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 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.

High Mobility Group Box Chromosomal Protein-1 Induces Myofibroblast Differentiation and Extracellular Matrix Production via RAGE, p38, JNK and AP-1 Signaling Pathways in Nasal Fibroblasts

2021 ◽  
pp. 194589242199814
Author(s):  
Soo-Hyung Lee ◽  
Jae Hoon Cho ◽  
Joo-Hoo Park ◽  
Jung-Sun Cho ◽  
Heung-Man Lee
Keyword(s):  
Extracellular Matrix ◽  
Chronic Rhinosinusitis ◽  
Smooth Muscle Actin ◽  
High Mobility ◽  
Signal Pathway ◽  
High Mobility Group ◽  
Myofibroblast Differentiation ◽  
Chromosomal Protein ◽  
Protein Levels ◽  
Matrix Production

Background Chronic rhinosinusitis is involved in myofibroblast differentiation and extracellular matrix (ECM) accumulation. High mobility group box chromosomal protein 1 (HMGB-1) is known to stimulate lung fibroblast to produce ECM in lung fibrosis. The aim of this study was to investigate whether HMGB-1 induces myofibroblast differentiation and ECM production in nasal fibroblasts and to identify the signal pathway. Methods Human nasal fibroblasts were cultured. After stimulation with HMGB-1, expressions of α-smooth muscle actin (α-SMA) and fibronectin were determined by real-time PCR and western blot. Total collagen was measured by Sircol assay. To investigate signal pathway, various signal inhibitors and RAGE siRNA were used. Results HMGB-1 increased α-SMA and fibronectin in mRNA and protein levels. It also increased collagen production. RAGE siRNA inhibited HMGB-1-induced α-SMA and fibronectin, and production of collagen. Furthermore, the inhibitors of RAGE downstream molecules such as p38, JNK and AP-1 also blocked the HMGB-1-induced effects. Conclusions HMGB-1 induces myofibroblast differentiation and ECM production in nasal fibroblast, which is mediated by RAGE, p38, JNK and AP-1 signal pathway. These results suggest that HMGB-1 may play an important role in tissue remodeling during chronic rhinosinusitis progression.

scholarly journals Intracellular chloride channel protein CLIC1 regulates macrophage function through modulation of phagosomal acidification

2012 ◽  
Vol 125 (22) ◽  
pp. 5479-5488 ◽  
Author(s):  
Lele Jiang ◽  
Kanin Salao ◽  
Hui Li ◽  
Joanna M. Rybicka ◽  
Robin M. Yates ◽  
...  
Keyword(s):  
Chloride Channel ◽  
Macrophage Function ◽  
Channel Protein ◽  
Intracellular Chloride

Abstract 19602: Characterization of Tgfβ/BMP-axis in Multiple Animal Models of PH

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Chris Happé ◽  
Nina Rol ◽  
Denielli Da Silva Goncalves Bos ◽  
Cathelijne van der Bruggen ◽  
Anton Vonk-Noordegraaf ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Animal Models ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Downstream Signaling ◽  
Protein Levels ◽  
Morphogenetic Protein ◽  
Cardiac Adaptation ◽  
Bmp Pathway ◽  
Β Receptor

