Abstract
Previous studies have reported the roles of Ca 2C in steroidogenesis. The present study has investigated an inhibitory effect of Ca 2C 
2C
channels on gene expression of steroidogenic acute regulatory (STAR) protein that regulates the transfer of substrate cholesterol to the inner mitochondrial membrane for steroidogenesis. Blocking Ca 2C influx through L-type Ca 2C channels using the selective Ca 2C channel blocker, nifedipine, markedly enhanced cAMP-induced STAR protein expression and progesterone production in MA-10 mouse Leydig cells. This was confirmed by utilization of different L-type Ca It was also observed that there is a synergistic interaction between nifedipine and cAMP. Normally, sub-threshold levels of cAMP are unable to induce steroidogenesis, but in the presence of the L-type Ca 2C channel blocker, they increased STAR protein and steroid hormone to the maximal levels. However, in the absence of minimal levels of cAMP, none of the L-type Ca 2C channel blockers are able to induce Star gene expression. These observations indicate that Ca 2C influx through L-type CaIntroduction Steroidogenesis in testicular Leydig cells is mainly regulated by LH secreted from the pituitary gland. Binding of LH to its receptor on Leydig cells induces cAMP-dependent signaling and cAMP-independent signaling. These signaling pathways act in concert to regulate steroidogenesis through their effects on the expression or activity of steroidogenic proteins in Leydig cells (Payne & Youngblood 1995 , Cooke 1999 , Wang & Stocco 1999 .
2C
In these signaling pathways, Ca
2C
-mediated signaling has been reported to play an important role in steroidogenesis. LH or human chorionic gonadotropin (hCG) stimulation of Leydig cells increases Ca 2C influx (Sullivan & Cooke 1986 , Kumar et al. 1994 . In the absence of Ca 2C , LH-or cAMP-stimulated testosterone production is reduced. Also, the reduced testosterone levels could be restored by the addition of Ca 2C to the culture medium (Janszen et al. 1976 , Ramnath et al. 1997 .
2C
Further studies suggest that Ca 2C affect the transfer of the substrate cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis (Hall et al. 1981 , Meikle et al. 1991 . This was confirmed by a study reporting a Ca 2C -induced increase in steroidogenic acute regulatory (STAR) protein (Manna et al. 1999) , which is critical for the cholesterol transfer to the inner mitochondrial membrane to initiate steroidogenesis (Clark et al. 1994 , Lin et al. 1995 , Wang et al. 1998 . While hCG-induced STAR protein expression and steroid production are enhanced by Ca 2C , the increases in STAR protein and steroid hormone are reversed by calcium chelators. In addition, the effect of Ca 2C on hCG-induced Star mRNA expression is suppressed by blocking L-type Ca 2C channels using a blocker, verapamil (Manna et al. 1999) .
