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Wu Q, Leng X, Zhang Q, Zhu YZ, Zhou R, Liu Y, Mei C, Zhang D, Liu S, Chen S, Wang X, Lin A, Lin X, Liang T, Shen L, Feng XH, Xia B, Xu P. IRF3 activates RB to authorize cGAS-STING-induced senescence and mitigate liver fibrosis. SCIENCE ADVANCES 2024; 10:eadj2102. [PMID: 38416816 PMCID: PMC10901380 DOI: 10.1126/sciadv.adj2102] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/14/2023] [Accepted: 01/23/2024] [Indexed: 03/01/2024]
Abstract
Cytosolic double-stranded DNA surveillance by cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (STING) signaling triggers cellular senescence, autophagy, biased mRNA translation, and interferon-mediated immune responses. However, detailed mechanisms and physiological relevance of STING-induced senescence are not fully understood. Here, we unexpectedly found that interferon regulatory factor 3 (IRF3), activated during innate DNA sensing, forms substantial endogenous complexes in the nucleus with retinoblastoma (RB), a key cell cycle regulator. The IRF3-RB interaction attenuates cyclin-dependent kinase 4/6 (CDK4/6)-mediated RB hyperphosphorylation that mobilizes RB to deactivate E2 family (E2F) transcription factors, thereby driving cells into senescence. STING-IRF3-RB signaling plays a notable role in hepatic stellate cells (HSCs) within various murine models, pushing activated HSCs toward senescence. Accordingly, IRF3 global knockout or conditional deletion in HSCs aggravated liver fibrosis, a process mitigated by the CDK4/6 inhibitor. These findings underscore a straightforward yet vital mechanism of cGAS-STING signaling in inducing cellular senescence and unveil its unexpected biology in limiting liver fibrosis.
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Affiliation(s)
- Qirou Wu
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
| | - Xiaohong Leng
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
| | - Qian Zhang
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
- Department of Hepatobiliary and Pancreatic Surgery and Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, University School of Medicine, Zhejiang University, Hangzhou 310058, China
| | - Ye-Zhang Zhu
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
| | - Ruyuan Zhou
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
- Department of Hepatobiliary and Pancreatic Surgery and Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, University School of Medicine, Zhejiang University, Hangzhou 310058, China
| | - Yutong Liu
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
| | - Chen Mei
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
| | - Dan Zhang
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
| | - Shengduo Liu
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
- Department of Hepatobiliary and Pancreatic Surgery and Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, University School of Medicine, Zhejiang University, Hangzhou 310058, China
- ZJU-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou 310058, China
| | - Shasha Chen
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
| | - Xiaojian Wang
- Institute of Immunology, Zhejiang University School of Medicine, Hangzhou 310058, China
| | - Aifu Lin
- MOE Laboratory of Biosystem Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou 310058, China
| | - Xia Lin
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
| | - Tingbo Liang
- Department of Hepatobiliary and Pancreatic Surgery and Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, University School of Medicine, Zhejiang University, Hangzhou 310058, China
- Cancer Center, Zhejiang University, Hangzhou 310058, China
| | - Li Shen
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
| | - Xin-Hua Feng
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
- Department of Thoracic Cancer, Affiliated Hangzhou Cancer Hospital, School of Medicine, Zhejiang University, Hangzhou 310058, China
| | - Bing Xia
- Cancer Center, Zhejiang University, Hangzhou 310058, China
| | - Pinglong Xu
- MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
- Department of Hepatobiliary and Pancreatic Surgery and Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, University School of Medicine, Zhejiang University, Hangzhou 310058, China
- ZJU-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou 310058, China
- Department of Thoracic Cancer, Affiliated Hangzhou Cancer Hospital, School of Medicine, Zhejiang University, Hangzhou 310058, China
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Urushima H, Yuasa H, Matsubara T, Kuroda N, Hara Y, Inoue K, Wake K, Sato T, Friedman SL, Ikeda K. Activation of Hepatic Stellate Cells Requires Dissociation of E-Cadherin-Containing Adherens Junctions with Hepatocytes. THE AMERICAN JOURNAL OF PATHOLOGY 2020; 191:438-453. [PMID: 33345995 DOI: 10.1016/j.ajpath.2020.12.007] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/22/2020] [Revised: 12/02/2020] [Accepted: 12/03/2020] [Indexed: 12/12/2022]
Abstract
Hepatic stellate cells (HSCs) are resident mesenchymal cells in the space of Disse interposed between liver sinusoidal endothelial cells and hepatocytes. Thorn-like microprojections, or spines, project out from the cell surface of HSCs, crossing the space of Disse, to establish adherens junctions with neighboring hepatocytes. Although HSC activation is initiated largely from stimulation by adjacent cells, isolated HSCs also activate spontaneously in primary culture on plastic. Therefore, other unknown HSC-initiating factors apart from paracrine stimuli may promote activation. The dissociation of adherens junctions between HSCs and hepatocytes as an activating signal for HSCs was explored, establishing epithelial cadherin (E-cadherin) as an adhesion molecule linking hepatocytes and HSCs. In vivo, following carbon tetrachloride-induced liver injury, HSCs lost their spines and dissociated from adherens junctions in the early stages of injury, and were subsequently activated along with an increase in YAP/TAZ expression. After abrogation of liver injury, HSCs reconstructed their spines and adherens junctions. In vitro, reconstitution of E-cadherin-containing adherens junctions by forced E-cadherin expression quiesced HSCs and suppressed TAZ expression. Additionally, increase of TAZ expression leading to the activation of HSCs by autocrine stimulation of transforming growth factor-β, was revealed as a mechanism of spontaneous activation. Thus, we have uncovered a critical event required for HSC activation through enhanced TAZ-mediated mechanotransduction after the loss of adherens junctions between HSCs and hepatocytes.
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Affiliation(s)
- Hayato Urushima
- Department of Anatomy and Regenerative Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan.
| | - Hideto Yuasa
- Department of Anatomy and Regenerative Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan
| | - Tsutomu Matsubara
- Department of Anatomy and Regenerative Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan
| | - Noriyuki Kuroda
- Department of Anatomy, Tissue and Cell Biology, School of Dental Medicine, Tsurumi University, Yokohama, Japan
| | - Yaiko Hara
- Department of Anatomy, Tissue and Cell Biology, School of Dental Medicine, Tsurumi University, Yokohama, Japan
| | - Kouji Inoue
- Research Center of Electron Microscopy, School of Dental Medicine, Tsurumi University, Yokohama, Japan
| | - Kenjiro Wake
- Department of Anatomy and Regenerative Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan; Research Center of Electron Microscopy, School of Dental Medicine, Tsurumi University, Yokohama, Japan; Liver Research Unit, Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan
| | - Tetsuji Sato
- Department of Anatomy, Tissue and Cell Biology, School of Dental Medicine, Tsurumi University, Yokohama, Japan
| | - Scott L Friedman
- Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, New York
| | - Kazuo Ikeda
- Department of Anatomy and Regenerative Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan.