Introduction: Mutations in the bone morphogenetic protein recptor type 2 (BMPR2) comprise a large portion of familial Pulmonary Arterial Hypertension (PAH) cases. The transforming growth factor-β (TGF-β)/BMP-axis in PAH has been of interest, which is hypothesized to favor TGF-β signaling due to defective BMPR2 signaling, consequently leading to pro-proliferative signaling within the lung. In addition, it has been proposed that the BMPRII mutations might affect cardiac adaptation. To date none of the available animal models have been fully characterized with regard to the TGF-β/BMP pathway. This study assessed the lung and heart TGF-β/BMP-axis in multiple rat animal models of pulmonary hypertension to ensure translational capability. Methods: Heart and lung TGF-β/BMP-axis was assessed by qPCR, western blot and immunofluorescence in the the monocrotaline (MCT), Sugen-hypoxia (SuHx), Sugen-Pneumonectomy (SuPnx) and Pulmonary artery banding model (PAB) and compared to control and PAH patient tissues. Circulating ligands, TGF-β receptor (TGFβR) type 1 and 2 and BMPR2, and canonical downstream signaling (Smad2/3, Smad1/5/8, and transcription factors) were investigated. Results: BMPR2 was down-regulated at both transcription and protein levels in the lung of all PH animal models (p<0.05). Transcription of pulmonary TGFβR1 and -2 were increased in the SuPNx-model, compared to control (P<0.001). In both SuHx and SuPnx models an increase in protein Smad2/3 expression was observed by immunofluorescence implying overactivation of TGF-β signaling. Cardiac TGFβR1 was decreased in PAB model, compared to control (P<0.05), while TGFβR2 was decreased in both the MCT and PAB model. Conclusion: Early indications reveal differences between several pulmonary hypertension animal models, with regard to the TGF-β/BMP pathway. Additional analysis is needed to fully characterize the regulation of TGF-β and BMP in the rat models.

scholarly journals CXXC5 Attenuates Pulmonary Fibrosis in a Bleomycin-Induced Mouse Model and MLFs by Suppression of the CD40/CD40L Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-15 ◽  
Author(s):  
Wei Cheng ◽  
Fangfang Wang ◽  
Airan Feng ◽  
Xiaodan Li ◽  
Wencheng Yu
Keyword(s):  
Pulmonary Fibrosis ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Feedback Regulation ◽  
Collagen Type I ◽  
Mouse Lung ◽  
Type I ◽  
Negative Feedback Regulation ◽  
Protein Levels ◽  
Proliferation And Apoptosis

Objective. To investigate the role of CXXC5 and the CD40/CD40L pathway in lung fibrosis. Methods. (1) We constructed mouse models of bleomycin-induced pulmonary fibrosis and transfected them with a CXXC5 overexpression vector to evaluate the severity of pulmonary fibrosis. (2) Mouse lung fibroblast (MLF) models stably overexpressed or knockout of CXXC5 vector were constructed. After transforming growth factor-β1 (TGF-β1) stimulation, we examined the proliferation and apoptosis of the MLF model and evaluated the expression of mesenchymal markers and the CXXC5/CD40/CD40L pathway. Results. (1) Compared with other groups, the overexpressed CXXC5 group had less alveolar structure destruction, thinner alveolar septum, and lower Ashcroft score. (2) In bleomycin-induced mice, the expression of CD40 and CD40L increased at both transcriptional and protein levels, and the same changes were observed in α-smooth muscle actin (α-SMA) and collagen type I (Colla I). After upregulation of CXXC5, the increase in CD40, CD40L, α-SMA, and Colla I was attenuated. (3) Stimulated with TGF-β1, MLF proliferation was activated, apoptosis was suppressed, and the expression of CD40, CD40L, α-SMA, and Colla I was increased at both transcriptional and protein levels. After upregulation of CXXC5, these changes were attenuated. Conclusion. CXXC5 inhibits pulmonary fibrosis and transformation to myofibroblasts by negative feedback regulation of the CD40/CD40L pathway.

PTH regulates expression of ClC-5 chloride channel in the kidney

2000 ◽  
Vol 278 (2) ◽  
pp. F238-F245 ◽  
Author(s):  
Ian V. Silva ◽  
Carol J. Blaisdell ◽  
Sandra E. Guggino ◽  
William B. Guggino
Keyword(s):  
Vitamin D ◽  
Protein Expression ◽  
Chloride Channel ◽  
Urinary Calcium ◽  
Kidney Cortex ◽  
Lower Serum ◽  
Protein Levels ◽  
Calcium Reabsorption ◽  
Mrna And Protein Expression ◽  
Serum Pth