2C
However, observations on the roles of L-type Ca 2C channels in Star gene expression and steroidogenesis were not consistent in the previous studies. While blocking Ca 2C influx through L-type Ca 2C channels attenuates the progesterone production enhanced by the flavonoid, quercetin, it does not affect the increase in Star promoter activity in MA-10 mouse Leydig cells (Chen et al. 2007) . Also, a previous study reported that androgen production is unaffected by L-type Ca 2C channel blockers at concentrations !0 . 1 mM (Moger 1983) . The data from the study show that blocking L-type Ca 2C channel produces a biphasic effect on steroidogenesis, with LH-induced androgen production being increased to the maximal levels and then reduced, as the concentration of verapamil in the cultures is increased. It is possible that the concentrations of L-type Ca 2C channel blockers, levels of tropic hormones or extraand intra-cellular Ca 2C status alter the effects of Ca 2C influx through L-type Ca 2C channels on Star gene transcription and steroid production. It was also observed that Ca 2C is an inhibitor of hCG-activated adenylate cyclase activity in MA-10 mouse Leydig cells (Pereira et al. 1988 ). This inhibitory effect may reduce cAMP formation and attenuate hCG-stimulated Star gene expression. Therefore, further studies are required to understand the roles of L-type Ca 2C channel in Star gene expression. The present study has investigated an inhibitory effect generated by Ca 2C influx through L-type Ca 2C channel on Star gene transcription in MA-10 mouse Leydig cells. N 6 ,2 0 -dibutyryl cAMP (dbcAMP), H89, and Waymouth's MB/752 medium were purchased from Sigma. H8 was purchased from EMD Chemicals (Gibbstown, NJ, USA). Nifedipine, verapamil, isradipine, and diltiazem were purchased from BIOMOL (Plymouth Meeting, PA, USA). Rabbit antiserum generated against STAR protein was a generous gift from Dr D B Hales (University of Illinois, Chicago; Hales et al. 2000) . The monoclonal antibody against DAX-1 (NR0B1 as listed in the MGI Database) protein was a generous gift from Dr E Lalli (Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France). Donkey antirabbit IgG antibody conjugated with HRP was purchased from Biosource (Camarillo, CA, USA). Horse serum was purchased from Invitrogen. The Dual-Luciferase Reporter Assay System was purchased from Promega. Other common chemicals used in this study were obtained from either Sigma or Fisher Chemicals (Pittsburgh, PA, USA).
Materials and Methods

Reagents

MA-10 cell culture
The MA-10 mouse Leydig tumor cell line was a generous gift from Dr Mario Ascoli (University of Iowa, Iowa City). The cells were cultured in 12-well culture plates in Waymouth's MB/752 medium containing 15% horse serum as previously described (Ascoli 1981) , in an incubator at 37 8C and 5% CO 2 . Before each experiment, the medium was replaced with serum-free Waymouth's medium.
Steroid hormone production
MA-10 cells were cultured for 30 min in serum-free Waymouth's medium with or without L-type Ca 2C channel blockers (as described in the figure legends), and then 0 . 05 mM dbcAMP was added to the culture for 6 h.
Steroid hormone production
The medium was collected at the end of each experiment and stored at K20 8C. The concentration of progesterone in the medium was determined by RIA (Resko et al. 1974) .
Western blot analysis
Western blot analyses were performed to detect STAR protein and DAX-1 protein in MA-10 cells as previously described (Townson et al. 1996) . Western blot analysis experiments were repeated at least thrice, and the results of one representative experiment are shown for each figure. Transfections also included 6 . 0 ng pRL-SV40 vector DNA (a plasmid, which constitutively expresses Renilla luciferase under the control of the SV40 promoter, Promega). Transfections were performed using FuGENE HD Transfection Reagent (Roche) following the manufacturer's instructions. After 48-h culture, the cells were utilized for further experiments.
Luciferase assays
Following experiments, the cells were washed with cold PBS and lysed with Passive Lysis Buffer (Promega). The supernatants were utilized for luciferase assays using a Dual Luciferase Reporter Assay System following the manufacturer's instructions (Promega). The Relative Light Units (RLU, determined by dividing the reading from the PGL2/Star promoter by the reading from Renilla luciferase) were measured using a TD-20/20 luminometer (Turner Designers, Sunnyvale, CA, USA). The Star promoter activities were expressed as fold over RLU of control.
Reverse transcription-PCR
Experiments were performed for reverse transcription (RT)-PCR analyses of Star mRNA and Dax-1 (Nr0b1) mRNA expressions. After experiments, MA-10 cells were washed with cold PBS and used for total RNA purification using TRIzol reagent in accordance with the manufacturer's instructions (Invitrogen). The first-strand cDNA was synthesized from the RNA using the Reverse Transcription System (Promega). PCR was performed as previously described for analyses of Star mRNA (Rao et al. 2003) and Dax-1 mRNA (Jana et al. 2008) . b-Actin was used as an internal marker.
