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Li X, Wang Y, Ren M, Liu Q, Li J, Zhang L, Yao S, Tang L, Wen G, An J, Jin H, Tuo B. The role of chloride intracellular channel 4 in tumors. Cancer Cell Int 2025; 25:118. [PMID: 40140845 PMCID: PMC11948840 DOI: 10.1186/s12935-025-03737-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2024] [Accepted: 03/07/2025] [Indexed: 03/28/2025] Open
Abstract
Tumors are among the most predominant health problems in the world, and the annual incidence of cancer is increasing globally; therefore, there is an urgent need to identify effective therapeutic targets. Chloride intracellular channel 4 (CLIC4) belongs to the family of chloride intracellular channels (CLICs), which are widely expressed in various tissues and organs, such as the brain, lung, pancreas, colorectum, and ovary, and play important roles in promoting apoptosis, promoting angiogenesis, maintaining normal proliferation of endothelial cells, and regulating the assembly and reconstruction of the cytoskeleton. The expression and function of CLIC4 in tumors varies. It has been reported that CLIC4 is low expressed in gastric cancer, skin cancer and prostate cancer, suggesting a tumor suppressor role. Interestingly, CLIC4 is overexpressed in pancreatic, ovarian and breast cancers, indicating a cancer-promoting role. CLIC4 expression is dysregulated in some solid tumors, which may be because CLIC4 is involved in the growth, migration or invasion of some cancer cells through various mechanisms. Regulation of CLIC4 expression may be a potential therapeutic strategy for some tumors. CLIC4 may be a promising therapeutic target and a biomarker for some cancers. In this study, we review the role of CLIC4 in several cancers and its value in the diagnosis and treatment of tumors.
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Affiliation(s)
- Xin Li
- Department of Gastroenterology, Digestive Disease Hospital, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Huichuan, Zunyi, 563003, Guizhou, China
| | - Yongfeng Wang
- Department of Gastroenterology, Digestive Disease Hospital, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Huichuan, Zunyi, 563003, Guizhou, China
| | - Minmin Ren
- Department of General Surgery, Digestive Disease Hospital, Affiliated Hospital of Zunyi Medical University, Zunyi, 563003, Guizhou Province, China
- Nursing School of Zunyi Medical University, Zunyi, 563003, Guizhou Province, China
| | - Qian Liu
- Department of Gastroenterology, Digestive Disease Hospital, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Huichuan, Zunyi, 563003, Guizhou, China
| | - Jiajia Li
- Department of Gastroenterology, Digestive Disease Hospital, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Huichuan, Zunyi, 563003, Guizhou, China
| | - Li Zhang
- Department of Gastroenterology, Digestive Disease Hospital, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Huichuan, Zunyi, 563003, Guizhou, China
| | - Shun Yao
- Department of Gastroenterology, Digestive Disease Hospital, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Huichuan, Zunyi, 563003, Guizhou, China
| | - Lulu Tang
- Department of Gastroenterology, Digestive Disease Hospital, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Huichuan, Zunyi, 563003, Guizhou, China
| | - Guorong Wen
- Department of Gastroenterology, Digestive Disease Hospital, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Huichuan, Zunyi, 563003, Guizhou, China
| | - Jiaxing An
- Department of Gastroenterology, Digestive Disease Hospital, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Huichuan, Zunyi, 563003, Guizhou, China
| | - Hai Jin
- Department of Gastroenterology, Digestive Disease Hospital, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Huichuan, Zunyi, 563003, Guizhou, China.
- The Collaborative Innovation Center of Tissue Damage Repair and Regenerative Medicine, Zunyi Medical University, Zunyi, 563003, China.
| | - Biguang Tuo
- Department of Gastroenterology, Digestive Disease Hospital, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Huichuan, Zunyi, 563003, Guizhou, China.
- The Collaborative Innovation Center of Tissue Damage Repair and Regenerative Medicine, Zunyi Medical University, Zunyi, 563003, China.
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Liu Z, Liu J, Chen Z, Zhu X, Ding R, Huang S, Xu H. CLIC4 Is a New Biomarker for Glioma Prognosis. Biomedicines 2024; 12:2579. [PMID: 39595145 PMCID: PMC11591648 DOI: 10.3390/biomedicines12112579] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2024] [Revised: 11/05/2024] [Accepted: 11/06/2024] [Indexed: 11/28/2024] Open
Abstract
BACKGROUND Chloride Intracellular Channel 4 (CLIC4) plays a versatile role in cellular functions beyond its role in primary chloride ion transport. Notably, many studies found an association between CLIC4 expression and cancers. However, the correlation between CLIC4 and glioma remains to be uncovered. METHODS A total of 3162 samples from nine public datasets were analyzed to reveal the relationship between CLIC4 expression and glioma malignancy or prognosis. Immunohistochemistry (IHC) staining was performed to examine the results in an in-house cohort. A nomogram model was constructed to predict the prognosis. Functional enrichment analysis was employed to find CLIC4-associated differentially expressed genes in glioma. Immune infiltration analysis, correlation analysis, and IHC staining were employed, aiming to examine the correlation between CLIC4 expression, immune cell infiltration, and ECM (extracellular matrix)-related genes. RESULTS The expression level of CLIC4 was correlated with the malignancy of glioma and the prognosis of patients. More aggressive gliomas and mesenchymal GBM are associated with a high expression of CLIC4. Gliomas with IDH mutation or 1p19q codeletion express a low level of CLIC4, and a high expression of CLIC4 correlates with poor prognosis. The nomogram model shows a good predictive performance. The DEGs (differentially expressed genes) in gliomas with high and low CLIC4 expression are enriched in extracellular matrix and immune functions. On the one hand, gliomas with high CLIC4 expression have a greater presence of macrophages, neutrophils, and eosinophils; on the other hand, a high CLIC4 expression in gliomas is positively associated with ECM-related genes. CONCLUSIONS Compared to glioma cells with low CLIC4 expression, gliomas with high CLIC4 expression exhibit greater malignancy and poorer prognosis. Our findings indicate that a high level of CLIC4 correlates with high expression of ECM-related genes and the infiltration of macrophages, neutrophils, and eosinophils within glioma tissues.
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Affiliation(s)
| | | | | | | | | | - Shulan Huang
- Department of Neurosurgery, Renmin Hospital of Wuhan University, Wuhan 430060, China; (Z.L.); (J.L.); (Z.C.); (X.Z.); (R.D.)
| | - Haitao Xu
- Department of Neurosurgery, Renmin Hospital of Wuhan University, Wuhan 430060, China; (Z.L.); (J.L.); (Z.C.); (X.Z.); (R.D.)
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Wang ZZ, Wang K, Xu LF, Su C, Gong JS, Shi JS, Ma XD, Xie N, Qian JY. Unlocking the Potential of Collagenases: Structures, Functions, and Emerging Therapeutic Horizons. BIODESIGN RESEARCH 2024; 6:0050. [PMID: 39381623 PMCID: PMC11458858 DOI: 10.34133/bdr.0050] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2024] [Revised: 08/06/2024] [Accepted: 08/31/2024] [Indexed: 10/10/2024] Open
Abstract
Collagenases, a class of enzymes that are specifically responsible for collagen degradation, have garnered substantial attention because of their pivotal roles in tissue repair, remodeling, and medical interventions. This comprehensive review investigates the diversity, structures, and mechanisms of collagenases and highlights their therapeutic potential. First, it provides an overview of the biochemical properties of collagen and highlights its importance in extracellular matrix function. Subsequently, it meticulously analyzes the sources of collagenases and their applications in tissue engineering and food processing. Notably, this review emphasizes the predominant role played by microbial collagenases in commercial settings while discussing their production and screening methods. Furthermore, this study elucidates the methodology employed for determining collagenase activity and underscores the importance of an accurate evaluation for both research purposes and clinical applications. Finally, this review highlights the future research prospects for collagenases, with a particular focus on promoting wound healing and treating scar tissue formation and fibrotic diseases.
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Affiliation(s)
- Zhen-Zhen Wang
- School of Life Sciences and Health Engineering,
Jiangnan University, Wuxi 214122, PR China
| | - Kang Wang
- School of Life Sciences and Health Engineering,
Jiangnan University, Wuxi 214122, PR China
| | - Ling-Feng Xu
- School of Life Sciences and Health Engineering,
Jiangnan University, Wuxi 214122, PR China
| | - Chang Su
- School of Life Sciences and Health Engineering,
Jiangnan University, Wuxi 214122, PR China
| | - Jin-Song Gong
- School of Life Sciences and Health Engineering,
Jiangnan University, Wuxi 214122, PR China
| | - Jin-Song Shi
- School of Life Sciences and Health Engineering,
Jiangnan University, Wuxi 214122, PR China
| | - Xu-Dong Ma
- Cytori Therapeutics LLC., Shanghai 201802, PR China
| | - Nan Xie
- Cytori Therapeutics LLC., Shanghai 201802, PR China
| | - Jian-Ying Qian
- School of Life Sciences and Health Engineering,
Jiangnan University, Wuxi 214122, PR China
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Ozaki S, Mikami K, Kunieda T, Tanaka J. Chloride Intracellular Channel Proteins (CLICs) and Malignant Tumor Progression: A Focus on the Preventive Role of CLIC2 in Invasion and Metastasis. Cancers (Basel) 2022; 14:cancers14194890. [PMID: 36230813 PMCID: PMC9562003 DOI: 10.3390/cancers14194890] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2022] [Revised: 10/03/2022] [Accepted: 10/03/2022] [Indexed: 11/27/2022] Open
Abstract
Simple Summary Although chloride intracellular channel proteins (CLICs) have been identified as ion channel proteins, their true functions are still elusive. Recent in silico analyses show that CLICs may be prognostic markers in cancer. This review focuses on CLIC2 that plays preventive roles in malignant cell invasion and metastasis. CLIC2 is secreted extracellularly and binds to matrix metalloproteinase 14 (MMP14), while inhibiting its activity. As a result, CLIC2 may contribute to the development/maintenance of junctions between blood vessel endothelial cells and the inhibition of invasion and metastasis of tumor cells. CLIC2 may be a novel therapeutic target for malignancies. Abstract CLICs are the dimorphic protein present in both soluble and membrane fractions. As an integral membrane protein, CLICs potentially possess ion channel activity. However, it is not fully clarified what kinds of roles CLICs play in physiological and pathological conditions. In vertebrates, CLICs are classified into six classes: CLIC1, 2, 3, 4, 5, and 6. Recently, in silico analyses have revealed that the expression level of CLICs may have prognostic significance in cancer. In this review, we focus on CLIC2, which has received less attention than other CLICs, and discuss its role in the metastasis and invasion of malignant tumor cells. CLIC2 is expressed at higher levels in benign tumors than in malignant ones, most likely preventing tumor cell invasion into surrounding tissues. CLIC2 is also expressed in the vascular endothelial cells of normal tissues and maintains their intercellular adhesive junctions, presumably suppressing the hematogenous metastasis of malignant tumor cells. Surprisingly, CLIC2 is localized in secretory granules and secreted into the extracellular milieu. Secreted CLIC2 binds to MMP14 and inhibits its activity, leading to suppressed MMP2 activity. CLIC4, on the other hand, promotes MMP14 activity. These findings challenge the assumption that CLICs are ion channels, implying that they could be potential new targets for the treatment of malignant tumors.
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Affiliation(s)
- Saya Ozaki
- Department of Neurosurgery, Graduate School of Medicine, Ehime University, Toon 791-0295, Japan
- Department of Neurosurgery, National Cerebral and Cardiovascular Center Hospital, Suita 564-8565, Japan
- Correspondence: (S.O.); (J.T.)
| | - Kanta Mikami
- Department of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime University, Toon 791-0295, Japan
| | - Takeharu Kunieda
- Department of Neurosurgery, Graduate School of Medicine, Ehime University, Toon 791-0295, Japan
| | - Junya Tanaka
- Department of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime University, Toon 791-0295, Japan
- Correspondence: (S.O.); (J.T.)
