scholarly journals P149 The intracellular chloride channel 4 (CLIC4) plays an important role in systemic sclerosis fibroblast activation

Rheumatology ◽  
2021 ◽  
Vol 60 (Supplement_1) ◽  
Author(s):  
Christopher Wasson ◽  
Rebecca Ross ◽  
Ruth Morton ◽  
Jamel Mankouri ◽  
Francesco Del Galdo
Keyword(s):  
Systemic Sclerosis ◽  
Ion Channel ◽  
Smooth Muscle Actin ◽  
Chloride Ion ◽  
Dermal Fibroblasts ◽  
Cancer Associated Fibroblasts ◽  
Alpha Smooth Muscle Actin ◽  
Direct Role ◽  
Fibroblast Activation ◽  
Intracellular Chloride

Abstract Background/Aims  The intracellular chloride ion channel CLIC4 mediates the activation of cancer associated fibroblasts. Interestingly, systemic sclerosis (SSc) fibroblasts display a number of similar properties to cancer associated fibroblasts. Tissue fibrosis in SSc is driven by active fibroblasts (myofibroblasts). Therefore in this study we investigated the role of CLIC4 in SSc fibroblast activation. Methods  Dermal fibroblasts were obtained from full thickness skin biopsies from SSc patients (early-diffuse). RNA and protein were collected from the fibroblasts and CLIC4 transcript and protein levels were assessed by qPCR and western blot. SSc patient fibroblasts were treated with the chloride ion channel inhibitors NPPB and IAA-94. Results  CLIC4 was found to be expressed at significantly higher levels in SSc patients fibroblasts compared to healthy controls, at both the transcript (3.7 fold) and protein (1.7 fold) levels. Inhibition of the TGF-β signalling pathway led to reduced CLIC4 expression in SSc fibroblasts, confirming this pathway as the main driver of CLIC4 expression. Finally, treatment of SSc fibroblasts with small molecule inhibitors that target the channel led to reduced expression of the myofibroblast markers collagen type 1 and alpha-smooth muscle actin, suggesting a direct role for CLIC4 in SSc associated skin fibrosis. Conclusion  We have identified a novel role for CLIC4 in SSc myofibroblast activation, which further strengthen the similarities between SSc fibroblasts and cancer associated fibroblasts. Furthermore this study highlights this channel as a novel target for therapeutic intervention. Disclosure  C. Wasson: None. R. Ross: None. R. Morton: None. J. Mankouri: None. F. Del Galdo: None.

The intracellular chloride channel 4 (CLIC4) activates systemic sclerosis fibroblasts

Rheumatology ◽  
2020 ◽  
Author(s):  
Christopher W Wasson ◽  
Rebecca L Ross ◽  
Ruth Morton ◽  
Jamel Mankouri ◽  
Francesco Del Galdo
Keyword(s):  
Chloride Channel ◽  
Smooth Muscle Actin ◽  
Cancer Associated Fibroblasts ◽  
Direct Role ◽  
Intracellular Chloride ◽  
Protein Levels ◽  
Thickness Skin ◽  
Main Driver ◽  
Β Receptor ◽  
Novel Target

Abstract Objectives Tissue fibrosis in SSc is driven by active fibroblasts (myofibroblasts). Previous studies have shown the intracellular chloride channel 4 (CLIC4) mediates the activation of cancer-associated fibroblasts. In this study we investigated the role of CLIC4 in SSc fibroblast activation. Methods Fibroblasts were obtained from full thickness skin biopsies from SSc patients (early-diffuse). RNA and protein were collected from the fibroblasts and CLIC4 transcript and protein levels were assessed by qPCR and western blot. SSc patient fibroblasts were treated with the chloride channel inhibitors nitro-2-(3-phenylpropylamino)benzoic acid and indyanyloxyacetic acid 94. Results CLIC4 was expressed at significantly higher levels in SSc patients’ fibroblasts compared with healthy controls, at both the transcript (3.7-fold) and protein (1.7-fold) levels. Inhibition of the TGF-β receptor and its downstream transcription factor SMAD3 led to a reduction in CLIC4 expression, confirming this pathway as the main driver of CLIC4 expression. Importantly, treatment of SSc fibroblasts with known pharmacological inhibitors of CLIC4 led to reduced expression of the myofibroblast markers collagen type 1 and α-smooth muscle actin, inferring a direct role for CLIC4 in disease pathogenesis. Conclusions We have identified a novel role for CLIC4 in SSc myofibroblast activation, which strengthens the similarities of SSc fibroblasts with cancer-associated fibroblasts and highlights this channel as a novel target for therapeutic intervention.

