Induction of Pro-Fibrotic CLIC4 in Dermal Fibroblasts by TGF-β/Wnt3a Is Mediated by GLI2 Upregulation.

Begoña Caballero-Ruiz,Natalia A. Riobo-Del Galdo,Jamel Mankouri,Francesco Del Galdo,Justin Gillespie,Christopher P. Denton,Gianluca Canettieri,Christopher W. Wasson,Emma Derrett-Smith

doi:10.3390/cells11030530

Abstract

Chloride intracellular channel 4 (CLIC4) is a recently discovered driver of fibroblast activation in Scleroderma (SSc) and cancer-associated fibroblasts (CAF). CLIC4 expression and activity are regulated by TGF-β signalling through the SMAD3 transcription factor. In view of the aberrant activation of canonical Wnt-3a and Hedgehog (Hh) signalling in fibrosis, we investigated their role in CLIC4 upregulation. Here, we show that TGF-β/SMAD3 co-operates with Wnt3a/β-catenin and Smoothened/GLI signalling to drive CLIC4 expression in normal dermal fibroblasts, and that the inhibition of β-catenin and GLI expression or activity abolishes TGF-β/SMAD3-dependent CLIC4 induction. We further show that the expression of the pro-fibrotic marker α-smooth muscle actin strongly correlates with CLIC4 expression in dermal fibroblasts. Further investigations revealed that the inhibition of CLIC4 reverses morphogen-dependent fibroblast activation. Our data highlights that CLIC4 is a common downstream target of TGF-β, Hh, and Wnt-3a through signalling crosstalk and we propose a potential therapeutic avenue using CLIC4 inhibitors

Highlights

Chloride intracellular channel 4 (CLIC4) can be found in soluble or membraneassociated forms and has multiple functions acting as a chloride channel and as a regulator of internal membrane and cytoskeletal dynamics
Given the interconnection of TGF-β signalling with Hh and Wnt3a, we investigated the possibility that Hh and Wnt-3a signalling regulate CLIC4 expression in systemic sclerosis (SSc) fibroblasts
To investigate the role of morphogen signalling in the regulation of CLIC4 expression, healthy human immortalized dermal fibroblasts were stimulated with TGF-β, Wnt-3a, or Smoothened agonist (SAG) for 48 h

Summary

Introduction

Chloride intracellular channel 4 (CLIC4) can be found in soluble or membraneassociated forms and has multiple functions acting as a chloride channel and as a regulator of internal membrane and cytoskeletal dynamics. CLIC4 is a SMAD3 target gene in fibroblasts, exerting a positive feedback loop to amplify TGF-β signalling [10]. TGF-β has been shown to stimulate canonical Wnt-3a/β-catenin signalling in a p38 MAPK-dependent manner in dermal fibroblasts [17]. Direct interactions between SMAD3 and β-catenin have been identified at numerous promoter sites, [19] including the GLI2 promoter [20] These findings are of interest in the context of SSc, where both Hh and Wnt-3a signalling are hyper-activated and play important roles in the activation of SSc fibroblasts [21,22]. In the context of SSc, we further demonstrate that CLIC4 expression can be supressed through the inhibition of β-catenin and GLI1/GLI2-dependent transcription in patient dermal fibroblasts

Patient Cells

Cell Culture

Immunohistochemistry

Morphogen Pathway Stimulation

Western Blotting

Quantitative Real Time PCR

Results

Wnt3a and Hh Signalling Cooperate with SMAD3 to Enhance CLIC4 Expression

The regulation

Enhanced CLIC4 Levels in SSc Fibroblasts Are Mediated by GLI2 Expression