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Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA
(Requests for offprints should be addressed to D M Stocco; Email: doug.stocco{at}ttuhsc.edu)
* (P R Manna and Y Jo contributed equally to this work) 
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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10–15% of steroid synthesis appears to be mediated through StAR-independent mechanisms (Lin et al. 1995, Stocco & Clark 1996, Clark et al. 1997, Manna et al. 2001). Two putative PKA phosphorylation sites have been identified in the mouse StAR protein at Ser56 and Ser194, and mutations (Ser
Ala) in these sites demonstrated the importance of Ser194 in the biological activity of StAR in steroid synthesis (Arakane et al. 1997). Whereas the cAMP/PKA pathway is unquestionably the major signaling cascade for tropic hormone-induced StAR expression and steroidogenesis, PKC has also been shown to be involved in these processes (Jo et al. 2005, Stocco et al. 2005, Manna et al. 2006a, Renlund et al. 2006). We have recently reported that the PKC signaling pathway plays an integral role in regulating the steroidogenic response in Leydig cells. While PKC-dependent induction of steroid synthesis is low when compared with that observed with PKA signaling, it is capable of potentiating gonadotropin and/or cAMP-stimulated steroidogenic responsiveness (Jo et al. 2005, Stocco et al. 2005, Manna et al. 2006a).
DAX-1 (also called Nr0b1 or Ahch), an orphan member of the nuclear hormone receptor superfamily of transcription factors, is expressed in the hypothalamic–pituitary–adrenal/gonadal axis and has been demonstrated to have roles in the regulation of steroidogenesis (Zazopoulos et al. 1997, Lalli et al. 1998, Osman et al. 2002, Tajima et al. 2003, Jo & Stocco 2004, Manna et al. 2006a) as well as in the gonadal development and function (Yu et al. 1998, Meeks et al. 2003a,b, Niakan & McCabe 2005). Targeted disruption of DAX-1 in mice results in testicular dysgenesis, which is manifested by decreased testicular size and decreased spermatogenesis, impaired Sertoli and Leydig cell function, and infertility (Yu et al. 1998, Jeffs et al. 2001, Meeks et al. 2003a,b). Another protein considered in the present studies was SR-B1, a high-density lipoprotein (HDL) receptor that binds various lipoprotein particles, facilitates HDL-derived cellular cholesterol influx, and thus replenishes cholesterol stores for steroid hormone biosynthesis (Ji et al. 1997, Azhar et al. 1998, Fidge 1999). In previous studies, both in vivo and in vitro approaches have demonstrated a striking correlation between the hormonal induction of SR-B1 expression (accompanied by enhanced lipid uptake from HDL) and steroid biosynthesis in steroidogenic cells (Landschulz et al. 1996, Rigotti et al. 1996, Azhar et al. 1998, Sun et al. 1999, Manna et al. 2006b).
The mitogen activated protein kinase (MAPK) signaling cascade has been implicated in the regulation of cell cycle progression, is used as a biochemical marker in evaluating mitogenic effects of a variety of stimuli, and is a point of convergence for diverse signaling pathways (Seger & Krebs 1995, Lewis et al. 1998, Le & Schimmer 2001, Manna et al. 2006b). Extracellular signal-regulated kinase 1/2 (ERK1/2), a memberof the familyof MAPKs, has been shown to be involved in StAR expression and steroid biosynthesis. However, its mode of action is poorly understood as a consequence of conflicting reports demonstrating stimulation, inhibition, or no effect in different steroidogenic cells (Gyles et al. 2001, Seger et al. 2001, Martinelle et al. 2004, Martinat et al. 2005, Tajima et al. 2005, Manna et al. 2006b). Despite the importance of the PKA and PKC signaling pathways in steroidogenesis, their precise roles in the regulation of MAPK/ERK-mediated steroidogenic responsiveness remain elusive. Utilizing MA-10 mouse Leydig tumor cells (that possess many characteristics of normal Leydig cells including low levels of LH receptors) as an experimental model, the present studies were aimed at elucidating these roles and demonstrated that ERK1/2 signaling has a variety of effects on the regulation of StAR expression and steroidogenesis in Leydig cells that are dependent on the specific experimental conditions and stimuli utilized.