scholarly journals Global gene expression analysis of systemic sclerosis myofibroblasts demonstrates a marked increase in the expression of multiple NBPF genes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Giuseppina Abignano ◽  
Heidi Hermes ◽  
Sonsoles Piera-Velazquez ◽  
Sankar Addya ◽  
Francesco Del Galdo ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Smooth Muscle Actin ◽  
Cell Lineage ◽  
Global Gene Expression ◽  
Dermal Fibroblasts ◽  
Alpha Smooth Muscle Actin ◽  
Western Blots ◽  
Effector Cells ◽  
Recent Onset

AbstractMyofibroblasts are the key effector cells responsible for the exaggerated tissue fibrosis in Systemic Sclerosis (SSc). Despite their importance to SSc pathogenesis, the specific transcriptome of SSc myofibroblasts has not been described. The purpose of this study was to identify transcriptome differences between SSc myofibroblasts and non-myofibroblastic cells. Alpha smooth muscle actin (α-SMA) expressing myofibroblasts and α-SMA negative cells were isolated employing laser capture microdissection from dermal cell cultures from four patients with diffuse SSc of recent onset. Total mRNA was extracted from both cell populations, amplified and analyzed employing microarrays. Results for specific genes were validated by Western blots and by immunohistochemistry. Transcriptome analysis revealed 97 differentially expressed transcripts in SSc myofibroblasts compared with non-myofibroblasts. Annotation clustering of the SSc myofibroblast-specific transcripts failed to show a TGF-β signature. The most represented transcripts corresponded to several different genes from the Neuroblastoma Breakpoint Family (NBPF) of genes. NBPF genes are highly expanded in humans but are not present in murine or rat genomes. In vitro studies employing cultured SSc dermal fibroblasts and immunohistochemistry of affected SSc skin confirmed increased NBPF expression in SSc. These results indicate that SSc myofibroblasts represent a unique cell lineage expressing a specific transcriptome that includes very high levels of transcripts corresponding to numerous NBPF genes. Elevated expression of NBPF genes in SSc myofibroblasts suggests that NBPF gene products may play a role in SSc pathogenesis and may represent a novel therapeutic target.

scholarly journals Chloride intracellular channel 4 (CLIC4) expression is transcriptionally regulated by crosstalk of the TGF-beta, Wnt and Hedgehog signalling pathways

2021 ◽  
Author(s):  
Christopher W Wasson ◽  
Begoňa Caballero-Ruiz ◽  
Jamel Mankouri ◽  
Gianluca Canettieri ◽  
Natalia A Riobo-Del Galdo ◽  
...  
Keyword(s):  
Smooth Muscle Actin ◽  
Dermal Fibroblasts ◽  
Beta Catenin ◽  
Tgf Beta ◽  
Alpha Smooth Muscle Actin ◽  
Hedgehog Signalling ◽  
Fibroblast Activation ◽  
Smad3 Expression ◽  
Canonical Wnt ◽  
Intracellular Channel

Chloride intracellular channel 4 (CLIC4) is a recently discovered driver of fibroblast activation in Scleroderma (SSc) and cancer-associated fibroblasts. CLIC4 expression and activity are regulated by TGF-beta signalling through the SMAD3 transcription factor. In view of the aberrant activation of canonical Wnt and Hedgehog (Hh) signalling in fibrosis, we investigated their role in CLIC4 upregulation. Here, we show Wnt3a/beta-catenin and Smoothened/GLI signalling cooperate with SMAD3 to regulate CLIC4 expression in normal dermal fibroblasts, and that inhibition of SMAD3 expression or activity abolishes Wnt and Hh-dependent CLIC4 induction. We further show that expression of the profibrotic marker alpha-smooth muscle actin strongly correlates with CLIC4 expression in dermal fibroblasts. Our data highlight novel mechanisms that regulate CLIC4 expression that present targetable pathways to prevent fibroblast activation in SSc and other fibrotic conditions.

