Identification of vimentin as a novel target of HSF4 in lens development and cataract by proteomic analysis

Abstract

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.