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Mori K, Toiyama Y, Okugawa Y, Ichikawa T, Nagano Y, Oki S, Shimura T, Fujikawa H, Hiro J, Kobayash M, Araki T, Inoue Y, Mohri Y, Kusunoki M. Preoperative heat shock protein 47 levels identify colorectal cancer patients with lymph node metastasis and poor prognosis. Oncol Lett 2020; 20:333. [PMID: 33123244 PMCID: PMC7583735 DOI: 10.3892/ol.2020.12196] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2019] [Accepted: 05/28/2020] [Indexed: 11/23/2022] Open
Abstract
Accumulating evidence suggests that overexpression of heat shock protein 47 (HSP47) increases cancer progression, and that HSP47 level in the tumor-associated stroma may serve as a diagnostic marker in various cancers. The present study aimed to evaluate whether HSP47 gene expression in colorectal cancer (CRC) tissues could be used to identify lymph node (LN) metastasis status preoperatively in patients with CRC. To do so, HSP47 gene expression was determined and its association with the clinicopathological characteristics of patients with CRC was analyzed. A total of 139 surgical specimens from patients with CRC and 36 patients with benign colonic disease undergoing surgery at Mie University Hospital were analyzed. HSP47 gene expression was determined by reverse transcription quantitative PCR using Power SYBR Green PCR methods. Expression level of HSP47 was significantly higher in CRC tissues compared with normal tissue from patients with benign colonic disease. Furthermore, high HSP47 expression was significantly associated with tumor progression, including high T stage, lymph node metastasis and venous invasion, and high TNM stage. High HSP47 expression may therefore serve as a novel predictive biomarker for determining patients with CRC and LN metastasis. According to Kaplan-Meier analysis, patients with high HSP47 expression level had significantly poorer overall survival than those with low HSP47 expression level. Furthermore, multivariate analyses identified HSP47 expression as an independent predictive marker for LN metastasis and poor overall survival in patients with CRC. In summary, the present study demonstrated that HSP47 expression may be considered as a novel biomarker for predicting LN metastasis status and prognosis in patients with CRC.
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Affiliation(s)
- Koichiro Mori
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Yuji Toiyama
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Yoshinaga Okugawa
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Takashi Ichikawa
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Yuka Nagano
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Satoshi Oki
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Tadanobu Shimura
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Hiroyuki Fujikawa
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Junichiro Hiro
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Minako Kobayash
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Toshimitsu Araki
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Yasuhiro Inoue
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Yasuhiko Mohri
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
| | - Masato Kusunoki
- Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
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Yuan Q, Hou S, Zhai J, Tian T, Wu Y, Wu Z, He J, Chen Z, Zhang J. S100A4 promotes inflammation but suppresses lipid accumulation via the STAT3 pathway in chronic ethanol-induced fatty liver. J Mol Med (Berl) 2019; 97:1399-1412. [PMID: 31321478 DOI: 10.1007/s00109-019-01808-7] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2018] [Revised: 05/24/2019] [Accepted: 05/31/2019] [Indexed: 02/07/2023]
Abstract
S100A4, a member of the S100 calcium-binding protein family, has been identified in a subpopulation of liver macrophages and promotes liver fibrosis via hepatic stellate cell activation. However, the specific role of S100A4 in alcoholic liver disease (ALD) has not been well investigated. Here, S100A4 knockout (S100A4-/-) mice were used in a chronic-binge ethanol model for studying the role of S100A4 and its related molecular mechanism in ALD. S100A4 expression was increased in ethanol-induced liver tissues of wild-type (WT) mice. Macrophage-derived S100A4 promoted liver inflammation but suppressed lipid accumulation under the ethanol feeding condition. S100A4 deficiency promoted ethanol-induced liver injury and hepatic fat accumulation. Further mechanistic studies found that S100A4 inhibited liver fat accumulation mainly by activating the STAT3 pathway and downregulating lipogenic gene expression, especially that of SREBP-1c. In AML-12 cells, a STAT3 inhibitor abolished STAT3 levels and decreased the expression of SREBP1c. Furthermore, the administration of a neutralizing S100A4 antibody to WT mice significantly promoted ethanol-induced liver injury and fatty accumulation. Thus, S100A4 may represent a potential candidate target for the prevention and treatment of ethanol-induced fatty liver. In this study, we discovered the special role of S100A4 in alcoholic liver disease. S100A4 deficiency attenuated ethanol-induced hepatitis and promoted hepatic fat accumulation in ethanol-induced liver tissues. Further mechanistic studies have found that S100A4 promotes early alcoholic hepatitis mainly by activating the STAT3 pathway and its downstream proinflammatory gene expression. Interestingly, activation of the STAT3 pathway downregulates lipogenic gene expression, especially SREBP-1c. KEY MESSAGES: In this study, we discovered the special role of S100A4 in alcoholic liver disease. S100A4 deficiency attenuated ethanol-induced hepatitis and promoted hepatic fat accumulation in ethanol-induced liver tissues. Further mechanistic studies have found that S100A4 promotes early alcoholic hepatitis mainly by activating the STAT3 pathway and its downstream proinflammatory gene expression. Interestingly, activation of the STAT3 pathway downregulates lipogenic gene expression, especially SREBP-1c.
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Affiliation(s)
- Qi Yuan
- The College of Life Science and Bioengineering, Beijing Jiaotong University, No.3 Shangyuancun Road, Beijing, 100044, People's Republic of China
| | - Shasha Hou
- The College of Life Science and Bioengineering, Beijing Jiaotong University, No.3 Shangyuancun Road, Beijing, 100044, People's Republic of China
| | - Junfeng Zhai
- The Chinese Academy of Inspection and Quarantine, Beijing, People's Republic of China
| | - Tian Tian
- The College of Life Science and Bioengineering, Beijing Jiaotong University, No.3 Shangyuancun Road, Beijing, 100044, People's Republic of China
| | - Yingjie Wu
- The State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, People's Republic of China
| | - Zhenlong Wu
- The State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, People's Republic of China
| | - Jinsheng He
- The College of Life Science and Bioengineering, Beijing Jiaotong University, No.3 Shangyuancun Road, Beijing, 100044, People's Republic of China
| | - Zhinan Chen
- The College of Life Science and Bioengineering, Beijing Jiaotong University, No.3 Shangyuancun Road, Beijing, 100044, People's Republic of China.,The Cell Engineering Research Center and Department of Cell Biology, State Key Laboratory of Cancer, Fourth Military Medical University, Xi'an, People's Republic of China
| | - Jinhua Zhang
- The College of Life Science and Bioengineering, Beijing Jiaotong University, No.3 Shangyuancun Road, Beijing, 100044, People's Republic of China.
