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M. H. Strungaru, T. Footz, E. Deilhes, P. Belleau, V. Raymond, F. Berry, M. A. Walter; The Axenfeld-Rieger PITX2 Transcription Factor Directly Regulates the Expression of Solute Carrier Family 13 Sodium-Dependent Dicarboxylate Transporter Member 3 (SLC13A3). Invest. Ophthalmol. Vis. Sci. 2009;50(13):2074.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations of the PITX2 transcription factor cause Axenfeld-Rieger Syndrome (ARS) and glaucoma. We hypothesize that by studying the genes directly regulated by PITX2, we will elucidate genetic mechanisms underlying ocular development and disorders including ARS and glaucoma. Here, we have identified and characterized the Solute carrier family 13 sodium-dependent dicarboxylate transporter member 3 (SLC13A3) as gene directly regulated by of PITX2.
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. In silico analyses revealed that the SLC13A3 is highly conserved among species, contains PITX2 binding sites in its upstream region and it is expressed in the eye and brain. Molecular analyses indicated that PITX2 significantly activated transcription from reporter plasmids containing SLC13A3 upstream elements, while mutated PITX2 significantly decreased transcription from reporter plasmids containing SLC13A3 upstream elements. Chromatin immunoprecipitation (ChIP) showed that PITX2 directly binds to a SLC13A3 upstream element. Reducing PITX2 expression in human trabecular meshwork (HTM) cells by silencing PITX2 expression resulted in reduced expression of SLC13A3. Over expression of PITX2 in HTM cells resulted in increased expression of SLC13A3.
Our results indicate that SLC13A3 is a direct downstream target of PITX2. Interestingly, it has been suggested that SLC13A3 is involved in transporting glutathione, a known defense mechanism molecule against reactive oxygen species. Since cellular stresses have been implicated in the pathology of glaucoma, analyses of the involvement of the PITX2-> SLC13A3 regulatory pathway in the ocular cellular stress response is underway.
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