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. 2008 Jul 24;149(11):5549–5556. doi: 10.1210/en.2008-0618

The Luteinizing Hormone Receptor-Activated Extracellularly Regulated Kinase-1/2 Cascade Stimulates Epiregulin Release from Granulosa Cells

Nebojsa Andric 1, Mario Ascoli 1
PMCID: PMC2584583  PMID: 18653716

Abstract

We examine the pathways involved in the luteinizing hormone receptor (LHR)-dependent activation of the epidermal growth factor (EGF) network using cocultures of LHR-positive granulosa cells and LHR-negative test cells expressing an EGF receptor (EGFR)-green fluorescent protein fusion protein. Activation of the LHR in granulosa cells results in the release of EGF-like growth factors that are detected by measuring the phosphorylation of the EGFR-green fluorescent protein expressed only in the LHR-negative test cells. Using neutralizing antibodies and real-time PCR, we identified epiregulin as the main EGF-like growth factor produced upon activation of the LHR expressed in immature rat granulosa cells, and we show that exclusive inhibition or activation of the ERK1/2 cascade in granulosa cells prevents or enhances epiregulin release, respectively, with little or no effect on epiregulin expression. These results show that the LHR-stimulated ERK1/2 pathway stimulates epiregulin release.

THE GONADOTROPIN-SENSITIVE ERK1/2 cascade plays important roles in the regulation of ovarian functions such as oocyte maturation (1) and steroid biosynthesis in granulosa (2,3,4,5) and theca cells (6). The initial studies on the LH receptor (LHR)-stimulated phosphorylation of ERK1/2 in the ovary emphasized the importance of the cAMP/protein kinase A (PKA) pathway as a mediator of this response (2,7,8,9,10), but more recent studies have shown that phospholipase C and members of the EGF family are also involved (3,11,12). This latter finding is of particular interest because members of the EGF-like family are now emerging as important mediators of the ovulatory actions of LH (11,13,14,15,16).

In a recent series of studies, Conti and co-workers (11,13,14,15) showed that LH/chorionic gonadotropin (CG) increase the expression of three EGF-like growth factors (amphiregulin, epiregulin, and betacellulin) in mural granulosa cells and that one or more of these growth factors activate the epidermal growth factor receptor (EGFR) in cumulus granulosa cells to induce cumulus expansion and the resumption of meiosis in oocytes. These experiments show that an increase in the expression of the EGF-like growth factors is an important component of the LHR-mediated ovulatory response (11,13) and reemphasize the importance of cAMP/PKA because inhibitors of this pathway attenuate the LHR-provoked trans-activation of the EGFR (11). These data are consistent with a model whereby the classical cAMP/PKA pathway activated by the LHR is responsible for the increase in the expression and release of EGF-like growth factors that then phosphorylate the EGF receptor and activate the Ras/Raf/MAPK kinase (MEK)/ERK1/2 cascade (17,18).

We have shown that the LHR-provoked activation of the ERK1/2 cascade in cultures of granulosa cells is biphasic with a fast wave that reaches a maximum within 15–30 min of human CG (hCG) addition and a slow one that reaches a maximum 6–9 h later (3). We also showed that both of these waves are sensitive to PKA, metalloprotease, or EGFR kinase inhibitors (3). More recently, we reported that two mutants of the LHR that fail to stimulate the two waves of ERK1/2 phosphorylation also fail to increase the accumulation of bioactive EGF-like growth factors in granulosa cells (12). These results are consistent with the model described above whereby activation of the LHR results in an increase in the synthesis and release of EGF-like growth factors that then activate the EGFR and thus enhance ERK1/2 phosphorylation. Conversely, because MAPK signaling can regulate the shedding of EGF-like growth factors in transfected cells (19,20), it is also possible that the lack of stimulation of the ERK1/2 cascade by the LHR mutants is responsible for the lack of accumulation of EGF-like growth factors. In this case, the ERK1/2 response would be downstream of the activation of the LHR but upstream of the activation of the EGFR. To distinguish between these two possibilities, we employed a previously described coculture system whereby LHR-positive granulosa cells are cultured with LHR-negative test cells expressing an EGFR-green fluorescent protein (GFP) fusion protein (12,21). In these cocultures, the LHR-dependent production of extracellular EGF-like growth factors released from granulosa cells is detected by measuring the phosphorylation of the EGFR-GFP fusion protein that is expressed only in the LHR-negative test cells. Using this experimental paradigm, we show that ERK1/2 phosphorylation activates epiregulin release without affecting epiregulin synthesis and that the LHR- dependent early wave of ERK1/2 phosphorylation is necessary for epiregulin release in granulosa cells.

