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First published online May 23, 2008
doi: 10.1242/10.1242/dev.020560
1 Department of Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX77030, USA.
2 Department of Applied Animal Science, Graduate School of Biosphere Science,
Hiroshima University, Higashi-Hiroshima, 739-8528, Japan.
3 Breast Cancer Center, Baylor College of Medicine, Houston, TX77030, USA.
4 NV Organon, part of the Schering-Plough Corporation, Target Discovery Oss,
Molenstraat 110, 5340 BH Oss, The Netherlands.
* Author for correspondence (e-mail: joanner{at}bcm.edu)
Accepted 22 April 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Ovary, Ovulation, Granulosa cell, Kras (K-ras), Signal transduction, MKP3 (DUSP6)
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Cespedes et al., 2006;
Gupta et al., 2007;
Rocks et al., 2006). In
response to growth-regulatory molecules, transient activation of RAS can
stimulate controlled proliferation as well as differentiation of cells.
Uncontrolled activation of RAS is often associated with oncogenic
transformation or senescence of cells
(Jackson et al., 2001;
Lin et al., 1998;
Serrano et al., 1997;
Shaw et al., 2007).
Specifically, RAS family members become oncogenic by single-point mutations,
mainly at codons 12 or 13 (Bourne et al.,
1990), leading to constitutive signaling and cell transformation
with changes in morphology, increased proliferation and/or inhibition of
apoptosis. Tissue-specific activation of oncogenic
KrasG12D causes mammary gland, lung and endometrioid
ovarian carcinoma in mouse (Dinulescu et
al., 2005; Jackson et al.,
2001; Sarkisian et al.,
2007). Mutations of KRAS or BRAF in non-invasive
and invasive carcinomas of the ovary [involving the ovarian surface epithelium
(OSE)] have been reported (Gemignani et
al., 2003; Mayr et al.,
2006).
In this study, we sought to determine the impact of RAS activation in
granulosa cells in vivo. The constitutively active
KrasG12D mutation
(Johnson et al., 2001) was
selectively expressed in mouse granulosa cells using a Cre-mediated DNA
recombination approach. The inappropriate, premature expression of
KrasG12D in granulosa cells blocked granulosa cell
differentiation at an early stage, leading to the formation of abnormal
follicle-like structures containing non-mitotic, non-apoptotic,
non-differentiated and non-tumorigenic cells. Moreover, those follicles that
reached the antral stage exhibited impaired responses to hormones, leading to
ovulation failure. Thus, transient but not sustained activation of RAS in
granulosa cells is crucial for normal follicle growth and successful
completion of the ovulation process.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Tuveson et al.,
2004). Although Amhr2 is highly expressed in granulosa
cells of growing follicles it is also known to be expressed in other
reproductive tissues, including ovarian surface epithelial cells and the
uterus (Arango et al., 2008)
(our unpublished observations). Therefore, we sought to obtain a
Cre-expressing mouse model that would be more highly specific for granulosa
cells. Cyp19-Cre transgenic mice were generated by oocyte
microinjection of a DNA fragment in which the 304 bp Cyp19 promoter
(GenBank S85356, bp -278 to +26) was ligated to iCre cDNA. To study
ovarian responses to exogenous gonadotropins, 21-day-old immature females were
analyzed to avoid the complexity of ovarian functions associated with estrous
cycles and endogenous surges of gonadotropins. Specifically, immature mice
were injected intraperitoneally (ip) with 4 IU eCG (equine chorionic
gonadotropin; Calbiochem) followed 48 hours later with 5 IU hCG (human
chorionic gonadotropin; American Pharmaceutical Partners, Schaumburg, IL).
Ovulated COCs were collected from oviducts 16 hours after hCG injection.
Animals were treated in accordance with the NIH Guide for the Care and Use of
Laboratory Animals.
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BrdU incorporation and TUNEL assays
Mice were injected ip with 50 mg/kg of BrdU in PBS, and were killed 2 hours
later. Ovaries were isolated and fixed with 4% paraformaldehyde (PFA)
overnight. Incorporated BrdU was detected by immunohistochemistry using BrdU
antibody according to manufacturer's instructions (Sigma, St Louis, MO). TUNEL
assays were performed on PFA-fixed paraffin-embedded sections using the
ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Serologicals
Corporation, Norcross, GA) according to manufacturer's instructions.
