Luteinizing Hormone Receptor mRNA Down-Regulation Is Mediated through ERK-Dependent Induction of RNA Binding Protein

Luteinzing Hormone (LH)-regulated ERK½ signaling is required for the LHR mRNA binding protein-mediated downregulation of LH Receptor mRNA.

Abstract

The ligand-induced down-regulation of LH receptor (LHR) expression in the ovaries, at least in part, is regulated by a posttranscriptional process mediated by a specific LH receptor mRNA binding protein (LRBP). The LH-mediated signaling pathways involved in this process were examined in primary cultures of human granulosa cells. Treatment with 10 IU human chorionic gonadotropin (hCG) for 12 h resulted in the down-regulation of LHR mRNA expression while producing an increase in LHR mRNA binding to LRBP as well as a 2-fold increase in LRBP levels. The activation of ERK½ pathway in LH-mediated LHR mRNA down-regulation was also established by demonstrating the translocation of ERK½ from the cytosol to the nucleus using confocal microcopy. Inhibition of protein kinase A using H-89 or ERK½ by U0126 abolished the LH-induced LHR mRNA down-regulation. These treatments also abrogated both the increases in LRBP levels as well as the LHR mRNA binding activity. The abolishment of the hCG-induced increase in LRBP levels and LHR mRNA binding activity was further confirmed by transfecting granulosa cells with ERK½ specific small interfering RNA. This treatment also reversed the hCG-induced down-regulation of LHR mRNA. These data show that LH-regulated ERK½ signaling is required for the LRBP-mediated down-regulation of LHR mRNA.

LH/human chorionic gonadotropin (hCG) receptor, a member of the rhodopsin-like family of G protein–coupled receptors (GPCR), undergoes down-regulation in response to exposure to pharmacological dose of the ligand (1). Extensive studies from our laboratory using ovarian cells have shown that the down-regulation seen in response to LH surge or pharmacological doses of hCG occurs predominantly through the accelerated degradation of LH receptor (LHR) mRNA (2–3). We have identified a protein designated as LH receptor mRNA binding protein (LRBP) that binds to the coding region of the LHR mRNA and acts as a trans factor regulating its steady state levels (4). Subsequent studies showed that the LHR mRNA expression and mRNA binding activity of LRBP exhibit a reciprocal relationship during follicle maturation, and that increasing intracellular cAMP levels can mimic LH/hCG-induced LHR mRNA down-regulation (5–6). The protein has been purified and its identity was established as being mevalonate kinase (MVK) (7). LRBP, purified to homogeneity, was able to bind LHR mRNA directly and was recognized by rat MVK antibody in Western blots performed with one- and two-dimensional SDS-PAGE (7). Recombinant MVK produced in human embryonic kidney cells (293 cells) showed all of the characteristics of LRBP with respect to specificity of the LH receptor mRNA binding sequence (7). The functional role of MVK in LH receptor mRNA down-regulation has also been confirmed independently by others (8). Detailed investigations into the molecular mechanisms of LH/hCG-induced down-regulation showed that LRBP translocates to ribosomes, associates with LHR mRNA to form an untranslatable ribonucleoprotein complex, and inhibits LHR mRNA translation, paving the way to its degradation (9). Furthermore, using yeast two hybrid screens, we showed that direct interaction of LRBP with ribosomal protein S20 might play a role in the formation of an untranslatable complex, and that sumoylation of LRBP might be involved in targeting the untranslatable mRNP complex to the decay machinery (10).

The goal of the present study was to identify the signaling pathways that participate in the LH-mediated increase in LRBP expression that ultimately leads to LHR mRNA down-regulation. Because down-regulation of LHR expression follows the initial hormone-receptor interaction, we hypothesized that protein kinase A (PKA) and ERK½ pathways, downstream targets of LH activation in granulosa cells, might play an important role in regulating the expression and binding activity of LRBP. It has been shown that cAMP and PKA are mediators of the LH-generated signaling cascade (11–16). The ERK family, consisting mainly of ERK1 (p44 MAPK) and ERK2 (p42 MAPK), is well known to exert a broad regulatory influence over a wide range of processes, including LH-induced regulation of ovarian function (17–18). While activation of ERK½ pathway appears to be required for eliciting LH/hCG-induced responses in ovarian granulosa cells, we show here a role of the ERK½ pathway in the regulated degradation of LHR mRNA during ligand-induced down-regulation of LHR mRNA expression.

