Involvement of Protein Kinase C and Cyclic Adenosine 3′,5′-Monophosphate-Dependent Kinase in Steroidogenic Acute Regulatory Protein Expression and Steroid Biosynthesis in Leydig Cells1

Abstract

This study investigated the roles of the protein kinase C (PKC) and protein kinase A (PKA) pathways in regulating constitutive steroidogenesis and steroidogenic acute regulatory (STAR; herein designated by its common name, StAR) protein in R2C Leydig tumor cells. Inhibition of PKC and phospholipase C resulted in significant decreases in steroid production, phosphorylation of cAMP-responsive element binding (CREB) protein, and Star gene transcription under basal conditions in R2C cells. These observations were corroborated in MA-10 and mLTC-1 Leydig tumor cell lines, in which activation of PKC by phorbol-12-myristate-13-acetate (PMA, 10 nM) increased CREB phosphorylation and total StAR (tot-StAR) protein expression. However, induction of StAR protein by PMA did not result in the expected concomitant increase in steroids because PKC failed to phosphorylate StAR, the biologically active form of the protein. However, in conjunction with PMA, minor increases in PKA activity using submaximal doses of (Bu)2cAMP (0.05-0.1 mM; a concentration range insufficient for induction of StAR), were able to stimulate dramatic increases in both phospho-StAR (P-StAR) and steroid production. Human chorionic gonadotropin stimulation also resulted in a further enhancement in P-StAR and progesterone production when added to PMA-treated MA-10 cells. Similar results for tot-StAR and P-StAR expression were observed in primary cultures of immature rat Leydig cells treated with PMA and submaximal doses of (Bu)2cAMP. In summary, the present study demonstrates that basal activities of both PKC and PKA play important roles in the constitutive steroidogenic characteristics of R2C cells. This study also demonstrates for the first time a role for PMA-induced PKC in StAR protein regulation and the requirement for submaximal doses of cAMP to produce steroids in Leydig cells.

Estradiol regulation of progesterone synthesis in the brain

“…In peripheral steroidogenic tissues, steroid production is regulated by gonadotropins released from the anterior pituitary that activate G protein-coupled receptor (GPCR) that induce the phosphorylation of protein kinases (Alila et al, 1990;Rao et al, 1987;Shalem et al, 1988;Shemesh et al, 1984) and lead to activation of enzyme activity and transcription (Momoi et al, 1992;Waterman, 1994). Phosphorylation of StAR and PBR are part of a rapid phase of steroidogenesis that does not involve gene transcription (Arakane et al, 1997;Jo et al, 2005;Papadopoulos et al, 1997a;Papadopoulos et al, 1997b;Stocco et al, 2005).…”

Delineation of Type I Protein Kinase A-selective Signaling Events Using an RI Anchoring Disruptor

“…This process is facilitated by the steroid acute response protein (StAR), which is a PKA substrate. StAR phosphorylation is a marker for acute hormonal regulation of steroid biosynthesis by adrenocorticotropic hormone (ACTH) or luteinizing hormone in adrenocortical cells and ovarian theca cells, respectively (47,48). Type I PKA anchoring to the mitochondria via its interaction with PAP7 is thought to be necessary for this regulation (17).…”

Inhibition of Cyclooxygenase-2 Down-regulates Aromatase Activity and Decreases Proliferation of Leydig Tumor Cells

“…Several observations indicate that constitutively active mutants of LHR could be involved in Leydig cell transformation (55). Indeed, the constitutive activation of the cAMP-PKA pathway in R2C Leydig tumor cells allows for a steroidogenic phenotype (56), which could contribute to aromatase overexpression in Leydig cell tumors. For these reasons we believe that other molecular mechanisms could be involved in constitutive activation of CREB-dependent aromatase activation.…”