Immunohistochemistry and immunofluorescence
Immunohistochemistry was performed on 4% PFA-fixed paraffin-embedded
5-µm sections using the VectaStain Elite Avidin-Biotin Complex Kit as
directed by the manufacturer (Vector Labs, Burlingame, CA). Sections were
probed with primary antibodies against FOXO1 or PCNA (Cell Signaling, CA) and
visualized using a 3,3V-Diaminobenzidine Peroxidase Substrate Kit (Vector
Labs). For immunofluorescence, ovaries were PFA fixed, embedded in OCT
compound (Sakura Finetek USA, Torrance, CA) and stored at -80°C before
sectioning. Sections were probed with anti-KRAS (Santa Cruz Biotechnology,
Santa Cruz, CA), anti-phospho-AKT, anti-phospho-ERK1/2, anti-cleaved caspase
3, or anti-phospho-histone H3 (Cell Signaling Technology) antibodies and
visualized with Alexa Fluor 594-conjugated goat anti-rabbit IgG (Molecular
Probes, Eugene, OR). Digital images were captured using a Zeiss Axiphot
microscope with 5-40x objectives. For all the experiments, exposure time
was kept the same for control and Kras mutant samples.
In situ hybridization
Plasmids for Nr5a2 and Cyp11a1 probes were as described
previously (Boerboom et al.,
2005; Falender et al.,
2003). A cDNA fragment of Mkp3 was amplified by RT-PCR
from mouse ovary total cDNA and subcloned into the pCR-TOPO4 vector
(Invitrogen, Carlsbad, CA). In situ hybridization was performed as previously
reported (Falender et al.,
2003; Hsieh et al.,
2005). Tissue histology and the radioactive probe were visualized
under light- and dark-field illumination, respectively.
RT-PCR and real-time RT-PCR
Reverse transcription (RT)-PCR was performed using the SuperScript One-Step
RT-PCR System with the Platinum Taq Kit (Invitrogen) and 100 ng samples of
ovarian total RNA that had been isolated using the RNeasy Mini Kit (Qiagen,
Germantown, MD). Approximately 0.625 µCi of [
-32P]dCTP
(3000 Ci/mmol; MP Biomedicals, Irvine, CA) were added to each reaction to
generate radioactive signals. Primer sequences and amplification conditions
used are available upon request. Samples were separated by electrophoresis on
5% PAGE gels, dried and exposed to Biomax XAR film (Eastman Kodak, Rochester,
NY) to generate the presented images.
Quantitative (q) RT-PCR was performed using the Rotor-Gene 3000 thermocycler (Corbett Research, Sydney, Australia). Relative levels of gene expression were normalized to β-actin.
RAS activity assay
The RAS-binding domain (RBD) of the mouse PI3K p110 subunit (PIK3CA;
aa 220-311) (Rodriguez-Viciana et al.,
1996) and of mouse RAF1 (aa 55-131)
(Campbell-Valois and Michnick,
2007) were PCR amplified from a mouse ovary cDNA pool and
subcloned into pGEX 4T1 vector. Recombinant GST-PI3K RBD and GST-RAF1 RBD were
expressed in the Rosetta-pLysS E. coli strain (Novagen) and affinity
purified using glutathione-agarose beads (Sigma). Ovaries were homogenized in
lysis buffer (20 mM NaF, 10 mM MgCl2, 100 mM NaCl, 10% glycerol,
0.5% Triton X-100, 20 mM HEPES, pH7.5). The lysates were incubated with
agarose slurries linked with mouse RAF1 RBD or PI3K RBD to bind RAS-GTP. The
agarose beads were washed and resuspended in Laemmli sample buffer prior to
western blot analysis.
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RNAi of Mkp3
Mkp3 siRNA (sc-39001) was purchased from Santa Cruz Biotechnology.
