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Y. Ito, F. Berry, M. A. Walter; Analysis of Foxc1 Direct Target Genes That Are Associated With Cellular Stress Response. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5837.
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FOXC1 mutations cause human Axenfeld-Rieger Syndrome (ARS), an autosomal-dominant disease that affects the anterior segment of the eye. ARS patients have an elevated risk to develop early-onset glaucoma, which is usually associated with increased intraocular pressure (IOP). Structures of the anterior segment of the eye, such as the trabecular meshwork (TM), are important in maintaining the aqueous flow pathway. The TM outflow pathway tissue is subjected to constant stress. Chronic exposure to stress may overwhelm cellular defense mechanisms, resulting in progressive diseases such as glaucoma. The identification and characterization of direct target genes of FOXC1, specifically those genes that stress-responsive, are essential in understanding how disruptions in FOXC1 can result in ARS and glaucoma. The purpose of this investigation was to examine molecular chaperones that are potential FOXC1 target genes and to determine whether FOXC1 plays a role in stress response of the cell.
Previous work in our laboratory using microarray technology has led to the identification of potential genes that are regulated by FOXC1 including stress responsive genes such as heat shock protein 27 (HSP27) and heat shock protein 70 (HSP70). Experiments to validate the genes as bona fide target genes of FOXC1 were carried out. FOXC1 was knocked down in human trabecular meshwork (HTM) cells and the RNA and protein levels of the molecular chaperons were analyzed by Northern blot analysis, RT-PCR, qPCR, and Western blot analysis. Also, chromatin immunoprecipitaiton (ChIP) analysis was carried out to identify FOXC1 binding sites in the upstream promoter region of target genes.
HSP27 and HSP70 were validated as authentic target genes of FOXC1. FOXC1 knockdown by siRNA resulted in altered RNA and protein levels of HSP27 and HSP70. In addition, ChIP analysis identified a bona fide FOXC1 binding site in the upstream promoter region of the HSP27 gene, all consistent with HSP27 being a direct target gene of FOXC1.
Validation of HSP27 and HSP70 as direct target genes of FOXC1 supports the idea that FOXC1 plays a role in responding to stress. Molecular chaperones such as HSP27 and HSP70 interact with unfolded or partially folded protein intermediates to promote efficient protein folding in vivo. Dysregulation of molecular chaperones by FOXC1 may disrupt the protein quality control system eventually overloading the protein degradation pathway. The resulting stress may lead to abnormal cell function and apoptosis, thus underlying part of the pathophysiology of ARS.
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