Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Invest Ophthalmol Vis Sci. 2010 Jan;51(1):396-404. doi: 10.1167/iovs.09-3772. Epub 2009 Jul 23.
To explore the target genes of HSF4, especially those involved in lens developmental processes and cataract formation.
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.
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.
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.
探索 HSF4 的靶基因，特别是那些参与晶状体发育过程和白内障形成的靶基因。
对 Hsf4(tm1Xyk)-敲除小鼠和野生型小鼠进行裂隙灯生物显微镜检查。采用二维电泳结合质谱技术鉴定野生型和 Hsf4(tm1Xyk)-敲除小鼠之间差异表达的晶状体蛋白，并通过 Western blot 和免疫组化进一步验证。组织学分析用于分析晶状体纤维细胞的去核过程。此外，还进行了电泳迁移率变动分析（EMSA）、荧光素酶测定和染色质免疫沉淀（ChIP）测定，以验证 HSF4 对波形蛋白表达的影响。
Hsf4(tm1Xyk)-敲除小鼠的晶状体异常，并发展为白内障。下调的蛋白主要是结构蛋白，包括α-和β-晶状体蛋白，而上调的蛋白主要是酶和中间丝蛋白波形蛋白。Western blot、Q-PCR 和免疫荧光进一步证实了波形蛋白表达水平的上调。EMSA、荧光素酶测定和 ChIP 测定验证了 HSF4 对波形蛋白启动子具有 DNA 结合能力，并抑制了波形蛋白的表达。
这些发现表明，HSF4 通过 HSE 样元件抑制波形蛋白基因表达。HSF4 功能丧失导致 Hsf4(tm1Xyk)-敲除小鼠中波形蛋白表达增加，并影响晶状体分化，特别是损害晶状体纤维细胞的去核。这些事件似乎暗示了 Hsf4(tm1Xyk)-敲除小鼠中异常晶状体发育和白内障形成的分子机制。HSF4-波形蛋白轴似乎是开发抗白内障药物的新靶点，特别是对于那些由于 HSF4 表达异常引起的白内障。
