title: 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.
abstract: The steroidogenic acute regulatory (StAR) protein is indispensable for maximal 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 mechanisms subserving this LH-insulin (IGF-I) augmentation. We corroborate LH's amplification of insulin as well as IGF-I-stimulated granulosa-luteal cell progesterone and cAMP accumulation (P < 0.001). Insulin or IGF-I elevated LH receptor transcript accumulation, and LH did not alter this effect. To determine 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 plasmid containing the full-length porcine 5'-flanking region of StAR (pStAR1423/luc) showed superadditive stimulation by LH and insulin or IGF-I after 24 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 supraadditive StAR promoter expression. To further explore the role of cAMP in 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 pStAR1423/luc promoter construct. Cotransfection of PKI, but not PKImut, with pStAR1423/luc significantly attenuated LH's stimulation of luciferase activity and also reduced the magnitude of the transcriptional amplification exerted by LH and insulin or IGF-I. In corollary analyses of the protein kinase A (PKA) pathway, cotransfection of full-length pStAR1423/luc and a complementary DNA encoding a constitutively activated PKA catalytic subunit elevated 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 evaluated by Western blotting. Together, these investigations document a significant role for insulin or IGF-I in enhancing LH-stimulated progesterone and cAMP biosynthesis and endogenous StAR message and protein accumulation and in augmenting cAMP-PKA-dependent transcriptional activation of the exogenous StAR promoter.
authors.affiliation: Department of Internal Medicine, National Institutes of Health Specialized Cooperative Center in Reproductive Research, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
authors.forename: N
authors.initials: N
authors.lastname: Sekar
authors.forename: H A
authors.initials: HA
authors.lastname: Lavoie
authors.forename: J D
authors.initials: JD
authors.lastname: Veldhuis
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004357:Drug Synergism; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D006107:Granulosa Cells / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D008184:Luteal Cells / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000494:pharmacology*; D010750:Phosphoproteins / Q000235:genetics*; D011374:Progesterone / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011974:Receptors, LH / Q000235:genetics; D011993:Recombinant Fusion Proteins; D013552:Swine; D014162:Transfection; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: D007328:Insulin; D010750:Phosphoproteins; D012333:RNA, Messenger; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D000098682:Steroidogenic Acute Regulatory Protein; D015124:8-Bromo Cyclic Adenosine Monophosphate; D011374:Progesterone; D007334:Insulin-Like Growth Factor I; D007986:Luteinizing Hormone; D000242:Cyclic AMP; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases