The Calcium Signaling Pathway Regulates Leydig Cell ...

REPRODUCTION-DEVELOPMENT

The Calcium Signaling Pathway Regulates Leydig Cell Steroidogenesis through a Transcriptional Cascade Involving the Nuclear Receptor NR4A1 and the Steroidogenic Acute Regulatory Protein Houssein S. Abdou, Gabrielle Villeneuve, and Jacques J. Tremblay Reproduction, Mother and Youth Health (H.S.A., G.V., J.J.T.), Centre Hospitalier Universitaire de Québec Research Centre, Québec City, Québec, Canada G1V 4G2; and Centre for Research in Biology of Reproduction (J.J.T.), Department of Obstetrics, Gynecology, and Reproduction, Faculty of Medicine, Université Laval, Québec City, Québec, Canada G1V 0A6

In the gonads and adrenal glands, the transient increase in steroidogenesis after hormonal stimulation requires modulation of steroidogenic acute regulatory protein (Star) expression and activity in a tightly regulated process involving cAMP and Ca2⫹. In Leydig cells, the cAMP and Ca2⫹ pathways account for most if not all of LH-induced steroidogenesis. Although the cAMP-activated molecular network has been well characterized in Leydig cells, little is known about the molecular cascade triggered by the Ca2⫹ signaling pathway and the transcription factors responsible for mediating the genomic response. It is established that LH induces an increase in cytoplasmic Ca2⫹ from the endoplasmic reticulum primarily through the ryanodine receptors. Previous reports also suggested a role of the Ca2⫹ signaling pathway in Star expression based on the fact that inhibition of the Ca2⫹/calmodulin (CaM) protein kinase pathway greatly impaired Star expression in Leydig and adrenal cells. In this study, we used ryanodine receptors and CaM antagonists to show that the increase in intracellular Ca2⫹ level is an essential modulator of progesterone synthesis through the regulation of Star gene expression in MA-10 Leydig cells. Furthermore, we mapped a Ca2⫹/CaMsensitive element in the Star promoter, which led to the identification of the nuclear receptor 4A1 (NR4A1) as a key mediator of the Ca2⫹/CaM signaling pathway in these cells. These data provide new insights into the Ca2⫹ molecular pathway essential for steroidogenesis in Leydig cells. (Endocrinology 154: 511–520, 2013)

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estosterone synthesis is regulated in a pulsatile manner by modulating circulating LH levels. In the testis, it is established that binding of LH to its G protein-coupled receptor [LH receptor (LHR)] on Leydig cells acutely increases the levels of cAMP and cytoplasmic Ca2⫹, which are both required for steroidogenesis (reviewed in Ref. 1). The transient synthesis of steroids in response to LH occurs through modulation of a key factor, steroidogenic acute regulatory protein (STAR), whose expression highly correlates with testosterone level (2). STAR is the ratelimiting factor in hormone-dependent steroidogenesis because it is essential for the transport of cholesterol, pre-

cursor to all steroids, from the outer to the inner mitochondrial membrane (2, 3). The importance of STAR is emphasized by naturally occurring mutations in humans that suppress gonadal and adrenal steroidogenesis and cause lipoid congenital adrenal hyperplasia and pseudohermaphroditism in male (4), phenotypes also observed in Star⫺/⫺ mice (5). The cAMP pathway induces Star expression and steroidogenesis through activation by posttranslational modifications of several transcription factors including the cAMP-response element binding protein/cAMP-response element modulator (6) and GATA binding protein

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2013 by The Endocrine Society doi: 10.1210/en.2012-1767 Received July 22, 2012. Accepted November 7, 2012. First Published Online November 26, 2012

Abbreviations: CaM, Calmodulin; CaMKI, Ca2⫹/CaM-dependent kinase I; DMSO, dimethylsulfoxide; ER, endoplasmic reticulum; Fsk, forskolin; hCG, human chorionic gonadotropin; IP3R, inositol triphosphate receptor; LHR, LH receptor; NR4A1, nuclear receptor 4A1; OH-Chol, 22(R)-hydroxycholesterol; PKA, protein kinase A; RyR, ryanodine receptor; SF1, steroidogenic factor 1; STAR, steroidogenic acute regulatory protein.

