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A Delayed Gonadotropin-Dependent and Growth Factor-Mediated Activation of the Extracellular Signal-Regulated Kinase 1/2 Cascade Negatively Regulates Aromatase Expression in Granulosa Cells

Department of Pharmacology, Carver College of Medicine, The University of Iowa, Iowa City, Iowa 52242

Address all correspondence and requests for reprints to: Mario Ascoli, Ph.D., Department of Pharmacology, 2–319B BSB, 51 Newton Road, The University of Iowa, Iowa City, Iowa 52242. E-mail: mario-ascoli{at}uiowa.edu.

	ABSTRACT

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Human chorionic gonadotropin and human FSH (hFSH) elicit a transient increase in ERK1/2 phosphorylation lasting less than 60 min in immature granulosa cells expressing a low density of gonadotropin receptors. In cells expressing a high density of receptors, human chorionic gonadotropin and human FSH elicit this fast transient increase in ERK1/2 phosphorylation and also a delayed and more sustained increase that is detectable after 6–9 h. Both the early and delayed increases in ERK1/2 phosphorylation can be blocked with inhibitors of protein kinase A, the epidermal growth factor receptor kinase, metalloproteases, and MAPK kinase. The delayed effect, but not the early effect, can also be blocked with an inhibitor of protein kinase C. Because the delayed increase in ERK1/2 phosphorylation correlates with low aromatase expression in response to gonadotropins, we tested the effects of these inhibitors on aromatase expression. These inhibitors had little or no effect on gonadotropin-induced aromatase expression in cells expressing a low density of receptors, but they enhanced gonadotropin-induced aromatase expression in cells expressing a high density of receptors. Phorbol esters also induced a prolonged increase in ERK1/2 phosphorylation and, when added together with hFSH, blocked the induction of aromatase expression by hFSH in cells expressing a low density of hFSH receptor. A MAPK kinase inhibitor reversed the inhibitory effect of the phorbol ester on aromatase induction. We conclude that the effects of gonadotropins on ERK1/2 phosphorylation are mediated by epidermal growth factor-like growth factors and that the delayed effect is partially mediated by protein kinase C and acts as a negative regulator of aromatase expression.

	INTRODUCTION

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THE RECEPTORS for FSH (FSHR) and LH (LHR) are members of the G protein-coupled family of receptors, and their expression in granulosa cells depends on the stage of cell differentiation. The FSHR is expressed in both immature and mature cells, but the LHR is expressed only in the mature cell type. The FSHR promotes the proliferation of immature granulosa cells and induces the expression of aromatase and the LHR. The LHR promotes cell cycle arrest, induces luteinization and progesterone synthesis, and suppresses its own expression as well as the expression of aromatase. These divergent effects of LH and FSH stand in contrast with the high degree of amino acid sequence homology between the two hormones (1, 2, 3) and between their two receptors (3, 4, 5, 6, 7), and with the fact that both the LH/LHR and the FSH/FSHR complexes use Gs/adenylyl cyclase/cAMP as their main signaling pathway (3, 4, 5, 6, 7).

Using adenovirus-mediated expression of the recombinant LHR in immature granulosa cells, Zeleznik and co-workers (8, 9) showed that two of the hallmark responses of FSH action (i.e. the induction of aromatase and the LHR) are likely due to differences in the signaling properties of the LHR and the FSHR rather than to their expression at different stages of maturation of the granulosa cells. Two hypotheses have been put forward to explain the divergent actions of LH and FSH on aromatase expression in immature granulosa cells expressing the recombinant gonadotropin receptors. One hypothesis (9) states that FSH and LH stimulate the cAMP signaling pathway but that FSH also stimulates the protein kinase B (PKB)/Akt pathway and that this activation of the PKB/Akt pathway is essential for aromatase induction. There are several lines of evidence that support this hypothesis (9, 10, 11). In more recent experiments we reported that the LHR and the FSHR can both activate the PKB/Akt pathway and we proposed an alternative hypothesis (12). Our hypothesis states that the stimulation of the cAMP signaling pathway (alone or together with the PKB/Akt pathway) by the FSHR and LHR is sufficient for aromatase induction but that at high receptor densities the LHR can also preferentially activate the inositol phosphate cascade (and/or other unknown signaling pathways) that antagonize the actions of cAMP on aromatase induction. Note that we do not propose that the ability of the LHR to activate the inositol phosphate cascade is unique. We simply propose that it is a function of receptor density. In fact, our data show that, at low LHR densities, when LH/CG can induce only cAMP accumulation it can also induce aromatase expression. Likewise, at high FSHR density, when FSH can induce cAMP and inositol phosphate accumulation it cannot induce aromatase expression (12).

