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Departments of Molecular Cell Biology (K.T., A.D., A.A.) and Biological Regulation (Z.Y., R.S.), Weizmann Institute of Science, Rehovot 71600, Israel; and Department of Obstetrics and Gynecology (F.K.), Fukui Medical University, Fukui 910-1193, Japan
Address all correspondence and requests for reprints to: Abraham Amsterdam, Ph.D., Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: abraham.amsterdam{at}weizmann.ac.il.
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Introduction |
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The cAMP and e steroidogenic responses to gonadotropin stimulation become refractory in the presence of high doses of hormone and prolonged stimulation by a mechanism that is not completely understood (14, 23, 28). Moreover, even during a short exposure to the hormone, the steroidogenic response to the hormone is much lower than direct challenging of the cells with cAMP or forskolin (FK), which nonspecifically activates the adenylate cyclase (22, 23).
Activation of alternative pathways by the gonadotropin receptors was described in the last decade (for review see Ref.19). These include calcium ion mobilization, activation of the phosphoinositol pathway, and chloride ion pump activation (29). They were demonstrated to occur in gonadotropin-responsive cells; however, activation of the alternative pathways by gonadotropin was never implicated with desensitization to the hormone-stimulated steroidogenesis (30, 31, 32, 33, 34).
We have demonstrated that in immortalized steroidogenic granulosa cells, gonadotropin-stimulated steroidogenesis is attenuated to a large extent through the activation of MAPK and the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1 and -2) (35). Moreover, treatment of the cells with potent and specific MAPK inhibitions increases the intracellular level of the steroidogenic acute regulatory protein (StAR) without affecting the steroidogenic enzymes. As StAR is a key regulatory protein in the rapid modulation of steroidogenesis (36), our recent findings may explain a novel mechanism for desensitization to gonadotropin/cAMP stimulation.
StAR expression is regulated by the steroidogenic factor 1 (SF-1/Ad4BP), a transcription factor whose activity is believed to be exerted by phosphorylation of SF-1 after binding to the promoter of the StAR gene (37, 38). On the other hand, SF-1 activity is blocked by DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenita, critical region on the X chromosome gene-1), another transcription factor whose expression is mostly restricted to steroidogenic tissues, such as adrenal cortex, ovary, and Leydig cells (39, 40). In addition, DAX-1 is expressed in testicular Sertoli cells, pituitary gonadotropes, and the ventromedial hypothalamic nucleus (39, 40). Mutations in the DAX-1 gene cause adrenal hyperplasia congenita, which is usually associated with hypogonadotropic hypogonadism (41).
In recent studies it was suggested that in addition to the direct interaction of DAX-1 with SF-1, DAX-1 could affect the steroidogenic cascade at multiple levels by inhibition of the expression of enzymes involved in different steps of steroidogenesis (42). Comparative localization of DAX-1 and SF-1 during development of the hypothalamic-pituitary gonadal axis suggested their closely related and distinct functions. Moreover, it seems likely that the ratio between these two transcription factors will determine whether their combined effect will either enhance or inhibit steroidogenesis in different steroidogenic cell types and at different stages of development.
Human granulosa cells are subject to extensive research due to their crucial role in successful reproduction. Surrounding and nursing the oocyte, they support its maturation, and in addition, the large amount of steroid hormones secreted by these cells ensures a congenial environment for the implantation and development of the early embryo (for reviews, see Refs.43, 44). Numerous reports on granulosa cells obtained from women participating in in vitro fertilization (IVF) programs confirm that these cells become highly steroidogenic due to their previous overstimulation with gonadotropic hormones (46, 47, 48). Freshly prepared cells fail to show a consistent response to human chorionic gonadotropin (hCG)/LH, and no response to FSH was observed in short-term cultures (49, 50, 51). However, prolonged culture of the cells in gonadotropin-free medium was demonstrated to reestablish responsiveness to FSH stimulation.
Activation of MAPK by GnRH (52), ATP (53), and hCG (53) in primary cultures of human granulosa cells obtained from an IVF program was recently demonstrated, but its possible effect on steroidogenesis was not completely explored. In the present paper we demonstrate for the first time that attenuation of steroidogenesis by activation of MAPK exerted by FSH, LH, and hCG involves down-regulation of StAR expression. Moreover, modulation of DAX-1 and SF-1 intracellular levels may participate in MAPK regulation of steroidogenesis in these cells.
