Regulation of porcine granulosa cell steroidogenic acute regulatory protein (StAR) by insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist.
The transfer of cholesterol from the outer to the inner mitochondrial membrane, where side-chain cleavage occurs to form pregnenolone, is a crucial event in the regulation of steroidogenesis and recently has been demonstrated to be mediated by steroidogenic acute regulatory protein (StAR). We generated a partial porcine StAR complementary DNA (280 bp) by RT-PCR and used the corresponding antisense riboprobe to quantify the control of StAR gene expression by FSH and insulin-like growth factor I (IGF-I) in hormonally responsive swine granulosa cells, which typically manifest synergistic steroidogenic stimulation by these two dominant intrafollicular regulators. RNase protection assays were implemented to investigate the time course of the actions of FSH (100 ng/ml), IGF-I (100 ng/ml), and FSH plus IGF-I on StAR messenger RNA accumulation in serum-free cultures granulosa cells. Treatment with FSH (1.6-fold) or IGF-I (2.7-fold) alone had a small but consistent stimulatory effect on StAR message accumulation (corrected for 18S ribosomal RNA in each lane) at 48 h, whereas only IGF-I stimulated StAR protein expression (at least 6-fold as assessed by Western blot). Notably, the combined effect of FSH plus IGF-I was strongly synergistic and already significant by 24 h and maximal at 48 h (P < 0.001). Protein kinase A agonist, 8-bromoadenosine 3',5'-cAMP (8-bromo-cAMP) (1 mM) alone elicited a 3.5-fold increase in StAR message and more than 3.7-fold increase in StAR protein expression by 48 h. The combination of IGF-I and FSH or 8-bromo-cAMP evoked a 26- to 40-fold (P < 0.001) synergistic rise in StAR message accumulation. StAR protein also showed a similar synergistic pattern of expression driven by IGF-I and FSH or 8-bromo-cAMP, namely a greater than 56- to 60-fold increase. In summary, two distinct first messenger regulatory molecules, FSH and IGF-I, interact synergistically to induce amplification of StAR messenger RNA and protein expression in serum-free monolayer cultures of immature (swine) granulosa cells.
Balasubramanian
K
K
Division of Endocrinology and Metabolism, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Lavoie
H A
HA
Garmey
J C
JC
Stocco
D M
DM
Veldhuis
J D
JD
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000819:agonists*; D004357:Drug Synergism; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006107:Granulosa Cells / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D013552:Swine; D000098682:Steroidogenic Acute Regulatory Protein
D010750:Phosphoproteins; D012333:RNA, Messenger; D000098682:Steroidogenic Acute Regulatory Protein; D015124:8-Bromo Cyclic Adenosine Monophosphate; D007334:Insulin-Like Growth Factor I; D005640:Follicle Stimulating Hormone; D017868:Cyclic AMP-Dependent Protein Kinases