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M. A. Walter, J. Skarie, B. A. Link, F. B. Berry; FOXC1 Is Required for Cell Viability and Resistance to Oxidative Stress in the Eye. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5915.
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To identify patho-physiological pathways responsible for Axenfeld-Rieger malformations resulting from FOXC1 mutations. We have determined that FOXC1 directly regulates FOXO1A expression. Since FOXO1A is involved regulation of cell death and resistance to cellular stress, we examined the role of FOXC1 in cell viability in response to oxidative damage.
Expression of FOXC1 was reduced in human trabecular meshwork (HTM) cells by siRNA transfection. Cells were treated with H2O2 to evoke oxidative stress. Cell death was measured by trypan blue uptake and by monitoring cleavage of Caspase 7.
HTM cells transfected with FOXC1-specific siRNAs displayed reduced FOXC1 protein levels and resulted in a concomitant decrease in FOXO1A expression, consistent with the observation that FOXO1A is a target of FOXC1 transcriptional regulation. We demonstrate that reducing FOXC1 expression by siRNA transfection resulted in an increased percentage of cells immunoreactive for cleaved Caspase 7. This increase was augmented when cells were treated with 500 uM H2O2 for 2 hours. Through immunoblot analysis, we observed a decrease in FOXO1A expression when cells were treated with FOXC1 siRNAs and an increase in the cleavage of Caspase 7, which was again elevated when cells were treated with H2O2. Furthermore increased acridine orange staining was also observed in the developing zebrafish eye when foxC1 levels are knocked-down with morpholino antisense oligonucleotides, indicative of an increase in cell death.
These studies have uncovered a novel role for FOXC1 as a survival factor in the eye. Loss of FOXC1 expression reduces TM cell number through increased apoptosis and such a loss in cell number would impair the aqueous humor out flow capacity. Our results are consistent with the notion that a decreased resistance to oxidative stress underlies AR-glaucoma pathogenesis.
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