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Materials and Methods |
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Mouse monoclonal anti diphospho-ERK (anti-active ERK/MAPK) antibodies (anti-DP-ERK Ab) and anti-general ERK antibodies (anti-G-ERK Ab) were obtained from Sigma Israel Chemical Ltd. (Rehovot, Israel). Antibodies to progesterone were provided by Dr. F. Kohen (Weizmann Institute of Science, Rehovot, Israel). Antihuman StAR antibodies and antihuman cytochrome P450scc antibodies were provided by Dr. J. F. Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA). Anti-SF-1 antibodies were provided by Dr. K. Morohashi (National Institute for Basic Biology, Okazaki, Japan). Antiadrenodoxin (anti-ADX) antibodies were provided by Dr. W. L. Miller (University of California, San Francisco, CA). Anti-DAX-1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Both goat antimouse and antirabbit IgG coupled to horseradish peroxidase were obtained from Biomakor (Rehovot, Israel). Antirabbit immunoglobulin G (IgG) labeled with fluorescein isothiocyanate was purchased from Sigma Israel Chemical Ltd.
Reagents
Human (h) FSH, hLH, and hCG were provided by the NIH and Dr. A. F. Parlow. FK (a potent activator of adenylate cyclase) and 8-bromo-cAMP (8-Br-cAMP) were purchased from Sigma Israel Chemical Ltd. PD98059 and UO126 were purchased from Calbiochem (San Diego, CA). Moloney murine leukemia virus reverse transcriptase, Taq polymerase, and deoxy-NTPs were obtained from Promega Corp. (Madison, WI). An expression vector for human StAR (StAR sport) was provided by Dr. J. F. Strauss III.
Cell cultures
Primary human granulosa cells were obtained from women undergoing IVF at Sheba Medical Center (Tel-Hashomer, Israel). Patients received a GnRH analog in combination with FSH or human menopausal gonadotropin, followed by administration of hCG. Granulosa cells were isolated as described previously (54) from aspirated follicular fluid after ovum retrieval. The follicular fluid was centrifuged at 300 x g for 5 min to separate granulosa cells from red blood cells. The resulting pellet was resuspended and cultured in Nunc tissue culture dishes (35 mm) with DMEM/Ham’s F-12 (1:1) containing 10% fetal calf serum (FCS), penicillin (100 IU/ml), and streptomycin (100 µg/ml), for 48 h. Cells were maintained at 37 C in 5% CO2 in humidified incubators. For stimulation, cells were washed on d 7 of culture with PBS and incubated in serum-free medium or serum-supplemented medium (7.5%) containing the desired stimulant in saturating concentrations. The primary culture cells contained at least 93% granulosa cells, as shown by StAR immunofluorescent staining (not shown). The rest contained mainly fibroblasts and monocytes.
Primary rat granulosa cells of preantral follicles were obtained from 23-d-old female Wistar-derived rats that were treated with diethylstilbestrol (1.5 mg/rat) for 3 consecutive d (55). Primary granulosa cells of the preovulatory follicle were obtained from 25-d-old female rats subsequent to 48 h of injection of 15 IU pregnant mare’s serum gonadotropin (56). Cells were plated in DMEM/F-12 (1:1) containing 5% FCS on plastic tissue culture dishes. Cells were maintained at 37 C in 5% CO2 in humidified incubators.
Western blot analysis
Western blot analysis was carried out essentially as described previously (35, 57). Briefly primary cultures at the end of incubation with the appropriate stimulant or with no stimulation as indicated in each experiment were rinsed with ice-cold PBS and once with buffer A [50 mM ß-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM sodium vanadate] and were subsequently harvested in buffer A plus proteinase inhibitors (35). Cell lysates were centrifuged at 20,000 x g for 20 min. The supernatant was assayed for protein content and subjected to Western blot analysis to detect DP-ERK and G-ERK. Alternatively cells were rinsed with cold PBS and harvested in lysis buffer containing 50 mM HEPES (pH 7.2), 150 mM NaF, 30 mM sodium pyrophosphate, 1 mM orthovanadate, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 5 µg/ml aprotinin and subjected to Western blot analysis to detect P450scc, DAX-1, SF-1, StAR, and ADX. Samples containing equal amounts of protein (40 µg) were separated by 10% (to detect DP-ERK, G-ERK, P450scc, DAX-1, and SF-1), 12% (to detect StAR), or 15% (to detect ADX) acrylamide SDS-PAGE. The relevant proteins were detected on blots using their specific antibodies.
Determination of progesterone and protein levels
Progesterone was determined by RIA at the end of the stimulation (23, 58). Protein was quantified by the Bradford method (59).