Results

Inhibition of protein kinase A inhibits hCG-induced down-regulation of LHR mRNA in granulosa cells

Because we have shown that cAMP plays an intermediary role in LH-activated LHR mRNA down-regulation in the ovary, the role of PKA in this process was first examined. In granulosa cells collected from in vitro fertilization (IVF) retrieval fluids, LHR is down-regulated at the time of collection due to exposure to high doses of hCG used for inducing ovulation (19). Incubation with serum-containing media for 48 h has been shown to abate LHR down-regulation. Previous studies from the laboratory have demonstrated that in human granulosa cells, as in the case of rat ovary, there is a loss of LHR mRNA transcripts with a corresponding increase in LRBP levels, as well as LHR mRNA binding activity of LRBP, during down-regulation (19–20). Because these cells are readily available, we used this as a model system for the present study. Subsequently, the human granulosa cell cultures were incubated in serum free media for 24 h, then treated with PKA inhibitor H-89 (10 μm) for 1 h followed by 10 IU hCG for 12 h to down-regulate the LH receptor. Total RNA was isolated and LHR mRNA levels were examined using real-time PCR. In agreement with our previous results, treatment with hCG alone caused a 50% reduction in LHR mRNA (19–20) due to down-regulation. The inclusion of the PKA inhibitor, H-89, abolished this inhibitory effect of hCG. The fold change vs. control was 0.47 ± 0.12 for hCG alone and 1 ± 0.13 for H-89+ hCG (P < 0.05, n = 4; Fig. 1A). hCG treatment also produced a 2-fold increase in LRBP levels, which was significantly reduced when cells were treated with H-89. The fold change vs. control was 2.16 ± 0.1 for hCG and 1.17 ± 0.07 for H-89 plus hCG (P < 0.05, n = 3; Fig. 1B). These data show that the inhibition of the PKA pathway blocked hCG-induced down-regulation of LHR mRNA.

Fig. 1.

Effect of PKA inhibitor H-89 on the expression of LHR mRNA and LRBP protein in human granulosa cells. Day 3 granulosa cells were serum-starved, treated with hCG (10 IU/ml) with or without the PKA inhibitor H-89 (10 μm; 1 h pretreatment) for a total of 12 h, and were either processed for total RNA isolation or lysed using RIPA buffer. A, Total RNAs were reverse transcribed, and the resulting cDNAs were subjected to real-time PCR quantitation using specific primers and probes for LHR. The graph represents changes in mRNA levels normalized to 18S rRNA, shown as fold change vs. control. Error bars, mean ± se. *, P < 0.05 vs. CTL; ^#, P < 0.05 vs. hCG; n = 4. B, Cell lysates were subjected to Western blot analysis to detect LRBP using LRBP antibody. The membranes were then stripped and reprobed for β-tubulin. The lower panel represents densitometric scanning of the LRBP normalized for tubulin and expressed as fold change vs. CTL. The blot shown is representative of three independent experiments, and the results in the bar graph are average and se of three experiments. *, P < 0.05 vs. CTL; ^#, P < 0.05 vs. hCG.

hCG activates ERK½ and promotes its translocation to the nucleus in human granulosa cells