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Zhang J, Song K, Wang J, Li Y, Liu S, Dai C, Chen L, Wang S, Qin Z. S100A4 blockage alleviates agonistic anti-CD137 antibody-induced liver pathology without disruption of antitumor immunity. Oncoimmunology 2018; 7:e1296996. [PMID: 29632708 PMCID: PMC5889198 DOI: 10.1080/2162402x.2017.1296996] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2016] [Revised: 02/11/2017] [Accepted: 02/14/2017] [Indexed: 01/01/2023] Open
Abstract
Liver-related autoimmune toxicities triggered by agonistic anti-CD137 antibodies have greatly limited their use in clinical applications. Here, we found that anti-CD137 monoclonal antibody (mAb) treatment in mice induced the infiltration of a large number of S100A4+ macrophages into the liver. Depletion of these cells or deficiency of S100A4 decreased inflammatory cytokine profiles and drastically reduced the number of liver pathogenic CD8+ T cells. Mechanistically, soluble S100A4 directly activated the Akt pathway and specifically prolonged CD8+ T cell survival. Interestingly, one S100A4 neutralizing mAb selectively alleviated liver abnormalities but did not affect the antitumor immunity induced by anti-CD137 mAb therapy. Thus, our study presents a novel molecular link to the liver pathology induced by an immune stimulatory antibody and proposes that combinational immunotherapies targeting those pathways could potentially elicit optimal antitumor immunity with minimal side effects.
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Affiliation(s)
- Jinhua Zhang
- Key Laboratory of Protein and Peptide Pharmaceuticals, CAS Center for Excellence in Biomacromolecules, Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Kun Song
- Key Laboratory of Protein and Peptide Pharmaceuticals, CAS Center for Excellence in Biomacromolecules, Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Jun Wang
- Department of Immunobiology and Yale Cancer Center, Yale University School of Medicine, New Haven, CT, USA
| | - Yanan Li
- Key Laboratory of Protein and Peptide Pharmaceuticals, CAS Center for Excellence in Biomacromolecules, Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Shuangqing Liu
- Key Laboratory of Protein and Peptide Pharmaceuticals, CAS Center for Excellence in Biomacromolecules, Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Chengliang Dai
- Key Laboratory of Protein and Peptide Pharmaceuticals, CAS Center for Excellence in Biomacromolecules, Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Lieping Chen
- Department of Immunobiology and Yale Cancer Center, Yale University School of Medicine, New Haven, CT, USA
| | - Shengdian Wang
- Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Zhihai Qin
- Key Laboratory of Protein and Peptide Pharmaceuticals, CAS Center for Excellence in Biomacromolecules, Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.,Medical Research Center, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, China
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Chatani N, Kamada Y, Kizu T, Ogura S, Furuta K, Egawa M, Hamano M, Ezaki H, Kiso S, Shimono A, Ouchi N, Yoshida Y, Takehara T. Secreted frizzled-related protein 5 (Sfrp5) decreases hepatic stellate cell activation and liver fibrosis. Liver Int 2015; 35:2017-26. [PMID: 25488180 DOI: 10.1111/liv.12757] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/16/2014] [Accepted: 12/03/2014] [Indexed: 02/13/2023]
Abstract
BACKGROUND & AIMS Obesity-related adipocytokine dysregulation is known to accelerate liver fibrosis progression. Recently, a natural Wnt5a inhibitor, secreted frizzled-related protein 5 (Sfrp5), was identified as a novel adipocytokine that has reduced expression in obese adipose tissue in both rodents and human. In addition, hepatic gene expression of Wnt5a and its receptor frizzled 2 (Fz2) is elevated during fibrosis progression. Therefore, Sfrp5 could have biological significance in liver fibrosis. METHODS We first investigated the effects of Sfrp5 on primary cultured mouse hepatic stellate cells (HSCs) in vitro. Next, to elucidate the roles of Sfrp5 in liver fibrosis, we investigated a carbon-tetrachloride (CCl4 )-induced liver fibrosis model using Sfrp5 knockout (KO) and wild type (WT) mice in vivo. Each mouse was injected intraperitoneally with CCl4 (0.5 ml/kg) or olive oil as a single dose (acute liver injury model), or twice a week for 6 weeks (liver fibrosis model). RESULTS In in vitro studies, Wnt5a enhanced both proliferation and migration of HSCs, and these effects could be completely blocked by Sfrp5. Moreover, siRNA knockdown of Fz2 in HSCs could block the effects of Wnt5a on both HSC proliferation and migration. In in vivo studies, there were no differences in the CCl4 -induced liver injury between KO and WT mice. Hepatic Wnt5a gene expression and plasma Wnt5a levels significantly increased after a single CCl4 injection in both mice. Sfrp5 knockout significantly enhanced CCl4 -induced liver fibrosis. CONCLUSIONS Our findings demonstrate that Sfrp5 may ameliorate mouse liver fibrosis through inhibition of Wnt5a/Fz2 signalling.
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Affiliation(s)
- Norihiro Chatani
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Osaka, Japan
| | - Yoshihiro Kamada
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Osaka, Japan.,Department of Molecular Biochemistry & Clinical Investigation, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
| | - Takashi Kizu
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Osaka, Japan
| | - Satoshi Ogura
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Osaka, Japan
| | - Kunimaro Furuta
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Osaka, Japan
| | - Mayumi Egawa
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Osaka, Japan
| | - Mina Hamano
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Osaka, Japan
| | - Hisao Ezaki
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Osaka, Japan
| | - Shinichi Kiso
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Osaka, Japan
| | | | - Noriyuki Ouchi
- Department of Molecular Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Yuichi Yoshida
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Osaka, Japan
| | - Tetsuo Takehara
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Osaka, Japan
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7
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Harth Y. Painless, safe, and efficacious noninvasive skin tightening, body contouring, and cellulite reduction using multisource 3DEEP radiofrequency. J Cosmet Dermatol 2015; 14:70-5. [PMID: 25598274 DOI: 10.1111/jocd.12124] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/06/2013] [Indexed: 11/28/2022]
Abstract
In the last decade, Radiofrequency (RF) energy has proven to be safe and highly efficacious for face and neck skin tightening, body contouring, and cellulite reduction. In contrast to first-generation Monopolar/Bipolar and "X -Polar" RF systems which use one RF generator connected to one or more skin electrodes, multisource radiofrequency devices use six independent RF generators allowing efficient dermal heating to 52-55°C, with no pain or risk of other side effects. In this review, the basic science and clinical results of body contouring and cellulite treatment using multisource radiofrequency system (Endymed PRO, Endymed, Cesarea, Israel) will be discussed and analyzed.
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Affiliation(s)
- Yoram Harth
- Medical OR Center, Herzlya, Israel; EndyMed Medical, Cesarea, Israel
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8
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Chen L, Li J, Zhang J, Dai C, Liu X, Wang J, Gao Z, Guo H, Wang R, Lu S, Wang F, Zhang H, Chen H, Fan X, Wang S, Qin Z. S100A4 promotes liver fibrosis via activation of hepatic stellate cells. J Hepatol 2015; 62:156-64. [PMID: 25111176 DOI: 10.1016/j.jhep.2014.07.035] [Citation(s) in RCA: 117] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/16/2014] [Revised: 07/29/2014] [Accepted: 07/29/2014] [Indexed: 12/13/2022]
Abstract
BACKGROUND & AIMS S100A4 has been linked to the fibrosis of several organs due to its role as a fibroblast-specific marker. However, the role of S100A4 itself in the development of fibrosis has not been much investigated. Here, we determined whether S100A4 regulates liver fibrogenesis and examined its mechanism by focusing on the activation of hepatic stellate cells (HSCs). METHODS S100A4 deficient mice were used to determine the role of S100A4 in liver fibrogenesis. The effect of S100A4 on HSC activation was estimated by using primary mouse HSCs and the human HSC cell line LX-2. Serum levels of S100A4 in cirrhotic patients were determined by ELISA. RESULTS S100A4 was found to be secreted by a subpopulation of macrophages and to promote the development of liver fibrosis. It accumulated in the liver during the progression of liver fibrosis and activated HSCs in mice. In vitro studies demonstrated that S100A4 induced the overexpression of alpha-smooth muscle actin through c-Myb in HSCs. Both, the selective depletion of S100A4-expressing cells and knockdown of S100A4 in the liver by RNA interference, resulted in a reduction of liver fibrosis following injury. Importantly, increased S100A4 levels in both the liver tissue and serum correlated positively with liver fibrosis in humans. CONCLUSIONS S100A4 promotes liver fibrosis by activating HSCs, which may represent a potential target for anti-fibrotic therapies.