Scrambled siRNA duplex (Ambion) was used as control. Transfection of siRNA (50
nM) into cultured granulosa cells was accomplished using the HVJ Envelope
Vector Kit (Ishihara Sangyo, Tokyo, Japan) as previously reported
(Shimada et al., 2007). The
culture medium was replaced 5 hours after transfection and the cells were
treated with 250 ng/ml amphiregulin (R&D Systems) for up to 4 hours.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or with
transgenic mice in which Cre expression is driven by the Cyp19
promoter (Cyp19-Cre). The ovarian expression pattern of the
Amhr2-Cre allele has been described previously
(Jorgez et al., 2004;
Pangas et al., 2006;
Boerboom et al., 2006). The
generation of the Cyp19-Cre mouse strain is described in
Materials and methods. To monitor the Cre activity in the ovaries, the
Cyp19-Cre mice were crossed to the ROSA26 reporter mouse
strain that expresses β-galactosidase (β-gal) only in Cre-expressing
cells (Soriano, 1999). Ovaries
were stained for β-gal activity using X-Gal substrate as previously
reported (Jorgez et al.,
2004). No β-gal activity was detected in ovaries of
ROSA26 mice lacking Cre (Fig.
1A). In the ROSA26;Cyp19-Cre mice, β-gal was
detected at low levels in granulosa cells of small follicles at postnatal day
10 (Fig. 1D) and at increased
levels in granulosa cells of all antral follicles
(Fig. 1B,E). Injections of
ROSA26;Cyp19-Cre mice with equine chorionic gonadotropin (eCG), a
known inducer of endogenous Cyp19 expression, stimulated follicle
growth and increased Cre activity (Fig.
1C,F). Cre activity was not detected in theca cells or oocytes
throughout postnatal development (Fig.
1D-F). These results indicate that the Cyp19-Cre mouse
strain exhibits specific expression of Cre in the granulosa cells, with
minimal leakage in other cell types. Examination of KrasG12D mRNA in immature LSL-KrasG12D;Amhr2-Cre and LSL-KrasG12D;Cyp19-Cre mice demonstrated that the KrasG12D allele was efficiently recombined and expressed at levels comparable to the endogenous Kras gene (Fig. 1G). Since endogenous KRAS protein is highly expressed in granulosa cells of growing follicles, expression of the mutant allele is being induced in the same cell type as the endogenous gene (Fig. 1H).
Granulosa cell expression of KrasG12D impairs ovulation and female fertility
For fertility tests, LSL-KrasG12D;Amhr2-Cre and
LSL-KrasG12D;Cyp19-Cre females were bred to wild-type
males continuously for 6 months. The average number of 
7-8 pups per
litter for the control mice (LSL-KrasG12D) was not
different from that of our C57BL/6J mouse colony. However, the
LSL-KrasG12D;Amhr2-Cre and
KrasG12D;Cyp19-Cre females (n=6, respectively)
were subfertile over the 6-month period, with most pups being born in the
first 2 months (Fig. 2A).
To determine the cause of reduced fertility in the KrasG12D mutant mice, we tested their ability to ovulate by injecting immature mice with 4 IU of eCG and 46 hours later 5 IU of human chorionic gonadotropin (hCG). Whereas the control littermates ovulated many COCs at 16 hours after hCG injection, most LSL-KrasG12D;Amhr2- Cre mice did not ovulate at all, and only a few COCs were observed in the oviducts of LSL-KrasG12D;Cyp19-Cre mice (Fig. 2B). The histological data from the LSL-KrasG12D;Amhr2-Cre mice are presented because the block of ovulation was more complete in this strain. However, the overall histological patterns in the two mutant strains were similar. Specifically, ovulation failure in the mutant mice was associated with defects of COC expansion and with the germinal vesicle breakdown of oocytes (Fig. 2D,F), whereas expanded COCs and meiotic oocytes with condensed chromosomes were present in the preovulatory follicles of control mice, at 8 hours post-hCG (Fig. 2C,E). In control mice, most large antral follicles ovulated by 16 hours after hCG (Fig. 2G) and had well developed corpora lutea (CLs) at 48 hours post-hCG (Fig. 2I). By contrast, antral follicles containing unovulated COCs remained in the mutant mouse ovaries at 16 hours post-hCG (Fig. 2H) and unovulated oocytes were trapped at the center of the CL at 48 hours post-hCG (Fig. 2J). More than 40 sections of KrasG12D mutant ovaries (n=4) were examined, and trapped oocytes were present in 80-90% of the newly formed CLs. Because comparative analyses of the two mutant mouse strains revealed similar phenotypes and because expression of the Cyp19-Cre transgene is more specific for granulosa cells than is Amhr2-Cre, data from the KrasG12D;Cyp19-Cre mice (C-Cre) mice are presented.