In an attempt to identify the downstream targets of PKA, we examined the activation of several protein kinases that are downstream in cAMP/PKA signaling. We examined the activation of ERK½, because ERK cascade has also been implicated in the signaling of GPCRs (21). Treatment of human granulosa cells with hCG markedly increased the phosphorylation of ERK½ in a time-dependent manner, starting as early as 5 min and sustaining up to 30 min but decreasing thereafter to control levels (Fig. 2A). The maximum increase was seen at 5 and 15 min (2.5-fold vs. CTL). LH/hCG has also been shown to stimulate the ERK pathway through PKA activation (22–23). Consistent with these reports, the hCG-induced increase in the levels of p-ERK½ was inhibited by PKA inhibitor H-89. The fold change vs. CTL was 1.08 for H-89 + hCG compared with 2.5-fold for hCG alone (P < 0.05, n = 3; Fig. 2B). ERK½ phosphorylation by GPCRs is known to transmit signals to the nucleus for transcriptional activation (24). In view of this, subcellular localization of ERK½ after hCG-treatment in human granulosa cells was examined. For this, cells were treated with hCG for 15 and 30 min, and the changes in the levels of ERK½ and p-ERK½ in the cytoplasm and nucleus were analyzed using two different approaches. The isolation of nuclear and cytoplasmic compartments followed by Western blot analysis revealed that hCG treatment significantly increased the levels of ERK½ protein in the nucleus. This increase was observed as early as 15 min of hCG treatment and further increased by 30 min (Fig. 3A). The purity of the nuclear and cytoplasmic fractions was assessed by probing the membrane with antibodies against nuclear specific CREB protein and cytoplasmic marker, heat shock protein 90β (HSP90β). As evident from Fig. 3A, both the nuclear and cytoplasmic fractions were free of contamination from the other. To confirm these findings, the localization of phosphorylated ERK½ was examined using immunofluorescence microscopy. Consistent with the Western blot data, under control conditions a minimal pERK½ localization was observed in the cytosol (Fig. 3B; left panel); whereas upon treatment with hCG, a detectable amount of pERK½ could be detected in the nuclei (Fig. 3B; right panel). Thus, these results point to the possibility that ERK½-mediated activation of nuclear events after hCG treatment might lead to changes in LRBP protein expression and subsequent LHR mRNA binding activity. Although phosphatidylinositol 3 kinase/Akt pathway is known to be activated by LH (25), we were not able to detect any major changes in Akt activation under the present experimental conditions.

Fig. 2.

Activation of ERK½ in human granulosa cells. Day 4 granulosa cells were serum-starved, treated with hCG (10 IU/ml) alone for different time intervals (5 min, 15 min, 30 min, and 1 h; A), or in the presence of H-89 (10 μm; 1 h pretreatment) for 15 min (B) and were lysed using RIPA buffer. The cell lysates were subjected to Western blot analysis to detect p-ERK½. The membranes were then stripped and reprobed for total ERK2. Lower panels represent densitometric scanning of the p-ERK½ signals normalized with ERK2 and expressed as fold change vs. CTL. The blots shown are representative of three independent experiments, and the results in the bar graphs are average and se of three experiments. *, P < 0.05 vs. CTL; ^#, P < 0.05 vs. hCG.

Fig. 3.

Nuclear translocation of ERK½ in response to hCG treatment in human granulosa cells. A, Day 3 granulosa cells were serum-starved, then treated with hCG (10 IU/ml) for 15 min and 30 min. The nuclear and cytoplasmic fractions were separated and subjected to Western blot analysis using ERK 2 antibody (upper panel). The membranes were stripped and reprobed for CREB (middle panel) and HSP90β (lower panel). The blots shown are representative of three independent experiments. B, Immunofluorescence analysis of control (CTL) and hCG-treated (30 min) human granulosa cells incubated with antibody against p-ERK½, then incubated with secondary antibody (Alexa fluor 594-labeled goat anti-mouse IgG) and mounted with a DAPI containing anti-FADE reagent. Using confocal microscopy, the top panels show DAPI (A; blue fluorescence) and p-ERK½ (B; red fluorescence) labeling. Bottom panel (C) shows the merged images of the above two.

The effect of ERK½ inhibitor U0126 on hCG-induced LHR mRNA down-regulation and increase in the expression and LHR mRNA binding activity of LRBP

