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 2

TRPV4 calcium channel is expressed and functional in HLFs and murine lung fibroblasts.

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TRPV4 calcium channel is expressed and functional in HLFs and murine lun...
(A) Representative immunoblots of TRPV4 protein (~88 kDa) and GAPDH loading control in normal and IPF lung fibroblast lysates with or without TGF-β1 (0, 2, and 10 ng/ml, 48 hours). n = 6 per group. (B) Single cell recording of fura-2 dye–loaded normal HLFs shows that TRPV4 agonist GSK induces Ca2+ influx, which is abrogate d by its antagonist, AB1. (C) Concentration-dependent inhibition of TRPV4 activity (Ca2+ influx) by AB1. Ca2+ influx is shown by relative fluorescence units (RFUs) measuring ΔF/F (max-min), using Calcium 5 dye on intact fibroblast monolayers (FlexStation 3, Molecular Devices). Results are expressed as mean ± SEM. *P < 0.05 compared with no AB1 conditions by ANOVA. (D) Loss of GSK-inducible Ca2+ influx in Trpv4 KO mouse lung fibroblasts. Fibroblast monolayers were incubated with the indicated concentration of GSK, and Ca2+ influx was measured as in C. All experiments were repeated ≥3 times in quadruplicate. Results are expressed as mean ± SEM. (E) Augmentation of GSK-inducible Ca2+ influx in lung fibroblasts from patients with IPF (IPF) compared with normal lung fibroblasts (NL). Fibroblast monolayers were incubated with GSK (10 nM), and Ca2+ influx was measured as in C. Results are expressed as mean ± SEM. *P < 0.05 for IPF vs. NL; n = 5 per group.