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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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 2

Fasudil promotes myofibroblast apoptosis by BCL-2 downregulation.

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Fasudil promotes myofibroblast apoptosis by BCL-2 downregulation. (A) IP...
(A) IPF lung myofibroblasts were treated with 25 μM fasudil for 24 hours. Cell lysates were collected at the indicated time points. Release of cytochrome c (Cyto c) from mitochondria was evaluated by a change in the levels of cytochrome c in the mitochondrial (Mito) and cytoplasmic (Cyto) fractions. VDAC and GAPDH were used as loading controls for mitochondrial and cytoplasmic proteins, respectively. (B) IPF lung myofibroblasts were treated with 0–50 μM fasudil for 24 hours. Relative levels of BCL2, Bcl-xL, and Mlc-1 mRNA were determined by real-time RT-PCR. 18s rRNA was used as internal reference control. Data are mean ± SD of 3 separate experiments. (C) Protein levels of BCL-2, Bcl-xL, and Mlc-1 (24 hours after treatment) were determined by immunoblot. GAPDH was used as loading control. (D) IPF lung myofibroblasts were treated with 25 μM fasudil or an equal volume of PBS for 24 hours. Cleavage of caspase 9 and caspase 3 was determined by immunoblot analysis. GAPDH was used as loading control. Caspase activities were measured with a colorimetric assay. Data are mean ± SD of 3 separate experiments. (E) Constitutive expression of BCL-2 protein in IPF myofibroblasts and non-IPF control fibroblasts were determined by immunoblot analyses. GAPDH was used as loading control. Densitometry was performed using ImageJ. Relative density of BCL-2 was normalized to GAPDH. Data are mean ± SD (n = 4 per group). *P < 0.05, **P < 0.01.