 5. The effect of staurosporine on insulin-dependent expression of
c-fos and c-jun. The GCs from fractions 3 and 4 were cultured for 5 h in
the presence of control medium or medium supplemented with insulin and/
or staurosporine. GCs were then stained for either c-fos or c-jun. The percentage of cells that stained for each treatment was determined by visual
observation under brightfield optics. The net change from control was then
calculated. Values are expressed as the mean net change + SE. This entire
experiment was repeated four times on four separate days. The mean percentage of small GCs in the control group that express c-fos and c-jun is
shown at the top of the graph.
with insulin. After a 2-h incubation, culture medium was
supplemented with either a sense (control) or antisense
oligonucleotide to c-fos or c-jun, respectively. After 24 h of
culture in the presence of either c-fos or cjun sense oligonucleotide, insulin stimulated a 20-30% increase in small
GC mitosis. In contrast, exposure to either antisense oligonucleotide attenuated insulin's mitogenic capability (Fig.
7).
DISCUSSION
The present study demonstrates that 30-40% of GCs isolated in fraction 3 undergo mitosis in response to insulin
or TPA. While this increase in GC numbers may appear unremarkable, several factors must be considered. First, unlike transformed cell lines, GCs have a limited mitogenic
capacity [22]. Second, fraction 3 is composed of approximately 70% small GCs. Since only small GCs undergo insulin-induced mitosis, the maximum mitogenic response
(MMR) is reduced to 70%. Third, the cell viability of these
Percoll fractions is - 60%. Finally, about 70% of freshly
isolated GCs are in the Go/G 1 phase of the cycle [23]. This
further reduces the MMR, since insulin and IGF-1 only act
on G0/G, cells in facilitating their transition into the S-phase
of the cell cycle [24]. Given these considerations, the MMR
can be estimated using the following equation: MMR (%
increase) = 100% of initial value x % viable cells x %
small cells x % cells in Go/Gj. Thus, the MMR is estimated
mQ
-5
0.
-10
IN
IN + cAMP
IN + P4
FIG. 6. The effect of 8-br-cAMP (cAMP) and progesterone (P
4) on insulin-dependent expression of c-fos and c-jun. The GCs from fractions 3
and 4 were cultured for 5 h in the presence of control medium or medium
supplemented with either 8-br-cAMP or P4. GCs were then stained for c-fos
or c-jun. This experiment was repeated four times and c-fos and c-jun values are expressed as described for Figure 5.
to be about 29%, which indicates that both insulin and TPA
induce a near MMR for GCs within fraction 3.
The mitogenic response to insulin and TPA diminishes
for GCs collected in the higher fractions. Failure to observe
an MMR in the higher fractions could indicate that the large
GCs that predominate in the higher fractions are not capable of undergoing mitosis. This putative loss of "mitotic
competence" correlates with the fact that these larger GCs
are more differentiated as indicated by their increased ability to secrete estradiol-173 and progesterone (P4) [19, 21].
However, it could be argued that there are two populations
of viable large GCs: those that attach and those that do not
attach to the culture dish within 2 h. The data generated
through use of the in situ cell counting procedure clearly
demonstrate that large GCs that attach do not undergo mitosis in response to insulin. Similarly, insulin does not promote mitosis in unattached large GCs. Collectively, these
studies support our conclusion that large GCs do not
undergo insulin-dependent mitosis.
It is possible that the mitogenic stimuli could change
with increasing GC size, since these fractions contain many
GCs that are in the S- and G2/M-phases of the cell cycle
[25]. For example, large GCs may proliferate in response
to other growth factors, such as FSH/TGF3 [8] or EGF/TGF3
[9], since these treatments stimulate GC DNA synthesis. If
non-insulin-like growth factors can stimulate mitogenic
pathways in large GCs, their mechanism most likely would
not include the activation of PKC, since the mitogenic response to TPA also decreases for GCs isolated from fractions 5 and greater.
129
MECHANISM OF INSULIN-DEPENDENT GRANULOSA CELL MITOSIS
The present study is the first attempt to elucidate the
cellular mechanism involved in insulin-dependent GC proliferation. These initial studies suggest that insulin mediates
its action through a PKC-dependent pathway. This concept
is supported by the observation that TPA does not have an
additive effect on the number of GCs that undergo insulindependent mitosis. Thus, both mitogens stimulate the same
population of "mitotically competent" GCs. Further, staurosporine blocks insulin-dependent proliferation when used
at a concentration that specifically inhibits PKC activity in
several different cell types.