Endocrinology, January 2013, 154(1):511–520

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4 (7). Maximal Star activation also requires de novo synthesis of the nuclear receptor 4A1 (NR4A1) also known as NUR77 and nerve growth factor induced B (NGFI-B) transcription factor (8). Furthermore, induction of NR4A1 and STAR involves the Ca2⫹ pathway because inhibition of the Ca2⫹/calmodulin (CaM)-dependent kinase I (CaMKI) greatly impaired LH- and forskolin (Fsk)induced Nr4a1 and Star gene expression in the MA-10 Leydig cell line (8, 9). The classical cAMP pathway has long been known to account for most of the LH-induced steroidogenesis (reviewed in Ref. 10). An increase in intracellular Ca2⫹ is nonetheless required. Indeed, cytosolic Ca2⫹ concentration increases in parallel with testosterone production (11). The presence of Ca2⫹ in the culture media was also found to further increase the human chorionic gonadotropin (hCG)-induced Star mRNA and STAR protein levels as well as progesterone production in the mLTC-1 mouse Leydig cell line (12). Conversely, in the absence of extracellular Ca2⫹, LH/hCG-induced steroidogenesis was decreased (13, 14). It is hypothesized that the decrease in steroidogenesis observed when blocking extracellular Ca2⫹ influx using divalent metals is due to an impairment of LH- and cAMPmediated increase in intracellular Ca2⫹ from internal stores [i.e. endoplasmic reticulum (ER)] (15). The ER of murine Leydig cells contains two L-type Ca2⫹ channels receptors: the ryanodine receptors (RyRs) I, II, and III and inositol triphosphate receptors (IP3Rs) I, II, and III (16). The RyRI, some of the RyRIII and IP3RII are localized near the plasma membrane, whereas the RyRII, RyRIII, IP3RI, IP3RIII are localized throughout the cytoplasm (16). Although both types of receptors (RyRs and IP3Rs) are required for the LH-induced rise in intracellular Ca2⫹, activation of the RyRs is of primary importance. Costa et al. (16) showed that the acute (0-200 min) induction of testosterone synthesis in response to LH was reduced by approximately 50% when Leydig cells were incubated with the RyR inhibitor ryanodine (100 ␮M), whereas the IP3R inhibitor 2-aminoethoxydiphenyl borate had no effect. Interestingly, ryanodine had no inhibitory effect on basal testosterone production (16), thus suggesting that the Ca2⫹ signaling pathway initiated by the internal stores is mostly required for the acute LH-induced steroidogenesis. Although Ca2⫹ is essential for hormone-induced steroidogenesis, its mechanisms of action remain poorly understood. In the present study, we sought to determine the molecular mechanisms by which the Ca2⫹ signaling pathway participates in the cAMP-induced steroidogenesis. Here we show that the inhibitory effect of ryanodine (RyR inhibitor) and W7 hydrochloride (CaM inhibitor) on the cAMP-induced steroidogenesis in Leydig MA-10 cells is


due to a decrease in STAR expression and promoter activity. Furthermore, expression and promoter activity of NR4A1, a mediator of hormone-stimulated Star expression, was also compromised by ryanodine. Our data identify a transcriptional cascade initiated by ER Ca2⫹ store release in the regulation of steroid hormone biosynthesis.

Materials and Methods Cell culture Mouse tumor MA-10 Leydig cells (17) were provided by Dr. Mario Ascoli (University of Iowa, Iowa City, IA) and maintained in DMEM-F12 supplemented with penicillin and streptomycin and 15% horse serum and incubated at 37 C in 5% CO2.

Chemicals The RyR inhibitors ryanodine and dantrolene and the CaM inhibitor W7 hydroxychloride were obtained from Tocris Bioscience (Minneapolis, MN). Fsk, 22(R)-hydroxycholesterol (OH-Chol), and 8-bromo-cAMP (8Br-cAMP) were purchased from Sigma-Aldrich Canada (Oakville, Ontario, Canada). Ryanodine binds to all three RyRs, whereas dantrolene inhibits mainly RyRI and RyRIII (18). Dantrolene and ryanodine target RyRs located in the ER and in the plasma membrane. There is currently no specific antagonist for a given RyR.

Plasmids The ⫺980, ⫺195, ⫺144, ⫺120, ⫺95, and ⫺70-bp Star reporter constructs and the ⫺980-bp construct harboring a mutation in the NUR77/NGFI-B response element/steroidogenic factor 1 (SF1) (NR4A1/5A1) element at ⫺100 bp were previously described (8). The ⫺747 to ⫹50-bp Nr4a1 reporter construct was previously described (9).

Protein purification and Western blots Nuclear extracts, whole-cell lysates, and Western blots were performed as previously described (8). For each treatment, cells were cultured in serum-free medium in the presence of vehicle [dimethylsulfoxide (DMSO) or ethanol)], Fsk (10 ␮M) alone, or Fsk plus ryanodine (75 ␮M) or W7 hydrochloride (20 ␮M) for either 1 h (nuclear extracts for NR4A1 detection) or 4 h (wholecell lysates for STAR detection). The antibodies used for detecting NR4A1 (M-210), STAR (FL-285), and lamin B (C-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies used for NR5A1 and tubulin were purchased from BD Bioscience PharMingen (San Diego, CA) and SigmaAldrich Canada (Oakville, Ontario, Canada), respectively. Data from Western blots were analyzed using the ImageJ software.