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Affiliation(s)
- Lin Chen
- Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China
| | - Jie Li
- Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China
| | - Jinhua Zhang
- Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Chengliang Dai
- Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China
| | - Xiaoman Liu
- Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Jun Wang
- Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA
| | - Zhitao Gao
- Xinxiang Medical University, Xinxiang, China
| | - Hongyan Guo
- Beijing YouAn Hospital, Capital Medical University, Beijing, China
| | - Rui Wang
- Beijing YouAn Hospital, Capital Medical University, Beijing, China
| | - Shichun Lu
- Beijing YouAn Hospital, Capital Medical University, Beijing, China
| | - Fusheng Wang
- Department of Infectious Diseases, Beijing 302 Hospital, Beijing, China
| | - Henghui Zhang
- Peking University People's Hospital, Peking University Hepatology Institute, Beijing, China
| | - Hongsong Chen
- Peking University People's Hospital, Peking University Hepatology Institute, Beijing, China
| | - Xiaolong Fan
- Beijing Key Laboratory of Gene Resource and Molecular Development, Laboratory of Neuroscience and Brain Development, Beijing Normal University, Beijing, China
| | - Shengdian Wang
- Key Laboratory for Infection and Immunity, Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Zhihai Qin
- Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; Chinese Academy of Sciences-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
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Expression of heat shock protein 47, transforming growth factor-beta 1, and connective tissue growth factor in liver tissue of patients with Schistosoma japonicum-induced hepatic fibrosis. Parasitology 2014; 142:341-51. [PMID: 25111595 DOI: 10.1017/s0031182014001115] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
SUMMARY To detect the expression of pro-fibrotic molecules, such as heat shock protein 47 (Hsp47), transforming growth factor-beta 1 (TGF-β1) and connective tissue growth factor (CTGF) in liver specimens, and analyse their correlations with the progression of schistosomal hepatic fibrosis, liver biopsy was performed in 42 chronic schistosomiasis (CS) patients, 16 chronic hepatitis B (CHB) patients and five healthy individuals (HI). Immunohistochemistry (IHC) analyses displayed that the expression of Hsp47, TGF-β1 and CTGF was increased in CS and CHB patients compared with HI. Using real-time PCR, the mRNA levels of Hsp47, TGF-β1 and CTGF were higher in CS patients compared with HI. In CS patients, the mRNA levels of these genes were correlated with the stage of fibrosis, and TGF-β1 mRNA expression was associated with the grade of inflammation. Additional analyses indicated that the mRNA levels of Hsp47 and CTGF were highly correlated with liver stiffness value and spleen thickness diameter, both of which represented the severity of fibrosis. In conclusion, the three molecules are involved in the pathogenesis of hepatic fibrosis infected by Schistosoma japonicum. TGF-β1 participates not only in the inflammatory process, but also in the fibrotic process in which Hsp47 and CTGF probably play a key role.
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10
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Padden J, Megger DA, Bracht T, Reis H, Ahrens M, Kohl M, Eisenacher M, Schlaak JF, Canbay AE, Weber F, Hoffmann AC, Kuhlmann K, Meyer HE, Baba HA, Sitek B. Identification of novel biomarker candidates for the immunohistochemical diagnosis of cholangiocellular carcinoma. Mol Cell Proteomics 2014; 13:2661-72. [PMID: 25034945 PMCID: PMC4188994 DOI: 10.1074/mcp.m113.034942] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell-type-specific expression profile via immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma, and stress-induced phosphoprotein 1 in tumorous cholangiocytes relative to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Because stress-induced phosphoprotein 1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as a potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases, as well as metastatic malignancies in the liver.
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Affiliation(s)
- Juliet Padden
- From the ‡Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801 Bochum, Germany;
| | - Dominik A Megger
- From the ‡Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801 Bochum, Germany
| | - Thilo Bracht
- From the ‡Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801 Bochum, Germany
| | - Henning Reis
- ¶Institut für Pathologie, Universitätsklinikum Essen, Universität Duisburg-Essen, 45141 Essen, Germany
| | - Maike Ahrens
- From the ‡Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801 Bochum, Germany
| | - Michael Kohl
- From the ‡Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801 Bochum, Germany
| | - Martin Eisenacher
- From the ‡Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801 Bochum, Germany
| | - Jörg F Schlaak
- ‖Klinik für Gastroenterologie und Hepatologie, Universitätsklinikum Essen, 45141 Essen, Universität Duisburg-Essen, 45141 Essen, Germany
| | - Ali E Canbay
- ‖Klinik für Gastroenterologie und Hepatologie, Universitätsklinikum Essen, 45141 Essen, Universität Duisburg-Essen, 45141 Essen, Germany
| | - Frank Weber
- **Klinik für Allgemeinchirurgie, Viszeral- und Transplantationschirurgie, Universitätsklinikum Essen, Universität Duisburg-Essen, 45141 Essen, Germany
| | - Andreas-Claudius Hoffmann
- ‡‡Innere Klinik (Tumorforschung), Westdeutsches Tumorzentrum, Universitätsklinikum Essen, Universität Duisburg-Essen, 45141 Essen, Germany
| | - Katja Kuhlmann
- From the ‡Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801 Bochum, Germany
| | - Helmut E Meyer
- From the ‡Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801 Bochum, Germany; §§Leibniz Institute for Analytical Sciences - ISAS, 44139 Dortmund, Germany
| | - Hideo A Baba
- ¶Institut für Pathologie, Universitätsklinikum Essen, Universität Duisburg-Essen, 45141 Essen, Germany
| | - Barbara Sitek
- From the ‡Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801 Bochum, Germany;
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11
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Chang W, Yang M, Song L, Shen K, Wang H, Gao X, Li M, Niu W, Qin X. Isolation and culture of hepatic stellate cells from mouse liver. Acta Biochim Biophys Sin (Shanghai) 2014; 46:291-8. [PMID: 24389643 DOI: 10.1093/abbs/gmt143] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Hepatic stellate cells (HSCs) are the primary extracellular matrix-producing cells within the liver and have numerous vital functions. A robust protocol for the isolation and culture of HSCs is important for further investigations of cell functions and related mechanisms in liver disease. The volume of the mouse liver is much smaller than that of the rat liver, which makes it much more difficult to isolate mouse HSCs (mHSCs) than rat HSCs. At present, isolating mHSCs is still a challenge because there is no efficient, robust method to isolate and culture these cells. In the present study, C57BL/6J mice were intravenously injected with liposome-encapsulated dichloromethylene diphosphate (CL2MDP) to selectively eliminate Kupffer cells from the liver. The mouse livers were then perfused in situ, and the mHSCs were isolated with an optimized density gradient centrifugation technique. In the phosphate buffer solution (PBS)-liposome group, the yield of mHSCs was (1.37 ± 0.23) × 10(6)/g liver, the cell purity was (90.18 ± 1.61)%, and the cell survival rate was (94.51 ± 1.61)%. While in the CL2MDP-liposome group, the yield of mHSCs was (1.62 ± 0.34) × 10(6)/g liver, the cell purity was (94.44 ± 1.89)%, and the cell survival rate was (94.41 ± 1.50)%. Based on the yield and purity of mHSCs, the CL2MDP-liposome treatment was superior to the PBS-liposome treatment (P < 0.05, P < 0.01). This study established successfully a robust and efficient protocol for the separation and purification of mHSCs, and both a high purity and an adequate yield of mHSCs were obtained.
