Luteinizing Hormone-Induced Extracellular-Signal Regulated Kinase Activation Differently Modulates Progesterone and Androstenedione Producti
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内分泌学杂志 2005年第7期
Departments of Obstetrics and Gynecology (K.T., K.Y., S.F., M.O., F.K.) and Biochemistry (K.M.), Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan; and Department of Molecular Cell Biology (A.A.), Weizmann Institute of Science, Rehovot 71600, Israel
Address all correspondence and requests for reprints to: Kimihisa Tajima, M.D., Department of Obstetrics and Gynecology, Faculty of Medical Sciences, University of Fukui, 23 Shimoaizuki, Matsuoka, Fukui 910-1193, Japan. E-mail: kimihisa@fmsrsa.fukui-med.ac.jp.
Abstract
It has been reported that gonadotropins promoted phosphorylation of ERK/MAPK in granulosa cells. However, little is known about the effects of gonadotropin on ERK activity in theca cells. This study explores how LH/forskolin controls ERK phosphorylation in cultured bovine theca cells. Effects of ERK on steroidogenesis were also investigated. Phosphorylation of ERK in bovine theca cells was augmented by LH and forskolin in 5 min; it decreased thereafter below basal levels in 20 min. Nevertheless, phosphorylation of the ERK kinase, MEK, was unaffected. Addition of H89 (a protein kinase A inhibitor) significantly reduced the effect of LH/forskolin on ERK phosphorylation. A potent MEK inhibitor PD98059 eliminated ERK phosphorylation and augmented progesterone production concomitantly with the elevation of intracellular steroidogenic acute regulatory protein mRNA in LH/forskolin-stimulated theca cells. In contrast to progesterone production, androgen production was diminished significantly by inhibition of ERK with decreased intracellular P450c17 mRNA levels. Taking these results together, we conclude that LH/cAMP leads to phosphorylation of ERK in a biphasic manner through MEK-independent pathway in bovine theca cells. Protein kinase A-induced phosphatase could possibly contribute to the phosphorylation process. Furthermore, modulation of ERK phosphorylation involves control of thecal steroidogenesis via modulation of the expression of steroidogenic acute regulatory protein and P450c17.
Introduction
THECA CELLS PLAY an important role in controlling ovarian steroidogenesis by providing essential androgens for granulosa cell steroidogenesis (1). Androgens also work as local regulators of ovarian steroidogenesis upon binding androgen receptors that are expressed in granulosa and theca cells (2). Therefore, normal ovarian function requires accurate regulation of steroidogenic activity of theca cells through extraovarian and intraovarian mechanisms. Indeed, under pathological conditions such as polycystic ovary syndrome, thecal steroidogenic hyperactivity causes ovarian dysfunction (3).
It is now well established that the steroidogenic activity of theca cells is regulated by LH in concert with local paracrine factors (4, 5, 6). In a broad range of species including rat, rabbit, cattle, and humans, LH stimulates theca cells to produce androgens (7, 8, 9, 10). LH also maintains progesterone production in theca cells (11). The above effects of LH are caused by the induction of genes involved in steroidogenesis: CYP11A [cytochrome P450 side-chain cleavage enzyme (P450scc)], 3?-hydroxysteroid dehydrogenase, CYP17 (P450c17), and steroidogenic acute regulatory protein (StAR) (12, 13, 14, 15). The actions of LH are mediated in major part through the second-messenger cAMP-protein kinase A (PKA) pathway (7, 16).
Studies over the last decade have revealed that a wide range of extracellular signals including growth factors, hormones, and cytokines activate ERKs (reviewed in Refs. 17 , 18). ERKs belong to the family of the MAPKs that transduce extracellular stimuli into intracellular signals and affect the expression of genes linked to cell proliferation and differentiation. The cell surface signals converge toward activation of the small G protein, Ras, which activates the serine/threonine kinase, Raf. The signal is subsequently amplified via two downstream kinases, MAPK kinase or ERK kinase (MEK) and ERK, both of which are activated uniquely via phosphorylation. Raf phosphorylates two serine residues of MEK; then MEK dually phosphorylates threonine and tyrosine residues of ERK1/2 (sequence TEY). MEK appears to be the only kinase that can phosphorylate the two activating residues of ERK.
It has been demonstrated that LH and FSH activate ERK cascade in ovarian granulosa cells. These effects were mimicked by elevation of intracellular cAMP and inhibited by PKA inhibitors, indicating that ERK transduces signals downstream of PKA in gonadotropin-induced granulosa cells (19, 20). Our previous studies have shown that, in immortalized steroidogenic granulosa cells (20) and primary rat and human granulosa cells (21), gonadotropin-stimulated progesterone production is attenuated to a large extent through activation of ERK1/2. In addition, treatment of the cells with potent and specific ERK inhibitors increased progesterone production concurrent with the elevations of StAR expression. StAR is a key regulatory protein in the rapid modulation of steroidogenesis (22, 23). Therefore, our findings suggested a novel mechanism for acute down-regulation of steroidogenesis (i.e. desensitization) via cross-talk between cAMP and ERK pathway in ovarian granulosa cells.
In contrast to granulosa cells, the effect of gonadotropin on ERK activity in ovarian theca cells and its role in the regulation of thecal steroidogenesis remains elusive. Thus, we decided to explore how LH/forskolin controls ERK phosphorylation using bovine primary theca cells. Moreover, effects of ERK on thecal steroidogenesis were investigated.
Materials and Methods
Antibodies
Mouse monoclonal antidiphospho (DP)-ERK (i.e. active ERK) antibodies (Abs) (anti-DP-ERK Ab), antigeneral (G) ERK antibodies (anti-G-ERK Ab), goat antimouse and antirabbit IgG coupled to horseradish peroxidase, and antirabbit IgG labeled with fluorescein isothiocyanate were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-G MEK antibodies and anti-P450scc antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Dr. J. F. Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA) provided antihuman StAR antibodies. Rabbit polyclonal antiphospho-MEK (antiactive MEK) antibodies were purchased from New England Biolabs Inc./Cell Signaling Technologies (Beverly, MA).
Reagents
The National Institutes of Health and Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA) provided human LH. Forskolin (a potent activator of adenylate cyclase), okadaic acid, phorbol myristic acid (PMA), and sodium orthovanadate (VA) were purchased from Sigma; PD98059 and U0126 were purchased from Calbiochem-Novabiochem AG (San Diego, CA).
Bovine theca cell culture
Bovine ovaries were collected less than 15 min after slaughter at a local abattoir. The ovaries were placed in an ice-cold buffered salt solution and transferred to the laboratory less than 90 min after collection. The estrous cycle stage was determined morphologically, as previously described by Ireland et al. (24); only those ovaries with a regressing corpus luteum were used for this study. Theca cells were isolated from the ovaries under sterile conditions, as described previously (25). Briefly, follicles with clear surfaces were cut into halves and theca interna removed in situ with fine forceps. Granulosa cells, together with part of the theca cell layer, were removed by scraping with a scalpel under a stereomicroscope. The resultant thin thecal layer was minced and then treated with a Hanks’-HEPES buffer containing collagenase (2150 U/ml, type 1; Sigma) and DNase (100 U/ml; Sigma), 0.4% (vol/vol) BSA, and 0.2% (wt/vol) glucose (pH 7.4). Cell dissociation was allowed to continue for 30–60 min at 37 C with continuous stirring at 80 rpm and 0.25% (wt/vol) pancreatin (Sigma) in a Hanks’-HEPES buffer for 7 min. Dispersed cells were washed three times; cell viability, as determined using the trypan blue-dye exclusion test, was 90–93%. Purity of the theca cell preparation used in this study was substantiated by the secretion of estradiol; prepared theca cells did not secret estradiol in the presence or absence of forskolin, whereas granulosa cells obtained from the same follicle secret significant estradiol (control, 0.63 ± 0.11 pg/μg protein; forskolin-treated, 0.92 ± 0.06 pg/μg protein). Furthermore, the theca cells responded in ERK phosphorylation to LH but not to FSH, whereas granulosa cells obtained from the same follicle responded to FSH but not to LH.