RNA isolation and quantitative RT-PCR RNA isolation, cDNA synthesis, and quantitative RT-PCR were performed as previously described (8). Briefly, for each treatment, cells were cultured in serum-free medium in the presence of vehicle (DMSO/ethanol), Fsk (10 ␮M) alone, or Fsk plus ryanodine (75 ␮M) for either 1 h (for Nr4a1 mRNA quantification) or 2.5 h (for Star mRNA quantification).


Transfections and reporter assays MA-10 Leydig cells were transfected and lysed, and lysates were analyzed as previously described (8, 9). Before lysis, cells were treated with vehicle (DMSO or ethanol), ryanodine (150 ␮M), 8Br-cAMP (0.5 mM), or 8Br-cAMP plus ryanodine for 4 h. 8Br-cAMP, or its analog dibutyryl cAMP, are usually used by us (8, 9) and others (6, 19) for promoter stimulation because it leads to more consistent and reliable promoter activation than Fsk. These experiments were performed at least three times, each time in duplicate.

Calcium quantification Intracellular Ca2⫹ levels were measured using the Fluo-8 calcium assay kit (Abcam, Toronto, Ontario, Canada), as recommended by the manufacturer. Briefly, MA-10 Leydig cells were seeded in 96-well black Corning plates with clear bottom at 40,000 cells per well. Two days later, the cells were washed once with 100 ␮l serum-free DMEM/F12 medium and then treated with DMSO, Fsk (10 ␮M), or Fsk plus ryanodine (150 ␮M) in serum-free medium for 15 min. The medium was then replaced with 100 ␮l of the Fluo-8 dye, and fluorescence was read at 485 nm excitation/530 nm emission using the Infinite 200 PRO reader (Tecan, Männedorf, Switzerland).

Progesterone quantification ELISAs for progesterone quantification were performed as recommended by the manufacturer (Cayman Chemical, Ann Arbor, MI). Briefly, MA-10 cells were seeded in 24-well plates at 150,000 cells per well. Two days later (⬃500,000 cells), the medium was replaced with serum-free medium in the presence of vehicle (DMSO or ethanol), OH-Chol (40 ␮M), OH-Chol plus ryanodine (75 ␮M), Fsk (10 ␮M) alone, Fsk plus W7 hydrochloride (40-80 ␮M), or Fsk plus ryanodine for 4 h. The medium (500 ␮l) was then transferred to 1.5-ml tubes and stored at ⫺80 C until ELISAs were performed. For basal progesterone quantification (without Fsk stimulation), 50 ␮l of medium was diluted with 950 ␮l EIA buffer (1:20), 50 ␮l of which was then used in the ELISA. For Fsk-stimulated cells, 20 ␮l of the medium was diluted with 980 ␮l EIA buffer (1:50), 50 ␮l of which was subsequently used in the ELISA.

Statistical analyses For all single comparisons between two experimental groups, paired Student’s t tests were performed. For all statistical analyses, P ⬍ 0.05 was considered significant. For multiple group comparisons, statistical analyses were done using one-way ANOVA followed by the Bonferroni posttest. All statistical analyses were done using the SigmaStat software package (Systat Software Inc., San Jose, CA).

Results Inhibition of RyRs in MA-10 Leydig cells impairs steroidogenesis by blunting cAMP-induced Star expression Ryanodine inhibits all RyRs (RyRI, -II, and -III) located mainly on the ER of the Leydig cells (16). To evaluate the