Mutations in the chloride channel, ClC-5, have been described in several inherited diseases that result in the formation of kidney stones. To determine whether ClC-5 is also involved in calcium homeostasis, we investigated whether ClC-5 mRNA and protein expression are modulated in rats deficient in 1α,25(OH)2 vitamin D3 with and without thyroparathyroidectomy. Parathyroid hormone (PTH) was replaced in some animals. Vitamin D-deficient, thyroparathyrodectomized rats had lower serum and higher urinary calcium concentrations compared with control animals as well as lower serum PTH and calcitonin concentrations. ClC-5 mRNA and protein levels in the cortex decrease in vitamin D-deficient, thyroparathyroidectomized rats compared with both control and vitamin D-deficient animals. ClC-5 mRNA and protein expression increase near to control levels in vitamin D-deficient, thyroparathyroidectomized rats injected with PTH. No significant changes in ClC-5 mRNA and protein expression in the medulla were detected in any experimental group. Our results suggest that PTH modulates the expression of ClC-5 in the kidney cortex and that neither 1α,25(OH)2 vitamin D3 nor PTH regulates ClC-5 expression in the medulla. The pattern of expression of ClC-5 varies with urinary calcium. Animals with higher urinary calcium concentrations have lower levels of ClC-5 mRNA and protein expression, suggesting that the ClC-5 chloride channel plays a role in calcium reabsorption.

Adipolin/CTRP12 protects against pathological vascular remodelling through suppression of smooth muscle cell growth and macrophage inflammatory response

2019 ◽  
Vol 116 (1) ◽  
pp. 237-249 ◽  
Author(s):  
Hayato Ogawa ◽  
Koji Ohashi ◽  
Masanori Ito ◽  
Rei Shibata ◽  
Noriyoshi Kanemura ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Inflammatory Response ◽  
Transforming Growth Factor ◽  
Inflammatory Responses ◽  
Transforming Growth Factor Β ◽  
Vascular Remodelling ◽  
Vsmc Proliferation ◽  
Protein Levels ◽  
Β Receptor

AbstractAimsSecreted factors produced by adipose tissue are involved in the pathogenesis of cardiovascular disease. We previously identified adipolin, also known as C1q/TNF-related protein 12, as an insulin-sensitizing adipokine. However, the role of adipolin in vascular disease remains unknown. Here, we investigated whether adipolin modulates pathological vascular remodelling.Methods and resultsAdipolin-knockout (APL-KO) and wild-type (WT) mice were subjected to wire-induced injury of the femoral artery. APL-KO mice showed increased neointimal thickening after vascular injury compared with WT mice, which was accompanied by an enhanced inflammatory response and vascular cell proliferation in injured arteries. Adipolin deficiency also led to a reduction in transforming growth factor-β (TGF-β) 1 protein levels in injured arteries. Treatment of cultured macrophages with adipolin protein led to a reduction in lipopolysaccharide-stimulated expression of inflammatory mediators, including tumour necrosis factor (TNF)-α, interleukin (IL) 6, and monocyte chemotactic protein (MCP)-1. These effects were reversed by inhibition of TGF-β receptor II (TGF-βRII)/Smad2 signalling. Adipolin also reduced platelet-derived growth factor (PDGF)-BB-stimulated proliferation of vascular smooth muscle cells (VSMCs) through a TGF-βRII/Smad2-dependent pathway. Furthermore, adipolin treatment significantly increased TGF-β1 concentration in media from cultured VSMCs and macrophages.ConclusionThese data indicate that adipolin protects against the development of pathological vascular remodelling by attenuating macrophage inflammatory responses and VSMC proliferation.

scholarly journals TBK1 regulates YAP/TAZ and fibrogenic fibroblast activation

2020 ◽  
Vol 318 (5) ◽  
pp. L852-L863 ◽  
Author(s):  
Aja Aravamudhan ◽  
Andrew J. Haak ◽  
Kyoung Moo Choi ◽  
Jeffrey A. Meridew ◽  
Nunzia Caporarello ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Large Scale ◽  
Smooth Muscle Actin ◽  
Lung Fibroblasts ◽  
Cell Contact ◽  
Actin Stress Fiber ◽  
Fibroblast Activation ◽  
Protein Levels ◽  
Major Cell Type ◽  
Interfering Rna