RNA extraction and RT-PCR
Total RNA was isolated using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the instructions of the manufacturer. RNA pellets were ethanol-precipitated, washed, and resuspended in sterile ribonuclease-free water. The quality of the RNA was assessed by fractionating it on 1% agarose gel and observing the presence of the typical 28S and 18S rRNA under UV light. Total RNA (5 µg) was reverse transcribed to first strand cDNA with 200 U Moloney murine leukemia virus reverse transcriptase in the presence of 0.5 mM deoxy-NTPs and 25 µg/ml oligo(deoxythymidine) in a total volume of 20 µl for 60 min at 37 C. The amplification of human StAR cDNA was carried out as previously described (24). Briefly, the sense primer (5'-AAGAACTTGTGGACCGCATGG-3') was from bases 474–493, and the antisense primer (5'-GGTGGTTGGCGAACTCTATCT-3') spanned the region 860–880 bp of the human StAR cDNA. The expected size of the amplified fragment was 408 bp. PCR was performed in a total volume of 50 µl with 1 µl reverse transcribed product, 2.5 U Taq polymerase, 50 µM deoxy-NTP, 0.6 µM primers in 1x PCR buffer [10 mM Tris (pH 9), 50 mM KCl, 1 mM MgCl2, and Triton X-100]. Amplification was performed using one cycle at 95 C for 2 min, followed by 27 cycles at 94 C for 30 sec, 50 C for 30 sec, 72 C for 30 sec, and then final extension at 72 C for 5 min (GeneAmp, PerkinElmer, Norwalk, CT). Five microliters of the PCR product were analyzed on 1% agarose gel containing 0.5 µg/ml ethidium bromide using TAE buffer (24).
Quantitative RT-PCR
Real time-quantitative PCR was used to determine the amounts of rat StAR mRNA. This PCR method monitors the progress of the PCR via detection of fluorescent signal released by the action of Taq polymerase from a specific probe that contains both fluorescent dye and quencher. In the present experiments the amount of specific amplicon present was related to ribosomal 18S and subsequently to an internal control. RNA was extracted from primary cultures of preovulatory rat granulosa cells as described above for human granulosa cells. RNA was subjected to deoxyribonuclease (DNase) treatment using 1 U DNase I (amp grade)/µg RNA in DNase reaction buffer for 15 min at room temperature (Invitrogen, San Diego, CA) to remove genomic DNA contamination. RNA was reverse transcribed to first cDNA as described for human granulosa cell RNA. Real-time PCR was repeated three times for each sample using StAR primers [5'-ACATTCAAGCTGTGT GC-3'(sense) and 5'-CTGGTCACT GTAGAGTGTT-3' (antisense)]. Real-time PCR was also repeated three times for a housekeeping gene, ribosome 18S. The primers used were as follows: sense, 5'-AAACGGCTACCACAT CCAAG-3'; and antisense, 5-CCTCCAATGGATCCTC-GTTA-3'. The LightCycler Fast Start DNA Master SYBR Green I kit (catalogue no. 3003230, Roche, Mannheim, Germany) was used in a LightCycler System (Roche). Data are expressed as the amount of specific PCR products (StAR) in stimulated cells over that in control (nonstimulated cells) after normalization according to the housekeeping gene product 18S (which did not show any significant difference in any of the treatments).
Microscopy
Cells were cultured on 24 x 24-mm coverglasses placed in 35-mm plastic tissue culture dishes. Cells were fixed with 3% paraformaldehyde subsequent to 24-h incubation at 37 C with the appropriate stimulants and were visualized in a Zeiss florescent microscope (Carl Zeiss, New York, NY) after incubation with a 1:200 dilution of antiserum to human StAR and goat antirabbit antibodies conjugated to fluorescein (57, 60). No mitochondrial straining could be detected using nonimmune serum.
Statistical analysis
All experiments were repeated at least three times with granulosa cells obtained from separate groups of rats or granulosa cells obtained from separate groups of women. All values were expressed as the mean ± SD (n = 3). Data were subjected to ANOVA. Means were contrasted using post hoc Tukey’s multiple comparison test. P < 0.05 was considered significant.
