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The ERK Signaling Cascade Inhibits Gonadotropin-stimulated Steroidogenesis

2001; Elsevier BV; Volume: 276; Issue: 17 Linguagem: Inglês

10.1074/jbc.m006852200

1083-351X

Rony Seger, Tamar Hanoch, Revital Rosenberg, Ada Dantes, Wolfgang E. Merz, Jerome F. Strauss, Abraham Amsterdam,

Reproductive Physiology in Livestock

The response of granulosa cells to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the steroidogenic acute regulatory protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis. The response of granulosa cells to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the steroidogenic acute regulatory protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis. The ERK signaling cascade inhibits gonadotropin-stimulated steroidogenesis.Journal of Biological ChemistryVol. 292Issue 21PreviewThis article has been withdrawn by the authors. The authors were recently made aware of an issue in Fig. 4, in which the sequence of treatment groups in the blot of rFSHR-17 cells was reorganized by cut-and-paste to align treatment groups with the companion blot of rLHR-4 cells. This rearrangement was not acknowledged in the original figure legend. Because the original data generated 16 years ago are no longer available, in the interest of maintaining accuracy in the published scientific literature, the authors wish to withdraw this article. Full-Text PDF Open Access Gonadotropic hormones, follicle-stimulating hormone (FSH)1 and luteinizing hormone (LH), which are released from the pituitary, play a crucial role in controlling reproductive function in males and females. The pleotropic effects of gonadotropins are manifested in various cells of the reproductive system including LH and FSH in ovarian granulosa cells, LH in theca interna cells, FSH in testicular Sertoli cells, and LH in Leydig cells (1Sprengel R. Braun T. Nikolics K. Segaloff D.L. Seeburg P.H. Mol Endocrinol.. 1990; 4: 525-530Google Scholar, 2Amsterdam A. Plehn-Dujowich D. Suh B.S. Biol. Reprod... 1992; 46: 513-522Google Scholar, 3Segaloff D.L. Ascoli M. Endocr. Rev... 1993; 14: 324-347Google Scholar). One of the main effects of both LH and FSH on the ovary is the stimulation of the production of estradiol and progesterone, which play important roles in ovarian function and control of the reproductive cycle (reviewed in Ref. 4Amsterdam A. Selvaraj N. Endocr. Rev... 1997; 18: 435-461Google Scholar). The mechanisms involved in the regulation of progesterone production by ovarian granulosa cells have been characterized in detail. Gonadotropins exert their stimulatory activity via interaction with specific seven-transmembrane receptors, the LH receptor and FSH receptor. Upon binding of the gonadotropins, both receptors stimulate the Gs protein, which activates the membrane-associated adenylyl cyclase, causing an elevation of intracellular cAMP (5Cooke B.A. Mol. Cell. Endocrinol... 1999; 151: 25-35Google Scholar). This cyclic nucleotide serves as a second messenger for the up-regulation of the steroidogenic acute regulatory protein (StAR) and the cytochrome P450 (P450scc) enzyme system (reviewed in Refs. 6Stocco D.M. J. Endocrinol... 2000; 164: 247-253Google Scholar and 7Strauss III, J.F. Kallen C.B. Christenson L.K. Watari H. Devoto L. Arakane F. Kiriakidou M. Sugawara T. Recent Prog. Horm. Res... 1999; 54: 369-394Google Scholar). Activation of alternative signaling pathways by the gonadotropin receptors was described in the last decade, including calcium ion mobilization, activation of the phosphoinositol pathway, and stimulation of chloride ion influx (reviewed in Ref. 8Amsterdam A. Gold R.S. Hosokawa K. Yoshida Y. Sasson R. Jung Y. Kotsuji F. Trends Endocrinol... 1999; 10: 255-262Google Scholar). However, these gonadotropin-induced signaling processes were not previously implicated in the modulation of steroidogenesis (5Cooke B.A. Mol. Cell. Endocrinol... 