Materials and Methods

Materials

Purified recombinant hCG was kindly provided by Ares Serono (Randolph, MA). Cell culture medium and other cell culture reagents were obtained from Invitrogen Corp. (Carlsbad, CA). GM6001 and UO126 were purchased from Calbiochem (La Jolla, CA). Neutralizing antibodies to epiregulin and anti-TGFα (catalog nos. AF1068 and AF-239-NA, respectively) were obtained from R&D Systems (Minneapolis, MN). All other chemicals were obtained from commonly used suppliers.

Viruses, plasmids, and cells

Recombinant adenoviral particles coding for the hLHR (Ad-hLHR) and β-galactosidase (Ad-βgal) have been described (3,22). Adenoviruses coding for dominant-negative (DN) MEK (Ad-DNMEK) or constitutively active (CA) MEK (Ad-CAMEK) were donated to us by Dr. Stefan Strack (The University of Iowa) and Dr. Tony Zeleznik (University of Pittsburgh), respectively. An expression vector for a C-terminally GFP-tagged form of the human EGFR (EGFR-GFP) was donated to us by Dr. John Koland (The University of Iowa).

I-10 cells (CCL-83), a clonal strain of Leydig tumor cells that lack the LHR (23,24), were purchased from the American Type Culture Collection (Rockville, MD) and maintained in DMEM/F12 medium supplemented with 15% horse serum, 20 mm HEPES, and 50 μg/ml gentamicin (pH 7.4).

The methods used to isolate, maintain, and infect primary cultures of immature rat granulosa cells have been previously described (3,22). These protocols were approved by the Institutional Animal Care and Use Committee of The University of Iowa.

Bioassay for EGF-like growth factors

The bioassay used for detection of EGF-like growth factors has been previously described by us (12,21). Briefly, hLHR-positive granulosa cells were cocultured with LHR-negative I-10 Leydig tumor cells expressing the recombinant EGFR-GFP (henceforth referred to as test cells). Test cells were transfected with the EGFR-GFP using Lipofectamine as described elsewhere (21,25). The transfected test cells were trypsinized and plated (4 × 105 cells) on six-well plates already containing 2 × 105 granulosa cells that had been infected 1 d earlier with different adenoviral constructs as described earlier (22) and as indicated in the figure legends. Ad-hLHR, Ad-DNMEK, or Ad-CAMEK were used at a multiplicity of infection (MOI) or 200, 100, and 100, respectively, but the total amount of virus used for each infection was adjusted as needed with Ad-βgal to give a constant MOI of 300. When Ad-hLHR is used at a MOI of 200, the density of hLHR expressed in the granulosa cells is similar to that expressed in granulosa cells in intact rats at the time of ovulation (3,12,22).

The cocultures were maintained for 8 h in DMEM/F12 containing 10 mm HEPES, 0.1% BSA, insulin (1 μg/ml), transferrin (1 μg/ml), selenium (1 ng/ml), penicillin (100 IU/ml), and streptomycin (0.1 mg/ml). The medium was then changed to DMEM/F12 containing 10 mm HEPES and 0.1% BSA, and the cells were incubated overnight before use.

Western blots

Lysates were prepared, resolved on SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride membranes as described before (3,12,25). The blots were first incubated overnight at 4 C with a 1:1000 dilution of a rabbit antibody to phosphotyrosine 1068 of the EGFR (Cell Signaling Technology, Beverly, MA; catalog no. 2234) followed by a second 1-h incubation with a 1:3000 dilution of a secondary antibody covalently coupled to horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA; catalog no. 170-6515). The immune complexes in the Western blots were visualized using the Super Signal West Femto Maximum Sensitivity detection system (Pierce Chemical, Rockford, IL). Images were captured digitally and quantitated with a Kodak Digital Imaging system (Eastman Kodak, Rochester, NY). After detection of the phospho-EGFR signal, the blots were stripped (12) and incubated with a 1:2000 dilution of a goat anti-GFP antibody coupled to horseradish peroxidase (from Abcam, Cambridge, MA; catalog no. ab6663-1000) during an overnight incubation at 4 C. This immune complex was directly detected as described above. The phospho-EGFR-GFP signal was corrected for the amount of EGFR-GFP present in the blot. Note that this assay does not detect the phosphorylation of the endogenous EGFR in granulosa or test cells because the GFP-tagged EGFR has a higher molecular weight than the endogenous EGFR, and these two products can be readily separated using SDS-PAGE (21).