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effects of blocking the RyRs with its antagonist ryanodine on the cAMP-stimulated internal Ca2⫹ release, we performed a fluorescence Ca2⫹ quantification assay. Stimulation of MA-10 Leydig cells with Fsk for 15 min increased intracellular Ca2⫹ by about 3-fold (Fig. 1A). This increase was blocked in the presence of the RyR inhibitor ryanodine (Fig. 1A), indicating that functional RyRs are essential for the Fsk-induced intracellular Ca2⫹ increase in MA-10 Leydig cells. We next tested whether blocking the internal Ca2⫹ release with ryanodine could affect steroidogenesis in MA-10 Leydig cells by measuring progesterone levels using ELISA. Progesterone is the main steroid hormone produced by this cell line because of a mutation in the CYP17A1 (cytochrome P450, family 17, subfamily A, polypeptide 1) enzyme, which prevents progesterone hydroxylation and thus stops steroidogenesis (17, 20-22). Stimulation of MA-10 cells for 4 h with Fsk resulted in an approximately 30-fold increase in progesterone level (Fig. 1B). Blocking the RyRs with ryanodine reduced the Fskinduced progesterone level between 40 and 60% according to the dose of inhibitor used (Fig. 1B), with 75 ␮M being the most effective as previously reported (16). Moreover, treatment of MA-10 cells with 75 ␮M ryanodine at an earlier (2 h) or later (8 h) time point also impaired the Fsk-dependent increase in steroidogenesis (Fig. 1D). Inhibition of the RyRs with dantrolene, another widely used RyR inhibitor (23, 24) that preferentially targets RyRI and RyRIII (18), also decreased the Fsk-induced steroidogenesis (Fig. 1C), albeit to a lesser extent (compare Fig. 1, B and C). These data are in agreement with a recently published report using mouse primary Leydig cells (16) and validate the MA-10 Leydig cell line as an adequate model to study the molecular mechanisms of Ca2⫹ action in Leydig cells. Inhibiting the RyRs could decrease steroidogenesis by preventing the translocation of cholesterol from the outer to the inner mitochondrial membrane and/or by affecting the expression/activity of any of the downstream steroidogenic enzymes. To evaluate whether inhibiting the RyRs decreases steroidogenesis by affecting cholesterol translocation, we used the cholesterol analog OH-Chol, a substrate for CYP11A1 (cytochrome P450, family 17, subfamily A, polypeptide 1) known to freely translocate to the inner mitochondrial membrane, thus bypassing the requirement of transporters such as STAR. Treating MA-10 cells with OH-Chol led to an approximately 10-fold increase in steroidogenesis (Fig. 2A). Inhibition of the RyRs with ryanodine did not repress the OH-Chol-induced steroidogenesis (Fig. 2A). In fact, combining ryanodine with OH-Chol resulted in a 1.7-fold increase in progesterone level compared with OH-Chol alone (Fig. 2A). These results indicate that ryanodine inhibits the Fsk-increased ste-

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FIG. 1. Blocking internal Ca2⫹ release impairs steroidogenesis. A, Intracellular Ca2⫹ from MA-10 Leydig cells was quantified after a 15-min treatment with vehicle (DMSO, white bar), Fsk (10 ␮M, black bar), or Fsk plus ryanodine (Rya) (150 ␮M, gray bar). The assay was performed in duplicate in three independent experiments. **, P ⬍ 0.02. B, ELISA was used to quantify progesterone secreted by MA-10 Leydig cells after a 4-h exposure to vehicle (DMSO, white bar), Fsk (10 ␮M, black bar), or Fsk plus ryanodine (20, 40, 75, 150 ␮M, gray bars). Results represent data obtained from three independent experiments, each performed in duplicate. C, Progesterone levels from MA-10 Leydig cells were measured after a 4-h exposure to vehicle (DMSO, white bar), Fsk (10 ␮M, black bar), or Fsk plus dantrolene (Dtrl) (40, 80 ␮M, gray bars). D, Progesterone levels from MA-10 Leydig cells were measured after treatments with vehicle (DMSO, white bar), Fsk (10 ␮M, black bars), or Fsk plus ryanodine (75 ␮M, gray bars) for 2, 4, and 8 h as indicated. For B, C and D, results represent data obtained from three independent experiments each performed in duplicate. P ⬍ 0.05 was considered statistically significant using a one-way ANOVA test. Different lowercase letters indicate a statistically significant difference.

roidogenesis by preventing cholesterol translocation from the outer to the inner mitochondrial membrane. STAR is an essential component of the cholesterol transport complex and required for the acute Fsk/cAMPinduced steroidogenesis (1-3). Because ryanodine did not impair steroidogenesis when cholesterol transport was bypassed (Fig. 2A), we evaluated whether blocking the RyR Ca2⫹ channels impaired STAR expression in MA-10 Leydig cells. As expected, STAR protein level was induced in MA-10 Leydig cells treated with Fsk for 4 h (Fig. 2B), a time point where the STAR protein is maximally induced after cAMP (8) and Fsk (data not shown) treatment. Interestingly, the Fsk-mediated stimulation was inhibited by about 50% in the presence of ryanodine (Fig. 2B). These

data suggest that blocking Ca2⫹ influx from the ER through the RyRs impairs steroidogenesis by modulating STAR protein level. To test whether the decrease in STAR protein was due to impaired Star gene expression, we first evaluated Star mRNA level. MA-10 Leydig cells were stimulated with Fsk for 2.5 h to ensure an optimal increase in Star transcription (8). This led to a 5-fold increase in Star mRNA level, which was significantly decreased in the presence of ryanodine (Fig. 2C). Next, to determine whether the inhibitory effects observed when blocking the RyRs were caused by a decrease in Star transcription, we analyzed the activity of a ⫺980-bp mouse Star reporter by transient transfections in MA-10 Leydig cells in the presence or absence of two different RyR antagonists. As shown in Fig. 2D, 8Br-cAMP activated the Star promoter by approximately 8-fold, as previously reported (8). Addition of the RyR inhibitors ryanodine or dantrolene (18, 25) impaired the cAMP-induced Star promoter activity by approximately 50% (Fig. 2D). Altogether, these results indicate that the cAMP-dependent increase in steroidogenesis and Star gene expression require Ca2⫹ release from the ER through activated RyRs.