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Since the cells in the aberrant ovarian lesions were non-mitotic, the
progressive enlargement of Kras mutant ovaries and the increased
number of abnormal follicle-like structures might be caused by repression of
apoptosis, a common feature in the mammalian ovary that serves to eliminate
atretic follicles (Wang et al.,
2006). Both the TUNEL assay and immunostaining for cleaved caspase
3 (CC3) were analyzed in the Kras mutant ovaries. Immature control
and Kras mutant mice were primed with eCG and hCG to stimulate
increased follicle growth. At 2 hours after hCG, DNA fragmentation
(Fig. 3M) and caspase 3
cleavage (Fig. 3O) were
detected in multiple pre- and early-antral follicles in control ovaries. By
contrast, these apoptosis markers were markedly reduced in the Kras
mutant ovaries, where the abnormal follicle-like structures were completely
devoid of fragmented DNA and cleaved caspase 3
(Fig. 3N,P). These results show
that apoptosis was repressed by the KrasG12D mutation in
granulosa cells.
KrasG12D downregulates genes essential for granulosa cell differentiation and ovulation
Because the KrasG12D knock-in mice failed to ovulate,
the expression of genes crucial for granulosa/cumulus cell differentiation and
ovulation was analyzed in wild-type and mutant ovaries. As shown in
Fig. 4A, Fshr mRNA was
readily detected in ovaries of immature control mice and increased
2.5-fold in response to eCG and was associated with the growth of
preovulatory follicles. Other genes highly induced by eCG were Lhcgr,
a marker of differentiated granulosa cells in preovulatory follicles,
Areg, which encodes an EGF-like factor, and Cyp11a1, which
encodes the steroidogenic enzyme leading to progesterone biosynthesis. Whereas
expression of Fshr and Lhcgr was selectively reduced by the
ovulatory stimulus of hCG, genes associated with ovulation (Areg,
Ptgs2 and Tnfaip6) and luteinization (Cyp11a1) were
upregulated markedly by hCG (Fig.
4A). By contrast, the induced expression of these genes was
reduced/altered in ovaries of the KrasG12D mutant mice.
Notably, levels of Fshr mRNA were reduced in the ovaries of immature
(untreated) Kras mutant mice indicating that constitutively active
KRAS impairs the expression of this gene at an early stage of granulosa cell
differentiation.
To determine whether the decreased levels of Fshr and Lhcgr mRNAs were the direct effect of mutant KrasG12D expression, we isolated undifferentiated granulosa cells from immature LSL-KrasG12D mice and cultured them in serum-free medium followed by infection with an adenoviral vector expressing Cre recombinase driven by the CMV promoter (Ad-CMV-Cre). In control cells, addition of FSH to the medium upregulated Fshr and induced the expression of Lhcgr mRNAs. However, the Fshr mRNA level decreased in the granulosa cells expressing KrasG12D, and the inductive effect of FSH on Lhcgr mRNA was totally abolished (Fig. 4B). This experiment confirmed our observations in vivo (Fig. 4A) and provided direct evidence that KRASG12D reduces Fshr mRNA levels and blocks FSH-mediated induction of luteinizing hormone (LH) receptors in granulosa cells.
KRASG12D activates both RAF1/MAPK and PI3K/AKT pathways in granulosa cells
FSH and LH transiently activate ERK1/2 and PI3K pathways in granulosa cells
(Cottom et al., 2003;
Gonzalez-Robayna et al.,
2000). Recently, FSH has been shown to activate RAS, indicating
that granulosa cells have factors that mediate G-protein receptor coupling to
RAS (Wayne et al., 2007).
Therefore, we analyzed components of the RAF1/MEK1/ERK1/2 and PI3K/AKT
cascades in both control and KrasG12D mutant ovaries.
First, we measured RAS activity by RAS-GTP pull-down assay. Whereas levels of
total RAS did not change throughout the ovulation period, levels of active,
GTP-bound RAS were undetectable in ovaries at postnatal day 23, increased
slightly in response to eCG (48 hours) and then increased markedly (but
transiently) 2 hours after hCG (Fig.
5A). When RAS-GTP was measured by GST-RAF1 and GST-p110
pull-down assays in Kras mutant ovaries, high levels of RAS-GTP were
present compared with control mice (Fig.
5B). These results indicate that KrasG12D
mutant protein interacts with both PI3K and RAF1 in ovaries.
