Regulation of steroidogenesis in NCI-H295 cells: a cellular model of the human fetal adrenal.
Regulation of steroidogenesis in NCI-H295 cells: a cellular model of the human fetal adrenal.
复制标题
DOI：
10.1210/mend.7.3.8387159
复制
发表时间：
1993-03
影响因子：
--
通讯作者：
B. Staels;D. Hum;W. Miller
中科院分区：
文献类型：
--
作者：
B. Staels;D. Hum;W. Miller
NCI-H295是最近描述的人肾上腺皮质癌细胞系，其产生多种类固醇激素。我们试图确定这些细胞中的类固醇生成是否采用与正常肾上腺类固醇生成中使用的酶相同的酶，以及编码这些酶的基因是否对细胞内第二信使的蛋白激酶-A和-C途径的激活剂表现出特征性反应。北方印迹显示NCI-H295细胞含有三种关键类固醇生成酶细胞色素P450 SCC、细胞色素P450 c17和细胞色素P450 c21的丰富mRNA。这些mRNA以时间和剂量依赖性的方式积累，以响应8-溴-cAMP（8Br-cAMP），毛喉素，霍乱毒素和3-异丁基-1-甲基黄嘌呤，所有蛋白激酶-A途径的激活剂。核运行的分析和放线菌素D的转录抑制实验表明，cAMP调节所有三个基因的表达主要在转录水平。用放线菌酮抑制蛋白质合成并不能阻止cAMP诱导的P450 SCC或P450 c17 mRNA的积累，但确实抑制了P450 c21 mRNA的积累，这表明cAMP是通过依赖于蛋白质合成的机制来促进P450 c21 mRNA的积累。用佛波酯刺激蛋白激酶C通路降低P450 SCC和P450 c17 mRNA，但刺激P450 c21 mRNA的积累。RNA酶保护实验、北方印迹杂交和逆转录-聚合酶链反应显示NCI-H295细胞表达由P450 c11 B1基因编码的11 β-羟化酶（P450 c11 beta）和由P450 c11 B2基因编码的醛固酮合成酶（P450 c11 AS）。8Br-cAMP以相似的动力学增加这两种mRNA的丰度，在约24 h后两者的积累最大。NCI-H295细胞还含有芳香化酶和胰岛素样生长因子-II的mRNA。8 Br-cAMP使芳香化酶mRNA丰度增加，IGF-II mRNA丰度降低。这些研究表明，NCI-H295细胞表达人类肾上腺类固醇生成所需的大多数酶，并且编码这些酶的基因以与人类肾上腺相似的方式响应第二信使途径的刺激。NCI-H295细胞似乎是研究人肾上腺类固醇生成的分子调控的良好模型。
NCI-H295 is a recently described human adrenocortical carcinoma cell line that makes a variety of steroid hormones. We sought to determine if steroidogenesis in these cells employs the same enzymes as those used in normal adrenal steroidogenesis, and if the genes encoding those enzymes exhibit characteristic responsiveness to activators of the protein kinase-A and -C pathways of intracellular second messengers. Northern blots show that NCI-H295 cells contain abundant mRNAs for three key steroidogenic enzymes, cytochrome P450scc, cytochrome P450c17, and cytochrome P450c21. These mRNAs accumulated in a time- and dose-dependent fashion in response to 8-bromo-cAMP (8Br-cAMP), forskolin, cholera toxin, and 3-isobutyl-1-methylxanthine, all activators of the protein kinase-A pathway. Nuclear run-on assays and actinomycin-D transcriptional inhibition experiments show that cAMP regulates the expression of all three genes primarily at the transcriptional level. Inhibition of protein synthesis with cycloheximide did not prevent the cAMP-induced accumulation of P450scc or P450c17 mRNAs, but did inhibit accumulation of P450c21 mRNA, suggesting that cAMP is acting through a mechanism dependent on protein synthesis to promote accumulation of P450c21 mRNA. Stimulation of the protein kinase-C pathway with phorbol ester decreased P450scc and P450c17 mRNAs, but stimulated the accumulation of P450c21 mRNA. RNase protection experiments, Northern blot hybridizations, and reverse transcription-polymerase chain reaction show that NCI-H295 cells express both the 11 beta-hydroxylase (P450c11 beta) encoded by the P450c11B1 gene and the aldosterone synthetase (P450c11AS) encoded by the P450c11B2 gene. 8Br-cAMP increased the abundance of both of these mRNAs with similar kinetics, with maximal accumulation of both after about 24 h. NCI-H295 cells also contain the mRNAs for aromatase and insulin-like growth factor-II. 8Br-cAMP increased the abundance of aromatase mRNA and decreased the abundance of IGF-II mRNA. These studies show that NCI-H295 cells express most of the enzymes needed for human adrenal steroidogenesis, and that the genes encoding these enzymes respond to stimulation of second messenger pathways in a manner similar to that of human adrenals. NCI-H295 cells appear to be a good model for studying the molecular regulation of human adrenal steroidogenesis.