Document Detail

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.
MedLine Citation:
PMID:  11089528     Owner:  NLM     Status:  MEDLINE    
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.
N Sekar; H A Lavoie; J D Veldhuis
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Endocrinology     Volume:  141     ISSN:  0013-7227     ISO Abbreviation:  Endocrinology     Publication Date:  2000 Nov 
Date Detail:
Created Date:  2000-11-29     Completed Date:  2000-12-22     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  0375040     Medline TA:  Endocrinology     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  3983-92     Citation Subset:  AIM; IM    
Department of Internal Medicine, National Institutes of Health Specialized Cooperative Center in Reproductive Research, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
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MeSH Terms
8-Bromo Cyclic Adenosine Monophosphate / pharmacology
Cells, Cultured
Cyclic AMP / metabolism
Cyclic AMP-Dependent Protein Kinases / genetics,  metabolism
Drug Synergism
Gene Expression Regulation / drug effects*
Granulosa Cells / metabolism*
Insulin / pharmacology*
Insulin-Like Growth Factor I / pharmacology
Luciferases / genetics
Luteal Cells / metabolism*
Luteinizing Hormone / pharmacology*
Phosphoproteins / genetics*
Progesterone / metabolism
Promoter Regions, Genetic
RNA, Messenger / metabolism
Receptors, LH / genetics
Recombinant Fusion Proteins
Grant Support
Reg. No./Substance:
0/Phosphoproteins; 0/RNA, Messenger; 0/Receptors, LH; 0/Recombinant Fusion Proteins; 0/steroidogenic acute regulatory protein; 11061-68-0/Insulin; 23583-48-4/8-Bromo Cyclic Adenosine Monophosphate; 57-83-0/Progesterone; 60-92-4/Cyclic AMP; 67763-96-6/Insulin-Like Growth Factor I; 9002-67-9/Luteinizing Hormone; EC 1.13.12.-/Luciferases; EC AMP-Dependent Protein Kinases

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