scholarly journals Myofibroblast transcriptome indicates SFRP2hi fibroblast progenitors in systemic sclerosis skin

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tracy Tabib ◽  
Mengqi Huang ◽  
Nina Morse ◽  
Anna Papazoglou ◽  
Rithika Behera ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Bioinformatics Analysis ◽  
Dermal Fibroblast ◽  
Smooth Muscle Actin ◽  
Cell Types ◽  
Dermal Fibroblasts ◽  
Muscle Actin ◽  
Alpha Smooth Muscle Actin ◽  
Modified Rodnan Skin Score ◽  
Different Cell Types

AbstractSkin and lung fibrosis in systemic sclerosis (SSc) is driven by myofibroblasts, alpha-smooth muscle actin expressing cells. The number of myofibroblasts in SSc skin correlates with the modified Rodnan skin score, the most widely used clinical measure of skin disease severity. Murine fibrosis models indicate that myofibroblasts can arise from a variety of different cell types, but their origin in SSc skin has remained uncertain. Utilizing single cell RNA-sequencing, we define different dermal fibroblast populations and transcriptome changes, comparing SSc to healthy dermal fibroblasts. Here, we show that SSc dermal myofibroblasts arise in two steps from an SFRP2hi/DPP4-expressing progenitor fibroblast population. In the first step, SSc fibroblasts show globally upregulated expression of transcriptome markers, such as PRSS23 and THBS1. A subset of these cells shows markers indicating that they are proliferating. Only a fraction of SFRP2hi SSc fibroblasts differentiate into myofibroblasts, as shown by expression of additional markers, SFRP4 and FNDC1. Bioinformatics analysis of the SSc fibroblast transcriptomes implicated upstream transcription factors, including FOSL2, RUNX1, STAT1, FOXP1, IRF7 and CREB3L1, as well as SMAD3, driving SSc myofibroblast differentiation.

scholarly journals The Intracellular Chloride Ion Channel Protein CLIC1 Undergoes a Redox-controlled Structural Transition

2003 ◽  
Vol 279 (10) ◽  
pp. 9298-9305 ◽  
Author(s):  
Dene R. Littler ◽  
Stephen J. Harrop ◽  
W. Douglas Fairlie ◽  
Louise J. Brown ◽  
Greg J. Pankhurst ◽  
...  
Keyword(s):  
Ion Channel ◽  
Structural Transition ◽  
Chloride Ion ◽  
Channel Protein ◽  
Intracellular Chloride

Role of Alpha-Smooth Muscle Actin and Fibroblast Activation Protein Alpha in Ovarian Neoplasms

2018 ◽  
Vol 83 (4) ◽  
pp. 381-387 ◽  
Author(s):  
Ana Carolinne da Silva ◽  
Millena Prata Jammal ◽  
Renata Margarida Etchebehere ◽  
Eddie Fernando Candido Murta ◽  
Rosekeila Simões Nomelini
Keyword(s):  
Smooth Muscle ◽  
Ovarian Neoplasms ◽  
Smooth Muscle Actin ◽  
Fibroblast Activation Protein ◽  
Muscle Actin ◽  
Alpha Smooth Muscle Actin ◽  
Fibroblast Activation ◽  
Fibroblast Activation Protein Alpha

scholarly journals Machine learning integration of scleroderma histology and gene expression identifies fibroblast polarisation as a hallmark of clinical severity and improvement

