TRPV4 ion channel is a novel regulator of dermal myofibroblast differentiation

Abstract

Scleroderma is a multisystem fibroproliferative disease with no effective medical treatment. Myofibroblasts are critical to the fibrogenic tissue repair process in the skin and many internal organs. Emerging data support a role for both matrix stiffness, and transforming growth factor β1 (TGFβ1), in myofibroblast differentiation. Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive ion channel activated by both mechanical and biochemical stimuli. The objective of this study was to determine the role of TRPV4 in TGFβ1- and matrix stiffness-induced differentiation of dermal fibroblasts. We found that TRPV4 channels are expressed and functional in both human (HDF) and mouse (MDF) dermal fibroblasts. TRPV4 activity (agonist-induced Ca influx) was induced by both matrix stiffness and TGFβ1 in dermal fibroblasts. TGFβ1 induced expression of TRPV4 proteins in a dose-dependent manner. Genetic ablation or pharmacological antagonism of TRPV4 channel abrogated Ca influx and both TGFβ1-induced and matrix stiffness-induced myofibroblast differentiation as assessed by ) α-smooth muscle actin expression/incorporation into stress fibers,) generation of polymerized actin, and ) expression of collagen-1. We found that TRPV4 inhibition abrogated TGFβ1-induced activation of AKT but not of Smad2/3, suggesting that the mechanism by which profibrotic TGFβ1 signaling in dermal fibroblasts is modified by TRPV4 may be through non-Smad pathways. Altogether, these data identify a novel reciprocal functional link between TRPV4 activation and TGFβ1 signals regulating dermal myofibroblast differentiation. These findings suggest that therapeutic inhibition of TRPV4 activity may provide a targeted approach to the treatment of scleroderma.

TRPV4 regulates matrix stiffness and TGFβ1‐induced epithelial‐mesenchymal transition

“…Transforming growth factor β1 is known to induce EMT in keratinocytes . Recent evidence from our group suggests a link between TRPV4 activation and TGFβ1 signals that mediate fibroblast differentiation . Previous reports have documented that calcium signalling is involved in EMT in many cell types .…”

“…The identification of the role of specific Ca 2+ ‐permeable ion channels in the induction of EMT in the context of fibrosis and oncogenesis suggests that these channels may be appropriate therapeutic targets for control of EMT‐mediated disease progression. Our recently published reports suggested that TRPV4‐dependent Ca 2+ influx potentiates TGFβ1‐induced lung and dermal myofibroblast differentiation in response to matrix stiffness, and showed that TRPV4 KO mice were protected from bleomycin‐induced fibrosis in skin and lungs . Although TGFβ1 and matrix stiffness were shown to play important roles in EMT and fibrosis, the role of TRPV4 mechanosensing in EMT has not been reported.…”

“…Plates were incubated in the dark for another 10 minutes and then transferred to the FlexStation3 system to measure fluorescence. Calcium influx was induced by the TRPV4 agonist GSK1016790A (GSK101) in vehicle‐pretreated or GSK219‐pretreated NMEKs, and cytosolic Ca 2+ influx was recorded by measuring ΔF/F (Max‐Min) as described previously . Data are shown as relative fluorescence units.…”

“…Recently, we reported that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive Ca 2+ ‐permeable channel, is associated with skin and lung fibrosis. TRPV4 regulates both biochemical (TGFβ1)‐ and mechanical (matrix stiffness) stimulus‐induced lung and dermal myofibroblast differentiation and contributes to the development of in vivo pulmonary and skin fibrosis in murine models . However, the specific role of TRPV4 in EMT in normal primary epidermal keratinocytes has not been determined.…”

“…TRPV4 was shown to function as a mechanosensor in response to cyclical stretching of cells, mechanical forces applied to β1 integrins in cell surface or dynamic loading in chondrocytes . Structurally, TRPV4 contains a number of potential regulatory domains and protein‐interaction sites, including phosphoinositide 3‐kinase (PI3K) and Src homology 2 recognition domains, putative protein kinase C phosphorylation sites, PDZ domains and an N‐terminal ankyrin repeat region . Numerous mutations in TRPV4 have been associated with human diseases including skeletal dysplasia and sensory and motor neuropathies .…”