Idiopathic pulmonary fibrosis (IPF) results in scarring of the lungs by excessive extracellular matrix (ECM) production. Resident fibroblasts are the major cell type involved in ECM deposition. The biochemical pathways that facilitate pathological fibroblast activation leading to aberrant ECM deposition are not fully understood. Tank binding protein kinase-1 (TBK1) is a kinase that regulates multiple signaling pathways and was recently identified as a candidate regulator of fibroblast activation in a large-scale small-interfering RNA (siRNA) screen. To determine the effect of TBK1 on fibroblast activation, TBK1 was inhibited pharmacologically (MRT-68601) and genetically (siRNA) in normal and IPF human lung fibroblasts. Reducing the activity or expression of TBK1 led to reduction in α-smooth muscle actin stress fiber levels by 40–60% and deposition of ECM components collagen I and fibronectin by 50% in TGF-β-stimulated normal and IPF fibroblasts. YAP and TAZ are homologous mechanoregulatory profibrotic transcription cofactors known to regulate fibroblast activation. TBK1 knockdown or inhibition decreased the total and nuclear protein levels of YAP/TAZ. Additionally, low cell-cell contact and increased ECM substrate stiffness augmented the phosphorylation and activation of TBK1, consistent with cues that regulate YAP/TAZ. The action of TBK1 toward YAP/TAZ activation was independent of LATS1/2 and canonical downstream TBK1 signaling mediator IRF3 but dependent on proteasomal machinery of the cell. This study identifies TBK1 as a fibrogenic activator of human pulmonary fibroblasts, suggesting TBK1 may be a novel therapeutic target in pulmonary fibrosis.

scholarly journals ERK1/2 Signaling Pathway Activated by EGF Promotes Proliferation, Transdifferentiation, and Migration of Cultured Primary Newborn Rat Lung Fibroblasts

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Yu Hu ◽  
JianHua Fu ◽  
XueYan Liu ◽  
XinDong Xue
Keyword(s):  
Western Blotting ◽  
Cell Cycle Progression ◽  
Cellular Proliferation ◽  
Smooth Muscle Actin ◽  
S Phase ◽  
Cell Counting ◽  
Lung Fibroblasts ◽  
Newborn Rats ◽  
Protein Levels ◽  
And Migration

Background. Bronchopulmonary dysplasia (BPD) is a common and serious complication in premature infants. Lung fibroblasts (LFs) are present in the extracellular matrix and participate in pulmonary development in response to BPD. The aim of this study was to investigate the effect of extracellular signal-regulated kinase (ERK) on LFs cultured from newborn rats. Material and Methods. Primary LFs were isolated and treated with epidermal growth factor (EGF, 20 ng/mL) in the presence or absence of an ERK inhibitor, PD98059 (10 μmol/L). Phosphorylated ERK1/2 (p-ERK1/2) protein levels were determined using immunocytochemistry, western blotting, and real-time reverse transcription quantitative (RT–q)PCR. LF proliferation was examined by flow cytometry and a cell counting kit-8 assay. LF transdifferentiation was examined by protein and mRNA expression of α-smooth muscle actin (α-SMA) by immunocytochemistry, western blotting, and RT–qPCR. LF migration was examined by the transwell method. Results. Phosphorylated ERK1/2, which was activated by EGF, promoted LF proliferation by accelerating cell-cycle progression from the G1 to S phase. After treatment with PD98059, the expression of p-ERK1/2 in LFs, cellular proliferation, and the percentage of cells in S phase were significantly decreased. Phosphorylated ERK1/2 also promoted the differentiation of LFs into myofibroblasts through increased α-SMA synthesis and migration. Conclusion. The activation of ERK promotes proliferation, transdifferentiation, and migration of lung fibroblasts from newborn rats.

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