Mechanisms of protein kinase C signaling in the modulation of 3',5'-cyclic adenosine monophosphate-mediated steroidogenesis in mouse gonadal cells

Abstract

The protein kinase C (PKC) signaling pathway plays integral roles in the expression of the steroidogenic acute regulatory (StAR) protein that regulates steroid biosynthesis in steroidogenic cells. PKC can modulate the activity of cAMP/protein kinase A signaling involved in steroidogenesis; however, its mechanism remains obscure. In the present study, we demonstrate that activation of the PKC pathway, by phorbol 12-myristate 13-acetate (PMA), was capable of potentiating dibutyryl cAMP [(Bu)(2)cAMP]-stimulated StAR expression, StAR phosphorylation, and progesterone synthesis in both mouse Leydig (MA-10) and granulosa (KK-1) tumor cells. The steroidogenic potential of PMA and (Bu)(2)cAMP was linked with phosphorylation of ERK 1/2; however, inhibition of the latter demonstrated varying effects on steroidogenesis. Transcriptional activation of the StAR gene by PMA and (Bu)(2)cAMP was influenced by several factors, its up-regulation being dependent on phosphorylation of the cAMP response element binding protein (CREB). An oligonucleotide probe containing a CREB/activating transcription factor binding region in the StAR promoter was found to bind nuclear proteins in PMA and (Bu)(2)cAMP-treated MA-10 and KK-1 cells. Chromatin immunoprecipitation studies revealed that the induction of phosphorylated CREB was tightly correlated with in vivo protein-DNA interactions and recruitment of CREB binding protein to the StAR promoter. Ectopic expression of CREB binding protein enhanced CREB-mediated transcription of the StAR gene, an event that was markedly repressed by the adenovirus E1A oncoprotein. Further studies demonstrated that the activation of StAR expression and steroid synthesis by PMA and (Bu)(2)cAMP was associated with expression of the nuclear receptor Nur77, indicating its essential role in hormone-regulated steroidogenesis. Collectively, these findings provide insight into the mechanisms by which PKC modulates cAMP/protein kinase A responsiveness involved in regulating the steroidogenic response in mouse gonadal cells.

Figure 1

Role of PKC in the modulation of (Bu)₂cAMP-stimulated StAR expression, P-StAR, and progesterone synthesis. MA-10 (A, D, and F) and KK-1 (B, C, E, and F) cells were pretreated without or with PKC (GFX, 20 μm) and PKA (H-89, 20 μm) inhibitors for 30 min and then treated with PMA (10 nm) in the absence or presence of either fixed (0.1 mm) or increasing amounts of (Bu)₂cAMP (0.05–0.5 mm) for 6 h, as indicated. Representative immunoblots show StAR, P-StAR, and CYP11A1 levels in different treatment groups using 20–30 μg of total cellular protein. Total RNA isolated from the specific treatment groups was subjected to RT-PCR analysis for determining StAR mRNA expression, and representative autoradiograms are illustrated in D and E. Integrated OD (IOD) values of each band were quantified, normalized with the corresponding L19 bands, and presented as StAR/L19. Data shown in immunoblotting and RT-PCR analyses are representative of three to five independent experiments. Actin and L19 expression was used as loading controls in Western and RT-PCR analyses, respectively. Accumulation of progesterone in the media was determined and expressed as nanograms per milligram protein (F), which represent the mean ± se of four independent experiments. *, P < 0.05 vs. control.

Figure 2

Role of ERK1/2 signaling in the activation of PMA- and (Bu)₂cAMP-mediated StAR expression and steroid synthesis. MA-10 (A) and KK-1 (B and C) cells were pretreated without or with a MAPK/ERK inhibitor U0126 (U0, 10 μm) for 30 min and then incubated with PMA (10 nm), (Bu)₂cAMP (0.1 mm), or their combination for an additional 30 min (A and B) or 6 h (C). Representative immunoblots illustrate P-ERK1/2 and ERK1/2 (A and B) and StAR and P-StAR (C) using 25–30 μg of total cellular protein. Accumulation of progesterone in media was determined and expressed as nanograms per milligram protein (n = 4). Actin expression was assessed as a loading control. Letters above the bars indicate that these groups differ significantly from each other at least at P < 0.05. Con, Control.

Figure 3

Assessment of different transcription factors in PMA and (Bu)₂cAMP responsiveness. Promoter sequences (−151/−1 bp) of the mouse StAR gene were studied for several elements by generating mutations (A). MA-10 (B) and KK-1 (C) cells were transfected with either the −151/−1 StAR reporter segment (−151/−1 wt) or the −151/−1 StAR containing mutations in each of the binding sites as indicated, in the presence of pRL-SV40 vector. After 36 h of transfection, cells were incubated without (basal) or with PMA (10 nm), (Bu)₂cAMP (0.1 mm), and PMA plus (Bu)₂cAMP for an additional 6 h. Luciferase activity in the cell lysates was determined and expressed as relative light unit (RLU, luciferase/renilla). Data represent the mean ± se five independent experiments. MA-10 cells were treated without (Con) or with PMA (10 nm), (Bu)₂cAMP (0.1 mm), and PMA plus (Bu)₂cAMP for 6 h (D). Expression of the StAR, Sp1, C/EBPβ, SF-1, cFos, cJun, GATA-4, and SREBP-1 proteins was determined by immunoblotting using 25–30 μg of total protein. Immunoblots shown are representative of three to six independent experiments. Actin expression was assessed as a loading control. Con, Control.