Insulin also stimulates a 15-20% increase in the percentage of GCs that express c-fos and cjun. This increase
approximates the percentage of GCs that undergo insulindependent mitosis. It is interesting that staurosporine not
only blocks insulin-dependent GC mitosis but also attenuates insulin's ability to induce c-fos and c-jun protein
expression. These findings suggest that PKC activation induces functional AP-1 transcription complexes (i.e.,fos/jun
heterodimers andjun/jun homodimers) within those GCs
that eventually undergo mitosis. The antisense studies support this concept by demonstrating that both c-fos and cjun
proteins are essential to insulin's mitogenic action.
The importance of c-fos and cjun is further emphasized
by the finding that pathways that negatively regulate GC mitosis do so in part by suppressing the expression of these
proto-oncogene products. The site at which each of these
negative regulators inhibits the expression of c-fos and cjun
is unknown and could be at either the nuclear or cytoplasmic level. It is likely that progesterone's action is nuclear. The progesterone receptor is a transcription factor
that when ligand activated generally decreases the levels of
c-fos and cjun mRNAs [26]. In avian oviductal cells, this
inhibitory action on c-jun expression is mediated through
a DNA sequence located within the cjun promoter [27].
These published reports support the idea that progesterone has a nuclear site of action. However, progesterone
increases intracellular concentrations of cAMP and downregulates agonist-induced inositol phosphate metabolism in
human term placenta [28]. If progesterone has a similar action within GCs, then progesterone could mediate its action
indirectly by stimulating cAMP synthesis. Thus, more experiments must be conducted to assess the mechanism by
which progesterone inhibits insulin's mitogenic action.
As previously mentioned, cAMP negatively influences insulin-dependent proto-oncogene expression (present study)
and GC mitosis [13, 29, 30]. Cyclic AMP could mediate its
anti-mitogenic action by stimulating progesterone synthesis
[31], which in turn would block insulin-dependent mitosis.
This is unlikely since cAMP prevents GC mitosis in the presence of aminoglutethimide (AG), which inhibits progesterone synthesis (i.e., a 29 + 3% increase in GC numbers for
insulin + AG vs. a 5 + 2% increase for insulin + AG +
cAMP treatment; p < 0.05; unpublished observations). It is
also unlikely that cAMP directly interacts with either the c-fos
45
I
a
40
35
30
(1
.)
25
U
20
a)
15
10
5
C-Fos
C-Jun
FIG. 7. The effect of sense and antisense oligonucleotides on the insulin-dependent proliferation of small GCs. GCs were exposed to insulin
for 2 h and then treated with either sense or antisense oligonucleotides for
c-fos and c-jun for an additional 22 h. GC proliferation was assessed as
described in Materials and Methods and expressed as the mean percent
increase + SE. This experiment was repeated three times.
or c-jun promoter to inhibit their transcription, since agents
that increase intracellular levels of cAMP (i.e., FSH, 8-brcAMP, forskolin) either have no effect or increase mRNA
levels of c-fos [15, 16] and c-jun [16] in cultured rat GCs. A
third possibility could be that cAMP interferes with insulin's
ability to interact with its receptor and stimulate inositol
phospholipid metabolism [32]. This is also unlikely, since
8-br-cAMP blocks TPA-induced GC mitosis [13].
The most likely site of cAMP's action appears to be at or
distal to PKC activation. In other systems TPA activates Ras
[33]. Ras then activates Raf-1, which in turn stimulates the
sequential activation of two mitogen-activated protein kinases, p4 4mapk and p42 mapk, and ultimately the gene cascade
that leads to mitosis [34]. The results of several studies have
also shown that Ras/Raf-1 activation enhances the transcription activity of c-jun by inducing phosphorylation of its
transcriptional activation domain [35, 36]. This would result
in the increased expression of both c-fos and c-jun [37].
Data presented from three separate laboratories demonstrate that cAMP interferes with the ability of Ras to activate
Raf-1 [38-40], thereby blocking the mitogen-activated kinase cascade and subsequent mitosis. The possibility that
cAMP has a similar mode of action in GCs remains to be
investigated.
In summary, it appears that insulin stimulates rat GC mitosis by activating PKC, which stimulates a series of kinases
that ultimately leads to the expression of the proto-oncogene products, c-fos and c-jun. These proto-oncogene
products play an essential role in the mitogenic pathway,
presumably by forming AP-1 transcription complexes that
in turn induce the mitogenic gene cascade. Finally, factors
that inhibit GC mitosis do so in part by attenuating the insulin-induced expression of c-fos and c-jun.
130
PELUSO ET AL.
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