Phosphorylated ERK1/2 was detected as described previously (3). The expression of MEK1/2 was detected by an overnight incubation (4 C) of the blots with a 1:5000 dilution of MEK1/2 antibody (Cell Signaling; catalog no. 9122) followed by a 1-h incubation with a 1:10,000 dilution of a secondary antibody covalently coupled to horseradish peroxidase.

Real-time PCR

Detection of mRNA for epiregulin, amphiregulin, betacellulin, and GAPDH was done using real-time PCR as previously described (3,22). The sequences of the primers used to amplify epiregulin and GAPDH have been published (3,22). Amphiregulin primers were 5′-TTCGCTGAACCTCTCAGT-3′ (forward) and 5′-CCAACCCGACTGCATAATGG-3′ (reverse), and betacellulin primers were 5-GGTGCCCCAAGCAGTCCAAG-3′ (forward) and 5′-TTGCAATTCCACCACGAA-3′ (reverse). The relative expression of epiregulin and amphiregulin was calculated using the Δ-Δ Ct method (26).

Statistical analysis

Results were analyzed using ANOVA followed by Tukey’s post hoc test or paired t test as described in the figure legends using the Prism software package (GraphPad Software, San Diego, CA). In all cases, statistical significance was taken at P < 0.05.

Results

Activation of the hLHR in granulosa cells provokes phosphorylation of the EGFR on adjacent test cells

To examine the molecular basis of the LHR-induced shedding of EGF-like growth factors from granulosa cells, we employed a previously described coculture system where soluble EGF-like growth factors released by gonadotropin stimulation of gonadal cells are detected by an increase in the phosphorylation of the EGFR in adjacent cells that are insensitive to gonadotropin stimulation (12,21). For this purpose, primary cultures of immature rat granulosa cells were first infected with Ad-hLHR to express a density of hLHR similar to that detected in intact rats at the time of ovulation (3,12,22). These cells were then cocultured with a test cell line (I-10 cells) that do not express the LHR but express a GFP-tagged form of the EGFR (12,21). Because the test cells are unresponsive to hCG (21,23,24) but express the EGFR-GFP, any hCG-induced phosphorylation of the EGFR-GFP in the test cells must originate from extracellular EGF-like growth factors that are produced by the actions of hCG on the granulosa cells. We used this system instead of analyzing the phosphorylation of the endogenous EGFR in granulosa cells because gonadotropins activate the Src family of kinases (27,28,29,30), and these kinases can directly phosphorylate the EGFR (31). Thus, an hCG-mediated increase in the phosphorylation of the endogenous EGFR in granulosa cells is not necessarily mediated by the actions of extracellular EGF-like growth factors. In contrast, the hCG-induced phosphorylation of the EGFR-GFP in the test cells cannot occur by intracellular signaling pathways, and it must be mediated by extracellular factors.

Figure 1A shows that hCG-induced activation of the LHR in granulosa cells provokes phosphorylation of the EGFR on adjacent test cells. This is a relatively slow process with maximal phosphorylation occurring after 9 h (longer incubation times were not tested). As a control, cultures of test cells alone were stimulated with hCG or hFSH for 9 h (to reproduce the conditions used in the cocultures) or with EGF for 15 min. As expected, only EGF provoked a strong phosphorylation of the EGFR-GFP in cultures of test cells (Fig. 1B). A shorter incubation of test cells with hCG (15 min) was also ineffective in stimulating the phosphorylation of the EGFR-GFP (21), and longer incubations with EGF resulted in suboptimal phosphorylation of the EGFR over that shown (data not presented).

Figure 1.