Western blot analysis
Western blot analysis was carried out as described previously (20, 21, 26). Briefly, primary cultures at the end of incubation with the appropriate stimulant or no stimulation as indicated in each experiment were rinsed with ice-cold PBS and once with buffer A [50 mM ?-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM sodium vanadate] and were subsequently harvested in buffer A plus proteinase inhibitors. Cell lysates were centrifuged at 20,000 x g for 20 min. The supernatant was assayed for protein content and subjected to Western blot analysis to detect DP-ERK, G-ERK, phospho-MEK (P-MEK), and G-MEK. Alternatively, cells were rinsed with cold PBS and harvested in lysis buffer containing 50 mM HEPES (pH 7.2), 150 mM NaF, 30 mM sodium pyrophosphate, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 5 μg/ml aprotinin and subjected to Western blot analysis to detect P450scc and StAR. Samples containing equal amounts of protein (40 μg) were separated by 10% (to detect DP-ERK, G-ERK, P-MEK, G-MEK, and P450scc) or 12% (to detect StAR) acrylamide SDS-PAGE. The relevant proteins were detected on blots using their specific antibodies.
Determination of progesterone, androstenedione, and protein levels
Progesterone and androstenedione were determined by RIA at the end of the stimulation (27). Protein was quantified using Bradford method (28).
RNA extraction and RT-PCR
Total RNA was isolated using TRIzol (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s instructions. RNA pellets were ethanol precipitated, washed, and resuspended in sterile ribonuclease-free water. Quality of the RNA was assessed by fractionating it on 1% agarose gel and observing the presence of the typical 28S and 18S rRNA under UV light. RT-PCR analyses for bovine StAR, P450c17, P450scc, and 36B4 (an acidic ribosomal phosphoprotein as an internal control) (29) were performed on total RNAs from cultured theca cells using specific primers. Primers used for bovine StAR were 5'-TCGCGGCTCTCTCCTAGGT-3' and 5'-CTGCCGGCTCTCCTTCTTC-3', and those for bovine P450c17 were 5'-TCAGAGAAGTGCTCCGAATCC-3' and 5'-TGCCACTCCTTCTCACTGTGA-3'; those for bovine P450scc were 5'-CTTCATCCCACTGCTGAATCC-3' and 5'-GGTGATGGACTCAAAGGCAAA-3', and those for bovine 36B4 were 5'-GGCGACCTGGAAGTCCAACT-3' and 5'-GGATCTGCTGCATCTGCTTG-3'. In each case, RNAs were reverse transcribed in a final volume of 40 μl solution containing 1x first-strand buffer [3 mM MgCl2, 75 mM KCl, 50 mM Tris-HCl (pH 8.3)], 500 μM each deoxynucleotide triphosphate, 10 mM dithiothreitol, 200 U SuperScript III RNase H-free reverse transcriptase (Invitrogen), 200 ng random hexamers, and 2 μg total RNA. The target cDNAs were amplified, respectively, for 30 cycles and 25 cycles (36B4, internal control) (94 C for 20 sec, 60 C for 30 sec, and 72 C for 60 sec) in a thermal cycler using deoxynucleotide triphosphate (0.2 mM) and 1.5 U of TaKaRa Ex Taq (Takara Shuzo Co. Ltd., Kyoto, Japan). Aliquots of PCR products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide. The relative integrated density of each band was scanned and digitized using FluorChem (Alpha Innotech Co., San Leandro, CA); the ratios of densitometric readings of the amplified target cDNA and internal control, 36B4, DNA were analyzed.
Microscopy
Cells were cultured on 24 x 24-mm coverglasses placed in 35-mm plastic tissue culture dishes. Cells were fixed with 3% paraformaldehyde after 24 h incubation at 37 C with the appropriate stimulants and were viewed with a Zeiss fluorescent microscope (Carl Zeiss Inc., New York, NY) after incubation with a 1:200 dilution of antiserum to human StAR and goat antirabbit antibodies conjugated to fluorescein (30). No mitochondrial straining was detected using nonimmune serum.
Statistical analysis
All experiments were repeated at least three times with theca cells obtained from separate groups of bovines. Data were subjected to ANOVA. Group means were contrasted using post hoc Tukey’s multiple comparison test. P < 0.05 was considered significant. Furthermore, results were confirmed by nonparametric Kruskal-Wallis tests. All values are expressed as mean ± SD.
Results
Effect of LH and forskolin on ERK and MEK phosphorylation
Serum-starved bovine theca cells were stimulated with LH, forskolin, or PMA, and phosphorylation of the activation TEY motif of ERK was then assessed using Western blot analysis with anti-DP-ERK antibodies (31) (Fig. 1). Staining of two bands at 42 and 44 kDa, respectively, representing ERK2 and ERK1 (32, 33), was detected in the nonstimulated cells. The intensity of DP-ERK1/2 staining was enhanced (approximately twice that of basal levels) 5 min after LH treatment. It decreased rapidly below basal level at 20 min and then increased again by 120 min (Fig. 1A). Intracellular levels of ERK1/2 remained unchanged during the stimulation, as determined using general antibodies to ERK1/2 (G-ERK). The same pattern of ERK phosphorylation was observed when cells were stimulated with forskolin (Fig. 1B) or 8-bromoadenosine-cAMP (data not shown). In contrast to ERK, MEK phosphorylation was unaffected by addition of LH/forskolin (P-MEK in Fig. 1, A and B).
FIG. 1. Time-course effects of LH, forskolin, and PMA on ERK phosphorylation in bovine theca cells. Theca cells were plated onto serum-coated dishes with serum-free medium for 24 h and then stimulated with LH (1 IU/ml, A), forskolin (FK, 25 μM, B), and PMA (30 nM, C) for the indicated times. Cytosolic extracts (40 μg) were subjected to immunoblotting with antidiphospho-ERK antibody (DP-ERK; upper panel), anti-general ERK antibody (G-ERK; second panel), antiphospho-MEK antibody (P-MEK; third panel), and anti-general MEK antibody (G-MEK; bottom panel). The positions of ERK1 (44 kDa), ERK2 (42 kDa), and MEK are indicated. Top, Values for phospho-ERK2 are expressed as arbitrary densitometric units (ADU). Each experiment was reproduced at least three times.
Effect of PMA and basic fibroblast growth factor on ERK and MEK phosphorylation
PMA is known to activate the Raf/MEK/ERK pathway through protein kinase C-mediated stimulation of Raf-1 (33, 34). Compared with LH/forskolin, PMA enhanced ERK phosphorylation more intensively at 5 min (approximately 10-fold over basal levels). Thereafter, ERK phosphorylation declined more gradually with the concomitant increase of MEK phosphorylation (Fig. 1C). Basic fibroblast growth factor, which is known to activate the Raf/MEK/ERK pathway (35), demonstrated the same pattern of ERK and MEK phosphorylation, as shown in PMA-treated cells (data not shown).
Additional effect of LH/forskolin and PMA on ERK phosphorylation
This study explored the additional effect of LH/forskolin on PMA-induced ERK phosphorylation. LH/forskolin alone activated ERK (twice that of basal levels) 5 min after stimulation, whereas PMA alone augmented it more intensively (10-fold over basal levels) (Fig. 2A). Addition of LH/FK to PMA-stimulated theca cells did not affect the ERK activity at that time. In contrast, 20 min after stimulation, during which LH/forskolin diminished ERK phosphorylation and PMA augmented its phosphorylation (Fig. 2B), addition of LH/forskolin decreased PMA-induced ERK phosphorylation significantly.
FIG. 2. Effects of PMA and MEK inhibitors on ERK phosphorylation. Subconfluent cultures were pretreated with PMA (30 nM) or MEK inhibitors (25 μM PD98059 or 10 μM U0126) for 15 min and were consequently stimulated with LH (1 IU/ml) or forskolin (FK, 25 μM) for 5 (A) or 20 min (B). Cell lysates (40 μg) were subjected to SDS-PAGE and Western blotting using antidiphospho-ERK antibody (DP-ERK; upper panel) or anti-general ERK antibody (G-ERK; bottom panel). C, Control.
