The ERK Signaling Cascade Inhibits Gonadotropin-stimulated SteroidogenesisRony Seger, Tamar Hanoch, Revital Rosenberg et al.|Journal of Biological Chemistry|2001 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 and ERK were from was from to human StAR were in Kallen C.B. Arakane F. Kiriakidou M. III, J.F. J. 1997; Scholar). and were from and were from and were from rLHR-4 cell was by of granulosa cells with and the receptor B.S. R. S. A. J. 1992; Scholar). The rFSHR-17 cell was by of granulosa cells via of cells with and an were with the FSH receptor A. R. A. Mol. Cell. Endocrinol... 1993; Scholar). The cells were in were for 16 and for with the agents in the or of various stimulation, cells were with and with A and Y. T. R. J. Biol. 2000; and were in A inhibitors Y. T. R. J. Biol. 2000; Scholar). were for The was for protein content and to a blot or to as the of StAR, cells were in Y. T. R. J. Biol. 2000; and to blot The rLHR-4 and rFSHR-17 cells were in with to The were and J. Biol. from Dr. R. A. Health and H. T. R. J. Biol. 1999; Scholar). The were into the two cell to the was in the two cell lines a the rLHR-4 cells were in for and in for an additional The rFSHR-17 cells were in for The cells were and as which proteins were and from were on by with the appropriate proteins were in and to and The were with or or of were to with monoclonal as described the of were with in of and with of protein and of and The phosphorylation were by the addition of and and the were by and as previously described Y. T. R. J. Biol. 2000; Scholar). into the was by as previously described D. R. A. 1997; Scholar). were on in were with to with the appropriate and in a with a of to human StAR and to cells were with by the second of granulosa cells with the gonadotropin LH or FSH induces cellular including of the signaling pathways that gonadotropin receptors to the regulation of progesterone production, we two granulosa cell lines or FSH rLHR-4 and The addition of the appropriate gonadotropins to these cells has previously been shown to stimulate cAMP production, activation of PKA, and of steroidogenesis A. R. A. Mol. Cell. Endocrinol... 1993; Scholar and data not the ERK cascade was implicated in the signaling of receptors R. Trends 2000; Scholar), we first the ERK cascade is also activated in the rLHR-4 and rFSHR-17 cell lines. rLHR-4 cells were with which signals via the LH receptor (3Segaloff D.L. Ascoli M. Endocr. Rev... 1993; 14: 324-347Google Scholar), and phosphorylation of the activation of ERK was a blot with Y. Y. H. D. T. E. D. R. 1997; Scholar). of three and and Y. T. R. J. Biol. 2000; was in the cells. The of of and was enhanced the addition of hCG and to The of is of has been to in and human Y. T. R. J. Biol. 2000; Scholar), and activity as with that of and are Interestingly, the activity of in rLHR-4 cells was as as that of only increased stimulation and it to The of which are from of and a of regulation as recently in cells Y. T. R. J. Biol. 2000; Scholar). We LH, via the receptors. The effect of LH on ERK activity was the as that of hCG not which has previously been to the for binding to the LH receptor as the hormone but only a activity for stimulation of steroidogenesis S. J.F. W.E. J. Endocrinol... 1995; Scholar), also activation of However, this activation was that by the hormone activation treatment as with hCG treatment LH and hCG have previously shown to their via Gs and cAMP S. J.F. W.E. J. Endocrinol... 1995; Scholar), we the role of agents on the ERK both and which activate adenylyl cyclase, activated ERK phosphorylation in the rLHR-4 cells not indicating that the ERK activation may be on elevation of intracellular ERK phosphorylation of the which mainly MEK we also the activity of ERK This was by with by phosphorylation of the Y. T. R. J. Biol. 2000; Scholar). this that the activity of ERK correlated well with the regulatory phosphorylation of ERK two that both hCG and a activation of activity in rLHR-4 cells. The addition of the MEK both and activity of ERK to and a reduction was for and activity of ERK and data not of the in the of the ERKs as by with an ERK which both and We the of FSH to stimulate ERK activity in the granulosa-derived cell to the rLHR-4 was of three ERK and in from of cells. This was enhanced by the addition of FSH to the cells, in that were the of hCG stimulation in rLHR-4 cells The of the three ERK was enhanced FSH stimulation, stimulation, and in these cells, the and enhanced the phosphorylation of the three ERK and of the in the of the ERK as by the with a that as for the hCG the by the are due to in ERK phosphorylation and not due to of ERK expression. In we ERK activity by and phosphorylation of We found that not only ERK phosphorylation but also ERK activity was by FSH, and and was by that both LH and FSH receptors can signals to the ERK pathway in the cell lines. One of the important cellular processes that is by gonadotropins in granulosa cells is T. Kiriakidou M. Arakane F. III, J.F. 1997; Scholar). a in progesterone production was and LH stimulation of rLHR-4 cell hCG a effect to that of LH not while a and a of progesterone production the activated cascade is also involved in the of progesterone production, we the rLHR-4 cells with the MEK This inhibitor no effect by on progesterone production by rLHR-4 cells. However, the cells were with for to LH was a in progesterone production ERK activity was A stimulatory effect on progesterone production was the MEK inhibitor was to stimulation