Most Good, Least Harm: Isoform-Specific Targeting of ROCK in Pulmonary Fibrosis.

Rho-associated coiled-coil kinases (ROCKs), downstream effectors of the Rho family GTPases, play a central role in the regulation of actin cytoskeleton dynamics. They regulate a wide range of fundamental cellular functions, including generation of contractile forces, adhesion, migration, and phagocytosis (1). Both biochemical stimuli (e.g., transforming growth factor b, lysophosphatidic acid, and thrombin) and mechanical cues (e.g., increased matrix stiffness) activate ROCKs in lung fibrosis (2, 3). Pharmacological inhibition of ROCK activity by fasudil, a clinically approved ROCK inhibitor, protects mice against bleomycin injury-induced experimental lung fibrosis (4). Human and mouse ROCKs consist of two isoforms, ROCK1 and ROCK2, which share a 65% overall identity and an z90% identity in the kinase domain (5). Whether these two isoforms have redundant functions remains controversial and appears to depend on the cells in which they are expressed. Earlier studies have reported that ROCK1 and ROCK2 play distinct roles in regulating destabilization of the actin cytoskeleton, cell adhesion/detachment, and differentiation of mouse embryonic fibroblasts and keratinocytes (6, 7). In contrast, a recent study showed that ROCK1 and ROCK2 act redundantly in the regulation of actomyosin contractility in mouse embryonic fibroblasts, and that this function is key to cell-cycle progression and proliferation in vitro and in vivo (8). Additionally, it has been shown that ROCK1 is critical for the formation of stress fibers and focal adhesions, whereas ROCK2 controls cortical contractility and phagocytosis of fibronectin by fibroblasts (9, 10). ROCK activation is central to vascular smooth muscle cell contraction and the control of blood pressure (11). Because ROCK2 has higher expression and activity than ROCK1 in vascular smooth muscle cells (12), preferential inhibition of ROCK2 may be predicted to cause hypotension. Thus, dissecting the functions of ROCK isoforms in lung fibrosis will provide essential information for the development of effective and safer antifibrotic therapies based on targeting of ROCK. In this issue of the Journal, Knipe and colleagues (pp. 471–481) report their findings regarding the role of ROCK isoforms in the development of lung fibrosis in a murine model of bleomycin injury (13). The authors employed a genetic approach by using haploinsufficient mice for selective inhibition of ROCK1, ROCK2, or both isoforms. Previous studies have shown that mice bearing a homozygous deletion of ROCK1 or ROCK2 die during embryogenesis. Haploinsufficient mice are viable into adulthood and exhibit an z50% reduction in protein expression of each ROCK isoform. In this study, the authors first characterized ROCK1 and ROCK2 expression in haploinsufficient mice. In addition to confirming the previous findings, they observed a small compensatory increase in the alternate isoform in each single-isoform haploinsufficient line (a statistically significant increase of ROCK1 in ROCK2 mice, and an insignificant increase of ROCK2 in ROCK1 mice as compared with wildtype mice). The haploinsufficient mice displayed no obvious abnormalities in lung architecture at baseline. The authors observed that both singleand double-isoform haploinsufficient mice were protected from the development of pulmonary fibrosis at Day 14 after bleomycin lung injury. There appeared to be additional protection in the double-isoform haploinsufficient mice, although the difference between singleand double-isoform haploinsufficiency was not statistically significant. ROCK haploinsufficient mice also demonstrated protection against weight loss. Next, the authors examined ROCK isoform haploinsufficiency in the profibrotic responses of alveolar epithelial cells (AECs), endothelial cells, and fibroblasts. The authors found that both ROCK1 and ROCK2 haploinsufficiency protected AECs (primarily type I AECs) from apoptosis at Day 3 after bleomycin challenge. ROCK1 haploinsufficiency demonstrated greater protection of AEC apoptosis than ROCK2 haploinsufficiency. Similarly, protection from bleomycin-induced vascular leak was observed in ROCK1 and ROCK2 haploinsufficient mice at Day 7 after bleomycin challenge, and a reduction inmyofibroblast differentiation was observed at Day 14 after bleomycin injury. Interestingly, in vitro studies showed a reduction in fibroblast-to-myofibroblast differentiation when expression of ROCK1 and ROCK2 was simultaneously knocked down by siRNAs, whereas silencing of each isoform independently was insufficient. This study is significant because it demonstrates that each of the two ROCK isoforms has nonredundant functions in the development of lung fibrosis, at least in the bleomycin murine model. The finding that both isoforms contribute to the profibrotic responses of AEC apoptosis, loss of endothelial barrier function, and myofibroblast differentiation is novel. In particular, the finding that ROCK1 plays a greater role than ROCK2 in the profibrotic response of bleomycin-induced AEC death provides mechanistic insights into the differential functions of ROCK isoforms in lung fibrosis. The study by Knipe and colleagues not only confirms the results of prior studies and supports targeting of ROCK activity as a promising therapeutic strategy for pulmonary fibrosis, but also suggests that selective inhibition of specific ROCK isoforms may provide antifibrotic effects similar to those observed for nonselective ROCK inhibitors such as fasudil while minimizing unwanted on-target adverse effects. A major limitation of this study is that the relevance of the findings to human fibrotic lung diseases, in particular idiopathic pulmonary fibrosis, remains largely unknown, as most of the experiments involved bleomycin-induced mouse lung fibrosis and the results were not validated in human idiopathic pulmonary fibrosis tissues and/or cells. The mechanisms underlying the role of ROCK isoforms in regulating the profibrotic responses of AECs,