Effects of phosphatase inhibitors on LH/FK-mediated ERK phosphorylation
We examined the possibility that LH/forskolin controls ERK activation by affecting phosphatase activity. We explored the effects of okadaic acid (OA; a protein phosphatase 2A inhibitor) and VA (a broad-spectrum inhibitor of tyrosine phosphatases) on ERK activity of both nonstimulated and LH/forskolin-stimulated cells. OA did not affect ERK phosphorylation in any LH/forskolin-stimulated cells (Fig. 3B). VA also had no effect on ERK phosphorylation of LH/forskolin-stimulated cells, but it significantly increased the ERK activity in nonstimulated cells.
FIG. 3. Effects of PKA inhibitor and phosphatase inhibitors on ERK phosphorylation. Subconfluent cultures were pretreated with a PKA inhibitor (3 μM H89) or phosphatases inhibitors (OA, 100 nM; VA, 80 μM) for 15 min and then stimulated with LH (1 IU/ml) or forskolin (FK, 25 μM) for 5 (A) or 20 min (B). Intracellular levels of DP-ERK, G-ERK, P-MEK, and G-MEK were analyzed using Western blotting. Top, Densitometric tracing of diphospho-ERK2 protein. Values are the mean ± SD for three experiments. Different letters indicate a significant difference of means (P < 0.05). C, Control.
Effect of PKA inhibitor on ERK phosphorylation
This study explored the possibility that cAMP/PKA pathway, which is considered to be a major mediator of the LH-generated signaling, is involved in LH/forskolin-induced ERK phosphorylation. We observed the way in which H89, a potent and selective inhibitor of PKA, affects LH/forskolin-induced change in ERK. Use of either LH or forskolin significantly increased phosphorylation of ERK in 5 min, whereas the addition of H89 significantly inhibited this effect (Fig. 3A). H89 also inhibited LH/forskolin-mediated modulation of ERK in 20 min (Fig. 3B). However, H89 did not totally eliminate LH/FK-mediated dephosphorylation of ERK.
Effect of MEK inhibitor on progesterone and androstenedione production in bovine theca cells
MEK inhibitors, PD98059 (36) and U0126 (37), are useful tools to facilitate the study of involvement of ERK activity in cell function. Indeed, these inhibitors completely eliminated ERK1/2 activity in LH/FK-treated theca cells (Fig. 2, A and B; DP-ERK). No treatments caused a significant change in the total amount of ERKs, as judged by staining with an anti-G-ERK antibody (Fig. 2). Bovine theca cells were incubated with or without PD98059 in the presence or absence of LH/forskolin for 24 h to examine whether ERK activity is involved in theca cell steroidogenesis. LH and forskolin alone significantly increased progesterone and androstenedione production in bovine theca cells (Fig. 4, A and B). The MEK inhibitor had no solitary effect on progesterone production in theca cells. However, when the inhibitor was incubated with LH/forskolin, progesterone production increased significantly (Fig. 4A; 136 and 159% above control, respectively). In contrast, PD98059 alone suppressed androstenedione production. Moreover, addition of the MEK inhibitor significantly decreased LH- and forskolin-induced androstenedione production (Fig. 4A; 54 and 50% of control, respectively).
FIG. 4. Effects of MEK inhibitor on progesterone and androstenedione production in bovine theca cells. Bovine theca cell were stimulated with LH (1 IU/ml), forskolin (FK, 25 μM), PD98059 (25 μM), or their combination in serum-containing medium (5%) at 37 C for 24 h. Culture media were assayed for progesterone (A) and androstenedione (B) by RIA. Values are the mean ± SD for three experiments. Different letters indicate a significant difference of means (P < 0.05). C, Control.
Effects of MEK inhibitor on the expression of StAR, P450scc, and P450c17 in bovine theca cells
Along with examination of progesterone and androstenedione production, we used Western blot analysis to analyze the contents of StAR and the cytochrome P450scc in cell lysates (Fig. 5A). The StAR level in control cells was extremely low, probably because bovine theca cells in the early stage of follicular development do not fully express this protein (38, 39). In contrast, a pronounced elevation of StAR was evident on incubation of the cells with the MEK inhibitor U0126. A higher level of StAR was evident on LH or FK stimulation; it was further elevated in the presence of U0126. Modulation of the 30-kDa protein accompanied modulation of 37-kDa StAR precursors. In contrast to StAR, intracellular levels of cytochrome P450scc showed no significant changes among the different treatments.
FIG. 5. Effects of MEK inhibitor on expression of StAR, P450scc, and P450c17 in bovine theca cells. Cells were incubated with LH or forskolin (FK) in the presence or absence of U0126 (10 μM) in serum-coated dishes with serum-free medium for 12 h (for RT-PCR) or 24 h (for Western blotting). Cell lysates (40 μg) were subjected to SDS-PAGE and Western blotting using anti-StAR, anti-P50scc, or antiactin antibodies. The respective positions of mature StAR at 30 kDa, cytosolic StAR at 37 kDa, P450scc, and actin (internal control) are indicated (panel B). Values for 30 kDa StAR are expressed as arbitrary densitometric units (panel A). RT-PCR was carried out for 27 cycles with StAR, P450c17, P450scc, and 36B4 (internal control) primers using total RNA isolated from the cells. The products were fractionated on 1% agarose gel and stained with ethidium bromide (panel D). Values for P450c17 mRNA levels are expressed as arbitrary densitometric units (panel C). Data are the mean ± SD (n = 3). Different letters indicate statistically significant differences in the mean (P < 0.05). C, Control.
Effect of U0126 on mRNA levels of StAR, P450c17, and P450scc
Because we found elevated intracellular StAR levels in the presence of U0126, we examined whether these changes involved de novo synthesis of StAR, which is associated with increased StAR mRNA. RT-PCR analysis revealed elevation of StAR mRNA levels by U0126 alone and LH/FK stimulation and augmentation in the presence of LH plus U0126, compared with LH/FK stimulation alone (Fig. 5). Expression of P450c17 was increased by the addition of LH and FK, whereas U0126 treatment significantly suppressed its expression. Intracellular levels of cytochrome P450scc mRNA demonstrated no significant changes among these different treatments.
Subcellular localization of StAR on induction with LH and MEK inhibitor
Bovine primary theca cells were incubated with the MEK inhibitor in the absence or presence of forskolin to verify whether U0126 engenders accumulation of the steroidogenic protein in mitochondria. Immunocytochemistry using specific antibodies to StAR (Fig. 6) revealed that StAR was undetectable in mitochondria in nonstimulated cells (Fig. 6A). In contrast, clear elevation in mitochondrial StAR was evident after 24 h of treatment with U0126 (Fig. 6B). Forskolin clearly increased the StAR content in the mitochondria (Fig. 6C), whereas U0126 dramatically augmented forskolin-induced mitochondrial StAR content (Fig. 6D). Therefore, immunocytochemical observations confirmed the data obtained by Western blot and RT-PCR analyses regarding elevation of StAR expression by the MEK inhibitor.
FIG. 6. Subcellular localization of StAR on induction with forskolin and MEK inhibitor. Theca cells on coverslips were treated 24 h with forskolin (25 μM), U0126 (10 μM), or their combination. The cells were then subjected to immunofluorescence using anti-StAR antibodies, followed by goat antirabbit IgG conjugated to fluorescein (green). Their nuclear DNA was stained with DAPI (blue). A, No treatment; B, 24-h incubation with U0126; C, 24-h incubation with forskolin; D, 24-h incubation with U0126 and FK; note the localization of StAR in mitochondria (dotted green fluorescence). Bar, 10 μm.
Discussion
Recently activation of ERK pathway by LH/cAMP was demonstrated in rat theca cells in vivo (40) and in vitro (41). However, details of the kinetic regulation of ERK and MEK activity by LH stimulation have not been evaluated. The present study demonstrated that LH/cAMP activated ERK in a biphasic manner in bovine theca cells and that LH/forskolin stimulated ERK phosphorylation without phosphorylating MEK.