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Results |
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When human granulosa cells were incubated with hLH (3 IU/ml) or FK (50 µM) for 24 h at 37 C, there was a clear increase in progesterone secretion into the medium. An increase in progesterone in LH-stimulated cells was clearly evident 4 h after the onset of stimulation (717% above the control) and continued to increase sharply 8 h after the onset of stimulation (833% above the control; Fig. 1
). This increase in progesterone was significantly augmented in the presence of a MAPK inhibitor, PD98059 (Fig. 2). There was also moderate, but significant, elevation of progesterone production in PD98059-treated cells compared with that in control culture when examined after 20 min, 3 h, and 24 h of stimulation (Fig. 2, B and C). In parallel to the examination of progesterone production we analyzed the contents of StAR and the cytochrome P450scc in cell lysates by Western blots (Fig. 2). A clear elevation of 30-kDa StAR in the cultured cells was evident when comparing the intracellular levels in LH or FK to those after similar treatment in the presence of PD98059. The StAR level in control cells was extremely low, probably due to the 7-d incubation in monolayer in hormone-free medium to release the cells from desensitization to gonadotropin stimulation that took place in the IVF patients before oocytes and granulosa cell retrieval (54). In contrast, a pronounced elevation of StAR was evident upon incubation of the cells with PD98059 or UO126. A higher level of StAR was evident upon LH or FK stimulation, and it was further elevated in the presence of PD98059. The modulation of the 30-kDa protein accompanied modulation of the 37-kDa StAR precursors. Interestingly, although at 3 h of stimulation the intracellular levels of 37-kDa protein were about equal in stimulated cells, the intracellular levels of 37-kDa protein increased dramatically after 24 h of stimulation. In contrast to the modulation of StAR, intracellular levels of cytochrome P450scc were high without significant changes among the different treatments (Fig. 2B).
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). Attenuation of ERK1 and ERK2 phosphorylation was evident upon incubation of the cells with 25 µM PD98059 for 20 min at 37 C. A dramatic increase in phosphorylation of ERK1 and ERK2 was detected after 20 min of incubation of the cells with either LH or FK, which was clearly suppressed in the presence of the MAPK inhibitor PD98059 (Fig. 3). There was no change in the intracellular levels of ERK1/2 during the 20-min stimulation, as determined using general antibodies to ERK1/2 (61).
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The human granulosa cells analyzed in this study were obtained from IVF patients and therefore were highly luteinized in their final stage of differentiation (54). To analyze granulosa cells isolated from different stages of differentiation, we analyzed preantral and preovulatory rat granulosa cells for their gonadotropic response in terms of MAPK activation and regulation of steroidogenesis. In preantral (Fig. 5) as well as preovulatory (Fig. 6) cells phosphorylations of ERK1/2 were already evident in nonstimulated cells. Also, phosphorylation of the recently discovered ERK1b (62) was evident in both primary cultures. However, a marked decrease upon incubation of the cells with PD98059 was evident mainly in ERK1/2 and not in ERK1b (Fig. 5). Upon stimulation with LH, FSH, or FK, preantral cells responded to FSH or FK (but not to LH), and preovulatory cells responded to both LH and FSH as well as to FK (not shown) with a sharp increase in ERK1 and ERK2 phosphorylation, which markedly decreased in the presence of PD98059. No response to LH stimulation in preantral cells was expected, because preantral cells, in contrast to preovulatory cells, do not yet express the LHR (for review, see Refs.3 ,44 , and45) and therefore can serve as a reliable negative control to gonadotropin-induced ERK1 and ERK2 phosphorylation. No dramatic changes in ERK1b phosphorylation were noticed in any of these conditions in preantral or preovulatory cells. No change in the general amount of ERK1/2 was observed during the 20 min of stimulation with gonadotropin/cAMP. Although no significant change in progesterone secretion in rat preovulatory granulosa cells was evident upon incubation of control cultures with PD98059 for 24 h, a clear increase in progesterone production was evident in FSH- and LH-stimulated cells, with a significant further increase in progesterone secretion in the presence of PD98059 (Fig. 6). A significant increase in intracellular levels of StAR was observed in the presence of PD98059 compared with the same treatment devoid of MAPK inhibitor. No significant change in the intracellular levels of the P450scc enzyme system were recorded, as revealed by Western blot of ADX, which is an intrinsic component of the P450scc enzyme system, in control cells and FSH-stimulated preovulatory cells, whereas some elevation in ADX content was evident in the presence of LH plus PD98059 (Fig. 6).
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. In all conditions, i.e. in control or LH-, FSH-, hCG-, FK-, or 8-Br-cAMP-stimulated cells, StAR intracellular levels were significantly elevated in the presence of PD98059 compared with the same treatments devoid of MAPK inhibitor. There was a parallel modulation of 32-kDa precursor protein in all treatments; unfortunately, the large excess of 30-kDa protein did not allow clear visualization of the 32- and 37-kDa proteins in gel electrophoresis and Western blot. To determine whether a specific inhibitor of MAPK elevates mRNA coding for rat StAR, a quantitative real-time PCR was performed after isolation of RNA from cultured preovulatory rat granulosa cells incubated for 24 h at 37 C in the absence or presence of PD98059, UO 126, LH, LH plus PD98059, and LH plus UO 126 (Fig. 8). PD98059, UO 126, and LH elevated mRNA levels of StAR only moderately (140–150% above control). LH plus PD98059 elevated mRNA to StAR by 172%, and LH plus UO 126 elevated it by 280% compared with parallel treatments without the MAPK inhibitors.