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5
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Al Khamici H, Sanchez VC, Yan H, Cataisson C, Michalowski AM, Yang HH, Li L, Lee MP, Huang J, Yuspa SH. The oxidoreductase CLIC4 is required to maintain mitochondrial function and resistance to exogenous oxidants in breast cancer cells. J Biol Chem 2022; 298:102275. [PMID: 35863434 PMCID: PMC9418444 DOI: 10.1016/j.jbc.2022.102275] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2022] [Revised: 06/01/2022] [Accepted: 06/02/2022] [Indexed: 02/07/2023] Open
Abstract
The chloride intracellular channel-4 (CLIC4) is one of the six highly conserved proteins in the CLIC family that share high structural homology with GST-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H2O2 as a ROS-inducing agent. In intact cells, H2O2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H2O2-induced apoptosis was associated with low expression and degradation of the antiapoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H2O2-treated control and CLIC4-null cells revealed upregulation of genes associated with ROS-induced apoptosis and downregulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4-deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells.
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Affiliation(s)
- Heba Al Khamici
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health. Bethesda, Maryland, USA
| | - Vanesa C Sanchez
- Office of Science, Division of Nonclinical Science, Center for Tobacco Products, U.S. Food and Drug Administration, Silver Spring, Maryland, USA
| | - Hualong Yan
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health. Bethesda, Maryland, USA
| | - Christophe Cataisson
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health. Bethesda, Maryland, USA
| | - Aleksandra M Michalowski
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health. Bethesda, Maryland, USA
| | - Howard H Yang
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health. Bethesda, Maryland, USA
| | - Luowei Li
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health. Bethesda, Maryland, USA
| | - Maxwell P Lee
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health. Bethesda, Maryland, USA
| | - Jing Huang
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health. Bethesda, Maryland, USA
| | - Stuart H Yuspa
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health. Bethesda, Maryland, USA.
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Sanchez VC, Yang HH, Craig-Lucas A, Dubois W, Carofino BL, Lack J, Dwyer JE, Simpson RM, Cataisson C, Lee MP, Luo J, Hunter KW, Yuspa SH. Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence. PLoS Genet 2022; 18:e1010271. [PMID: 35727842 PMCID: PMC9249210 DOI: 10.1371/journal.pgen.1010271] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2022] [Revised: 07/01/2022] [Accepted: 05/22/2022] [Indexed: 11/18/2022] Open
Abstract
The TGF-β-regulated Chloride Intracellular Channel 4 (CLIC4) is an essential participant in the formation of breast cancer stroma. Here, we used data available from the TCGA and METABRIC datasets to show that CLIC4 expression was higher in breast cancers from younger women and those with early-stage metastatic disease. Elevated CLIC4 predicted poor outcome in breast cancer patients and was linked to the TGF-β pathway. However, these associations did not reveal the underlying biological contribution of CLIC4 to breast cancer progression. Constitutive ablation of host Clic4 in two murine metastatic breast cancer models nearly eliminated lung metastases without reducing primary tumor weight, while tumor cells ablated of Clic4 retained metastatic capability in wildtype hosts. Thus, CLIC4 was required for host metastatic competence. Pre- and post-metastatic proteomic analysis identified circulating pro-metastatic soluble factors that differed in tumor-bearing CLIC4-deficient and wildtype hosts. Vascular abnormalities and necrosis increased in primary tumors from CLIC4-deficient hosts. Transcriptional profiles of both primary tumors and pre-metastatic lungs of tumor-bearing CLIC4-deficient hosts were consistent with a microenvironment where inflammatory pathways were elevated. Altogether, CLIC4 expression in human breast cancers may serve as a prognostic biomarker; therapeutic targeting of CLIC4 could reduce primary tumor viability and host metastatic competence.
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Affiliation(s)
- Vanesa C. Sanchez
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Howard H. Yang
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Alayna Craig-Lucas
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Wendy Dubois
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Brandi L. Carofino
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Justin Lack
- NIAID Collaborative Bioinformatics Resource (NCBR), National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, United States of America
- Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, Maryland, United States of America
| | - Jennifer E. Dwyer
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health, Bethesda, Maryland, United States of America
| | - R. Mark Simpson
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Christophe Cataisson
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Max P. Lee
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Ji Luo
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Kent W. Hunter
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Stuart H. Yuspa
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Center, National Institutes of Health, Bethesda, Maryland, United States of America
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Wasson CW, Caballero-Ruiz B, Gillespie J, Derrett-Smith E, Mankouri J, Denton CP, Canettieri G, Riobo-Del Galdo NA, Del Galdo F. Induction of Pro-Fibrotic CLIC4 in Dermal Fibroblasts by TGF-β/Wnt3a Is Mediated by GLI2 Upregulation. Cells 2022; 11:cells11030530. [PMID: 35159339 PMCID: PMC8834396 DOI: 10.3390/cells11030530] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2022] [Revised: 01/21/2022] [Accepted: 02/01/2022] [Indexed: 11/16/2022] Open
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
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Affiliation(s)
- Christopher W. Wasson
- Leeds Institute of Rheumatic and Musculoskeletal Medicine, Faculty of Medicine and Health, University of Leeds, Leeds LS29JT, UK; (J.G.); (F.D.G.)
- Correspondence:
| | - Begoña Caballero-Ruiz
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS29JT, UK; (B.C.-R.); (J.M.); (N.A.R.-D.G.)
- Department of Molecular Medicine, Sapienza University of Rome, 00196 Rome, Italy;
| | - Justin Gillespie
- Leeds Institute of Rheumatic and Musculoskeletal Medicine, Faculty of Medicine and Health, University of Leeds, Leeds LS29JT, UK; (J.G.); (F.D.G.)
| | - Emma Derrett-Smith
- Centre for Rheumatology and Connective Tissue Diseases, UCL Division of Medicine, London NW32PF, UK; (E.D.-S.); (C.P.D.)
| | - Jamel Mankouri
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS29JT, UK; (B.C.-R.); (J.M.); (N.A.R.-D.G.)
| | - Christopher P. Denton
- Centre for Rheumatology and Connective Tissue Diseases, UCL Division of Medicine, London NW32PF, UK; (E.D.-S.); (C.P.D.)
| | - Gianluca Canettieri
- Department of Molecular Medicine, Sapienza University of Rome, 00196 Rome, Italy;
| | - Natalia A. Riobo-Del Galdo
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS29JT, UK; (B.C.-R.); (J.M.); (N.A.R.-D.G.)
- Leeds Institute of Medical Research, Faculty of Medicine and Health, University of Leeds, Leeds LS29JT, UK
| | - Francesco Del Galdo
- Leeds Institute of Rheumatic and Musculoskeletal Medicine, Faculty of Medicine and Health, University of Leeds, Leeds LS29JT, UK; (J.G.); (F.D.G.)
- Scleroderma Programme, NIHR Leeds Musculoskeletal Biomedical Research Centre, Leeds LS29JT, UK
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Zhou W, Menkhorst E, Dimitriadis E. Characterization of chloride intracellular channel 4 in the regulation of human trophoblast function. Placenta 2022; 119:24-30. [DOI: 10.1016/j.placenta.2022.01.012] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/18/2021] [Revised: 12/20/2021] [Accepted: 01/17/2022] [Indexed: 11/27/2022]
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Wang H, An J, He S, Liao C, Wang J, Tuo B. Chloride intracellular channels as novel biomarkers for digestive system tumors (Review). Mol Med Rep 2021; 24:630. [PMID: 34278487 DOI: 10.3892/mmr.2021.12269] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2021] [Accepted: 05/19/2021] [Indexed: 11/06/2022] Open
Abstract
Digestive system malignant tumors are common tumors, and the traditional treatment methods for these tumors include surgical resection, radiotherapy, chemotherapy, and molecularly targeted drugs. However, diagnosis remains challenging, and the early detection of postoperative recurrence is complicated. Therefore, it is necessary to explore novel biomarkers to facilitate clinical diagnosis and treatment. Accumulating evidence supports the crucial role of chloride channels in the development of multiple types of cancers. Given that chloride channels are widely expressed and involved in cell proliferation, apoptosis and cell cycle, among other processes, they may serve as a promising diagnostic and therapeutic target. Chloride intracellular channels (CLICs) are a class of chloride channels that are upregulated or downregulated in certain types of cancer. Furthermore, in certain cases, during cell cycle progression, the localization and function of the cytosolic form of the transmembrane proteins of CLICs are also altered, which may provide a key target for cancer therapy. The aim of the present review was to focus on CLICs as biomarkers for digestive system tumors.
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Affiliation(s)
- Hui Wang
- Department of Gastroenterology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou 563003, P.R. China
| | - Jiaxing An
- Department of Gastroenterology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou 563003, P.R. China
| | - Suyu He
- The Fourth Department of the Digestive Disease Center, Suining Central Hospital, Suining, Sichuan 629000, P.R. China
| | - Chengcheng Liao
- Special Key Laboratory of Oral Disease Research, Higher Education Institution in Guizhou Province, School of Stomatology, Zunyi Medical University, Zunyi, Guizhou 563006, P.R. China
| | - Juan Wang
- Department of Gastroenterology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou 563003, P.R. China
| | - Biguang Tuo
- Department of Gastroenterology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou 563003, P.R. China
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10
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Weston RM, Schmitt RE, Grotewiel M, Miles MF. Transcriptome analysis of chloride intracellular channel knockdown in Drosophila identifies oxidation-reduction function as possible mechanism of altered sensitivity to ethanol sedation. PLoS One 2021; 16:e0246224. [PMID: 34228751 PMCID: PMC8259981 DOI: 10.1371/journal.pone.0246224] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2021] [Accepted: 06/18/2021] [Indexed: 01/22/2023] Open
Abstract
Chloride intracellular channels (CLICs) are a unique family of evolutionarily conserved metamorphic proteins, switching between stable conformations based on redox conditions. CLICs have been implicated in a wide variety biological processes including ion channel activity, apoptosis, membrane trafficking, and enzymatic oxidoreductase activity. Understanding the molecular mechanisms by which CLICs engage in these activities is an area of active research. Here, the sole Drosophila melanogaster ortholog, Clic, was targeted for RNAi knockdown to identify genes and biological processes associated with Clic expression. Clic knockdown had a substantial impact on global transcription, altering expression of over 7% of transcribed Drosophila genes. Overrepresentation analysis of differentially expressed genes identified enrichment of Gene Ontology terms including Cytoplasmic Translation, Oxidation-Reduction Process, Heme Binding, Membrane, Cell Junction, and Nucleolus. The top term, Cytoplasmic Translation, was enriched almost exclusively with downregulated genes. Drosophila Clic and vertebrate ortholog Clic4 have previously been tied to ethanol sensitivity and ethanol-regulated expression. Clic knockdown-responsive genes from the present study were found to overlap significantly with gene sets from 4 independently published studies related to ethanol exposure and sensitivity in Drosophila. Bioinformatic analysis of genes shared between these studies revealed an enrichment of genes related to amino acid metabolism, protein processing, oxidation-reduction processes, and lipid particles among others. To determine whether the modulation of ethanol sensitivity by Clic may be related to co-regulated oxidation-reduction processes, we evaluated the effect of hyperoxia on ethanol sedation in Clic knockdown flies. Consistent with previous findings, Clic knockdown reduced acute ethanol sedation sensitivity in flies housed under normoxia. However, this effect was reversed by exposure to hyperoxia, suggesting a common set of molecular-genetic mechanism may modulate each of these processes. This study suggests that Drosophila Clic has a major influence on regulation of oxidative stress signaling and that this function overlaps with the molecular mechanisms of acute ethanol sensitivity in the fly.