To examine the possible role of ERK½ phosphorylation in hCG-induced LHR mRNA down-regulation, a widely used pharmacological agent U0126 was used, which reduces ERK½ activity by specifically inhibiting MEK, a dual specificity kinase which phosphorylates and activates ERK (26). Pretreatment with U0126 (10 μm) for 1 h followed by treatment with hCG for additional 12 h showed that inhibition of ERK½ phosphorylation by U0126 resulted in a significant reversal of hCG-induced down-regulation of LHR mRNA levels (Fig. 4A). The LHR mRNA level was reduced significantly by hCG, and U0126 treatment reversed this inhibition. The fold change vs. control was 0.47 ± 0.12 for hCG and 1.03 ± 0.14 for U0126 plus hCG (P < 0.05, n = 4). Whether the inhibition of down-regulation exerted by the ERK inhibitor is a result of changes in the expression and/or activity of LRBP was examined by measuring the LRBP levels and LHR mRNA binding activity. As expected, there was an increase in the amount of LRBP protein in cells treated with hCG (1.95-fold vs. CTL, P < 0.05, n = 3) as shown in Fig. 4B. However, when cells were treated with hCG in the presence of U0126, there was a significant reduction in the LRBP protein levels (1.04-fold vs. CTL, P < 0.05, n = 3). The changes in the LRBP mRNA levels were also examined. As shown previously (20), hCG increased LRBP mRNA levels significantly but this increase was abolished in the presence of the ERK inhibitor, U0126 (Fig. 4C). The effect of ERK inhibition on the LHR mRNA binding activity of LRBP was then examined by RNA EMSA (REMSA). The binding of LRBP to the labeled short (40 nt) LRBP binding sequence (LBS) of LHR mRNA was significantly increased in the hCG–down-regulated samples when compared with controls, as evident from the increase in the intensity of the band at the position corresponding to molecular weight 42 (Fig. 4D). However, ERK inhibition by U0126 completely abolished this increase, suggesting that the activation of ERK signaling leads to an increase in the LHR mRNA binding activity. Taken together, the above results show that the PKA/ERK signaling is necessary to down-regulate LHR mRNA by increasing the mRNA and protein expression of LRBP and LHR mRNA binding to LRBP.

Fig. 4.

U0126 inhibits hCG-induced LHR mRNA down-regulation and hCG-induced increase in LRBP mRNA levels, protein expression, and binding activity. Day 3 granulosa cells were serum-starved, treated with hCG (10 IU/ml) with or without the MEK inhibitor U0126 (10 μm; 1 h pretreatment), and were processed for total RNA isolation, Western blot analysis, or REMSA. A, After 12 h of hCG, total RNAs were reverse transcribed, and the resulting cDNAs were subjected to real-time PCR quantitation using specific primers and probes for LHR. The graph represents changes in mRNA levels normalized to 18S rRNA and shown as fold-change vs. control. Error bars, mean ± se. *, P < 0.05 vs. CTL; ^#, P < 0.05 vs. hCG; n = 4. B, Cell lysates after 5 min (panels 1 and 2) or 12 h (panels 3 and 4) of hCG treatment were subjected to Western blot analysis using P-ERK ½ (panel 1) or LRBP (panel 3) antibody, respectively. The membranes were then stripped and reprobed for β-tubulin (panels 2 and 4). The graph represents densitometric scanning of the LRBP signals normalized with tubulin and expressed as fold-change vs. CTL. The blot shown is a representative of three independent experiments, and the results in the bar graph are average and se of three experiments. *, P < 0.05 vs. CTL; ^#, P < 0.05 vs. hCG. C, After 6 h of hCG, total RNAs were reverse transcribed, and the resulting cDNAs were subjected to real-time PCR quantitation using specific primers and probes for MVK (LRBP). The graph represents changes in mRNA levels normalized to 18S rRNA and shown as fold-change vs. control. Error bars, mean ± se. *, P < 0.05 vs. CTL; ^#, P < 0.05 vs. hCG; n = 3. D, Gel mobility shift analysis was performed with [³²P]-labeled LBS (1.5 × 10⁵ c.p.m) and S100 fractions containing equal amounts of total protein, extracted from the different treatment groups. The autoradiogram shown is representative of three independent experiments.

Knockdown of ERK½ expression by specific ERK½ small interfering RNA (siRNA) abolishes hCG-induced LHR mRNA down-regulation

The above results were further confirmed by inhibiting the expression of ERK½ protein in human granulosa cells using ERK½ specific siRNA. The knockdown of ERK½ in the transfected cells was confirmed by Western blot analysis of the cell lysates. A significant reduction in the ERK protein levels was seen in the ERK½ siRNA transfected cells (ERKsi) compared with the control siRNA transfected cells (CTLsi), as shown in Fig. 5A. The LHR mRNA levels in the control and ERK½ siRNA transfected cells with or without hCG treatment was then examined. The results were consistent with the data obtained when ERK½ phosphorylation was inhibited by U0126, shown in Fig. 4A. There was no significant change in the LHR mRNA levels in cells transfected with ERK½ siRNA compared with cells transfected with control siRNA. However, a 60% reduction in LHR mRNA levels was seen in the CTLsi transfected cells treated with hCG compared with those not treated with hCG (CTLsi+hCG, 0.42 ± 0.03-fold vs. CTLsi; P < 0.05, n = 3). On the other hand, LHR mRNA levels in the ERK½ siRNA transfected cells treated with hCG were comparable to controls (ERKsi + hCG, 1.11 ± 015-fold vs. CTLsi; P < 0.05, n = 3; Fig. 5B). These results confirmed the results obtained using U0126 which showed that ERK½-mediated signaling is required for hCG-mediated down-regulation of LHR mRNA expression.