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To verify whether PD98059 leads to accumulation of the steroidogenic protein in mitochondria, preovulatory human granulosa cells were incubated with the MAPK inhibitor in the absence or the presence of LH. As revealed by immunocytochemistry using specific antibodies to StAR (Fig. 9), PD98059 alone led to accumulation of StAR, whereas mitochondrial StAR was further accumulated in the presence of LH plus PD98059 compared with cells stimulated by LH alone, suggesting a de novo synthesis of StAR in the presence of MAPK inhibitor as demonstrated for both the protein and mRNA in human and rat granulosa cells by RT-PCR Western blot and immunocytochemistry.
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SF-1 is a critical transcriptional factor in the induction of steroidogenesis and is believed to bind to and activate StAR expression by binding in a phosphorylated form to the promoter of the StAR gene. DAX-1 was reported to function as a transcriptional suppressor of SF-1 in steroidogenic cells other than human granulosa cells. Therefore, we analyzed in human granulosa cells the intracellular levels of SF-1 and DAX-1 upon treatment of cells with PD98059, LH, and LH plus PD98059. Using specific antibodies to DAX-1, the 53-kDa protein could be detected in all treatments. Intracellular levels of DAX-1 were essentially the same in all treatments after 3 h of stimulation. In contrast, intracellular levels of DAX-1 were significantly reduced after 24 h of stimulation with PD98059 or UO126 compared with either control or LH-stimulated cells (Fig. 10A). Using cell lysates and SF-1 antibodies only a weak band at 53 kDa could be detected in nonstimulated cells after 3 h of incubation (Fig. 10B). This band seemed to disappear after 24 h of stimulation with LH and was clearly intensified in LH-stimulated cells in the presence of UO126 or PD98059. Some increase in SF-1 was also noted in control cells incubated with UO126 or PD98059 alone for 24 h (not shown for PD98059).
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). In control cells DAX-1 was located in the nucleus, but was absent in the nucleoli. In cells treated with UO126 for 24 h, a clear decrease in nuclear staining was evident. Treatment of cells with LH for 24 h clearly augmented nuclear staining, whereas UO126 lead to a decrease in nuclear staining. The tiny fluorescent speckles of fluorescent occasionally appearing in the cytoplasm may indicate sites of DAX-1 synthesis on polyribosomes. No such speckles were visible using nonimmune serum for staining the cells (Fig. 11).
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). This is demonstrated by both RT-PCR and quantitative RT-PCR using mRNA of human granulosa cells and rat granulosa cells, respectively, and by Western blot and immunocytochemistry using rat preantral and rat and human preovulatory cells. We have recently demonstrated similar results in immortalized rat granulosa cells expressing either LH/CG or FSH receptors (35). Moreover, we could show in the immortalized granulosa cells that the main signal transmitted by MAPK activation is mediated by protein kinase A (PKA) similarly to the activation of steroidogenesis. These conclusions could be drawn as the result of marked attenuation of ERK1/2 phosphorylation using a specific inhibitor to PKA, H-89 (35). In complementary experiments we demonstrated that transfection of immortalized steroidogenic granulosa cells with a plasmid coding for protein kinase inhibitor (63), which is known to interact and to block PKA activity, also depresses to a large extent LH- and FSH-induced MAPK activation (35).
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Recently, it was demonstrated that MAPK can be activated in human granulosa lutein cells not only by the hCG/cAMP cascade, but also by GnRH (52) and ATP (53), which demonstrate a modest inhibitory effect on progesterone production in these cells. These observations are in line with the idea that activation of the MAPK cascade attenuates steroidogenesis via suppression of StAR expression (Fig. 12
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In another recent study it was demonstrated that in human granulosa cells obtained from an IVF program there was no change in LH/CG-stimulated progesterone production in the presence of MAPK inhibitors (64). These studies were performed in granulosa-lutein cells only 3 d after the onset of culture, which probably was not sufficient to completely release them from refractoriness to gonadotropin administration before retrieval of the oocyte and collection of granulosa cells in the regimen of IVF treatment (54), as evident from the high basal level of progesterone secretion and the very modest increase in gonadotropin-stimulated progesterone production. Therefore, it is most likely that StAR was not a limiting factor in that particular culture. In contrast, our studies were performed after 7 d in culture, which was previously demonstrated (54) to dramatically reduce basal progesterone and StAR intracellular levels that can be clearly up-regulated by LH/CG, leading to a pronounced stimulation of StAR expression, which was shown to be modulated by MAPK activation in the present work.