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Materials and Methods |
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Phorbol-12-myristate-13-acetate (PMA), dibutyryl cyclic AMP (dbcAMP), hCG, and H89 were purchased from Sigma. GF-10923X (GFX) was obtained from AG Scientific (San Diego, CA, USA). U0126 and ERK1/2 antibody were purchased from Cell Signaling (Beverly, MA, USA). Phosphorylated ERK1/2 monoclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell culture and treatments
MA-10 mouse Leydig tumor cells (Ascoli 1981) were grown in Waymouth MB 752/1 medium supplemented with 15% horse serum (Invitrogen) and antibiotics, as described previously (Manna et al. 2004a, Jo et al. 2005). Prior to experiments, cells were washed with 0.01 M PBS and incubated with or without additives (stimulators, inhibitors, etc.), which were freshly prepared and applied to the cells. Media were collected from different treatment groups and the accumulation of progesterone (increased several hundredfold over basal in response to tropic hormone or cAMP analog in 4–6 h) was determined (Manna et al. 2002b, Rao et al. 2003, Jo et al. 2005, Manna et al. 2006b). Some caution should be taken in measuring progesterone as the sole product of steroidogenesis in MA-10 cells, as a previous study has demonstrated that additional steroid metabolites are also present in these cells (Rommerts et al. 2001). However, the levels of such metabolites are low when compared with progesterone production in MA-10 cells.
Western blot analysis
Following the experiments, cells were washed, homogenized in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 1% NP-40), centrifuged at 12 000 g for 10 min, and the supernatant was assayed for total protein (Jo & Stocco 2004, Manna et al. 2006b). Equal amounts of protein were loaded on 10% SDS-PAGE gels and following electrophoresis were transferred to methanol-activated polyvinylidene difluoride membranes. Membranes were probed with specific antibodies that recognize phosphorylated (P)-ERK1/2, total ERK1/2, DAX-1 (Tamai et al. 1996; a gift from Dr Paolo Sassone-Corsi, University Louis Pasteur, Strasbourg, France), P-StAR (Manna et al. 2006b; a gift from Dr Steven R King, Baylor College of Medicine, Houston, TX, USA), and total StAR (Bose et al. 1999; a gift from Dr Walter L Miller, University of California, San Francisco, CA, USA). The antibody detecting P-StAR was generated against amino acids 190–199 of mouse StAR with phosphorylated Ser194. Horseradish peroxidase-conjugated secondary antibodies against rabbit, mouse, or goat IgG were used to recognize relevant primary antibodies. Immunodetection of different proteins was determined by chemiluminescence following exposure to X-ray film (Marsh Bio Products, Inc., Rochester, NY, USA), and the immunoblots were quantified using a computer-assisted image analyzer (Visage 2000, BioImage Corp., Ann Arbor, MI, USA). Expression of phosphorylated and total proteins was assessed using identically processed membranes; however, where appropriate, the same membranes were also analyzed by stripping and reprobing.
Quantitative reverse transcription and PCR (RT-PCR)
Total RNA was isolated from different treatment groups using Trizol reagent (Invitrogen). RT-PCR was run sequentially in the same assay tube using 2 µg of total RNA, as described previously (Manna et al. 1999a, 2002b, 2004b). Isolation and amplification of StAR and SR-B1 cDNAs were carried out utilizing the following primer pairs: StAR sense, 5'-GACCTTGAAAGGCTCAGGAAGAAC-3' and StAR antisense, 5'-TAGCTGAAGATGGACAGACTTGC-3'; SR-B1 sense, 5'-CTCATCAAGCAGCAGGTGCTCAAGAA-3' and SR-B1 antisense, 5'-GGTGGGTAGACGGACCCGTTGGCAAACA-3'. The variation in RT-PCR efficiency was assessed using L19 ribosomal protein gene using the sense primer 5'-GAAATCGCCAATGCCAACTC-3' and the antisense primer 5'-TCTTAGACCTGCGAGCCTCA-3'. The cDNAs generated were further amplified by PCR using the primer pairs listed above. The parameters used were optimized to be in the exponential phase of the PCR. The molecular sizes of StAR, SR-B1, and L19 were determined on 1.2% agarose gels. Gels were vacuum dried, exposed to X-ray film (Marsh Bio Products) for 1–3 h, and the resulting signals were quantified (Visage 2000). Integrated optical density (IOD) values of each StAR band were quantified and normalized with the corresponding L19 bands.
