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Hamid Ahmadieh, Hamidreza Moazzeni, Elahe Elahi, Mohammad Taghi Akbari, Shahin Yazdani, Mozhgan Rezaeikanavi, Mehraban Mirrahimi, Hassan paylakhi; Bidirectional regulation of PITX2 and TGF-β signaling in human trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4681.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is a leading cause of blindness, and elevated intraocular pressure (IOP) is its most important risk factor. The trabecular meshwork (TM) is very relevant to glaucoma pathogenesis. It affects IOP by contributing to aqueous humor outflow resistance. Pituitary homeobox2 (PITX2) is a transcription factor that affects eye development and is implicated in glaucoma because mutations in its gene can cause Axenfeld-Rieger syndrome that is often accompanied by glaucoma. We aimed to identify PITX2 target genes in the TM that may be involved in glaucoma by using microarray TM gene expression data, in silico bioinformics tools, and empirical studies.
SiRNA knockdowns and microarray expression analysis were performed to identify genes in human TM primary cultures that are affected by PITX2. Among genes identified, those with PITX2 binding sites within their promoters and therefore potentially directly regulated by PITX2 were selected using DECODE and TRANSFAC databases. Evolutionary conservation of the binding sites was confirmed with PipMaker. DISGENET, KEGG, STRING and Gencard were used to further select candidate genes with roles specifically in glaucoma. Promoters of these genes were cloned into the PGL4-14 vector. Co-transfection studies with the PGL4-14 vectors and a PITX2 expression vector were performed in three cell lines, and dual luciferase assay data were used to confirm the validity of the bioinformatics based results.
Knockdown experiments and microarray data analysis identified 41 genes that may be affected by PITX2. Among these, 34 had evolutionarily conserved PITX2 binding sites within their promoters. Four, NOMO2, ALDH1A1, CXCL6 and ADAMTS5, were selected as candidate genes with roles in glaucoma. The dual luciferase assay confirmed that NOMO2 and CXCL6 are direct targets of PITX2.
The combined bioinformatics empirical studies showed that the expression of two potentially glaucoma related genes, NOMO2 and CXCL6, is directly regulated by PITX2. CXCL6 is a cytokine implicated in aqueous fluid outflow regulation. NOMO2 is a known modulator of the nodal signaling pathway. Nodal signaling, like TGF-β signaling, can activate SMAD2 and SMAD3 which in turn affect PITX2 expression. Our finding on regulation of NOMO2 by PITX2 suggests that there is a bidirectional regulation between PITX2 and TGF-β signaling.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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