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Affiliation(s)
- Wenju Chang
- Department of General Surgery, Zhongshan Hospital, Shanghai Medical College of Fudan University, Shanghai 200032, China
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12
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Westra IM, Oosterhuis D, Groothuis GMM, Olinga P. Precision-cut liver slices as a model for the early onset of liver fibrosis to test antifibrotic drugs. Toxicol Appl Pharmacol 2014; 274:328-38. [PMID: 24321339 DOI: 10.1016/j.taap.2013.11.017] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2013] [Revised: 11/19/2013] [Accepted: 11/25/2013] [Indexed: 01/26/2023]
Abstract
Induction of fibrosis during prolonged culture of precision-cut liver slices (PCLS) was reported. In this study, the use of rat PCLS was investigated to further characterize the mechanism of early onset of fibrosis in this model and the effects of antifibrotic compounds. Rat PCLS were incubated for 48h, viability was assessed by ATP and gene expression of PDGF-B and TGF-β1 and the fibrosis markers Hsp47, αSma and Pcol1A1 and collagen1 protein expressions were determined. The effects of the antifibrotic drugs imatinib, sorafenib and sunitinib, PDGF-pathway inhibitors, and perindopril, valproic acid, rosmarinic acid, tetrandrine and pirfenidone, TGFβ-pathway inhibitors, were determined. After 48h of incubation, viability of the PCLS was maintained and gene expression of PDGF-B was increased while TGF-β1 was not changed. Hsp47, αSma and Pcol1A1 gene expressions were significantly elevated in PCLS after 48h, which was further increased by PDGF-BB and TGF-β1. The increased gene expression of fibrosis markers was inhibited by all three PDGF-inhibitors, while TGFβ-inhibitors showed marginal effects. The protein expression of collagen 1 was inhibited by imatinib, perindopril, tetrandrine and pirfenidone. In conclusion, the increased gene expression of PDGF-B and the down-regulation of fibrosis markers by PDGF-pathway inhibitors, together with the absence of elevated TGF-β1 gene expression and the limited effect of the TGFβ-pathway inhibitors, indicated the predominance of the PDGF pathway in the early onset of fibrosis in PCLS. PCLS appear a useful model for research of the early onset of fibrosis and for testing of antifibrotic drugs acting on the PDGF pathway.
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Affiliation(s)
- Inge M Westra
- Division of Pharmacokinetics, Toxicology and Targeting, Department of Pharmacy, University of Groningen, The Netherlands
| | - Dorenda Oosterhuis
- Division of Pharmaceutical Technology and Biopharmacy, Department of Pharmacy, University of Groningen, The Netherlands
| | - Geny M M Groothuis
- Division of Pharmacokinetics, Toxicology and Targeting, Department of Pharmacy, University of Groningen, The Netherlands
| | - Peter Olinga
- Division of Pharmaceutical Technology and Biopharmacy, Department of Pharmacy, University of Groningen, The Netherlands.
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13
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Involvement of heat shock protein 47 in Schistosoma japonicum-induced hepatic fibrosis in mice. Int J Parasitol 2014; 44:23-35. [DOI: 10.1016/j.ijpara.2013.08.009] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2013] [Revised: 08/14/2013] [Accepted: 08/15/2013] [Indexed: 12/15/2022]
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14
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Takemura T, Yoshida Y, Kiso S, Kizu T, Furuta K, Ezaki H, Hamano M, Egawa M, Chatani N, Kamada Y, Imai Y, Higashiyama S, Iwamoto R, Mekada E, Takehara T. Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice. Biochem Biophys Res Commun 2013; 437:185-91. [PMID: 23743191 DOI: 10.1016/j.bbrc.2013.05.097] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2013] [Accepted: 05/23/2013] [Indexed: 12/23/2022]
Abstract
Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.
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Affiliation(s)
- Takayo Takemura
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka, Japan
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15
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Lin X, Yang H, Zhang H, Zhou L, Guo Z. A novel transcription mechanism activated by ethanol: induction of Slc7a11 gene expression via inhibition of the DNA-binding activity of transcriptional repressor octamer-binding transcription factor 1 (OCT-1). J Biol Chem 2013; 288:14815-23. [PMID: 23592778 DOI: 10.1074/jbc.m113.466565] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Solute carrier family 7, member 11 (Slc7a11) is a plasma membrane cystine/glutamate exchanger that provides intracellular cystine to produce glutathione, a major cellular antioxidant. Oxidative and endoplasmic reticulum stresses up-regulate Slc7a11 expression by activation of nuclear factor erythroid 2-related factor 2 and transcription factor 4. This study examined the effect of ethanol on Slc7a11 expression and the underlying mechanism involved. Treatment of mouse hepatic stellate cells with ethanol significantly increased Slc7a11 mRNA and protein levels. Deletion of a 20-bp DNA sequence between -2044 to -2024 upstream of the transcription start site significantly increased basal activity and completely abolished the ethanol-induced activity of the Slc7a11 promoter. This deletion did not affect Slc7a11 promoter activity induced by oxidative or endoplasmic reticulum stress. DNA sequence analysis revealed a binding motif for octamer-binding transcription factor 1 (OCT-1) in the deleted fragment. Mutation of this OCT-1 binding motif resulted in a similar effect as the deletion experiment, i.e. it increased the basal promoter activity and abolished the response to ethanol. Ethanol exposure significantly inhibited OCT-1 binding to the Slc7a11 promoter region, although it did not alter OCT-1 mRNA and protein levels. OCT-1 reportedly functions as either a transcriptional enhancer or repressor, depending on the target genes. Results from this study suggest that OCT-1 functions as a repressor on the Slc7a11 promoter and that ethanol inhibits OCT-1 binding to the Slc7a11 promoter, thereby increasing Slc7a11 expression. Taken together, inhibition of the DNA binding activity of transcriptional repressor OCT-1 is a mechanism by which ethanol up-regulates Slc711 expression.