1999; 151: 25-35Google Scholar). Another process that plays an important role in inhibiting gonadotropin-induced steroidogenesis is the desensitization of the gonadotropin receptor (3Segaloff D.L. Ascoli M. Endocr. Rev... 1993; 14: 324-347Google Scholar). G-protein-coupled receptor kinase phosphorylation of the gonadotropin receptors, the adaptor protein arrestin, and massive internalization of the receptors are thought to play a role in the down-regulation of gonadotropin signaling. However, since desensitization precedes the internalization of the gonadotropin receptor (9Amsterdam A. Berkowitz A. Nimrod A. Kohen F. Proc. Natl. Acad. Sci. U. S. A... 1980; 77: 3440-3444Google Scholar), additional mechanisms are likely to participate in the rapid attenuation of gonadotropin signals downstream of the receptors. The extracellular signal-regulated kinases (ERKs) include three kinases (p42ERK2, p44ERK1, p46ERK1b) that belong to the family of the signaling mitogen-activated protein kinases (MAPKs). Upon extracellular stimulation, the ERKs are activated by a network of interacting proteins, which funnel the signals into a multitier kinase cascade (reviewed in Refs. 10Seger R. Krebs E.G. FASEB. J... 1995; 9: 726-735Google Scholar and 11Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res... 1998; 74: 49-139Google Scholar). The activated ERKs in turn regulate additional signaling kinases (e.g. RSK) or can by themselves phosphorylate and activate target regulatory proteins (e.g.Elk1) that govern various cellular processes. Although the ERKs were first implicated in the regulation of proliferation and differentiation, it is presently known that these kinases participate also in the control of cellular morphology, learning and memory in the central nervous system, apoptosis, and carcinogenesis (11Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res... 1998; 74: 49-139Google Scholar). It has previously been shown that ovarian granulosa cell ERK is activated (2–5-fold) in response to LH and FSH (12Cameron M.R. Foster J.S. Bukovsky A. Wimalasena J. Biol. Reprod... 1996; 55: 111-119Google Scholar, 13Das S. Maizels E.T. DeManno D. St. Clair E. Adam S.A. Hunzicker-Dunn M. Endocrinology.. 1996; 137: 967-974Google Scholar). These effects were mimicked by elevation of intracellular cAMP, and the FSH effect was inhibited by inhibitors of PKA, indicating that ERK transduces signals downstream of PKA in gonadotropin-induced granulosa cells. In the present work, we show that gonadotropins induce ERK activation and progesterone production via cAMP in immortalized granulosa cell lines. These cell lines are homogeneous populations, unlike follicular granulosa cells, which represent a heterogeneous population with respect to LH receptor content and the degree of maturation (14Amsterdam A. Rotmensch S. Endocr. Rev... 1987; 8: 309-337Google Scholar). Interestingly, inhibition of ERK activation causes an elevation in gonadotropin-cAMP-induced progesterone production, while activation of ERK inhibits this process. Moreover, the addition of a MEK inhibitor elevated the intracellular content of StAR, which operates downstream of cAMP, suggesting that the inhibitory effect of the ERK on steroidogenesis may be mediated by the reduction in the expression of StAR. Therefore, it is likely that gonadotropin-induced progesterone formation is regulated by PKA, which induces not only the expression of StAR but also a counteracting down-regulating mechanism. These two mechanisms are simultaneously brought into play by the activation of ERK, which reduces StAR expression. Human FSH, human LH, and human chorionic gonadotropin (hCG) were kindly provided by the National Institutes of Health and Dr. Parlow. Deglycosylated hCG was enzymatically prepared as previously described (15Merz W.E. Biochem. Biophys. Res. Commun... 