The Rho kinases: critical mediators of multiple profibrotic processes and rational targets for new therapies for pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive lung scarring, short median survival, and limited therapeutic options, creating great need for new pharmacologic therapies. IPF is thought to result from repetitive environmental injury to the lung epithelium, in the context of aberrant host wound healing responses. Tissue responses to injury fundamentally involve reorganization of the actin cytoskeleton of participating cells, including epithelial cells, fibroblasts, endothelial cells, and macrophages. Actin filament assembly and actomyosin contraction are directed by the Rho-associated coiled-coil forming protein kinase (ROCK) family of serine/threonine kinases (ROCK1 and ROCK2). As would therefore be expected, lung ROCK activation has been demonstrated in humans with IPF and in animal models of this disease. ROCK inhibitors can prevent fibrosis in these models, and more importantly, induce the regression of already established fibrosis. Here we review ROCK structure and function, upstream activators and downstream targets of ROCKs in pulmonary fibrosis, contributions of ROCKs to profibrotic cellular responses to lung injury, ROCK inhibitors and their efficacy in animal models of pulmonary fibrosis, and potential toxicities of ROCK inhibitors in humans, as well as involvement of ROCKs in fibrosis in other organs. As we discuss, ROCK activation is required for multiple profibrotic responses, in the lung and multiple other organs, suggesting ROCK participation in fundamental pathways that contribute to the pathogenesis of a broad array of fibrotic diseases. Multiple lines of evidence therefore indicate that ROCK inhibition has great potential to be a powerful therapeutic tool in the treatment of fibrosis, both in the lung and beyond.

Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Figures

Fig. 1.

Structure and activation of the two ROCK isoforms. In the closed inactive conformation, the RhoA-binding domains (RBDs) and the kinase domains of ROCK1 and ROCK2 fold over on each other (left). Activated GTP-bound RhoA is able to bind the RBDs, shifting the ROCKs to an open conformation (right), exposing and activating the kinase domains.

Fig. 2.

ROCK control of cytoskeletal dynamics. ROCK isoforms are activated by GTP-bound RhoA (as in Fig. 1) downstream of G protein–coupled receptors, such as LPA1 and PAR1. Activated ROCKs phosphorylate MLC phosphatase, inhibiting its ability to dephosphorylate (and inactivate) MLC. Persistently phosphorylated active MLC is then able to induce stress fiber and focal adhesion formation, and cell contraction.

Fig. 3.

ROCK activation of MTRF-SRF–directed profibrotic gene expression. ROCK activation has been shown to induce profibrotic gene expression through its ability to drive actin polymerization, which induces the nuclear translocation of G-actin–binding transcriptional coactivators, such as MRTF-A and MRTF-B. In this pathway, activation of G protein–coupled receptors, such as LPA1, or matrix stiffening, induces ROCK activation, which, in turn, drives G-actin polymerization into F-actin. G-Actin binds MRTF-A and MRTF-B and sequesters them in the cytoplasm. Actin polymerization into F-actin liberates G-actin–bound MRTF-A and MRTF-B, allowing their translocation to the nucleus where they transactivate SRF-dependent transcription of important profibrotic genes containing serum response elements (SREs) in their promoters, such as CTGF.

Fig. 4.

ROCK activation of YAP/TAZ-TEAD–directed profibrotic gene expression. In addition to the MRTF-SRF pathway, ROCK has recently been shown to regulate gene expression through the downstream effectors of the Hippo pathway, the transcriptional coactivators YAP and TAZ. YAP and TAZ bind and activate the TEAD family of transcription factors, which directs the expression of genes that promote cell proliferation, including CTGF. The subcellular localization of YAP and TAZ is determined by their phosphorylation state. Phosphorylation of YAP and TAZ by Lats 1/2 kinases results in their sequestration in the cytoplasm due to binding by 14-3-3 regulatory proteins; inhibition of Lats 1/2 kinases results in YAP/TAZ dephosphorylation and nuclear translocation. LPA activation of its G protein–coupled receptors has been shown to inhibit Lats 1/2 kinases, and promote YAP/TAZ dephosphorylation, as has matrix stiffening, through a signaling pathway that also involves ROCK activation and actin polymerization.

Fig. 5.

Role of ROCK activation in aberrant responses to lung injury implicated in IPF pathogenesis. This schematic indicates the sequential profibrotic processes implicated in the currently prevailing paradigm of IPF pathogenesis, in which recurrent or persistent injury to the alveolar epithelium is thought to drive aberrant wound healing responses, resulting in fibrosis rather than repair. (Figure was adapted from Ahluwalia et al., 2014, and inspired by Selman et al., 2001.) Proposed roles of ROCK activation in cells participating in IPF pathogenesis are placed in the context of the profibrotic process(es) they are thought to mediate.