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Affiliation(s)
- Rory M. Weston
- Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia, United States of America
- VCU Alcohol Research Center, Virginia Commonwealth University, Richmond, Virginia, United States of America
| | - Rebecca E. Schmitt
- VCU Alcohol Research Center, Virginia Commonwealth University, Richmond, Virginia, United States of America
- Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia, United States of America
| | - Mike Grotewiel
- VCU Alcohol Research Center, Virginia Commonwealth University, Richmond, Virginia, United States of America
- Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia, United States of America
| | - Michael F. Miles
- Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia, United States of America
- VCU Alcohol Research Center, Virginia Commonwealth University, Richmond, Virginia, United States of America
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11
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Wang B, Zheng J, Chen Q, Wu C, Li Y, Yu XY, Liu B, Liang C, Liu SB, Ding H, Wang S, Xue T, Song D, Lei Z, Amin HM, Song YH, Zhou J. CLIC4 abrogation promotes epithelial-mesenchymal transition in gastric cancer. Carcinogenesis 2020; 41:841-849. [PMID: 31560739 DOI: 10.1093/carcin/bgz156] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2019] [Revised: 09/04/2019] [Accepted: 09/23/2019] [Indexed: 12/15/2022] Open
Abstract
Chloride intracellular channel protein 4 (CLIC4) has been implicated in different types of cancers, but the role of CLIC4 in the development of gastric cancer (GC) remains unknown. We analyzed the expression of CLIC4 in 102 pairs of gastric adenocarcinomas by western blot and real-time PCR. Our data revealed that the expression of CLIC4 is reduced in GC tumor tissues compared with adjacent normal tissues. The expression levels of CLIC4 correlate inversely with the clinical stage of GC. CLIC4 expression is lowest in MKN45 cells, which have the highest tumorigenic potential and express the highest levels of cancer stem cell markers CD44 and OCT4, compared with N87 and AGS cells. Exogenous overexpression of CLIC4 downregulated the expression of CD44 and OCT4, and inhibited migration, invasion and epithelial-mesenchymal transition (EMT). Moreover, anchorage-independent growth of GC cells was decreased and the cells became more sensitive to 5-fluorouracil and etoposide treatment when CLIC4 was overexpressed. The ability of N87 cells to form tumors in nude mice was enhanced when CLIC4 was silenced. We, for the first time, demonstrate that CLIC4 suppresses tumor growth by inhibiting cancer cell stemness and EMT.
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Affiliation(s)
- Baolong Wang
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, P. R. China
| | - Jiqing Zheng
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, P. R. China
| | - Qiongyuan Chen
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, P. R. China
| | - Chaofan Wu
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, P. R. China
| | - Yangxin Li
- Department of Cardiovascular Surgery and Institute of Cardiovascular Science, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, P. R. China
| | - Xi-Yong Yu
- Key Laboratory of Molecular Clinical Pharmacology and Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China
| | - Bin Liu
- Department of Cardiology, Second Hospital of Jilin University, Changchun, Jilin, P. R. China
| | - Chun Liang
- Department of Cardiology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, P. R. China
| | - Song-Bai Liu
- Suzhou Vocational Health College, Suzhou Key Laboratory of Biotechnology for Laboratory Medicine, Suzhou, Jiangsu Province, China
| | - Hui Ding
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, P. R. China
| | - Shuochen Wang
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, P. R. China
| | - Ting Xue
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, P. R. China
| | - David Song
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, P. R. China
| | - Zhangni Lei
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, P. R. China
| | - Hesham M Amin
- Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Yao-Hua Song
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou, P. R. China
| | - Jin Zhou
- Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou, P. R. China
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12
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Changes in Proteome of Fibroblasts Isolated from Psoriatic Skin Lesions. Int J Mol Sci 2020; 21:ijms21155363. [PMID: 32731552 PMCID: PMC7432102 DOI: 10.3390/ijms21155363] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2020] [Revised: 07/23/2020] [Accepted: 07/27/2020] [Indexed: 11/16/2022] Open
Abstract
The dermal fibroblasts are in constant contact with the cells of the immune system and skin epidermis. Therefore, they are essential for the development of lesions in psoriasis. The aim of this study was to assess the changes in the proteomic profile of fibroblasts in the dermis of psoriasis patients, and to discuss the most significant changes and their potential consequences. The proteomic results indicate that fibroblast dysfunction arises from the upregulation of proinflammatory factors and antioxidant proteins, as well as those involved in signal transduction and participating in proteolytic processes. Moreover, downregulated proteins in psoriatic fibroblasts are mainly responsible for the transcription/translation processes, glycolysis/ adenosine triphosphate synthesis and structural molecules. These changes can directly affect intercellular signaling and promote the hyperproliferation of epidermal cells. A better understanding of the metabolic effects of the proteomic changes observed could guide the development of new pharmacotherapies for psoriasis.
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Lima FJ, Lopes MLDDS, Barros CCDS, Nonaka CFW, Silveira ÉJDD. Modification in CLIC4 Expression is Associated with P53, TGF-β, TNF-α and Myofibroblasts in Lip Carcinogenesis. Braz Dent J 2020; 31:290-297. [PMID: 32667519 DOI: 10.1590/0103-6440202003104] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2019] [Accepted: 12/11/2019] [Indexed: 11/21/2022] Open
Abstract
Chloride intracellular channel-4 (CLIC4) is regulated by p53 and tumor necrosis factor-α (TNF-α), it is linked to the increase of transforming growth factor-β (TGF-β), and myofibroblastic differentiation in skin carcinogenesis. This study analyzed the immunoexpression of CLIC4, p53, TGF-β, TNF-α, and α-SMA in 50 actinic cheilitis (AC) and 50 lower lip squamous cell carcinoma (LLSCC). AC and LLSCC immunoexpression were categorized as score 1 (<5% positive cells), 2 (5-50%) or 3 (>50%). For CLIC4, nuclear and cytoplasmic immunostaining of epithelial cells was considered individually. For morphologic analysis, the World Health Organization criteria were used to epithelial dysplasia grade of ACs, and Bryne grading of malignancy system was applied for LLSCC. Higher nuclear CLIC4 (CLIC4n) and TGF-β were observed in ACs with low-risk of transformation, while cytoplasmic CLIC4 (CLIC4c), p53 and TNF-α were higher in the high-risk cases (p<0.05). In LLSCCs, CLIC4c was higher in cases with lymph node metastasis, advanced clinical stages, and histological high-grade malignancy. p53 expression was higher in high-grade LLSCCs, whereas TGF-β decreased as the clinical stage and morphological grade progressed (p<0.05). ACs showed an increased expression of CLIC4n and TGF-β, while CLIC4c and α-SMA were higher in LLSCCs (p<0.0001). Both lesions showed negative correlation between CLIC4n and CLIC4c, while in LLSCCs, negative correlation was also verified between CLIC4c and p53, as well as CLIC4c and TGF-β (p<0.05). Change of CLIC4 from the nucleus to cytoplasm and alterations in p53, TGF-β, TNF-α, and α-SMA expression are involved in lip carcinogenesis.
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Affiliation(s)
- Francisco Jadson Lima
- Department of Dentistry, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
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14
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Scorpion Toxins and Ion Channels: Potential Applications in Cancer Therapy. Toxins (Basel) 2020; 12:toxins12050326. [PMID: 32429050 PMCID: PMC7290751 DOI: 10.3390/toxins12050326] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2020] [Revised: 04/14/2020] [Accepted: 04/15/2020] [Indexed: 12/24/2022] Open
Abstract
Apoptosis, a genetically directed process of cell death, has been studied for many years, and the biochemical mechanisms that surround it are well known and described. There are at least three pathways by which apoptosis occurs, and each pathway depends on extra or intracellular processes for activation. Apoptosis is a vital process, but disturbances in proliferation and cell death rates can lead to the development of diseases like cancer. Several compounds, isolated from scorpion venoms, exhibit inhibitory effects on different cancer cells. Indeed, some of these compounds can differentiate between healthy and cancer cells within the same tissue. During the carcinogenic process, morphological, biochemical, and biological changes occur that enable these compounds to modulate cancer but not healthy cells. This review highlights cancer cell features that enable modulation by scorpion neurotoxins. The properties of the isolated scorpion neurotoxins in cancer cells and the potential uses of these compounds as alternative treatments for cancer are discussed.
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15
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Gururaja Rao S, Patel NJ, Singh H. Intracellular Chloride Channels: Novel Biomarkers in Diseases. Front Physiol 2020; 11:96. [PMID: 32116799 PMCID: PMC7034325 DOI: 10.3389/fphys.2020.00096] [Citation(s) in RCA: 78] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2019] [Accepted: 01/27/2020] [Indexed: 12/27/2022] Open
Abstract
Ion channels are integral membrane proteins present on the plasma membrane as well as intracellular membranes. In the human genome, there are more than 400 known genes encoding ion channel proteins. Ion channels are known to regulate several cellular, organellar, and physiological processes. Any mutation or disruption in their function can result in pathological disorders, both common or rare. Ion channels present on the plasma membrane are widely acknowledged for their role in various biological processes, but in recent years, several studies have pointed out the importance of ion channels located in intracellular organelles. However, ion channels located in intracellular organelles are not well-understood in the context of physiological conditions, such as the generation of cellular excitability and ionic homeostasis. Due to the lack of information regarding their molecular identity and technical limitations of studying them, intracellular organelle ion channels have thus far been overlooked as potential therapeutic targets. In this review, we focus on a novel class of intracellular organelle ion channels, Chloride Intracellular Ion Channels (CLICs), mainly documented for their role in cardiovascular, neurophysiology, and tumor biology. CLICs have a single transmembrane domain, and in cells, they exist in cytosolic as well as membranous forms. They are predominantly present in intracellular organelles and have recently been shown to be localized to cardiomyocyte mitochondria as well as exosomes. In fact, a member of this family, CLIC5, is the first mitochondrial chloride channel to be identified on the molecular level in the inner mitochondrial membrane, while another member, CLIC4, is located predominantly in the outer mitochondrial membrane. In this review, we discuss this unique class of intracellular chloride channels, their role in pathologies, such as cardiovascular, cancer, and neurodegenerative diseases, and the recent developments concerning their usage as theraputic targets.
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Affiliation(s)
- Shubha Gururaja Rao
- Department of Physiology and Cell Biology, The Ohio State University Wexner Medical Center, Columbus, OH, United States
| | - Neel J Patel
- Department of Cardiology, Hospital of the University of Pennsylvania, Philadelphia, PA, United States
| | - Harpreet Singh
- Department of Physiology and Cell Biology, The Ohio State University Wexner Medical Center, Columbus, OH, United States
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16
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Carofino BL, Dinshaw KM, Ho PY, Cataisson C, Michalowski AM, Ryscavage A, Alkhas A, Wong NW, Koparde V, Yuspa SH. Head and neck squamous cancer progression is marked by CLIC4 attenuation in tumor epithelium and reciprocal stromal upregulation of miR-142-3p, a novel post-transcriptional regulator of CLIC4. Oncotarget 2019; 10:7251-7275. [PMID: 31921386 PMCID: PMC6944452 DOI: 10.18632/oncotarget.27387] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2019] [Accepted: 12/02/2019] [Indexed: 02/06/2023] Open
Abstract
Chloride intracellular channel 4 (CLIC4) is a tumor suppressor implicated in processes including growth arrest, differentiation, and apoptosis. CLIC4 protein expression is diminished in the tumor parenchyma during progression in squamous cell carcinoma (SCC) and other neoplasms, but the underlying mechanisms have not been identified. Data from The Cancer Genome Atlas suggest this is not driven by genomic alterations. However, screening and functional assays identified miR-142-3p as a regulator of CLIC4. CLIC4 and miR-142-3p expression are inversely correlated in head and neck (HN) SCC and cervical SCC, particularly in advanced stage cancers. In situ localization revealed that stromal immune cells, not tumor cells, are the predominant source of miR-142-3p in HNSCC. Furthermore, HNSCC single-cell expression data demonstrated that CLIC4 is lower in tumor epithelial cells than in stromal fibroblasts and endothelial cells. Tumor-specific downregulation of CLIC4 was confirmed in an SCC xenograft model concurrent with immune cell infiltration and miR-142-3p upregulation. These findings provide the first evidence of CLIC4 regulation by miRNA. Furthermore, the distinct localization of CLIC4 and miR-142-3p within the HNSCC tumor milieu highlight the limitations of bulk tumor analysis and provide critical considerations for both future mechanistic studies and use of miR-142-3p as a HNSCC biomarker.
