Involvement of Protein Kinase C and Cyclic Adenosine: RESULTS

RESULTS

Constitutive StAR and Steroid Production in R2C Cells Requires PLC and PKC Activity

In an effort to determine whether constitutive steroidogenesis in R2C cells is the result of increased activity of the PKC pathway, the possibility that PLC, the enzyme that produces the DAG activator of PKC, was constitutively activated in R2C cells was examined. U73122, a specific inhibitor of PLC, inhibited R2C cell steroid production by 50% (Fig. 1A). To determine whether this observation was the result of nonspecific toxicity of U73122, cells were incubated with 22(R)-hydroxycholesterol (22R-OHC) in the presence of U73122. The hydrophilic 22R-OHC can readily diffuse unassisted across the mitochondrial membranes and reach the P450scc enzyme system where it is converted to pregnenolone and then to progesterone by 3p-hydroxyste-roid dehydrogenase (HSD3B). U73122 had no effect on progesterone synthesis when 22R-OHC was used as a substrate; therefore, its inhibitory effects were located upstream of P450scc. In parallel with the observed decrease in steroid production, RT-PCR was used to demonstrate a concomitant 40% reduction in Sfar mRNA levels in R2C cells treated with U73122 (Fig. 1B), while the levels of RpH9 were not changed.

To determine whether PKC is also constitutively active in the R2C cell line, cells were treated with two PKC inhibitors, GF-109203X (GFX) and Go6983. Following 6 h of treatment with either inhibitor, the high basal levels of steroid normally observed in R2C cells were reduced by 70% (Fig. 2A). Furthermore, whereas R2C cells cultured in the presence or absence of either PMA (10 nM) or (Bu)2cAMP (1 mM) for 6 h showed little change in steroid output, in the presence of either inhibitor, steroid levels decreased by 70% in PMA-treated cells and by 40%-45% in (Bu)2cAMP-treated cells. Using the same treatments followed by RT-PCR, transcriptional levels of Sfar in R2C cells decreased significantly following PKC inhibition even in the presence of PMA or (Bu)2cAMP (Fig. 2B).

To understand the mechanism through which PKC affects Star gene expression, we examined the expression patterns and phosphorylation states of CREB using the same treatments described above. Results from these experiments revealed that R2C cells had constitutively increased levels of P-CREB under basal conditions as shown in Figure 2C, and stimulation of the PKA pathway resulted in a slight increase in P-CREB levels. The levels of P-CREB in basal and PMA-stimulated R2C cells were significantly decreased by the PKC inhibitors (70%-80% reduction). The same PKC inhibitors were not as effective in reducing the levels of (Bu)2cAMP-induced P-CREB (45% reduction). When stained for tot-CREB, no changes were observed following any of the treatments. The basal level of P-CREB in R2C cells was assessed following H89 treatment and was found to be significantly decreased (data not shown). These results indicate that constitutive steroid and StAR production in R2C cells are, in part, due to the constitutive phosphorylation of CREB that occurs as a result of the intrinsically activated PLC/PKC or PKA pathways (or both).

PKC Stimulates StAR Expression but Not Steroid Production in MA-10 Cells

To further explore the role of the PKC pathway in steroidogenesis, MA-10 cells were stimulated with (Bu)2cAMP or PMA. MA-10 cells expressed extremely low basal levels of StAR and P-CREB, but stimulation with either compound strongly increased the levels of these proteins (Fig. 3A, top and middle panels). Concomitant with these changes, steroid production increased only in cells stimulated with (Bu)2cAMP (Fig. 3B). When PKA or PKC activity was blocked with H89 or GFX, respectively, the levels of StAR and P-CREB were strongly attenuated as was (Bu)2cAMP-stimulated progesterone synthesis.

PMA Stimulation Requires Submaximal Doses of (Bu)2cAMP to Synthesize Steroids in MA-10 Cells

To better understand the roles of PKA and PKC, the levels of StAR protein and steroid synthesis induced with different doses of PMA were compared with those obtained with different doses of (Bu)2cAMp (Fig. 4A) in MA-10 cells. Concentrations of (Bu)2cAMP below 0.1 mM did not appreciably affect StAR protein and steroid synthesis. Treatment with 10 and 50 nM of PMA stimulated StAR protein production to levels comparable to those observed with 0.5 and 1.0 mM (Bu)2cAMP, respectively, but again, without significantly affecting steroid production. Thus, stimulation of PKC activity using PMA can induce expression of StAR protein, but this induction is insufficient to drive steroid biosynthesis.

Because of these results, MA-10 cells were manipulated in an attempt to mimic the PKC and PKA activities found in R2C cells. MA-10 cells were stimulated with 10 nM PMA in the presence of increasing but low doses of (Bu)2cAMP (0.005-0.1 mM, which by themselves did not affect StAR protein and steroid synthesis appreciably). As shown in Figure 4B, compared to the controls, a significant increase in StAR protein occurred following PMA stimulation with little change in steroid biosynthesis. In the presence of 0.005 and 0.01 mM (Bu)2cAMP, PMA-treated MA-10 cells showed no further induction of StAR protein and a small and gradual increase in steroid production. However, in the presence of 0.05 and 0.1 mM (Bu)2cAMP (still submaximal concentrations), StAR protein levels were further increased compared with that of PMA-only treatment, and dramatic increases in steroids were observed in the presence of PMA. Under these conditions, steroid levels were comparable to those measured after stimulation with 0.5 mM (Bu)2cAMP only. These results indicate that the production of steroids in response to PMA requires a minimal PKA activity, which, by itself is insufficient to elicit steroid synthesis or StAR expression.

PMA Stimulates Star Gene Transcription

To determine whether PMA affected the abundance of Star mRNA, experiments on Star promoter activity and Northern blot analyses were performed. Different mouse Star promoter-luciferase constructs were transfected into MA-10 cells and promoter activity was assessed using a dual luciferase reporter assay. PMA alone slightly increased Star promoter activity compared with that of control groups (Fig. 5A). The activity of the Star promoter was also stimulated when both 10 nM PMA and 0.05 mM (Bu)2cAMP were used, but the activities were less than those of 1 mM (Bu)2cAMP. It was also noted that PMA-responsive elements were likely present within the —151/—110 region of the Star promoter. Also, the activity of the — Q66/— 1 promoter construct was always lower than those of the —254/ — 1 or —151/—1 constructs, a pattern previously observed. This unexplained observation could indicate the presence of an inhibitory element between —Q66 and —254 in the Star promoter. The result of Figure 5A paralleled those obtained with Northern blot analysis (Fig. 5B). While PMA alone increased Star mRNA, 0.05 mM (Bu)2cAMP alone was not able to induce Star mRNA expression. Simultaneous treatment with PMA plus 0.05 mM (Bu)2cAMP increased Star mRNA levels beyond those observed with PMA alone or with 1 mM (Bu)2cAMP Even 1 mM (Bu)2cAMP-induced Star mRNA levels were further increased by costimulation with PMA.

In the Presence of PMA, a Submaximal Dose of (Bu)2cAMP Is Required for the Phosphorylation of StAR in MA-10 Cells

It was consistently observed that while PMA can induce StAR expression, it requires the addition of (Bu)2cAMP to induce a steroidogenic response. Because our previous data suggested that PMA potentiates both Star gene transcription and translation, we hypothesized that the lack of steroidogenesis in PMA-treated MA-10 cells is due to its inability to activate StAR activity at a posttranslational level. More specifically, in the presence of PMA, a submaximal dose of (Bu)2cAMP was required to induce PKA activity required for the phosphorylation and full activation of the StAR protein. To discriminate between tot-StAR and P-StAR, two different antibodies were used to recognize P-StAR protein; one recognizes the phospho-Ser/Thr PKA substrate and the other detects phosphorylated Ser194 on StAR. Stimulation of MA-10 cells with PMA alone results in abundant synthesis of nonphosphorylated tot-StAR (Fig. 6A). Importantly, the submaximal dose of (Bu)2cAMP (0.05 mM), when used alone, was unable to induce either tot-StAR or P-StAR. In contrast, treatment of MA-10 cells with both PMA and submaximal (Bu)2cAMP generated high levels of tot-StAR protein, and the protein was phos-phorylated. As expected, cells stimulated with 1 mM of (Bu)2cAMP showed high levels of tot-StAR and P-StAR, and the levels could be further increased when cells were stimulated simultaneously with PMA. The results were similar using both antibodies to recognize P-StAR.