These results suggest that LH/forskolin stimulates phosphorylation of ERK through the pathway independent to MEK. So far, however, MEK is the only known kinase that directly activates ERK (17). Accordingly, we designed the following two studies.
First, we confirmed the effect of MEK activity on ERK phosphorylation in bovine theca cells using PMA, which is known to activate the Raf/MEK/ERK pathway through protein kinase C-mediated stimulation of Raf-1 (33, 34). Indeed, PMA stimulated phosphorylation of both MEK and ERK. The effect of PMA on ERK phosphorylation was much stronger than that of LH/forskolin and rather persistent, whereas the effect of LH/forskolin on ERK activity was transient. Moreover, LH and PMA/growth factors showed additive effects on ERK phosphorylation 20 min after stimulation. These results suggest that LH/FK and PMA regulate ERK activity through different pathways and that those pathways are cross-talking in theca cells (Fig. 7). No additive effect was observed 5 min after the addition of two agents, probably because augmentation of ERK phosphorylation by PMA is much more intensive than that by LH/forskolin at that time point.
FIG. 7. Schematic model showing involvement of ERK activity in the control of thecal steroidogenesis. Upon binding its seven-transmembrane receptor (48 ) LH activates ERK through the PKA-dependent but MEK-independent pathway. G, G-stimulatory protein; AC, adenylate cyclase; Pase, phosphatase; Chol, cholesterol; P4, progesterone; Adione, androstenedione. Arrows indicate stimulatory signals; the blocked line indicates an inhibitory signal.
Second, we explored the possibility that LH-/cAMP modulated ERK phosphatase activity, resulting in a change in ERK phosphorylation. Cottom et al. (42) demonstrated recently that FSH stimulated ERK activation but not MEK activation in rat primary granulosa cells. They showed that FSH promoted phosphorylation of a 100-kDa phosphotyrosine phosphatase that promotes dephosphorylation of regulatory Tyr residue of ERK, resulting in ERK inactivation. They concluded that FSH stimulates ERK activity in immature rat granulosa cells by relieving an inhibition imposed by phosphotyrosine phosphatase. In the present study, however, OA and VA, which are respective specific inhibitors of phosphoserine/phosphothreonine phosphatase and tyrosine phosphatase, did not affect LH/forskolin-induced dephosphorylation of ERK. These results suggest that LH/forskolin does not modulate phosphoserine/phosphothreonine phosphatase and tyrosine phosphatase; however, further study is needed to determine the possibility that LH/forskolin stimulates other phosphatases. In addition, we cannot reject the possible existence of an unknown kinase through which LH/forskolin controls ERK phosphorylation. Mechanisms by which LH/forskolin stimulated ERK phosphorylation in this case remain elusive. In this study, VA significantly increased the basal level of ERK activity without affecting MEK activity, suggesting that protein tyrosine phosphatase serves a pivotal role in maintaining basal activity of ERK.
cAMP and PKA are known to be major mediators of LH-generated signaling. Therefore, we explored the possibility that cAMP/PKA pathway is involved in LH/forskolin-induced ERK phosphorylation. A potent and selective inhibitor of PKA, H89 (43), significantly inhibited the effect of LH/forskolin on ERK phosphorylation, indicating that LH-mediated ERK phosphorylation is PKA-dependent. Recent studies have demonstrated that gonadotropin-mediated ERK phosphorylation is PKA dependent in granulosa cells (20, 42). In the present work, H89 did not completely eliminate the LH/forskolin-mediated dephosphorylation of ERK. Therefore, we cannot rule out the possibility that the ERK cascade is also modulated by an alternative signaling mechanism such as cAMP-responsive guanine nucleotide exchange factors (Epac1 and Epac2). Upon binding of cAMP, Epac1/2 rapidly activate Rap1, which subsequently promotes activation of B-raf and the rest of the ERK cascade (44). Recently Gonzalez-Robayna et al. (45) showed expression of Epac 1 and 2 in primary rat granulosa cells, Epac 1 and 2 both have a role in activation of phosphatydilinositol-3-kinase (PI3-kinase) and p38 MAPK pathway by FSH/cAMP stimulation. Therefore, it can be inferred that theca cells also express these regulatory components and modulate the ERK pathway. Further studies are needed to elucidate the presence and role of the PKA-independent pathway in theca cells.
We also explored the way in which the change in ERK activation affects steroidogenesis in cultured theca cells. We demonstrated that inhibition of ERK pathway in LH-stimulated primary bovine theca cells augmented progesterone production via increased StAR expression. This phenomenon was demonstrated using RT-PCR, Western blot, and immunocytochemistry analyses using bovine theca cells obtained from small follicles. We recently demonstrated similar results in immortalized rat granulosa cells expressing either LH/chorionic gonadotropin or FSH receptors (20) and primary rat and human granulosa cells (21). In the present study, the increase of progesterone production by ERK inhibition was smaller than the elevation of intracellular StAR protein. Multiple effects of inhibitors on thecal steroidogenic process might explain this discrepancy. In contrast to progesterone production, inhibition of ERK activity, produced by MEK inhibitor U0126, significantly decreased the expression of P450c17 with concomitant reduction of androstenedione production. These data suggest that ERK activity plays a crucial role in promotion of steroidogenesis in thecal cells. We speculate that ERK activation engenders a decrease in progesterone production and an increase in androstenedione production. It appears that ERK activation directed steroidogenesis toward P450c17 activation and androgen secretion, whereas its inhibition represses this secretion, thereby increasing progesterone secretion by these cells (Fig. 7). Actually, LH/forskolin augmented both progesterone and androstenedione production with increased expression of StAR and P450c17. It has been reported that regulatory elements such as steroidogenic factor-1, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1, CCAAT/enhancer-binding protein, GATA binding protein 4, and specificity protein 1 are involved in the cAMP/PKA-dependent regulation of StAR and P450c17 expression (46, 47). We infer that ERK phosphorylation contributes to activation or inactivation of these regulatory elements in the control of thecal steroidogenesis.
Contrary to our result, Munir et al. (41) reported recently that U0126 did not increase forskolin-stimulated 17-hydroxylase activity (i.e. conversion of progesterone to -hydroxyprogesterone) in human theca cells. That study found that the ERK cascade is not involved in forskolin-mediated stimulation of -hydroxylase activity (41). Instead, they showed that PI3-kinase inhibitor LY294002 block insulin-stimulated P450c17 activity completely, consequently concluding that insulin stimulates both PI3-kinase and ERK activity in human theca cells but only PI3-kinase mediated insulin augmentation of forskolin-stimulated p450c17 activity. Explanations for this apparently contradictory result may include differences in species, cell preparation, incubation time with agents, and assay procedures for androgen production. Indeed, we used freshly prepared bovine theca cells and stimulated cells with the inhibitor for 12 or 24 h. Then the effect of ERK inhibition on thecal steroidogenesis was assessed using direct measurement of androstenedione concentration and expression of P450c17 mRNA. On the other hand, the study mentioned above used frozen-preserved human theca cells and stimulated the cells with forskolin, insulin, U0126, or their combination for 3 d. Thereafter effects were analyzed according to the change in 17-hydroxylase activities. Further study is necessary to clarify the potential role of ERK signaling pathway in the control of thecal steroidogenesis.
Taking this evidence together, we conclude that, in bovine theca cells, LH/cAMP activates ERK in a biphasic manner through MEK-independent pathway. Furthermore, inhibition of ERK activity increases progesterone production and decreases androstenedione production with concomitant modulation of expression of StAR and P450c17. Findings of this study provide new evidence that LH-mediated ERK activity plays a pivotal role in the regulation of steroidogenesis in ovarian theca cells.
Acknowledgments
We thank Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA) and the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD) for providing the human LH; Dr. J. F. Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA) for antihuman StAR antibodies; and the Kanazawa Meat Inspection Office (Kanazawa, Japan) for allowing us to collect the bovine ovaries used in these experiments. We also thank Dr. N. Suganuma (Department of Environmental Health, University of Fukui, Fukui, Japan) for help in statistics; and Mikiko Misawa and Yuka Sugimoto for excellent technical assistance.