1988; 156: 1271-1278Google Scholar). Mouse monoclonal anti-diphospho-ERK (anti-active ERK/MAPK) antibodies (DP-ERK Ab) and anti-general ERK antibody were obtained from Sigma, Israel (Rehovot, Israel). Anti C-terminal ERK1 antibody (C16) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal antibodies to human StAR were raised in rabbits (16Pollack S.E. Furth E.E. Kallen C.B. Arakane F. Kiriakidou M. Kozarsky K.F. Strauss III, J.F. J. Clin. Endocrinol. Metab... 1997; 82: 4243-4251Google Scholar). Alkaline phosphatase-, horseradish peroxidase-, and flourescein-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). PD98059 and U0126 were purchased from Calbiochem. H89, forskolin, and 8-Br-cAMP were obtained from Sigma. rLHR-4 cell line was established by cotransfection of rat preovulatory granulosa cells with mutated p53 (Val135-p53), Ha-ras genes and plasmid expressing the rat LH/CG receptor (17Suh B.S. Sprengel R. Keren-Tal I. Himmelhoch S. Amsterdam A. J. Cell Biol... 1992; 119: 439-450Google Scholar). The rFSHR-17 cell line was established by immortalization of preovulatory rat granulosa cells via cotransfection of primary cells with SV40 DNA and an HA-rasgene. Cells were transfected with plasmid expressing the rat FSH receptor (18Keren-Tal I. Dantes A. Sprengel R. Amsterdam A. Mol. Cell. Endocrinol... 1993; 95: R1-R10Google Scholar). The cells were maintained in F-12/DMEM medium (1:1) containing 5% fetal calf serum. Subconfluent cultures were serum-starved for 16 h and subsequently incubated for selected time intervals with the indicated agents in the presence or absence of various inhibitors. Following stimulation, cells were washed twice with ice-cold phosphate-buffered saline and once with buffer A (50 mm β-glycerophosphate, pH 7.3, 1.5 mmEGTA, 1 mm EDTA, 1 mm dithiothreitol, and 0.1 mm sodium vanadate (19Yung Y. Yao Z. Hanoch T. Seger R. J. Biol. Chem... 2000; 256: 15799-15808Google Scholar)) and were subsequently harvested in ice-cold buffer A plus proteinase inhibitors (19Yung Y. Yao Z. Hanoch T. Seger R. J. Biol. Chem... 2000; 256: 15799-15808Google Scholar). Cell lysates were centrifuged at 20,000 × g, for 20 min. The supernatant was assayed for protein content and subjected to a Western blot analysis or to immunoprecipitation as below. For the detection of StAR, cells were lysed in radioimmune precipitation buffer (19Yung Y. Yao Z. Hanoch T. Seger R. J. Biol. Chem... 2000; 256: 15799-15808Google Scholar) and subjected to Western blot analysis. The rLHR-4 and rFSHR-17 cells were grown in DMEM supplemented with 10% fetal calf serum up to 70% confluency. The plasmids used were RSV-PKI and RSV-PKImutant (20Day R.N. Walder J.A. Maurer R.A. J. Biol. Chem... 1989; 264: 431-436Google Scholar) (a generous gift from Dr. R. A. Maurer; Oregon Health Sciences University, Portland, OR) and pGFP-ERK2 (21Rubinfeld H. Hanoch T. Seger R. J. Biol. Chem... 1999; 274: 30349-30352Google Scholar). The plasmids were introduced into the two cell types using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instruction. About 15–20% transfection was observed in the two cell lines using a Zeiss fluorescent microscope. After transfection, the rLHR-4 cells were grown in DMEM plus 10% fetal calf serum for 6 h and then starved in DMEM plus 0.1% fetal calf serum for an additional 14 h. The rFSHR-17 cells were grown in DMEM plus 10% fetal calf serum for 20 h. The transfected cells were then stimulated and harvested as above. Cell supernatants, which contained cytosolic proteins were collected, and aliquots from each sample (30 μg) were separated on 10% SDS-polyacrylamide gel electrophoresis followed by Western blotting with the appropriate antibodies. Alternatively, immunoprecipitated proteins were boiled in sample buffer and subjected to SDS-polyacrylamide gel electrophoresis and Western blotting. The blots were developed with alkaline phosphatase or horseradish peroxidase-conjugated anti-mouse or anti-rabbit Fab antibodies. Cell supernatants (200 μg of