Sekar N, Lavoie H A, Veldhuis J D
Department of Internal Medicine, National Institutes of Health Specialized Cooperative Center in Reproductive Research, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Endocrinology. 2000 Nov;141(11):3983-92. doi: 10.1210/endo.141.11.7763.
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.
类固醇生成急性调节（StAR）蛋白对于肾上腺、睾丸和卵巢在促性腺激素最大刺激下的类固醇生成必不可少。最近，我们实验室开发了一种猪颗粒黄体细胞的体外原代培养系统，该系统保留了对促黄体生成素（LH）的反应性，并在刺激StAR信使核糖核酸积累方面显示出LH与胰岛素[或胰岛素样生长因子（IGF-I）]的协同作用。在此，我们研究了支持这种LH-胰岛素（IGF-I）增强作用的机制。我们证实了LH对胰岛素以及IGF-I刺激的颗粒黄体细胞孕酮和环磷酸腺苷（cAMP）积累的放大作用（P<0.001）。胰岛素或IGF-I提高了LH受体转录物的积累，而LH并未改变这种作用。为了确定StAR启动子的激素反应性，将猪基因-1423至+130 bp上游序列的截短区域连接到萤火虫荧光素酶报告质粒中。含有StAR全长猪5'-侧翼区的StAR质粒（pStAR1423/luc）的瞬时转染显示，24小时后LH与胰岛素或IGF-I具有超加性刺激作用。单独的LH刺激pStAR1423/luc活性，但胰岛素或IGF-I则无此作用。近端假定的类固醇生成因子-1（-48至-41）位点的缺失消除了激素驱动的StAR启动子活性。一种稳定的cAMP类似物8-溴-cAMP（1 mM）以及胰岛素/IGF-I也引起了超加性的StAR启动子表达。为了进一步探讨cAMP在LH-胰岛素（或IGF-I）作用中的作用，我们将编码cAMP依赖性蛋白激酶热稳定抑制剂的劳氏肉瘤病毒（RSV）驱动的小基因（RSV/PKI）或突变体质粒（RSV/PKImut）与pStAR1423/luc启动子构建体共转染。PKI与pStAR1423/luc共转染（而非PKImut）显著减弱了LH对荧光素酶活性的刺激，并降低了LH与胰岛素或IGF-I施加的转录放大程度。在蛋白激酶A（PKA）途径的相关分析中，全长pStAR1423/luc与编码组成型激活的PKA催化亚基的互补DNA共转染提高了基础以及胰岛素（或IGF-I）刺激的StAR启动子表达。通过同源逆转录-聚合酶链反应（RT-PCR）评估，LH与胰岛素（或IGF-I）也提高了StAR转录物的稳态水平，通过蛋白质印迹法评估，它们还提高了StAR蛋白浓度。总之，这些研究证明胰岛素或IGF-I在增强LH刺激的孕酮和cAMP生物合成以及内源性StAR信息和蛋白质积累，以及增强cAMP-PKA依赖性的外源性StAR启动子转录激活方面具有重要作用。
