Department of Ophthalmology and Visual Sciences, SUNY Upstate Medical University, Syracuse, NY, USA.
Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY, USA.
Curr Eye Res. 2022 Aug;47(8):1165-1178. doi: 10.1080/02713683.2022.2071943. Epub 2022 May 19.
Transforming growth factor-beta 2 (TGFβ2) is a major contributor to the pathologic changes occurring in human trabecular meshwork (HTM) cells in primary open-angle glaucoma (POAG). TGFβ2 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 TGFβ2-induced pathologic HTM cell contractility, and the crosstalk between ERK and ROCK signaling pathways with different culture substrates.
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.
HTM cell morphology and filamentous (F)-actin organization were affected by the underlying culture substrates. TGFβ2 increased HTM cell contractility ERK and ROCK signaling pathways by differentially regulating F-actin, α-smooth muscle actin (αSMA), fibronectin (FN), and p-MLC in HTM cells. ERK inhibition, even as short as 4 h, further increased TGFβ2-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 TGFβ2-induced hydrogels.
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 .
转化生长因子-β2（TGFβ2）是导致原发性开角型青光眼（POAG）患者小梁网（HTM）细胞发生病理变化的主要因素。TGFβ2 激活细胞外信号调节激酶（ERK）和 Rho 相关激酶（ROCK）信号通路，这两种信号通路都影响 HTM 细胞的行为。然而，这些信号通路如何协同调节 HTM 细胞收缩力尚不清楚。本研究旨在探讨 TGFβ2 诱导病理性 HTM 细胞收缩力的分子机制，以及 ERK 和 ROCK 信号通路在不同培养底物中的相互作用。
通过混合含有光活性官能团的 I 型胶原蛋白、弹性蛋白样多肽和透明质酸来制备水凝胶，然后进行 UV 交联。将原代 HTM 细胞接种在预先形成的水凝胶上（与玻璃相比）或包埋在水凝胶中，以比较其形态变化、细胞外基质（ECM）产生、磷酸化肌球蛋白轻链（p-MLC）水平和水凝胶收缩情况。
HTM 细胞形态和丝状（F）肌动蛋白的组织均受到培养底物的影响。TGFβ2 通过差异调节 HTM 细胞中的 F-肌动蛋白、α-平滑肌肌动蛋白（αSMA）、纤维连接蛋白（FN）和 p-MLC，激活 ERK 和 ROCK 信号通路。ERK 抑制（即使只有 4 小时）也会进一步增加 TGFβ2 诱导的 HTM 细胞在水凝胶上的 p-MLC，但在玻璃上没有这种作用。这导致 HTM 细胞包埋水凝胶的过度收缩。ROCK 抑制则产生相反的效果，强烈缓解 TGFβ2 诱导的水凝胶收缩。
我们的数据表明，ERK 信号负调节 ROCK 介导的 HTM 细胞收缩力。这些发现强调了使用组织模拟 ECM 底物对于研究 HTM 细胞生理学和青光眼病理生理学的重要性。
