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Michael A. Walter, Tim Footz, Vincent Raymond, Fred Berry, Pascal Belleau, Marcela H. Strungaru; Involvement Of Pitx2 In Ocular Cellular Stress Response Through Regulation Of SLC13A3. Invest. Ophthalmol. Vis. Sci. 2011;52(14):76.
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Mutations of the PITX2 transcription factor gene cause Axenfeld-Rieger Syndrome (ARS) and glaucoma. By studying the genes directly regulated by PITX2, we gain insight into the mechanisms underlying these disorders. Here we have identified and characterized solute carrier family 13 sodium-dependent dicarboxylate transporter member 3 (SLC13A3) as a gene directly regulated by PITX2 using a hormone-inducible PITX2 expression system.
A hormone-inducible PITX2 expression system was generated to identify genes directly regulated by PITX2. RNA from non-pigmented ciliary body cells (NPCEs) transfected with hormone-inducible PITX2, or a negative control, was subjected to microarray analyses using Affymetrix U133A arrays. Data were analyzed using D-CHIP algorithms to detect significant differences in expression. Genes with significantly altered expression in multiple microarray experiments in the presence of PITX2 were subjected to in silico and biochemical analyses.
SLC13A3 was identified as a potential PITX2 target gene by microarray analysis and RT-PCR. PITX2 directly regulates SLC13A3 expression as demonstrated by luciferase reporter and chromatin immunoprecipitation (ChIP) assays. Since oxidative stresses have been implicated in the pathology of glaucoma, the involvement of the PITX2-> SLC13A3 regulatory pathway in the ocular oxidative stress response was determined. Reduction in PITX2 or SLC13A3 levels produced an increased in HTM cell death when the cells were exposed to oxidative stress such as hydrogen peroxide.
These results indicate that SLC13A3 is a direct downstream target of PITX2 and indicate for the first time that PITX2, through SLC13A3, is involved in ocular oxidative stress pathways.
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