Glaucoma is a leading cause of blindness. Elevated intraocular pressure (IOP) and the trabecular meshwork (TM) are important components in glaucoma etiology. FOXC1 and PITX2 are transcription factors that affect eye development. Their genes can cause Axenfeld-Rieger Syndrome (ARS), and are implicated in glaucoma because glaucoma often manifests in ARS. We aimed to identify genes in the TM affected by these factors and the molecular pathways implicated.

PITX2 and FOXC1 siRNA knockdowns and microarray analysis were performed in human TM primary cultures. In order to identify genes directly affected by the encoded factors, evolutionarily conserved DNA binding sites in promoters were sought using DECODE, TRANSFAC, and Pipmaker bioinformatics tools. Subsequently, genes relevant to the TM and glaucoma were identified using KEGG, STRING, and DISGENET. Finally, GEMEMANIA, CHEMBlE, and BINGO were used to identify molecular pathways implicated by the genes. A model of gene interaction was derived and parts of the model were assessed by real time PCR, immunoblotting, and dual luciferase assays.

Microarrays identified 41 and 849 genes, respectively, affected by PITX2 and FOXC1 knockdowns. Bioinformatic analysis identified 34 and 170 genes, respectively, with PITX2 and FOXC1 binding sites. Among these genes, NOMO2, ALDH1A1, CXCL6 and ADAMTS5 for PITX2 and PLEKHG5, CXCL6 and MEIS2 for FOXC1 have functions in the TM and are related to glaucoma. Three important pathways, TGF? signaling, a pathway involving MEIS2 and PAX6, and Rho signaling, were implicated by these genes. Real time PCR, immunoblotting and luciferase assays confirmed predicted effects of the genes in Hela cells, Hek-293 and TM primary cultures.

The results suggest that PITX2 can through NOMO2 regulate expression of SMADS that are components of the TGFβ signaling pathway. As SMADS regulates PITX2 expression, it becomes evident that PITX2 and the SMADS regulate each other bidirectionally. FOXC1 enhanced PLEKHG5 expression. PLEKHG5 is a regulator of the Rho signaling pathway, a pathway is known to affect IOP. It is suggested that FOXC1 affects glaucoma through its effect on IOP mediated by PLEKHG5. Finally, FOXC1 affected the expression of MEIS2 and PAX6, transcription factors important in the development of the TM.