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Affiliation(s)
- Xinghua Lin
- Department of Physiology, Meharry Medical College, Nashville, Tennessee 37208, USA
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16
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Zhang HF, Lin XH, Yang H, Zhou LC, Guo YL, Barnett JV, Guo ZM. Regulation of the activity and expression of aryl hydrocarbon receptor by ethanol in mouse hepatic stellate cells. Alcohol Clin Exp Res 2012; 36:1873-81. [PMID: 22486318 DOI: 10.1111/j.1530-0277.2012.01787.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2011] [Accepted: 02/09/2012] [Indexed: 11/29/2022]
Abstract
BACKGROUND During the course of alcohol-induced liver damage, hepatic stellate cells are transformed into proliferative, fibrogenic, and contractile myofibroblasts. Aryl hydrocarbon receptor (AhR) is a transcription factor that controls the expression of genes involved in the metabolism of xenobiotics, inflammation, cell proliferation, and death. METHODS Immortal mouse hepatic stellate cells (MHSCs) were isolated from transgenic mice that expressed a thermolabile SV40 tumor antigen. Quantitative real-time reverse transcription polymerase chain reaction assays, Western blot analysis, promoter activity assays, and chromatin immunoprecipitation analyses were performed for studying the effect of ethanol (EtOH) on AhR expression and transcriptional activity. RESULTS Treatment of MHSCs with 50 to 200 mM EtOH for 6 hours induced AhR nuclear translocation, enhanced the promoter activity of cytochrome P450 (CYP) 1A1, increased the amount of AhR bound to the promoter of CYP1A1 and 1B1, and up-regulated the mRNA expression of these AhR target genes in a dose-dependent manner. In contrast, EtOH exposure down-regulated AhR mRNA and protein expression. Similarly, benzo(a)pyrene (BaP) at 10 nM reduced AhR and increased CYP1A1 and 1B1 mRNAs. Pretreatment of MHSCs with 50 mM EtOH for 7 days diminished the capacity of MHSCs to express CYP1A1 and 1B1 induced by a 200 mM EtOH challenge, or by 10 nM BaP. However, the up-regulatory effect of EtOH on solute carrier family 16, member 6 (SLC16a6) was unaffected by EtOH pretreatment. Similar to EtOH, dimethyl sulfoxide (DMSO) at concentrations of 50 to 100 mM down-regulated AhR and up-regulated CYP1A1 mRNA expression in a dose-dependent manner. CONCLUSIONS These data, for the first time, demonstrate that EtOH activates MHSC AhR and down-regulates its expression. Chronic EtOH pretreatment lowers the availability of AhR, and specifically diminishes the inducibility of CYP genes. The effect on AhR appears to not be an EtOH-specific response, as DMSO alone (and possibly other organic solvents) was also able to activate AhR.
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Affiliation(s)
- Hong Feng Zhang
- Department of Physiology, Meharry Medical College, Nashville, TN 37208, USA
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17
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Fascin, a novel marker of human hepatic stellate cells, may regulate their proliferation, migration, and collagen gene expression through the FAK-PI3K-Akt pathway. J Transl Med 2012; 92:57-71. [PMID: 22005766 DOI: 10.1038/labinvest.2011.150] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Fascin is a component of actin bundles and may regulate various cellular events. The expression and function of fascin in human hepatic stellate cells (HSCs) has remained largely uncharacterized. Fascin expression in human liver tissue was studied using immunohistochemistry. To identify cells expressing fascin, double immunofluorescent staining with vimentin, α-smooth muscle actin (α-SMA), or fibulin-2 was performed and analyzed with confocal microscopy. In culture experiments, fascin expression and the phosphorylation of focal adhesion kinase (FAK) and Akt in LX-2 cells, a cell line of human HSCs, were investigated using western blot. Specific siRNAs were used to reduce the expression of fascin in LX-2 cells. Proliferation and migration were assayed with a CyQuant assay kit and a Matrigel-coated culture insert system, respectively. Levels of matrix metalloproteinase (MMP)-2 and collagen mRNAs were examined using quantitative RT-PCR. Immunohistochemistry revealed the expression of fascin along sinusoids and overlapping with vimentin and α-SMA in both non-fibrotic and fibrotic liver tissue, but it was almost absent in periportal myofibroblastic cells and did not colocalize with fibulin-2, a marker of portal myofibroblasts. In addition, fascin immunoreactivity was almost undetectable in septa of fibrotic human liver tissue. The expression of fascin in LX-2 cells was confirmed using western blot. Two different specific siRNAs against fascin significantly reduced the number of viable LX-2 cells to 65% compared with control cultures and downregulated the mRNAs levels of types I and III collagen and MMP-2 to 62%, 65%, and 70% of control levels, respectively. This condition also reduced the migration activity of LX-2 cells to 46% of control cells and the phosphorylation level of both FAK and Akt. Fascin may be an excellent novel marker of human HSCs that distinguishes HSCs from periportal myofibroblasts. Fascin may regulate functions of human HSCs through the FAK-phosphoinositide 3-kinase-Akt pathway.
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Rios ECS, Moretti AIS, de Souza HP, Velasco IT, Soriano FG. Hypertonic saline reduces metalloproteinase expression in liver during pancreatitis. Clin Exp Pharmacol Physiol 2009; 37:35-9. [PMID: 19515067 DOI: 10.1111/j.1440-1681.2009.05220.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
1. We recently demonstrated that hypertonic saline reduces inflammation and mortality in acute pancreatitis. The present study investigated the effects of hypertonic saline in metalloproteinase (MMP) regulation and pancreatitis-associated hepatic injury. 2. Wistar rats were divided into four groups: (i) control, not subjected to insult or treatment; (ii) no treatment (NT), induction of pancreatitis (retrograde infusion of 2.5% sodium taurocholate (1.0 mL/kg)), but no further treatment; (iii) normal saline (NS), induction of pancreatitis and treatment with normal saline (0.9% NaCl, 34 mL/kg, i.v. bolus, 1 h after the induction of pancreatitis); and (iv) hypertonic saline (HS), induction of pancreatitis and treatment with hypertonic saline (7.5% NaCl, 4 mL/kg administered over a period of 5 min, 1 h after the induction of pancreatitis). In all four groups, 4, 12 and 24 h after the induction of pancreatitis, liver tissue samples were assayed to determine levels of MMP-2, MMP-9, 47 kDa heat shock protein (HSP47) and collagen (Type I and III). 3. Compared with the control group, MMP-9 expression and activity was increased twofold in the NS and NT groups 4 and 12 h after the induction of pancreatitis, but remained at basal levels in the HS group. In contrast, MMP-2 expression was increased twofold 12 h after the induction of pancreatitis only in the NS group, whereas the expression of HSP47 was increased 4 h after the induction of pancreatitis in the NS and NT groups. Greater extracellular matrix remodelling occurred in the NS and NT groups compared with the HS group, probably as a result of the hepatic wound-healing response to repeated injury. However, the collagen content in hepatic tissue remained at basal levels in the HS group. 4. In conclusion, the results of the present study indicate that hypertonic saline is hepatoprotective and reduces hepatic remodelling, maintaining the integrity of the hepatic extracellular matrix during pancreatitis. Hypertonic saline-mediated regulation of MMP expression may have clinical relevance in pancreatitis-associated liver injury.