proteins) were subjected to immunoprecipitation with monoclonal anti-ERK C-terminal antibodies (C16; Santa Cruz Biotechnology) as described above. During the final step of immunoprecipitation, pellets were washed with buffer A, resuspended in 15 μl of buffer A, and incubated (20 min, 30 °C) with 5 μl of 2 mg/ml myelin basic protein (MBP) and 10 μl of 3× reaction mix (30 mm MgCl2, 4.5 mmdithiothreitol, 75 mm β-glycerophosphate, pH 7.3, 0.15 mm Na3VO4, 3.75 mmEGTA, 30 μm calmidazolium, 2.5 mg/ml bovine serum albumin, and 100 μm [γ-32P]ATP (2 cpm/fmol)). The phosphorylation reactions were terminated by the addition of sample buffer and boiling (5 min), and the samples were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography as previously described (19Yung Y. Yao Z. Hanoch T. Seger R. J. Biol. Chem... 2000; 256: 15799-15808Google Scholar). Progesterone secreted into the culture medium was assayed by radioimmunoassay as previously described (22Aharoni D. Meiri I. Atzmon R. Vlodavsky I. Amsterdam A. Curr. Biol... 1997; 7: 43-51Google Scholar). Cells were cultured on 24 × 24-mm cover glasses 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 visualized in a Zeiss fluorescent microscope following incubation with a 1:200 dilution of antiserum to human StAR and goat anti-rabbit antibodies conjugated to fluorescein. For negative controls, cells were incubated with nonimmune rabbit serum followed by the second antibodies. Stimulation of granulosa cells with the gonadotropin LH or FSH induces several cellular processes, including de novosynthesis of steroid hormones. To study the signaling pathways that couple gonadotropin receptors to the regulation of progesterone production, we used two distinct granulosa cell lines expressing either LH/CG or FSH receptors: rLHR-4 and rFSHR-17. The addition of the appropriate gonadotropins to these cells has previously been shown to stimulate cAMP production, activation of PKA, and induction of steroidogenesis (Ref. 18Keren-Tal I. Dantes A. Sprengel R. Amsterdam A. Mol. Cell. Endocrinol... 1993; 95: R1-R10Google Scholar and data not shown). Since the ERK cascade was implicated in the signaling of G protein-coupled receptors (23Naor Z. Benard O. Seger R. Trends Endocrinol. Metab... 2000; 11: 91-99Google Scholar), we first examined whether the ERK cascade is also activated in the rLHR-4 and rFSHR-17 cell lines. Serum-starved rLHR-4 cells were stimulated with hCG, which signals via the LH receptor (3Segaloff D.L. Ascoli M. Endocr. Rev... 1993; 14: 324-347Google Scholar), and phosphorylation of the activation TEY motif of ERK was then assessed using a Western blot analysis with DP-ERK Ab (24Yung Y. Dolginov Y. Yao Z. Rubinfeld H. Michael D. Hanoch T. Roubini E. Lando Z. Zharhary D. Seger R. FEBS Lett... 1997; 408: 292-296Google Scholar). Considerable staining of three bands at 42, 44, and 46 kDa (ERK2, ERK1, and ERK1b respectively (19Yung Y. Yao Z. Hanoch T. Seger R. J. Biol. Chem... 2000; 256: 15799-15808Google Scholar)) was detected in the resting, nonstimulated cells. The intensity of staining of ERK2 and ERK1 was enhanced (∼5-fold) 5–20 min after the addition of hCG and remained high (∼3-fold) up to 60 min after stimulation. The appearance of p46 ERK1b is of particular interest, because although ERK1b has been reported to exist in rat and human (19Yung Y. Yao Z. Hanoch T. Seger R. J. Biol. Chem... 2000; 256: 15799-15808Google Scholar), its abundance and relative activity as compared with that of ERK1 and ERK2 are usually small. Interestingly, the basal activity of ERK1b in rLHR-4 cells was as high as that of ERK1, only modestly increased 5–20 min after stimulation (∼2-fold), and it declined back to basal level 40 min later. The kinetics of activation, which are different from those of ERK1 and ERK2, suggests a differential mode of ERK1b regulation as recently demonstrated in EJ cells (19Yung Y. Yao Z. Hanoch T. Seger R. J. Biol. Chem... 2000; 256: 15799-15808Google Scholar). We next examined LH, which, like hCG, specifically acts via the LH/CG receptors. The effect of LH on ERK activity was essentially the same as that of hCG under all conditions examined (data not shown). dghCG, which has previously been reported to maintain the same affinity for binding to the LH receptor as the intact hormone but retains only a residual activity for stimulation of steroidogenesis (25van Loenen H.J. van Gelderen-Boele S. Flinterman J.F. Merz W.E. Rommerts F.F. J. Endocrinol... 