TGFβ2 Regulates Human Trabecular Meshwork Cell Contractility via ERK and ROCK Pathways with Distinct Signaling Crosstalk Dependent on the Culture Substrate

Purpose: Transforming growth factor-beta 2 (TGF beta 2) is a major contributor to the pathologic changes occurring in human trabecular meshwork (HTM) cells in primary open-angle glaucoma (POAG). TGF beta 2 activates extracellular-signal-regulated kinase (ERK) and Rho-associated kinase (ROCK) signaling pathways, both affecting HTM cell behavior. However, exactly how these signaling pathways converge to regulate HTM cell contractility is unclear. Here, we investigated the molecular mechanism underlying TGF beta 2-induced pathologic HTM cell contractility, and the cross-talk between ERK and ROCK signaling pathways with different culture substrates. Methods: Hydrogels were engineered by mixing collagen type I, elastin-like polypeptide, and hyaluronic acid, each containing photoactive functional groups, followed by UV crosslinking. Primary HTM cells were seeded atop pre-formed hydrogels for comparisons with glass, or encapsulated within the hydrogels. Changes in actin cytoskeleton, extracellular matrix (ECM) production, phospho-myosin light chain (p-MLC) levels, and hydrogel contraction were assessed. Results: HTM cell morphology and filamentous (F)-actin organization were affected by the underlying culture substrates. TGF beta 2 increased HTM cell contractility via ERK and ROCK signaling pathways by differentially regulating F-actin, alpha-smooth muscle actin (alpha SMA), fibronectin (FN), and p-MLC in HTM cells. ERK inhibition, even as short as 4 h, further increased TGF beta 2-induced p-MLC in HTM cells on hydrogels, but not on glass. This translated into hypercontractility of HTM cell-laden hydrogels. ROCK inhibition had precisely the opposite effects and potently relaxed the TGF beta 2-induced hydrogels. Conclusions: Our data suggest that ERK signaling negatively regulates ROCK-mediated HTM cell contractility. These findings emphasize the critical importance of using tissue-mimetic ECM substrates for investigating HTM cell physiology and glaucomatous pathophysiology in vitro.

Automated summary

This study investigated the molecular mechanism underlying TGF beta 2-induced pathologic HTM cell contractility and found that ERK signaling negatively regulates ROCK-mediated HTM cell contractility.