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Affiliation(s)
- Brandi L. Carofino
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
| | - Kayla M. Dinshaw
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
- Department of Molecular and Cellular Biology, University of California, Berkeley, Berkeley, CA, USA
| | - Pui Yan Ho
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
- Department of Pediatrics, Division of Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - Christophe Cataisson
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
| | - Aleksandra M. Michalowski
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
| | - Andrew Ryscavage
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
| | | | - Nathan W. Wong
- CCR Collaborative Bioinformatics Resource (CCBR), Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
- Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research, Frederick, MD, USA
| | - Vishal Koparde
- CCR Collaborative Bioinformatics Resource (CCBR), Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
- Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research, Frederick, MD, USA
| | - Stuart H. Yuspa
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
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17
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Albrechtsen R, Wewer Albrechtsen NJ, Gnosa S, Schwarz J, Dyrskjøt L, Kveiborg M. Identification of ADAM12 as a Novel Basigin Sheddase. Int J Mol Sci 2019; 20:ijms20081957. [PMID: 31013576 PMCID: PMC6514901 DOI: 10.3390/ijms20081957] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2019] [Revised: 04/16/2019] [Accepted: 04/17/2019] [Indexed: 02/08/2023] Open
Abstract
The transmembrane glycoprotein basigin, a member of the immunoglobulin superfamily, stimulates matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) degradation and thereby drives cancer cell invasion. Basigin is proteolytically shed from the cell surface and high concentrations of soluble basigin in the blood dictates poor prognosis in cancer patients. A positive correlation between basigin and a disintegrin and metalloproteinase (ADAM)-12 in serum from prostate cancer patients has been reported. Yet, the functional relevance of this correlation is unknown. Here, we show that ADAM12 interacts with basigin and cleaves it in the juxtamembrane region. Specifically, overexpression of ADAM12 increases ectodomain shedding of an alkaline phosphatase-tagged basigin reporter protein from the cell surface. Moreover, CRISPR/Cas9-mediated knockout of ADAM12 in human HeLa carcinoma cells results in reduced shedding of the basigin reporter, which can be rescued by ADAM12 re-expression. We detected endogenous basigin fragments, corresponding to the expected size of the ADAM12-generated ectodomain, in conditioned media from ADAM12 expressing cancer cell-lines, as well as serum samples from a healthy pregnant donor and five bladder cancer patients, known to contain high ADAM12 levels. Supporting the cancer relevance of our findings, we identified several cancer-associated mutations in the basigin membrane proximal region. Subsequent in vitro expression showed that some of these mutants are more prone to ADAM12-mediated shedding and that the shed ectodomain can enhance gelatin degradation by cancer cells. In conclusion, we identified ADAM12 as a novel basigin sheddase with a potential implication in cancer.
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Affiliation(s)
- Reidar Albrechtsen
- Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
| | - Nicolai J Wewer Albrechtsen
- Department of Biomedical Sciences and Department of Clinical Biochemistry, Rigshospitalet, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
| | - Sebastian Gnosa
- Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
| | - Jeanette Schwarz
- Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
| | - Lars Dyrskjøt
- Department of Molecular Medicine (MOMA), Aarhus University Hospital, 8200 Aarhus, Denmark.
| | - Marie Kveiborg
- Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
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18
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CLIC1 and CLIC4 complement CA125 as a diagnostic biomarker panel for all subtypes of epithelial ovarian cancer. Sci Rep 2018; 8:14725. [PMID: 30282979 PMCID: PMC6170428 DOI: 10.1038/s41598-018-32885-2] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2018] [Accepted: 09/18/2018] [Indexed: 01/20/2023] Open
Abstract
New plasma and tissue biomarkers of epithelial ovarian cancer (EOC) could improve early diagnosis and post-diagnosis clinical management. Here we investigated tissue staining and tissue secretion of CLIC1 and CLIC4 across EOC subtypes. CLIC1 and CLIC4 are two promising biomarkers we previously showed were elevated in EOC patient sera. Individually, CLIC1 or CLIC4 stained larger percentages of malignant tumors across all EOC subtypes compared with CA125, particularly early stage and mucinous tumors. CLIC4 also stained benign tumors but staining was limited to nuclei; whereas malignant tumors showed diffuse cellular staining of stromal and tumor cells. Both proteins were shed by all EOC subtypes tumors in short term organ culture at more consistent levels than CA125, supporting their potential as pan-subtype serum and tissue biomarkers. Elevated CLIC4 expression, but not CLIC1 expression, was a negative indicator of patient survival, and CLIC4 knockdown in cultured cells decreased cell proliferation and migration indicating a potential role in tumor progression. These results suggest CLIC1 and CLIC4 are promising serum and tissue biomarkers as well as potential therapeutic targets for all EOC subtypes. This justifies development of high throughput serum/plasma biomarker assays to evaluate utility of a biomarker panel consisting of CLIC1, CLIC4 and CA125.
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19
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Huang MC, Chu IT, Wang ZF, Lin S, Chang TC, Chen CT. A G-Quadruplex Structure in the Promoter Region of CLIC4 Functions as a Regulatory Element for Gene Expression. Int J Mol Sci 2018; 19:ijms19092678. [PMID: 30201851 PMCID: PMC6165315 DOI: 10.3390/ijms19092678] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2018] [Revised: 09/05/2018] [Accepted: 09/06/2018] [Indexed: 12/25/2022] Open
Abstract
The differential transcriptional expression of CLIC4 between tumor cells and the surrounding stroma during cancer progression has been suggested to have a tumor-promoting effect. However, little is known about the transcriptional regulation of CLIC4. To better understand how this gene is regulated, the promoter region of CLIC4 was analyzed. We found that a high GC content near the transcriptional start site (TSS) might form an alternative G-quadruplex (G4) structure. Nuclear magnetic resonance spectroscopy (NMR) confirmed their formation in vitro. The reporter assay showed that one of the G4 structures exerted a regulatory role in gene transcription. When the G4-forming sequence was mutated to disrupt the G4 structure, the transcription activity dropped. To examine whether this G4 structure actually has an influence on gene transcription in the chromosome, we utilized the CRISPR/Cas9 system to edit the G4-forming sequence within the CLIC4 promoter in the cell genome. The pop-in/pop-out strategy was adopted to isolate the precisely-edited A375 cell clone. In CRISPR-modified A375 cell clones whose G4 was disrupted, there was a decrease in the endogenous CLIC4 messenger RNA (mRNA) expression level. In conclusion, we found that the G4 structure in the CLIC4 promoter might play an important role in regulating the level of transcription.
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Affiliation(s)
- Mu-Ching Huang
- Department of Biochemical Science and Technology, National Taiwan University, Taipei 106, Taiwan.
| | - I-Te Chu
- Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 115, Taiwan.
| | - Zi-Fu Wang
- Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 115, Taiwan.
| | - Steven Lin
- Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
| | - Ta-Chau Chang
- Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 115, Taiwan.
| | - Chin-Tin Chen
- Department of Biochemical Science and Technology, National Taiwan University, Taipei 106, Taiwan.
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20
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Liang J, De Castro A, Flores L. Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans. J Vis Exp 2018. [PMID: 30102267 DOI: 10.3791/57914] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and γ-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.
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Affiliation(s)
- Jun Liang
- Department of Science, Borough of Manhattan Community College/CUNY;
| | - Aijo De Castro
- Department of Science, Borough of Manhattan Community College/CUNY
| | - Lizette Flores
- Department of Science, Borough of Manhattan Community College/CUNY
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21
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The 3'UTR signature defines a highly metastatic subgroup of triple-negative breast cancer. Oncotarget 2018; 7:59834-59844. [PMID: 27494850 PMCID: PMC5312352 DOI: 10.18632/oncotarget.10975] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2016] [Accepted: 07/18/2016] [Indexed: 01/13/2023] Open
Abstract
Triple-negative breast cancer (TNBC) is a highly heterogeneous disease with an aggressive clinical course. Prognostic models are needed to chart potential patient outcomes. To address this, we used alternative 3′UTR patterns to improve postoperative risk stratification. We collected 327 publicly available microarrays and generated the 3′UTR landscape based on expression ratios of alternative 3′UTR. After initial feature filtering, we built a 17-3′UTR-based classifier using an elastic net model. Time-dependent ROC comparisons and Kaplan–Meier analyses confirmed an outstanding discriminating power of our prognostic model for TNBC patients. In the training cohort, 5-year event-free survival (EFS) was 78.6% (95% CI 71.2–86.0) for the low-risk group, and 16.3% (95% CI 2.3–30.4) for the high-risk group (log-rank p<0.0001; hazard ratio [HR] 8.29, 95% CI 4.78–14.4), In the validation set, 5-year EFS was 75.6% (95% CI 68.0–83.2) for the low-risk group, and 33.2% (95% CI 17.1–49.3) for the high-risk group (log-rank p<0.0001; HR 3.17, 95% CI 1.66–5.42). In conclusion, the 17-3′UTR-based classifier provides a superior prognostic performance for estimating disease recurrence and metastasis in TNBC patients and it may permit personalized management strategies.
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22
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Zou Q, Yang Z, Li D, Liu Z, Yuan Y. Association of chloride intracellular channel 4 and Indian hedgehog proteins with survival of patients with pancreatic ductal adenocarcinoma. Int J Exp Pathol 2017; 97:422-429. [PMID: 28205343 DOI: 10.1111/iep.12213] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2016] [Accepted: 10/18/2016] [Indexed: 12/30/2022] Open
Abstract
Pancreatic cancer is the fourth most common cause of cancer-related mortality. Novel molecular biomarkers need to be identified for personalized medicine and to improve survival. The aim of this study was to examine chloride intracellular channel 4 (CLIC4) and Indian Hedgehog (Ihh) expression in benign and malignant lesions of the pancreas and to examine the eventual association between CLIC4 and Ihh expression, with clinicopathological features and prognosis of pancreatic cancer. A retrospective study of specimens collected from January 2000 to December 2011 at the Department of Pathology of the Second and Third Xiangya Hospitals, Central South University was undertaken to explore this question. Immunohistochemistry of CLIC4 and Ihh was performed with EnVision™ in 106 pancreatic ductal adenocarcinoma specimens, 35 paracancer samples (2 cm away from the tumour, when possible or available), 55 benign lesions and 13 normal tissue samples. CLIC4 and Ihh expression in pancreatic ductal adenocarcinoma were significantly higher than in paracancer tissue and benign lesions (CLIC4: P = 0.009 and Ihh: P < 0.0001; CLIC4: P = 0.0004 and Ihh: P = 0.0001 respectively). CLIC4 and Ihh expression was negative in normal pancreatic tissues. The expression of CLIC4 and Ihh was associated significantly with tumour grade, lymph node metastasis, tumour invasion and poor overall survival. Thus CLIC4 and Ihh could serve as biological markers for the progression, metastasis and/or invasiveness of pancreatic ductal adenocarcinoma.
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Affiliation(s)
- Qiong Zou
- Department of Pathology, Third Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Zhulin Yang
- Research Laboratory of Hepatobiliary Diseases, Second Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Daiqiang Li
- Department of Pathology, Second Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Ziru Liu
- Research Laboratory of Hepatobiliary Diseases, Second Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Yuan Yuan
- Department of Pathology, Third Xiangya Hospital, Central South University, Changsha, Hunan, China
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Maternal Plane of Nutrition during Late Gestation and Weaning Age Alter Angus × Simmental Offspring Longissimus Muscle Transcriptome and Intramuscular Fat. PLoS One 2015; 10:e0131478. [PMID: 26153887 PMCID: PMC4496061 DOI: 10.1371/journal.pone.0131478] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2015] [Accepted: 06/01/2015] [Indexed: 12/29/2022] Open
Abstract
In model organisms both the nutrition of the mother and the young offspring could induce long-lasting transcriptional changes in tissues. In livestock, such changes could have important roles in determining nutrient use and meat quality. The main objective was to evaluate if plane of maternal nutrition during late-gestation and weaning age alter the offspring's Longissimus muscle (LM) transcriptome, animal performance, and metabolic hormones. Whole-transcriptome microarray analysis was performed on LM samples of early (EW) and normal weaned (NW) Angus × Simmental calves born to grazing cows receiving no supplement [low plane of nutrition (LPN)] or 2.3 kg high-grain mix/day [medium plane of nutrition (MPN)] during the last 105 days of gestation. Biopsies of LM were harvested at 78 (EW), 187 (NW) and 354 (before slaughter) days of age. Despite greater feed intake in MPN offspring, blood insulin was greater in LPN offspring. Carcass intramuscular fat content was greater in EW offspring. Bioinformatics analysis of the transcriptome highlighted a modest overall response to maternal plane of nutrition, resulting in only 35 differentially expressed genes (DEG). However, weaning age and a high-grain diet (EW) strongly impacted the transcriptome (DEG = 167), especially causing a lipogenic program activation. In addition, between 78 and 187 days of age, EW steers had an activation of the innate immune system due presumably to macrophage infiltration of intramuscular fat. Between 187 and 354 days of age (the "finishing" phase), NW steers had an activation of the lipogenic transcriptome machinery, while EW steers had a clear inhibition through the epigenetic control of histone acetylases. Results underscored the need to conduct further studies to understand better the functional outcome of transcriptome changes induced in the offspring by pre- and post-natal nutrition. Additional knowledge on molecular and functional outcomes would help produce more efficient beef cattle.
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Patel D, Ythier D, Brozzi F, Eizirik DL, Thorens B. Clic4, a novel protein that sensitizes β-cells to apoptosis. Mol Metab 2015; 4:253-64. [PMID: 25830089 PMCID: PMC4354924 DOI: 10.1016/j.molmet.2015.01.003] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/17/2014] [Revised: 01/06/2015] [Accepted: 01/09/2015] [Indexed: 01/09/2023] Open
Abstract
Objectives Chloride intracellular channel protein 4 (Clic4) is a ubiquitously expressed protein involved in multiple cellular processes including cell-cycle control, cell differentiation, and apoptosis. Here, we investigated the role of Clic4 in pancreatic β-cell apoptosis. Methods We used βTC-tet cells and islets from β-cell specific Clic4 knockout mice (βClic4KO) and assessed cytokine-induced apoptosis, Bcl2 family protein expression and stability, and identified Clic4-interacting proteins by co-immunoprecipitation and mass spectrometry analysis. Results We show that cytokines increased Clic4 expression in βTC-tet cells and in mouse islets and siRNA-mediated silencing of Clic4 expression in βTC-tet cells or its genetic inactivation in islets β-cells, reduced cytokine-induced apoptosis. This was associated with increased expression of Bcl-2 and increased expression and phosphorylation of Bad. Measurement of Bcl-2 and Bad half-lives in βTC-tet cells showed that Clic4 silencing increased the stability of these proteins. In primary islets β-cells, absence of Clic4 expression increased Bcl-2 and Bcl-xL expression as well as expression and phosphorylation of Bad. Mass-spectrometry analysis of proteins co-immunoprecipitated with Clic4 from βTC-tet cells showed no association of Clic4 with Bcl-2 family proteins. However, Clic4 co-purified with proteins from the proteasome suggesting a possible role for Clic4 in regulating protein degradation. Conclusions Collectively, our data show that Clic4 is a cytokine-induced gene that sensitizes β-cells to apoptosis by reducing the steady state levels of Bcl-2, Bad and phosphorylated Bad.
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Affiliation(s)
- Dhaval Patel
- Center for Integrative Genomics, University of Lausanne, Genopode Building, CH-1015 Lausanne, Switzerland
| | - Damien Ythier
- Center for Integrative Genomics, University of Lausanne, Genopode Building, CH-1015 Lausanne, Switzerland
| | - Flora Brozzi
- ULB Center for Diabetes Research, Université Libre de Bruxelles (ULB), Brussels, Belgium
| | - Decio L Eizirik
- ULB Center for Diabetes Research, Université Libre de Bruxelles (ULB), Brussels, Belgium
| | - Bernard Thorens
- Center for Integrative Genomics, University of Lausanne, Genopode Building, CH-1015 Lausanne, Switzerland
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25
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Al Khamici H, Brown LJ, Hossain KR, Hudson AL, Sinclair-Burton AA, Ng JPM, Daniel EL, Hare JE, Cornell BA, Curmi PMG, Davey MW, Valenzuela SM. Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity. PLoS One 2015; 10:e115699. [PMID: 25581026 PMCID: PMC4291220 DOI: 10.1371/journal.pone.0115699] [Citation(s) in RCA: 67] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2014] [Accepted: 11/26/2014] [Indexed: 01/07/2023] Open
Abstract
The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.
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Affiliation(s)
- Heba Al Khamici
- School of Medical and Molecular Biosciences, University of Technology Sydney, Sydney, New South Wales 2007, Australia
| | - Louise J. Brown
- Department of Chemistry and Bimolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia
| | - Khondker R. Hossain
- School of Medical and Molecular Biosciences, University of Technology Sydney, Sydney, New South Wales 2007, Australia
- Bragg Institute, Australian Nuclear Science and Technology Organisation, Sydney, New South Wales 2234, Australia
| | - Amanda L. Hudson
- School of Medical and Molecular Biosciences, University of Technology Sydney, Sydney, New South Wales 2007, Australia
| | - Alxcia A. Sinclair-Burton
- School of Medical and Molecular Biosciences, University of Technology Sydney, Sydney, New South Wales 2007, Australia
| | - Jane Phui Mun Ng
- School of Medical and Molecular Biosciences, University of Technology Sydney, Sydney, New South Wales 2007, Australia
| | - Elizabeth L. Daniel
- Department of Chemistry and Bimolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia
| | - Joanna E. Hare
- Department of Chemistry and Bimolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia
| | - Bruce A. Cornell
- Surgical Diagnostics, Roseville, Sydney, New South Wales 2069, Australia
| | - Paul M. G. Curmi
- School of Physics, University of New South Wales, Sydney, New South Wales 2052, Australia
- Centre for Applied Medical Research, St Vincent's Hospital, Sydney, New South Wales 2010, Australia
| | - Mary W. Davey
- School of Medical and Molecular Biosciences, University of Technology Sydney, Sydney, New South Wales 2007, Australia
| | - Stella M. Valenzuela
- School of Medical and Molecular Biosciences, University of Technology Sydney, Sydney, New South Wales 2007, Australia
- Centre for Health Technologies, University of Technology Sydney, Sydney, New South Wales 2007, Australia
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26
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Peretti M, Angelini M, Savalli N, Florio T, Yuspa SH, Mazzanti M. Chloride channels in cancer: Focus on chloride intracellular channel 1 and 4 (CLIC1 AND CLIC4) proteins in tumor development and as novel therapeutic targets. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2014; 1848:2523-31. [PMID: 25546839 DOI: 10.1016/j.bbamem.2014.12.012] [Citation(s) in RCA: 107] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/16/2014] [Revised: 12/05/2014] [Accepted: 12/11/2014] [Indexed: 02/07/2023]
Abstract
In recent decades, growing scientific evidence supports the role of ion channels in the development of different cancers. Both potassium selective pores and chloride permeabilities are considered the most active channels during tumorigenesis. High rate of proliferation, active migration, and invasiveness into non-neoplastic tissues are specific properties of neoplastic transformation. All these actions require partial or total involvement of chloride channel activity. In this context, this class of membrane proteins could represent valuable therapeutic targets for the treatment of resistant tumors. However, this encouraging premise has not so far produced any valid new channel-targeted antitumoral molecule for cancer treatment. Problematic for drug design targeting ion channels is their vital role in normal cells for essential physiological functions. By targeting these membrane proteins involved in pathological conditions, it is inevitable to cause relevant side effects in healthy organs. In light of this, a new protein family, the chloride intracellular channels (CLICs), could be a promising class of therapeutic targets for its intrinsic individualities: CLIC1 and CLIC4, in particular, not only are overexpressed in specific tumor types or their corresponding stroma but also change localization and function from hydrophilic cytosolic to integral transmembrane proteins as active ionic channels or signal transducers during cell cycle progression in certain cases. These changes in intracellular localization, tissue compartments, and channel function, uniquely associated with malignant transformation, may offer a unique target for cancer therapy, likely able to spare normal cells. This article is part of a special issue itled "Membrane Channels and Transporters in Cancers."
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Affiliation(s)
- Marta Peretti
- Department of Life Science, University of Milan, Milano I-20133, Italy
| | - Marina Angelini
- Department of Life Science, University of Milan, Milano I-20133, Italy
| | - Nicoletta Savalli
- Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90075, USA
| | - Tullio Florio
- Sezione di Farmacologia, Dipartimento di Medicina Interna and Centro di Eccellenza per la Ricerca Biomedica (CEBR), University of Genova, Genova, Italy
| | - Stuart H Yuspa
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, NCI, Bethesda, MD 20892, USA
| | - Michele Mazzanti
- Department of Life Science, University of Milan, Milano I-20133, Italy.
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Siani A, Tirelli N. Myofibroblast differentiation: main features, biomedical relevance, and the role of reactive oxygen species. Antioxid Redox Signal 2014; 21:768-85. [PMID: 24279926 DOI: 10.1089/ars.2013.5724] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
SIGNIFICANCE Myofibroblasts are prototypical fibrotic cells, which are involved in a number of more or less pathological conditions, from foreign body reactions to scarring, from liver, kidney, or lung fibrosis to neoplastic phenomena. The differentiation of precursor cells (not only of fibroblastic nature) is characterized by a complex interplay between soluble factors (growth factors such as transforming growth factor β1, reactive oxygen species [ROS]) and material properties (matrix stiffness). RECENT ADVANCES The last 15 years have seen very significant advances in the identification of appropriate differentiation markers, in the understanding of the differentiation mechanism, and above all, the involvement of ROS as causative and persistence factors. CRITICAL ISSUES The specific mechanisms of action of ROS remain largely unknown, although evidence suggests that both intracellular and extracellular phenomena play a role. FUTURE DIRECTIONS Approaches based on antioxidant (ROS-scavenging) principles and on the potentiation of nitric oxide signaling hold much promise in view of a pharmacological therapy of fibrotic phenomena. However, how to make the active principles available at the target sites is yet a largely neglected issue.
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Affiliation(s)
- Alessandro Siani
- 1 School of Pharmacy and Pharmaceutical Sciences, University of Manchester , Manchester, United Kingdom
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28
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Padmakumar V, Masiuk KE, Luger D, Lee C, Coppola V, Tessarollo L, Hoover SB, Karavanova I, Buonanno A, Simpson RM, Yuspa SH. Detection of differential fetal and adult expression of chloride intracellular channel 4 (CLIC4) protein by analysis of a green fluorescent protein knock-in mouse line. BMC DEVELOPMENTAL BIOLOGY 2014; 14:24. [PMID: 24886590 PMCID: PMC4073518 DOI: 10.1186/1471-213x-14-24] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/31/2014] [Accepted: 05/13/2014] [Indexed: 01/17/2023]
Abstract
Background Chloride Intracellular Channel 4 (CLIC4) is one of seven members in the closely related CLIC protein family. CLIC4 is involved in multiple cellular processes including apoptosis, cellular differentiation, inflammation and endothelial tubulogenesis. Despite over a decade of research, no comprehensive in situ expression analysis of CLIC4 in a living organism has been reported. In order to fulfill this goal, we generated a knock-in mouse to express Green Fluorescent Protein (GFP) from the CLIC4 locus, thus substituting the GFP coding region for CLIC4. We used GFP protein expression to eliminate cross reaction with other CLIC family members. Results We analyzed CLIC4 expression during embryonic development and adult organs. During mid and late gestation, CLIC4 expression is modulated particularly in fetal brain, heart, thymus, liver and kidney as well as in developing brown adipose tissue and stratifying epidermis. In the adult mouse, CLIC4 is highly expressed globally in vascular endothelial cells as well as in liver, lung alveolar septae, pancreatic acini, spermatogonia, renal proximal tubules, cardiomyocytes and thymic epithelial cells. Neural expression included axonal tracks, olfactory bulb, Purkinje cell layer and dentate gyrus. Renal CLIC4 expression was most pronounced in proximal tubules, although altered renal function was not detected in the absence of CLIC4. Myeloid cells and B cells of the spleen are rich in CLIC4 expression as are CD4 and CD8 positive T cells. Conclusions In a comprehensive study detailing CLIC4 expression in situ in a mouse model that excludes cross reaction with other family members, we were able to document previously unreported expression for CLIC4 in developing fetus, particularly the brain. In addition, compartmentalized expression of CLIC4 in specific adult tissues and cells provides a focus to explore potential functions of this protein not addressed previously.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | | | - Stuart H Yuspa
- Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD, USA.