Fig. 5.

ERK½ silencing inhibits hCG-induced decrease in LHR mRNA levels and increases in LRBP protein expression and binding activity. Granulosa cells were transfected with either control siRNA (CTLsi) or ERK ½ siRNA (ERKsi) and cultured for 48 h. After serum-starving for another 24 h, cells were treated with hCG (10 IU/ml) for 12 h and processed for total RNA isolation, for Western blot analysis, or for REMSA. A, ERK½ silencing was confirmed by the Western Blot analysis of cell lysates using total ERK2 antibody. B, Total RNAs were reverse transcribed, and the resulting cDNAs were subjected to real-time PCR quantitation using LHR-specific primers and probes. The graph represents changes in mRNA levels normalized to 18S rRNA and are shown as fold change vs. control. Error bars, mean ± se. *, P < 0.05 vs. CTL; ^#, P < 0.05 vs. hCG; n = 3. C, Cell lysates were subjected to Western blot analysis to detect LRBP using specific antibody. The same membranes were then stripped and reprobed for ERK2 and β-tubulin. The blot shown is a representative of three independent experiments. D, Gel mobility shift analysis was performed with [³²P]-labeled rat LBS (1.5 × 10⁵ c.p.m) and S100 fractions containing equal amounts of total protein extracted from the different treatment groups. The autoradiogram shown is representative of three independent experiments.

ERK½ siRNA abolishes the hCG-induced increase in LRBP expression and activity

The role of ERK½ activation in LHR mRNA down-regulation was further examined by determining the effect of ERK½ knockdown on LRBP protein expression and its LHR mRNA binding activity. Western blot analysis of the cell lysates from control siRNA or ERK½ siRNA transfected cells treated with hCG showed that ERK inhibition abrogated the hCG-induced increase in LRBP protein expression (Fig. 5C, top panel). The same membrane was then stripped and reprobed with ERK2 antibody to demonstrate the knockdown of ERK½ protein in the ERK½ siRNA transfected cells (Fig. 5C, middle panel, lanes 3 and 4). The LHR mRNA binding activity of LRBP was also examined using REMSA. In Fig. 5D, the band indicated by the line represents the LRBP-³²P labeled LBS complex. There was a clear increase in the intensity of this band in the hCG-treated samples when compared with control. However, the intensity of this band was decreased to the control level when ERK½ siRNA cells were treated with hCG, suggesting a role for ERK½ activation in hCG-induced increase in the LHR mRNA binding activity. These experiments confirm our findings that ERK½ signaling plays an important role in hCG-induced LHR mRNA down-regulation mediated by LRBP.

Discussion

In normal ovarian cycle, the expression of LH receptor increases during follicle maturation in response to increasing levels of FSH (27). After preovulatory LH surge, the LHR undergoes a transient down-regulation followed by full recovery. Although the physiological role of the down-regulation is not understood, it is likely that it might prevent ovarian hyperstimulation. Our previous studies using a rodent model, as well as human granulosa cells, demonstrated that a novel posttranscriptional mechanism involving the participation of LHR mRNA binding protein (28) is responsible for this down-regulation. We have shown that this protein, identified as mevalonate kinase, binds to the coding region of LHR mRNA and that the RNA binding region overlaps the catalytic center of LRBP (4, 7, 29). The present study is an extension of this work to delineate the mechanism underlying the LRBP-mediated LHR mRNA down-regulation (Fig. 6).

Fig. 6.

Schematic model depicting the proposed signaling pathway in LH/hCG-induced LHR mRNA down-regulation. Binding of ligand to LH receptor induces activation of ERK½ through the cAMP/PKA pathway. This leads to an increase in the expression of LRBP and thereby its LHR mRNA binding activity, which ultimately results in LHR mRNA degradation.

