Involvement of Protein Kinase C and Cyclic Adenosine: DISCUSSION

In the present study, we continued investigations on the factors that render R2C cells constitutively steroidogenic. The importance of the PKA pathway was suggested when R2C cells were treated with H8Q, an inhibitor of PKA, and decreases in both Star mRNA levels and steroid production were observed. Given the complex phenotype exhibited by R2C cells described in earlier studies, we perceived that in addition to PKA, other regulatory mechanisms were likely involved in steroidogenesis. In this study the functional impact of the PKC pathway in R2C cells was examined by testing both PKC itself and PLC, the enzyme that generates the PKC activator, DAG. Inhibition of either PLC or PKC in R2C cells resulted in significant decreases in Star mRNA and steroid production without any adverse effects on the activity of the P450scc enzyme. The basal levels of P-CREB in R2C cells were, interestingly, also decreased by PKC inhibition, indicating that phosphorylation of CREB may result from activation of both the PKA and the PKC pathways. Studies showed that PMA, like (Bu)2cAMP, was able to increase P-CREB levels in MA-10 cells and suggested that upon activation, both the PKA and PKC pathways converged at CREB phosphorylation. The mouse Star promoter has been well characterized and shown to contain three separate cAMP-responsive element (CRE) half sites. The regulation of those CRE half sites by the ATF/CREB/CREM family of transcription factors has been thoroughly studied [36-3Q].

The phosphorylation of CREB by PKA and PKC pathways was first reported in 1QQ1 by Sakurai et al., who used an in vitro system to test the specific phosphorylation sites of recombinant human CREB-1. Their results illustrated that PKA phosphorylated Ser62, and PKC phosphorylated Ser340 and Ser367 in CREB-1. Later, Xie and Rothstein showed that PKC phosphorylates CREB on Ser133 in B cells. Recently, it was demonstrated that phosphorylation of CREB at Ser133 in PC12 cells was increased within 15 min in response to forskolin, PMA, epidermal growth factor, and nerve growth factor. Consistent with these observations, we conclude that R2C and MA-10 cells express CREB that is phosphorylated on Ser133 by PKA (stimulated by (Bu)2cAMP) and PKC (stimulated by PMA), because the antibody used in the present study was specific for phospho-Ser133 in CREB.

PMA itself, although capable of increasing StAR protein levels, was not able to stimulate steroid production in MA-10 cells, confirming earlier observations that described PMA as a weak stimulator of steroidogenesis in rat Leydig cells and Y-1 adrenocortical cells. Similar results from other studies also suggested that the inability of PMA to induce a comparable increase in steroid production to that of StAR protein was due to a requirement for a PKA-dependent posttranslational modification. In accordance with these observations we demonstrated that PMA treatment alone could not increase steroid production significantly, because the PMA-induced StAR protein was not activated by phosphorylation.

Phosphorylation of the StAR protein requires the action of the PKA pathway as demonstrated in an earlier observation that showed that PMA failed to generate phospho-protein species of StAR that are present in hCG-, (Bu^cAMP-, or forskolin-stimulated MA-10 cells. Studies evaluating the role of phosphorylation in StAR activity demonstrated that the phosphorylation of StAR at Ser1Q4 was functionally more important than the phosphorylation at other potential PKA sites in the StAR protein. A 50% decrease in StAR activity occurred when Ser1Q4 was mutated to alanine. Loss of this site in heterologous cell systems reduced StAR activity by 70%-80% [4Q, 50]. In agreement with these observations, results of the present study demonstrated that the PMA-stimulated PKC pathway increased StAR protein levels, but the protein was not phosphorylated, and consequently, was incapable of mediating steroid synthesis. However, when PMA-treat-ed MA-10 and mLTC-1 cells were exposed to submaximal doses of (Bu)2cAMP, high levels of steroid production were obtained, presumably through PKA-mediated phosphorylation of the PMA-induced StAR protein. As seen in Figure 6B, under basal conditions, R2C cells contain high levels of P-StAR that were dramatically decreased following inhibition of either the PKC or PKA pathways. These results indicate that R2C cells can produce P-StAR basally because of the presence of constitutive levels of PLC/PKC and PKA activities not observed in unstimulated MA-10 cells. It was also noted that the sensitivity of MA-10 and mLTC-1 cells to submaximal doses of (Bu)2cAMP was quite different. Mouse LTC-1 cells were more sensitive to low doses of (Bu)2cAMP than were MA-10 cells (Fig. 7). The reasons for this difference are not presently clear.

A role for other factors in sensitizing Leydig cells to low levels of PKA stimulation has been previously described. For example, stimulation of Leydig cells with LH, hCG, or cAMP was linked to increased chloride ion conductance. Removal of extracellular chloride ions significantly enhanced 0.1 mM (Bu)2cAMP-stimulated StAR and steroid production in MA-10 cells. The authors suggested that when PKA is submaximally activated, cAMP-dependent regulation of steroidogenesis is likely to be influenced by the synthesis and specific phosphorylation of proteins, perhaps one of them being StAR. Wang et al. reported that submaximal doses of (Bu)2cAMP were involved in steroid production and StAR expression following the production of arachidonic acid metabolites via one of its metabolizing pathways. In MA-10 cells, the addition of arachidonic acid alone had no effect on StAR protein levels and steroid production, whereas both parameters were significantly increased when submaximal doses of (Bu)2cAMP were added simultaneously with arachidonic acid. The hypothesis that minimal levels of PKA are critical for steroidogenesis was also addressed by Wang et al. in another study. MA-10 cells treated with a cyclooxy-genase 2-specific inhibitor displayed no change in StAR protein or steroid synthesis, but significant increases in these two parameters were observed when a submaximal dose of (Bu)2cAMP was added in conjunction with the inhibitor. The precise mechanism involved in this observation is not yet fully understood. However, the present study clearly demonstrates the absolute requirement for a low level of PKA activity for the phosphorylation of StAR protein in PMA-induced steroidogenesis, as illustrated in the diagram in Figure 8.