Ca2ⴙ-induced Star transcription requires the NR4A1/5A1 element To identify the regulatory elements on the Star promoter involved in the Ca2⫹ response and thus targeted by the RyR antagonist, MA-10 Leydig cells were transiently transfected with a series of 5⬘ progressive deletions of the Star promoter, and cells were treated with vehicle, cAMP alone, or cAMP plus dantrolene. As shown in Fig. 3A, dantrolene repressed the cAMP-induced activity of the ⫺195-, ⫺144-, and ⫺120-bp Star reporters. However, the ⫺95- and ⫺70-bp Star reporters were insensitive to dantrolene (Fig. 2D). These data indicate that the dantrolenesensitive elements are located between ⫺120 and ⫺95 bp of the Star promoter. The promoter region encompassing ⫺120 to ⫺95 bp was previously shown to contain a nuclear receptor binding site at ⫺100 bp (ATCCTTGA) that is recognized by


FIG. 2. Blocking internal Ca2⫹ release reduces Star expression in MA10 cells. A, Progesterone levels were measured from MA-10 Leydig cells treated for 4 h with vehicle (DMSO, white bar), OH-Chol (40 ␮M, black bar), or OH-Chol plus ryanodine (Rya) (75 ␮M, gray bar). B, STAR protein levels were determined by Western blot using whole-cell extracts from MA-10 Leydig cells treated for 4 h with vehicle, Fsk (10 ␮M), or Fsk plus Rya (75 ␮M). A representative blot from three independent experiments is shown. The bands were quantified using ImageJ software and analyzed using Student’s t test: *, P ⬍ 0.05 was considered statistically significant. C, Star mRNA levels were determined by quantitative RT-PCR using total RNA isolated from MA10 Leydig cells treated for 2.5 h with vehicle (white bar), Fsk (10 ␮M, black bar), or Fsk plus Rya (75 ␮M, gray bar). Values were normalized to Rpl19 level, and results are shown as fold induction over vehicle ⫾ SD. *, Statistically significant difference (P ⬍ 0.05). D, Transient transfections of the murine Star promoter (⫺980 to ⫹16 bp) reporter were performed in MA-10 Leydig cells treated for 4 h with vehicle (white bar), 8Br-cAMP (0.5 mM, black bar), 8Br-cAMP plus Rya (75 ␮M, striped bar), or 8Br-cAMP plus dantrolene (Dtrl) (150 ␮M, gray bar). Previously characterized NR5A1 (⫺890, ⫺135, ⫺100, and ⫺42 bp) and NR4A1/5A1 (⫺100 bp) elements are indicated. Results are shown as fold induction over vehicle ⫾ SD. *, P ⬍ 0.05; *** P ⬍ 0.01.

the nuclear receptors NR5A1 (SF1) (19) and NR4A1 (NUR77) (8). In addition, this NR4A1/5A1 element is required for maximal cAMP-dependent activation of the Star promoter (Fig. 3B) (8). To verify whether this NR4A1/5A1 element could mediate the stimulatory effects of ER Ca2⫹ influx on Star expression, a mutation known to prevent binding of NR4A1 and NR5A1 (ATc-


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FIG. 3. Ca2⫹-induced Star transcription requires the NR4A1/5A1 element. A, To locate the Ca2⫹-responsive element within the murine Star promoter, MA-10 cells were transiently transfected with a series of 5⬘ deletion constructs (⫺980, ⫺195, ⫺144, ⫺120, ⫺95, and ⫺70 bp) of the murine Star promoter and cells were treated with vehicle (white bars), 8Br-cAMP (0.5 mM, black bars), or 8Br-cAMP plus dantrolene (150 ␮M, gray bars) for 4 h. Results from four independent experiments each in duplicate are shown as fold induction over vehicle ⫾ SD. *, P ⬍ 0.05; **, P ⬍ 0.02. B, MA-10 cells were transiently transfected with a ⫺980-bp mouse Star reporter, either wild type or harboring a mutation in the NR4A1/5A1 element at ⫺100 bp. Cells were then treated with vehicle (white bars), 8Br-cAMP (0.5 mM, black bars), or 8Br-cAMP plus ryanodine (75 ␮M, gray bars) for 4 h. Results represent data obtained from three independent experiments each performed in duplicate. P ⬍ 0.05 was considered statistically significant using a one-way ANOVA test. Different lowercase letters indicate a statistically significant difference.

