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

Abstract

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.

Figure 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 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.

Figure 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 DMSO (Buffer control) or AG1478 (10 μM), AG43 (10 μM), GM6001 (20 μM), H89 (50 μM) or UO126 (25 μM) for 1h. 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.

Figure 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.

Figure 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 1h. 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.

Figure 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 1h with 25 μM UO126 (panel A), 20 μM GM6001 (panel B), or 10 μM AG1478 or AG43 (panel C). Cells preincubated without inhibitors received DMSO only. The cells were then stimulated with hFSH (100 ng/ml) or hCG (100 ng/ml) for 48h 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-βgal 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 indicated 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.

Figure 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 1h with 25 μM UO126 (panel A), 20 μM GM6001 (panel B), or 10 μM AG1478 or AG43 (panel C). Cells preincubated without inhibitors received DMSO only. The cells were then stimulated with hFSH (100 ng/ml) or hCG (100 ng/ml) for 48h 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-βgal 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 indicated 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.

Figure 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 1h with 25 μM UO126 (panel A), 20 μM GM6001 (panel B), or 10 μM AG1478 or AG43 (panel C). Cells preincubated without inhibitors received DMSO only. The cells were then stimulated with hFSH (100 ng/ml) or hCG (100 ng/ml) for 48h 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-βgal 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 indicated 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.

Figure 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-βgal as described in Figure 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 Figure 5 all data are expressed relative to the hFSH-induced aromatase response in cells infected with β-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-βgal as described in Materials and Methods and preincubated with 25 μM UO126 (B) or 10 μM Ro-318820 (C) for 1h 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.

Figure 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 9h 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 β-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.