of the cells with and not to the rLHR-4 cells, MEK inhibitor increased steroidogenesis in rFSHR-17 cells. in these cells, FSH and a elevation of progesterone production and which was by the addition of In to the by the MEK which is a known of the ERK cascade R. Y. R. T. A. Y. J. Biol. 1995; Scholar), a effect on the production of progesterone in both cell lines and these results suggest that the ERK signaling cascade gonadotropin-stimulated progesterone StAR plays a crucial role in the regulation of from the to the cytochrome as a enzyme in into 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 of StAR and downstream effects are likely to be processes as for gonadotropin-induced steroidogenesis in the and steroidogenesis in the cells of the 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 R. D.M. Endocrinology.. 1997; Scholar), we studied down-regulation of StAR may the effect of the ERK cascade on progesterone rLHR-4 cells were with the various agents described and for the expression of StAR LH, and to a induced the expression of StAR the an of StAR by but the cells with this MEK inhibitor to the addition of LH, and was a elevation in the production of StAR. results were also in the rFSHR-17 cells, increased the and FSH-induced expression of StAR. the ERK cascade may regulate and this can be by the attenuation of StAR which may be the regulatory that the signals from both the cAMP and the ERK pathway to regulate the of steroidogenesis. the results with we an additional specific MEK the F. J. Biol. 1998; Scholar). with the the addition of this inhibitor to both rLHR-4 and rFSHR-17 cells an elevation in the of StAR F. Kallen C.B. Watari H. Foster D. M. III, J.F. J. Biol. 1998; The addition of the gonadotropins also elevated this but the inhibitor was with the appropriate gonadotropins, the expression of StAR was and to expression This was with the from the expression induced by and gonadotropin Interestingly, in of the a F. Kallen C.B. Watari H. Foster D. M. III, J.F. J. Biol. 1998; was by the This protein is known to be in a it into the of StAR in the F. Kallen C.B. Watari H. Foster D. M. III, J.F. J. Biol. 1998; Scholar). the StAR, the of this protein not the addition of LH, FSH, or MEK inhibitors We studied the effect of on steroidogenesis in the rLHR-4 and the rFSHR-17 cells. to the results of not induce steroidogenesis by but with the gonadotropins to of progesterone results that MEK inhibitors gonadotropin-induced StAR expression and steroidogenesis. However, the MEK inhibitors themselves induced elevation of StAR expression elevation in progesterone This is due to the that in the immortalized granulosa cell lines no of the cytochrome the activity of which is for the of to can be Suh B.S. S. A. J. 1990; Scholar). This is by that in granulosa cells from and by increased progesterone the other MEK inhibitors with stimulation of of of the cytochrome which is by in the granulosa cell lines B.S. R. S. A. J. 1992; Suh B.S. S. A. J. 1990; Scholar). the of StAR expression by gonadotropins, and agents is mainly in T. D. III, J.F. 1995; Scholar), we rFSHR-17 cells with to or FSH, and stimulation In cells, StAR not be in In elevation in StAR was of treatment with LH increased the StAR content in the while increased StAR content StAR while enhanced StAR content in the the the data by blot on the elevation of StAR expression by Although we that an elevation of cAMP is to activate ERK, it was not cAMP and PKA are the of the signaling to Therefore, we which is a and inhibitor of PKA to the involvement of in the activation of ERK in rLHR-4 and rFSHR-17 cells. The addition of to gonadotropin stimulation not the activity of the three ERKs but the of ERK by hCG in rLHR-4 cells and by FSH in rFSHR-17 cells ERK activation by and in both cell lines was also inhibited by not indicating that ERK activation is mediated mainly by PKA and not via nucleotide J. A. 1998; Scholar). the involvement of PKA in the activation of ERK by gonadotropins, we H. T. R. J. Biol. 1999; with the PKA inhibitor or J. Biol. Scholar). activation of ERK in the cells was by the of into the activation of the with ERK with inhibition of PKA with inhibited ERK activation by gonadotropins and by these results that the activation of ERK by gonadotropin in the cell lines is We StAR activation is mediated by PKA the PKA inhibitor was to rLHR-4 and rFSHR-17 cells, it inhibited and StAR expression also StAR expression indicating that StAR production is regulated by PKA in the cell lines progesterone production was also inhibited by the inhibitor not indicating that the processes may function mainly downstream of PKA. However, progesterone production downstream of PKA and of StAR, ERK, activated by PKA, serves as a regulator of this pathway due to of StAR of the signaling pathways controlling gonadotropin-induced In this we a mechanism for two signaling the and the ERK cascade in a The interaction these two has been studied in cellular the years Krebs E.G. 1999; Scholar). In as in J. T. A. 1993; or human cells R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A... 1993; Scholar), it was shown that cAMP inhibits the activation of the ERK cascade. This inhibition to by inhibitory phosphorylation of J. T. A. 1993; or by activation of the which with for the activation of F. 1993; Scholar). In other cell as cells, cAMP not only not the ERK cascade but in activates it to induce various or processes. One mechanism that activates the ERK cascade by PKA includes the activation