It was demonstrated earlier that MAPK activation led to an increase in steroidogenesis in Y-1 adrenocortical cells. It was suggested that this up-regulation of StAR occurs via the induction of phosphorylation of SF-1 that in its phosphorylated form binds and trans-activates the StAR gene leading to the de novo synthesis of the StAR protein and enhanced steroidogenesis. In contrast to granulosa cells that express DAX-1 (Ref.65 and our present observations), which was reported to function as a transcription suppressor of SF-1, Y-1 cells do not express DAX-1 (66), and therefore, this transcription factor cannot negate SF-1 activity. As DAX-1 is reported here for the first time to be expressed in human granulosa cells, and its expression is enhanced by LH and reduced by MAPK inhibitors, it seems likely that the elevation of DAX-1 may exert a dominant effect on StAR expression and attenuation of steroidogenesis in granulosa cells, in contrast to Y-1 cells, which lack this transcription factor. Moreover, gonadotropin signaling, as demonstrated in the present work, led to a reduction of intracellular levels of SF-1, which may abrogate the stimulatory effect of this transcription factor upon prolonged gonadotropin action. The protein phosphatase inhibitor cantharidin has recently shown to inhibit StAR expression and steroidogenesis in rat preovulatory follicles (67). This suggests that phosphatase activity is stimulatory for steroidogenesis by neutralizing the MAPK activity cascade, which is in line with and complementary to our observations.
DAX-1 is probably not the only factor that may affect StAR transcription. There is increasing evidence that in addition to the orphan nuclear receptor SF-1, which modulates cAMP-dependent responsiveness of the rat StAR promoter in rat luteal cells, SF-1 and sterol regulatory element-binding protein-1 can synergistically activate the high density lipoprotein receptor gene, demonstrating the combined action of two regulatory pathways to provide cholesterol as a substrate for steroidogenesis (68). Recent studies suggest the cooperation between sterol regulatory element-binding protein-1a and nuclear factor-Y in regulation of the StAR promoter, suggesting that the StAR gene is responsive to selective combinations of multiple regulatory pathways to enhance or attenuate StAR gene transcription (68). The contribution of MAPK activation to the regulation of intracellular levels of StAR may involve a phosphorylation/dephosphorylation cascade of reactions that lead to the regulation of genes whose products may serve as coactivators or corepressors of the StAR gene. We exclude the possibility that MAPK can directly phosphorylate StAR, because we obtained negative results when such a possibility was examined in vitro using highly purified StAR (Seger, R., and A. Amsterdam, unpublished observations).
DAX-1 was not phosphorylated by the MAPK/ERK system either in vivo or in vitro. Nevertheless, analysis of the amino acid sequence of DAX-1 reveals a potential site for phosphorylation on the KSP motif by ERK or on the RRRET motif by PKA and ribosomal S6 protein kinase, which are downstream of ERK (69). Whether such phosphorylation will activate DAX-1 is not yet clear. However, it should be explored in the future whether the 55-kDa band represents a phosphorylation form of DAX-1, and its absence in the presence of MAPK inhibitor could indicate that MAPK phosphorylates DAX-1.
As gonadotropins are able to activate MAPK via the cAMP cascade, this raises the possibility that the cross-talk between gonadotropin/cAMP and the MAPK signaling cascade may play an important role in the mitogenic effect of FSH in folliculogenesis. Therefore, a stronger signal would be expected in gonadotropin/cAMP phosphorylation of ERK1 and -2 in granulosa cells obtained from preantral follicles compared with cells derived from preovulatory follicles. Our observations did not demonstrate greater activation of gonadotropin-induced ERK1 and -2 phosphorylation in primary cultures of rat preantral or preovulatory cells. However, it is well known that rat granulosa cells ceased to divide in vitro even in the presence of estradiol (reviewed in Ref.3) and thus may require as yet unknown cofactors to initiate or preserve their mitogenic activity in vitro. Therefore, we cannot exclude the possibility that FSH stimulation of MAPK contributes to the mitogenic signal of granulosa cell proliferation during follicular development in vivo.