Quantitative real-time PCR
Total RNA was prepared from MA-10 cells after experiments, and cDNA was synthesized from the RNA as described above. Real-time PCR was performed with the TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA), for 10 min at 95 8C for initial denaturing, followed by 40 cycles of 95 8C for 15 s and 60 8C for 1 min in the ABI 7900HT Fast Real-Time PCR system (Applied Biosystems). The primer sets for the real-time PCR of mouse Dax-1, Star, and b-actin (Applied Biosystems' Assays ID: Mm00431729_m1, Mm00441558_m1, and Mm02619580_g1 respectively) were chosen from the collection of the TaqMan Gene Expression Assays (Applied Biosystems). The method of relative quantitation was used for the real-time PCR following the manufacturer's instructions.
Statistical analysis
Each experiment was repeated at least thrice. Statistical analysis of the data was performed with ANOVA followed by Tukey's significant difference test using the GraphPad Prism 4 system (GraphPad Software, San Diego, CA, USA). The data are shown as the meanGS.E.M.
Results

Steroid hormone production
MA-10 mouse Leydig cells were incubated with increasing concentrations of the selective L-type Ca 2C channel blocker, nifedipine, for 30 min, followed by the addition of 0 . 05 mM dbcAMP for 6 h to determine the effect of Ca 2C influx through L-type Ca 2C channels on steroidogenesis. The stimulation with 0 . 05 mM dbAMP alone did not induce significant increase in steroidogenesis in MA-10 mouse Leydig cells, but co-incubation with nifedipine significantly increased the progesterone production in a concentration-dependent manner. Progesterone concentration in the culture medium was increased from 9 . 4 to 97 . 9 ng/ml, as nifedipine in the culture was increased from 0 to 50 mM (Fig. 1) .
STAR protein expression
Western blot analyses were performed to determine whether blocking Ca 2C influx through L-type Ca 2C channels affects STAR protein expression in MA-10 cells. As shown in Fig. 1 , when L-type Ca 2C channel was blocked with the increasing concentrations of nifedipine, STAR protein expression was markedly elevated in MA-10 cells cultured in the medium containing 0 . 05 mM dbcAMP. The increase in STAR protein expression also occurred in a concentration-dependent manner, which was concomitant with the increase in progesterone production. (Fig. 2) .
Star gene transcription
Luciferase assays of Star promoter activity and RT-PCR analyses of Star mRNA levels were performed to understand the mechanism responsible for the inhibitory effect of Ca 2C influx through L-type Ca 2C channels on STAR protein expression. Incubation of MA-10 cells with increasing levels of nifedipine resulted in a concentration-dependent increase in Star promoter activity. The Star promoter activity increased 2 . 6-fold over controls when the concentration of nifedipine in the cultures was increased to 50 mM. Similarly, the results obtained from RT-PCR analyses of Star mRNA showed that Star mRNA levels were increased as the levels of nifedipine in the cultures increased (Fig. 3) .
Synergistic interaction between nifedipine and cAMP
To examine the interaction between cAMP and nifedipine, MA-10 cells were treated with or without 20 mM nifedipine for 30 min, and then increasing concentrations of dbcAMP were added to the culture medium for 6 h. The results shown in Fig. 4 demonstrate that the sub-threshold level of dbcAMP (0 . 05 or 0 . 1 mM) alone was unable to stimulate significant increases in STAR protein expression and steroidogenesis. However, in the presence of 20 mM nifedipine, these low levels of dbcAMP increased STAR protein expression and steroid hormone production. Similarly, while nifedipine itself did not increase STAR protein and steroid hormone synthesis, in the presence of sub-threshold levels of dbcAMP (0 . 05 or 0 . 1 mM), nifedipine dramatically increased STAR protein expression and progesterone production in MA-10 cells. In the cells treated with 0 . 1 mM dbcAMP, nifedipine increased progesterone production 6 . 6-fold over that of cells treated with 0 . 1 mM dbcAMP alone. Although blocking Ca 2C influx through L-type Ca 2C channels enhanced cAMP-induced steroidogenesis, this effect was reduced when the levels of cAMP were higher than the threshold of STAR protein expression. When dbcAMP level in the cultures increased to 0 . 5 mM, nifedipine failed to increase STAR protein expression and steroid production (Fig. 4) . MA-10 cells were transfected with a Star promoter/luciferase plasmid (PGL2/Star) and a pRLSV40 vector, a plasmid that constitutively expresses Renilla luciferase. The cells were then treated as described above, and the cell lysate was used for luciferase assays using a Dual-Luciferase Reporter Assay System. The Relative Light Unit (RLU) was determined by dividing the reading from the PGL2/Star promoter by the reading from Renilla luciferase. The promoter activities were expressed as fold over RLU of control. ***P!0 . 001, nZ3; compared with 0 . 05 mM dbcAMP alone. 
