Hosokawa K, Dantes A, Schere-Levy C, Barash A, Yoshida Y, Kotsuji F, Vlodavsky I, Amsterdam A
Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, Israel.
Endocrinology. 1998 Nov;139(11):4679-87. doi: 10.1210/endo.139.11.6279.
We have established immortalized human granulosa cells by triple transfection of primary cells obtained from in vitro fertilization patients with SV40 DNA, Ha-ras oncogene, and a temperature sensitive (ts) mutant of the tumor suppressor gene p53 (p53val135). Forty-one clones were isolated, and their steroidogenic responses were analyzed. While all the cell lines proliferate rapidly and show only traces of progesterone production, upon stimulation with 50 microM of forskolin (FK), which elevates intracellular cAMP, they become steroidogenic as evidenced by progesterone production. The steroidogenic response of the cell lines was stable even after 20 generations and several cycles of freezing and thawing. A highly responsive cell line (HO-23) was further examined for characteristics of the steroidogenic response. Cells stimulated with FK and 8-Br-cAMP produced high levels of pregnenolone, progesterone, and 20alpha-hydroxy-4-pregnen-3-one (20alpha-OH-progesterone) comparable with amounts produced by highly differentiated primary human granulosa-luteal cells. Hydrocortisone and dexamethasone highly augment the cAMP-stimulated progesterone production, whereas testosterone and PRL enhanced cAMP-induced progesterone synthesis only moderately. Estradiol, insulin-like growth factor I, and insulin showed no significant effect on cAMP-induced steroidogenesis. The phorbol ester TPA, and basic fibroblast growth factor, dramatically suppress cAMP-induced production of progesterone, whereas bovine corneal endothelial cell ECM (BCE/ECM) enhanced cAMP-induced progesterone and antagonized basic fibroblast growth factor suppression of cAMP-induced steroidogenesis. Steroidogenic factor 1 (Ad4BP/SF-1) was expressed in control cells, and its expression was augmented by FK, whereas the steroidogenic acute regulatory protein showed low expression in the nonstimulated cells but was clearly elevated upon cAMP stimulation and was slightly decreased by TPA in cAMP-stimulated cells. Expression of the electron carrier adrenodoxin (ADX), which is a part of the cytochrome P450scc enzyme system, was very low in nonstimulated cells but was dramatically elevated in FK- and 8-Br-cAMP-stimulated cells, whereas no reduction of ADX was evident in cells costimulated with FK and TPA. Immunocytochemical studies revealed a weak staining of ADX in mitochondria of nonstimulated cells and intensive staining in highly clustered mitochondria of FK- or 8-Br-cAMP-stimulated cells. Only moderate reduction in ADX staining was evident in cells costimulated with FK and TPA. These unique cell lines can provide a useful model for the investigation of induced steroidogenesis in human granulosa cells.
我们通过将从体外受精患者获取的原代细胞与SV40 DNA、Ha-ras癌基因以及肿瘤抑制基因p53的温度敏感（ts）突变体（p53val135）进行三重转染，建立了永生化人颗粒细胞。分离出41个克隆，并分析了它们的类固醇生成反应。虽然所有细胞系增殖迅速且仅显示微量的孕酮生成，但在用50 microM的福司可林（FK）刺激后，细胞内cAMP升高，它们开始产生类固醇，这通过孕酮生成得以证明。即使经过20代以及几个冻融循环，细胞系的类固醇生成反应仍然稳定。对一个高反应性细胞系（HO-23）进一步检测其类固醇生成反应的特征。用FK和8-溴-cAMP刺激的细胞产生高水平的孕烯醇酮、孕酮和20α-羟基-4-孕烯-3-酮（20α-OH-孕酮），与高度分化的原代人颗粒黄体细胞产生的量相当。氢化可的松和地塞米松极大地增强了cAMP刺激的孕酮生成，而睾酮和催乳素仅适度增强了cAMP诱导的孕酮合成。雌二醇、胰岛素样生长因子I和胰岛素对cAMP诱导的类固醇生成无显著影响。佛波酯TPA和碱性成纤维细胞生长因子显著抑制cAMP诱导的孕酮生成，而牛角膜内皮细胞细胞外基质（BCE/ECM）增强了cAMP诱导的孕酮生成，并拮抗碱性成纤维细胞生长因子对cAMP诱导的类固醇生成的抑制作用。类固醇生成因子1（Ad4BP/SF-1）在对照细胞中表达，其表达被FK增强，而类固醇生成急性调节蛋白在未刺激的细胞中表达较低，但在cAMP刺激后明显升高，并且在cAMP刺激的细胞中被TPA轻微降低。电子载体肾上腺皮质铁氧还蛋白（ADX）是细胞色素P450scc酶系统的一部分，其在未刺激的细胞中表达非常低，但在FK和8-溴-cAMP刺激的细胞中显著升高，而在用FK和TPA共同刺激的细胞中未观察到ADX的明显减少。免疫细胞化学研究显示，未刺激细胞的线粒体中ADX染色较弱，而在FK或8-溴-cAMP刺激的细胞高度聚集的线粒体中染色强烈。在用FK和TPA共同刺激的细胞中，ADX染色仅适度减少。这些独特的细胞系可为研究人颗粒细胞中诱导的类固醇生成提供有用的模型。