1995; 147: 367-375Google Scholar), also caused activation of ERK. However, this activation was significantly lower than that achieved by the intact hormone (2.5-fold activation 20 min after dghCG treatment as compared with 4.5-fold 20 min after hCG treatment (Fig.1)). Since LH and hCG have previously shown to transmit their signal via Gs and cAMP (25van Loenen H.J. van Gelderen-Boele S. Flinterman J.F. Merz W.E. Rommerts F.F. J. Endocrinol... 1995; 147: 367-375Google Scholar), we examined the role of cAMP-elevating agents on the ERK activity. Indeed, both 8-Br-cAMP, and forskolin, which activate adenylyl cyclase, significantly activated ERK phosphorylation in the rLHR-4 cells (data not shown), indicating that the hCG-induced ERK activation may be dependent on elevation of intracellular cAMP. Besides ERK phosphorylation of the TEY motif, which mainly reflects MEK activity, we also measured the activity of ERK itself. This was performed by immunoprecipitation with anti C-terminal ERK1 antibody followed by phosphorylation of the general substrate, MBP (19Yung Y. Yao Z. Hanoch T. Seger R. J. Biol. Chem... 2000; 256: 15799-15808Google Scholar). As expected, this method revealed that the activity of ERK correlated well with the regulatory phosphorylation of ERK (Fig. 1, bottom two panels), verifying that both hCG and dghCG cause a 4–5-fold activation of ERK1 activity in rLHR-4 cells. The addition of the MEK inhibitor, PD98059, reduced both hCG-stimulated and -nonstimulated activity of ERK to below basal levels, and a similar reduction was observed for dghCG-, forskolin-, and 8-Br-cAMP-stimulated activity of ERK (Fig. 1 and data not shown). None of the treatments caused any significant change in the total amount of the ERKs as judged by staining with an anti-general ERK antibody (7884), which recognizes both ERK1 and ERK2 much better than ERK1b (Fig. 1;G-ERK). We tested the ability of FSH to stimulate ERK activity in the rat granulosa-derived cell line, rFSHR-17. Similarly to the rLHR-4 line, there was considerable staining of all three ERK isoforms, ERK2, ERK1, and ERK1b, in Western blots from extracts of serum-starved cells. This staining was enhanced by the addition of FSH to the cells, in kinetics that were slightly slower than the kinetics of hCG stimulation in rLHR-4 cells (Fig. 2, upper lanes). The staining of the three ERK isoforms was enhanced 5 min after FSH stimulation, peaked (5-fold above basal level) at 20 min after stimulation, and slightly decreased at 60 min. Also in these cells, the cAMP-stimulating agents, forskolin and 8-Br-cAMP, enhanced the phosphorylation of the three ERK isoforms (3- and 5-fold above basal level, respectively). None of the treatments caused any change in the amount of the ERK isoforms as judged by the staining with a general anti-ERK antibody, confirming that as for the hCG experiment, the changes detected by the DP-ERK Ab are indeed due to changes in ERK phosphorylation and not due to induction of ERK expression. In addition, we examined ERK activity by immunoprecipitation and phosphorylation of MBP. We found (Fig. 2, bottom) that not only ERK phosphorylation but also ERK activity was stimulated by FSH, forskolin and 8-Br-cAMP and was attenuated by PD98059, confirming that both LH and FSH receptors can transmit signals to the ERK pathway in the examined cell lines. One of the important cellular processes that is stimulated by gonadotropins in granulosa cells is stereoidogenesis (26Sugawara T. Kiriakidou M. McAllister J.M. Holt J.A. Arakane F. Strauss III, J.F. Steroids.. 