The Axenfeld-Rieger PITX2 Transcription Factor Directly Regulates the Expression of the Cellular-Stress Responsive PDP2 Gene

Abstract

Purpose:: The pituitary homeobox 2 (PITX2) transcription factor is involved in the development and regulation of several organs, including the eye. PITX2 mutations result in Axenfeld-Rieger malformations and glaucoma. The identification of direct targets of PITX2 is necessary for understanding the genetic mechanisms underlying ocular development and disorders including glaucoma. Here, we identified PITX2 target genes in the eye and characterized Pyruvate dehydrogenase phosphatase (PDP2) as a target gene directly regulated by PITX2.

Methods:: 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 the Affymetrix U133A arrays. Data was analyzed using D-CHIP algorithms and Bioconductor 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.

Results:: Microarray experiments revealed that PITX2 reproducibly and significantly altered the expression of 498 genes in NPCEs. Ten of the 15 genes tested by Northern assays displayed altered expression in the presence of PITX2. One of these potential PITX2 target genes, PDP2, was selected for further analyses. In silico analyses revealed that PDP2 gene is highly conserved among species and it is expressed in the eye and brain. Molecular analyses indicated that PITX2 significantly activated transcription from reporter plasmids containing PDP2 upstream elements.

Conclusions:: Our analyses indicate that PITX2 activates genes in the anterior segment of the eye involved in a variety of key processes including response to cellular stress, development and visual function. Our results also indicate that PDP2 is a direct downstream target of PITX2. Interestingly, PDP2 activity is affected by chemical stresses, which decrease PDP2 activity altering cell function and viability. Since cellular stresses have been implicated in the pathology of glaucoma, analyses of the involvement of the PITX2-> PDP2 regulatory pathway in the ocular cellular stress response is underway.

Keywords: gene microarray • transcription factors • candidate gene analysis