TRANSCRIPTION FACTOR AP-2 REGULATES HUMAN INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-5 GENE-EXPRESSION
TRANSCRIPTION FACTOR AP-2 REGULATES HUMAN INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-5 GENE-EXPRESSION
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DOI：
10.1074/jbc.270.42.24844
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发表时间：
1995-10-20
影响因子：
4.8
通讯作者：
CLEMMONS, DR
中科院分区：
文献类型：
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作者：
DUAN, CM;CLEMMONS, DR
胰岛素样生长因子结合蛋白-5（IGFBP-5）是IGF作用的重要调节因子。IGFBP-5 mRNA在人成纤维细胞中丰富，并受cAMP调节。为了理解这种细胞类型特异性表达和调控的分子机制，我们分离了人IGFBP-5基因的5 '侧翼区，并将其融合到编码荧光素酶的无启动子报告质粒上。瞬时转染成纤维细胞的构建体显示组成型和cAMP诱导的启动子活性的方向特异性的方式。序列分析表明，存在的远端和近端的共识AP-2识别位点位于5'从TATA框。通过凝胶迁移率测定，这两个序列在体外特异性结合人AP-2。AP-2的可能作用通过用IGFBP-5启动子构建体和人AP-2表达构建体共转染AP-2缺陷型HepG 2细胞来检查。与AP-2共转染显著提高IGFBP-5启动子活性。这种反式激活是IGFBP-5启动子和AP-2特异性的。在AP-2丰富的成纤维细胞中，AP-2B（AP-2的显性负性抑制剂）的表达抑制IGFBP-5启动子活性。在HepG 2细胞中，单独AP-2B没有显著作用，但AP-2B以剂量依赖性方式抑制AP α诱导的启动子活性的激活。使用一些缺失突变体和点突变的相对功能的重要性的推定AP-2结合位点进行了检查。当前两个远端CCCCACCC样推定的AP-2位点被删除或突变时，AP-2诱导的反式激活没有变化。然而，近端GCCNNNGGC样序列的缺失或突变消除了AP-2诱导的激活。这些结果表明AP-2通过近端GCCNNNGGC样序列调控IGFBP-5基因的表达。这种AP-2介导的反式激活至少部分地有助于IGFBP-5在成纤维细胞中的组成性高表达和该基因的cAMP响应性。
Insulin-like growth factor binding protein-5 (IGFBP-5) is an important modulator of IGF actions. IGFBP-5 mRNA is abundant in human fibroblasts and is regulated by cAMP. To understand the molecular mechanism underlying this cell type-specific expression and regulation, we isolated the 5'-flanking region of the human IGFBP-5 gene and fused it to a promoter-less reporter plasmid encoding luciferase. Transient transfection of the construct into fibroblasts displayed both constitutive and cAMP-induced promoter activity in an orientation-specific manner. Sequence analysis revealed the existence of distal and proximal consensus AP-2 recognition sites located 5' from the TATA box. Both sequences bound specifically to human AP-2 in vitro by gel shift mobility assay. The possible role of AP-2 was examined by cotransfection of AP-2-deficient HepG2 cells with the IGFBP-5 promoter construct and a human AP-2 expression construct. Cotransfection with AP-2 significantly elevated IGFBP-5 promoter activity. This trans-activation was IGFBP-5 promoter and AP-2 specific. In AP-2 abundant fibroblasts, expression of AP-2B, a dominant-negative inhibitor of AP-2, suppressed IGFBP-5 promoter activity. In HepG2 cells, AP-2B alone had no significant effect, but the AP a-induced activation of promoter activity was inhibited by AP-2B in a dose-dependent manner. The relative functional importance of the putative AP-2 binding sites was examined using a number of deletion mutants and point mutations. When the first two distal CCCCACCC-like putative AP-2 sites were deleted or mutated, there was no change in AP-2-induced trans-activation. Deletion or mutation of the proximal GCCNNNGGC-like sequences, however, abolished the AP-2-induced activation. These results suggest that AP-2 regulates the IGFBP-5 gene expression through the proximal GCCNNNGGC-like sequences. This AP-2-mediated trans-activation contributes at least in part to the constitutively high expression of IGFBP-5 in fibroblasts and to the cAMP responsiveness of this gene.