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Affiliation(s)
- Ester C S Rios
- Laboratory of Medical Investigation, Department of Emergency Medicine, University of São Paulo School of Medicine, São Paulo, Brazil.
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Xia Z, Abe K, Furusu A, Miyazaki M, Obata Y, Tabata Y, Koji T, Kohno S. Suppression of renal tubulointerstitial fibrosis by small interfering RNA targeting heat shock protein 47. Am J Nephrol 2007; 28:34-46. [PMID: 17890856 DOI: 10.1159/000108759] [Citation(s) in RCA: 56] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2007] [Accepted: 08/09/2007] [Indexed: 11/19/2022]
Abstract
BACKGROUND/AIM Unilateral ureteral obstruction (UUO) is a well-established model for tubulointerstitial fibrosis. During the progression of tubulointerstitial fibrosis, upregulation of collagen synthesis and subsequent accumulation of collagen were observed in the tubulointerstitial area. Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone and plays an essential role in regulating collagen synthesis. We designed small interfering RNA (siRNA) sequences for HSP47 mRNA to examine whether HSP47 is involved in the progression of renal tubulointerstitial fibrosis in a mouse UUO model. METHODS The HSP47 siRNA was injected once via the ureter at the time of UUO preparation. We also applied a new gene delivery system for siRNA using cationized gelatin microspheres. The kidneys were harvested 7 and 14 days after UUO. The HSP47 and type I, III, and IV collagen expression levels were analyzed by immunohistochemistry and Western blotting. RESULTS Seven days after UUO, the expression levels of HSP47 and type I, III, and IV collagens were markedly upregulated in obstructed kidneys or green fluorescent protein siRNA treated obstructed kidneys. HSP47 siRNA injection significantly reduced the protein expression levels and significantly diminished the accompanying interstitial fibrosis. Moreover, cationized gelatin microspheres as a delivery system enhanced and lengthened the antifibrotic effect of HSP47 siRNA. CONCLUSIONS Our results indicate that HSP47 is a candidate target for the prevention of tubulointerstitial fibrosis and that selective blockade of the HSP47 expression by using siRNA could be a potentially useful therapeutic approach for patients with renal disease.
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Affiliation(s)
- Zhiyin Xia
- Second Department of Internal Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
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Song JY, Li L, Ahn JB, Park JG, Jo JS, Park DH, Jang HK, Jang JJ, Lee MJ. Acute liver toxicity by carbon tetrachloride in HSP70 knock out mice. ACTA ACUST UNITED AC 2007; 59:29-34. [PMID: 17582750 DOI: 10.1016/j.etp.2007.02.005] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2006] [Accepted: 02/19/2007] [Indexed: 02/06/2023]
Abstract
The effects of carbon tetrachloride (CCl(4)) treatment on acute liver damage in knock out (heat shock proteins -- HSP70-/-) mice and wild-type (C57BL/6) mice were examined. Acute liver injury was induced by a single intraperitoneal injection of 0.3 ML/kg CCl(4) in olive oil. Mice were sacrificed at 12, 24, 48 and 72 h after treatment. To assess hepatotoxicity, alanine transaminase, neutrophil infiltration and degree of necrosis were measured. Western blot analysis was employed for heat shock proteins. The result revealed that HSP70-/- mice showed higher alanine transaminase levels and a more severe degree of neutrophilic infiltration and necrosis than those of wild-type mice. Furthermore, HSP70-/- mice recovered more slowly from CCl(4) treatment. In HSP70-/- mice, HSP47 was overexpressed. Therefore, HSP70-/- mice could be an adequate model of acute liver toxicity study.
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Affiliation(s)
- Ji-Ye Song
- Department of Veterinary Lab Animal Medicine & Science, School of Veterinary Medicine, Kangwon National University, Chuncheon, Kangwon-Do, Korea
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Van de Bovenkamp M, Groothuis GMM, Meijer DKF, Olinga P. Liver fibrosis in vitro: Cell culture models and precision-cut liver slices. Toxicol In Vitro 2007; 21:545-57. [PMID: 17289342 DOI: 10.1016/j.tiv.2006.12.009] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2006] [Revised: 12/07/2006] [Accepted: 12/18/2006] [Indexed: 01/27/2023]
Abstract
Chronic liver injury of various etiologies can cause liver fibrosis, which is characterized by the progressive accumulation of connective tissue in the liver. As no effective treatment for liver fibrosis is available yet, extensive research is ongoing to further study the mechanisms underlying the development of disease- or toxicity-induced liver fibrosis and to identify potential pro- or anti-fibrotic properties of compounds. This review gives an overview of the in vitro methods that are currently available for this purpose. The first focus is on cell culture models, since the majority of in vitro research uses these systems. Both primary cells and cell lines as well as the use of different culture matrices and co-culture models are discussed. Second, the use of precision-cut liver slices, which recently came into attention as in vitro model for the study of fibrosis, is discussed. The overview clearly shows that continuous optimization and adaptation have extended the potential of in vitro models for liver fibrosis during the past years. By combining the use of the different cell and tissue culture models, the mechanisms underlying multicellular fibrosis development can be studied in vitro and potential pro- or anti-fibrotic properties of compounds can be identified both on single liver cell types and in human liver tissue.
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Affiliation(s)
- M Van de Bovenkamp
- Department of Pharmacokinetics and Drug Delivery, University Center for Pharmacy, Groningen, The Netherlands
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22
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van de Bovenkamp M, Groothuis GMM, Meijer DKF, Slooff MJH, Olinga P. Human liver slices as an in vitro model to study toxicity-induced hepatic stellate cell activation in a multicellular milieu. Chem Biol Interact 2006; 162:62-69. [PMID: 16815347 DOI: 10.1016/j.cbi.2006.05.006] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2006] [Accepted: 05/11/2006] [Indexed: 11/20/2022]
Abstract
INTRODUCTION Hepatic stellate cell (HSC) activation is a key event in wound healing as well as in fibrosis development in the liver. Previously we developed a technique to induce HSC activation in slices from rat liver. Although this model provides a physiologic, multicellular milieu that is not present in current in vitro models it might still be of limited predictive value for the human situation due to species-differences. Therefore, we now aimed to evaluate the applicability of human liver slices for the study of HSC activation. METHOD Liver slices (8 mm diameter, 250 microm thickness) were generated from human liver tissue and incubated for 3 or 16 h with 0-15 microl of carbon tetrachloride (CCl4) after which ATP-content and expression levels of HSC (activation) markers was determined. RESULTS Human liver slices remained viable during incubation as shown by constant ATP levels. Incubation with CCl(4) caused a dose-dependent decrease in viability and an increase in mRNA expression of the early HSC activation markers HSP47 and alphaB-crystallin, but not the late markers for HSC activation, alphaSMA and pro-collagen 1a1. Synaptophysin mRNA expression remained constant during incubation with or without CCl4, indicating a constant number of HSC in the liver slices. CONCLUSION We developed a technique to induce early toxicity-induced HSC activation in human liver slices. This in vitro model provides a multicellular, physiologic milieu to study mechanisms underlying toxicity-induced HSC activation in human liver tissue.
