Identification of ERK and JNK as signaling mediators on protein kinase C activation in cultured granulosa cells

https://doi.org/10.1016/j.mce.2008.07.011Get rights and content

Abstract

PKC signaling is critical for follicular development and the induction of ovulatory genes including Pgr, Prkg2, and Cyp11a1 (SCC). We investigated PKC signaling mechanisms in the JC-410 porcine granulosa cell line stably expressing an SCC-luciferase reporter gene containing 2 kb of the porcine SCC promoter. Addition of phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C, induced the promoter ∼6-fold over the basal levels in 4 h. This effect was predominantly mediated by the PKC β and δ isoforms. PMA-mediated induction of the SCC promoter was sensitive to inhibition of ERK1/2 or JNK. Inhibition of p38 MAP kinase or Src tyrosine kinase did not alter the PMA-mediated inducibility of the promoter. SCC promoter induction in response to PMA treatment required basal EGF-receptor activity, but did not involve ectodomain shedding. Western blot analyses using phospho-specific antibodies showed that PMA treatment of JC-410 cells induced phosphorylation of MEK1/2, ERK1/2, and its downstream target p90 RSK at 15 min. We also documented the rapid phosphorylation of JNK1/2 in response to PMA treatment. Phosphorylation of ERK and JNK was robust and sustained in contrast to activation of PKA and EGF-receptor signaling in these cells. Pretreatment of JC-410 granulosa cells with IGF-1 had a synergistic effect on PMA-mediated induction of the SCC promoter. We demonstrated the importance of PMA activation of ERK signaling and the synergism with IGF-1 by showing similar responses for Prkg2 expression in primary granulosa cells. In conclusion, our studies demonstrated PMA activation of ERK and JNK signaling which is relevant in the regulation of gene expression during follicular development, ovulation, and luteinization.

Introduction

Follicular development, ovulation, and luteinization are primarily mediated by the gonadotropins and involve cross-talk between different signaling mechanisms (Richards et al., 2002). The role of cAMP signaling in follicular development and ovulation has been well investigated (Richards, 2001). Recent evidence also indicates a functional role for EGF-like growth factor (Hsieh et al., 2007, Park et al., 2004, Wayne et al., 2007) and cGMP signaling in these processes (LaPolt et al., 2003, Sriraman et al., 2006). However, the role of protein kinase C (PKC) signaling in this process is poorly understood. There are several isoforms of protein kinase C, which are divided into three subtypes based on allosteric activators (Corbalan-Garcia and Gomez-Fernandez, 2006): (a) conventional isoforms that require Ca2+ and diacylglycerol (DAG) for their activation (PKC α, β1, β2, and γ), (b) novel isoforms that require DAG for activation (PKC μ, η, θ, ɛ, and δ), and (c) atypical isoforms that require neither Ca2+ nor DAG for activation (PKC ι, λ, and ζ).

Earlier studies documented the importance of PKC signaling in ovulation in rabbits by administering calphostin C with hCG, which resulted in the inhibition of ovulation (Kaufman et al., 1992). In addition, the administration of the PKC antagonist H-7 to hormone-primed rats blocked ovulation, which was attributed to PKC effects on tissue plasminogen activator expression (Shimamoto et al., 1993). Also, blocking PKC action in cultured granulosa cells inhibited the gonodotropin-induced expression of Ptgs2, an enzyme that is critical for the induction of ovulation (Morris and Richards, 1995). This inhibition was also associated with blocking the luteinization of granulosa cells. In addition to the induction of Ptgs2, PKC activation is critical for the maximal induction of LH-induced ovulatory genes in primary granulosa cells, namely Pgr (Sriraman et al., 2003), Adamts-1 (Doyle et al., 2004), Snap25 (Shimada et al., 2007), and Prkg2 (Sriraman et al., 2006). Interestingly, studies on primary granulosa cells by Salvador et al. (1992) indicated that acute signaling of the LH receptor is independent of PKC activation (Salvador et al., 2002). In their studies, PKCs were partially activated in the absence of LH receptor activation, and LH receptor activation did not elicit further detectable PKC activation (Salvador et al., 2002). However, studies of primary granulosa cells subjected to longer LH treatment times showed delayed activation of ERK, partially due to PKC activation in response to LH (Andric and Ascoli, 2006). PKC δ was found to be induced when corpora lutea form and may transduce prolactin signaling in granulosa cells (Peters et al., 1999).