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Peter B, Polyansky AA, Fanucchi S, Dirr HW. A Lys-Trp cation-π interaction mediates the dimerization and function of the chloride intracellular channel protein 1 transmembrane domain. Biochemistry 2013; 53:57-67. [PMID: 24328417 DOI: 10.1021/bi401433f] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. The oligomerization of the transmembrane domain (TMD) remains speculative despite it being implicated in pore formation. The extent to which electrostatic and van der Waals interactions drive folding and association of the dimorphic TMD is unknown and is complicated by the requirement of interactions favorable in both aqueous and membrane environments. Here we report a putative Lys37-Trp35 cation-π interaction and show that it stabilizes the dimeric form of the CLIC1 TMD in membranes. A synthetic 30-mer peptide comprising a K37M TMD mutant was examined in 2,2,2-trifluoroethanol, sodium dodecyl sulfate micelles, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes using far-ultraviolet (UV) circular dichroism, fluorescence, and UV absorbance spectroscopy. Our data suggest that Lys37 is not implicated in the folding, stability, or membrane insertion of the TMD peptide. However, removal of this residue impairs the formation of dimers and higher-order oligomers. This is accompanied by a 30-fold loss of chloride influx activity, suggesting that dimerization modulates the rate of chloride conductance. We propose that, within membranes, individual TMD helices associate via a Lys37-mediated cation-π interaction to form active dimers. The latter findings are also supported by results of modeling a putative TMD dimer conformation in which Lys37 and Trp35 form cation-π pairs at the dimer interface. Dimeric helix bundles may then associate to form fully active ion channels. Thus, within a membrane-like environment, aromatic interactions involving a polar lysine side chain provide a thermodynamic driving force for helix-helix association.
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Affiliation(s)
- Bradley Peter
- Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand , Johannesburg 2050, South Africa
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30
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Proteome of human stem cells from periodontal ligament and dental pulp. PLoS One 2013; 8:e71101. [PMID: 23940696 PMCID: PMC3733711 DOI: 10.1371/journal.pone.0071101] [Citation(s) in RCA: 61] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2013] [Accepted: 06/25/2013] [Indexed: 12/12/2022] Open
Abstract
Background Many adult tissues contain a population of stem cells with the ability to regenerate structures similar to the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical disorder. Human adult stem cells (SCs) including bone marrow stem cells (BMSCs), dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) have been characterized for their high proliferative potential, expression of characteristic SC-associated markers and for the plasticity to differentiate in different lineage in vitro. Methodology/Principal Findings The aim of this study is to define the molecular features of stem cells from oral tissue by comparing the proteomic profiles obtained with 2-DE followed by MALDI-TOF/TOF of ex-vivo cultured human PDLSCs, DPSCs and BMSCs. Our results showed qualitative similarities in the proteome profiles among the SCs examined including some significant quantitative differences. To enrich the knowledge of oral SCs proteome we performed an analysis in narrow range pH 4–7 and 6–9, and we found that DPSCs vs PDLSCs express differentially regulated proteins that are potentially related to growth, regulation and genesis of neuronal cells, suggesting that SCs derived from oral tissue source populations may possess the potential ability of neuronal differentiation which is very consistent with their neural crest origin. Conclusion/Significance This study identifies some differentially expressed proteins by using comparative analysis between DPSCs and PDLSCs and BMSCs and suggests that stem cells from oral tissue could have a different cell lineage potency compared to BMSCs.
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Wu MP, Huang KH, Long CY, Yang CC, Tong YC. In vitro extracellular matrix model to evaluate stroma cell response to transvaginal mesh. Neurourol Urodyn 2013; 33:449-54. [PMID: 23775843 DOI: 10.1002/nau.22425] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2013] [Accepted: 04/15/2013] [Indexed: 11/07/2022]
Abstract
AIMS The use of surgical mesh for female pelvic floor reconstruction has increased in recent years. However, there is paucity of information about the biological responses of host stroma cells to different meshes. This study was aimed to establish an in vitro experimental model to study the micro-environment of extracellular matrix (ECM) with embedded mesh and the stroma cell behaviors to different synthetic meshes. METHODS Matrigel multi-cellular co-culture system with embedded mesh was used to evaluate the interaction of stroma cells and synthetic mesh in a simulated ECM environment. Human umbilical vein endothelial cells (HUVEC) and NIH3T3 fibroblasts were inoculated in the system. The established multi-cellular Matrigel co-culture system was used to detect stroma cell recruitment and tube formation ability for different synthetic meshes. RESULTS HUVEC and NIH3T3 cells were recruited into the mesh interstices and organized into tube-like structures in type I mesh material from Perigee, Marlex and Prolift 24 hr after cell inoculation. On the contrary, there was little recruitment of HUVEC and NIH3T3 cells into the type III mesh of intra-vaginal sling (IVS). CONCLUSIONS The Matrigel multi-cellular co-culture system with embedded mesh offers a useful in vitro model to study the biological behaviors of stroma cells in response to different types of synthetic meshes. The system can help to select ideal mesh candidates before actual implantation into the human body.
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Affiliation(s)
- Ming-Ping Wu
- Division of Urogynecology and Pelvic Floor Reconstruction, Department of Obstetrics and Gynecology, Chi Mei Foundation Hospital, Tainan, Taiwan; Center of General Education, Chia Nan University of Pharmacy and Science, Tainan, Taiwan
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Peter B, Ngubane NCML, Fanucchi S, Dirr HW. Membrane Mimetics Induce Helix Formation and Oligomerization of the Chloride Intracellular Channel Protein 1 Transmembrane Domain. Biochemistry 2013; 52:2739-49. [DOI: 10.1021/bi4002776] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Affiliation(s)
- Bradley Peter
- Protein Structure-Function Research
Unit, School of
Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa
| | - Nomxolisi Chloë Mina-Liz Ngubane
- Protein Structure-Function Research
Unit, School of
Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa
| | - Sylvia Fanucchi
- Protein Structure-Function Research
Unit, School of
Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa
| | - Heini W. Dirr
- Protein Structure-Function Research
Unit, School of
Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa
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CLIC4 regulates TGF-β-dependent myofibroblast differentiation to produce a cancer stroma. Oncogene 2013; 33:842-50. [PMID: 23416981 PMCID: PMC3912213 DOI: 10.1038/onc.2013.18] [Citation(s) in RCA: 58] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2012] [Revised: 12/10/2012] [Accepted: 12/14/2012] [Indexed: 01/27/2023]
Abstract
Cancer stroma has a profound influence on tumor development and progression. The conversion of fibroblasts to activated myofibroblasts is a hallmark of reactive tumor stroma. Among a number of factors involved in this conversion, transforming growth factor (TGF)-β has emerged as a major regulator. CLIC4, an integral protein in TGF-β signaling, is highly upregulated in stroma of multiple human cancers, and overexpression of CLIC4 in stromal cells enhances the growth of cancer xenografts. In this study, we show that conditioned media from tumor cell lines induces expression of both CLIC4 and the myofibroblast marker alpha smooth muscle actin (α-SMA) in stromal fibroblasts via TGF-β signaling. Genetic ablation of CLIC4 in primary fibroblasts prevents or reduces constitutive or TGF-β-induced expression of α-SMA and extracellular matrix components that are markers of myofibroblasts. CLIC4 is required for the activation of p38 map kinase by TGF-β, a pathway that signals myofibroblast conversion in stromal cells. This requirement involves the interaction of CLIC4 with PPM1a, the selective phosphatase of activated p38. Conditioned media from fibroblasts overexpressing CLIC4 increases tumor cell migration and invasion in a TGF-β-dependent manner and promotes epithelial to mesenchymal transition indicating that high stromal CLIC4 serves to enhance tumor invasiveness and progression. Thus, CLIC4 is significantly involved in the development of a nurturing tumor microenvironment by enhancing TGF-β signaling in a positive feedback loop. Targeting CLIC4 in tumor stroma should be considered as a strategy to mitigate some of the tumor enhancing effects of the cancer stroma.
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Albrechtsen R, Kveiborg M, Stautz D, Vikeså J, Noer JB, Kotzsh A, Nielsen FC, Wewer U, Fröhlich C. ADAM12 redistributes and activates MMP-14, resulting in gelatin degradation, reduced apoptosis, and increased tumor growth. J Cell Sci 2013; 126:4707-20. [DOI: 10.1242/jcs.129510] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Matrix metalloproteases (MMPs), in particular MMP-2, -9, and -14, play a key role in various aspects of cancer pathology. Likewise, ADAMs (A Disintegrin And Metalloproteases), including ADAM12, are upregulated in malignant tumors and contribute to the pathology of cancers. Here we showed a positive correlation between MMP-14 and ADAM12 expression in human breast cancer. We demonstrated that in 293-VnR and human breast cancer cells expressing ADAM12 at the cell surface, endogenous MMP-14 was recruited to the cell surface, resulting in its activation. Subsequent to this activation, gelatin degradation was stimulated and tumor-cell apoptosis was decreased, with reduced expression of the pro-apoptotic proteins BCL2L11 and BIK. The effect on gelatin degradation was abrogated by inhibition of the MMP-14 activity and appeared to be dependent on cell-surface αVβ3 integrin localization, but neither the catalytic activity of ADAM12 nor the cytoplasmic tail of ADAM12 were required. The significance of ADAM12-induced activation of MMP-14 was underscored by a reduction in MMP-14–mediated gelatin degradation and abolition of apoptosis-protective effects by specific monoclonal antibodies against ADAM12. Furthermore, orthotopic implantation of ADAM12-expressing MCF7 cells in nude mice produced tumors with increased levels of activated MMP-14 and confirmed that ADAM12 protects tumor cells against apoptosis, leading to increased tumor progression. In conclusion, our data suggest that a ternary protein complex composed of ADAM12, αVβ3 integrin, and MMP-14 at the tumor cell surface regulates MMP-14 functions. This interaction may point to a novel concept for the development of MMP-14–targeting drugs in treating cancer.
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Labbus K, Henning M, Borkham-Kamphorst E, Geisler C, Berger T, Mak TW, Knüchel R, Meyer HE, Weiskirchen R, Henkel C. Proteomic profiling in Lipocalin 2 deficient mice under normal and inflammatory conditions. J Proteomics 2012; 78:188-96. [PMID: 23219901 DOI: 10.1016/j.jprot.2012.11.021] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2012] [Revised: 10/31/2012] [Accepted: 11/12/2012] [Indexed: 01/17/2023]
Abstract
Lipocalin 2 (LCN2) belongs to the superfamily of lipocalins which represent a group of small secreted proteins classified as extracellular transport proteins expressed in many tissues. LCN2 is strongly increased in experimental models of acute and chronic liver injuries. To investigate the function of LCN2 in normal liver homeostasis and under conditions of inflammatory liver injury, we comparatively analyzed hepatic extracts taken from Lcn2-deficient and wild type mice under basal conditions and after stimulation with lipopolysaccharides. Liver was chemically and mechanically lysed and extracts were subjected to 2-D-DIGE after minimal labeling (G200 and G300 dyes) using an appropriate internal standard (G100). Afterwards MALDI TOF MS and MS/MS were used to identify differentially expressed proteins. Proteins that were identified to be differentially expressed include for example the chloride intracellular channel protein 4 (CLIC4), aminoacylase 1 and transketolase. The altered expression of respective genes was confirmed by Western blot analysis and further validated by quantitative real time PCR. Altogether, the complex expression alterations in mice lacking LCN2 under normal conditions and after exposure to inflammatory stimuli reveal that LCN2 has essential function in liver homeostasis and in the onset of inflammatory responses in which LCN2 expression dramatically increases.
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Affiliation(s)
- Kirsten Labbus
- Institute of Pathology, RWTH University Hospital Aachen, Germany
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36
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Zhong J, Kong X, Zhang H, Yu C, Xu Y, Kang J, Yu H, Yi H, Yang X, Sun L. Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation. PLoS One 2012; 7:e39378. [PMID: 22761775 PMCID: PMC3382619 DOI: 10.1371/journal.pone.0039378] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2012] [Accepted: 05/21/2012] [Indexed: 12/19/2022] Open
Abstract
CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.
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Affiliation(s)
- Jiateng Zhong
- Department of Pathophysiology, Norman Bethune College of Medicine, Jilin University, Changchun, Jilin, China
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Padmakumar VC, Speer K, Pal-Ghosh S, Masiuk KE, Ryscavage A, Dengler SL, Hwang S, Edwards JC, Coppola V, Tessarollo L, Stepp MA, Yuspa SH. Spontaneous skin erosions and reduced skin and corneal wound healing characterize CLIC4(NULL) mice. THE AMERICAN JOURNAL OF PATHOLOGY 2012; 181:74-84. [PMID: 22613027 DOI: 10.1016/j.ajpath.2012.03.025] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/09/2011] [Revised: 02/21/2012] [Accepted: 03/20/2012] [Indexed: 01/29/2023]
Abstract
Cutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-β (TGF-β) signaling, and mice lacking components of the TGF-β signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-β pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4(NULL) mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4(NULL) mice, resolution of inflammation was delayed, and expression of β4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4(NULL) skin. The induced level of phosphorylated Smad2 in response to TGF-β was reduced in cultured CLIC4(NULL) keratinocytes relative to in wild-type cells, and CLIC4(NULL) keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-β. CLIC4(NULL) keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-β pathway.
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Affiliation(s)
- V C Padmakumar
- Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD 20892, USA
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Wang L, He S, Tu Y, Ji P, Zong J, Zhang J, Feng F, Zhao J, Zhang Y, Gao G. Elevated expression of chloride intracellular channel 1 is correlated with poor prognosis in human gliomas. J Exp Clin Cancer Res 2012; 31:44. [PMID: 22578365 PMCID: PMC3441274 DOI: 10.1186/1756-9966-31-44] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2012] [Accepted: 05/05/2012] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Chloride intracellular channel 1 (CLIC1) is expressed ubiquitously in human tissues and is involved in the regulation of cell cycle, cell proliferation and differentiation. Recent studies have shown that CLIC1 is highly expressed in several human malignant tumors. However, its roles in human gliomas are still unclear. The aim of this study was to investigate the clinicopathological significance and prognostic value of CLIC1 expression in human gliomas. METHODS CLIC1 expression in human gliomas and nonneoplastic brain tissues was measured by real-time quantitative RT-PCR assay and immunohistochemistry. Its association with clinicopathological factors or prognosis in patients with gliomas was statistically analyzed. RESULTS The expression of CLIC1 at both mRNA and protein levels was significantly increased in high-grade (Grade III~IV) glioma tissues compared with that in low-grade (Grade I~II) and nonneoplastic brain tissues, and was up-regulated with ascending tumor World Health Organization (WHO) grades. The elevated expression of CLIC1 protein was also significantly correlated with low Karnofsky performance score (KPS) (P=0.008). Moreover, both univariate and multivariate analysis shown that high CLIC1 expression was significantly associated with poor prognosis in patients with gliomas (P<0.001 and P=0.01, respectively). In particular, the elevated CLIC1 expression also correlated with shorter overall survival in different glioma subgroups stratified according to the WHO grading. CONCLUSIONS Our data provide the first evidence that CLIC1 expression might play an important role in the regulation of aggressiveness in human gliomas. The elevated expression of CLIC1 might represent a valuable prognostic marker for this disease.
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Affiliation(s)
- Liang Wang
- Department of neurosurgery, Tangdu hospital, Fourth Military Medical University of PLA, No,569, Xinsi Road, Baqiao District, Xi'an City, 710038, China
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Inducible NOS-induced chloride intracellular channel 4 (CLIC4) nuclear translocation regulates macrophage deactivation. Proc Natl Acad Sci U S A 2012; 109:6130-5. [PMID: 22474389 DOI: 10.1073/pnas.1201351109] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Nuclear translocation of cytosolic CLIC4 is an essential feature of its proapoptotic and prodifferentiation functions. Here we demonstrate that CLIC4 is induced concurrently with inducible nitric oxide synthase (iNOS) and S-nitrosylated in proinflammatory peritoneal macrophages. Chemical inhibition or genetic ablation of iNOS inhibits S-nitrosylation and nuclear translocation of CLIC4. In macrophages, iNOS-induced nuclear CLIC4 coincides with the pro- to anti-inflammatory transition of the cells because IL-1β and CXCL1 mRNA remain elevated in CLIC4 and iNOS knockout macrophages at late time points, whereas TNFα mRNA is elevated only in the iNOS knockout macrophages. Active IL-1β remains elevated in CLIC4 knockout macrophages and in macrophages in which CLIC4 nuclear translocation is prevented by the NOS inhibitor l-NAME. Moreover, overexpression of nuclear-targeted CLIC4 down-regulates IL-1β in stimulated macrophages. In mice, genetically null for CLIC4, the number of phagocytosing macrophages stimulated by LPS is reduced. Thus, iNOS-induced nuclear CLIC4 is an essential part of the macrophage deactivation program.
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Suh KS, Malik M, Shukla A, Ryscavage A, Wright L, Jividen K, Crutchley JM, Dumont RA, Fernandez-Salas E, Webster JD, Simpson RM, Yuspa SH. CLIC4 is a tumor suppressor for cutaneous squamous cell cancer. Carcinogenesis 2012; 33:986-95. [PMID: 22387366 DOI: 10.1093/carcin/bgs115] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Chloride intracellular channel (CLIC) 4 is a member of a redox-regulated, metamorphic multifunctional protein family, first characterized as intracellular chloride channels. Current knowledge indicates that CLICs participate in signaling, cytoskeleton integrity and differentiation functions of multiple tissues. In metabolically stressed skin keratinocytes, cytoplasmic CLIC4 is S-nitrosylated and translocates to the nucleus where it enhances transforming growth factor-β (TGF-β) signaling by protecting phospho-Smad 2 and 3 from dephosphorylation. CLIC4 expression is diminished in multiple human epithelial cancers, and the protein is excluded from the nucleus. We now show that CLIC4 expression is reduced in chemically induced mouse skin papillomas, mouse and human squamous carcinomas and squamous cancer cell lines, and the protein is excluded from the nucleus. The extent of reduction in CLIC4 coincides with progression of squamous tumors from benign to malignant. Inhibiting antioxidant defense in tumor cells increases S-nitrosylation and nuclear translocation of CLIC4. Adenoviral-mediated reconstitution of nuclear CLIC4 in squamous cancer cells enhances TGF-β-dependent transcriptional activity and inhibits growth. Adenoviral targeting of CLIC4 to the nucleus of tumor cells in orthografts inhibits tumor growth, whereas elevation of CLIC4 in transgenic epidermis reduces de novo chemically induced skin tumor formation. In parallel, overexpression of exogenous CLIC4 in squamous tumor orthografts suppresses tumor growth and enhances TGF-β signaling. These results indicate that CLIC4 suppresses the growth of squamous cancers, that reduced CLIC4 expression and nuclear residence detected in cancer cells is associated with the altered redox state of tumor cells and the absence of detectable nuclear CLIC4 in cancers contributes to TGF-β resistance and enhances tumor development.
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Affiliation(s)
- K Stephen Suh
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA
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Gerolymos M, Karagianni F, Papasotiriou M, Kalliakmani P, Sotsiou F, Charonis A, Goumenos D. Expression of Transgelin in Human Glomerulonephritis of Various Etiology. ACTA ACUST UNITED AC 2011; 119:c74-82. [DOI: 10.1159/000324655] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2010] [Accepted: 01/24/2011] [Indexed: 11/19/2022]
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Inhibition of pancreatic stellate cell activation by halofuginone prevents pancreatic xenograft tumor development. Pancreas 2010; 39:1008-15. [PMID: 20442678 DOI: 10.1097/mpa.0b013e3181da8aa3] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
OBJECTIVES Most solid tumors consist of neoplastic and nonneoplastic cells and extracellular matrix components. In the pancreas, activated stellate cells (PSCs) are the source of the extracellular matrix proteins. We evaluated the significance of PSC activation in tumor establishment and development in mouse xenografts. METHODS Xenografts were established by implanting human pancreatic cancer cells (MiaPaca-2) subcutaneously or orthotopically by injecting them into the spleen. Fibrosis was induced by cerulein. Collagen level was evaluated by Sirius red staining. Prolyl 4-hydroxylase β and stellate cell activation-associated protein (Cygb/STAP) were determined by immunohistochemistry. RESULTS Halofuginone inhibited subcutaneous tumor development implanted with Matrigel and reduced collagen and prolyl 4-hydroxylase β levels. Few tumors, which developed slowly, were observed after MiaPaca-2 implantation without Matrigel. Increase in tumor number and rate of development were observed with addition of PSCs from control pancreas, and further increase was observed when the PSCs were from cerulein-treated mice. Preincubation of the PSCs with halofuginone elicited Cygb/STAP level reduction and tumor growth inhibition. More tumors developed orthotopically in cerulein-treated mice than in controls; this was prevented by halofuginone. CONCLUSIONS Extracellular matrix production by activated PSCs is essential for tumor establishment and growth. Thus, inhibition of PSC activation is a viable means of reducing pancreatic tumor development.
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43
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Unique precursors for the mesenchymal cells involved in injury response and fibrosis. Proc Natl Acad Sci U S A 2010; 107:13730-5. [PMID: 20634425 DOI: 10.1073/pnas.0910382107] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
We investigated an alternative pathway for emergence of the mesenchymal cells involved in epithelial sheet wound healing and a source of myofibroblasts that cause fibrosis. Using a mock cataract surgery model, we discovered a unique subpopulation of polyploid mesenchymal progenitors nestled in small niches among lens epithelial cells that expressed the surface antigen G8 and mRNA for the myogenic transcription factor MyoD. These cells rapidly responded to wounding of the lens epithelium with population expansion, acquisition of a mesenchymal phenotype, and migration to the wound edges where they regulate the wound response of the epithelium. These mesenchymal cells also were a principal source of myofibroblasts that emerged following lens injury and were responsible for fibrotic disease of the lens that occurs following cataract surgery. These studies provide insight into the mechanisms of wound-healing and fibrosis.