Constitutive Activation of PKC and PKA Pathways Are Responsible for the R2C Cells’ Steroidogenic Phenotypes

R2C cells were treated with inhibitors of the PKC (GFX and Go6983) and PKA (H89) pathways to determine whether both pathways are critical for constitutive steroid production and whether P-StAR and tot-StAR are significantly altered by these inhibitors. As demonstrated in Figure 6B, P-StAR was constitutively present under basal conditions in R2C cells. Inhibition of both pathways resulted in significant decreases in the levels of P-StAR as well as corresponding decreases in steroid production. Tot-StAR levels were slightly decreased in R2C cells in response to the inhibitors, indicating that StAR protein was not yet significantly degraded within 6 h of treatment. These studies once again demonstrated the importance of the phosphor-ylated form of StAR in steroid synthesis.

PMA Requires a Submaximal Dose of (Bu)2cAMP to Induce Steroid Production in mLTC-1 Cells

The combined effects of PMA and (Bu)2cAMP on steroid production were also tested in another cell line. The mLTC-1, mouse Leydig tumor cell line, was treated with or without PMA in the presence or absence of either 0.05 or 0.5 mM (Bu)2cAMP. As shown in Figure 7, while PMA alone induced the expression of unphosphorylated StAR, no steroid production was observed. Treatment of mLTC-1 cells with 0.05 mM (Bu)2cAMP alone was able to induce a slight increase in unphosphorylated StAR expression but only slight increases in P-StAR and steroid production. However, PMA plus 0.05 mM (Bu)2cAMP resulted in high levels of P-StAR and steroids. Using the same experimental design the levels of P-CREB were also determined by Western blot analysis and showed that P-CREB was also increased by PMA stimulation (data not shown). In summary, both the PKA and PKC pathways appear to act in concert to regulate StAR protein expression and steroidogenesis in Leydig tumor cell lines. A general schematic summarizing how PMA and a submaximal dose of (Bu)2cAMP appear to work together in MA-10 cells is depicted in Figure 8.

PMA and hCG Synergistically Increase Progesterone Production in MA-10 Cells

Additional studies attempted to determine whether the PMA-induced PKC pathway has a synergistic effect on StAR expression and steroid production in the presence of trophic hormones. As observed earlier, PMA stimulated strong expression of unphosphorylated StAR in MA-10 cells without a comparable increase in steroid production (Fig. 9). Stimulation with hCG induced both total and P-StAR production within 6 h and resulted in a corresponding increase in progesterone synthesis. Addition of hCG in combination with PMA resulted in increases in tot-StAR and P-StAR levels (2.4-fold and 4-fold, respectively), and an increase in progesterone of 2.6-fold over treatment with hCG only. This result demonstrates that PMA significantly enhances trophic hormone-stimulated StAR and steroid production.Effects of PMA on Steroidogenic Enzymes in Isolated Immature Rat Leydig Cells

