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Tropic hormones acutely stimulate adrenal and gonadal steroidogenesis by activation of the cAMP-dependent protein kinase A (PKA) signaling pathway and subsequent induction of Steroidogenic Acute Regulatory (StAR) protein (StAR) expression. We present a comparative study of StAR regulation in mouse adrenocortical Y1 and the derived PKA mutant Kin-8 cell lines to evaluate the PKA requirement for StAR expression. A parallel increase in StAR steady-state mRNA and protein was observed in Y1 cells. StAR mRNA was induced in 8-Br-cAMP-treated Kin-8 cells with maximal expression levels approx. 50% of that observed in Y1 cells. However, a corresponding increase in StAR protein, as detected by Western analysis, was absent in the Kin-8 cells. A similar distribution of StAR mRNA in active polysome fractions was observed for both 8-Br-cAMP-treated Y1 and Kin-8 cells, as well as a 2-fold increase in incorporation of [35S]methionine into StAR, which indicated translation was not blocked in Kin-8 cells. Together these data indicate that PKA functions at the post-translational level to regulate StAR expression and we propose that phosphorylation of StAR by PKA contributes to protein stability

Adrenal steroid production is stimulated by adrenocorticotropin hormone activation of the cAMP-dependent protein kinase A (PKA) signaling pathway and subsequent induction of Steroidogenic Acute Regulatory (StAR) protein expression. Herein we have compared StAR mRNA and protein levels in 8-Br-cAMP-treated mouse adrenocortical Y1 and the derived PKA mutant Kin-8 cell lines to evaluate the PKA requirement in StAR expression. StAR mRNA was induced by 8-Br-cAMP-treatment of both Y1 and Kin-8 cells with maximal expression levels in Kin-8 cells approximately 50% of that observed in Y1 cells. StAR protein levels, as detected by Western analysis, were concomitantly increased in Y1 cells but were not detected in the Kin-8 cells. StAR mRNA colocalized with the active polysome fractions in both 8-Br-cAMP-treated Y1 and Kin-8 cells, indicating translation was not blocked in Kin-8 cells. Consistent with this data, a 2-fold increase in incorporation of [35S]methionine into StAR was also observed after 8-Br-cAMP treatment of both cell lines. Since StAR protein levels were not sufficient to detect by Western analysis, these data indicate that PKA functions at the post-translational level to regulate StAR expression and we propose that phosphorylation of StAR by PKA contributes to protein stability.

Steroidogenic acute regulatory protein (StAR) synthesis and steroidogenesis are stimulated by activation of divergent signaling pathways in the adrenal cortex. The two major physiological regulators of aldosterone synthesis in the adrenal zona glomerulosa are angiotensin II (AII) and extracellular K+, which both mediate an increase in intracellular calcium levels, although by distinct mechanisms. Previously, we demonstrated that increased mineralocorticoid synthesis by N6,2'-O-dibutyryl cAMP (Bt2cAMP), AII, and K+ treatment is paralleled by an increase in StAR protein in the H295R human adrenocortical cell line. We now show that StAR steady state messenger RNA levels are increased by Bt2cAMP and AII, but not by K+ or 12-O-tetradecanoylphorbol-13-acetate, treatment of H295R cells. Northern analysis detected two major transcripts of 1.7 and 2.7 kb present in H295R cells, with the most prominent effect of agonist treatment on induction of the 1.7-kb messenger RNA. Similarly, StAR promoter activity in transient transfections of H295R cells with a luciferase reporter gene driven by 1.3 kb of the human promoter was increased only by Bt2cAMP and AII treatment. 5'-Deletion analysis of the StAR promoter indicates that both the cAMP- and AII-responsive elements are within 150 bases of the transcriptional start site. Mutation of a steroidogenic factor-1/AdBP4 element localized at -95 abolishes both Bt2cAMP- and AII-induced luciferase activity in these transient transfection assays. Thus, transcriptional activation of the StAR gene by a steroidogenic factor-1-dependent mechanism may represent a common pathway for ACTH (protein kinase A) and AII action in stimulating steroid production in the adrenal fasciculata and glomerulosa.

Hormonal induction of steroidogenesis in the adrenal and gonads is dependent on the synthesis and function of the steroidogenic acute regulatory protein (StAR). As a first approach to investigate the role of translation in the control of StAR expression, we examined StAR protein synthesis and steroid production in MA-10 mouse Leydig tumor cells in the presence of the transcriptional inhibitor, actinomycin D. We show that human CG (hCG)-induced StAR synthesis, as determined by radiolabeling MA-10 cells with [35S]methionine and immunoprecipitation of StAR, is blocked by actinomycin D. The rate of hCG-stimulated progesterone production is also decreased, but not completely blocked, suggesting a possible StAR-independent mechanism that may contribute approximately 10-20% of the acute steroidogenic potential of the cells. When MA-10 cells were pretreated with hCG to increase StAR messenger RNA levels and then the proteins radiolabeled in the presence of hCG or hCG plus actinomycin D, no difference was observed in the amount of the 30-kDa StAR protein synthesized. However, a 50% increase in the precursor form of StAR protein was detected with hCG treatment alone. These data suggest that ongoing StAR protein synthesis is not inhibited by actinomycin D, but that continued synthesis requires transcriptional activity. Progesterone production was inhibited by actinomycin D in the hCG-pretreated cells, supporting the proposal that maintaining StAR protein synthesis is required for optimal steroid production in MA-10 mouse Leydig tumor cells.