Critical role of protein kinase A in verapamil-enhanced steroidogenesis
The role of protein kinase A (PKA) in the verapamilenhanced steroidogenesis was investigated using the PKA inhibitor H89 or H8. While co-action of 15 mM verapamil and 0 . 05 mM cAMP dramatically increased STAR protein, inhibition of PKA activity with either H89 or H8 reversed this increase. Similarly, while progesterone concentration was increased from 6 . 6 to 86 . 0 ng/ml by verapamil in the same culture, it was reduced to 27 . 4 or 40 . 6 ng/ml by inhibition of PKA activity using H89 or H8 respectively (Fig. 5) . When 22(R)hydroxylcholesterol was added to the cultures, there was no significant difference in progesterone production among the groups, indicating that the activities of steroidogenic enzymes were not affected by the treatments.
Dax-1 gene transcription
To further understand how Ca (Fig. 6 ). The observations were further confirmed by quantitative real-time PCR analyses of Dax-1 mRNA and Star mRNA, with Dax-1 mRNA level being reduced and Star mRNA level being increased significantly by blocking L-type Ca 2C channels (Fig. 7) .
Discussion
Previous studies suggested that Ca 2C influx through L-type Ca 2C channels may produce differential effects on Star gene expression in Leydig cells (Manna et al. 1999 , Chen et al. 2007 ). The present study further indicates that Ca 2C influx through L-type Ca 2C channels is involved in an inhibitory effect on Star gene expression and regulates the sensitivity of Leydig cells to cAMP stimulation.
Discussion
The inhibitory effect generated by Ca 2C influx through L-type Ca 2C channels on Star gene expression was demonstrated by three lines of evidence: 1) blocking Ca 2C influx through L-type Ca 2C channels with increasing levels of a selective blocker, nifedipine, induced a concentrationdependent increase in STAR protein expression in the cells ) and then treated as described above. The medium was collected, and progesterone concentration in the medium was assessed by RIA. *P!0 . 05; **P!0 . 01; nZ3; compared with the cells treated with 0 . 05 mM dbcAMP alone. ***P!0 . 001; nZ3;
Discussion
compared with the cells treated with H89 or H8. In the presence of 22(R)hydroxylcholesterol in the cultures, there was no significant difference in progesterone production among the treatments. treated with 0 . 05 mM dbcAMP. Progesterone production by the cells was also increased, as STAR protein expression was enhanced by nifedipine; 2) the inhibitory effect generated by Ca 2C influx through L-type Ca 2C channels on STAR protein expression was confirmed by the experiments utilizing three different selective L-type Ca 2C channel blockers, verapamil, isradipine, and diltiazem respectively. Incubation of MA-10 cells with 25 mM each of the blockers produced similar steroidogenic results, with each of them dramatically enhancing STAR protein expression and progesterone production; 3) the observations with nifedipine were corroborated by RT-PCR analyses of Star mRNA and also by the luciferase assays of Star promoter activity in the cells co-incubated with nifedipine and 0 . 05 mM dbcAMP, with both Star mRNA level and promoter activity being increased by the treatments. The results indicated that blocking L-type Ca 2C channels enhanced Star gene transcription. Similar inhibitory effect of Ca 2C influx through L-type Ca 2C channel on steroidogenesis was also reported in other types of steroidogenic cells (Barrett et al. 1991 , Rossier et al. 1996 .