Recent studies have implicated a novel gonadotropin-responsive ovarian paracrine pathway that leads to cell differentiation and modulation of gene expression. This pathway involves an LH-dependent intraovarian expression of epidermal growth factor (EGF)-like factors such as amphiregulin, epiregulin, and ß-cellulin, which are proteolytically processed and released from the cell surface to activate EGF receptors (EGFR) in a paracrine fashion, leading to oocyte nuclear maturation, cumulus expansion, enzyme expression, and ovulation (reviewed in Refs. 10 and 13). A common consequence of the engagement of the EGFR in many cell types is the activation of the ERK1/2 cascade, which in turn regulates various cellular processes through activation of additional kinases or transcription factors (reviewed in Refs. 14 and 15). Because it has been shown that the ERK1/2 signaling cascade regulates the expression of steroidogenic acute regulatory protein in immortalized preovulatory rat granulosa cells (16) and bovine theca cells (17) and the expression of aromatase expression in immature rat Sertoli cells (11), we decided to test for the involvement of a gonadotropin-dependent autocrine/paracrine pathway on the regulation of the ERK1/2 cascade in the regulation of aromatase expression in granulosa cells.

We show that the gonadotropin-induced activation of the ERK1/2 cascade in immature granulosa cells is indirectly mediated by the release of EGF-like factors, which in turn activate the EGFR, and that it occurs in two phases. A rapid and transient activation of the ERK1/2 cascade is a common event in immature granulosa cells regardless of the density of gonadotropin receptors expressed. At high densities, the LHR and FSHR also provoke a delayed and more sustained activation of ERK1/2. This delayed activation of ERK1/2 requires protein kinase C (PKC) activation and inhibits aromatase expression.

	RESULTS

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ERK1/2 Phosphorylation Is Monophasic in Granulosa Cells Expressing a Low Density of Gonadotropin Receptors But Is Biphasic in Cells Expressing a High Density of Gonadotropin Receptors
All experiments described below were done using four different sets of immature granulosa cells that can be classified into two groups. Cells infected with 200 multiplicity of infection (MOI) Ad-ßgal and cells infected with 20 MOI of Ad-hLHR comprise the low receptor density group. The cells infected with Ad-ßgal express the endogenous FSHR and were used to measure human FSH (hFSH) responses at a low FSHR density (1 ng hFSH/10⁶ cells). The cells infected with 20 MOI Ad-hLHR bind approximately 0.1 ng human chorionic gonadotropin (hCG)/10⁶ cells, and they were used to measure hCG responses at a low hLHR density. Cells infected with 100 MOI of Ad-hFSHR bind approximately 10 ng hFSH/10⁶ cells, whereas those infected with 200 MOI Ad-hLHR bind approximately 4 ng hCG/10⁶ cells (12), and they were used to measure hFSH or hCG-induced responses, respectively, at a high receptor density. These groups were chosen because the low receptor density cells respond to hFSH or hCG with an increase in aromatase expression, whereas the high receptor density cells do not (12). It is important to recognize that the levels of ¹²⁵I-hCG binding observed in immature granulosa cells expressing a low or a high density of the recombinant hLHR are within the limits of expression of the endogenous LHR attained when its expression is induced by stimulation of these cells with FSH or cAMP analogs (18). Others have shown that immature rat granulosa cells maintained for 3 d under the same culture conditions used here but in the presence of maximally effective concentrations of FSH or 8-bromo-cAMP bind 1–4 ng ¹²⁵I-hCG/10⁶ cells (18). Lastly, all experiments described below were done using a maximally effective concentration of hormone or other stimuli such as forskolin or phorbol 12-myristate 13-acetate (PMA) (see Materials and Methods and figure and table legends). These concentrations were chosen based on preliminary experiments done using several concentrations of each of the hormones or pharmacological stimuli tested (data not shown).

Although hFSH and hCG have been previously shown to induce ERK1/2 phosphorylation in a number of granulosa cell models (16, 19, 20), the effect of receptor density on this response has not been examined. Figure 1, A and B, shows that, in the low receptor density cells, changes in ERK1/2 phosphorylation are detectable only after short-term stimulation with hFSH or hCG, and they are transient. Maximal effects were attained within 15–30 min of stimulation with hFSH or hCG. These elevated levels quickly returned toward basal levels and remained at or below basal levels after several hours of stimulation. Cells expressing a high density of receptors respond to hCG or hFSH with a biphasic ERK1/2 phosphorylation pattern consisting of a transient increase similar to that detected in the low receptor density cells followed by a second and more pronounced increase after 6 or 9 h of incubation (Fig. 1, C and D).

