BMP4 INDUCED ID PROTEIN PROTECTS TM FROM GLAUCOMATOUS EFFECTS OF TGFβ-2

Insight into the BMP4 pathways in various disease models and different cell types has shown BMP4 to be a potent inducer of inhibitor of DNA binding proteins (ID1 and ID3). ID1 and ID3 are negative regulators of basic Helix loop Helix (bHLH) transcription factors and are known to control specific gene expression, including extracellular cellular matrix (ECM) genes. We previously have shown that BMP4 attenuates the pathogenic effects of TGFβ2 in the TM, an ocular tissue involved in regulation of intraocular pressure in glaucoma. We hypothesize that BMP-4 attenuates the effects TGFβ2 in the TM by inducing ID1 and ID3 expression. In our current study, we show that BMP4 induces ID1 and ID3 expression in TM cells. Over-expression and knockdown of ID1 and ID3 in TM cells show that ID1 and ID3 play a crucial role in attenuating the profibrotic effects of TGFβ2 in TM cells. Purpose (a): Increased aqueous humor (AH) outflow resistance causes high intraocular pressure (IOP), which is a critical risk factor in primary open-angle glaucoma. Elevated transforming growth factor b2 (TGFβ2) in the AH of glaucoma patients increases extracellular matrix (ECM) protein deposition in the trabecular meshwork (TM), thereby elevating IOP. Bone morphogenetic protein 4 (BMP4) inhibits the pathogenic effects of TGFβ2 in the TM. However, the underlying molecular mechanism for this BMP4 inhibition remains unknown. BMP4 regulates various cellular processes by induction of inhibitors of DNA binding proteins (ID1, ID3), which are transcriptional regulators that bind specific transcription factors and suppress their functions. This study will determine whether ID1/ID3 are downstream targets of BMP4, attenuating the TGFb-2 effects on TM cells. Methods (b): Cultured primary human TM cells and the GTM3 cell line were treated with BMP4 (5-10ng/ml) for 1-48 hrs. Q-PCR and western immunoblotting were performed to determine ID1 and ID3 expression. GTM3 and primary TM cells were transfected with ID1 and ID3 expression plasmids vectors or ID1 and ID3 siRNA to determine the effects of ID1 and ID3 on TGFβ2 induced extracellular matrix (ECM) proteins. The expression of fibronectin and plasminogen activator inhibitor-1 (PAI-1) was studied by western immunoblotting. Results (c): BMP4 induced ID1 and ID3 expression in TM cells. ID1 and ID3 suppressed the TGFβ2 induction of ECM proteins in TM cells, and therefore are key signaling molecules involved in the BMP4 suppression of TGFβ2 profibrotic activity. These specific regulators controlling TGFβ2 effects in the TM may lead to the development of potential new IOP lowering therapies for the treatment of glaucoma. Conclusions (d): BMP4 induced ID1 and ID3 expression in TM cells. ID1 and ID3 suppressed the TGFβ2 induction of ECM proteins in TM cells, and therefore are key signaling molecules involved in the BMP4 suppression of TGFβ2 profibrotic activity. These specific regulators controlling TGFβ2 effects in the TM may lead to the development of potential new IOP lowering therapies for the treatment of glaucoma.