Follicle stimulating hormone (FSH) induces c-fos mRNA expression by PKC-dependent mechanisms (Pennybacker and Herman, 1991). Recent studies indicate that the FSH-mediated sustained phosphorylation of ERK requires PKC activation independent of EGF-receptor kinase, which involves RAF, MEK, and phospholipase A1 (Yang and Roy, 2006). Despite the fact that LH and FSH do not induce remarkable PKC activation in treated granulosa cells, follicular development and ovulation are dependent on PKC activation, and PKC signaling exhibits differential effects based on the stage of differentiation of granulosa cells.

As mentioned earlier, our studies document that the maximal induction of Prkg2 in primary granulosa cell culture requires the activation of PKC. Activation of EGF-like growth factor signaling did not induce the expression of Prkg2 similar to PKC activation and, on the contrary, activation of protein kinase A had no effects on Prkg2 inducibility (Sriraman et al., 2006). These observations and the fact that PKC has a critical role in follicular maturation and ovulation prompted us to determine the activators and mediators of PKC signaling in granulosa cells. Acute activation of PKC has been shown to induce Cyp11a1 (SCC), the rate limiting step in entry of cholesterol into the steroidogenic pathway, in porcine granulosa cells (Lahav et al., 1995), and we chose this promoter to understand the effects of PKC activation on gene expression. Toward this goal we employed the JC-410 porcine granulosa cell line, in which 2 kb of the SCC promoter is linked to a luciferase reporter stably integrated into chromosomal DNA. Specific pharmacological inhibitors were employed to determine the signaling mechanisms that induce the promoter in response to PKC activation by PMA. We also extended our studies to determine phoshphorylation-dependent signaling cascades that are activated in response to PMA treatment in JC-410 granulosa cells, and supported the importance of these observations by analyzing Prkg2 expression in primary mouse granulosa cells.

Section snippets

Hormones and chemicals

Pregnant mare serum gonadotropin (PMSG) was purchased from Calbiochem (San Diego, CA) and human chorionic gonadotropin (hCG) was obtained from the UTMB pharmacy (Galveston, TX). Forskolin (Fo) and phorbol 12-myristate 13-acetate (PMA) were purchased from Calbiochem (San Diego, CA); DMEM:F12 medium and penicillin–streptomycin were from Invitrogen (Carlsbad, CA); fetal bovine serum (FBS) and newborn calf serum (NBCS) were obtained from Life Technologies Inc. (Grand Island, NY); Oligonucleotide

Induction of SCC promoter in response to Fo and PMA treatment

Earlier studies have documented the induction of SCC mRNA in response to PKC activation by acute treatment with TPA in porcine granulosa cells (Lahav et al., 1995). In an attempt to understand the PKC signaling mechanisms in granulosa cells we determined the induction of the SCC promoter in response to short-term activation of PKC by the phorbol ester PMA. For this we employed a JC-410 granulosa cell line that is stably integrated with 2 kb of the porcine SCC promoter ligated to a luciferase

Discussion

The results of our studies demonstrate mechanisms regulating induction of the SCC promoter in the engineered JC-410 porcine granulosa cells and the induction of Prkg2 in primary mouse granulosa cells following activation of PKC by PMA. Induction of SCC promoter was dependent on both ERK and JNK activations, in contrast to the induction of Prkg2 which was dependent on ERK but not JNK. Earlier studies have characterized the porcine SCC promoter and documented a GC-rich region, CTGAGTCTGGGAGG (IGF

Acknowledgements

This research was supported by NIH grant R01 HD044566-01 (R.J.U. and L.A.D.). V.S. was supported in part by the NICHD, NIAID, and ORWH as a BIRCWH scholar (HD052023) and ASRM/Organon grant.

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