Regulation of steroidogenic acute regulatory (StAR) protein and evidence that constitutive steroidogenesis in ovine large luteal cells is mediated by tonically active protein kinase A

Abstract

Progesterone is required for maintenance of pregnancy in all-mammalian species and is produced by the corpus luteum (CL) during estrous/menstrual cycles and early pregnancy. The rate-limiting step in steroidogenesis and the step most acutely regulated by second messengers is the transport of cholesterol across the mitochondrial membrane, a process mediated by the steroidogenic acute regulatory protein (StAR). Factors activating the cyclic AMP (cAMP)/protein kinase-A (PKA) pathway stimulate steroidogenesis by increasing expression, and activity of StAR via phosphorylation. The CL contains two steroidogenic cell types; large cells which have high constitutive progesterone secretion, and small cells that have low basal progesterone production, which increases in response to stimulation by tropic hormones. To determine if tonically active PKA causes constitutive steroidogenesis by large cells, purified large and small ovine luteal cells were treated with a specific PKA inhibitor (PKI) and the adenylate cylase activator forskolin. Effects on progesterone and cAMP production, and the expression and phosphorylation state of StAR, were quantified. Additionally, the mechanism of StAR-associated cholesterol transport may involve the peripheral-type benzodiazepine receptor (PBR). Bioluminescence resonance energy transfer (BRET) was used to test StAR/PBR interactions. Treating small cells with PKI prevented forskolin-induced increases in progesterone secretion without affecting cAMP production. In large cells, basal secretion of progesterone and relative StAR phosphorylation were significantly decreased following PKI treatment, indicating the presence of tonically active PKA. There was a significant correlation between StAR phosphorylation and progesterone secretion, but no correlation between the quantity of StAR and secretion of progesterone. This indicates that phosphorylation, but not changes in quantity, is the primary effect of PKA to regulate StAR's activity. No StAR/PBR interactions were detected by BRET analysis, although data were obtained indicating that PBR aggregates in the mitochondrial membrane and may form a pore through which cholesterol can pass. In summary, a novel assay for quantifying concentrations and relative phosphorylation states of StAR was developed and used to provide evidence that constitutive steroidogenesis in ovine large luteal cells is mediated by tonically active PKA. Additionally, it appears that PBR aggregates in the mitochondrial membrane, but no evidence was obtained that StAR and PBR interact.