cTTGA to ATaaTTGA) was introduced in the context of the ⫺980-bp Star reporter. This mutation abrogated the inhibitory effects of ryanodine on cAMP-induced Star promoter activity (Fig. 3B), thus suggesting the involvement of either NR4A1 or NR5A1 in mediating the Ca2⫹-stimulatory effect on Star expression. cAMP-mediated Ca2ⴙ release by the ER is essential for NR4A1 expression To verify whether NR4A1 and NR5A1 expression is modulated by changes in intracellular Ca2⫹ level, Western blots were performed. As shown in Fig. 4A, treatment of MA-10 Leydig cells with Fsk for 1 h induced the NR4A1 protein without affecting NR5A1, as previously reported

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Intracellular Ca2ⴙ-induced steroidogenesis requires an intact NR4A1/5A1 element in the Star promoter Because blocking Ca2⫹ release from intracellular stores inhibits Fsk-induced progesterone production, expression of STAR and its regulator NR4A1, we tested whether inhibiting the activity of CaM, a Ca2⫹ sensor protein and an immediate Ca2⫹ effector (26), would mimic the effect of ryanodine. Treatment of MA-10 Leydig cells with two concentrations of W7 hydrochloride, a potent CaM antagonist, abolished the Fsk-induced steroidogenesis as measured by progesterone production (Fig. 5). Inhibition of CaM also suppressed the Fsk-mediated induction in STAR protein (Fig. 6A, left panel) and reduced the NR4A1 protein (Fig. 6A, right FIG. 4. NR4A1 expression is modulated by changes in internal Ca2⫹ level. A, NR4A1 and NR5A1 protein levels were determined by Western blot using nuclear extracts from MA-10 panel) while having no effect on Leydig cells treated for 1 h with vehicle, Fsk (10 ␮M), or Fsk plus ryanodine (Rya) (75 ␮M). A NR5A1 (Fig. 6A, right panel). Similar representative blot from three independent experiments is shown. Detection of lamin B was results were observed at the transcripused as loading control. The bands were quantified using ImageJ software and analyzed using Student’s t test. *, P ⬍ 0.05 was considered statistically significant. B, Nr4a1 mRNA level was tional level on the Star (⫺980-bp redetermined by quantitative RT-PCR using RNA isolated from MA-10 Leydig cells treated for porter) and Nr4a1 (⫺747-bp reporter) 1 h with vehicle, Fsk (10 ␮M), or Fsk plus Rya (75 ␮M). Values were normalized to Rpl19 level, promoters by transient transfections in and results are shown as fold activation over vehicle ⫾ SD. *, Statistically significant difference (P ⬍ 0.05). C, Transient transfections of the rat Nr4a1 promoter (⫺747 to ⫹50 bp) reporter MA-10 Leydig cells (Fig. 6B). Furtherwere performed in MA-10 Leydig cells treated with vehicle (white bar), cAMP (0.5 mM, black more, mutation of the NR4A1/5A1 elbar), or cAMP plus Rya (75 ␮M, gray bar). Results are shown as fold induction over vehicle ⫾ ement at ⫺100 bp abolished the inhibSD. *, Statistically significant difference (P ⬍ 0.05). D, MA-10 cells were transiently transfected itory effects of W7 on the Star promoter with either an empty vector (⫺) or an expression vector encoding NR4A1 (⫹), and 48 h later, cells were treated with vehicle, Fsk (10 ␮M), or Fsk plus Rya (75 ␮M) for 4 h. Whole-cell after cAMP stimulation (Fig. 6C). extracts were prepared, and STAR protein was detected by Western blot. Tubulin was used as Taken together, these results suggest a loading control. A representative blot from three independent experiments is shown. that the Fsk/cAMP-dependent induction of steroidogenesis requires, at least (8, 9). Blocking ER Ca2⫹ release with ryanodine impaired in part, induction of Star expression through an intact the Fsk-mediated induction of the NR4A1 protein by ap- NR4A1/5A1 element and that NR4A1 is a target and a proximately 60% while having no effect on NR5A1 pro- mediator of Ca2⫹/CaM signaling in these cells. tein (Fig. 4A). Similar results were observed at the mRNA level by quantitative PCR (Fig. 4B). Using transient transfections of a proximal rat ⫺747-bp Nr4a1 reporter in Discussion MA-10 Leydig cells, we found that the cAMP-induced The importance of the Ca2⫹ signaling pathway is well Nr4a1 promoter activity was inhibited by 40% in the presestablished in many cellular processes such as gene reguence of ryanodine (Fig. 4C). These results show that lation, apoptosis, cell differentiation, and proliferation as Nr4a1 expression is regulated at the transcriptional level well as in steroid hormone synthesis and male fertility (11, by intracellular Ca2⫹ release and thus identify this gene as 13, 27, 28). Several studies have shown the requirement of a novel target of Ca2⫹ signaling that modulates steroido- Ca2⫹ in steroidogenesis after tropic hormone stimulation genesis in Leydig cells. This is further supported by the fact that causes a cytoplasmic Ca2⫹ influx in a biphasic manner that overexpression of NR4A1 in Fsk-treated MA-10 Ley- involving first cytoplasmic T-type channels and second ER dig cells eliminated the repressive effects of ryanodine and L-type and associated channels (11, 13, 15, 16, 29). Furrestored STAR protein levels (Fig. 4D). thermore, clinical use of an L-type Ca2⫹ channel antago-



