甲羟戊酸途径在晚期糖基化终末产物诱导角质形成细胞中基质金属蛋白酶9表达及激活过程中特定CpG位点去甲基化的作用。
Role of the mevalonate pathway in specific CpG site demethylation on AGEs-induced MMP9 expression and activation in keratinocytes.
作者信息
Lu Wan, Li Jin, Ren Meng, Zeng Yinjuan, Zhu Pin, Lin Li, Lin Diaozhu, Hao Shaoyun, Gao Qi, Liang Junqiang, Yan Li, Yang Chuan
机构信息
Department of Endocrinology and Metabolism, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China.
Department of Endocrinology and Metabolism, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai 519000, China.
出版信息
Mol Cell Endocrinol. 2015 Aug 15;411:121-9. doi: 10.1016/j.mce.2015.04.019. Epub 2015 Apr 23.
BACKGROUND
Advanced glycation end products (AGEs) played an important role for the development of diabetic foot. In the present study we tried to show the mevalonate pathway and the key demethylation site(s) in the MMP-9 cis-promoter to the component of MMP-9 by AGEs in keratinocyte.
METHOD
Human keratinocyte cell line (HaCaT) cells were exposed to AGE-BSA. The plasmid construction and site-directed mutagenesis, dual-luciferase reporter assays, immunoblot, zymography, pull down, bisulfite sequencing PCR analysis and Western blotting were applied.
RESULTS
The AGE-BSA could increase and more activate the MMP9 in keratinocyte. The RhoA and ROCK1 also could be activated. These affects were blocked by the simvastatin. Meanwhile, the CpG site at -562 site was largely demethylated with AGE-BSA treatment. The cis-promoter sequences with -562 bp site methylated had a lower activity change, which had a highest expression activity and was decreased by simvastatin. Moreover, site-directed mutagenesis of CpG site (-562 bp) in the recombinant plasmid pCpGL-571 brought more reduction in activity, and the activity of methylated mutation pCpGL-571 remains decreased.
CONCLUSION
The cis-promoter regions of MMP9 would be methylated by AGE-BSA in keratinocyte through the mevalonate pathway, especially the -562 bp site.
背景
晚期糖基化终产物（AGEs）在糖尿病足的发展中起重要作用。在本研究中，我们试图揭示甲羟戊酸途径以及基质金属蛋白酶-9（MMP-9）顺式启动子中的关键去甲基化位点对角质形成细胞中MMP-9成分的影响。
方法
将人角质形成细胞系（HaCaT）细胞暴露于AGE-牛血清白蛋白（AGE-BSA）。应用质粒构建和定点诱变、双荧光素酶报告基因检测、免疫印迹、酶谱分析、下拉实验、亚硫酸氢盐测序PCR分析和蛋白质免疫印迹法。
结果
AGE-BSA可增加并进一步激活角质形成细胞中的MMP9。RhoA和ROCK1也可被激活。这些作用被辛伐他汀阻断。同时，经AGE-BSA处理后，-562位点的CpG位点大量去甲基化。-562 bp位点甲基化的顺式启动子序列活性变化较小，其具有最高的表达活性且被辛伐他汀降低。此外，重组质粒pCpGL-571中CpG位点（-562 bp）的定点诱变使活性降低更多，且甲基化突变体pCpGL-571的活性仍然降低。
结论
MMP9的顺式启动子区域在角质形成细胞中会通过甲羟戊酸途径被AGE-BSA甲基化，尤其是-562 bp位点。
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