TRPV2 Channel Inhibitors Attenuate Fibroblast Differentiation and Contraction Mediated by Keratinocyte-Derived TGF-β1 in an In Vitro Wound Healing Model of Rats

Abstract

Background: Keratinocytes release several factors involved in wound contracture and scar formation. A three-dimensional reconstruction model derived from rat skin represents a robust wound healing model.

Objective: To characterize the role of transient receptor potential (TRP) channels in the release of transforming growth factor (TGF)-β1 from keratinocytes and the differentiation of fibroblasts, identifying potential pharmacological approaches to prevent scar formation and contractures.

Methods: A three-dimensional culture model was created from rat keratinocytes seeded on a collagen gel in which dermal fibroblasts were embedded.

Results: Among TRP channel inhibitors tested, TRPV2 inhibitors SKF96365 and tranilast most potently attenuated keratinocyte-dependent and -independent collagen gel contraction due to TGF-β signaling, as well as TGF-β1 release from keratinocytes and α-smooth muscle actin (α-SMA) production in myofibroblasts. TRPV2 mRNA and protein levels increased after fibroblasts were embedded in the gel. TRPV2 was also expressed in the epidermis and keratinocyte layers of the model. Both inhibitors and TRPV2 siRNA attenuated the intracellular Ca²⁺ increase induced by the TRPV agonist 2-aminoethoxydiphenyl borate in TGF-β1-pretreated fibroblasts.

Conclusion: This is the first study to show that compounds targeting TRPV2 channels ameliorate wound contraction through inhibition of TGF-β1 release and differentiation of dermal fibroblasts in a culture model.

Introduction

Wound healing in skin proceeds through inflammation, proliferation, and scar maturation, involving sequential interactions of different cell types. Granulation tissue contains myofibroblasts, which acquire a contractile phenotype via α-SMA expression, contributing to pathogenic scarring and fibrosis. Pharmacological intervention in fibroblast differentiation may help prevent hypertrophic scar formation and contractures.

Keratinocytes at the wound edge migrate over granulation tissue, producing new stratified layers (re-epithelialization) and releasing growth factors and cytokines, including TGF-βs. TGF-βs, secreted by platelets, fibroblasts, macrophages, and keratinocytes, stimulate granulation tissue formation and myofibroblast production. Previous work showed that keratinocytes secrete latent TGF-β1, which upregulates α-SMA in fibroblasts after activation.

Targeting ion channels involved in hypertrophic scar formation is a promising approach. TRP channels (nonselective ion channels) contribute to wound healing responses. TRPV1, TRPV4, TRPA1, TRPC3, TRPC6, and TRPM7 have all been implicated in fibroblast differentiation and wound healing, often via TGF-β1 signaling. However, it is unclear whether ion channel-mediated wound healing is based on keratinocyte-fibroblast interactions during re-epithelialization.

We previously reported a 3D reconstruction model of rat skin using keratinocytes and fibroblasts/myofibroblasts, which is suitable for studying wound contraction and cell interactions. Using this model, we aimed to clarify the mechanism of TGF-β1 release from keratinocytes and myofibroblast contraction, seeking pharmacological strategies to prevent scar formation and contractures.

Materials and Methods
Antibodies and Reagents:

Primary antibodies: mouse anti-α-SMA, rabbit anti-TRPV2. Recombinant human TGF-β1, TRP channel inhibitors (SKF96365, AMG9810, HC067047, tranilast, 2-APB, capsaicin, GSK1016790A, RN1734), and others were sourced from commercial suppliers.

Cell Isolation:

Dorsal skin from 2-day-old Wistar rats was used. Keratinocytes were isolated from epidermal tissue by enzymatic digestion and filtration. Dermal fibroblasts were cultured from minced dermal tissue.

3D Skin Model Construction:

Collagen gels containing dermal fibroblasts were prepared and overlaid with keratinocytes. The gels were cultured at an air-liquid interface to induce keratinocyte stratification.

Immunohistochemistry:

Cryostat sections of the model and rat tissues were stained for TRPV2 and α-SMA, visualized by confocal microscopy.

Real-Time PCR:

Total RNA from keratinocytes and fibroblasts/myofibroblasts was analyzed for α-SMA, TRPV2, TRPV3, TRPV4, and GAPDH expression.

ELISA for TGF-β1:

Supernatants from stratified keratinocyte cultures were analyzed for TGF-β1 by ELISA after acid activation.

Ca²⁺ Imaging:

Fibroblasts loaded with Fluo-4 AM were imaged for intracellular Ca²⁺ changes after treatment with TRPV agonists or inhibitors.

siRNA Knockdown:

TRPV2 expression in fibroblasts was silenced using specific siRNAs, and effects on Ca²⁺ influx and gene expression were assessed.

Statistical Analysis:

Data were analyzed using t-tests or ANOVA with Bonferroni’s post-hoc test; P < 0.05 was considered significant.

Results
Keratinocyte-Dependent Gel Contraction:

The 3D model showed that keratinocytes seeded on fibroblast-containing collagen gels induced gel contraction, reaching 40% of initial size over 9 days. Inhibition of TGF-β signaling (LY364947) partially reduced contraction, indicating TGF-β involvement. Exogenous TGF-β1 increased contraction in gels without keratinocytes.

TRP Channel Inhibitors:

SKF96365 (TRPV2 inhibitor) inhibited gel contraction in both keratinocyte-dependent and -independent models, with more pronounced effects in the absence of keratinocytes. Other TRP inhibitors and activators had smaller effects. Cell viability was not affected by these compounds.

Inhibition of Fibroblast Differentiation and TGF-β1 Secretion:

SKF96365 and tranilast (another TRPV2 inhibitor) dose-dependently inhibited gel contraction and reduced α-SMA mRNA levels, indicating inhibition of myofibroblast differentiation. TRPV2 siRNA knockdown also reduced contraction. Both inhibitors significantly reduced TGF-β1 secretion from stratified keratinocytes.

TGF-β1-Induced Contraction:

In gels without keratinocytes but treated with exogenous TGF-β1, SKF96365 and tranilast restored gel size and inhibited TGF-β1-induced contraction, suggesting that their effects are mediated via the TGF-β pathway.

Expression of TRPV Channels:

TRPV2 and TRPV3 mRNA were low in normal dermis but increased in fibroblasts during model construction, especially after keratinocyte overlay. TRPV4 was high in normal dermis but decreased during fibroblast differentiation. TRPV2 protein was localized to the lower epidermis and increased in fibroblasts and keratinocytes in the model.

Discussion

This study demonstrates that TRPV2 channels play a critical role in keratinocyte-mediated fibroblast differentiation and wound contraction via TGF-β1 signaling. Inhibitors of TRPV2, such as SKF96365 and tranilast, effectively attenuate myofibroblast formation, collagen gel contraction, and TGF-β1 release from keratinocytes. TRPV2 expression is upregulated during fibroblast differentiation and is present in both keratinocytes and fibroblasts in the wound healing model.

Targeting TRPV2 channels may offer a promising pharmacological approach to prevent hypertrophic scar formation and contractures by disrupting the TGF-β1-dependent differentiation of fibroblasts into myofibroblasts. The findings also highlight the importance of TRP channels in skin wound healing and fibrosis.

Conclusion

Compounds targeting TRPV2 channels, such as SKF96365 and tranilast, ameliorate wound contraction by inhibiting TGF-β1 release from keratinocytes and differentiation of dermal fibroblasts. TRPV2 is upregulated during fibroblast differentiation and is a promising target for preventing scar formation and contractures in wound healing.