Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis
Yong Zhou, … , Karen Bernard, Victor J. Thannickal
Yong Zhou, … , Karen Bernard, Victor J. Thannickal
Published February 22, 2013
Citation Information: J Clin Invest. 2013;123(3):1096-1108. https://doi.org/10.1172/JCI66700.
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Research Article

Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis

Abstract

Matrix stiffening and myofibroblast resistance to apoptosis are cardinal features of chronic fibrotic diseases involving diverse organ systems. The interactions between altered tissue biomechanics and cellular signaling that sustain progressive fibrosis are not well defined. In this study, we used ex vivo and in vivo approaches to define a mechanotransduction pathway involving Rho/Rho kinase (Rho/ROCK), actin cytoskeletal remodeling, and a mechanosensitive transcription factor, megakaryoblastic leukemia 1 (MKL1), that coordinately regulate myofibroblast differentiation and survival. Both in an experimental mouse model of lung fibrosis and in human subjects with idiopathic pulmonary fibrosis (IPF), we observed activation of the Rho/ROCK pathway, enhanced actin cytoskeletal polymerization, and MKL1 cytoplasmic-nuclear shuttling. Pharmacologic disruption of this mechanotransduction pathway with the ROCK inhibitor fasudil induced myofibroblast apoptosis through a mechanism involving downregulation of BCL-2 and activation of the intrinsic mitochondrial apoptotic pathway. Treatment with fasudil during the postinflammatory fibrotic phase of lung injury or genetic ablation of Mkl1 protected mice from experimental lung fibrosis. These studies indicate that targeting mechanosensitive signaling in myofibroblasts to trigger the intrinsic apoptosis pathway may be an effective approach for treatment of fibrotic disorders.

Authors

Yong Zhou, Xiangwei Huang, Louise Hecker, Deepali Kurundkar, Ashish Kurundkar, Hui Liu, Tong-Huan Jin, Leena Desai, Karen Bernard, Victor J. Thannickal

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Figure 5

RhoA/ROCK signaling is enhanced in regions of active fibrosis in human IPF and in mice subjected to bleomycin-induced lung injury, as well as in (myo)fibroblasts isolated from fibrotic lungs.

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RhoA/ROCK signaling is enhanced in regions of active fibrosis in human I...
(A) Paraffin-embedded lung tissue sections from non-IPF control subjects (n = 3) and patients with IPF (n = 5) were subjected to immunohistochemical staining for phosphorylated MLC20. Frozen mouse lung tissue sections from saline- or bleomycin-treated mice (n = 5 per group) were subjected to double staining for α-SMA (red) and phosphorylated MYPT-1 (green). Nuclei were stained by DAPI. Confocal microscopic images are overlaid to show the correlation of MYPT-1 phosphorylation with fibrosis. Arrowheads denote relatively normal lung area; arrows denote fibrotic lung area. Scale bars: 50 μm; 200 μm (higher-magnification view of boxed region in IPF panel). Levels of phosphorylated and total MYPT-1 in mouse lung homogenates were also determined by immunoblot. GAPDH was used for loading control. Data are mean ± SD (n = 5 per group). (B) (Myo)fibroblasts isolated from lungs of patients with IPF (n = 8), non-IPF control subjects (n = 6), bleomycin-treated mice (n = 8), and saline-treated mice (n = 8) were subjected to ROCK activity assays using a colorimetric approach. Data are mean ± SD. (C) Relative protein levels of phosphorylated versus total ERM in isolated (myo)fibroblasts by densitometric analysis. Data are mean ± SD (n = 6 or 8). Representative immunoblots are shown. (D) Level of active RhoA (normalized by GAPDH) in isolated (myo)fibroblasts by densitometric analysis. Data are mean ± SD (n = 8 per group). Representative immunoblots are shown. *P < 0.05, **P < 0.01, ***P < 0.001.