View larger version (37K): [in this window] [in a new window]   Fig. 1. Time Course of ERK1/2 Phosphorylation in Immature Granulosa Cells Expressing Different Densities of Gonadotropin Receptors Primary cultures of immature granulosa cells were infected with the indicated adenoviral constructs as described in Materials and Methods. Two days after infection cells were incubated with vehicle only or 100 ng/ml hFSH, 100 ng/ml hCG, 10 µM forskolin, or 100 nM PMA as indicated. Phospho-ERK1/2 and total ERK1/2 were measured as described in Materials and Methods. Results are representative of two independent experiments.

In all cases examined, the levels of total ERK1/2 remained unchanged (Fig. 1, A–E).

The Early Increase in ERK1/2 Phosphorylation Elicited by Gonadotropins Is PKA Dependent and PKC Independent But the Late Increase Is PKA and PKC Dependent
It is well established that the early increase in ERK1/2 phosphorylation provoked by FSH in immature granulosa cells or by hCG in preovulatory granulosa cells is a PKA-dependent process (19, 20). In the experiments described below we investigated whether granulosa cells expressing a low or a high receptor density employ the same signaling cascades to induce the early and late increase in ERK1/2 phosphorylation described above.

We tested the effects of a PKA-selective inhibitor (H89; see Ref. 21), an inhibitor of the EGFR kinase and its inactive analog (AG1478 and AG43, respectively; See Ref. 22), and a broad spectrum metalloprotease inhibitor that blocks the release of the heparin-bound form of EGF-like growth factors (GM6001; see Ref. 23). These inhibitors of the EGF pathway were used because recent studies on a variety of cell types have shown that proteolytic processing of precursor forms of EGF-like factors and subsequent activation of EGFR-like receptors are the primary mechanism of autocrine transactivation of the ERK1/2 cascade (13, 14, 24, 25). Lastly, we also tested a MAPK kinase (MEK) inhibitor (UO126; see Ref. 21) to ensure that ERK1/2 phosphorylation was being effected by the classical Raf/MEK activation pathway. The effects of these and other inhibitors (see below) were initially ascertained using multiple inhibitor concentrations. The concentrations of each inhibitor used for the experiments presented here were chosen based on these initial results because they were the minimal concentrations of each inhibitor that produce maximal inhibitory effects.

As expected from previous studies (19, 20), H89 and UO126 were effective inhibitors of the early increase in ERK1/2 phosphorylation provoked by hCG or hFSH in cells expressing a low (Fig. 2, A and B) or a high density (Fig. 2, C and D) of gonadotropin receptors. An inhibitory effect of AG1478 on the hFSH-induced early increase in ERK1/2 phosphorylation in immature granulosa cells has been reported before (20), and our data (Fig. 2, A–D) show that this inhibitor (but not its inactive analog AG43) is effective against the early increase in ERK1/2 phosphorylation provoked by hCG or hFSH in low or high receptor density cells. GM6001 is effective as well (Fig. 2, A–D). The results presented in Fig. 2, E and F, show that the EGFR, MEK, and metalloprotease inhibitors also inhibit the early increase in ERK1/2 phosphorylation induced by forskolin or PMA, indicating some overlap between the pathways activated by the gonadotropins and those activated by these pharmacological agents.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Effects of Inhibitors on the Early ERK1/2 Phosphorylation in Immature Granulosa Cells Expressing Different Densities of Gonadotropin Receptors Primary cultures of immature granulosa cells were infected with the indicated adenoviral constructs as described in Materials and Methods. Two days after infection the cells were preincubated with dimethyl sulfoxide (DMSO) (buffer control) or AG1478 (10 µM), AG43 (10 µM), GM6001 (20 µM), H89 (50 µM), or UO126 (25 µM) for 1 h. The cells were then stimulated with FSH (100 ng/ml), hCG (100 ng/ml), 10 µM forskolin, or 100 nM PMA as indicated. Phospho-ERK1/2 and total ERK1/2 were measured as described in Materials and Methods. Results are representative of two independent experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Effects of Ro-318220 on the Early ERK1/2 Phosphorylation in Immature Granulosa Cells Expressing Different Densities of Gonadotropin Receptors Primary cultures of immature granulosa cells were infected with the indicated adenoviral constructs as described in Materials and Methods. Two days after infection cells were incubated with Ro-318220 (10 µM) or without Ro-318220 (i.e. DMSO only) for 30 min. Cells were then incubated with hFSH (100 ng/ml); hCG (100 ng/ml), 10 µM forskolin or 100 nM PMA as indicated. Phospho-ERK1/2 and total ERK1/2 were measured as described in Materials and Methods. Results are representative of two independent experiments.

