Identification of vimentin as a novel target of HSF4 in lens development and cataract by proteomic analysis.
Identification of vimentin as a novel target of HSF4 in lens development and cataract by proteomic analysis.
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DOI：
10.1167/iovs.09-3772
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发表时间：
2010
影响因子：
4.4
通讯作者：
L. Mou;Jing-ying Xu;Weiye Li;X. Lei;Yalan Wu;Guoxu Xu;Xiangyin Kong;Guo-Tong Xu
中科院分区：
文献类型：
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作者：
L. Mou;Jing-ying Xu;Weiye Li;X. Lei;Yalan Wu;Guoxu Xu;Xiangyin Kong;Guo-Tong Xu
目的研究HSF 4的靶基因，特别是与透镜发育和白内障形成相关的基因。方法应用裂隙灯显微镜观察Hsf 4（tm 1Xyk）基因敲除小鼠和野生型小鼠的心肌细胞凋亡情况。双向电泳结合质谱鉴定野生型和Hsf 4（tm 1 Xyk）基因敲除小鼠之间透镜蛋白的差异表达，并通过Western blot和免疫组化进一步证实。用组织学方法分析透镜纤维细胞的脱核过程。此外，电泳迁移率变动分析（EMSA），荧光素酶测定，染色质免疫沉淀（ChIP）测定用于验证HSF 4对波形蛋白表达的影响。结果Hsf 4（tm 1Xyk）基因敲除小鼠晶状体异常，并发生白内障。下调的蛋白质是主要的结构蛋白，包括α-和β-晶体蛋白，而上调的蛋白质主要是酶和中间丝蛋白，波形蛋白。通过Western印迹、Q-PCR和免疫荧光进一步证实上调的波形蛋白表达水平。EMSA、荧光素酶试验和ChIP试验验证了HSF 4具有与波形蛋白启动子DNA结合的能力并抑制波形蛋白表达。结论HSF 4通过HSE样元件抑制波形蛋白基因表达。HSF 4功能的丧失导致HSF 4（tm 1 Xyk）敲除小鼠中波形蛋白表达增加，并影响透镜分化，特别是损害透镜纤维细胞的去核。这些事件似乎暗示了Hsf 4（tm 1Xyk）基因敲除小鼠异常透镜发育和白内障形成的分子机制。HSF 4-波形蛋白轴似乎是开发抗白内障药物的新靶点，特别是对于那些因HSF 4表达异常而导致的白内障。
PURPOSE To explore the target genes of HSF4, especially those involved in lens developmental processes and cataract formation. METHODS A slit lamp biomicroscopy examination was performed on Hsf4(tm1Xyk)-knockout mice and wild-type mice. Two-dimensional electrophoresis combined with mass spectrometry was used to identify differentially expressed lens proteins between wild-type and Hsf4(tm1Xyk)-knockout mice and further confirmed by Western blot and immunohistochemistry. Histologic analysis was used to analyze the denucleation process of lens fiber cells. Moreover, an electrophoretic mobility shift assay (EMSA), luciferase assay, and chromatin immunoprecipitation (ChIP) assay were used to validate the effects of HSF4 on vimentin expression. RESULTS Hsf4(tm1Xyk)-knockout mice had abnormal lenses and developed cataract. The downregulated proteins were major structural proteins including alpha- and beta-crystallins, whereas the upregulated proteins were mainly enzymes and an intermediate filament protein, vimentin. The upregulated vimentin expression level was further confirmed by Western blot, Q-PCR, and immunofluorescence. EMSA, luciferase assay, and ChIP assay validated that HSF4 had DNA-binding ability to vimentin promoter and repressed vimentin expression. CONCLUSIONS These findings indicate that HSF4 represses vimentin gene expression via the HSE-like element. The loss of HSF4 function results in an increase in vimentin expression in Hsf4(tm1Xyk)-knockout mice and affects lens differentiation, particularly impairing the denucleation of lens fiber cells. These events appear to implicate a molecular mechanism in abnormal lens development and cataract formation in Hsf4(tm1Xyk)-knockout mice. The HSF4-vimentin axis appears to be a new target for developing anti-cataract drugs, especially for those cataracts resulting from aberrations in HSF4 expression.