Statistical analysis
All experiments were repeated at least three times. Data were analyzed by either Student’s t-test or ANOVA followed by Fisher’s least significant difference tests using the StatView program (Abacus Concepts, Inc., Berkeley, CA, USA). Data were expressed as the mean ± S.E.M. and the P<0.05 values were considered significant.
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Results |
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The mechanism of the ERK1/2 signaling cascade in the regulation of StAR expression and steroidogenesis was studied in MA-10 cells. Cells were treated with PMA and dbcAMP in order to activate the PKC and PKA pathways respectively, and phosphorylation and expression of ERK1/2 were determined. Phosphorylation of ERK1/2 (P-ERK1/2), following PMA treatment increased by 3 min, was maximal between 10 and 120 min (~4.6-fold) and decreased by 180 min, the longest time tested (Fig. 1A
). Also, dbcAMP (0.5 mM) resulted in an increase in P-ERK1/2 by 3 min (P<0.05) and a 5.8 ± 0.9-fold increase by 30 min, an induction that was sustained for 180 min (Fig. 1B). The total amount of ERK1/2 protein was unaltered in these groups. Pretreatment with a MAPK/ERK inhibitor, U0126 (10 µM), for 30 min with either PMA or dbcAMP markedly inhibited P-ERK1/2 levels at a concentration shown to inhibit MAPK/ERK activity in mouse Leydig and adrenal cells (Gyles et al. 2001, Manna et al. 2002a, 2006a). These results demonstrate that activation of ERK1/2 by PMA and dbcAMP involves protein phosphorylation rather than protein synthesis.
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, U0126 was capable of increasing (P<0.05) StAR protein expression by 6 h but not its phosphorylation (P-StAR). PMA (10 nM) resulted in a 5.2 ± 1.4-fold elevation in StAR expression but not P-StAR, and a slight increase (3.2 ± 0.8-fold) in progesterone synthesis when compared with controls. Addition of 0.05 mM dbcAMP (a dose incapable of increasing StAR expression and steroid synthesis by itself (Jo et al. 2005)) to PMA-treated cells dramatically elevated StAR, P-StAR, and progesterone to levels that are quantitatively similar to those seen with 0.5 mM dbcAMP (Fig. 2A and B). Notably, while U0126 decreased PMA and PMA plus dbcAMP-stimulated StAR and P-StAR, it increased (P<0.05) dbcAMP-mediated responses (Fig. 2A). However, progesterone levels in response to these stimuli were significantly decreased (P<0.01) by U0126. This inhibitor also decreased basal secretion of progesterone by 44% (control, 1.58 ± 0.21 vs U0126, 0.87 ± 0.14 ng/mg protein). U0126 had no apparent effect on 22R-hydroxycholesterol (22R-OHC, 25 µM)-mediated progesterone synthesis, indicating that the cytochrome P450 side-chain cleavage (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) enzyme activities were not affected (Fig. 2B). These findings suggest that activation of ERK1/2 via PKC and PKA signaling influences StAR expression and steroid synthesis differently without affecting the activity of P450scc and 3ß-HSD.
