Concerted regulation of steroidogenic acute regulatory gene expression by luteinizing hormone and insulin (or insulin-like growth factor I) in primary cultures of porcine granulosa-luteal cells

The steroidogenic acute regulatory (StAR) protein is indispensable for maxi mal trophic hormone-stimulated steroidogenesis by the adrenal gland, testis , and ovary. Recently, our laboratory developed an in vitro primary culture system of porcine granulosa-luteal cells that retain responsiveness to LH and show LH and insulin [or insulin-like growth factor (IGF-I)] synergy in stimulating StAR messenger RNA accumulation. Here, we examine the mechanism s subserving this LH-insulin (IGF-I) augmentation. We corroborate LH's ampl ification of insulin as well as IGF-I-stimulated granulosa-luteal cell prog esterone and cAMP accumulation (P < 0.001). Insulin or ICF-I elevated LH re ceptor transcript accumulation, and LH did not alter this effect. To determ ine the hormonal responsiveness of StAR promoter, truncated regions of the -1423 to +130 bp upstream sequence of the porcine gene were ligated into a firefly luciferase reporter plasmid. Transient transfection of the StAR pla smid containing the full-length porcine 5'-flanking region of StAR (pStAR14 23/luc) showed superadditive stimulation by LH and insulin or IGF-I after 2 4 h. LH, but not insulin or IGF-I alone, stimulated pStAR1423/luc activity. Deletion of the proximal putative steroidogenic factor-1 (-48 to -41) site abolished hormonally driven StAR promoter activity. A stable cAMP analog, 8-bromo-cAMP (1 mM), and insulin/IGF-I also evoked su praadditive StAR promoter expression. To further explore the role of cAMP i n LH-insulin (or IGF-I) actions, we cotransfected a Rous sarcoma virus (RSV )-driven minigene encoding the heat-stable inhibitor of the cAMP-dependent protein kinase (RSV/ PKI) or a mutant plasmid (RSV/PKImut) along with the p StAR1423/luc promoter construct. Cotransfection of PKI, but not PKImut, wit h pStAR1423/luc significantly attenuated LH's stimulation of luciferase act ivity and also reduced the magnitude of the transcriptional amplification e xerted by LH and insulin or IGF-I. In corollary analyses of the protein kin ase A (PKA) pathway, cotransfection of full-length pStAR1423/luc and a comp lementary DNA encoding a constitutively activated PKA catalytic subunit ele vated basal and insulin (or IGF-I)-stimulated StAR promoter expression. LH and insulin (or IGF-I) also augmented steady state StAR transcript levels, as assessed by homologous RT-PCR, and StAR protein concentrations, as evalu ated by Western blotting. Together, these investigations document a significant role for insulin or I GF-I in enhancing LH-stimulated progesterone and cAMP biosynthesis and endo genous StAR message and protein accumulation and in augmenting cAMP-PKA-dep endent transcriptional activation of the exogenous StAR promoter.