Ogawa Kenji, Chen Feifei, Kuang Chenzhong, Chen Yan
Department of Medical and Molecular Genetics, Walther Oncology Center, Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis, IN 46202, USA.
Biochem J. 2004 Jul 15;381(Pt 2):413-22. doi: 10.1042/BJ20040058.
TGF-beta (transforming growth factor-beta) plays a critical role in modulating the inflammatory response and other biological processes through its regulation of the production of MMPs (matrix metalloproteinases). In both Mono-Mac-6 and RAW264.7 monocyte/macrophage cells, TGF-beta abrogated lipopolysaccharide-induced increases in the enzymic activity and mRNA level of MMP-9. A fragment of the human MMP-9 promoter was used to characterize its regulation by TGF-beta signalling. In RAW264.7 cells, TGF-beta or its downstream signalling protein, Smad3 (Sma- and Mad-related protein 3), inhibited lipopolysaccharide-stimulated promoter activity. The suppressive activity of TGF-beta on the MMP-9 promoter was abrogated by an inhibitory Smad, Smad7. The MMP-9 promoter contains a putative TIE (TGF-beta inhibitory element). However, neither mutation nor deletion of the TIE had any effect on the inhibitory activity of TGF-beta on MMP-9 transcription, indicating that the consensus TIE is not required for this effect of TGF-beta. Analysis using a series of deletion mutants of the MMP-9 promoter revealed that a region containing a consensus NF-kappaB (nuclear factor-kappaB) site is required for the basal activity and TGF-beta-mediated suppression of the promoter. Mutation of the putative NF-kappaB site not only markedly reduced the basal transcriptional activity of the promoter, but also abrogated the responsiveness of the promoter to TGF-beta. In addition, a minimal promoter containing one copy of the NF-kappaB sequence was responsive to TGF-beta treatment. Furthermore, an electrophoretic mobility shift assay was performed with the nuclear extracts from RAW264.7 cells, and it was found that TGF-beta treatment did not disrupt the binding of NF-kappaB p50 and p65 proteins to the NF-kappaB sequence. Taken together, these studies indicate that the NF-kappaB site is indispensable for the suppressive activity of TGF-beta in the regulation of MMP-9 transcription.
转化生长因子-β（TGF-β）通过调节基质金属蛋白酶（MMPs）的产生，在调节炎症反应和其他生物学过程中发挥关键作用。在单核细胞系Mono-Mac-6和RAW264.7单核细胞/巨噬细胞中，TGF-β均可消除脂多糖诱导的MMP-9酶活性和mRNA水平的升高。利用人MMP-9启动子的一个片段来表征其受TGF-β信号通路的调控情况。在RAW264.7细胞中，TGF-β或其下游信号蛋白Smad3（Sma和Mad相关蛋白3）可抑制脂多糖刺激的启动子活性。TGF-β对MMP-9启动子的抑制活性可被抑制性Smad蛋白Smad7消除。MMP-9启动子含有一个假定的TGF-β抑制元件（TIE）。然而，TIE的突变或缺失对TGF-β对MMP-9转录的抑制活性均无影响，这表明该共有TIE并非TGF-β发挥此效应所必需。对MMP-9启动子的一系列缺失突变体进行分析发现，一个含有共有核因子-κB（NF-κB）位点的区域是启动子基础活性和TGF-β介导的启动子抑制所必需的。假定的NF-κB位点的突变不仅显著降低了启动子的基础转录活性，还消除了启动子对TGF-β的反应性。此外，含有一份NF-κB序列的最小启动子对TGF-β处理有反应。此外，对RAW264.7细胞的核提取物进行了电泳迁移率变动分析，发现TGF-β处理并未破坏NF-κB p50和p65蛋白与NF-κB序列的结合。综上所述，这些研究表明NF-κB位点对于TGF-β在调节MMP-9转录中的抑制活性不可或缺。
