Wan Lu1, Jin Li1, Meng Ren1, Yinjuan Zeng2, Pin Zhu3, Li Lin1, Diaozhu Lin1, Shaoyun Hao1, Qi Gao1, Junqiang Liang1, Li Yan4, Chuan Yang5. 1. Department of Endocrinology and Metabolism, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China. 2. Department of Endocrinology and Metabolism, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai 519000, China. 3. Department of Endocrinology and Metabolism, Guangzhou Red Cross Hospital, Medical College of Jinan University, Guangzhou 510220, China. 4. Department of Endocrinology and Metabolism, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China. Electronic address: bear3151@sohu.com. 5. Department of Endocrinology and Metabolism, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China. Electronic address: bear3151@sina.com.
Abstract
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.
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.