The effects of the same pharmacological inhibitors on the delayed increase in ERK1/2 phosphorylation elicited by FSH and LH in granulosa cells expressing a high density of receptors are shown in Fig. 4. Figure 4, A and B, shows that, like the early increase in ERK1/2 phosphorylation (cf. Fig. 2), the delayed response is sensitive to inhibitors of the EGFR kinase (AG1478), metalloproteases (GM6001), PKA (H89), and MEK (UO126). In contrast to the early increase in ERK1/2 phosphorylation (cf. Fig. 3), however, the delayed response is readily blocked by Ro-318220, a PKC inhibitor (Fig. 4, C and D). Again, the effectiveness of Ro-318220 was documented by testing its ability to inhibit the delayed stimulation of ERK1/2 phosphorylation provoked by PMA (Fig. 4E).

View larger version (22K): [in this window] [in a new window]   Fig. 4. Effects of Inhibitors on the Delayed ERK1/2 Phosphorylation Response in Immature Granulosa Cells Expressing a High Density of Gonadotropin Receptors Primary cultures of immature granulosa cells were infected with the indicated adenoviral constructs as described in Materials and Methods. Two days after infection the cells were preincubated with DMSO (control) or AG1478 (10 µM), AG43 (10 µM), GM6001 (20 µM), H89 (50 µM), UO126 (25 µM), or Ro-318220 (10 µM) for 1 h. The cells were then stimulated with 100 ng/ml hFSH, 100 ng/ml hCG, or 100 nM PMA as indicated. Phospho-ERK1/2 and total ERK1/2 were measured as described in Materials and Methods. Results are representative of four to five independent experiments.

Inhibitors of EGFR Signaling and ERK1/2 Phosphorylation Overcome the Inability of Gonadotropins to Enhance Aromatase Expression in Cells Expressing a High Density of Gonadotropin Receptors
In a recent study we showed that immature granulosa cells expressing a low density of FSHR or LHR respond to hFSH or hCG, respectively, with an increase in aromatase expression, whereas those expressing a high density of FSHR or LHR do not (12). Because the ERK1/2 signaling cascade has been implicated in the negative regulation of steroidogenesis in ovarian cells (16, 17) and the negative regulation of aromatase expression in Sertoli cells (11) we sought to determine whether the gonadotropin-induced activation of the ERK1/2 cascade is also involved in the regulation of aromatase expression in granulosa cells. We directly examined this possibility by testing the effects of a MEK inhibitor (UO126) on aromatase expression (Fig. 5A). Because the gonadotropin-induced ERK1/2 phosphorylation is also sensitive to a metalloprotease inhibitor (GM6001) and an EGFR kinase inhibitor (AG1478), we also tested the effects of these compounds on aromatase expression (Fig. 5, B and C).

View larger version (25K): [in this window] [in a new window]   Fig. 5. EGFR Signaling Network and MEK Inhibitors Restore the Ability of Gonadotropins to Enhance Aromatase Expression in Immature Granulosa Cells Expressing a High Density of their Cognate Receptors Primary cultures of immature granulosa cells were infected with the indicated adenoviral constructs as described in Materials and Methods. Two days after infection the cells were pretreated for 1 h with 25 µM UO126 (A), 20 µM GM6001 (B), or 10 µM AG1478 or AG43 (C). Cells preincubated without inhibitors received DMSO only. The cells were then stimulated with hFSH (100 ng/ml) or hCG (100 ng/ml) for 48 h as indicated. Total RNA was collected and used to quantitate aromatase mRNA using real-time PCR amplification as described in Materials and Methods. For easy comparison and to avoid inherent variability in absolute values associated with use of primary cultures, all data are expressed relative to FSH-induced aromatase response of cells infected with Ad-ßgal and incubated with hFSH (see first white bar on the left panel of each figure) because the levels of aromatase mRNA in cells incubated with buffer only were often undetectable. Each bar is the mean ± SEM of four to six independent experiments. Asterisks indicate a statistically significant difference (P < 0.05, two-tailed t test) between the two groups of cells incubated with a given hormone but with or without a given inhibitor.

Because cAMP analogs and forskolin can induce aromatase expression in immature granulosa cells to about the same extent as that detected when the endogenous FSHR is activated by hFSH (12), we tested the effects of the aforementioned inhibitors on the forskolin induction of aromatase expression. These results are summarized in Table 1 and show that these inhibitors have variable effects on forskolin-induced aromatase expression, but none of them are statistically significant.

An Inhibitor of MEK Overcomes the Ability of PMA to Antagonize the Induction of hFSH-Induced Aromatase Expression in Cells Expressing a Low Density of FSHRs
Because addition of PMA induces a prolonged ERK1/2 phosphorylation response and because PMA antagonizes aromatase expression when provoked by hFSH in cells with a low density of FSHR (12), we tested the potential role of the PMA-induced ERK1/2 phosphorylation on the inhibition of aromatase expression.

