Possible bidirectional regulation of PITX2 and TGF-β signaling in human trabecular meshwork cells

Glaucoma is a leading cause of blindness, and elevated intraocular pressure (IOP) is its most important risk factor. The trabecular meshwork (TM) in the eye affects IOP by contributing to aqueous humor outflow resistance, and is thus very relevant to glaucoma pathogenesis. PITX2 is a transcription factor that affects development of the eye, and mutations in its encoding gene are a common cause of Axenfeld-Rieger syndrome (ARS). As ARS is very often accompanied with glaucoma, it is reasonable to consider that PITX2 malfunction or the malfunction of its target genes may contribute to glaucoma. In an earlier study, PITX2 (pituitary homeobox2) target genes were identified by microarray analysis, and NOMO that encodes NODAL modulator was among the identified genes. Effect on NOMO was then confirmed using a dual luciferase promoter assay. As NODAL signaling is a form of TGFB signaling, and TGFB signaling is known to be an important glaucoma related pathway. We aimed to further assess effect of PITX2 on NOMO and Nodal signaling molecules including NODAL, SMAD2 and SMAD3 in the TM cells.Methods: Knockdown of PITX2 with PITX2 siRNA was performed to determine whether NOMO is affected by PITX2 in human TM primary cultures. Expression of NOMO and Nodal signaling molecules including NODAL, SMAD2 and SMAD3 were examined by RT-qPCR and Western blotting in TM cells.Results: Knockdown experiments identified NOMO expression was affected by PITX2. In addition, expression of SMAD2 and SMAD3 were altered by PITX2 knockdown.Conclusion: Our results supported earlier findings and identified NOMO as a potentially glaucoma related gene that is directly regulated by PITX2. Furthermore, downstream effects of PITX2 knockdown on expression of SMAD2 and SMAD3 which are important nodal signaling molecules may disrupt this pathway. This scenario predicts bidirectional regulation between PITX2 and TGF-β signaling.