Title: BMP4 induced ID proteins inhibit the fibrotic effects of TGFβ2 in primary human TM cells.

Purpose: Elevated intraocular pressure (IOP) is a major risk factor associated with primary open angle glaucoma (POAG). Increase expression of transforming growth factor β2 (TGFβ2) in POAG aqueous humor (AH) and trabecular meshwork(TM) causes extracellular matrix (ECM) protein deposition in TM, leading to increase in outflow resistance and elevating IOP. However underlying mechanism of BMP4 pathway that regulates the inhibition of TGFβ2 induced fibrosis remains undetermined. BMP4 regulates variety of cellular processes by induction of inhibitor of DNA binding protein (ID1, ID3), which bind and suppress specific transcription factors complex and in turn regulates the gene function. This study will determine whether ID1/ID3 are downstream target of BMP4 that attenuates TGFβ2 induction of ECM proteins. Methods: Primary human TM cells were treated with BMP4 (10ng/ml) for 0-48hr and ID1 and ID3 mRNA and protein expression was determined by Q-PCR and western immunoblotting. Primary TM cells were treated with a BMPR inhibitor to confirm that BMP4 signaling is necessary for induction of ID1 and ID3 protein expression. Further GTM3 cells were transfected with ID1 or ID3 expression vectors to study inhibitory effects on TGFβ2 induced fibronectin and plasminogen activator inhibitor-I (PAI-1) protein expression. Additionally GTM3 cells were transfected with ID1 and ID3 siRNA to determine whether these ID proteins are responsible for the BMP4 suppression of TGFβ2 induced fibronectin expression. Results: BMP4 (10ng/ml) induced early expression of ID1 and ID3 in primary TM cells. Overexpression of ID1 and ID3 significantly inhibited TGFβ2 induced expression of fibronectin and PAI-1 in TM cells. Further, knockdown of ID1 and ID3 suppressed the inhibitory effects of BMP4 on the TGFβ2 induction of fibronectin. Conclusion: BMP4 induced ID1 and ID3 expression suppresses TGFβ2 pro-fibrotic activity in human TM cells. In the future, targeting specific regulators may control the TGFβ2 pro-fibrotic effects on the TM, leading to disease modifying IOP lowering therapies.