In agreement with our previous results, Fig. 6A shows that hFSH enhances aromatase expression in cells expressing a low density of FSHR and that this response can be abolished by coincubation with PMA. Remarkably, Fig. 6A also shows that addition of a MEK inhibitor (UO126) completely reverses the inhibitory effect of PMA on aromatase expression. Also note that, as shown above (cf. Fig. 5A), UO126 increases the expression of aromatase induced by hFSH alone, but this effect is not statistically significant.

View larger version (20K): [in this window] [in a new window]   Fig. 6. A MEK Inhibitor Reverses the PMA-Provoked Inhibition of Aromatase Expression in Cells Expressing a Low Density of FSHR and Incubated with hFSH. A, Primary cultures of immature granulosa cells were infected with Ad-ßgal as described in Fig. 1 and Materials and Methods. The cells were treated without or with 25 µM UO126 as indicated for 1 h and then incubated with buffer only, 100 ng/ml hFSH only, or 100 ng/ml hFSH plus 100 nM PMA for 48 h as indicated. Total RNA was collected and used to quantitate aromatase mRNA using real-time PCR amplification as described in Materials and Methods. As in Fig. 5, all data are expressed relative to the hFSH-induced aromatase response in cells infected with ß-gal (see white bar). Each bar is the mean ± SEM of four independent experiments. Within each panel, the asterisk denotes a statistically significant difference (P < 0.05, two-tailed t test) between the cells incubated with hFSH and those incubated with hFSH + UO126. B and C, Primary cultures of immature granulosa cells were infected with Ad-ßgal as described in Materials and Methods and were preincubated with 25 µM UO126 (B) or 10 µM Ro-318820 (C) for 1 h as indicated. They were then incubated with 100 ng/ml hFSH alone or 100 ng/ml hFSH plus100 nM PMA for 9 h as indicated. Phospho-ERK1/2 and total ERK1/2 were measured as described in Materials and Methods. Results are representative of four independent experiments.

Gonadotropins Induce Epiregulin mRNA in Immature Granulosa Cells Expressing a Low or a High Density of Gonadotropin Receptors
Because gonadotropins enhance the expression of EGF-like factors in the ovary (10) and because EGF-like factors appear to mediate the effects of gonadotropins on ERK1/2 phosphorylation in immature granulosa cells (see above), we compared the effects of hFSH and hCG on the expression of these factors in granulosa cells expressing low and high gonadotropin receptor densities. We limited our analysis to epiregulin mRNA because this is the most abundant and potent EGF-like factor detected in rat granulosa cells (28). Figure 7 shows that the expression of epiregulin mRNA is enhanced by gonadotropins in immature granulosa cells expressing a low or high density of gonadotropin receptors, but the levels of epiregulin mRNA are several-fold higher in cells expressing the high density of receptors.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Expression of Epiregulin in Immature Granulosa Cells Expressing Different Densities of Gonadotropin Receptors Primary cultures of immature granulosa cells were infected with the indicated adenoviral constructs as described in the legend to Fig. 1 and Materials and Methods. Two days after infection cells were incubated with buffer only or with 100 ng/ml hFSH or hCG for 9 h as indicated. Total RNA was collected and used to quantitate epiregulin mRNA using real-time PCR amplification as described in Materials and Methods. For easy comparison all data are expressed relative to the hFSH-induced epiregulin response of cells infected with ß-gal and incubated with hFSH (see white bar) because the levels of epiregulin mRNA in cells incubated with buffer only were often undetectable. Each bar is the mean ± SEM of four independent experiments. Within each panel, the asterisk denotes a statistically significant difference (P < 0.05, two-tailed t test) between the low and high density receptor cells incubated with a given hormone.

