Role of Rho-Associated Coiled-Coil Forming Kinase Isoforms in Regulation of Stiffness-Induced Myofibroblast Differentiation in Lung Fibrosis

Abstract

Fibrosis is a major cause of progressive organ dysfunction in several chronic pulmonary diseases. Rho-associated coiled-coil forming kinase (ROCK) has been shown to be involved in myofibroblast differentiation driven by altered matrix stiffness in a fibrotic state. There are two known ROCK isoforms in humans, ROCK1 and ROCK2, but the specific role of each isoform in myofibroblast differentiation in lung fibrosis remains unknown. To study this, we developed a gelatin methacryloyl hydrogel-based culture system with different stiffness levels relevant to healthy and fibrotic lungs. We have shown that stiff matrix, but not soft matrix, can induce myofibroblast differentiation with high smooth muscle actin isoform (αSMA) expression. Furthermore, our data confirmed that the inhibition of ROCK signaling by a pharmacological inhibitor (i.e., Y27632) attenuates stiffness-induced αSMA expression and fiber assembly in myofibroblasts. To assess the role of ROCK isoforms in this process, we used short interfering RNA to knock down the expression of each isoform. Our data showed that knocking down either ROCK1 or ROCK2 did not result in a reduction in αSMA expression in myofibroblasts on stiff matrix, as opposed to soft matrix, where αSMA expression was reduced significantly. Paradoxically, on stiff matrix, the absence of one isoform (particularly ROCK2) exaggerated αSMA expression and led to thick fiber assembly. Moreover, complete loss of αSMA fiber assembly was seen only in the absence of both ROCK isoforms, suggesting that both isoforms are implicated in this process. Overall, our results indicate the differential role of ROCK isoforms in myofibroblast differentiation on soft and stiff matrices.

The Role of the RhoA/ROCK Signaling Pathway in Mechanical Strain-Induced Scleral Myofibroblast Differentiation

“…ROCK1 and ROCK2 are downstream signals of RhoA and are reported to be involved in the mechanotransduction pathway. 59 This study focused mainly on the role of ROCK2, as ROCK2 had greater expression than ROCK1 in the eye. 19 Further studies are needed to explore whether ROCK1 has a role in myopia sclera remodeling.…”

Effect of Rho-kinase inhibition on complexity of breathing pattern in a guinea pig model of asthma

“…Results obtained from animal models indicate that Rho-kinase has a main role in the pathophysiology of asthma [ 20 – 23 ]. A previous study demonstrated that challenge with OVA can induce Rho-kinase activation [ 23 ] that is associated with increased collagen synthesis [ 47 ], α-SMA expression and fiber assembly, F-actin polymerization [ 48 ] and myosin filament assembly in myofibroblasts [ 49 ]. We found that the inhibition of Rho-kinase decreases AHR and airway remodeling in asthmatic animal.…”

Targeting extracellular matrix stiffness to attenuate disease: From molecular mechanisms to clinical trials

“…ROCK is the major downstream effector of Rho that drives cell contractility and is a mediator of fibrotic pathologies (93,107). ROCK phosphorylates myosin light chain and myosin light chain phosphatase to induce the generation of cellular force from actomyosin filament contraction.…”

“…ROCK phosphorylates myosin light chain and myosin light chain phosphatase to induce the generation of cellular force from actomyosin filament contraction. Recently, a stiffness-dependent role for ROCK in driving fibroblast to myofibroblast conversion in pulmonary fibrosis was identified, and ROCK inhibition attenuated increased endothelial permeability caused by age-related increased intima stiffness (107,108). ROCK inhibition can be pursued using traditional small-molecule discovery efforts but is also plagued by nonspecificity.…”