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Titolo:
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
Autore:
Sekar, N; Lavoie, HA; Veldhuis, JD;
Indirizzi:
Univ Virginia, Hlth Sci Ctr, Dept Internal Med, Div Endocrinol & Metab,NIH,Specialized Cooperat C, Charlottesville, VA 22908 USA Univ Virginia Charlottesville VA USA 22908 Charlottesville, VA 22908 USA Univ S Carolina, Sch Med, Dept Neurosci & Cell Biol, Columbia, SC 29208 USA Univ S Carolina Columbia SC USA 29208 & Cell Biol, Columbia, SC 29208 USA
Titolo Testata:
ENDOCRINOLOGY
fascicolo: 11, volume: 141, anno: 2000,
pagine: 3983 - 3992
SICI:
0013-7227(200011)141:11<3983:CROSAR>2.0.ZU;2-S
Fonte:
ISI
Lingua:
ENG
Soggetto:
FOLLICLE-STIMULATING-HORMONE; SIDE-CHAIN CLEAVAGE; LEYDIG TUMOR-CELLS; MESSENGER-RIBONUCLEIC-ACID; PROTEIN STAR GENE; POLYCYSTIC-OVARY-SYNDROME; ELEMENT-BINDING PROTEIN; NUCLEAR RECEPTOR SF-1; C/EBP-BETA; CATALYTIC SUBUNIT;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
62
Recensione:
Indirizzi per estratti:
Indirizzo: Veldhuis, JD Univ Virginia, Sch Med, Dept Internal Med, Div Endocrine, Box202, Charlottesville, VA 22908 USA Univ Virginia Box 202 Charlottesville VA USA 22908 22908 USA
Citazione:
N. Sekar et al., "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", ENDOCRINOL, 141(11), 2000, pp. 3983-3992

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 LHand show LH and insulin [or insulin-like growth factor (IGF-I)] synergy instimulating 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 ICF-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 afirefly 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-dependentprotein 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. LHand 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.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 29/09/20 alle ore 07:40:03