Figure 1

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hCG-induces trans-phosphorylation of the EGFR in test cells when cocultured with granulosa cells expressing the hLHR. A, Cocultures of test cells expressing the EGFR-GFP and primary granulosa cells expressing the hLHR were incubated with buffer or hCG (100 ng/ml) for the indicated times before measuring phosphorylated and total EGFR-GFP in the lysates. The blots shown are results of a representative experiment whereas the graphs show the average ± sem of four independent experiments. B, Cultures of test cells expressing the EGFR-GFP were incubated with buffer only (C), with 100 ng/ml hCG or hFSH for 9 h, or with 100 ng/ml EGF for 15 min as indicated. The blot shown is representative of four experiments with similar results. We have previously noted a reduction in the level of total EGFR after stimulation with EGF (28). This is likely due to EGF-induced internalization and degradation of the EGFR.

The time course of hCG-induced trans-activation of the EGFR in these cocultures is slower than that reported in the ovary upon injection of hCG (13) or when LH is added to cultures of ovarian follicles (11). We presume that this slow time course observed in the cocultures is a reflection of the levels of extracellular EGF-like growth factors that accumulate in the medium. As shown in Fig. 1B, it takes as little as 15 min to detect phosphorylation of the EGFR upon addition of EGF, but it may take several hours to reach a threshold level of extracellular EGF-like growth factors that can enhance the phosphorylation of the EGFR-GFP in test cells.

Activation of ERK1/2 in granulosa cells is required for phosphorylation of the EGFR on adjacent test cells

Recently, we showed that mutants of the hLHR that do not stimulate phospholipase C or ERK1/2 phosphorylation fail to provoke phosphorylation of the EGFR on adjacent test cells (12). The experiments described here were done to test for the potential involvement of the ERK1/2 pathway on the hCG-induced trans-phosphorylation of the EGFR on adjacent test cells.

We first inhibited ERK1/2 activation in the granulosa cells by expression of a DNMEK and stimulated the cells with hCG for 9 h. Figure 2A shows that DNMEK is robustly expressed in the infected granulosa cells and that it completely prevents the hCG-induced phosphorylation of ERK1/2. (Although only the late wave of ERK1/2 phosphorylation is shown, DNMEK also inhibits the early wave of ERK1/2 phosphorylation.) Furthermore, Fig. 2B shows that when granulosa cells expressing the DNMEK are cocultured with test cells, hCG no longer increases the phosphorylation of the EGFR-GFP in the test cells. We also tested the effects of DNMEK on hCG-induced epiregulin expression because hCG increases the expression of epiregulin in granulosa cells (3,11,12,14,32), and this increased expression of epiregulin has been implicated in EGFR trans-activation (11,32). Figure 2C shows that hCG can still robustly induce epiregulin expression in granulosa cells expressing DNMEK, but the induction is slightly reduced compared with that of cells not expressing DNMEK.

Figure 2.

Figure 2

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The selective inhibition of ERK1/2 phosphorylation in granulosa cells blocks the hCG-induced trans-phosphorylation of the EGFR in test cells. A, Primary cultures of granulosa cells expressing the hLHR alone or together with DNMEK were incubated with buffer only (white bars) or 100 ng/ml hCG (black bars) for 9 h before measuring phosphorylated ERK1/2, total ERK2, and total MEK1/2 in the lysates. The blots shown are results of a representative experiment, whereas the graphs show the average ± sem of four independent experiments. Means with different letters are significantly different from each other (P < 0.05, ANOVA). B, Test cells expressing the EGFR-GFP were cocultured with primary granulosa cells expressing the hLHR only, or the hLHR and DNMEK as indicated. The cocultures were then incubated with buffer only (white bars) or 100 ng/ml hCG (black bars) for 9 h before measuring the phosphorylated and total EGFR-GFP. The blots shown are results of a representative experiment, whereas the graphs show the average ± sem of three independent experiments. Means with different letters are significantly different from each other (P < 0.05, ANOVA). C, Primary cultures of granulosa cells expressing the hLHR alone or together with DNMEK were incubated with buffer only (white bars) or 100 ng/ml hCG (black bars) for 9 h. Epiregulin and GAPDH mRNA were quantitated using RT followed by real-time PCR as described in Materials and Methods. Each bar is the mean ± sem of five to six independent experiments. Means with different letters are significantly different from each other (P < 0.05, ANOVA).

Because hCG-induces two waves of ERK1/2 phosphorylation in granulosa cells (Fig. 3A) (3), we added a pharmacological inhibitor of ERK1/2 (UO126) to the cocultures at different times to gain a better understanding of the timing of the phospho-ERK1/2 response involved in the trans-phosphorylation of the EGFR in test cells (Fig. 3B). Addition of UO126 to the cocultures of granulosa and test cells inhibits the trans-phosphorylation of the EGFR in test cells only when the compound is added before hCG or up to 2 h after addition of hCG (shown by comparing the dashed line with the squares with asterisks in Fig. 3B). If UO126 is added 4 h after addition of hCG, it does not inhibit the trans-phosphorylation of the EGFR in test cells (shown by comparing the dashed line with the squares without asterisks in Fig. 3B). When UO126 is added before the addition of hCG or together with hCG, it effectively blocks the fast and slow waves of ERK1/2 phosphorylation (3). When added at later times after the addition of hCG, however, it blocks only the wave of phosphorylation of ERK1/2 that would be detectable after its addition. We note that the inhibitory effect of UO126 on the trans-phosphorylation of the EGFR in test cells (Fig. 3B) was partial, whereas the inhibitory effect of the expression of DNMEK in granulosa cells was complete (Fig. 2B). We did not seek an explanation for this difference. These results show that the early but not the late wave of ERK1/2 phosphorylation is upstream of the EGFR phosphorylation.

Figure 3.

Figure 3

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The hCG-induced trans-phosphorylation of the EGFR in test cells depends on the early wave of ERK1/2 phosphorylation in granulosa cells. A, Primary cultures of granulosa cells expressing the hLHR were incubated with 100 ng/ml hCG for the times indicated. The blot of phosphorylated ERK1/2 and total ERK2 is representative of four experiments. B, Cocultures of test cells expressing the EGFR-GFP and primary granulosa cells expressing the hLHR were incubated without a MEK inhibitor (dashed horizontal line) or with a MEK inhibitor (UO126, 10 μm) added at the times indicated. hCG (100 ng/ml) was added to all the cultures at time zero (indicated by the vertical arrow), and the phosphorylated and total EGFR-GFP was measured at 9 h. Each point is the mean ± sem of three to four experiments. Asterisks denote significant differences between the cocultures incubated with hCG and UO126 and those incubated with hCG but without UO126 (t test, P < 0.05).

The contribution of the ERK1/2 signaling cascade in the hLHR-induced trans-activation of the EGFR on adjacent test cells was further explored by infection of granulosa cells with an adenoviral construct encoding for a constitutively active form of MEK (Ad-CAMEK). Infection of granulosa cells with Ad-CAMEK provoked sustained ERK1/2 phosphorylation as shown in Fig. 4A. When the Ad-CAMEK-infected granulosa cells were cocultured with test cells, a sustained phosphorylation of the EGFR in the test cells was also readily detected (Fig. 4B). Ad-CAMEK did not stimulate epiregulin expression in granulosa cells, however (Fig. 4C).

Figure 4.

Figure 4

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The selective activation of ERK1/2 in granulosa cells provokes the trans-phosphorylation of the EGFR in test cells in the absence of hCG stimulation. A, Primary cultures of granulosa cells were infected with Ad-hLHR and Ad-βgal alone or Ad-hLHR and Ad-CAMEK as indicated. Phosphorylated ERK1/2, total ERK2, and total MEK1/2 were measured using Western blots at different times after infection. The blot shown is representative of four experiments with similar results. B, Test cells expressing the EGFR-GFP were cocultured with primary granulosa cells expressing the hLHR and βgal (white symbols) or the hLHR and CAMEK (black symbols) at time zero. The phosphorylated and total EGFR-GFP was measured at different times after the cocultures were initiated. Note that the three time points shown in this panel correspond to the same time points shown in A and C for the individual cultures of granulosa cells. Each point is the mean ± sem of three independent experiments. Asterisks denote significant differences between the cells expressing CAMEK or βgal at a given time point (t test, P < 0.05). C, Primary cultures of granulosa cells were infected with Ad-hLHR and Ad-bgal (white symbols) or Ad-hLHR and Ad-CAMEK (black symbols) as indicated. Epiregulin and GAPDH mRNA were quantitated using RT followed by real-time PCR as described in Materials and Methods. Each point is the mean ± sem of five to six independent experiments. Asterisks denote significant differences between the cells expressing CAMEK or βgal at a given time point (t test, P < 0.05).

