Shi D L, Savona C, Gagnon J, Cochet C, Chambaz E M, Feige J J
Laboratoire de Biochimie des Régulations Cellulaires Endocrines, Institut National de la Santé et de la Recherche Medicale, Grenoble, France.
J Biol Chem. 1990 Feb 15;265(5):2881-7.
Adrenocortical cell major secreted protein was purified from the conditioned medium of primary cultures of bovine adrenocortical (BAC) cells. Immunochemical analysis and N-terminal sequencing of the purified protein identified it to alpha 2-macroglobulin (alpha 2-M). It appeared that 15 out of the 17 N-terminal amino acids were conserved between adrenocortical cell major secreted protein and human alpha 2-M. Study of alpha 2-M production by BAC cells revealed that its secretion was stimulated severalfold by transforming growth factor-beta 1 (TGF-beta 1). The stimulation occurred in a time-dependent (reaching a plateau at 24 h) and dose-dependent (ED50 = 0.1 ng/ml TGF-beta 1) manner. It was blocked when BAC cells were exposed to 5,6-dichlorobenzimidazole riboside, a potent inhibitor of RNA polymerase II, suggesting that TGF-beta 1 acts as an activator of alpha 2-M gene expression at the transcriptional level. Northern blot analysis confirmed that the alpha 2-M mRNA level was increased (4-fold) in BAC cells following TGF-beta 1 treatment. TGF-beta 2, TGF-beta 1,2, basic fibroblast growth factor, and angiotensin II also appeared able to stimulate alpha 2-M secretion in BAC cells, whereas adrenocorticotropin was strongly inhibitory. Given the previous reports that TGF-beta 1 is a potent inhibitor of adrenocortical steroidogenesis (Feige J.J., Cochet, C., Rainey, W.E., Madani, C., and Chambaz, E. M. (1987) J. Biol. Chem. 262, 13491-13495) and that alpha 2-M is a TGF-beta 1-binding protein, these observations suggest that alpha 2-M may play an important role in conjunction with hormones and growth factors in the homeostatic regulation of adrenocortical functions.
从牛肾上腺皮质（BAC）细胞原代培养的条件培养基中纯化出肾上腺皮质细胞主要分泌蛋白。对纯化蛋白进行免疫化学分析和N端测序，确定其为α2-巨球蛋白（α2-M）。肾上腺皮质细胞主要分泌蛋白与人类α2-M之间17个N端氨基酸中有15个是保守的。对BAC细胞产生α2-M的研究表明，转化生长因子-β1（TGF-β1）可使其分泌增加数倍。这种刺激呈时间依赖性（24小时达到平台期）和剂量依赖性（ED50 = 0.1 ng/ml TGF-β1）。当BAC细胞暴露于RNA聚合酶II的强效抑制剂5,6-二氯苯并咪唑核糖苷时，刺激作用被阻断，这表明TGF-β1在转录水平上作为α2-M基因表达的激活剂发挥作用。Northern印迹分析证实，TGF-β1处理后BAC细胞中α2-M mRNA水平升高（4倍）。TGF-β2、TGF-β1,2、碱性成纤维细胞生长因子和血管紧张素II似乎也能够刺激BAC细胞中α2-M的分泌，而促肾上腺皮质激素则具有强烈的抑制作用。鉴于先前有报道称TGF-β1是肾上腺皮质类固醇生成的强效抑制剂（Feige J.J., Cochet, C., Rainey, W.E., Madani, C., and Chambaz, E. M. (1987) J. Biol. Chem. 262, 13491-13495），且α2-M是一种TGF-β1结合蛋白，这些观察结果表明α2-M可能在与激素和生长因子共同作用下，在肾上腺皮质功能的稳态调节中发挥重要作用。