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Address all correspondence and requests for reprints to: Kimihisa Tajima, M.D., Department of Obstetrics and Gynecology, Faculty of Medical Sciences, University of Fukui, 23 Shimoaizuki, Matsuoka, Fukui 910-1193, Japan. E-mail: kimihisa@fmsrsa.fukui-med.ac.jp.
Abstract
It has been reported that gonadotropins promoted phosphorylation of ERK/MAPK in granulosa cells. However, little is known about the effects of gonadotropin on ERK activity in theca cells. This study explores how LH/forskolin controls ERK phosphorylation in cultured bovine theca cells. Effects of ERK on steroidogenesis were also investigated. Phosphorylation of ERK in bovine theca cells was augmented by LH and forskolin in 5 min; it decreased thereafter below basal levels in 20 min. Nevertheless, phosphorylation of the ERK kinase, MEK, was unaffected. Addition of H89 (a protein kinase A inhibitor) significantly reduced the effect of LH/forskolin on ERK phosphorylation. A potent MEK inhibitor PD98059 eliminated ERK phosphorylation and augmented progesterone production concomitantly with the elevation of intracellular steroidogenic acute regulatory protein mRNA in LH/forskolin-stimulated theca cells. In contrast to progesterone production, androgen production was diminished significantly by inhibition of ERK with decreased intracellular P450c17 mRNA levels. Taking these results together, we conclude that LH/cAMP leads to phosphorylation of ERK in a biphasic manner through MEK-independent pathway in bovine theca cells. Protein kinase A-induced phosphatase could possibly contribute to the phosphorylation process. Furthermore, modulation of ERK phosphorylation involves control of thecal steroidogenesis via modulation of the expression of steroidogenic acute regulatory protein and P450c17.
Introduction
THECA CELLS PLAY an important role in controlling ovarian steroidogenesis by providing essential androgens for granulosa cell steroidogenesis (1). Androgens also work as local regulators of ovarian steroidogenesis upon binding androgen receptors that are expressed in granulosa and theca cells (2). Therefore, normal ovarian function requires accurate regulation of steroidogenic activity of theca cells through extraovarian and intraovarian mechanisms. Indeed, under pathological conditions such as polycystic ovary syndrome, thecal steroidogenic hyperactivity causes ovarian dysfunction (3).
It is now well established that the steroidogenic activity of theca cells is regulated by LH in concert with local paracrine factors (4, 5, 6). In a broad range of species including rat, rabbit, cattle, and humans, LH stimulates theca cells to produce androgens (7, 8, 9, 10). LH also maintains progesterone production in theca cells (11). The above effects of LH are caused by the induction of genes involved in steroidogenesis: CYP11A [cytochrome P450 side-chain cleavage enzyme (P450scc)], 3?-hydroxysteroid dehydrogenase, CYP17 (P450c17), and steroidogenic acute regulatory protein (StAR) (12, 13, 14, 15). The actions of LH are mediated in major part through the second-messenger cAMP-protein kinase A (PKA) pathway (7, 16).
Studies over the last decade have revealed that a wide range of extracellular signals including growth factors, hormones, and cytokines activate ERKs (reviewed in Refs. 17 , 18). ERKs belong to the family of the MAPKs that transduce extracellular stimuli into intracellular signals and affect the expression of genes linked to cell proliferation and differentiation. The cell surface signals converge toward activation of the small G protein, Ras, which activates the serine/threonine kinase, Raf. The signal is subsequently amplified via two downstream kinases, MAPK kinase or ERK kinase (MEK) and ERK, both of which are activated uniquely via phosphorylation. Raf phosphorylates two serine residues of MEK; then MEK dually phosphorylates threonine and tyrosine residues of ERK1/2 (sequence TEY). MEK appears to be the only kinase that can phosphorylate the two activating residues of ERK.
It has been demonstrated that LH and FSH activate ERK cascade in ovarian granulosa cells. These effects were mimicked by elevation of intracellular cAMP and inhibited by PKA inhibitors, indicating that ERK transduces signals downstream of PKA in gonadotropin-induced granulosa cells (19, 20). Our previous studies have shown that, in immortalized steroidogenic granulosa cells (20) and primary rat and human granulosa cells (21), gonadotropin-stimulated progesterone production is attenuated to a large extent through activation of ERK1/2. In addition, treatment of the cells with potent and specific ERK inhibitors increased progesterone production concurrent with the elevations of StAR expression. StAR is a key regulatory protein in the rapid modulation of steroidogenesis (22, 23). Therefore, our findings suggested a novel mechanism for acute down-regulation of steroidogenesis (i.e. desensitization) via cross-talk between cAMP and ERK pathway in ovarian granulosa cells.
In contrast to granulosa cells, the effect of gonadotropin on ERK activity in ovarian theca cells and its role in the regulation of thecal steroidogenesis remains elusive. Thus, we decided to explore how LH/forskolin controls ERK phosphorylation using bovine primary theca cells. Moreover, effects of ERK on thecal steroidogenesis were investigated.
Materials and Methods
Antibodies
Mouse monoclonal antidiphospho (DP)-ERK (i.e. active ERK) antibodies (Abs) (anti-DP-ERK Ab), antigeneral (G) ERK antibodies (anti-G-ERK Ab), goat antimouse and antirabbit IgG coupled to horseradish peroxidase, and antirabbit IgG labeled with fluorescein isothiocyanate were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-G MEK antibodies and anti-P450scc antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Dr. J. F. Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA) provided antihuman StAR antibodies. Rabbit polyclonal antiphospho-MEK (antiactive MEK) antibodies were purchased from New England Biolabs Inc./Cell Signaling Technologies (Beverly, MA).
Reagents
The National Institutes of Health and Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA) provided human LH. Forskolin (a potent activator of adenylate cyclase), okadaic acid, phorbol myristic acid (PMA), and sodium orthovanadate (VA) were purchased from Sigma; PD98059 and U0126 were purchased from Calbiochem-Novabiochem AG (San Diego, CA).
Bovine theca cell culture
Bovine ovaries were collected less than 15 min after slaughter at a local abattoir. The ovaries were placed in an ice-cold buffered salt solution and transferred to the laboratory less than 90 min after collection. The estrous cycle stage was determined morphologically, as previously described by Ireland et al. (24); only those ovaries with a regressing corpus luteum were used for this study. Theca cells were isolated from the ovaries under sterile conditions, as described previously (25). Briefly, follicles with clear surfaces were cut into halves and theca interna removed in situ with fine forceps. Granulosa cells, together with part of the theca cell layer, were removed by scraping with a scalpel under a stereomicroscope. The resultant thin thecal layer was minced and then treated with a Hanks’-HEPES buffer containing collagenase (2150 U/ml, type 1; Sigma) and DNase (100 U/ml; Sigma), 0.4% (vol/vol) BSA, and 0.2% (wt/vol) glucose (pH 7.4). Cell dissociation was allowed to continue for 30–60 min at 37 C with continuous stirring at 80 rpm and 0.25% (wt/vol) pancreatin (Sigma) in a Hanks’-HEPES buffer for 7 min. Dispersed cells were washed three times; cell viability, as determined using the trypan blue-dye exclusion test, was 90–93%. Purity of the theca cell preparation used in this study was substantiated by the secretion of estradiol; prepared theca cells did not secret estradiol in the presence or absence of forskolin, whereas granulosa cells obtained from the same follicle secret significant estradiol (control, 0.63 ± 0.11 pg/μg protein; forskolin-treated, 0.92 ± 0.06 pg/μg protein). Furthermore, the theca cells responded in ERK phosphorylation to LH but not to FSH, whereas granulosa cells obtained from the same follicle responded to FSH but not to LH.
