Identification of novel TGF-β/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach
Identification of novel TGF-β/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach
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DOI：
10.1074/jbc.m100754200
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发表时间：
2001-05-18
影响因子：
4.8
通讯作者：
Mauviel, A
中科院分区：
文献类型：
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作者：
Verrecchia, F;Chu, ML;Mauviel, A
尽管对通过Smad途径转化生长因子- β (tgf - β)信号传导的内在机制的理解取得了重大进展，但在确定直接靶基因方面进展甚微。在本报告中，我们利用cDNA微阵列技术，重点研究了tgf - β在人真皮成纤维细胞中快速诱导的细胞外基质相关基因的特征，并试图鉴定tgf - β上调的基因是smad介导的。对于一个有资格成为Smad直接靶标的基因，我们假设它必须满足以下标准：(1) tgf - β刺激后，稳态mRNA水平快速（30分钟）且显著（至少s倍）升高；(2)外源性tgf - β和共转染的Smad3表达载体均可激活启动子；(3)显性阴性Smad3和抑制性Smad7表达载体均可阻断tgf - β对启动子活性的上调；(4)在Smad3（-/-）小鼠胚胎成纤维细胞中，tgf - β无法反激活启动子。使用这种严格的方法，我们确定了COL1A2， COL3A1, COL6A1， COL6A3和组织金属蛋白酶抑制剂-1作为tgf - β /Smad3的明确靶点。将这种方法外推到其他细胞外基质相关基因启动子也发现COL1A1和COL5A2是新的Smad靶点，但COL6A2不是。总之，这些结果代表了在成纤维细胞中鉴定新的、早期诱导的smad依赖性tgf - β靶基因的重要一步。
Despite major advances in the understanding of the intimate mechanisms of transforming growth factor-beta (TGF-beta) signaling through the Smad pathway, little progress has been made in the identification of direct target genes. In this report, using cDNA microarrays, we have focussed our attention on the characterization of extracellular matrix-related genes rapidly induced by TGF-beta in human dermal fibroblasts and attempted to identify the ones whose up-regulation by TGF-beta is Smad-mediated. For a gene to qualify as a direct Smad target, we postulated that it had to meet the following criteria: (1) rapid (30 min) and significant (at least S-fold) elevation of steady-state mRNA levels upon TGF-beta stimulation, (2) activation of the promoter by both exogenous TGF-beta and co-transfected Smad3 expression vector, (3) up-regulation of promoter activity by TGF-beta blocked by both dominant-negative Smad3 and inhibitory Smad7 expression vectors, and (4) promoter transactivation by TGF-beta not possible in Smad3(-/-) mouse embryo fibroblasts. Using this stringent approach, we have identified COL1A2, COL3A1, COL6A1, COL6A3, and tissue inhibitor of metalloproteases-1 as definite TGF-beta /Smad3 targets. Extrapolation of this approach to other extracellular matrix-related gene promoters also identified COL1A1 and COL5A2, but not COL6A2, as novel Smad targets. Together, these results represent a significant step toward the identification of novel, early-induced Smad-dependent TGF-beta target genes in fibroblasts.