1997; 62: 5-9Google Scholar). Indeed, a significant increase in progesterone production was observed 24 and 48 h after LH stimulation of rLHR-4 cell line (Fig.3 A). hCG had a similar effect to that of LH (data not shown), while dghCG had a very small effect, and forskolin caused a 2-fold greater induction of progesterone production than LH. To examine whether the activated MAPK cascade is also involved in the induction of progesterone production, we incubated the rLHR-4 cells with the MEK inhibitor, PD98059. This inhibitor had no effect by itself on progesterone production by rLHR-4 cells. However, when the cells were incubated with PD98059 for 15 min prior to LH induction, there was a 3-fold increase in LH-induced progesterone production (Fig. 3), under conditions where ERK activity was completely abolished (Fig. 1). A similar stimulatory effect on progesterone production was observed when the MEK inhibitor was added prior to stimulation of the cells with forskolin (Fig. 3), hCG, and 8-Br-cAMP (data not shown). Similar to the rLHR-4 cells, MEK inhibitor significantly increased steroidogenesis in rFSHR-17 cells. Thus, in these cells, FSH and forskolin caused a significant elevation of progesterone production after 24 and 48 h, which was dramatically amplified by the addition of PD98059. In contrast to the induction by the MEK inhibitor, TPA, which is a known activator of the ERK cascade (27Seger R. Biener Y. Feinstein R. Hanoch T. Gazit A. Zick Y. J. Biol. Chem... 1995; 270: 28325-28330Google Scholar), had a negative effect on the forskolin-induced production of progesterone in both cell lines after 24 and 48 h. Taken together, these results suggest that the ERK signaling cascade suppresses gonadotropin-stimulated progesterone production. StAR plays a crucial role in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, where cytochrome P450scc participates as a rate-limiting enzyme in steroidogenesis, converting cholesterol into pregnenolone (7Strauss III, J.F. Kallen C.B. Christenson L.K. Watari H. Devoto L. Arakane F. Kiriakidou M. Sugawara T. Recent Prog. Horm. Res... 1999; 54: 369-394Google Scholar). The induction of StAR and its downstream effects are likely to be cAMP-dependent processes as reported for gonadotropin-induced steroidogenesis in the gonads and ACTH-stimulated steroidogenesis in the fasciculata cells of the adrenal (7Strauss III, J.F. Kallen C.B. Christenson L.K. Watari H. Devoto L. Arakane F. Kiriakidou M. Sugawara T. Recent Prog. Horm. Res... 1999; 54: 369-394Google Scholar). Moreover, since StAR is known to have a short functional half-life (28Clark B.J. Combs R. Hales K.H. Hales D.B. Stocco D.M. Endocrinology.. 1997; 138: 4893-4901Google Scholar), we studied whether down-regulation of StAR may explain the effect of the ERK cascade on progesterone production. Thus, rLHR-4 cells were treated with the various agents described above and examined for the expression of StAR 24 h after stimulation. As expected, LH, hCG, forskolin, and to a considerably lesser extent dghCG, induced the expression of StAR under the conditions examined (Fig. 4). PD98059 alone caused an induction of StAR by itself, but when the cells where preincubated with this MEK inhibitor prior to the addition of forskolin, LH, and hCG, there was a synergistic elevation in the production of StAR. Similar results were obtained also in the rFSHR-17 cells, where PD98059 dramatically increased the forskolin- and FSH-induced expression of StAR. Thus, the ERK cascade may negatively regulate steroidogenesis, and this can be explained by the attenuation of StAR expression, which may be the regulatory component that integrates the signals from both the cAMP and the ERK pathway to regulate the rate of steroidogenesis. To