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). The concentrations of GFX and H89 used were based on previous studies (Jo et al. 2005, Manna et al. 2006b). Total ERK1/2 protein levels were unaltered by any of these inhibitors. The results summarized in Fig. 3B demonstrate that treatment with PMA and dbcAMP for 6 h resulted in 5.4 ± 1.2- and 8.7 ± 1.9-fold increases in StAR mRNA levels over untreated cells respectively. Inclusion of GFX and H89 in these treatments decreased PMA- and dbcAMP-induced StAR mRNA expression by 68 and 74% respectively. Treatment with PMA and dbcAMP increased progesterone synthesis over basal levels by 3.6- and 337 ± 29-fold respectively. GFX and H89 decreased PMA- and dbcAMP-treated progesterone synthesis by 70 and 85% respectively.
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). Notably, while U0126 increased dbcAMP (0.1–0.5 mM)-stimulated StAR and P-StAR, it decreased progesterone synthesis approximately by 50% (Fig. 4B), an observation in agreement with previous studies (Renlund et al. 2006). By contrast, U0126 markedly inhibited PMA and PMA plus 0.05 mM dbcAMP-stimulated StAR/P-StAR and progesterone levels, demonstrating U0126 had effects on StAR expression and steroid synthesis in stimulus-specific manners.
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). U0126 abolished hCG-stimulated P-ERK1/2 when compared with the value at 10 min. The amount of ERK1/2 was unchanged during this time course. Also, hCG was capable of inducing StAR expression (4.8 ± 1.5-fold) and P-StAR (3.5 ± 1.2-fold) over their respective controls. Both GFX and H89 decreased hCG-stimulated StAR and P-StAR by 80–95%, and as a result progesterone levels were markedly attenuated (Fig. 5B). On the other hand, U0126 resulted in moderate but consistent increases (1.6- to 2.2-fold) in hCG-stimulated StAR and P-StAR, but significantly decreased (P<0.01) progesterone synthesis.
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To determine whether U0126 mediates the hormone-stimulated steroidogenic response at the level of transcription and/or translation, the effects of actinomycin D (Act, 4 µM) and cycloheximide (Chx, 5 µM) on dbcAMP-stimulated MA-10 cells were determined (Fig. 6). The increase in U0126-mediated StAR expression was repressed (P<0.05) by both Act and Chx, indicating U0126 induced StAR expression both at the transcriptional and translational levels (Fig. 6A). Act and Chx also diminished basal progesterone synthesis between 37 and 45%. Furthermore, Act and Chx decreased (P<0.01) dbcAMP (0.3 mM)-stimulated StAR, P-StAR, and progesterone synthesis (Fig. 6B). The effect of U0126 on dbcAMP-stimulated elevations in StAR and P-StAR was decreased by 86 and 65% with Act and 50 and 27% with Chx respectively. Furthermore, U0126 suppressed (P<0.05) dbcAMP-stimulated progesterone production, a response further attenuated in the presence of both Act and Chx.
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To obtain more insight into these mechanisms, the role of ERK1/2 signaling on PMA-, dbcAMP-, and hCG-mediated expression of DAX-1 and SR-B1 was studied and their relationship to StAR expression and progesterone synthesis was assessed. In MA-10 cells, the constitutive expression of DAX-1 was decreased to 42% by U0126 (10 µM), an event that increased expression of the StAR protein. Treatment with PMA, dbcAMP, and hCG demonstrated 68, 45, and 47% decreases in DAX-1 protein levels respectively, observations inversely correlated with StAR expression (Fig. 7A and B). Progesterone levels in response to these stimuli were inhibited by U0126 (Fig. 7C). While U0126 reversed the inhibition of PMA-treated DAX-1 expression, it further attenuated dbcAMP (~70%)- and hCG (~52%)-mediated DAX-1 protein levels.
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). Addition of U0126 to these treatments repressed SR-B1 mRNA expression between 40 and 55%, while at the same time progesterone levels were also significantly decreased (P<0.05). Thus, the suppression of progesterone synthesis by U0126 could be, at least in part, due to inhibition of SR-B1 expression resulting in an insufficient uptake of cholesterol into these cells.