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FIG. 5. Inhibition of the Ca2⫹-sensing protein CaM impairs steroidogenesis in MA-10 Leydig cells. ELISA was used to quantify progesterone levels secreted by MA-10 Leydig cells treated for 4 h with vehicle (DMSO, white bar), Fsk (10 ␮M, black bar), or Fsk plus W7 hydrochloride (40 and 80 ␮M, gray bars). Results represent data obtained from three independent experiments each performed in duplicate. P ⬍ 0.05 was considered statistically significant using a oneway ANOVA test. Different lowercase letters indicate a statistically significant difference.

nist as antihypertensive and antiarrhythmic medication has been shown to reversibly affect male fertility in humans by preventing sperm capacitation (reviewed in Ref. 30) and by inhibiting steroidogenesis and spermatogenesis in rodents (14, 27, 31). Thus, proper Ca2⫹ signaling is critical for male reproductive health. Ca2ⴙ influx increases Star expression: a generalized mechanism Several studies in steroidogenic tissues other than Leydig cells support the requirement for the Ca2⫹ signaling pathway in increasing STAR protein level. For example, in adrenal cells, it has been shown that STAR level and steroid production are induced by the L-type calcium channel agonist Bay K8644 in the human H295R adrenocortical cell line (32) and in bovine glomerulosa (33) and fasciculata (34, 35) cells. When these cells were stimulated with ACTH or angiotensin II, there was an increase in cytosolic calcium level, paralleled by an increase in STAR protein level and steroid synthesis (35). The ACTH-induced STAR protein and steroidogenesis were inhibited by a CaMK inhibitor, KN-93, suggesting the involvement of the CaMK pathway (35). More recently, regulation of STAR expression by the CaMK pathway was also reported in mouse Leydig cells, with CaMKI being identified as the most relevant kinase in these cells (8). Together these studies support a conserved stimulatory mechanism involving the Ca2⫹/CaMK pathway in hormone-induced STAR expression in the main steroidogenic cells. The molecular

FIG. 6. CaM inhibition decreases STAR and NR4A1 expression in MA10 Leydig cells. A, STAR (left panel) and NR4A1 and NR5A1 (right panel) protein levels were determined by Western blot using whole-cell extracts (left panel) or nuclear extracts (right panel) from MA-10 Leydig cells treated with vehicle, Fsk (10 ␮M), or Fsk plus W7 (80 ␮M). Representative blots from three independent experiments are shown. Tubulin and lamin B were used as loading controls. The bands were quantified using ImageJ software and analyzed using Student’s t test. *, P ⬍ 0.05 was considered statistically significant. B, Transient transfections of the murine ⫺980- to ⫹16-bp Star reporter (left panel) and of the rat ⫺747- to ⫹50-bp Nr4a1 reporter (right panel) were performed in MA-10 Leydig cells treated with vehicle (white bars), 8BrcAMP (black bars), or 8Br-cAMP plus W7 (80 ␮M, gray bars). Results are shown as fold induction over vehicle in the absence of cAMP ⫾ SD. *, P ⬍ 0.05; **, P ⬍ 0.01. C, MA-10 cells were transiently transfected with a ⫺980-bp murine Star promoter harboring a mutation in the NR4A1/5A1 element at ⫺100 bp. Cells were then treated with vehicle (white bar), 8Br-cAMP (0.5 mM, black bar), or 8Br-cAMP plus W7 (80 ␮M, gray bar) for 4 h. Results are shown as fold induction over vehicle ⫾ SD.

