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. 2014 Dec;124(12):5225-38.
doi: 10.1172/JCI75331. Epub 2014 Nov 3.

TRPV4 mediates myofibroblast differentiation and pulmonary fibrosis in mice

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

TRPV4 mediates myofibroblast differentiation and pulmonary fibrosis in mice

Shaik O Rahaman et al. J Clin Invest. 2014 Dec.

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.

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Figures

Figure 8
Figure 8. Inhibition of TRPV4 channel activity abrogates molecular signaling essential for myofibroblast differentiation.
(A–D) HLFs were incubated with or without TGF-β1 (2 ng/ml, 48 hours) as well as with or without indicated TRPV4 inhibitor (AB1) or with or without inhibitor (latrunculin B [Lat-B], 500 nM) or activator (jasplakinolide [Jak], 100 nM) of actin polymerization, as indicated. n = 3. Results are expressed as mean ± SEM. (A) Immunoblots of cell lysates were separated into F-actin (filamentous) and G-actin (monomeric) fractions, which show abrogation of F-actin formation upon TRPV4 inhibition by AB1. (B) Quantification of results in A. *P < 0.05 for TGF-β1–treated cells with or without AB1. (C) MRTF-A nuclear translocation is abrogated by TRPV4 inhibition. Nuclear and cytoplasmic fractions were separated, and an equal amount of protein per lane was immunoblotted for MRTF-A, a nuclear marker (lamin A/C), and a cytoplasmic marker (14-3-3B). (D) Quantification of results from C. *P < 0.05 for TGF-β1–treated cells with vs. without AB1. (E) As in C, immunoblots show MRTF-A nuclear translocation is abrogated in Trpv4 KO mouse lung fibroblasts. (F) Quantification of results from E. Results are expressed as mean ± SEM. *P < 0.05 for TGF-β1 WT cells vs. Trpv4 KO cells (n = 3). (G) G-LISA RhoA activation assay shows inhibition of TGF-β1–induced activation of RhoA by TRPV4 antagonist, AB1. *P < 0.05 for TGF-β1–treated cells with or without AB1 at 10 minutes; n = 3. Results are expressed as mean ± SEM. (H) HLFs were pretreated with or without AB1 (20 μM) followed by TGF-β1 (2 ng/ml) for indicated times. Sonicated cell lysates (n = 2) were immunoblotted for total MLC2 or activated MLC2 (p-MLC2).
Figure 9
Figure 9. Schematic model showing the mechanistic pathway by which TRPV4 mediates myofibroblast differentiation and pulmonary fibrosis.
Our data suggest that TRPV4-dependent Ca2+ influx activity is sensitized by stiff matrices within the pathophysiological range. Interaction between TRPV4 activity (Ca2+ influx) and the profibrotic TGF-β1 signals promote nuclear localization of α-SMA transcription factor, MRTF-A, via regulation of actomyosin remodeling to potentiate myofibroblast differentiation during fibrogenesis. TβR, TGF-βR.
Figure 7
Figure 7. TRPV4 activity potentiates TGF-β1 actions in a SMAD2/3-independent manner.
(A) TRPV4-mediated Ca2+ influx in HLFs (measured as in Figure 2C) is not blocked by supraphysiologic concentrations of the TGF-βRI kinase inhibitor, SD208. Results are expressed as mean ± SEM. (B) Immunoblots of fibroblast cell lysates, treated as indicated, show no inhibition of TGF-β1–induced phosphorylation of SMAD2/3 or of expression of total TRPV4 proteins by the TRPV4 antagonist, AB1. Total SMAD2/3 was used as loading control. The experiments were repeated 2 times.
Figure 6
Figure 6. Increased matrix stiffness augments TRPV4-induced Ca2+ influx and myofibroblast differentiation.
(A and B) HLFs were plated (15,000 cells per well) on collagen-coated (100 μg/ml) hydrogels with varying degrees of stiffness (1, 6, 8, and 25 kPa) under vehicle-treated or AB1-treated (50 μM) conditions, with or without TGF-β1 (2 ng/ml, 24 hours). (A) GSK-induced (10 nM) Ca2+ influx, measured as in Figure 2C, is increased by stiffness and abrogated by AB1. (B) Fluorescence microscopy analysis shows that the TGF-β1 response in myofibroblast differentiation seen with increasing stiffness of the matrix is dependent on TRPV4 channel activity. *P < 0.05, **P < 0.01 by ANOVA; n > 12 cells per group. Int., integrated. (C and D) HLFs were plated on normal or stiffer fibrotic lung tissue sections with or without TRPV4 antagonist, AB1 (50 μM). (C) Representative confocal micrographs (original magnification, ×63) of phalloidin-stained F-actin (red). (D) Quantification of actin stress fiber density from C. The results are expressed as mean ± SD. *P < 0.05 by ANOVA for vehicle-treated normal lung vs. fibrotic lung; †P < 0.05 by ANOVA for vehicle-treated vs. AB1-treated fibrotic lung.
Figure 5
Figure 5. TRPV4 small-molecule antagonists abrogate TGF-β1–induced myofibroblast differentiation in a concentration-dependent manner.