An increase in cAMP level is one of the early responses of ovarian cells to LH, which in turn activates cAMP-dependent protein kinase levels (12–16). Using a type IV phosphodiesterase inhibitor, we have shown that chronic elevation of ovarian cAMP mimicked the effects of LH/hCG, decreasing the expression of LHR mRNA while concomitantly increasing LRBP binding activity (6). Our present results show that during down-regulation, LHR mRNA degradation is mediated by PKA, possibly by relaying signals to downstream targets to increase the levels of LRBP. The role of PKA in mediating gonadotropin-mediated signaling events is well established in granulosa/luteal cells as well as in theca interstitial cells (15, 30–37). It is also noteworthy that cAMP/PKA and ERK½ are largely responsible for early events of LH signaling that drive granulosa cell differentiation and steroid synthesis (15, 38).

The present studies using PKA inhibitor H-89 demonstrated the role of PKA in hCG-mediated ERK½ phophorylation in human granulosa cells. This was not surprising because ligands that signal via GPCRs have been shown to activate the MAPK signaling cascade (21). Activated ERKs phosphorylate a multitude of targets throughout the cell, exerting broad regulatory influence over a wide range of processes including transcription, translation, cell cycle regulation, cytoskeletal remodeling, and apoptosis (17). There are several studies that substantiate the role of PKA in gonadotropin-mediated ERK phosphorylation (22–23, 39–40). The changes in gonadotropin-mediated ERK activation were either mimicked by elevation of intracellular cAMP or inhibited by PKA inhibitors, indicating that ERK transduces signals downstream of PKA in gonadotropin-induced granulosa cells (22, 39).

The results presented in the study show that activation of the ERK pathway is critical for the hCG-induced down-regulation of LHR mRNA in granulosa cells. A similar role for ERK in mRNA decay/stability has been reported in other systems (41–45). For example, inhibition of the ERK pathway with U0126 has been shown to decrease the stability of mRNAs encoding another GPCR, β-adrenergic receptor (β-AR) (41). In vascular smooth muscle cells, platelet-derived growth factor has been shown to down-regulate the mRNA levels of angiopoietin-2 and cyclin-dependent kinase inhibitor (p27), independently, through ERK-dependent pathways (42–43). On the other hand, the stability of focal adhesion kinase mRNA has been shown to increase with TGF-β treatment, in an ERK-dependent manner (44). In another example, heterogeneous nuclear ribonucleoprotein K increases the stability of thymidine phosphorylase mRNA, and ERK phosphorylation is critical for this (45).

The involvement of ERK½ on LRBP expression is further supported by the data showing translocation of ERK½ to the nucleus after its activation by hCG. Thus, it is conceivable that increases in LRBP levels are regulated by transcriptional activation, because MAPK signaling cascade has been shown to increase transcriptional activity via translocation of P42 and P44 MAPK from the cytosol into the nucleus (46), thereby phosphorylating various transcription factors (47–49). Thus, our results suggest that in response to hCG stimulation, ERK translocates to the nucleus and initiates nuclear events resulting in the increased expression of LRBP protein.

We conclude that LH/hCG activates ERK½ pathway through cAMP/PKA, and the activated ERK then translocates to the nucleus. Inhibition of either PKA or ERK inhibits the hCG-induced increases in LRBP expression and LHR mRNA binding activity of LRBP, and this blocks the down-regulation. Findings in this study provide evidence that PKA-dependent ERK activation plays a pivotal role in the hCG-mediated posttranscriptional regulation of LH receptor mRNA expression.

Materials and Methods

Materials

Highly purified hCG (CR 127) was purchased from Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA). [α-³²P] uridine triphosphate was obtained from Perkin Elmer Life Sciences (Waltham, MA), and Maxiscript T7 kit was from Ambion (Austin, TX). EDTA-free protease inhibitor mixture tablets and Quickspin (G-50 Sephadex) columns for radiolabeled RNA purification were purchased from Roche Applied Science (Indianapolis, IN). RNAse inhibitor (RNasin) was from Promega (Madison, WI). Primers specific for LH receptor and 18S rRNA (TaqMan Assay-on-Demand Gene Expression Product) and Multiscribe reverse transcriptase were from Applied Biosystems (Foster City, CA). Because LRBP was identified as MVK, anti–N-terminal mevalonate kinase IgG was raised against the first 15 N-terminal amino acids of MVK (MLSEVLLVSAPGKVI), and this antibody is referred to as the LRBP antibody in the text. Purified antibodies against ERK2, p-ERK½ (Santa Cruz, CA), CREB (Upstate, Chicago, IL), β tubulin (Sigma, St. Louis, MO), and HSP90β (Assay Designs, Ann Arbor, MI) were commercial products. The Super Signal West Femto chemiluminescence kit and anti-rabbit and anti-mouse IgG conjugated to horseradish peroxidase were obtained from Pierce (Rockford, IL). Bicinchonic acid protein assay reagents were purchased from Thermo Scientific (Rockford, IL). ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI), McCoy's 5A medium, fetal bovine serum, nonessential amino acids, penicillin, streptomycin, and fungizone were from Invitrogen (Grand Island, NY). The inhibitors U0126 and H-89 were purchased from Calbiochem (La Jolla, CA).