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In comparison to their phosphorylation patterns in wild-type mice, elevated levels of phospho-MEK1/2 and phospho-AKT were observed in ovaries of Kras mutant mice even without hormonal stimulation (Fig. 5C, NT), and were only marginally increased in response to hCG, indicating that KRASG12D exerted stimulatory effects on these pathways (Fig. 5C). By contrast, the levels of phospho-ERK1/2 were undetectable in the same Kras mutant ovaries and increased only marginally after hCG treatment. These data suggested that potent inhibitory factors selectively reduced ERK1/2 phosphorylation.
To determine the cell-specific pattern of phospho-ERK1/2 and phospho-AKT in ovaries, immunofluorescent staining was performed using phospho-specific antibodies. In wild-type ovaries, phospho-ERK1/2 was only detected in the large antral follicles 2 hours after hCG (Fig. 5E and see Fig. S2B in the supplementary material). By contrast, levels of phospho-ERK1/2 were markedly reduced in Kras mutant ovaries treated in the same manner (Fig. 5F). In control ovaries, phospho-AKT was first detected after eCG stimulation in the mural layer of granulosa cells in the large antral follicles, whereas the signal was weak in the cumulus cells (Fig. 5H and see Fig. S1C in the supplementary material). However, within 2 hours of hCG treatment, phospho-AKT was present in all granulosa/cumulus cells (Fig. 5I and see Fig. S1D in the supplementary material). By comparison, the phospho-AKT signal was already high in granulosa cells of the 23-day-old Kras mutant mice before eCG treatment. Phospho-AKT was also detected in some of the abnormal follicle-like structures (Fig. 5J, arrows). The progressive pattern of AKT phosphorylation was not seen in Kras mutant follicles. Rather, all granulosa/cumulus cells were positive for phospho-AKT after eCG treatment alone (Fig. 5K) or eCG/hCG treatment (2 hours) (Fig. 5L).
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Mkp3 is upregulated by KrasG12D in granulosa cells and negatively regulates ERK1/2 activity
To elucidate specific changes in ovarian gene expression associated with
the KrasG12D mutation, microarray analyses were undertaken
using RNA prepared from ovaries of LSL-KrasG12D;Amhr2-Cre
versus LSL-KrasG12D mice at 26 days of age. The microarray
data showed that the Mkp3 (Dusp6) gene was upregulated in
the Kras mutant ovaries, and this was confirmed by RT-PCR
(Fig. 7A). This gene encodes
MAPK phosphatase 3 (MKP3), which is an ERK1/2-specific protein phosphatase
(Camps et al., 2000;
Keyse, 2000;
Li et al., 2007;
Urness et al., 2007;
Woods and Johnson, 2006).
Expression of Mkp3 was induced in granulosa cells both in vivo (2-4
hours) and in vitro (1-2 hours) by hCG and AREG stimulation, respectively
(Fig. 7B,J). In situ
hybridization showed that Mkp3 mRNA is highly expressed in
pre-ovulatory follicles 4 hours after hCG treatment
(Fig. 7C,D), but is
undetectable in 23-day-old ovaries (Fig.
7E,F). Ovaries of LSL-KrasG12D;Amhr2-Cre mice
(23 days old) exhibited elevated expression of Mkp3 mRNA in growing
follicles, as compared with wild type (Fig.
7G,H, arrows).