Western blot analysis
Western blot analysis was carried out as described previously (20, 21, 26). Briefly, primary cultures at the end of incubation with the appropriate stimulant or no stimulation as indicated in each experiment were rinsed with ice-cold PBS and once with buffer A [50 mM ?-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM sodium vanadate] and were subsequently harvested in buffer A plus proteinase inhibitors. Cell lysates were centrifuged at 20,000 x g for 20 min. The supernatant was assayed for protein content and subjected to Western blot analysis to detect DP-ERK, G-ERK, phospho-MEK (P-MEK), and G-MEK. Alternatively, cells were rinsed with cold PBS and harvested in lysis buffer containing 50 mM HEPES (pH 7.2), 150 mM NaF, 30 mM sodium pyrophosphate, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 5 μg/ml aprotinin and subjected to Western blot analysis to detect P450scc and StAR. Samples containing equal amounts of protein (40 μg) were separated by 10% (to detect DP-ERK, G-ERK, P-MEK, G-MEK, and P450scc) or 12% (to detect StAR) acrylamide SDS-PAGE. The relevant proteins were detected on blots using their specific antibodies.
Determination of progesterone, androstenedione, and protein levels
Progesterone and androstenedione were determined by RIA at the end of the stimulation (27). Protein was quantified using Bradford method (28).
RNA extraction and RT-PCR
Total RNA was isolated using TRIzol (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s instructions. RNA pellets were ethanol precipitated, washed, and resuspended in sterile ribonuclease-free water. Quality of the RNA was assessed by fractionating it on 1% agarose gel and observing the presence of the typical 28S and 18S rRNA under UV light. RT-PCR analyses for bovine StAR, P450c17, P450scc, and 36B4 (an acidic ribosomal phosphoprotein as an internal control) (29) were performed on total RNAs from cultured theca cells using specific primers. Primers used for bovine StAR were 5'-TCGCGGCTCTCTCCTAGGT-3' and 5'-CTGCCGGCTCTCCTTCTTC-3', and those for bovine P450c17 were 5'-TCAGAGAAGTGCTCCGAATCC-3' and 5'-TGCCACTCCTTCTCACTGTGA-3'; those for bovine P450scc were 5'-CTTCATCCCACTGCTGAATCC-3' and 5'-GGTGATGGACTCAAAGGCAAA-3', and those for bovine 36B4 were 5'-GGCGACCTGGAAGTCCAACT-3' and 5'-GGATCTGCTGCATCTGCTTG-3'. In each case, RNAs were reverse transcribed in a final volume of 40 μl solution containing 1x first-strand buffer [3 mM MgCl2, 75 mM KCl, 50 mM Tris-HCl (pH 8.3)], 500 μM each deoxynucleotide triphosphate, 10 mM dithiothreitol, 200 U SuperScript III RNase H-free reverse transcriptase (Invitrogen), 200 ng random hexamers, and 2 μg total RNA. The target cDNAs were amplified, respectively, for 30 cycles and 25 cycles (36B4, internal control) (94 C for 20 sec, 60 C for 30 sec, and 72 C for 60 sec) in a thermal cycler using deoxynucleotide triphosphate (0.2 mM) and 1.5 U of TaKaRa Ex Taq (Takara Shuzo Co. Ltd., Kyoto, Japan). Aliquots of PCR products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide. The relative integrated density of each band was scanned and digitized using FluorChem (Alpha Innotech Co., San Leandro, CA); the ratios of densitometric readings of the amplified target cDNA and internal control, 36B4, DNA were analyzed.
Microscopy
Cells were cultured on 24 x 24-mm coverglasses placed in 35-mm plastic tissue culture dishes. Cells were fixed with 3% paraformaldehyde after 24 h incubation at 37 C with the appropriate stimulants and were viewed with a Zeiss fluorescent microscope (Carl Zeiss Inc., New York, NY) after incubation with a 1:200 dilution of antiserum to human StAR and goat antirabbit antibodies conjugated to fluorescein (30). No mitochondrial straining was detected using nonimmune serum.
Statistical analysis
All experiments were repeated at least three times with theca cells obtained from separate groups of bovines. Data were subjected to ANOVA. Group means were contrasted using post hoc Tukey’s multiple comparison test. P < 0.05 was considered significant. Furthermore, results were confirmed by nonparametric Kruskal-Wallis tests. All values are expressed as mean ± SD.
Results
Effect of LH and forskolin on ERK and MEK phosphorylation
Serum-starved bovine theca cells were stimulated with LH, forskolin, or PMA, and phosphorylation of the activation TEY motif of ERK was then assessed using Western blot analysis with anti-DP-ERK antibodies (31) (Fig. 1). Staining of two bands at 42 and 44 kDa, respectively, representing ERK2 and ERK1 (32, 33), was detected in the nonstimulated cells. The intensity of DP-ERK1/2 staining was enhanced (approximately twice that of basal levels) 5 min after LH treatment. It decreased rapidly below basal level at 20 min and then increased again by 120 min (Fig. 1A). Intracellular levels of ERK1/2 remained unchanged during the stimulation, as determined using general antibodies to ERK1/2 (G-ERK). The same pattern of ERK phosphorylation was observed when cells were stimulated with forskolin (Fig. 1B) or 8-bromoadenosine-cAMP (data not shown). In contrast to ERK, MEK phosphorylation was unaffected by addition of LH/forskolin (P-MEK in Fig. 1, A and B).
FIG. 1. Time-course effects of LH, forskolin, and PMA on ERK phosphorylation in bovine theca cells. Theca cells were plated onto serum-coated dishes with serum-free medium for 24 h and then stimulated with LH (1 IU/ml, A), forskolin (FK, 25 μM, B), and PMA (30 nM, C) for the indicated times. Cytosolic extracts (40 μg) were subjected to immunoblotting with antidiphospho-ERK antibody (DP-ERK; upper panel), anti-general ERK antibody (G-ERK; second panel), antiphospho-MEK antibody (P-MEK; third panel), and anti-general MEK antibody (G-MEK; bottom panel). The positions of ERK1 (44 kDa), ERK2 (42 kDa), and MEK are indicated. Top, Values for phospho-ERK2 are expressed as arbitrary densitometric units (ADU). Each experiment was reproduced at least three times.
Effect of PMA and basic fibroblast growth factor on ERK and MEK phosphorylation
PMA is known to activate the Raf/MEK/ERK pathway through protein kinase C-mediated stimulation of Raf-1 (33, 34). Compared with LH/forskolin, PMA enhanced ERK phosphorylation more intensively at 5 min (approximately 10-fold over basal levels). Thereafter, ERK phosphorylation declined more gradually with the concomitant increase of MEK phosphorylation (Fig. 1C). Basic fibroblast growth factor, which is known to activate the Raf/MEK/ERK pathway (35), demonstrated the same pattern of ERK and MEK phosphorylation, as shown in PMA-treated cells (data not shown).
Additional effect of LH/forskolin and PMA on ERK phosphorylation
This study explored the additional effect of LH/forskolin on PMA-induced ERK phosphorylation. LH/forskolin alone activated ERK (twice that of basal levels) 5 min after stimulation, whereas PMA alone augmented it more intensively (10-fold over basal levels) (Fig. 2A). Addition of LH/FK to PMA-stimulated theca cells did not affect the ERK activity at that time. In contrast, 20 min after stimulation, during which LH/forskolin diminished ERK phosphorylation and PMA augmented its phosphorylation (Fig. 2B), addition of LH/forskolin decreased PMA-induced ERK phosphorylation significantly.
FIG. 2. Effects of PMA and MEK inhibitors on ERK phosphorylation. Subconfluent cultures were pretreated with PMA (30 nM) or MEK inhibitors (25 μM PD98059 or 10 μM U0126) for 15 min and were consequently stimulated with LH (1 IU/ml) or forskolin (FK, 25 μM) for 5 (A) or 20 min (B). Cell lysates (40 μg) were subjected to SDS-PAGE and Western blotting using antidiphospho-ERK antibody (DP-ERK; upper panel) or anti-general ERK antibody (G-ERK; bottom panel). C, Control.