Cell isolation and culture

Human granulosa cells were isolated from the ovarian follicular aspirates obtained from women undergoing oocyte retrieval for in vitro fertilization at the University of Michigan Health System (Ann Arbor, MI) and IVF Michigan Laboratory (Rochester Hills, MI). The protocol for isolating granulosa cells has been described in detail previously (19–20). In brief, the isolated granulosa cells were plated onto 35-mm dishes, at a density of approximately 1 × 10⁶ viable cells, in McCoy's 5A medium supplemented with 10% fetal bovine serum, 1.65 mg/liter glutamine, nonessential amino acids, penicillin (100 U/ml), streptomycin (100 μg/ml), and fungizone (0.25 μg/ml). Media were replaced after 24 h, and incubations were continued for an additional 2–3 d. For immunofluorescence, cells were cultured on coverslips. These d 3–4 cultured cells were then treated with the necessary reagents and were used for total RNA isolation for real-time PCR, or subjected to subcellular fractionation, or processed for REMSA, immunostaining, or Western blot analysis, as required.

Real-Time PCR (qPCR) analysis

Total RNA was reverse-transcribed and subjected to real-time PCR quantitation as described before (20). The fold change in gene expression was calculated using the ΔΔCt method (50) with 18S rRNA as the internal control.

Western Blot analysis

Cell proteins were extracted using RIPA buffer. The cytosolic and nuclear fractions were separated by the procedure described by Sikora et al. (51). After determining protein content of total cell lysates as well as nuclear and cytoplasmic fractions using the BCA protein assay, equal amounts of total protein were subjected to 10% SDS-PAGE under reducing conditions followed by Western blot analysis as previously described (20).

Immunofluroscence analysis of human granulosa cells

Control granulosa cells as well as cells treated with hCG (15 min and 30 min) were fixed, permeabilized, and incubated with anti–p-ERK½ antibody (dilution 1:25 in PBS containing 0.1% BSA) at 4 C overnight. Cells were then incubated with the secondary antibody, Alexa Fluor 594-labeled goat anti-mouse IgG (dilution 1:100), at room temperature for 2 h. The coverslips were then mounted using ProLong Gold antifade reagent with DAPI and observed using a confocal microscope (Olympus FluoView FV500) and photographed.

REMSA

REMSA was performed by incubating S100 cytosolic fractions from control and down-regulated ovaries with a fixed concentration of [α-³²P]UTP-labeled LBS, as described previously (52). The labeled RNA for the binding assay was prepared using the Maxiscript kit and S100 fractions were prepared from the granulosa cells, as described in detail in our previous manuscripts (52). The RNA-protein complexes were resolved by 5% native polyacrylamide (70:1) gel electrophoresis and analyzed by autoradiography.

Transfection of human granulosa cells with siRNA

Human granulosa cells were transfected with the siRNA targeted against ERK½ protein using the Amaxa Cell line Nucleofector Kit V, by following the instructions of the manufacturer (Amaxa, Cologne, Germany). The cells after transfection were cultured on 35-mm dishes, and after 48 h the media were changed to normal growth medium. Transfection efficiency was calculated by performing Western blot analysis of the cultured cells and analyzing the changes in the ERK protein levels.

Statistical Analysis

Statistical analysis was carried out using one-way ANOVA followed by the Tukey multiple comparison test. Values were considered statistically significant for P < 0.05. Each experiment was repeated at least three times with similar results. Blots and autoradiograms shown are representative of a minimum of three experiments.