	DISCUSSION

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We show that hFSH and hCG provoke rapid (5–30 min) increases in ERK1/2 phosphorylation in immature granulosa cells expressing a low or a high density of their cognate receptors. Recent reports from other laboratories (16, 19, 20) showed that this rapid increase in ERK1/2 phosphorylation induced by hFSH or hCG in a number of granulosa cells models is dependent on the cAMP/PKA pathway, extracellular Ca²⁺, the Src family of kinases, MEK, and the EGFR but is independent of the PKC pathway. Using a number of pharmacological inhibitors, we confirmed and extended this knowledge. We confirm the involvement of the cAMP/PKA pathway by showing that the rapid gonadotropin-sensitive ERK1/2 response is inhibited by H89 and can be faithfully mimicked with forskolin. We also confirm the lack of involvement of PKC by showing that Ro-318220 does not inhibit this rapid gonadotropin response, and we confirm the involvement of MEK and the EGFR family by showing that UO126 and AG1478 inhibit this response. In addition, we expand these data by showing that a broad-spectrum metalloprotease inhibitor (GM6001) that inhibits the release of the precursor forms of EGF-like factors also inhibits the gonadotropin-induced rapid ERK1/2 response. Based on these results, we conclude that the main signaling pathway by which hCG and hFSH induce the rapid increase in ERK1/2 phosphorylation in immature granulosa cells is a PKA-dependent release of preexisting pro-EGF-like factors that subsequently activate endogenous EGFR-like receptors and the MEK/ERK signaling cascade. An intermediate role of precursor forms of EGF-like factors in the G protein-coupled receptor-induced activation of the ERK1/2 cascade has been previously demonstrated in a number of cells types (13, 29) but not in granulosa cells or in response to gonadotropins.

When hFSH or hCG bind to immature granulosa cells expressing a high density of their cognate receptors, they also provoke a delayed (6–9 h) increase in ERK1/2 phosphorylation. Like the rapid effect discussed above, this delayed effect is sensitive to inhibitors of PKA, MEK, the EGFR, and metalloproteases. In contrast to the rapid effect, which is insensitive to PKC inhibitors, this delayed effect is sensitive to PKC inhibitors. The delayed increase in ERK1/2 phosphorylation in immature granulosa cells expressing a high density of their cognate receptors is probably mediated by the gonadotropin-induced increase in the expression of the EGF-like factors as well as the release of these factors. Gonadotropin-induced epiregulin gene expression is higher in the high receptor density cells than in the low receptor density cells, and others have shown that there is delay of several hours needed for this effect to become detectable (30). Enhanced expression of the precursor forms of these EGF-like factors is likely to result in enhanced proteolysis by simple substrate availability, but this increase substrate availability is also likely to be potentiated by an increase in protease expression and/or an increase in their activity. Whereas cAMP is undoubtedly involved in the gonadotropin-induced expression of epiregulin (30, 31), the involvement of other signaling pathways in this gonadotropin-induced effect is also possible. As discussed above, a PKA-dependent pathway appears to be responsible for the early increase in proteolysis of the precursor forms of EGF-like factors, and this pathway also appears to be operative at later time points. It is presently unclear why the late increase in proteolysis is also dependent on PKC, however. One possibility is that different EGF-like factors mediate the early and delayed gonadotropin-sensitive ERK1/2 response and that there is a switch in the identity of the proteases that cleave the EGF-like precursors and the signaling pathways involved this release. There are several proteases that can cleave the different precursors of EGF-like factors (24), and the activation of these proteases and subsequent transactivation of EGFR-like receptors can occur through PKA- or PKC-dependent pathways, as reported elsewhere (13, 32, 33, 34, 35). Regardless of the locus of PKC in this process, its proposed involvement is consistent with the findings that 1) hCG and hFSH stimulate the inositol phosphate cascade only when acting on high receptor densities (12); 2) the delayed ERK1/2 response is detectable only in cells expressing a high receptor density or by adding hFSH and PMA to granulosa cells expressing a low density of the FSHR (this paper); 3) the delayed ERK1/2 response is not detectable in cells stimulated with cAMP analogs or forskolin (this paper); and 4) the delayed ERK1/2 response is always sensitive to PKC inhibitors (this paper).

Our results show that three distinct inhibitors that block the early and delayed gonadotropin-induced increase in ERK1/2 phosphorylation: UO126 (a MEK inhibitor), AG1478 (an EGFR kinase inhibitor), and GM6001 (a broad metalloprotease inhibitor), greatly enhance the low levels of gonadotropin-induced aromatase mRNA in immature granulosa cells expressing a high density of their cognate receptors. Perhaps more dramatically, UO126 was also shown to completely reverse the ability of PMA to prevent the induction of aromatase provoked by hFSH in immature granulosa cells expressing a low density of hFSHR. The same inhibitors tend to enhance the high levels of gonadotropin-induced aromatase mRNA in cells expressing a low density of their cognate receptors, but the magnitude of these effects was generally small and not statistically significant. These data suggest that the ERK1/2 cascade is a negative regulator of aromatase expression in granulosa cells. This conclusion is also consistent with a previous report showing that expression of constitutively active MEK suppresses FSH-stimulated estradiol synthesis in immature granulosa cells (9) and with recent data showing that inhibitors of MEK activation can enhance FSH-induced aromatase expression in Sertoli cells (11). The significant effects of all these inhibitors on aromatase expression are detected only in cells that display a delayed increase in ERK1/2 phosphorylation. In all the experiments presented here, we added the inhibitors before adding hFSH or hCG. Other experiments done with UO126 (not shown), however, showed that addition of this inhibitor up to 4 h after addition of the hormones still had the same effect on aromatase expression as that seen when the inhibitor was added before hormone addition. Together these results allow us to conclude that the delayed increase in ERK1/2 phosphorylation is ultimately responsible for the negative regulation of aromatase expression. These results also provide a direct link between the activation of the inositol phosphate cascade by high densities of gonadotropin receptors (12) and the delayed increase in ERK1/2 phosphorylation. Unfortunately, however, the effect of inhibitors of PKC on aromatase expression could not be tested because they are toxic during the long incubation times needed to detect aromatase expression.

