HSF4b基因敲除下调αB-晶状体蛋白表达介导白内障发生

Knockdown of HSF4b leads to cataract development through down-regulation ofαB-crystallin expression

目的阐明热休克转录因子4b(HSF4b)对αB-晶状体蛋白(αB-crystallin,CRYAB)的调控在白内障发病过程中的作用及其机制。方法采用qPCR、Western印迹法和免疫荧光检测Hsf4^(tm1Xyk)基因敲除小鼠晶状体中CRYAB和HSF4的表达情况;通过凝胶迁移实验、染色质免疫共沉淀、双报告基因检测等方法研究HSF4b对CRYAB的调控作用;通过免疫荧光分析和免疫共沉淀研究CRYAB与波形蛋白(vimentin,VIM)的相互作用。结果Hsf4^(tm1Xyk)基因敲除小鼠晶状体中CRYAB表达下调;体外试验证实HSF4b和CRYAB的表达呈现正相关性;HSF4b直接与CRYAB启动子结合,激活CRYAB表达的转录过程;CRYAB与VIM存在相互作用。结论阐明了Hsf4^(tm1Xyk)基因敲除小鼠中晶状体发育异常和白内障形成的分子机制。鉴定出CRYAB是HSF4b的下游调控分子。CRYAB对VIM具有稳定作用,有可能是抗白内障药物的作用靶点。

Objective To investigate the effect of HSF4b on expression ofαB-crystallin(CRYAB)and the development of cataract.Methods Quantitative real-time polymerase chain reaction(qPCR),Western blot and immunofluorescence were used to detect the expression ofαB-crystallin and HSF4 in the lens of Hsf4^(tm1Xyk)-knockout mice.The regulative effects of HSF4b onαB-crystallin were examined by electrophoretic mobility shift assay,chromatin immunoprecipitation assay,and luciferase activity analysis.The interaction betweenαB-crystallin and vimentin(VIM)was studied by co-immunopre-cipitation and immunofluorescence.Results The expression ofαB-crystallin was down-regulated in the lens of Hsf4^(tm1Xyk)-knockout mice.When HSF4b overexpressed or slienced in SRA01/04 cells,the expression ofαB-crystallin showed in similar tendency at both mRNA level and protein level.HSF4b was directly bound to the HSE inαB-crystallin promoter and activated the expression ofαB-crystallin.Meanwhile,αB-crystallin was able to interact with vimentin.Conclusion Our result shows thatαB-crystallin may be a novel downstream molecular of HSF4b.The interaction ofαB-crystallin and vimentin may be a key mechanism of cataract in Hsf4^(tm1Xyk) knockout mice.Based onαB-crystallin and vimentin interaction,it is possible to find a novel therapeutic target for anti-cataract drugs.