Crosstalk Between TGFβ2 and TLR-4 Signaling Pathways in the Glaucomatous Trabecular Meshwork

Purpose: Glaucoma is a heterogeneous group of optic neuropathies that increases extracellular matrix (ECM) proteins in the trabecular meshwork (TM) and leads to thinning of the retinal nerve fiber layer and vision loss. TM regulates aqueous humor outflow and intraocular pressure (IOP). In this study, we employ experimental cell culture methods to determine whether the crosstalk between transforming growth factor beta 2 (TGFβ2) and toll-like receptor 4 (TLR4) signaling pathways regulate ECM production. We hypothesize that TGFβ2-TLR4 crosstalk is a pathway that assist TLR4 ligands to augment TGFβ2 signaling and regulate ECM production in the TM. Methods: Transformed human TM (GTM3) and primary human TM (HTM) cells were grown to confluency. Cells were pre-treated with TAK-242 for 2 hours followed by treatment of TGFβ2 for 24 hours (RNA) or 48 hours (protein). To activate TLR4, HTM cells were plated on dishes pre-coated with cellular fibronectin (cFN) containing the FN-EDA isoform in the presence or absence of TGFβ2. ECM expression was assessed by western immunoblotting and quantified by densitometry. Results: These experiments revealed a TGFβ2-TLR4 signaling crosstalk that regulates ECM production. TGFβ2 treatment in GTM3 cells enhanced fibronectin (FN) and collagen-1 mRNA expression and FN protein expression in the condition medium and cell lysate, while TAK-242 significantly blocked this effect. Activating TLR4 using cFN-EDA alone increased FN expression in HTM3 cells. TGFβ2 with cFN-EDA treatment significantly enhanced FN expression compared to TGFβ2 alone. TLR4 inhibitor TAK-242 significantly blocked TGFβ2 and cFN-EDA induced ECM changes. For future studies, I will use low molecular weight hyaluronan (LMWH) as a TLR4 activator. I expect the LMWH to enhance the effect of TGFβ2 induced ECM production. Conclusions: These studies identified TGFβ2-TLR-4 crosstalk as a novel pathway involved in ECM regulation in the TM. These data provide novel targets to further explore the molecular mechanism involved in glaucomatous TM development.