Taken together, these results show that activation of ERK1/2 signaling in granulosa cells is required for the trans-activation of the EGFR on adjacent test cells. Furthermore, the results obtained with DNMEK (Fig. 3) and CAMEK (Fig. 4) show that the LHR-provoked trans-phosphorylation of the EGFR occurs independently of changes in epiregulin expression.

Epiregulin is responsible for the LHR-provoked trans-phosphorylation of the EGFR on adjacent test cells

Recent studies measuring the effects of gonadotropins on the expression of ovarian EGF-like growth factors (3,12,13,14,33,34), the ovulatory response of epiregulin or amphiregulin null mice (15), and the effects of mixtures of neutralizing antibodies on granulosa cell cultures (11) have implicated amphiregulin, epiregulin, and betacellulin as the EGF-like growth factors that mediate the ovarian actions of hCG. To characterize the EGF-like growth factors that may be present in immature rat granulosa cells, we first measured the expression of these three growth factors in primary cultures of immature rat granulosa cells expressing the hLHR and stimulated with hCG. The results presented in Table 1 show that the expression of epiregulin mRNA is about 2000-fold higher than that of amphiregulin and that betacellulin mRNA is not detectable.

Table 1.

Relative expression of EGF-like growth factors in immature rat granulosa cells

mRNA measured Relative expression (×100)
Epiregulin 29.4 ± 5.5
Amphiregulin 0.016 ± 0.003
Betacellulin NDa
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Primeary cultures of granulosa cells expressing the hLHR were incubated with 100 ng/ml hCG for 9 h. Epiregulin, amphiregulin, betacellulin, and GAPDH mRNA were quantitated using RT followed by real-time PCR as described in Materials and Methods. The relative expression of epiregulin and amphiregulin was calculated as described in Materials and Methods. The results presented are the mean ± sem of seven independent experiments. 

a

Not detectable at the maximal number of cycles (∼40) used. 

To define which growth factors participate in the hLHR-induced trans-activation of the EGFR on test cells, cocultures of granulosa and test cells were incubated with hCG for 9 h in the presence of neutralizing antibodies. Figure 5A shows that addition of epiregulin-neutralizing antibody completely blocked the hCG-induced trans-phosphorylation of the EGFR in test cells. In contrast, a neutralizing antibody to TGFα, did not prevent hCG-induced trans-activation of the EGFR on test cells (Fig. 5B).

Figure 5.

Figure 5

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The hCG-induced trans-phosphorylation of the EGFR in test cells is blocked by an epiregulin antibody. Cocultures of test cells expressing the EGFR-GFP and primary immature granulosa cells expressing the hLHR were incubated without or with neutralizing antibodies (12.5 μg/ml) to epiregulin (A) or TGFα (B) for 15 min as indicated. Buffer (white bars) or 100 ng/ml hCG (black bars) was then added, and the incubation was continued for 9 h before measuring the phosphorylated and total EGFR-GFP. The blots shown are results of a representative experiment, whereas the graphs show the average ± sem of three independent experiments. Means with different letters are significantly different from each other (P < 0.05, ANOVA).

To further test whether activation of ERK1/2 signaling is involved in the release of epiregulin, granulosa cells expressing CAMEK were cocultured with test cells in the presence of an epiregulin-neutralizing antibody. As shown in Fig. 6, addition of epiregulin-neutralizing antibody prevented CAMEK-induced trans-phosphorylation of EGFR on test cells.

Figure 6.

Figure 6

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The trans-phosphorylation of the EGFR in test cells provoked by the selective phosphorylation of ERK1/2 in granulosa cells is blocked by an epiregulin antibody. Test cells expressing the EGFR-GFP were cocultured with primary granulosa cells expressing βgal (white bars) or CAMEK (black bars). Buffer or an antibody to epiregulin (12.5 μg/ml) was added as indicated at the beginning of the coculture and then again 8 h later. The phosphorylated EGFR-GFP and total EGFR-GFP were measured 12 h after the second addition of antibody. The blots shown are results of a representative experiment, whereas the graphs show the average ± sem of three independent experiments. Means with different letters are significantly different from each other (P < 0.05, ANOVA).