The type of signaling pathways activated by G protein-coupled receptors in general and the gonadotropin receptors in particular can vary as the density of receptors or the amount of agonist used increase (12, 36, 37, 38). The inositol phosphate cascade is in fact a perfect example of this phenomenon. For example, in MA-10 cells or heterologous cells lines expressing the recombinant LHR, cAMP accumulation can be elicited by low concentrations of hCG at very low densities of the LHR, whereas inositol phosphate accumulation can be stimulated only at high receptor densities and with higher concentrations of hCG (36, 37, 38). Although not as well documented, the same appears to be true in granulosa cells (12). Therefore, at low signal strengths (i.e. low receptor density and/or agonist concentration) the two gonadotropins could activate common signaling pathways that are stimulatory to the expression of aromatase (such as cAMP and/or Akt) and that at high signal strengths (i.e. high receptor density and/or agonist concentration) they could also activate additional signaling pathways (such as the inositol phosphate cascade and a delayed ERK1/2 activation) that may be inhibitory to the expression of aromatase. So far our studies have focused only on aromatase, which is, of course, only one of many ovarian genes that are regulated by gonadotropins (39, 40, 41). We have not yet investigated whether the stimulatory and inhibitory pathways proposed here are unique to aromatase expression or more ubiquitously involved in the regulation of the expression of other ovarian genes.

The concentration of hCG (100 ng/ml, 2.5 nM) that was used in these experiments is similar to the dissociation constant of the hLHR for hCG (1–3 nM; see Ref. 42), and it was chosen because it is maximally effective for cAMP accumulation (which, according to our hypothesis, is the main stimulus for aromatase induction). We know little about the dose-response curves for the LHR-mediated activation of the different signaling cascades proposed to be involved as positive and negative regulators of aromatase induction, but based on studies done with the hLHR expressed in MA-10 Leydig tumor cells it is likely that they display different EC₅₀ values (38). As such, it may be possible to find concentrations of hCG that stimulate the cAMP pathway but have little or no effect on the inositol phosphate or ERK1/2 cascades. If this is indeed the case, then even at high densities of the LHR lower concentrations of hCG could, theoretically, induce aromatase expression. An alternative way to test this prediction is to express mutants of the hLHR that activate the cAMP pathway but are unable to activate the inositol phosphate or ERK1/2 cascades. These mutants should also mediate only stimulatory effect of hCG on aromatase induction. We are currently pursuing both of these avenues of investigation as an alternative means to further test our hypothesis.

In summary, the data presented here are consistent with our previously proposed hypothesis (12), but we have now identified the ERK1/2 cascade as a negative regulator of aromatase expression. Our hypothesis can thus be refined to state that the FSHR and LHR stimulate the cAMP signaling pathway, which (alone or together with Akt) is sufficient for aromatase induction, but that at high receptor densities the LHR and FSHR can also activate the inositol phosphate cascade, which mediates a delayed increase in ERK1/2 phosphorylation. The ERK1/2 cascade is the pathway that ultimately antagonizes the actions of cAMP on aromatase induction.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Viruses and Cells
The preparation of recombinant adenoviral particles coding for the hLHR (Ad-hLHR), hFSHR (Ad-hFSHR), and ß-galactosidase (Ad-ßgal) as well as the methods used to isolate, maintain, and infect primary cultures of immature rat granulosa cells have been described (12). Cells infected with Ad-ßgal at a MOI of 200 were used as controls. Other experimental groups included cells infected with Ad-hLHR at an MOI of 200 and cells infected with a mixture of the Ad-hLHR or Ad-hFSHR (at 20 MOI) and Ad-ßgal (at 180 MOI) or cells infected with a mixture of Ad-hFSHR (at 100 MOI) and Ad-ßgal (at 100 MOI). These conditions allowed us to keep a constant viral load (200 MOI) while changing the amount of recombinant receptors expressed (12).

The procedures used to isolate granulosa cells were approved by the Institutional Animal Care and Use Committee for the University of Iowa.