The involvement of the PKC pathway in Leydig cell steroidogenesis has been documented, and the mechanisms that regulate steroidogenesis and StAR expression via PKC action appear to be highly dependent on the cells and their tissue types, as well as the external stimuli influencing these cells. Several studies have shown that the PKC pathway is involved in the activation of steroid synthesis, while others have observed negative relationships or no effect in various cell types. It appears that the concentration of PMA is one critical factor in controlling trophic hormone effects on steroid production. An earlier study showed that 100 ng/ml (162 nM) PMA treatment had an inhibitory effect on hCG-stimulated progesterone production in MA-10 cells. This observation was consistent with cAMP content, because 162 nM PMA had an inhibitory effect on the accumulation of cAMP induced by hCG. However, in the presence of hCG, the 10 nM PMA used in the present study stimulated both P-StAR and progesterone production in MA-10 cells, indicating that in the presence of trophic hormone, low concentrations of PMA are stimulatory, whereas higher concentrations are inhibitory.

In the present study, stimulation of freshly isolated rat Leydig cells with 10 nM PMA and low doses of cAMP resulted in a decrease in testosterone levels (Fig. 10), whereas P-StAR levels were increased. However, levels of progesterone were greatly increased in these cells. These observations indicated that PMA had differential effects on the steroidogenic enzymes in the pathways that produce progesterone and testosterone. In an early study, Welsh et al. treated immature rat testicular cells with 10 ng/ml (16.2 nM) PMA in the presence of hCG and showed that 17a-hydroxyprogesterone, androstenedione, and testosterone production were inhibited by 80%-90%, while progesterone was increased in these cells. Because testosterone production was also inhibited by PMA following the exogenous addition of progesterone and 17a-hydroxyproges-terone under both basal and stimulated conditions, the authors suggested that PMA inhibited CYP17 activity. A similar report demonstrated that PMA inhibited cAMP-induced Cyp17 mRNA expression, the activity of CYP17, and testosterone production in mouse Leydig cells. Also, 10 nM PMA was shown to be stimulatory for expression of Hsd3b mRNA but inhibitory for expression of Cyp17 mRNA in human H295R adrenocortical cells. Thus, in our study, the decrease in testosterone and the concomitant increase in progesterone are undoubtedly due to an inhibition of CYP17 activity by PMA. This may mirror what occurs in ovarian theca cells after the LH surge in that they switch from producing androgens to progestagens even though stimulatory levels of LH remain in the ovary. When we measured the expression levels of CYP17 protein in immature rat Leydig cells, decreases in CYP17 protein were not observed (Fig. 12). Thus, as seen in previous studies, we concluded that PMA results in an inhibition of CYP17 enzyme activity, but not its expression. The mechanism of this inhibition remains unknown.

The PMA receptor, PKC, was identified more than two decades ago. Nikula and Huhtaniemi studied the effects of PKC activation by PMA on cAMP and testosterone levels using Percoll-purified rat Leydig cells. Activation of PKC with PMA or OAG (1-oleoyl-2-acetyl-sn-glycerol) alone did not affect levels of intracellular cAMP; however, when used in conjunction with other treatments, each was shown to have profound effects on cAMP levels.

They were inhibitory on hCG-stimulated cAMP production, but stimulatory with cholera toxin and forskolin. The authors concluded that PKC activation by LH might affect the coupling between the LH receptor and guanine nucleotide binding protein, alpha inhibiting (GNAI) or guanine nucleotide binding protein, alpha stimulating (GNAS). Another study supporting this idea resulted from the infection of Sf9 cells with LH receptor constructs. The data demonstrated the activation of adenylyl cyclase and PLC through interaction with GNAS and GNAI2, respectively, and resulted in increases in cAMP and IP3. This result suggests that LH/hCG binding to the LH receptor leads to the activation of the PKA and PKC pathways through the independent targeting or interaction of guanine nucleotide binding protein subfamilies. Moreover, independent stimulation of both pathways may allow for significant levels of testosterone synthesis without the need for high levels of LH. This could be useful, for instance, in maintaining high basal steroid production in the testes between LH pulses.

In summary, the present study indicates that constitutive stimulation of the PLC/PKC pathway in the presence of low basal PKA activity is primarily responsible for the high level of basal steroidogenesis observed in R2C cells and that activation of the PKC pathway potentiates steroidogenesis in the presence of hCG or cAMP analogs in Leydig cells. Collectively, the data demonstrate that while activation of PKC by PMA can induce transcription and translation of Star, low doses of cAMP are required to phos-phorylate and activate StAR protein before it can mediate cholesterol transfer and result in steroid synthesis. The increase in expression of StAR but lack of steroid synthesis following stimulation by PMA represents a novel observation in Leydig cells. The subsequent increase in steroids by submaximal levels of cAMP in PMA-treated cells demonstrates the absolute requirement for minimal PKA activity and phosphorylation of StAR in the regulation of steroidogenesis.

Category: Leydig Cells

Tags: signal transduction, StAR, steroid biosynthesis, testosterone