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Discussion |
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-mediated StAR expression but decreased steroid synthesis (Martinat et al. 2005, Manna et al. 2006a,b). Also, ERK1/2 signaling resulted in differential effects on LH-induced StAR and CYP17 levels, producing an increase in progesterone but a decrease in androsterone synthesis in bovine theca cells (Tajima et al. 2005). In granulosa lutein cells, while inhibition of ERK1/2 had no effect on LH/hCG-induced steroidogenesis, it decreased (Bu)2cAMP-, cholera toxin-, and forskolin-stimulated progesterone levels (Dewi et al. 2002). The present studies extend these observations by elucidating the molecular events in which the ERK1/2 cascade (in correlation with PKA and PKC signaling) acts to drive StAR expression and, thus, steroid biosynthesis in mouse Leydig cells. Our findings demonstrate that both the PKC (activated by PMA) and PKA (activated by dbcAMP/hCG) signaling pathways are capable of rapidly phosphorylating ERK1/2. Selective inhibition of these pathways using specific inhibitors markedly repressed ERK1/2 phosphorylation and had a variety of effects on StAR expression and steroidogenesis. In previous studies, we examined the effect of several doses of these inhibitors and found them to have no adverse effects on cell viability or cellular DNA synthesis when compared with control Leydig cells (Manna et al. 2001, 2002a, Rao et al. 2003, Jo et al. 2005, Manna et al. 2006a). In addition, these inhibitors have also been widely used for blocking relevant signaling pathways in other steroidogenic cells (Gyles et al. 2001, Seger et al. 2001, Martinelle et al. 2004). Interestingly, inhibition of ERK1/2 by U0126 decreased PMA-stimulated StAR expression but increased dbcAMP/hCG responsiveness. However, progesterone levels were decreased by these treatments. The decrease in progesterone synthesis was not associated with inhibition of P450scc and/or 3ß-HSD, as U0126 had no effect on 22R-OHC-mediated progesterone production. Consistent with this, ERK1/2 inhibition has been demonstrated to decrease forskolin-stimulated pregnenolone synthesis in MA-10 cells, but in those studies the expression of the StAR protein was not determined (Gyles et al. 2001). In human LH receptor expressing MA-10 cells, hCG was able to increase the PKA-mediated phosphorylation of Ras and ERK1/2 (Hirakawa & Ascoli 2003). Also, the effect of hCG on P-ERK1/2 was diminished by the inhibition of PKC and PKA responsiveness in immature rat Leydig cells (Martinelle et al. 2004). These observations indicate the complexity of the role of the ERK1/2 signaling cascade, which could be dependent on receptor–effector coupling and might involve signaling crosstalk and/or the involvement of additional factor(s) in the regulation of the steroidogenic response.
The regulation of StAR expression and steroidogenesis involves multiple second messenger systems including the PKA (Balasubramanian et al. 1997, Manna et al. 1999b, Sekar et al. 2000, Gyles et al. 2001, Martinat et al. 2005, Manna et al. 2006a) and PKC (Fiedler et al. 1999, Jo et al. 2005, Manna et al. 2006a,b, Renlund et al. 2006) pathways. It is noted, however, that the effect of PKC signaling on steroid synthesis is far smaller than that of PKA signaling, usually being 1% of that seen with PKA activation, and the inability of PKC to phosphorylate StAR has been demonstrated to be a critical event for this dramatic difference (Jo et al. 2005, Manna et al. 2006a). Our present data corroborate previous findings which indicated that both the PKA and PKC pathways are involved in regulating StAR expression and steroidogenesis (Jo et al. 2005, Martinat et al. 2005, Manna et al. 2006a,b), and demonstrated that inhibition of ERK1/2 by U0126 elevated StAR protein expression but not its phosphorylation. Activation of PKC by PMA markedly increased the expression of the StAR protein but not P-StAR, thus producing inactive StAR and resulting in only a modest increase in progesterone synthesis. Increasing the PKA activity with a low dose of dbcAMP (one that is ineffective in increasing the steroidogenic response) in PMA-treated cells dramatically enhanced P-StAR and progesterone levels. While ERK1/2 inhibition resulted in decreases in PMA and PMA plus dbcAMP-stimulated StAR/P-StAR and progesterone levels, it produced increases in dbcAMP/hCG-stimulated StAR expression and P-StAR, but diminished progesterone synthesis. These results are in support of the previous findings demonstrating that ERK1/2 inhibition increased interleukin-1 (IL-1)-stimulated StAR and P-StAR but decreased androgen synthesis in rat Leydig cells (Renlund et al. 2006). Thus, it appears that ERK1/2 signaling plays a discrete role in StAR expression and steroid synthesis in pathway- and/or stimulus-specific manners in mouse Leydig cells.