Figure 4

Effect of PMA in modulation of (Bu)₂cAMP-mediated CREB, P-CREB and CBP levels, P-CREB and CBP association with the StAR promoter, and their relevance to StAR gene expression. MA-10 (A and C–F) and KK-1 (A and B) cells were pretreated without or with GFX (20 μm) and H-89 (20 μm) for 30 min and then incubated in the absence or presence of PMA (10 nm), (Bu)₂cAMP (0.1 mm), and PMA plus (Bu)₂cAMP for 0–300 min (A), 6 h (B), or 1 h (C and D), and samples were processed for immunoblotting and ChIP analyses. Representative immunoblots show P-CREB, CREB, and CBP levels in response to different treatments using 25–30 μg of total protein. Integrated OD (IOD) values of each immuno-specific band in P-CREB, CREB, and CBP were quantified and either presented as a ratio of P-CREB/CREB (A) or compiled from three independent experiments (B, bottom panel). Cross-linked sheared chromatin obtained from different treatment groups was immunoprecipitated (IP) without or with anti-P-CREB (C) and anti-CBP (D) Abs. Recovered chromatin was subjected to PCR analysis using primers specific to the proximal region of the mouse StAR promoter. Representative autoradiograms (n = 3–5) illustrate the association of P-CREB and CBP with the StAR promoter. E, MA-10 cells were transfected with either empty vector (pcDNA) or a nonphosphorylatable CREB mutant (CREB-M1) in the presence of the −151/−1 StAR promoter segment. pGL3 basic (pGL3) was used as a control. Using the −151/−1 StAR reporter segment, cells were also transfected with pcDNA, wild-type CREB, CBP, and E1A expression plasmids, or a combination of them, as indicated (F). The pRL-SV40 plasmid was included in these experiments for normalization of transfection efficiency. After36 h of transfection, cells were treated without (Basal), or with PMA (10 nm), (Bu)₂cAMP (0.1 mm) and PMA plus (Bu)₂cAMP for an additional 6 h. Luciferase activity in the cell lysates was determined and expressed as relative light unit (RLU, luciferase/renilla). Data represent the mean ± se of four to six independent experiments. Letters above the bars indicate that these groups differ significantly from each other at least at P < 0.05.

Figure 5

Binding of the CREB/ATF motif (−83/−67 bp) of the StAR promoter to MA-10 and KK-1 NE, using EMSAs. NE (12–15 μg) obtained from control, PMA (10 nm), (Bu)₂cAMP (0.1 mm), and PMA plus (Bu)₂cAMP-treated MA-10 (A, lanes 2–7) and KK-1 (B, lanes 9–14) cells were incubated with the ³²P-labeled probe specific to the −83/−67 bp region of the StAR promoter. DNA-protein complexes (labeled as I and II) were challenged with unlabeled wild-type (−83/−67; lanes 6 and 13) or mutant (−83/−67 M; lanes 7 and 14) sequences. Cold competitors were used at 100-fold molar excess. Migration of free probes is shown in both cases. Data are representative of three independent experiments.

Figure 6

Role of PMA in the activation of (Bu)₂cAMP-mediated Nur77 protein expression and its relevance to StAR gene transcription and a schematic model on PKC-dependent modulation of cAMP-mediated steroidogenesis. MA-10 (A and B) and KK-1 (B and C) cells were pretreated without or with GFX (20 μm) for 30 min and then incubated with PMA (10 or 1–100 nm), (Bu)₂cAMP (0.1 mm), or their combination for 6 h, as indicated. Representative immunoblots (n = 3–4) show expression of Nur77 in different treatment groups using 25–30 μg of total cellular protein. Integrated OD (IOD) values of each band were quantified and presented (B). C, KK-1 cells were transfected with empty vector (pcDNA), wild-type Nur77, DN-Nur77, or a combination of them, in the presence of the −151/−1 StAR promoter segment. Cells were cotransfected in the presence of pRL-SV40 plasmid for normalization of transfection efficiency. After 36 h of transfection, cells were treated without (basal) or with PMA (10 nm), (Bu)₂cAMP (0.1 mm), and PMA plus (Bu)₂cAMP for an additional 6 h, and luciferase activity in the cell lysates was determined and expressed as relative light unit (RLU, luciferase/renilla). Data represent the mean ± se of four independent experiments. D, Illustration of how a number of factors may serve in the PKC-dependent potentiation of cAMP/PKA-mediated steroidogenesis. Induction of PKC by PMA can induce StAR expression but is incapable of P-StAR and thus produces a marginal response in progesterone synthesis. However, a nominal increase in PKA activity by means of a low dose of (Bu)₂cAMP, phosphorylates and activates PMA-induced StAR, increases its cholesterol-transferring capacity, and results in the modulation cAMP-mediated steroid synthesis.