To provide further evidence that MKP3 is functionally involved in the negative regulation of ERK1/2 activity, Mkp3 mRNA was depleted in cultured granulosa cells by RNAi. Mkp3 siRNA (50 nM) efficiently decreased Mkp3 mRNA in unstimulated cells or those exposed to AREG, the most potent stimulator of ERK1/2 in granulosa cells (Fig. 7I). AREG induced rapid but transient phosphorylation of ERK1/2 in control granulosa cells. However, in cells treated with Mkp3 siRNA, the dephosphorylation of ERK1/2 was significantly delayed (Fig. 7J,K). Lastly, ERK1/2 activity is required for the induction of Mkp3, because the MEK1/2 inhibitor PD98059 blocked the AREG-induced Mkp3 expression in cultured granulosa cells (Fig. 7M,N).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Based on
in vitro studies, the ERK1/2 pathway is presumed to be essential for COC
expansion and meiotic resumption of oocytes
(Diaz et al., 2006;
Fan et al., 2003;
Shimada et al., 2006;
Su et al., 2002). FSH-mediated
stimulation of the PI3K pathway is also presumed to impact follicular
development (Alam et al., 2004;
Alliston et al., 2000;
Park et al., 2005;
Richards et al., 2002;
Zeleznik et al., 2003). By
analyzing the effects of expressing a constitutively active form of KRAS
(KRASG12D) selectively in granulosa cells of two mouse models, we
report the first detailed investigation of the consequences of mutant KRAS
activation during mammalian follicle development and ovulation. In both
models, we observed two distinct follicular phenotypes, suggesting that the
impact of KrasG12D is dependent on the stage of granulosa
cell differentiation. If recombination occurred at early stages of follicle
development, many abnormal follicle-like structures were observed, whereas if
recombination occurred later, events associated with ovulation were impaired
(Fig. 8). These changes in
ovarian function caused the KrasG12D mutant mice to be
subfertile and to exhibit premature ovarian failure. Because
Amhr2-Cre is expressed earlier in follicular development than
Cyp19-Cre (our unpublished observations), the
KrasG12D;Amhr2-Cre mice exhibited more-severe ovulation
detects. The expression of the Cyp19-Cre transgene is highly specific
for granulosa cells. Although Cyp19-Cre is expressed in
follicles at slightly later stages of growth than Amhr2-Cre, the
KrasG12D;Cyp19-Cre and
KrasG12D;Amhr2-Cre phenotypes are very similar, verifying
that mutant KRASG12D impacts granulosa cell function in a
stage-specific manner.
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;
Sarkisian et al., 2007;
Shaw et al., 2007;
Tuveson et al., 2004), we
anticipated that expression of KRASG12D in granulosa cells might
lead to enhanced proliferation and oncogenic transformation of these cells.
Oncogenic transformation was not observed and alterations in proliferation
were critically dependent on when recombination and expression of
KRASG12D were initiated. In the abnormal follicle-like structures,
no evidence for proliferation was observed. However, in the large antral
follicles, proliferation was increased, indicating that the effects of mutant
KRAS were dependent on the stage of granulosa cell differentiation.
Furthermore, expression of KRASG12D led to impaired apoptosis of
granulosa cells in the abnormal follicle-like structures, whose growth
appeared to be self-limiting. Expression of KRASG12D also
profoundly altered the fate and differentiation of granulosa cells.
Specifically, expression of mutant KRASG12D in granulosa cells of
small growing follicles completely disrupted normal follicular development and
granulosa cell differentiation, as known markers of granulosa cell function
(Fshr and Nr5a2)
(Richards, 1994) were not
detected. This altered cell fate led to a novel and unexpected ovarian
phenotype, with follicle-like structures that were devoid of mitotic,
apoptotic and differentiated cells (Fig.
8). The behavior of the granulosa cells in these abnormal
follicle-like structures appears to be similar to the premature senescence
observed in primary cells in culture expressing mutant forms of HRAS
(Lin et al., 1998).
Follicles in which granulosa cells escaped the recombination events at an
early stage of development continued to grow to the antral stage. However,
follicles with granulosa cells expressing KRASG12D at this stage
also exhibited impaired function. Specifically, most antral follicles failed
to ovulate even if exposed to exogenous hormones. Ovulation failure was
associated with impairments in expansion of cumulus cells, in meiotic
maturation of the oocytes and in expression of ovulation-related genes. This
phenotype is similar to that of the mutant mouse model with an EGFR signaling
defect (Hsieh et al., 2007).
The altered response of KRASG12D-expressing granulosa cells to
LH/hCG appears to be related to low levels of Fshr and the inability
of FSH to induce expression of Lhcgr mRNA, and therefore to the loss
of the crucial LH-ERK1/2 signaling pathways. This conclusion is supported by
the reduced expression of specific genes known to be essential for COC
expansion and ovulation (Richards,
2005), including Ptgs2, Has2 and Tnfaip6.
|
), as well as the studies of others who have shown that
KRASG12D selectively activates RAF1 and/or PI3K in a cell- and
context-specific manner (Campbell et al.,
2007; Cespedes et al.,
2006; Gupta et al.,
2007; Tuveson et al.,
2004). However, KRASG12D-mediated ERK1/2
phosphorylation is transient and becomes markedly reduced in the mutant
granulosa cells in vivo and in culture. This transient activation of ERK1/2 is
mediated, at least in part, by the upregulation of MKP3, a specific ERK1/2
phosphatase (Camps et al.,
2000; Keyse, 2000;
Li et al., 2007;
Urness et al., 2007;
Woods and Johnson, 2006).