A metalloprotease is required for the LHR or ERK1/2-induced release of epiregulin

It has been shown that the LHR-induced trans-activation of the EGFR and other actions of LH/CG in granulosa cells can be inhibited with GM6001, a metalloprotease inhibitor (11,16). To determine whether enzymatic release of epiregulin is required for phosphorylation of the EGFR in adjacent test cells, we tested the effects of GM6001 on the trans-phosphorylation of the EGFR in hCG-stimulated cocultures and in cocultures of test cells and granulosa cells expressing CAMEK. GM6001 was an effective inhibitor of EGFR trans-phosphorylation in both cases (Fig. 7).

Figure 7.

Figure 7

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The hCG- or CAMEK-induced trans-phosphorylation of the EGFR in test cells is prevented by GM6001. A, Test cells expressing the EGFR-GFP were cocultured with primary granulosa cells expressing the hLHR. The cocultures were preincubated with GM6001 (20 μm) for 15 min before addition of buffer (white bars) or 100 ng/ml hCG (black bars). Phosphorylated and total EGFR-GFP was measured 9 h later. The blots shown are results of a representative experiment, whereas the graphs show the average ± sem of three independent experiments. Means with different letters are significantly different from each other (P < 0.05, ANOVA). B, Test cells expressing the EGFR-GFP were cocultured with primary rat granulosa cells expressing βgal (white bars) or CAMEK (black bars). Dimethylsulfoxide or GM6001 (20 μm) was added at the beginning of the coculture period and then again 8 h later. The phosphorylated EGFR-GFP and total EGFR-GFP were measured 12 h after the second addition of GM6001. The blots shown are results of a representative experiment, whereas the graphs show the average ± sem of three independent experiments. Means with different letters are significantly different from each other (P < 0.05, ANOVA).

Discussion

Although it is clear that EGF-like growth factors and the EGFR are important mediators of the actions of LH/CG in the ovary (11,13,14,15) much remains to be learned about the mechanisms by which the LHR activates the EGF network. Previous studies from a number of laboratories have shown that the cAMP/PKA and the inositol phosphate signaling systems as well as the EGF network are important mediators of the LHR-activated ERK1/2 response in the ovary (2,3,7,8,9,10,11,12). Our data show that these seemingly distinct signaling pathways act in concert to elicit the fast and slow waves of the LHR-dependent ERK1/2 response detected in granulosa cells (3).

The initial wave of the ERK1/2 response elicited by the LHR is detected within minutes of stimulation and is partly mediated by the increased levels of cAMP that follow activation of the LHR (2,3,7,8,9,10). Although the mechanisms by which cAMP activates the ovarian ERK1/2 cascade are not completely understood, all investigators agree that this pathway is activated by cAMP using PKA as an intermediate (2,3,7,8,9,10). Activation of the LHR also results in increased expression of amphiregulin, epiregulin, and betacellulin in granulosa cells (3,13,14,33,34) by a cAMP/PKA-dependent pathway (11,34,35). Although increased expression of these EGF-like growth factors can be detected within minutes of stimulation (11) maximal expression occurs several hours later (11,13,14). Thus, the initial stimulation of the cAMP/PKA pathway that follows activation of the LHR results in a rapid increase in the phosphorylation of ERK1/2 and a slower increase in the expression of EGF-like growth factors.

Our data show that activation of the LHR in granulosa cells results in an increase in both epiregulin expression and release and that selective inhibition of the ERK1/2 cascade leads to inhibition of the hCG-provoked epiregulin release with minimal effects on epiregulin expression. Moreover, we show that expression of a constitutively active MEK increases epiregulin release without major changes in epiregulin expression. We thus conclude that the increase in epiregulin release induced by addition of hCG is mediated by the ERK1/2 cascade, whereas the increase in epiregulin expression is not. We can also conclude that only the first wave of hCG-stimulated ERK1/2 phosphorylation is involved in stimulating epiregulin release because UO126 inhibits the stimulatory effect of hCG on epiregulin release only when added within about 2 h of addition of hCG. Because the cAMP-activated PKA is necessary for epiregulin expression and is involved in the early wave of ERK1/2 activation, our data are consistent with a central role for the involvement of the cAMP/PKA pathway as an upstream mediator of all these responses.