further verify the results obtained with PD98059, we used an additional specific MEK inhibitor, the U0126 (29Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem... 1998; 273: 18623-18632Google Scholar). As observed with the PD98059, the addition of this inhibitor to both rLHR-4 and rFSHR-17 cells caused an elevation in the amount of 30-kDa mature StAR (30Arakane F. Kallen C.B. Watari H. Foster J.A. Sepuri N.B. Pain D. Stayrook S.E. Lewis M. Gerton G.L. Strauss III, J.F. J. Biol. Chem... 1998; 273: 16339-16345Google Scholar) within 24 h (Fig. 5). The addition of the gonadotropins alone also elevated this expression, but when the inhibitor was added together with the appropriate gonadotropins, the expression of StAR was significantly higher and reached up to 10-fold above basal expression levels. This was significantly higher compared with the amounts expected from the expression induced by U0126 and gonadotropin alone. Interestingly, in some of the experiments, a 37-kDa pre-StAR (30Arakane F. Kallen C.B. Watari H. Foster J.A. Sepuri N.B. Pain D. Stayrook S.E. Lewis M. Gerton G.L. Strauss III, J.F. J. Biol. Chem... 1998; 273: 16339-16345Google Scholar) was detected by the anti-StAR antibody (Fig.5 A). This cytosolic protein is known to be maintained in a low steady state level, because it rapidly matures into the 30-kDa form of StAR in the mitochondria (30Arakane F. Kallen C.B. Watari H. Foster J.A. Sepuri N.B. Pain D. Stayrook S.E. Lewis M. Gerton G.L. Strauss III, J.F. J. Biol. Chem... 1998; 273: 16339-16345Google Scholar). Unlike the 30-kDa StAR, the relatively low amount of this protein did not change upon the addition of LH, FSH, or MEK inhibitors (Fig. 5). We then studied the effect of U0126 on steroidogenesis in the rLHR-4 and the rFSHR-17 cells. Similar to the results of PD98059, U0126 did not induce steroidogenesis by itself but synergized with the gonadotropins to produce high amounts of progesterone (Fig. 5). Taken together, our results indicate that MEK inhibitors dramatically increase gonadotropin-induced StAR expression and steroidogenesis. However, the MEK inhibitors themselves induced clear elevation of StAR expression without corresponding elevation in progesterone production. This is probably due to the fact that in the immortalized granulosa cell lines no basal levels of the cytochrome p450scc, the activity of which is obligatory for the conversion of cholesterol to pregnenolone, can be detected (31Hanukoglu I. Suh B.S. Himmelhoch S. Amsterdam A. J. Cell Biol... 1990; 111: 1373-1381Google Scholar). This notion is supported by our preliminary findings that in primary rat granulosa cells obtained from preovulatory follicles and containing p450scc, PD98059 by itself increased progesterone production. On the other hand, MEK inhibitors do synergize with gonadotropin/cAMP stimulation of stereoidogenesis because of thede novo synthesis of the cytochrome p450scc, which is stimulated by gonadotropin/cAMP in the granulosa cell lines (17Suh B.S. Sprengel R. Keren-Tal I. Himmelhoch S. Amsterdam A. J. Cell Biol... 1992; 119: 439-450Google Scholar,31Hanukoglu I. Suh B.S. Himmelhoch S. Amsterdam A. J. Cell Biol... 1990; 111: 1373-1381Google Scholar). To examine whether the enhancement of StAR expression by PD98059, gonadotropins, and cAMP-elevating agents is mainly located in mitochondria (32Sugawara T. Lin D. Holt J.A. Martin K.O. Javitt N.B. Miller W.L. Strauss III, J.F. Biochemistry.. 1995; 34: 12506-12512Google Scholar), we stained rFSHR-17 cells with anti-StAR antibodies prior to or following PD98059, FSH, and forskolin stimulation (Fig.6). In nonstimulated cells, StAR could not be detected in mitochondria (a). In contrast, clear elevation in mitochondrial StAR was evident following 24 h of treatment with PD98059 (b). LH clearly increased the StAR content in the mitochondria (c), while PD98059 dramatically increased mitochondrial StAR content (d).