Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis
Yong Zhou, Xiangwei Huang, Louise Hecker, Deepali Kurundkar, Ashish Kurundkar, Hui Liu, Tong-Huan Jin, Leena Desai, Karen Bernard, Victor J. Thannickal
Yong Zhou, Xiangwei Huang, Louise Hecker, Deepali Kurundkar, Ashish Kurundkar, Hui Liu, Tong-Huan Jin, Leena Desai, Karen Bernard, Victor J. Thannickal
View: Text | PDF
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

×

Figure 3

Fasudil downregulation of BCL-2 expression occurs by deactivation of MKL1-mediated intrinsic mechanotransduction.

Options: View larger image (or click on image) Download as PowerPoint
Fasudil downregulation of BCL-2 expression occurs by deactivation of MKL...
(A) Subcellular distribution of MKL1 in IPF lung myofibroblasts and control fibroblasts, determined by subcellular fractionation followed by immunoblot. Lamin A/C and GAPDH were used as loading controls for nucleic and cytoplasmic proteins, respectively. (B) F-actin (F) and G-actin (G) content in IPF lung fibroblasts and control fibroblasts, determined by immunoblot and densitometric analysis. (C and D) IPF myofibroblasts and control fibroblasts were treated with PBS or 25 μM fasudil for 24 hours. (C) MKL1 subcellular localization, determined as in A. (D) F-actin and G-actin content, determined as in B. (E) Binding of MKL1-SRF complex to BCL2 promoter was quantified by quantitative ChIP. Data are mean ± SD of 3 separate experiments. (F) WT and 3 mutated human BCL2 promoters: CArG box1 (b1 mut), CArG box2 (b2 mut), or both box1 and box2 (b1&2 mut). Promoter reporters were transfected into IPF myofibroblasts and treated with PBS or 25 μM fasudil for 24 hours. Promoter activity was determined by luciferase assay. Data are mean ± SD of 3 separate experiments, each performed in triplicate. EV, empty vector. (G) IPF myofibroblasts were treated with PBS or 25 μM fasudil in the presence or absence of 200 nM jasplakinolide (Jas) for 24 hours. A subset of cells was transfected with constitutively active MKL1 (caMKL1) or empty vector before fasudil treatment. Protein levels of BCL-2 and GAPDH were determined by immunoblot. (H) IPF myofibroblasts were cultured in the presence or absence of 1 μM latrunculin B (LatB) or 1 μM CCG-1423 (CCG) for 24 hours. A subset of cells was transfected with dnMKL1-expressing plasmid or empty vector. Protein levels of BCL-2 and GAPDH were determined by immunoblot. *P < 0.05, **P < 0.01.