Glomerular mesangial cell-specific transactivation of matrix metalloproteinase 2 transcription is mediated by YB-1
Glomerular mesangial cell-specific transactivation of matrix metalloproteinase 2 transcription is mediated by YB-1
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DOI：
10.1074/jbc.272.36.22905
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发表时间：
1997-09-05
影响因子：
4.8
通讯作者：
Lovett, DH
中科院分区：
文献类型：
--
作者：
Mertens, PR;Harendza, S;Lovett, DH
系膜细胞（MC）的激活在大多数慢性肾小球疾病终末期硬化病变的发展中起关键作用。我们之前已经证明RLC激活与基质金属蛋白酶-2 （MMP-2）酶的高水平表达直接相关（Turck， J., Pollock， A. S., Lee， L., Marti, h . p .）。（1996） [j]。化学，25,15074-15083)，其转录以组织特异性方式调节。最近的研究(Harendza， S., Pollock， A. Mertens， P. R., and Lovett， D. H.（1995）。Chem. 270, 18786-18796)在大鼠MMP-2基因的5'-侧翼区域描绘了一个强顺式作用的增强元件，称为MMP-2 RE1。凝胶位移、DNA足迹和转录分析将增强子元件定位到一个独特的40碱基对（bp）序列，位于相对于翻译起始位点的-1322至-1282 bp。溴脱氧尿嘧啶取代的40 bp增强子元件与MC核提取物交联得到单个52 kDa的蛋白，而MMP-2 RE1的西南印迹分析显示三个杂交核蛋白的大小分别为52、62和86 kDa。用MMP-2 RE1筛选人mccdna表达文库，获得与转录因子YB-1序列相同的克隆。用抗YB-1抗体对MC核提取物进行Western blot和supershift凝胶分析，证实在移位的复合物中存在YB-1。检测MMP-2 RE1序列发现一个不完整的Y-box序列（CTGCTGGGCAAG），该序列在DMS保护足迹分析中与重组YB-1特异性相互作用。YB-1蛋白优先结合40-bp MMP-2 RE1的单链组分，并随着浓度的增加形成多聚物复合物。在MC中共同转染YB-1可提高天然MMP-2启动子的增强子活性，而在非合成MMP-2的肾小球上皮细胞中转染YB-1可导致转录抑制。本研究表明，YB-1是肾小球系膜细胞MMP-2转录的一个主要的、细胞类型特异性的反激活因子。
Mesangial cell (MC) activation plays a pivotal role in the development of the end stage sclerotic lesion characteristic of most forms of chronic glomerular disease. We have previously demonstrated that RLC activation is directly linked to high level expression of the matrix metalloproteinase-2 (MMP-2) enzyme (Turck, J., Pollock, A. S., Lee, L., Marti, H.-P., and Lovett, D. H. (1996) J. Biol. Chem. 25, 15074-15083), the transcription of which is regulated in a tissue-specific fashion. Recent studies (Harendza, S., Pollock, A. Mertens, P. R., and Lovett, D. H. (1995) J. Biol. Chem. 270, 18786-18796) delineated a strong cis-acting enhancer element, designated MMP-2 RE1, within the 5'-flanking region of the rat MMP-2 gene. Gel shift, DNA footprint, and transcriptional analyses mapped the enhancer element to a unique 40-base pair (bp) sequence located at -1322 to -1282 bp relative to the translational start site. Bromodeoxyuridine-substituted UV cross-linking of the 40-bp enhancer element with MC nuclear extracts yielded a single protein of 52 kDa, while Southwestern blot analysis with MMP-2 RE1 demonstrated three hybridizing nuclear proteins of 52, 62, and 86 kDa size. Screening of a human MC cDNA expression library with MMP-2 RE1 exclusively yielded clones with the identical sequence of the transcription factor YB-1. Western blot and supershift gel analysis of MC nuclear extracts with an anti-YB-1 antibody confirmed the presence of YB-1 within the shifted complex. Examination of the MMP-2 RE1 sequence revealed an incomplete Y-box sequence (CTGCTGGGCAAG), which specifically interacted with recombinant YB-1 on DMS protection footprinting analysis. YB-1 protein preferentially bound the single-stranded components of the 40-bp MMP-2 RE1 and, with increasing concentrations, formed multimeric complexes. Co-transfection of YB-1 in MC increased the enhancer activity within the context of the native MMP-2 promoter, while transfection of non-MMP-2-synthesizing glomerular epithelial cells with YB-1 led to transcriptional suppression. This study indicates that YB-1 is a major, cell type-specific transactivator of MMP-2 transcription by glomerular mesangial cells.