SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor β
SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor β
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DOI：
10.1073/pnas.95.25.14769
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发表时间：
1998-12-08
影响因子：
11.1
通讯作者：
Mauviel, A
中科院分区：
文献类型：
--
作者：
Vindevoghel, L;Lechleider, RJ;Mauviel, A
人类III型胶原基因(COL7A1)最近被鉴定为转化生长因子β(TGF-β)/SMAD信号通路的即刻-早期反应基因。在这项研究中，通过使用MDA-MB-468 Smad4-/-乳腺癌细胞，我们证明了Smad4的表达是SMAD介导的启动子活性的绝对要求。我们还证明了代表COL7A1启动子-496/-444区转化生长因子-β反应元件的SMAD结合序列(SBS)在异源启动子的背景下作为增强子发挥作用。对转染Smads 1-5表达载体的COS-1细胞核提取物进行的凝胶迁移率分析表明，Smad3形成了一种与成纤维细胞提取物中观察到的内源性转化生长因子-β特异性复合体类似的迁移复合体。重组谷胱甘肽S-转移酶-SmadD融合蛋白的凝胶迁移率分析表明，Smad4和C端截短的Smad3都能与COL7A1SBS结合，但Smad2不能。Smad3和Smad4在COS-1细胞中的共表达导致了两个复合体的形成：一个是单独含有Smad3的DNA/蛋白质复合体，另一个是同时含有Smad3和Smad4的迁移较慢的复合体，后者在成纤维细胞中没有检测到。COL7A1 SBS驱动的启动子在MDA-MB-468癌细胞或成纤维细胞中的最大反式激活需要伴随的Smad3和Smad4的过表达。这些数据可能代表了首次发现了调节人类基因的功能同源SMAD3复合体。
The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor beta (TGF-beta)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4-/- breast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-beta response element in the region -496/-444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1-5 indicate that SMAD3 forms a complex with a migration similar to that of the endogenous TGF-beta-specific complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion proteins indicate that both SMAD4 and C-terminally truncated SMAD3 but not SMAD2, can bind the COL7A1 SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two complexes: a DNA/protein complex containing SMAD3 alone and another slower-migrating complex containing both SMAD3 and SMAD4, the latter complex not being detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 complex regulating a human gene.