In view of our present findings, it appears that the transcription factor DAX-1, which plays an important role in regulating StAR expression through the PKA pathway, is also regulated by PKC signaling. In addition to its role in the development of the adrenal and gonads, DAX-1 has been demonstrated to negatively regulate StAR expression and steroid synthesis both in Leydig and adrenal cells (Zazopoulos et al. 1997, Lalli et al. 1998, Manna et al. 1999a,b, Jo & Stocco 2004). In this study, we observed that inhibition of ERK1/2 was associated with a decrease in DAX-1 expression. While PMA, dbcAMP, and hCG increased StAR expression and progesterone synthesis, they inhibited expression of the DAX-1 levels. In support of this mechanism, an inverse relationship between DAX-1 and StAR expression has been reported in different steroidogenic cells (Tamai et al. 1996, Osman et al. 2002, Manna et al. 2006a,b). Furthermore, the inhibitory effect of PMA on DAX-1 expression could be reversed by U0126, which, in contrast, further diminished dbcAMP/hCG-mediated DAX-1 levels. Therefore, the differential effects of U0126 on PKC- and PKA-mediated StAR expression might be explained, at least in part, by alterations in DAX-1 expression.
The present results also demonstrate the importance of SR-B1 in regulating steroid biosynthesis. SR-B1 is involved in providing cholesterol substrate for steroidogenesis in steroidogenic tissues. In MA-10 cells, we observed relatively low levels of SR-B1 mRNA by RT-PCR analysis. PMA and dbcAMP/hCG treatments were found to markedly increase SR-B1 mRNA expression, an event decreased by ERK1/2 inhibition, and are in agreement with our recent findings in mLTC-1 cells (Manna et al. 2006a,b). Previous studies have also documented that the expression of SR-B1 increases with hCG/ACTH and decreases with dexamethasone and is coordinately regulated with steroid synthesis in Leydig and adrenocortical cells (Landschulz et al. 1996, Rigotti et al. 1996, Sun et al. 1999). In addition, the direct involvement of ACTH on the HDL-mediated cholesterol influx, the utilization of this cholesterol for steroid synthesis, and the induction of SR-B1 expression have been demonstrated in the adrenal gland (Rigotti et al. 1996). However, it has been shown that HDL binding to SR-B1 stimulates the ERK1/2 pathway following Ras activation (Grewal et al. 2003). Overall, it is conceivable that the decrease in progesterone synthesis in MA-10 cells caused by U0126 (even with elevated StAR) could be a result of a decreased uptake of cholesterol into cells due to the repression of SR-B1. Previously, U0126 has been shown to suppress IL-1-stimulated androgen production by dissipating the mitochondrial electrochemical potential in immature rat Leydig cells (Renlund et al. 2006). Hence, we cannot exclude the possibility that U0126 might inhibit PMA- and dbcAMP/hCG-stimulated progesterone production by affecting the integrity of the mitochondria.
Taken together, these findings provide insight into the mechanisms of ERK1/2 signaling in regulating PKC- and PKA-mediated StAR expression and steroidogenesis in mouse Leydig cells and illustrate the involvement of DAX-1 and SR-B1 in these processes. Thus, multiple and complex intracellular events appear to be involved in crosstalk among the different pathways and may function in regulating the steroidogenic response in Leydig cells. The exploration of the MAPK/ERK1/2 signaling pathway may help in understanding the mitogenic potential of a variety of hormones and growth factors in other steroidogenic cells.
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Acknowledgements |
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References |
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Received in final form 11 January 2007
Accepted 15 January 2007
Made available online as an Accepted Preprint 25 January 2007
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