TISSUE-SPECIFIC, CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-INDUCED, AND PHORBOL ESTER-REPRESSED TRANSCRIPTION FROM THE HUMAN P450C 17 PROMOTER IN MOUSE CELLS
TISSUE-SPECIFIC, CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-INDUCED, AND PHORBOL ESTER-REPRESSED TRANSCRIPTION FROM THE HUMAN P450C 17 PROMOTER IN MOUSE CELLS
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DOI：
10.1210/mend-4-12-1972
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发表时间：
1990-12-01
影响因子：
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通讯作者：
MILLER, WL
中科院分区：
文献类型：
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作者：
BRENTANO, ST;PICADOLEONARD, J;MILLER, WL
细胞色素P450c17是催化类固醇17-α-羟化酶和17 - 20-裂解酶活性的单微粒体酶。 它在人肾上腺和性腺中表达并受热带激素调节，但在胎盘中不表达。 为了研究人P450c17基因的转录调控，我们构建了11个含有氯霉素乙酰转移酶（CAT）报告基因的5 '非翻译区序列缺失的质粒。 将这些构建体转染到小鼠肾上腺Y1和睾丸MA-10细胞中，并与毛喉素、8-溴-cAMP或12-O-十四烷酰基-佛波醇-13-乙酸酯（TPA）孵育12小时。 从标准结构中解释结果是困难的，显然是因为载体中的DNA序列错误地启动了一些转录。 因此，我们构建了一个修饰的CAT报告载体，消除了可检测的通读转录。 在Y1细胞中，含有-82至-184碱基对（bp）的5 '侧翼DNA的构建体的基础活性在无启动子对照的80 - 150%之间。含有至少~235 bp该DNA的构建体表达的CAT为对照值的540%，但向~774添加序列没有进一步的影响。 在含有-184~-774bp DNA的构建体中，毛喉素使CAT活性的表达增加到高于基础的300%。含有-184至-310 bp的构建体在用TPA处理的细胞中以毛喉素诱导水平的50%表达CAT。 基础和cAMP诱导的表达在MA-10细胞中比在Y1细胞中低得多，并且随着启动子长度增加而增加至-774。 因此，cAMP诱导、TPA抑制和人P450c17基因的完全基础转录由启动子的不同区域介导。（分子内分泌学4：1972 - 1979，1990）
Cytochrome P450c17 is the single microsomal enzyme catalyzing steroid 17-alpha-hydroxylase and 17-20-lyase activities. It is expressed and regulated by tropic hormones in the human adrenal and gonads, but is not expressed in the placenta. To study the transcriptional regulation of the human P450c17 gene, we constructed 11 plasmids containing serial deletions of its 5' nontranslated region driving expression of the chloramphenicol acetyltransferase (CAT) reporter gene. These constructs were transfected into mouse adrenal Y1 and testis MA-10 cells and incubated with forskolin, 8-bromo-cAMP, or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) for 12 h. Interpretation of results from standard constructions was difficult, apparently because some transcription was incorrectly initiated by DNA sequences in the vector. Therefore, we built a modified CAT reporter vector that eliminated detectable read-through transcription. In Y1 cells, the basal activity of constructs containing from -82 to -184 basepairs (bp) of 5' flanking DNA was between 80-150% of the promoterless control. Constructs containing at least -235 bp of this DNA expressed CAT at 540% of the control value, but addition of sequences to -774 had no further effect. Forskolin increased the expression of CAT activity to 300% above basal with constructions containing DNA from -184 to -774 bp. Constructs containing between -184 and -310 bp expressed CAT at 50% of the forskolin-induced levels in cells treated with TPA. Both basal and cAMP-induced expression were much lower in MA-10 cells than in Y1 cells and increased with increasing promoter length to -774. Thus, cAMP induction, TPA repression, and full basal transcription from the human P450c17 gene are mediated by different regions of the promoter. (Molecular Endocrinology 4: 1972-1979, 1990)