TRPV4 ion channel is a novel regulator of dermal myofibroblast differentiation

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 Ca2+ 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 Ca2+ influx and both TGFβ1-induced and matrix stiffness-induced myofibroblast differentiation as assessed by 1) α-smooth muscle actin expression/incorporation into stress fibers, 2) generation of polymerized actin, and 3) 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.

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TRPV4 Plays a Role in Matrix Stiffness-Induced Macrophage Polarization

Frontiers in Immunology ◽

10.3389/fimmu.2020.570195 ◽

2020 ◽

Vol 11 ◽

Author(s):

Bidisha Dutta ◽

Rishov Goswami ◽

Shaik O. Rahaman

Keyword(s):

Transient Receptor Potential ◽

Macrophage Polarization ◽

Transient Receptor Potential Vanilloid ◽

Polarization Spectrum ◽

Dependent Manner ◽

Matrix Stiffness ◽

Soft Matrix ◽

Genetic Ablation ◽

M1 Macrophage

Phenotypic polarization of macrophages is deemed essential in innate immunity and various pathophysiological conditions. We have now determined key aspects of the molecular mechanism by which mechanical cues regulate macrophage polarization. We show that Transient Receptor Potential Vanilloid 4 (TRPV4), a mechanosensitive ion channel, mediates substrate stiffness-induced macrophage polarization. Using atomic force microscopy, we showed that genetic ablation of TRPV4 function abrogated fibrosis-induced matrix stiffness generation in skin tissues. We have determined that stiffer skin tissue promotes the M1 macrophage subtype in a TRPV4-dependent manner; soft tissue does not. These findings were further validated by our in vitro results which showed that stiff matrix (50 kPa) alone increased expression of macrophage M1 markers in a TRPV4-dependent manner, and this response was further augmented by the addition of soluble factors; neither of which occurred with soft matrix (1 kPa). A direct requirement for TRPV4 in M1 macrophage polarization spectrum in response to increased stiffness was evident from results of gain-of-function assays, where reintroduction of TRPV4 significantly upregulated the expression of M1 markers in TRPV4 KO macrophages. Together, these data provide new insights regarding the role of TRPV4 in matrix stiffness-induced macrophage polarization spectrum that may be explored in tissue engineering and regenerative medicine and targeted therapeutics.

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Acyltransferase skinny hedgehog regulates TGFβ-dependent fibroblast activation in SSc

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2019-215066 ◽

2019 ◽

Vol 78 (9) ◽

pp. 1269-1273 ◽

Cited By ~ 5

Author(s):

Ruifang Liang ◽

Rosebeth Kagwiria ◽

Ariella Zehender ◽

Clara Dees ◽

Christina Bergmann ◽

...

Keyword(s):

Long Range ◽

Transforming Growth Factor ◽

Human Fibroblasts ◽

Transforming Growth Factor Β ◽

Dermal Fibroblasts ◽

Myofibroblast Differentiation ◽

Dependent Manner ◽

Hedgehog Signalling ◽

Fibroblast Activation ◽

Novel Approach

ObjectivesSystemic sclerosis (SSc) is characterised by aberrant hedgehog signalling in fibrotic tissues. The hedgehog acyltransferase (HHAT) skinny hedgehog catalyses the attachment of palmitate onto sonic hedgehog (SHH). Palmitoylation of SHH is required for multimerisation of SHH proteins, which is thought to promote long-range, endocrine hedgehog signalling. The aim of this study was to evaluate the role of HHAT in the pathogenesis of SSc.MethodsExpression of HHAT was analysed by real-time polymerase chain reaction(RT-PCR), immunofluorescence and histomorphometry. The effects of HHAT knockdown were analysed by reporter assays, target gene studies and quantification of collagen release and myofibroblast differentiation in cultured human fibroblasts and in two mouse models.ResultsThe expression of HHAT was upregulated in dermal fibroblasts of patients with SSc in a transforming growth factor-β (TGFβ)/SMAD-dependent manner. Knockdown of HHAT reduced TGFβ-induced hedgehog signalling as well as myofibroblast differentiation and collagen release in human dermal fibroblasts. Knockdown of HHAT in the skin of mice ameliorated bleomycin-induced and topoisomerase-induced skin fibrosis.ConclusionHHAT is regulated in SSc in a TGFβ-dependent manner and in turn stimulates TGFβ-induced long-range hedgehog signalling to promote fibroblast activation and tissue fibrosis. Targeting of HHAT might be a novel approach to more selectively interfere with the profibrotic effects of long-range hedgehog signalling.

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