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mechanisms linking the hormonal stimuli, Ca2⫹, and increased Star expression remained to be fully characterized. Recently, all three RyRs were shown to be present in Leydig cells and reported to be critical players in mediating the Ca2⫹ release from intracellular stores in response to LH stimulation (16). Blocking RyR activity blunted LH-induced testosterone secretion (16). This is consistent with the fact that RyRs and L-type calcium channels are functionally coupled, at least in skeletal muscles (36) and neurons (37). In the present study, we found that functional RyRs are also essential for maximal Fsk-induced Star expression. We used two distinct RyR antagonists: ryanodine and dantrolene. Ryanodine binds to all three RyRs and induces allosteric changes affecting Ca2⫹ permeability (38). At high doses (ⱖ100 ␮M), ryanodine irreversibly locks the channel in a closed configuration (39). Dantrolene on the other hand inhibits mainly RyRI and RyRIII (18), although it can also inhibit RyRII under certain conditions (40). Dantrolene acts by stabilizing domain-domain interactions within the RyR, locking it in a closed conformation (41). Although both antagonists inhibited Fsk-induced progesterone production and Star expression, ryanodine was more effective than dantrolene. These data indicate that RyRII, along with RyRI and/or RyRIII, is essential for optimal Ca2⫹ release from internal stores in stimulated Leydig cells. The nuclear receptor NR4A1 is a downstream effector of Ca2ⴙ signaling in MA-10 Leydig cells Despite the requirement for Ca2⫹ release via RyRs in hormone-induced Star expression, the downstream molecular effector(s) of Ca2⫹ signaling remain poorly characterized. In this study, we have mapped the Ca2⫹-responsive region on the murine Star promoter to position ⫺100 bp. This element, also called NR4A1/5A1 (NBRE/SF1), is known to bind the nuclear receptors NR5A1 (SF1) and NR4A1 (NUR77) (8), which indicates that one or both nuclear receptors might be involved in Ca2⫹ signaling in MA-10 Leydig cells. In agreement with this, a previous study found that hCG- plus Ca2⫹-mediated stimulation of Star expression in mLTC-1 Leydig cells was correlated with a 2- to 3-fold increase in Nr5a1 mRNA level (12), suggesting a potential role for this nuclear receptor in Ca2⫹ signaling. Although not mutually exclusive, our present data would rather support a predominant role for NR4A1. Indeed, we found that Nr4a1 promoter activity, mRNA level, and NR4A1 protein level were modulated by intracellular Ca2⫹ influx from the ER in a manner that strikingly paralleled that of STAR. In addition, overexpression of NR4A1 restored the STAR level despite the presence of ryanodine. More importantly, we found that the NR5A1 protein level was unaffected either by changes in intracellular Ca2⫹ level or by inhibition of CaM. Fur-


thermore, the involvement of Ca2⫹ signaling in Nr4a1 expression has been reported in tissues other than steroidogenic cells. For instance, Nr4a1 expression is induced in PC12 adrenal medulla cells exposed to Ca2⫹ ionophores (42). Similarly, in the gonadotrope cell line ␣T3-1, the GnRH-induced NR4A1 protein was also shown to be dependent on extracellular calcium influx (43). In addition to NR4A1, another NR4A member, NR4A2, is present in Leydig cells, but NR4A1 is considerably more abundant (44). Thus, although NR5A1 or NR4A2 could be involved in the Ca2⫹-induced steroidogenesis, our present data along with evidence from the literature point to the nuclear receptor NR4A1 as a major effector of this pathway. Cross talk between the cAMP and Ca2ⴙ pathways in Leydig cells It has been demonstrated that the LH-dependent increase in cytosolic Ca2⫹ occurs in two phases: first via a calcium-induced calcium release that is initiated by the extracellular influx of Ca2⫹ through the T-type calcium channels CaV3 and second via the L-type and associated channels on the ER, which accounts for most of the cytosolic Ca2⫹ increase (16). The second phase of Ca2⫹ release occurs mainly through RyRs but also involves IP3Rs; it also requires protein kinase A (PKA) and to a lesser extent PKC (13, 29). Interestingly, it has been shown that ␤-adrenergic stimulation of cardiomyocytes activates the RyRs through direct phosphorylation by PKA (45-47), thus demonstrating that the cAMP pathway can enhance intracellular Ca2⫹ release. Furthermore, pharmacological inhibition of PKA activity using H89 was found to completely inhibit LH/hCG-dependent Ca2⫹ release in Leydig cells (13, 29). Thus, it is likely that PKA directly affects the activity of the RyRs in steroidogenic cells as in cardiac muscle cells, although this remains to be confirmed.

Acknowledgments We thank Dr. Mario Ascoli for generously providing the MA-10 cell line used in this study. Address all correspondence and requests for reprints to: Dr. Jacques J. Tremblay, Reproduction, Mother and Youth Health, Centre Hospitalier Universitaire de Québec Research Centre, Centre Hospitalier del’Université Laval Room T1-49, 2705 Laurier Boulevard, Québec City, Québec, Canada G1V 4G2. E-mail: [email protected]. J.J.T. holds a Chercheur-Boursier Scholarship from the Fonds de recherche du Québec–Santé. This work was funded by a grant from the Natural Sciences and Engineering Research Council of Canada (262224 to J.J.T.). Disclosure Summary: The authors have nothing to disclose.



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