In Leydig tumor cells, 10 nM PMA was sufficient to synthesize Sfar mRNA and StAR protein, but only in the presence of a low dose of (Bu)2cAMP or hCG was StAR phosphorylated and steroids synthesized. To investigate this phenomenon in primary cultures, Leydig cells were freshly isolated from 10-day-old rats. The cells were treated with PMA and two different doses of (Bu)2cAMP in a manner identical to the treatment of MA-10 and mLTC-1 cells with the exception that the incubation times were both 6 and 24 h. Primary cultures had very low levels of P-StAR and tot-StAR under basal conditions at both 6 (Fig. 10A) and 24 h (Fig. 10B). In primary cultures of rat Leydig cells, PMA was able to induce tot-StAR but not P-StAR, consequently resulting in no changes in steroid levels (Fig. 11). This situation was similar at both 6 (Fig. 11A) and 24 h (Fig. 11B). In contrast, addition of 0.05 mM (Bu)2cAMP to PMA-treated cells was able to induce the phosphorylation of StAR (Fig. 10), as observed in the Leydig tumor cell lines, and a corresponding increase in progesterone at both 6 and 24 h (Fig. 11, A and B). As anticipated, high doses of (Bu)2cAMP resulted in dramatic increases in both tot-StAR and P-StAR, which corresponded to increases in progesterone and testosterone. In these experiments we observed that costimulation of primary Leydig cells with 0.5 mM (Bu)2cAMP and PMA resulted in high levels of progesterone but significantly decreased levels of testosterone. Similar patterns were also observed in response to LH- or hCG-and-PMA-treated cells and are consistent with earlier observations that PMA can inhibit CYP17 enzyme activity in rat and mouse Leydig cells. In an attempt to determine whether PMA inhibited the expression of CYP17, Western blot analyses were performed. When the levels of CYP17 were normalized to actin, there was no significant difference in its expression at either 6 or 24 h (Fig. 12) in the absence or presence of PMA. These results demonstrated that PMA inhibited testosterone production by affecting the activity of CYP17 and not its expression. The manner in which PMA inhibits CYP17 activity remains unknown. Most importantly, however, these studies show that, as seen in Leydig tumor cells, PMA stimulation of primary cultures of Leydig cells can induce unphosphory-lated StAR protein expression, but a low dose of cAMP is required to phosphorylate and activate StAR and stimulate steroid synthesis.
Fig1Involvement of Protein Kinase-1
FIG. 1. Involvement of constitutive activation of PLC in steroid and StAR synthesis in R2C cells. A) Serum-free Waymouth media was added and treated with vehicle (dimethylsulfoxide, controls) and the PLC inhibitor (U73122, 100 ^M) without or with 22(R)-hydroxycholesterol (25 ^M, a positive control), for 6 h and tested for progesterone production. Statistical analysis was performed as described in Materials and Methods (*P < 0.05). Small bars in the graph indicate SEM. B) RT-PCR analysis for Star and Rpl19 (reaction control).

Fg2Involvement of Protein Kinase-2
FIG. 2. Involvement of constitutive PKC activation in steroid and StAR expression in R2C cells. R2C cells were treated with 25 of either GFX or Go6983 for 15 min followed by 10 nM PMA or 1 mM (Bu)2cAMP for 6 h. The media were analyzed for steroid production (A), and the cells were used for RT-PCR (B) to determine the transcriptional levels of Star and Rpl19 (loading control) genes and Western blot analyses (C) to observe the levels of phosphorylated CREB (P-CREB) and total CREB (tot-CREB). Small bars in the graph indicate SEM. Figures are representative of three independent experiments with similar results.

Fig3Involvement of Protein Kinase-3
FIG. 3. PMA induces StAR but not steroid production in MA-10 cells. Cells were incubated in serum-free Waymouth media, treated with the PKC- and PKA-specific inhibitors GFX (25 ^,M) and H89 (25 ^M), respectively, for 15 min, followed by PMA (10 nM) or (Bu)2cAMP (1 mM) stimulation for 6 h. To determine the levels of P-CREB, cells were stimulated for 1 h. Equal amounts of proteins were analyzed for StAR, P-CREB, and tot-CREB by Western blot analysis (A). Medium was analyzed for progesterone production (B). Small bars in the graph indicate SEM.

Fig4Involvement of Protein Kinase-4
FIG. 4. Comparison of StAR expression and steroid production in MA-10 cells stimulated with (Bu)2cAMP, PMA, or costimulated with both. A) Cells were stimulated with different concentrations of PMA or (Bu)2cAMP for 6 h and analyzed for StAR protein expression and progesterone production. B) Cells were costimulated with both PMA (10 nM) and increasing doses of (Bu)2cAMP for 6 h to determine the levels of StAR and steroids. Small bars in the graph indicate SEM.

Fig5Involvement of Protein Kinase-5
FIG. 5. Transcriptional regulation of the Star gene by PMA in MA-10 cells. A) Different lengths of the mouse Star promoters (—966, —254, — 151, and —110) containing a luciferase reporter gene transfected into MA-10 cells and treated with 10 nM PMA, PMA + 0.05 mM (Bu)2cAMP, and 1 mM (Bu)2cAMP for 6 h. The promoter activity was determined as described in Materials and Methods. Small bars in the graph indicate SEM. B) Northern blot analysis for Star mRNA was performed on RNA isolated from MA-10 cells treated with 0.05 mM (Bu)2cAMP and 1 mM (Bu)2cAMP in the absence and presence of PMA (10 nM) for 6 h. Upper and lower panels show different exposure times of the same blot to examine the StAR transcripts.