2020 ◽  
pp. annrheumdis-2020-217840 ◽  
Author(s):  
Kimberly Showalter ◽  
Robert Spiera ◽  
Cynthia Magro ◽  
Phaedra Agius ◽  
Viktor Martyanov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Machine Learning ◽  
Systemic Sclerosis ◽  
Clinical Improvement ◽  
Smooth Muscle Actin ◽  
Clinical Severity ◽  
Support Vector ◽  
Alpha Smooth Muscle Actin ◽  
Modified Rodnan Skin Score ◽  
Diffuse Cutaneous Systemic Sclerosis

ObjectiveWe sought to determine histologic and gene expression features of clinical improvement in early diffuse cutaneous systemic sclerosis (dcSSc; scleroderma).MethodsFifty-eight forearm biopsies were evaluated from 26 individuals with dcSSc in two clinical trials. Histologic/immunophenotypic assessments of global severity, alpha-smooth muscle actin (aSMA), CD34, collagen, inflammatory infiltrate, follicles and thickness were compared with gene expression and clinical data. Support vector machine learning was performed using scleroderma gene expression subset (normal-like, fibroproliferative, inflammatory) as classifiers and histology scores as inputs. Comparison of w-vector mean absolute weights was used to identify histologic features most predictive of gene expression subset. We then tested for differential gene expression according to histologic severity and compared those with clinical improvement (according to the Combined Response Index in Systemic Sclerosis).ResultsaSMA was highest and CD34 lowest in samples with highest local Modified Rodnan Skin Score. CD34 and aSMA changed significantly from baseline to 52 weeks in clinical improvers. CD34 and aSMA were the strongest predictors of gene expression subset, with highest CD34 staining in the normal-like subset (p<0.001) and highest aSMA staining in the inflammatory subset (p=0.016). Analysis of gene expression according to CD34 and aSMA binarised scores identified a 47-gene fibroblast polarisation signature that decreases over time only in improvers (vs non-improvers). Pathway analysis of these genes identified gene expression signatures of inflammatory fibroblasts.ConclusionCD34 and aSMA stains describe distinct fibroblast polarisation states, are associated with gene expression subsets and clinical assessments, and may be useful biomarkers of clinical severity and improvement in dcSSc.

Cartilage oligomeric matrix protein expression in systemic sclerosis reveals heterogeneity of dermal fibroblast responses to transforming growth factor β

2008 ◽  
Vol 68 (3) ◽  
pp. 435-441 ◽  
Author(s):  
G Farina ◽  
R Lemaire ◽  
P Pancari ◽  
J Bayle ◽  
R L Widom ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Growth Factor ◽  
Mrna Expression ◽  
Cartilage Oligomeric Matrix Protein ◽  
Matrix Protein ◽  
Transforming Growth Factor ◽  
Dermal Fibroblast ◽  
Smooth Muscle Actin ◽  
Dermal Fibroblasts ◽  
Cultured Fibroblasts

Objective:Cartilage oligomeric matrix protein (COMP) accumulates in systemic sclerosis (SSc) skin and is upregulated by transforming growth factor (TGF)β. To further characterise the response to TGFβ in SSc, we investigated TGFβ1 and COMP expression and myofibroblast staining in SSc skin.Methods:Skin biopsies from patients with diffuse cutaneous SSc (dSSc), limited cutaneous SSc (lSSc) and healthy controls were evaluated for COMP mRNA expression using real-time PCR. COMP, α-smooth muscle actin (SMA) and TGFβ were assessed in skin sections and in cultured fibroblasts by immunohistochemistry. Clinical disease status was assessed by the modified Rodnan skin score (mRSS).Results:Myofibroblasts expressing SMA and COMP were found coexpressed in many cells in dSSc dermis, but each also stained distinct cells in the dermis. Cultured SSc dermal fibroblasts also showed heterogeneity for COMP and SMA expression, with cells expressing SMA, COMP, both or neither. TGFβ treatment increased COMP and SMA-expressing cells. COMP mRNA expression in lesional skin from patients with dSSc correlated with the mRSS and TGFβ1 staining.Conclusion:These findings suggest that TGFβ upregulation of COMP and/or SMA expression in subpopulations of fibroblasts contributes to different pathways of fibrosis and that multiple TGFβ regulated genes may serve as biomarkers for the degree of SSc skin involvement.

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