Effects of phosphatase inhibitors on LH/FK-mediated ERK phosphorylation
We examined the possibility that LH/forskolin controls ERK activation by affecting phosphatase activity. We explored the effects of okadaic acid (OA; a protein phosphatase 2A inhibitor) and VA (a broad-spectrum inhibitor of tyrosine phosphatases) on ERK activity of both nonstimulated and LH/forskolin-stimulated cells. OA did not affect ERK phosphorylation in any LH/forskolin-stimulated cells (Fig. 3B). VA also had no effect on ERK phosphorylation of LH/forskolin-stimulated cells, but it significantly increased the ERK activity in nonstimulated cells.
FIG. 3. Effects of PKA inhibitor and phosphatase inhibitors on ERK phosphorylation. Subconfluent cultures were pretreated with a PKA inhibitor (3 μM H89) or phosphatases inhibitors (OA, 100 nM; VA, 80 μM) for 15 min and then stimulated with LH (1 IU/ml) or forskolin (FK, 25 μM) for 5 (A) or 20 min (B). Intracellular levels of DP-ERK, G-ERK, P-MEK, and G-MEK were analyzed using Western blotting. Top, Densitometric tracing of diphospho-ERK2 protein. Values are the mean ± SD for three experiments. Different letters indicate a significant difference of means (P < 0.05). C, Control.
Effect of PKA inhibitor on ERK phosphorylation
This study explored the possibility that cAMP/PKA pathway, which is considered to be a major mediator of the LH-generated signaling, is involved in LH/forskolin-induced ERK phosphorylation. We observed the way in which H89, a potent and selective inhibitor of PKA, affects LH/forskolin-induced change in ERK. Use of either LH or forskolin significantly increased phosphorylation of ERK in 5 min, whereas the addition of H89 significantly inhibited this effect (Fig. 3A). H89 also inhibited LH/forskolin-mediated modulation of ERK in 20 min (Fig. 3B). However, H89 did not totally eliminate LH/FK-mediated dephosphorylation of ERK.
Effect of MEK inhibitor on progesterone and androstenedione production in bovine theca cells
MEK inhibitors, PD98059 (36) and U0126 (37), are useful tools to facilitate the study of involvement of ERK activity in cell function. Indeed, these inhibitors completely eliminated ERK1/2 activity in LH/FK-treated theca cells (Fig. 2, A and B; DP-ERK). No treatments caused a significant change in the total amount of ERKs, as judged by staining with an anti-G-ERK antibody (Fig. 2). Bovine theca cells were incubated with or without PD98059 in the presence or absence of LH/forskolin for 24 h to examine whether ERK activity is involved in theca cell steroidogenesis. LH and forskolin alone significantly increased progesterone and androstenedione production in bovine theca cells (Fig. 4, A and B). The MEK inhibitor had no solitary effect on progesterone production in theca cells. However, when the inhibitor was incubated with LH/forskolin, progesterone production increased significantly (Fig. 4A; 136 and 159% above control, respectively). In contrast, PD98059 alone suppressed androstenedione production. Moreover, addition of the MEK inhibitor significantly decreased LH- and forskolin-induced androstenedione production (Fig. 4A; 54 and 50% of control, respectively).
FIG. 4. Effects of MEK inhibitor on progesterone and androstenedione production in bovine theca cells. Bovine theca cell were stimulated with LH (1 IU/ml), forskolin (FK, 25 μM), PD98059 (25 μM), or their combination in serum-containing medium (5%) at 37 C for 24 h. Culture media were assayed for progesterone (A) and androstenedione (B) by RIA. Values are the mean ± SD for three experiments. Different letters indicate a significant difference of means (P < 0.05). C, Control.
Effects of MEK inhibitor on the expression of StAR, P450scc, and P450c17 in bovine theca cells
Along with examination of progesterone and androstenedione production, we used Western blot analysis to analyze the contents of StAR and the cytochrome P450scc in cell lysates (Fig. 5A). The StAR level in control cells was extremely low, probably because bovine theca cells in the early stage of follicular development do not fully express this protein (38, 39). In contrast, a pronounced elevation of StAR was evident on incubation of the cells with the MEK inhibitor U0126. A higher level of StAR was evident on LH or FK stimulation; it was further elevated in the presence of U0126. Modulation of the 30-kDa protein accompanied modulation of 37-kDa StAR precursors. In contrast to StAR, intracellular levels of cytochrome P450scc showed no significant changes among the different treatments.
FIG. 5. Effects of MEK inhibitor on expression of StAR, P450scc, and P450c17 in bovine theca cells. Cells were incubated with LH or forskolin (FK) in the presence or absence of U0126 (10 μM) in serum-coated dishes with serum-free medium for 12 h (for RT-PCR) or 24 h (for Western blotting). Cell lysates (40 μg) were subjected to SDS-PAGE and Western blotting using anti-StAR, anti-P50scc, or antiactin antibodies. The respective positions of mature StAR at 30 kDa, cytosolic StAR at 37 kDa, P450scc, and actin (internal control) are indicated (panel B). Values for 30 kDa StAR are expressed as arbitrary densitometric units (panel A). RT-PCR was carried out for 27 cycles with StAR, P450c17, P450scc, and 36B4 (internal control) primers using total RNA isolated from the cells. The products were fractionated on 1% agarose gel and stained with ethidium bromide (panel D). Values for P450c17 mRNA levels are expressed as arbitrary densitometric units (panel C). Data are the mean ± SD (n = 3). Different letters indicate statistically significant differences in the mean (P < 0.05). C, Control.
Effect of U0126 on mRNA levels of StAR, P450c17, and P450scc
Because we found elevated intracellular StAR levels in the presence of U0126, we examined whether these changes involved de novo synthesis of StAR, which is associated with increased StAR mRNA. RT-PCR analysis revealed elevation of StAR mRNA levels by U0126 alone and LH/FK stimulation and augmentation in the presence of LH plus U0126, compared with LH/FK stimulation alone (Fig. 5). Expression of P450c17 was increased by the addition of LH and FK, whereas U0126 treatment significantly suppressed its expression. Intracellular levels of cytochrome P450scc mRNA demonstrated no significant changes among these different treatments.
Subcellular localization of StAR on induction with LH and MEK inhibitor
Bovine primary theca cells were incubated with the MEK inhibitor in the absence or presence of forskolin to verify whether U0126 engenders accumulation of the steroidogenic protein in mitochondria. Immunocytochemistry using specific antibodies to StAR (Fig. 6) revealed that StAR was undetectable in mitochondria in nonstimulated cells (Fig. 6A). In contrast, clear elevation in mitochondrial StAR was evident after 24 h of treatment with U0126 (Fig. 6B). Forskolin clearly increased the StAR content in the mitochondria (Fig. 6C), whereas U0126 dramatically augmented forskolin-induced mitochondrial StAR content (Fig. 6D). Therefore, immunocytochemical observations confirmed the data obtained by Western blot and RT-PCR analyses regarding elevation of StAR expression by the MEK inhibitor.
FIG. 6. Subcellular localization of StAR on induction with forskolin and MEK inhibitor. Theca cells on coverslips were treated 24 h with forskolin (25 μM), U0126 (10 μM), or their combination. The cells were then subjected to immunofluorescence using anti-StAR antibodies, followed by goat antirabbit IgG conjugated to fluorescein (green). Their nuclear DNA was stained with DAPI (blue). A, No treatment; B, 24-h incubation with U0126; C, 24-h incubation with forskolin; D, 24-h incubation with U0126 and FK; note the localization of StAR in mitochondria (dotted green fluorescence). Bar, 10 μm.
Discussion
Recently activation of ERK pathway by LH/cAMP was demonstrated in rat theca cells in vivo (40) and in vitro (41). However, details of the kinetic regulation of ERK and MEK activity by LH stimulation have not been evaluated. The present study demonstrated that LH/cAMP activated ERK in a biphasic manner in bovine theca cells and that LH/forskolin stimulated ERK phosphorylation without phosphorylating MEK.