ERK Phosphorylation Assay
Adenoviral-infected cells (in 12-well plates) were washed twice with DMEM/F-12 medium (1:1 vol/vol) containing 10 mM HEPES, 50 µg/ml gentamicin, and 0.1% BSA and placed in 1 ml of the same medium for 18 h before any experiment. The medium was then replaced with 0.5 ml of the same medium with or without inhibitors for 1 h as described in the figure and table legends. After this, cells were incubated for the indicated times (see figure and table legends) with hFSH (100 ng/ml), hCG (100 ng/ml), PMA (100 nM), forskolin (10 µM), or buffer only.

At the end of this incubation, the cells were placed on ice; quickly washed twice with 1 ml of a cold buffer containing 150 mM NaCl, 20 mM HEPES (pH 7.4); and lysed with 35 µl of lysis buffer (150 mM NaCl, 50 mM Tris, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM Na₃VO₄, and 1 mM NaF, pH 7.4) by gentle rocking at 4 C for 40 min. The resulting lysates were clarified by centrifugation and assayed for protein content using the BCA protein assay kit from Bio-Rad Laboratories Inc. (Hercules, CA). Twenty micrograms of protein from each lysate were then resolved on 12% polyacrylamide gels and transferred electrophoretically to polyvinylidene diflouride membranes (38). This was followed by an overnight incubation of the membrane in a 1:10,000 dilution of dual phospho-specific p44/p42 (Thr202/Tyr204) ERK1/2 antibody from Cell Signaling Technology (Beverly, MA) or a 1:20,000 dilution of total ERK1/2 antibody from Santa Cruz Biotechnology (Santa Cruz, CA) followed by a second 1-h incubation with a 1:10,000 dilution of a secondary antibody covalently coupled to horseradish peroxidase (Bio-Rad Laboratories). Finally, immune complexes were visualized using the enhanced chemiluminescence detection system (Amersham Bioscience, Little Chalfont, UK) and Kodak x-ray film.

Quantitation of mRNA by Real-Time PCR
The methods and primers used to quantitate aromatase mRNA have been described (12). Epiregulin mRNA was quantitated using the same methodology, but the incubation time with hormones or other stimuli was 9 h and the primers used were 5'-CCGAGAGAAGGATGGAGACTTTC-3' for the forward primer and 5'-GGGAACCAAGGCAAAGCA-3' for the reverse primer.

Statistical Methods
To account for experimental variability, all values for aromatase and epiregulin expression were calculated relative to the value obtained in cells infected with 200 MOI Ad-ßgal and treated with FSH (which was taken as 1.0) and are thus shown in the corresponding graphs. All statistical analyses were conducted using t tests or Dunnet’s test (Table 1). In all cases, statistical significance was considered at P < 0.05.

Materials
Purified hCG (CR-127) and purified hFSH (AFP-5720D) were purchased from Dr. A. Parlow of the National Hormone and Pituitary Agency (Torrance, CA). Purified recombinant hCG and hFSH were kindly provided by Ares Serono (Randolph, MA). AG1478, AG43, GM6001, H89, UO126, GF109203X, and Ro-318220 were purchased from Calbiochem (San Diego, CA). Forskolin and PMA were from Sigma-Aldrich Corp. (St. Louis, MO). Cell culture medium was obtained from Invitrogen Corp. (Carlsbad, CA). Other supplies and reagents used for granulosa cell extraction and culture were obtained from Sigma-Aldrich Corp., BD Biosciences (San Jose, CA), or Fisher (Hampton, NH). Molecular biology reagents were obtained from Invitrogen Corp. or Roche Diagnostics Corp. (Indianapolis, IN). All other chemicals were obtained from commonly used suppliers.

	FOOTNOTES

 
This work was supported by the National Institute of Child Health and Human Development (Grant HD-28962).

First Published Online September 14, 2006

¹ Addition of cAMP analogs such as 8Br- or 8CPT-cAMP were as effective as forskolin on aromatase induction (cf. Table 1 and Ref. 12 ). A robust early increase in ERK1/2 phosphorylation was also detectable with cAMP analogs and forskolin, but it was more reproducibly obtained with forskolin. Because of these findings, we opted to use forskolin instead of cAMP analogs for all experiments designed to mimic activation of the cAMP pathway.

Abbreviations: DMSO, Dimethyl sulfoxide; EGF, epidermal growth factor; EGFR, EGF receptor; FSHR, FSH receptor; hCG, human chorionic gonadotropin; LHR, LH receptor; MEK, MAPK kinase; MOI, multiplicity of infection; PKB, protein kinase B; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate.

Received for publication June 7, 2006. Accepted for publication September 1, 2006.

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