The release of epiregulin induced by hCG or by activation of the ERK1/2 cascade is metalloprotease dependent because it can be blocked with GM6001. It is, however, difficult to investigate the molecular basis of the ERK1/2-dependent release of epiregulin in granulosa cells because the enzyme involved in this process has not been identified. Because the effects of hCG or ERK1/2 activation on epiregulin release from granulosa cells are relatively slow, one could argue that the ERK1/2 cascade is involved in regulating the expression or transport rather than the activity of the metalloprotease involved. In fact, the expression of a number of metalloproteases is known to be under control of gonadotropins in the ovary (36,37). A direct effect of ERK1/2 on regulating the activity of ovarian metalloproteases should also be considered because ERK1/2 has been reported to directly phosphorylate and activate TNFα-converting enzyme (20), a metalloprotease involved in cleaving EGF-like growth factors in many cells (38,39). In addition, activation of MAPK cascades has been shown to stimulate the rapid release of the soluble form TGFα in transfected cells (19).

Amphiregulin, epiregulin, and betacellulin have all been implicated as mediators of the actions of LH/CG in the rodent ovary, but their relative contributions are not well understood (3,11,13,14,15,33,34). Studies with epiregulin and amphiregulin null mice show that both are involved in the actions of LH (15), and the only previous study using neutralizing antibodies to EGF-like growth factors does not provide information about this subject because it used a combination of antibodies to epiregulin, amphiregulin, and betacellulin rather than individual antibodies (11). Our data identify epiregulin as the most abundant EGF-like growth factor expressed in primary cultures of immature rat granulosa cells and as the main EGF-like growth factor released in response to hCG stimulation. Using semiquantitative PCR, others have also reported that the expression of epiregulin is higher than the expression of amphiregulin or betacellulin in rat preovulatory follicles (14). We have also recently shown by real-time PCR (data not presented) that the expression of epiregulin is about 10 times higher than that of amphiregulin in the ovaries of immature mice injected with pregnant mare serum gonadotropin (PMSG). Therefore, the preeminent role of epiregulin reported here may be related to the use of rat rather than mouse granulosa cells and to the state of differentiation of the granulosa cells. Whereas we have used granulosa cells isolated from immature rats for all our studies (this paper and Refs. 3,12, and 22), all other studies have been done with ovaries, follicles, or granulosa cells of rats or mice that have been injected with PMSG alone or PMSG and hCG (11,13,14,15,33,34).

The fast and slow waves of hCG-stimulated ERK1/2 phosphorylation in granulosa cells are dependent on multiple signaling pathways as shown by their sensitivity to PKA, metalloprotease, and EGFR kinase inhibitors and to LHR mutants that do not activate phospholipase C (2,3,7,8,9,10,11,12). In contrast to the fast wave, however, the slow wave is also sensitive to inhibitors of PKC (3). The involvement of PKC in this process has not yet been investigated in detail. Some possibilities, however, include a costimulatory role of PKC and PKA in the expression of epiregulin (37) (40) or a costimulatory role of PKC and ERK1/2 on the level or activity of enzymes that participate in epiregulin release (37).

In summary, our studies lead to the identification of epiregulin as the main EGF-like growth factor produced in response to activation of the LHR in granulosa cells and the LHR-sensitive ERK1/2 cascade as an upstream regulator of epiregulin release. Because the release of epiregulin activates the EGFR and the ERK1/2 cascade is a prominent downstream effector of EGFR activation (17,18), the epiregulin released could contribute to the late wave of ERK1/2 phosphorylation.

Acknowledgments

We thank Drs. John Koland, Stefan Strack, and Tony Zeleznik for providing some of the constructs used in these experiments. We also acknowledge the Gene Transfer and Vector Core of the University of Iowa for preparing the adenoviral particles.

Footnotes

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

Disclosure Statement: The authors have nothing to declare.

First Published Online July 24, 2008

Abbreviations: Ad, Adenoviral; CA, constitutively active; CG, chorionic gonadotropin; DN, dominant negative; EGFR, epidermal growth factor receptor; βgal, β-galactosidase; GFP, green fluorescent protein; hCG, human CG; MEK, MAPK kinase; MOI, multiplicity of infection; PKA, protein kinase A.

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