of the nucleotide for the and Upon binding of cAMP, these activate which the activation of not and the of the ERK cascade J. A. 1998; Scholar). However, in the rLHR-4 and rFSHR-17 cells in the activation of ERK to be downstream of PKA, indicating that the are not involved in the ERK This pathway may an activation of the by PKA, which causes the with and of the ERK cascade. it was shown that activation of ERK by cAMP in the via a K. J. H. L. J. Biol. 2000; Scholar). Although this specific not to be in granulosa cells, it is that or is involved in the of PKA signals to ERK in the rLHR-4 and rFSHR-17 cells. The involvement of PKA in ERK activation is in the present both by PKA inhibitor and by of cells with for The data both are in that PKA plays a role in gonadotropin signaling it be that ERK it not the gonadotropin-induced ERK Therefore, it is that the gonadotropin receptors are other proteins or the of Gs protein to activate the ERK cascade as was for other receptors and cell (reviewed in Refs. R. Trends 2000; Scholar and J.S. 1998; Scholar). Interestingly, we recently found that progesterone production in the granulosa cell lines not which suggest that may be pathways in these cells that steroidogenesis via the ERK cascade. the and the ERK pathways has been in cells. it was shown that cAMP causes activation of the ERK which is important for in cells H. A. J. Biol. 1998; Scholar). In human cells, elevation of cAMP causes a response that is mediated by the ERK cascade T. S. 2000; Scholar). However, it was also shown that processes to a down-regulation of processes. of this interaction of the which to the activity of the H. A. Y. R. J. Biol. 1999; Scholar), involvement in the down-regulation of and ERK In to this of we show that the activation of processes downstream of PKA may also be inhibited by an mechanism. The inhibition of progesterone production the of of proteins that play a role in the steroidogenic In the present we the expression of StAR, which is known to be on or N. D. A. Mol. Cell. Endocrinol... 1996; Scholar). Although StAR phosphorylation may play a role it not to with the of PKA or ERK and we not phosphorylation of StAR by ERK not However, we an ERK activity and StAR expression in the The of ERK activity an elevation in the of StAR, while activation of ERK by StAR expression in granulosa cells. Therefore, it is that the two to regulate StAR the mechanism for StAR expression in granulosa cells M. Sugawara T. III, J.F. J. 1996; Scholar). including steroidogenic protein, and the regulator E. E. D.M. 1997; Scholar, E. D.M. Endocrinology.. 1998; Scholar, D.M. Reprod... 1998; participate in the regulation of this StAR is by the steroidogenic and protein downstream of PKA, but it is that these participate in the down-regulation of StAR expression via the ERK both have been shown to be by ERK Y. K. Mol. 1999; Scholar, N. J. 1999; Scholar). Therefore, it is that the regulation of StAR expression the or to be StAR expression be by of that both the PKA and ERK phosphorylation of steroidogenic and protein or induce a system that reduces the of the StAR. Another for the mechanism by which ERK can steroidogenesis be involvement in desensitization of the gonadotropin receptors. of granulosa cells with has previously been shown to desensitization of the cells to stimulation, which is characterized by down-regulation of cAMP formation as well as of steroidogenesis (9Amsterdam A. Berkowitz A. Nimrod A. Kohen F. Proc. Natl. Acad. Sci. U. S. A... 1980; 77: 3440-3444Google Scholar). Moreover, it has previously been that the ERK cascade activate G-protein-coupled receptor kinase J. Biol. 1999; Scholar), which in turn induces down-regulation of receptors. However, it is that this is the mechanism in the inhibitory effects of ERK were cells were by this can the receptor to activate PKA, of the inhibitory signals are the gonadotropins play a key role in modulation of ERK Moreover, activation of ERK can the signals by FSH It is known that unlike the of which is to the and of cAMP production, activation of ERK can be as a of in intracellular This due to a mechanism of the ERK which a signaling by extracellular signals Trends Biochem. 1996; Scholar). The activity of ERK, which downstream of cAMP, may the of steroidogenesis which to steroidogenesis. Therefore, a may the of steroidogenesis induced by which is to induce only signals by the receptors. In the present that activation of signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery that the ERK cascade. This down-regulation machinery inhibits the gonadotropin-induced steroidogenic pathway by mechanisms that are from the well characterized receptor desensitization Activation of the ERK cascade downstream of PKA in turn the of StAR which is the key in these down-regulation processes. PKA not only gonadotropin-induced it also activates the down-regulation mechanism that can steroidogenesis Moreover, the that activation or inhibition of ERK by other pathways be an important mechanism for or of gonadotropin-stimulated steroidogenesis. This to a process in which granulosa cells show to LH of LH receptor or to up-regulation of the steroidogenic machinery of granulosa cells (reviewed in Ref. Trends 1999; 10: Scholar). follicle-stimulating hormone hCG extracellular signal-regulated kinase human chorionic gonadotropin mitogen-activated protein kinase protein luteinizing hormone protein kinase A steroidogenic acute regulatory protein chorionic gonadotropin human protein kinase inhibitor