(A) TRPV4 blockade reduces TGF-β1–induced expression of α-SMA protein in a concentration-dependent manner, without inhibiting total TRPV4 protein level. n = 3. (B) TRPV4 antagonist, AB1 (50 μM), blocks TGF-β1–induced myofibroblast differentiation. Representative photomicrographic merged images of α-SMA (green) and F-actin (red). UT, no TGF-β1 treatment. Original magnification, ×20. (C) Quantitation of results from B by Pearson’s coefficient. **P < 0.001 for TGF-β1–treated cells with vs. without AB1; n > 18 cells per condition. (D) HC inhibits Ca2+ influx in a concentration-dependent manner. Mouse lung fibroblast monolayers from WT mice were examined for their Ca2+ influx response to GSK (10 nM) with or without selective inhibitor HC at the indicated concentrations, using similar methods as in Figure 2C. All data were repeated more than 3 times in quadruplicate. (E) Representative photomicrographic images as in B, with or without indicated HC concentrations. Original magnification, ×20. (F) Quantitation of results from E by Pearson’s coefficient. **P < 0.01 for TGF-β1–treated cells with vs. without HC; n > 18 cells per condition. (G) AB1 (50 μM) inhibits collagen-1 production in HLFs by collagen/GAPDH protein band density in immunoblots. (H) Quantitation of results from G. *P < 0.05 for TGF-β1–treated cells with vs. without AB1. Results are expressed as mean ± SEM.
Figure 4
Figure 4. TRPV4-dependent Ca2+ influx from the extracellular space mediates TGF-β1–induced myofibroblast differentiation.
(A) Extracellular calcium is required for TRPV4-mediated intracellular calcium rise. HLF monolayers were compared for their Ca2+ influx in response to the indicated concentrations of GSK, as in Figure 2C, with or without extracellular calcium. A23187 (2 μM), a calcium ionophore, was used as a positive control. Ca2+ influx for each condition is shown by the respective red arrow. (B) TRPV4-dependent calcium rise is independent of intracellular pools and/or regulators of calcium. HLF monolayers were compared for their intracellular calcium rise after TRPV4 activation (GSK, 10 nM) with or without selective inhibitors (30 minutes of pretreatment before addition of GSK) to the inositol 1,4,5-trisphosphate receptor (xestospongin C [Xesto], 10 μM), ryanodine receptors (ryanodine [RyD], 10 μM), or sarco/endoplasmic reticulum calcium transport ATPase (cyclopiazonic acid [CPA], 10 μM). Results are expressed as mean ± SEM. (C) Representative photomicrographs. Extracellular calcium is required for myofibroblast differentiation, as measured by intracellular remodeling of F-actin and α-SMA by immunofluorescence. Original magnification, ×20. (D) Quantification of photomicrographs from C using ImageJ software. Results are expressed as mean ± SEM. *P < 0.001 for TGF-β1–treated cells with vs. without calcium; n > 10 cells per condition, 1-way ANOVA.
Figure 3
Figure 3. 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.
Figure 2
Figure 2. TRPV4 calcium channel is expressed and functional in HLFs and murine lung fibroblasts.
(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.
Figure 1
Figure 1. Trpv4 KO mice are protected from the profibrotic effects of bleomycin.
WT or Trpv4 KO mice were instilled with (A–F) 4 U/kg or (G and H) 1 U/kg bleomycin or saline. (A) Hydroxyproline content in the lungs of WT and Trpv4 KO mice (day 14; n ≥ 5 per group, *P < 0.05). (B) Representative immunoblots of total lung protein lysates show reduced expression of collagen-1. Bar graphs show collagen-1 band density normalized to GAPDH. *P < 0.05, WT vs. Trpv4 KO; n > 5 per group. (C) Representative photomicrographs of trichrome-stained lung tissue (original magnification, ×10). The bar graph shows the percentage of fibrotic area. **P < 0.01, WT vs. Trpv4 KO; n = 5 per group. (D) Static lung compliance (Cst) was measured using the FlexiVent (day 14, static P-V loop; *P < 0.05, WT vs. Trpv4 KO; n = 5 per group). (E) Representative immunoblots of total lung protein lysates show reduced expression of α-SMA protein. The bar graph shows α-SMA band density normalized to GAPDH. *P < 0.05; n > 5 per group. (F) Cells counts and differentials from lung lavage from mice given bleomycin (4 U/kg, day 7, n ≥ 5 per group). Macs, macrophages; PMNs, polymorphonuclear leucocytes; Lymphs, lymphocytes. (G) Hydroxyproline as in A. WT, 203% ± 67% vs. KO, 143% ± 44% of WT IT saline; *P < 0.004; n = 5 per group. (H) Lung compliance as in D. *P < 0.05, WT vs. Trpv4 KO mice; n = 5 per group. (A, F, and G) Results are expressed as mean ± SD. (B–E and H) Results are expressed as mean ± SEM.
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