Discussion
The results from the present study further indicated that this inhibition on Star gene expression by Ca 2C influx through L-type Ca 2C channels was involved in regulating the sensitivity of Leydig cells to cAMP stimulation. Under normal circumstances, sub-threshold levels of cAMP (0 . 05 and 0 . 1 mM cAMP) are unable to induce STAR protein expression or a significant increase in progesterone production in MA-10 cells. However, in the presence of L-type Ca 2C channel blockers, the effectiveness of cAMP was dramatically increased, with 0 . 05 mM cAMP being able to induce a marked increase in STAR protein expression and also a significant increase in steroid hormone production. On the other hand, in the absence of these low levels of cAMP, blocking Ca 2C influx through L-type Ca 2C channels did not induce Star gene expression and steroidogenesis in MA-10 cells. We also observed that inhibition of PKA activity reversed the verapamil-increased STAR protein and progesterone production. These observations suggest that blocking L-type Ca 2C channels alone is unable to stimulate steroidogenesis. However, the blockers did attenuate the inhibitory effect and lowered the threshold of cAMPstimulated STAR protein expression. Therefore, subthreshold levels of cAMP are able to induce maximal levels of Star gene expression and steroid hormone production.
Discussion
To understand how the Ca 2C influx through L-type Ca 2C channels acted at the transcriptional level of Dax-1 gene expression. As shown in the present and previous studies , DAX-1 protein is constitutively expressed and maintained at a high level in MA-10 cells. DAX-1 generates a tonic inhibition on Star gene expression by binding to a hairpin structure in the Star promoter and depressing Star gene transcription (Zazopoulos et al. 1997) . In addition, DAX-1 expression is able to reduce the level of steroidogenic factor-1 (SF-1) protein, an important transcriptional factor in Star gene expression, and also represses the transactivation potential of SF-1 on Star gene transcription (Manna et al. 2009 ). Thus, reduction in DAX-1 protein by blocking L-type Ca 2C channels attenuated the tonic inhibition of DAX-1 protein and increased the sensitivity of Leydig cells to cAMP stimulation.
Discussion
These observations and previous studies indicated that Ca 2C influx through L-type Ca 2C channels not only produced positive effect (Manna et al. 1999 ), but also produced negative effect on Star gene expression. As described above, the negative effect resulted from the role of Ca 2C influx through L-type Ca 2C channels in maintaining DAX-1 expression when cAMP levels were below the threshold of Star gene expression. This can be seen from the present study that blocking L-type Ca 2C channels using the blocker markedly reduced DAX-1 protein and increased Star gene expression. However, the steroidogenic effect of the L-type Ca 2C channel blocker is affected by the levels of cAMP. As concentrations of cAMP in the cultures increased to produce maximal level of STAR protein expression, cAMP stimulation reduces DAX-1 protein to the low levels (Jo & Stocco 2004 , Manna et al. 2009 , and also increases positive signals, including Ca 2C (Sullivan & Cooke 1986 ). Since DAX-1 protein was already reduced to such a low level, blocking L-type Ca 2C channel might not be able to effectively further reduce the inhibitory effect of DAX-1 protein, but reduced the positive effect of the Ca 2C influx through L-type Ca 2C channels on Star gene expression as previously reported (Manna et al. 1999) .
Discussion
In summary, the present study demonstrated that blocking L-type Ca 2C channels enhanced cAMP-induced Star gene transcription, STAR protein expression, and progesterone production in MA-10 mouse Leydig cells, indicating a role for Ca 2C influx through L-type Ca 2C channels in an inhibition of Star gene expression and steroidogenesis. This inhibitory effect may involve a role of Ca 2C influx through L-type Ca 2C channels in the regulation of Dax-1 gene expression.

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