Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disorder with marginally effective medical treatment. Myofibroblasts are critical to the fibrogenic lung repair process; however, the mechanism whereby the mechanical signal that leads to myofibroblast generation is sensed and transmitted remains to be determined. As transient receptor potential vanilloid 4 (TRPV4) ion channels are activated by plasma membrane stretch/matrix stiffness, we investigated whether TRPV4 plays a role in myofibroblast differentiation and/or in vivo lung fibrosis. TRPV4 inhibition by small-molecule inhibitors almost completely abrogated both the calcium influx response to TPRV4 agonist in a dose-dependent manner and the myofibroblast differentiation response to transforming growth factor-β. Similar findings were noted on TRPV4 deletion with small interfering RNA or in TRPV4 KO murine lung fibroblasts. Further, TRPV4 exhibited its mechanosensing and myofibroblast-differentiating effect under conditions of matrix stiffness in the pathophysiological range (1–25 kPa). Mechanistically, TRPV4 activity modulates transforming growth factor β actions in a Smad-independent manner, in part through enhanced actomyosin remodeling with resultant nuclear translocation of the α–smooth muscle actin transcription coactivator (myocardin-related transcription factor-A). The myofibroblast differentiation response to actual fibrotic lung tissue was also completely inhibited on TRPV4 blockade. Furthermore, TRPV4 deficiency protects mice from fibrosis-inducing effects of bleomycin (1 or 4 U/kg dose) at the biochemical (75% less hydroxyproline; P < 0.05), histologic, and physiologic levels (57% less impairment of compliance; P < 0.05). These data identify TRPV4 as a long-elusive mechanosensor/transducer that mediates myofibroblast differentiation and in vivo pulmonary fibrogenesis. Successful manipulation of TRPV4 channel activity may be a novel therapeutic approach for fibrotic diseases of the lung and other organs.
Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease whose underlying molecular mechanisms are largely unknown. Herein, we show that focal adhesion kinase-related nonkinase (FRNK) plays a key role in limiting the development of lung fibrosis. Loss of FRNK function in vivo leads to increased lung fibrosis in an experimental mouse model. The increase in lung fibrosis is confirmed at the histological, biochemical, and physiological levels. Concordantly, loss of FRNK function results in increased fibroblast migration and myofibroblast differentiation and activation of signaling proteins that drive these phenotypes. FRNK-deficient murine lung fibroblasts also have an increased capacity to produce and contract matrix proteins. Restoration of FRNK expression in vivo and in vitro reverses these profibrotic phenotypes. These data demonstrate the multiple antifibrotic actions of FRNK. More important, FRNK expression is down-regulated in human IPF, and down-regulation of FRNK in normal human lung fibroblasts recapitulates the profibrotic phenotype seen in FRNK-deficient cells. The effect of loss and gain of FRNK in the experimental model, when taken together with its down-regulation in human IPF, suggests that FRNK acts as an endogenous negative regulator of lung fibrosis by repressing multiple profibrotic responses.
Abstract Macrophage phagocytosis and cytokine production are sensitive to the surrounding matrix and thereby mediate host defense and lung tissue injury. The consequences and mechanism of this matrix sensitivity are unknown. We have determined that the mechanosensitive channel, TRPV4, responds to extracellular matrix biophysical properties and thereby modulates the macrophage response to pathogens. We undertook this study to determine the in vivo consequences and the intracellular signaling pathway by which TRPV4 modulates macrophage responses. In order to evaluate the role of TRPV4 in chronic pneumonia/lung injury, WT and TRPV4 KO mice were administered agarose bead embedded-Pseudomonas aeruginosa. Loss of TRPV4 led to decreased phagocytosis by macrophages (6-fold), and increased lung injury as measured by inflammatory cell infiltration (≥ 80 ± 3%), vascular permeability (total protein ≥ 63 ± 6%), and cytokine secretion (IL-1β ≥ 71 ± 4%). In vivo, lung alveolar macrophages predominantly expressed TRPV4 and were the key phagocytic cell, as assessed by FACS and immunofluorescence. Known LPS signaling pathways were investigated in vitro. Loss of TRPV4 abrogates LPS-induced p38 activation and phagocytosis of E. coli particles. Additionally, loss of TRPV4 increased basal pro-/active IL-1β expression (2-fold), thereby enhancing IL-1β secretion independent of caspase 1 cleavage and blockade. These findings demonstrate that TRPV4 is important for bacterial clearance, lung injury and cytokine production in macrophages. TRPV4 mediates these effects through p38 and through upregulation of IL-1β protein expression in an inflammasome-independent manner. The results implicate macrophage TRPV4 as a key component of the host defense.