Fig6Involvement of Protein Kinase-6
FIG. 6. Role of PKC and PKA (a submaximal dose of (Bu)2cAMP) in MA-10 and R2C cells. A) MA-10 cells were stimulated using 0, 0.05, and 1 mM (Bu)2cAMP in the presence or absence of 10 nM PMA for 6 h. Western blot analyses were performed using a specific antibody to detect tot-StAR and two separate antibodies to detect P-StAR. The top panel shows the 30-kDa protein bands detected by a phospho-Ser/Thr (p-Ser/Thr)-spe-cific PKA substrate antibody purchased from Cell Signaling. The middle panel represents the phosphorylated StAR using a phospho-Ser194-spe-cific antibody (p-Ser194) of StAR protein. The bottom panel shows the tot-StAR using an antibody to tot-StAR. B) R2C cells were treated with GFX (25 ^M) and Go6983 (25 ^M) to inhibit the PKC pathway and H89 (25 ^M) to inhibit the PKA pathway and followed by 6 h of incubation. The protein levels of P-StAR and tot-StAR were analyzed by Western blot, and the production of progesterone was tested by radioimmunoassay Small bars in the graph indicate SEM.

Fig7Involvement of Protein Kinase-7
FIG. 7. Regulation of StAR phosphorylation and steroid production in mLTC-1 cells using PMA and a low dose of (Bu)2cAMP. Cells were stimulated similar to those treated in Figure 6. Western blot analysis was performed using cell lysates to determine the levels of tot-StAR and phos-phorylated-Ser194 on StAR (P-StAR) using each specific antibody.The media were recovered and analyzed for progesterone production. Small bars in the graph indicate SEM. The data are representative of three separate experiments with similar results.

Fig8Involvement of Protein Kinase-8
FIG. 8. Diagram of the interaction of PMA-induced PKC pathways with and without a submaximal stimulation of the PKA pathway by (Bu)2cAMP in the regulation of StAR expression and steroidogenesis. Arrows with black bars indicate pathways that are not activated; arrows without black bars indicate activated pathways. Panels on the right correspond to the levels of P-StAR, tot-StAR, and progesterone obtained in each experimental paradigm using MA-10 cells.

Fig9Involvement of Protein Kinase-9
FIG. 9. PMA synergistically increases hCG-induced P-StAR and progesterone production in MA-10 cells. Cells were treated with or without hCG in the absence or presence of 10 nM PMA for 6 h. Proteins were analyzed for tot-StAR and P-StAR by Western blot analysis, and the media were recovered and analyzed for progesterone synthesis. Small bars in the graph indicate SEM. The data are representative of two separate experiments with identical results.

Fig10Involvement of Protein Kinase-10
FIG. 10. Differential effects of PMA on StAR expression in freshly isolated rat Leydig cells. Immature rat Leydig cells were treated with low (0.05 mM) and high (0.5 mM) doses of (Bu)2cAMP, respectively, in the absence and presence of 10 nM PMA for 6 (A) and 24 h (B). Total cell lysates were analyzed for P-StAR and tot-StAR by Western blotting. The data are representative of four independent experiments with similar results.

Fig11Involvement of Protein Kinase-10
FIG. 11. Effects of PMA on steroidogenesis in freshly isolated rat Leydig cells. Immature rat Leydig cells were treated with low (0.05 mM) and high (0.5 mM) doses of (Bu)2cAMP, respectively, in the absence and presence of 10 nM PMA for 6 (A) and 24 h (B). Medium was analyzed for progesterone and testosterone by radioimmunoassay. Small bars in the graph indicate SEM. The data are representative of four independent experiments with similar patterns.

Fif12Involvement of Protein Kinase-12
FIG. 12. Effects of PMA on CYP17 expression in freshly isolated rat Leydig cells. Immature rat Leydig cells were treated with low (0.05 mM) and high (0.5 mM) doses of (Bu)2cAMP, respectively, in the absence and presence of 10 nM PMA for 6 (A) and 24 h (B). Total cell lysates were analyzed for CYP17 by Western blotting. Levels of actin were also determined as a loading control. Relative integrated optical density of the ratio using CYP17:actin was represented on the bottom of each blot. The figures are representative of two different experiments using two different antibodies for CYP17 (anti-human CYP17 and anti-porcine CYP17) with identical results.


Category: Leydig Cells

Tags: signal transduction, StAR, steroid biosynthesis, testosterone