Attenuation of StAR expression by MAPK activation is a novel pathway, which suggests that it may play a significant role in the mechanism of gonadotropin-induced desensitization in primary granulosa cells. Our earlier studies showing that the cAMP response in immortalized granulosa cells is attenuated 20 min after gonadotropin stimulation support this idea, as MAPK can be fully activated even by a low concentration of intracellular cAMP (23). In contrast, this is not the case for cAMP-induced steroidogenesis, which clearly depends on intracellular cAMP (reviewed in Ref.3). Indeed, we have shown that deglycosylated hCG, which can barely activate the gonadotropin-induced cAMP response in granulosa cells (70, 71), can almost fully activate MAPK, resulting in phosphorylation of ERK1 and -2 (35). The elevation of DAX-1 content after prolonged stimulation of human granulosa cells with hCG and its attenuation by MAPK inhibitors may suggest that activation of MAPK by gonadotropins may lead to a prolonged period of desensitization to the gonadotropic hormone via modulation of the intracellular level of DAX-1. Specific growth factors, such as basic fibroblast growth factor, that cooperate with the gonadotropin/cAMP response in modulation of the steroidogenic response may exert their effects via activation of MAPK, which can lead to modulation of steroidogenesis via attenuation of StAR expression (57). This is most likely the case in immature and immortalized granulosa cells. However, the mechanism by which basic fibroblast growth factor enhances steroidogenesis in mature granulosa cells despite possible activation of the MAPK cascade (72, 73) needs further research.
Our data suggest that activation of the MAPK cascade in steroidogenic cells may either increase or decrease steroidogenesis as a consequence of the interaction between SF-1 and DAX-1 (Fig. 12). Moreover, the MAPK cascade may serve as an important target of cross-talk between the gonadotropin/cAMP and tyrosine kinase growth factors in modulation of growth and differentiation of granulosa cells in both humans and rats.
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Acknowledgments |
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A.A. is incumbent of the Joyce and Ben B. Eisenberg Professorial Chair in Molecular Endocrinology and Cancer Research.
Abbreviations: ADX, Adrenodoxin; 8-Br-cAMP, 8-bromo-cAMP; DAX-1, dosage-sensitive sex reversal adrenal hypoplasia congenita, critical region on the X chromosome gene-1; DNase, deoxyribonuclease; DP-, diphospho-; ERK, extracellular-signal regulated kinase; FCS, fetal calf serum; FK, forskolin; FSHR, FSH receptor; G-, general; h, human; hCG, human chorionic gonadotropin; Ig, immunoglobulin; IVF, in vitro fertilization; LHR, LH receptor; P450scc, cytochrome P450 side-chain cleavage enzyme; PKA, protein kinase A; SF-1, steroidogenic factor-1; StAR, steroidogenic acute regulatory protein.
Received June 12, 2002.
Accepted January 27, 2003.
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 D. L. Russell and R. L. Robker Molecular mechanisms of ovulation: co-ordination through the cumulus complex Hum. Reprod. Update, May 1, 2007; 13(3): 289 - 312. [Abstract] [Full Text] [PDF]
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 P. R Manna, Y. Jo, and D. M Stocco Regulation of Leydig cell steroidogenesis by extracellular signal-regulated kinase 1/2: role of protein kinase A and protein kinase C signaling J. Endocrinol., April 1, 2007; 193(1): 53 - 63. [Abstract] [Full Text] [PDF]
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J. Guo, S.-X. Tao, M. Chen, Y.-Q. Shi, Z.-Q. Zhang, Y.-C. Li, X.-S. Zhang, Z.-Y. Hu, and Y.-X. Liu Heat Treatment Induces Liver Receptor Homolog-1 Expression in Monkey and Rat Sertoli Cells Endocrinology, March 1, 2007; 148(3): 1255 - 1265. [Abstract] [Full Text] [PDF]
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T. Miyoshi, F. Otsuka, K. Inagaki, H. Otani, M. Takeda, J. Suzuki, J. Goto, T. Ogura, and H. Makino Differential Regulation of Steroidogenesis by Bone Morphogenetic Proteins in Granulosa Cells: Involvement of Extracellularly Regulated Kinase Signaling and Oocyte Actions in Follicle-Stimulating Hormone-Induced Estrogen Production Endocrinology, January 1, 2007; 148(1): 337 - 345. [Abstract] [Full Text] [PDF]
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Y.-Q. Su, M. Nyegaard, M. T. Overgaard, J. Qiao, and L. C. Giudice Participation of Mitogen-Activated Protein Kinase in Luteinizing Hormone-Induced Differential Regulation of Steroidogenesis and Steroidogenic Gene Expression in Mural and Cumulus Granulosa Cells of Mouse Preovulatory Follicles Biol Reprod, December 1, 2006; 75(6): 859 - 867. [Abstract] [Full Text] [PDF]