Mkp3 mRNA was rapidly induced in granulosa cells of wild-type mice in
response to hCG and was elevated in KrasG12D mutant
ovaries (Fig. 7) and cultured
granulosa cells expressing KRASG12D (data not shown). Because
induction of Mkp3 mRNA by AREG in granulosa cells was blocked by the
MEK1/2 inhibitor PD98059, these results reinforce the notion that ERK1/2
induces expression of this negative-regulatory factor in granulosa cells.
Conversely, reducing Mkp3 expression by a siRNA approach prolonged
the presence of phospho-ERK1/2 in response to AREG by up to 60 minutes.
Collectively, these results provide evidence that MKP3 is regulated in murine
ovarian granulosa cells and controls the duration of ERK1/2
phosphorylation.
Although Mkp3 is induced in granulosa cells of preovulatory
follicles and is initially elevated in these cells in the
KrasG12D mutant ovaries, Mkp3 mRNA was not
expressed in the granulosa cells contained within the abnormal follicle-like
structures. Thus, the absence of phospho-ERK1/2 in these cells also indicates
that other potent mechanisms impact and reduce ERK1/2 signaling in these
mutant cells. For example, RAS can mediate the epigenetic silencing of genes
via its ability to induce CpG methylation at promoter regions of certain genes
(Gazin et al., 2007).
Moreover, the mediators of RAS epigenetic silencing include Mapk1
(Erk2), Pdpk1 (Pdk1) and Dnmt1
(Gazin et al., 2007). Thus, it
is tempting to speculate that the cells within the abnormal follicle-like
structures have undergone specific epigenetic changes to prevent their
proliferation, apoptosis and differentiation.
In contrast to ERK1/2, phosphorylation of AKT in granulosa cells of growing
and large antral follicles was enhanced by the presence of KRASG12D
in vivo and in KRASG12D-expressing granulosa cells in culture.
Since our GST pull-down assays showed that KRASG12D interacts
directly with the p110 subunit of PI3K as previously reported
(Rodriguez-Viciana et al.,
1994; Rodriguez-Viciana et
al., 1996), it is likely that KRASG12D stimulates the
PI3K pathway directly, leading to the prolonged activation of AKT in granulosa
cells. KRASG12D also impairs the expression of FOXO1 that may be
mediated by prolonged activation of AKT. Since FOXO1 has been shown to impair
granulosa cell differentiation (Park et
al., 2005; Rudd et al.,
2007), one might have predicted that the mutant cells would
exhibit increased responsiveness to FSH, which is not the case. Rather, the
PI3K pathway appears to regulate additional functions in granulosa cells.
Because Pdpk1 is a factor implicated in RAS-mediated epigenetic gene
silencing (Gazin et al.,
2007), it is possible that the PI3K pathway is crucial for
dictating the fate of granulosa cells in small follicles.
In summary, transient activation of RAS and the phosphorylation of downstream targets, such as the RAF1/MEK1/ERK1/2 and PI3K/AKT cascades, appear to be crucial for mediating appropriate responses of granulosa cells to the gonadotropic hormones FSH and LH, leading to progressive follicular development and ovulation. Conversely, persistent expression of a constitutively active form of KRAS (KRASG12D) impairs ovulation and the expression of ovulation-related genes. Moreover, if expressed at an early stage in follicle development, KRASG12D dramatically alters granulosa cell fate by precluding granulosa cell differentiation, proliferation and apoptosis, thus impairing granulosa cell responses to gonadotropins and leading to premature ovarian failure (Fig. 8). This marked divergence in granulosa cell function suggests that the potent epigenetic silencing of the promoters of specific genes might provide the basis of how activation of RAS alone can cause quiescence/senescence, rather than transformation, of these cells. These results also provide novel evidence that granulosa cells in vivo possess mechanisms that make them extremely impervious to tumorous transformation and that instead lead to premature ovarian failure.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/12/2127/DC1
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ACKNOWLEDGMENTS |
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