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Liu Y, Yan S, Wondimu A, Bob D, Weiss M, Sliwinski K, Villar J, Notario V, Sutherland M, Colberg-Poley AM, Ladisch S. Ganglioside synthase knockout in oncogene-transformed fibroblasts depletes gangliosides and impairs tumor growth. Oncogene 2010; 29:3297-306. [PMID: 20305696 PMCID: PMC2880627 DOI: 10.1038/onc.2010.85] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2009] [Revised: 01/04/2010] [Accepted: 02/15/2010] [Indexed: 12/22/2022]
Abstract
Biologically active membrane gangliosides, expressed and released by many human tumors, are hypothesized to significantly impact tumor progression. Lack of a model of complete and specific tumor ganglioside depletion in vivo, however, has hampered elucidation of their role. Here, we report the creation of a novel, stable, genetically induced tumor cell system resulting in specific and complete blockade of ganglioside synthesis. Wild-type (WT) and GM3 synthase/GM2 synthase double knockout (DKO) murine embryonic fibroblasts were transformed using amphotropic retrovirus-transduced oncogenes (pBABE-c-Myc(T58A)+H-RasG12V). The transformed cells, WT(t) and DKO(t) respectively, evidenced comparable integrated copy numbers and oncogene expression. Ganglioside synthesis was completely blocked in the DKO(t) cells, importantly without triggering an alternate pathway of ganglioside synthesis. Ganglioside depletion (to <0.5 nmol/10(7) cells from 9 to 11 nmol/10(7) WT(t) or untransfected normal fibroblasts) did not adversely affect cell proliferation kinetics but did reduce cell migration on fibronectin-coated wells, consistent with our previous observations in ganglioside-depleted normal human fibroblasts. Strikingly, despite similar oncogene expression and growth kinetics, DKO(t) cells evidenced significantly impaired tumor growth in syngeneic immunocompetent mice, underscoring the pivotal role of tumor cell gangliosides and providing an ideal system for probing their mechanisms of action in vivo.
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Affiliation(s)
- Yihui Liu
- Center for Cancer and Immunology Research, Children's National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010
- Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, D.C. 20037
| | - Su Yan
- Center for Cancer and Immunology Research, Children's National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010
| | - Assefa Wondimu
- Center for Cancer and Immunology Research, Children's National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010
| | - Daniel Bob
- Center for Cancer and Immunology Research, Children's National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010
| | - Michael Weiss
- Center for Cancer and Immunology Research, Children's National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010
| | - Konrad Sliwinski
- Center for Cancer and Immunology Research, Children's National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010
| | - Joaquín Villar
- Department of Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3970 Reservoir Rd, NW, Washington, DC 20057
| | - Vicente Notario
- Department of Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3970 Reservoir Rd, NW, Washington, DC 20057
| | - Margaret Sutherland
- Center for Cancer and Immunology Research, Children's National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010
| | - Anamaris M. Colberg-Poley
- Center for Cancer and Immunology Research, Children's National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010
- Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, D.C. 20037
- Department of Biochemistry and Molecular Biology, George Washington University School of Medicine and Health Sciences, Washington, D.C. 20037
| | - Stephan Ladisch
- Center for Cancer and Immunology Research, Children's National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010
- Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, D.C. 20037
- Department of Biochemistry and Molecular Biology, George Washington University School of Medicine and Health Sciences, Washington, D.C. 20037
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Malik M, Shukla A, Amin P, Niedelman W, Lee J, Jividen K, Phang JM, Ding J, Suh KS, Curmi PMG, Yuspa SH. S-nitrosylation regulates nuclear translocation of chloride intracellular channel protein CLIC4. J Biol Chem 2010; 285:23818-28. [PMID: 20504765 DOI: 10.1074/jbc.m109.091611] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its role in Ca(2+)-induced differentiation, stress-induced apoptosis, and modulating TGF-beta signaling in mouse epidermal keratinocytes. However, post-translational modifications on CLIC4 that govern nuclear translocation and thus these activities remain to be elucidated. The structure of CLIC4 is dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. Here we show that NO directly induces nuclear translocation of CLIC4 that is independent of the NO-cGMP pathway. Indeed, CLIC4 is directly modified by NO through S-nitrosylation of a cysteine residue, as measured by the biotin switch assay. NO enhances association of CLIC4 with the nuclear import proteins importin alpha and Ran. This is likely a result of the conformational change induced by S-nitrosylated CLIC4 that leads to unfolding of the protein, as exhibited by CD spectra analysis and trypsinolysis of the modified protein. Cysteine mutants of CLIC4 exhibit altered nitrosylation, nuclear residence, and stability, compared with the wild type protein likely as a consequence of altered tertiary structure. Moreover, tumor necrosis factor alpha-induced nuclear translocation of CLIC4 is dependent on nitric-oxide synthase activity. Inhibition of nitric-oxide synthase activity inhibits tumor necrosis factor alpha-induced nitrosylation and association with importin alpha and Ran and ablates CLIC4 nuclear translocation. These results suggest that S-nitrosylation governs CLIC4 structure, its association with protein partners, and thus its intracellular distribution.
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Affiliation(s)
- Mariam Malik
- Laboratory of Cancer Biology and Genetics, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, MD 20892, USA
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Two decades with dimorphic Chloride Intracellular Channels (CLICs). FEBS Lett 2010; 584:2112-21. [DOI: 10.1016/j.febslet.2010.03.013] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2009] [Revised: 03/08/2010] [Accepted: 03/08/2010] [Indexed: 01/11/2023]
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Shukla A, Yuspa SH. CLIC4 and Schnurri-2: a dynamic duo in TGF-beta signaling with broader implications in cellular homeostasis and disease. Nucleus 2010; 1:144-9. [PMID: 20617112 PMCID: PMC2898211 DOI: 10.4161/nucl.1.2.10920] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2009] [Accepted: 12/02/2009] [Indexed: 12/19/2022] Open
Abstract
CLIC4 is a highly conserved, multifunctional member of the chloride intracellular channel family of proteins. The protein is largely cytoplasmic but translocates to the nucleus upon a variety of stimuli including TGF-beta, TNF-alpha and etoposide. Nuclear resident CLIC4 causes growth arrest, terminal differentiation and apoptosis. Recently, it was discovered that TGF-beta causes CLIC4 to associate with Schnurri-2 and together they translocate to the nucleus and dissociate thereafter. The nuclear function of CLIC4 was further illuminated by the discovery that CLIC4 enhances TGF-beta signaling by associating with phospho-Smad2 and 3 and preventing their dephosphorylation. Enhanced TGF-beta dependent gene expression and growth inhibition are downstream consequences of this activity of CLIC4. In this article, we speculate on other consequences of the CLIC4 relation to TGF-beta signaling and the potential for CLIC4 to participate in other cellular functions related to normal homeostasis and disease.
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Affiliation(s)
- Anjali Shukla
- Laboratory of Cancer Biology and Genetics, 37 Convent Drive, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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Chloride channels of intracellular membranes. FEBS Lett 2010; 584:2102-11. [PMID: 20100480 DOI: 10.1016/j.febslet.2010.01.037] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2009] [Revised: 01/13/2010] [Accepted: 01/19/2010] [Indexed: 11/20/2022]
Abstract
Proteins implicated as intracellular chloride channels include the intracellular ClC proteins, the bestrophins, the cystic fibrosis transmembrane conductance regulator, the CLICs, and the recently described Golgi pH regulator. This paper examines current hypotheses regarding roles of intracellular chloride channels and reviews the evidence supporting a role in intracellular chloride transport for each of these proteins.
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Peng J, Zhang Q, Ma Y, Wang Y, Huang L, Zhang P, Chen J, Qin H. A rat-to-human search for proteomic alterations reveals transgelin as a biomarker relevant to colorectal carcinogenesis and liver metastasis. Electrophoresis 2009; 30:2976-2987. [PMID: 19711377 DOI: 10.1002/elps.200900203] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
In this study, a modified rat model similar to the classic human evolution of colorectal cancer (CRC) was established. As such, the altered profiles of proteins involved in these processes were further verified in human specimens, so as to determine the potential biomarkers relevant to human CRC development. Protein samples of four specific stages involved in CRC progression ((i) normal mucosa, (ii) adenoma, (iii) carcinoma, and (iv) liver metastasis)) were investigated by 2-DE. One protein spot displayed sequential suppression in the course of colorectal malignant transformation and was identified as transgelin by mass spectrometry. A decrease in its expression in both the epithelium and lamina propria was further confirmed by Western blot and immunohistochemistry analyses. Clinical and pathological parameter analysis revealed that downregulation of transgelin was associated with poor differentiation, and subsequent Dukes Stage and lower survival rate. Paradoxically, its sera level was significantly higher in CRC patients than in healthy donors, and the rise became dramatic, particularly in later Dukes Stages. These results indicate that downregulation of transgelin, in both the epithelium and lamina propria and accompanied with colorectal carcinogenesis, is correlated with worse prognosis. Its elevated serum levels might be the result of pathological hyperplasia of myofibroblasts and smooth muscle cells together with deeper tumor invasion into muscle layers. This altered expression represents interactions between cancer epithelium and stroma, such that transgelin might be a potential marker for CRC genesis and progression.
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Affiliation(s)
- Jiayuan Peng
- The Department of Surgery, The Sixth People's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, P. R. China
| | - Qingfu Zhang
- The Department of Surgery, The Sixth People's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, P. R. China
| | - Yanlei Ma
- The Department of Surgery, The Sixth People's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, P. R. China
| | - Yu Wang
- The Department of Surgery, The Sixth People's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, P. R. China
| | - Long Huang
- The Department of Surgery, The Sixth People's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, P. R. China
| | - Peng Zhang
- The Department of Surgery, The Sixth People's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, P. R. China
| | - Jie Chen
- Department of Pathology, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai, P. R. China
| | - Huanlong Qin
- The Department of Surgery, The Sixth People's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, P. R. China
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Xiong X, Huang S, Zhang H, Li J, Shen J, Xiong J, Lin Y, Jiang L, Wang X, Liang S. Enrichment and proteomic analysis of plasma membrane from rat dorsal root ganglions. Proteome Sci 2009; 7:41. [PMID: 19889238 PMCID: PMC2780401 DOI: 10.1186/1477-5956-7-41] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2009] [Accepted: 11/05/2009] [Indexed: 12/04/2022] Open
Abstract
BACKGROUND Dorsal root ganglion (DRG) neurons are primary sensory neurons that conduct neuronal impulses related to pain, touch and temperature senses. Plasma membrane (PM) of DRG cells plays important roles in their functions. PM proteins are main performers of the functions. However, mainly due to the very low amount of DRG that leads to the difficulties in PM sample collection, few proteomic analyses on the PM have been reported and it is a subject that demands further investigation. RESULTS By using aqueous polymer two-phase partition in combination with high salt and high pH washing, PMs were efficiently enriched, demonstrated by western blot analysis. A total of 954 non-redundant proteins were identified from the plasma membrane-enriched preparation with CapLC-MS/MS analysis subsequent to protein separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or shotgun digestion. 205 (21.5%) of the identified proteins were unambiguously assigned as PM proteins, including a large number of signal proteins, receptors, ion channel and transporters. CONCLUSION The aqueous polymer two-phase partition is a simple, rapid and relatively inexpensive method. It is well suitable for the purification of PMs from small amount of tissues. Therefore, it is reasonable for the DRG PM to be enriched by using aqueous two-phase partition as a preferred method. Proteomic analysis showed that DRG PM was rich in proteins involved in the fundamental biological processes including material exchange, energy transformation and information transmission, etc. These data would help to our further understanding of the fundamental DRG functions.
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Affiliation(s)
- Xia Xiong
- College of Life Sciences, Hunan Normal University, Changsha 410081, PR China
| | - Sha Huang
- College of Life Sciences, Hunan Normal University, Changsha 410081, PR China
| | - Hai Zhang
- College of Life Sciences, Hunan Normal University, Changsha 410081, PR China
| | - Jianjun Li
- College of Life Sciences, Hunan Normal University, Changsha 410081, PR China
| | - Jianying Shen
- College of Life Sciences, Hunan Normal University, Changsha 410081, PR China
| | - Jixian Xiong
- College of Life Sciences, Hunan Normal University, Changsha 410081, PR China
| | - Yong Lin
- College of Life Sciences, Hunan Normal University, Changsha 410081, PR China
| | - Liping Jiang
- College of Life Sciences, Hunan Normal University, Changsha 410081, PR China
| | - Xianchun Wang
- College of Life Sciences, Hunan Normal University, Changsha 410081, PR China
| | - Sonping Liang
- College of Life Sciences, Hunan Normal University, Changsha 410081, PR China
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