TRPV4 mediates myofibroblast differentiation and pulmonary fibrosis in mice
Shaik O. Rahaman, … , Daniel J. Tschumperlin, Mitchell A. Olman
Shaik O. Rahaman, … , Daniel J. Tschumperlin, Mitchell A. Olman
Published November 3, 2014
Citation Information: J Clin Invest. 2014;124(12):5225-5238. https://doi.org/10.1172/JCI75331.
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Research Article Pulmonology

TRPV4 mediates myofibroblast differentiation and pulmonary fibrosis in mice

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disorder with no effective medical treatments available. The generation of myofibroblasts, which are critical for fibrogenesis, requires both a mechanical signal and activated TGF-β; however, it is not clear how fibroblasts sense and transmit the mechanical signal(s) that promote differentiation into myofibroblasts. As transient receptor potential vanilloid 4 (TRPV4) channels are activated in response to changes in plasma membrane stretch/matrix stiffness, we investigated whether TRPV4 contributes to generation of myofibroblasts and/or experimental lung fibrosis. We determined that TRPV4 activity is upregulated in lung fibroblasts derived from patients with IPF. Moreover, TRPV4-deficient mice were protected from fibrosis. Furthermore, genetic ablation or pharmacological inhibition of TRPV4 function abrogated myofibroblast differentiation, which was restored by TRPV4 reintroduction. TRPV4 channel activity was elevated when cells were plated on matrices of increasing stiffness or on fibrotic lung tissue, and matrix stiffness–dependent myofibroblast differentiation was reduced in response to TRVP4 inhibition. TRPV4 activity modulated TGF-β1–dependent actions in a SMAD-independent manner, enhanced actomyosin remodeling, and increased nuclear translocation of the α-SMA transcription coactivator (MRTF-A). Together, these data indicate that TRPV4 activity mediates pulmonary fibrogenesis and suggest that manipulation of TRPV4 channel activity has potential as a therapeutic approach for fibrotic diseases.

Authors

Shaik O. Rahaman, Lisa M. Grove, Sailaja Paruchuri, Brian D. Southern, Susamma Abraham, Kathryn A. Niese, Rachel G. Scheraga, Sudakshina Ghosh, Charles K. Thodeti, David X. Zhang, Magdalene M. Moran, William P. Schilling, Daniel J. Tschumperlin, Mitchell A. Olman

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Figure 3

TRPV4 is required for TGF-β1–induced lung myofibroblast differentiation.

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TRPV4 is required for TGF-β1–induced lung myofibroblast differentiation....
HLFs were plated on fibronectin-coated (10 μg/ml) plastic wells and incubated with or without TGF-β1 (2 ng/ml, 24 hours), TRPV4 siRNA, or scrambled siRNA. (A) Representative immunoblots show knockdown of TRPV4 proteins by TRPV4-specific siRNA and blocking of TGF-β1–induced α-SMA expression under conditions of TRPV4 knockdown. (B and C) Quantification of (B) TRPV4/GAPDH and (C) α-SMA/GAPDH protein bands from A. *P < 0.05 scrambled vs. TRPV4 siRNA-treated cells, #P < 0.05 TGF-β1–treated cells treated with scrambled siRNA vs. TRPV4 siRNA; n = 3. (D) Representative fluorescence micrographs (original magnification, ×20). Myofibroblast differentiation is reduced in fibroblasts from Trpv4 KO mice (colocalization of α-SMA and F-actin, orange). (E) Quantification of results from D by Pearson’s coefficient analysis. **P < 0.01; TGF-β1–treated WT vs. Trpv4 KO cells; n > 18 cells per group. UT, untreated. (F) Reconstitution of TRPV4 into Trpv4 KO mouse lung fibroblasts (MLFs) using a lentivirus expression system (lenti-TRPV4-GFP) restores myofibroblast differentiation in response to TGF-β1. Lenti-GFP–infected Trpv4 KO mouse lung fibroblasts were used as negative control; uninfected WT mouse lung fibroblasts were used as positive control. Original magnification, ×20. (G) TRPV4 blockade has a greater inhibitory effect on myofibroblast differentiation (α-SMA/GAPDH band density in immunoblots) in fibroblasts from patients with IPF than in normal fibroblasts. (H) Quantitation of results from G. *P < 0.05; n = 5 per group. Results are expressed as mean ± SEM.