These results suggest that LH/forskolin stimulates phosphorylation of ERK through the pathway independent to MEK. So far, however, MEK is the only known kinase that directly activates ERK (17). Accordingly, we designed the following two studies.
First, we confirmed the effect of MEK activity on ERK phosphorylation in bovine theca cells using PMA, which is known to activate the Raf/MEK/ERK pathway through protein kinase C-mediated stimulation of Raf-1 (33, 34). Indeed, PMA stimulated phosphorylation of both MEK and ERK. The effect of PMA on ERK phosphorylation was much stronger than that of LH/forskolin and rather persistent, whereas the effect of LH/forskolin on ERK activity was transient. Moreover, LH and PMA/growth factors showed additive effects on ERK phosphorylation 20 min after stimulation. These results suggest that LH/FK and PMA regulate ERK activity through different pathways and that those pathways are cross-talking in theca cells (Fig. 7). No additive effect was observed 5 min after the addition of two agents, probably because augmentation of ERK phosphorylation by PMA is much more intensive than that by LH/forskolin at that time point.
FIG. 7. Schematic model showing involvement of ERK activity in the control of thecal steroidogenesis. Upon binding its seven-transmembrane receptor (48 ) LH activates ERK through the PKA-dependent but MEK-independent pathway. G, G-stimulatory protein; AC, adenylate cyclase; Pase, phosphatase; Chol, cholesterol; P4, progesterone; Adione, androstenedione. Arrows indicate stimulatory signals; the blocked line indicates an inhibitory signal.
Second, we explored the possibility that LH-/cAMP modulated ERK phosphatase activity, resulting in a change in ERK phosphorylation. Cottom et al. (42) demonstrated recently that FSH stimulated ERK activation but not MEK activation in rat primary granulosa cells. They showed that FSH promoted phosphorylation of a 100-kDa phosphotyrosine phosphatase that promotes dephosphorylation of regulatory Tyr residue of ERK, resulting in ERK inactivation. They concluded that FSH stimulates ERK activity in immature rat granulosa cells by relieving an inhibition imposed by phosphotyrosine phosphatase. In the present study, however, OA and VA, which are respective specific inhibitors of phosphoserine/phosphothreonine phosphatase and tyrosine phosphatase, did not affect LH/forskolin-induced dephosphorylation of ERK. These results suggest that LH/forskolin does not modulate phosphoserine/phosphothreonine phosphatase and tyrosine phosphatase; however, further study is needed to determine the possibility that LH/forskolin stimulates other phosphatases. In addition, we cannot reject the possible existence of an unknown kinase through which LH/forskolin controls ERK phosphorylation. Mechanisms by which LH/forskolin stimulated ERK phosphorylation in this case remain elusive. In this study, VA significantly increased the basal level of ERK activity without affecting MEK activity, suggesting that protein tyrosine phosphatase serves a pivotal role in maintaining basal activity of ERK.
cAMP and PKA are known to be major mediators of LH-generated signaling. Therefore, we explored the possibility that cAMP/PKA pathway is involved in LH/forskolin-induced ERK phosphorylation. A potent and selective inhibitor of PKA, H89 (43), significantly inhibited the effect of LH/forskolin on ERK phosphorylation, indicating that LH-mediated ERK phosphorylation is PKA-dependent. Recent studies have demonstrated that gonadotropin-mediated ERK phosphorylation is PKA dependent in granulosa cells (20, 42). In the present work, H89 did not completely eliminate the LH/forskolin-mediated dephosphorylation of ERK. Therefore, we cannot rule out the possibility that the ERK cascade is also modulated by an alternative signaling mechanism such as cAMP-responsive guanine nucleotide exchange factors (Epac1 and Epac2). Upon binding of cAMP, Epac1/2 rapidly activate Rap1, which subsequently promotes activation of B-raf and the rest of the ERK cascade (44). Recently Gonzalez-Robayna et al. (45) showed expression of Epac 1 and 2 in primary rat granulosa cells, Epac 1 and 2 both have a role in activation of phosphatydilinositol-3-kinase (PI3-kinase) and p38 MAPK pathway by FSH/cAMP stimulation. Therefore, it can be inferred that theca cells also express these regulatory components and modulate the ERK pathway. Further studies are needed to elucidate the presence and role of the PKA-independent pathway in theca cells.
We also explored the way in which the change in ERK activation affects steroidogenesis in cultured theca cells. We demonstrated that inhibition of ERK pathway in LH-stimulated primary bovine theca cells augmented progesterone production via increased StAR expression. This phenomenon was demonstrated using RT-PCR, Western blot, and immunocytochemistry analyses using bovine theca cells obtained from small follicles. We recently demonstrated similar results in immortalized rat granulosa cells expressing either LH/chorionic gonadotropin or FSH receptors (20) and primary rat and human granulosa cells (21). In the present study, the increase of progesterone production by ERK inhibition was smaller than the elevation of intracellular StAR protein. Multiple effects of inhibitors on thecal steroidogenic process might explain this discrepancy. In contrast to progesterone production, inhibition of ERK activity, produced by MEK inhibitor U0126, significantly decreased the expression of P450c17 with concomitant reduction of androstenedione production. These data suggest that ERK activity plays a crucial role in promotion of steroidogenesis in thecal cells. We speculate that ERK activation engenders a decrease in progesterone production and an increase in androstenedione production. It appears that ERK activation directed steroidogenesis toward P450c17 activation and androgen secretion, whereas its inhibition represses this secretion, thereby increasing progesterone secretion by these cells (Fig. 7). Actually, LH/forskolin augmented both progesterone and androstenedione production with increased expression of StAR and P450c17. It has been reported that regulatory elements such as steroidogenic factor-1, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1, CCAAT/enhancer-binding protein, GATA binding protein 4, and specificity protein 1 are involved in the cAMP/PKA-dependent regulation of StAR and P450c17 expression (46, 47). We infer that ERK phosphorylation contributes to activation or inactivation of these regulatory elements in the control of thecal steroidogenesis.
Contrary to our result, Munir et al. (41) reported recently that U0126 did not increase forskolin-stimulated 17-hydroxylase activity (i.e. conversion of progesterone to -hydroxyprogesterone) in human theca cells. That study found that the ERK cascade is not involved in forskolin-mediated stimulation of -hydroxylase activity (41). Instead, they showed that PI3-kinase inhibitor LY294002 block insulin-stimulated P450c17 activity completely, consequently concluding that insulin stimulates both PI3-kinase and ERK activity in human theca cells but only PI3-kinase mediated insulin augmentation of forskolin-stimulated p450c17 activity. Explanations for this apparently contradictory result may include differences in species, cell preparation, incubation time with agents, and assay procedures for androgen production. Indeed, we used freshly prepared bovine theca cells and stimulated cells with the inhibitor for 12 or 24 h. Then the effect of ERK inhibition on thecal steroidogenesis was assessed using direct measurement of androstenedione concentration and expression of P450c17 mRNA. On the other hand, the study mentioned above used frozen-preserved human theca cells and stimulated the cells with forskolin, insulin, U0126, or their combination for 3 d. Thereafter effects were analyzed according to the change in 17-hydroxylase activities. Further study is necessary to clarify the potential role of ERK signaling pathway in the control of thecal steroidogenesis.
Taking this evidence together, we conclude that, in bovine theca cells, LH/cAMP activates ERK in a biphasic manner through MEK-independent pathway. Furthermore, inhibition of ERK activity increases progesterone production and decreases androstenedione production with concomitant modulation of expression of StAR and P450c17. Findings of this study provide new evidence that LH-mediated ERK activity plays a pivotal role in the regulation of steroidogenesis in ovarian theca cells.
Acknowledgments
We thank Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA) and the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD) for providing the human LH; Dr. J. F. Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA) for antihuman StAR antibodies; and the Kanazawa Meat Inspection Office (Kanazawa, Japan) for allowing us to collect the bovine ovaries used in these experiments. We also thank Dr. N. Suganuma (Department of Environmental Health, University of Fukui, Fukui, Japan) for help in statistics; and Mikiko Misawa and Yuka Sugimoto for excellent technical assistance.
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