Balasubramanian K, Lavoie H A, Garmey J C, Stocco D M, Veldhuis J D
Division of Endocrinology and Metabolism, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Endocrinology. 1997 Jan;138(1):433-9. doi: 10.1210/endo.138.1.4894.
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.
胆固醇从线粒体外膜转运至内膜（在此发生侧链裂解形成孕烯醇酮）是类固醇生成调节中的关键事件，最近已证明该过程由类固醇生成急性调节蛋白（StAR）介导。我们通过逆转录聚合酶链反应（RT-PCR）生成了部分猪StAR互补DNA（280 bp），并使用相应的反义核糖探针来量化促卵泡激素（FSH）和胰岛素样生长因子I（IGF-I）对激素反应性猪颗粒细胞中StAR基因表达的调控，这些细胞通常对这两种主要的卵泡内调节因子表现出协同的类固醇生成刺激作用。实施核糖核酸酶保护试验以研究FSH（100 ng/ml）、IGF-I（100 ng/ml）以及FSH加IGF-I对无血清培养的颗粒细胞中StAR信使核糖核酸积累的作用时间进程。单独用FSH（1.6倍）或IGF-I（2.7倍）处理在48小时时对StAR信息积累有微小但一致的刺激作用（每个泳道以18S核糖体RNA校正），而只有IGF-I刺激StAR蛋白表达（通过蛋白质印迹法评估至少为6倍）。值得注意的是，FSH加IGF-I的联合作用具有强烈的协同性，在24小时时就已显著，48小时时达到最大（P < 0.001）。蛋白激酶A激动剂8-溴腺苷3',5'-环磷酸腺苷（8-溴-cAMP）（1 mM）单独作用在48小时时使StAR信息增加3.5倍，StAR蛋白表达增加超过3.7倍。IGF-I与FSH或8-溴-cAMP的组合使StAR信息积累产生26至40倍（P < 0.001）协同性升高。StAR蛋白在由IGF-I与FSH或8-溴-cAMP驱动时也表现出类似的协同表达模式，即增加大于56至60倍。总之，两种不同的第一信使调节分子FSH和IGF-I协同相互作用，在未成熟（猪）颗粒细胞的无血清单层培养中诱导StAR信使核糖核酸和蛋白表达的放大。
