Glucocorticoid coordinate regulation of type I procollagen gene expression and procollagen DNA-binding proteins in chick skin fibroblasts.
Glucocorticoid coordinate regulation of type I procollagen gene expression and procollagen DNA-binding proteins in chick skin fibroblasts.
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DOI：
10.1021/bi00408a013
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发表时间：
1988-04
期刊：
影响因子：
2.9
通讯作者：
D. Cockayne;K. Cutroneo
中科院分区：
文献类型：
--
作者：
D. Cockayne;K. Cutroneo
从对照和地塞米松处理（2小时）的鸡胚皮肤成纤维细胞中分离细胞核，并在体外转录。从地塞米松处理的成纤维细胞中分离的细胞核转录较少的pro α 1（I）和pro α 2（I）mRNA，但不转录β-肌动蛋白mRNA。接受地塞米松和[5，6 - 3 H]尿苷的成纤维细胞也表现出核I型前胶原mRNA的合成减少，但β-肌动蛋白mRNA的合成没有减少。放线菌酮处理的成纤维细胞中，新合成的核I型前胶原mRNA种类显着减少。放线菌酮加地塞米松处理成纤维细胞时，观察到增强的抑制作用。由于上述研究表明，活性蛋白质合成是维持I型前胶原基因的组成型表达所必需的，我们确定了糖皮质激素是否调节对α 2（I）前胶原基因具有序列特异性的DNA结合蛋白。用32 P末端标记的pBR 322载体DNA和32 P末端标记的含有α 2（I）前胶原启动子的DNA探测核蛋白印迹。在0.05和0.1 M NaCl的严格洗涤下，非组蛋白蛋白仍与标记的DNA结合。随着离子强度增加到0.2和0.3 M NaCl，非组蛋白-蛋白质DNA结合优先丢失。只有低分子量蛋白质在最高离子强度下仍与标记的DNA结合，表明这些核蛋白的非特异性结合。地塞米松治疗导致的非组蛋白的结合载体和启动子标记的DNA的增加，在控制成纤维细胞中观察到的严格洗涤的0.05和0.1 M NaCl和在较小程度上在0.2 M NaCl。计算非组蛋白对含DNA的α 2（I）前胶原启动子的结合特异性。三个非组蛋白DNA结合蛋白的Mr 90，000，50，000和30，000的特异性地塞米松治疗后改变。
Nuclei were isolated from control and dexamethasone-treated (2 h) embryonic chick skin fibroblasts and transcribed in vitro. Nuclei isolated from dexamethasone-treated fibroblasts transcribed less pro alpha 1(I) and pro alpha 2(I) mRNAs but not beta-actin mRNA. Fibroblasts receiving dexamethasone and [5,6-3H]uridine also demonstrated decreased synthesis of nuclear type I procollagen mRNAs but not beta-actin mRNA. In fibroblasts treated with cycloheximide the newly synthesized nuclear type I procollagen mRNA species were markedly decreased. An enhanced inhibitory effect was observed when fibroblasts were treated with cycloheximide plus dexamethasone. Since the studies above demonstrate that active protein synthesis is required to maintain the constitutive expression of the type I procollagen genes, we determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the alpha 2(I) procollagen gene. Nuclear protein blots were probed with the 32P-end-labeled pBR322 vector DNA and 32P-end-labeled alpha 2(I) procollagen promoter containing DNA. Nonhistone proteins remained bound to labeled DNA at stringency washes of 0.05 and 0.1 M NaCl. As the ionic strength was increased to 0.2 and 0.3 M NaCl, the nonhistone-protein DNA binding was preferentially lost. Only the low molecular weight proteins remained bound to labeled DNA at the highest ionic strength, indicating nonspecific binding of these nuclear proteins. Dexamethasone treatment resulted in an increase of binding of nonhistone proteins to vector- and promoter-labeled DNAs over that observed in control fibroblasts at stringency washes of 0.05 and 0.1 M NaCl and to a lesser extent at 0.2 M NaCl. The binding specificities of nonhistone proteins for the alpha 2(I) procollagen promoter containing DNA were calculated. Three nonhistone DNA-binding proteins of Mr 90,000, 50,000, and 30,000 had altered specificities following dexamethasone treatment.