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 B. M. Gummow, J. O. Scheys, V. R. Cancelli, and G. D. Hammer Reciprocal Regulation of a Glucocorticoid Receptor-Steroidogenic Factor-1 Transcription Complex on the Dax-1 Promoter by Glucocorticoids and Adrenocorticotropic Hormone in the Adrenal Cortex Mol. Endocrinol., November 1, 2006; 20(11): 2711 - 2723. [Abstract] [Full Text] [PDF]
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 I. Ben-Ami, S. Freimann, L. Armon, A. Dantes, D. Strassburger, S. Friedler, A. Raziel, R. Seger, R. Ron-El, and A. Amsterdam PGE2 up-regulates EGF-like growth factor biosynthesis in human granulosa cells: new insights into the coordination between PGE2 and LH in ovulation Mol. Hum. Reprod., October 1, 2006; 12(10): 593 - 599. [Abstract] [Full Text] [PDF]
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 P. R Manna, S. P Chandrala, Y. Jo, and D. M Stocco cAMP-independent signaling regulates steroidogenesis in mouse Leydig cells in the absence of StAR phosphorylation. J. Mol. Endocrinol., August 1, 2006; 37(1): 81 - 95. [Abstract] [Full Text] [PDF]
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L. A. Helguero, M. Hedengran Faulds, C. Forster, J.-A. Gustafsson, and L.-A. Haldosen DAX-1 Expression Is Regulated during Mammary Epithelial Cell Differentiation Endocrinology, July 1, 2006; 147(7): 3249 - 3259. [Abstract] [Full Text] [PDF]
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N Renlund, Y Jo, I Svechnikova, M Holst, D M Stocco, O Soder, and K Svechnikov Induction of steroidogenesis in immature rat Leydig cells by interleukin-1alpha is dependent on extracellular signal-regulated kinases. J. Mol. Endocrinol., April 1, 2006; 36(2): 327 - 336. [Abstract] [Full Text] [PDF]
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C. A. McDonald, A. C. Millena, S. Reddy, S. Finlay, J. Vizcarra, S. A. Khan, and J. S. Davis Follicle-Stimulating Hormone-Induced Aromatase in Immature Rat Sertoli Cells Requires an Active Phosphatidylinositol 3-Kinase Pathway and Is Inhibited via the Mitogen-Activated Protein Kinase Signaling Pathway Mol. Endocrinol., March 1, 2006; 20(3): 608 - 618. [Abstract] [Full Text] [PDF]
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P. R. Manna, S. P. Chandrala, S. R. King, Y. Jo, R. Counis, I. T. Huhtaniemi, and D. M. Stocco Molecular Mechanisms of Insulin-like Growth Factor-I Mediated Regulation of the Steroidogenic Acute Regulatory Protein in Mouse Leydig Cells Mol. Endocrinol., February 1, 2006; 20(2): 362 - 378. [Abstract] [Full Text] [PDF]
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J. Mutoh, J. Taketoh, K. Okamura, T. Kagawa, T. Ishida, Y. Ishii, and H. Yamada Fetal Pituitary Gonadotropin as an Initial Target of Dioxin in Its Impairment of Cholesterol Transportation and Steroidogenesis in Rats Endocrinology, February 1, 2006; 147(2): 927 - 936. [Abstract] [Full Text] [PDF]
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F.-Q. Yu, C.-S. Han, W. Yang, X. Jin, Z.-Y. Hu, and Y.-X. Liu Activation of the p38 MAPK pathway by follicle-stimulating hormone regulates steroidogenesis in granulosa cells differentially J. Endocrinol., July 1, 2005; 186(1): 85 - 96. [Abstract] [Full Text] [PDF]
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K. Tajima, K. Yoshii, S. Fukuda, M. Orisaka, K. Miyamoto, A. Amsterdam, and F. Kotsuji Luteinizing Hormone-Induced Extracellular-Signal Regulated Kinase Activation Differently Modulates Progesterone and Androstenedione Production in Bovine Theca Cells Endocrinology, July 1, 2005; 146(7): 2903 - 2910. [Abstract] [Full Text] [PDF]
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V. L. Nelson-Degrave, J. K. Wickenheisser, K. L. Hendricks, T. Asano, M. Fujishiro, R. S. Legro, S. R. Kimball, J. F. Strauss III, and J. M. McAllister Alterations in Mitogen-Activated Protein Kinase Kinase and Extracellular Regulated Kinase Signaling in Theca Cells Contribute to Excessive Androgen Production in Polycystic Ovary Syndrome Mol. Endocrinol., February 1, 2005; 19(2): 379 - 390. [Abstract] [Full Text] [PDF]
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R. Sasson, E. Rimon, A. Dantes, T. Cohen, V. Shinder, A. Land-Bracha, and A. Amsterdam Gonadotrophin-induced gene regulation in human granulosa cells obtained from IVF patients. Modulation of steroidogenic genes, cytoskeletal genes and genes coding for apoptotic signalling and protein kinases Mol. Hum. Reprod., May 1, 2004; 10(5): 299 - 311. [Abstract] [Full Text] [PDF]
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