Cheng C Y, Hornsby P J
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912.
Endocrinology. 1992 May;130(5):2883-9. doi: 10.1210/endo.130.5.1572301.
In order to elucidate mechanisms for the loss of expression of 11 beta-hydroxylase and 21-hydroxylase, induction of these genes in long-term cultures of bovine adrenocortical cells was reassessed and compared with induction of 17 alpha-hydroxylase. We previously showed that both 11 beta- and 21-hydroxylases require insulin-like growth factor-I (IGF-I) as well as cAMP for induction; these are the only factors needed by primary cultures. Cells at population doubling level 10 grown on fibronectin-coated polystyrene dishes and incubated with cholera toxin and IGF-I did not express 11 beta-hydroxylase and 21-hydroxylase. They showed a truncated steroidogenic pathway, converting 25-hydroxycholesterol to some 11-deoxycortisol but little cortisol. However, when population doubling level 10 cells were grown for 5 days in extracellular matrix Matrigel, cholera toxin and IGF-I induced a complete steroidogenic pathway to cortisol. Northern blotting also showed that expression of 11 beta-hydroxylase messenger RNA (mRNA) after cholera toxin/IGF-I induction was observed only in cultures grown in Matrigel and was undetectable in cultures grown on plastic. 21-Hydroxylase mRNA was observed in cultures grown on plastic but was greatly enhanced by Matrigel; however, 17 alpha-hydroxylase mRNA was induced to a similar extent with and without Matrigel. In other middle passage cultures, whether grown as mass cultures, SV40 T antigen-transfected clones, or normal (nontransfected) clones, cells did not express 11 beta-hydroxylase except when grown in Matrigel; 21-hydroxylase was low and expression was enhanced by Matrigel, whereas 17 alpha-hydroxylase expression was unaffected. As previously determined, in late-passage cells and clones only side-chain cleavage enzyme and 3 beta-hydroxysteroid dehydrogenase activities were detected and 17 alpha-hydroxylase was not expressed. In such cells 11 beta-hydroxylase and 21-hydroxylase were also not expressed, even in the presence of Matrigel. Thus, prior to the previously described loss of expression of 17 alpha-hydroxylase, Matrigel permits the cholera toxin/IGF-I-induced expression of a complete steroidogenic pathway in bovine adrenocortical cells in long-term culture.
为了阐明11β-羟化酶和21-羟化酶表达缺失的机制，我们重新评估了牛肾上腺皮质细胞长期培养中这些基因的诱导情况，并与17α-羟化酶的诱导情况进行了比较。我们之前表明，11β-羟化酶和21-羟化酶的诱导都需要胰岛素样生长因子-I（IGF-I）以及cAMP；这些是原代培养所需的唯一因子。在纤连蛋白包被的聚苯乙烯培养皿上生长并与霍乱毒素和IGF-I一起孵育的群体倍增水平为10的细胞不表达11β-羟化酶和21-羟化酶。它们显示出一条截断的类固醇生成途径，将25-羟胆固醇转化为一些11-脱氧皮质醇，但皮质醇很少。然而，当群体倍增水平为10的细胞在细胞外基质基质胶中生长5天时，霍乱毒素和IGF-I诱导出一条完整的生成皮质醇的类固醇生成途径。Northern印迹分析还表明，霍乱毒素/IGF-I诱导后11β-羟化酶信使核糖核酸（mRNA）的表达仅在基质胶中生长的培养物中观察到，而在塑料培养物中未检测到。在塑料培养物中生长的培养物中观察到21-羟化酶mRNA，但基质胶可使其显著增强；然而，有无基质胶时17α-羟化酶mRNA的诱导程度相似。在其他中期传代培养物中，无论是作为大规模培养物、SV40 T抗原转染克隆还是正常（未转染）克隆生长，细胞都不表达11β-羟化酶，除非在基质胶中生长；21-羟化酶水平较低，基质胶可增强其表达，而17α-羟化酶的表达不受影响。如之前所确定的，在后期传代细胞和克隆中仅检测到侧链裂解酶和3β-羟类固醇脱氢酶活性，且不表达17α-羟化酶。在这类细胞中，即使存在基质胶，也不表达11β-羟化酶和21-羟化酶。因此，在之前描述的17α-羟化酶表达缺失之前，基质胶可使霍乱毒素/IGF-I诱导的长期培养的牛肾上腺皮质细胞中完整的类固醇生成途径得以表达。