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Affiliation(s)
- M van de Bovenkamp
- Department of Pharmacokinetics and Drug Delivery, University Center for Pharmacy, Groningen, The Netherlands.
| | - G M M Groothuis
- Department of Pharmacokinetics and Drug Delivery, University Center for Pharmacy, Groningen, The Netherlands
| | - D K F Meijer
- Department of Pharmacokinetics and Drug Delivery, University Center for Pharmacy, Groningen, The Netherlands
| | - M J H Slooff
- Department of Surgery, Division of Hepatobiliary Surgery and Liver Transplantation, University Hospital Groningen, The Netherlands
| | - P Olinga
- Department of Pharmacokinetics and Drug Delivery, University Center for Pharmacy, Groningen, The Netherlands
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Matsumoto H, Tamura S, Kamada Y, Kiso S, Fukushima J, Wada A, Maeda N, Kihara S, Funahashi T, Matsuzawa Y, Shimomura I, Hayashi N. Adiponectin deficiency exacerbates lipopolysaccharide/D-galactosamine-induced liver injury in mice. World J Gastroenterol 2006; 12:3352-8. [PMID: 16733851 PMCID: PMC4087865 DOI: 10.3748/wjg.v12.i21.3352] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To examine the effects of adiponectin on the functions of Kupffer cells, key modulators of lipopolysaccharide (LPS) -induced liver injury.
METHODS: D-galactosamine (GalN) and LPS were injected intraperitoneally into adiponectin-/- mice and wild type mice. Kupffer cells, isolated from Sprague-Dawley rats, were preincubated with or without adiponectin, and then treated with LPS.
RESULTS: In knockout mice, GalN/LPS injection significantly lowered the survival rate, significantly raised the plasma levels of alanine transaminase and tumor necrosis factor-α (TNF-α), and significantly reduced IL-10 levels compared with wild type mice. TNF-α gene expression in the liver was which higher and those of IL-10 were lower in knockout mice than in wild type mice. In cultured adiponectin-pre-treated Kupffer cells, LPS significantly lowered TNF-α levels and raised IL-10 levels in the culture media and their respective gene expression levels, compared with Kupffer cells without adiponectin-pre-treatment.
CONCLUSION: Adiponectin supresses TNF-α production and induces IL-10 production by Kupffer cells in response to LPS stimulation, and a lack of adiponectin enhances LPS-induced liver injury.
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Affiliation(s)
- Hitoshi Matsumoto
- Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Suita, Japan
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Ohba K, Miyata Y, Koga S, Nishikido M, Kanetake H, Nazneen A, Razzaque MS, Taguchi T. Interstitial expression of heat-shock protein 47 correlates with capillary deposition of complement split product C4d in chronic allograft nephropathy. Clin Transplant 2005; 19:810-6. [PMID: 16313330 DOI: 10.1111/j.1399-0012.2005.00426.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
BACKGROUND Chronic allograft nephropathy (CAN), associated with late-allograft dysfunction is caused by alloantigen-dependent and -independent mechanisms, and eventually leads to interstitial fibrosis (ci). Activation of complement cascade is considered to be a poor prognostic marker of graft survival. This study was designed to examine the relationship between the expression of C4d and heat-shock protein 47 (HSP47, a collagen-specific chaperone) in the development of interstitial fibroproliferative lesions in CAN. METHODS Sixty-three renal allograft biopsy specimens, obtained from 48 patients, were examined for the expression of C4d, HSP47, CD68 and alpha-smooth muscle actin (alpha-SMA) by immunohistochemistry. Double-staining was performed to determine the colocalization of C4d and HSP47. The relationship of between the expression of C4d, HSP47, CD68 and alpha-SMA and the clinical and histopathological parameters were statistically analysed. RESULTS No expression of C4d was noted in the tubulointerstitium including peritubular capillary (PTC) of the control kidney. C4d was expressed in PTC in one-third of allograft renal tissues with morphological evidences of CAN. The interstitial cells around the fibrotic areas of the PTC of CAN were positive for the expression of HSP47. The deposition of C4d in PTC correlated with interstitial expression of HSP47 around the PTC. Most HSP47 expressing cells were phenotypically altered myofibroblasts, as determined by the dual staining of alpha-SMA. CONCLUSIONS The increased expression of HSP47 positively correlated with the expression of C4d in PTC, and might contribute to the progression of interstitial ci in CAN.
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Affiliation(s)
- Kojiro Ohba
- Department of Urology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
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25
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Brown KE, Broadhurst KA, Mathahs MM, Brunt EM, Schmidt WN. Expression of HSP47, a collagen-specific chaperone, in normal and diseased human liver. J Transl Med 2005; 85:789-97. [PMID: 15806139 DOI: 10.1038/labinvest.3700271] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
HSP47 is a collagen-specific chaperone that is required for normal collagen synthesis. In animal models of liver injury, hepatic stellate cells (HSC) have been identified as a source of HSP47. Because expression of HSP47 has not been investigated in human liver, the aim of these studies was to characterize expression of HSP47 in human liver and to investigate its regulation in human HSC in vitro. Immunohistochemistry demonstrated staining for HSP47 along the sinusoids of normal and cirrhotic human livers and in fibrous septa. Dual fluorescence confocal microscopy showed colocalization of HSP47 with synaptophysin, a marker for HSC. Levels of immunoreactive HSP47 and its transcript tended to be higher in cirrhotic livers than in normal livers. The abundance of HSP47 protein was unchanged by treatment of cultured human HSC with TGF-beta1, angiotensin II, hypoxia and a number of other treatments intended to increase collagen synthesis. A modest reduction in HSP47 was achieved by transfection with antisense oligonucleotides and was associated with a significant decrease in procollagen synthesis. These observations suggest that HSP47 is constitutively expressed in human HSC and that HSP47 may be a target for antifibrotic therapy.
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Affiliation(s)
- Kyle E Brown
- Iowa City Veterans Administration Medical Center, Iowa City, IA 52242, USA.
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26
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Fujimoto M, Hamaguchi Y, Yazawa N, Komura K, Takehara K, Sato S. Autoantibodies to a collagen-specific molecular chaperone, heat-shock protein 47, in systemic sclerosis. Clin Exp Immunol 2005; 138:534-9. [PMID: 15544633 PMCID: PMC1809250 DOI: 10.1111/j.1365-2249.2004.02633.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
Heat-shock proteins are highly conserved and immunogenic proteins, which may be involved in the initiation and perpetuation of autoimmune diseases. Heat-shock protein 47 (HSP47) is expressed by collagen-secreting cells such as fibroblasts and serves as a collagen-specific molecular chaperone that plays a crucial role in collagen metabolism. Abnormal collagen accumulation and autoimmunity are characteristics of systemic sclerosis (SSc). We determined the presence and prevalence of autoantibodies to HSP47 in patients with SSc and also in tight-skin (TSK/+) mice, an animal model for SSc. Anti-HSP47 autoantibodies were present in SSc patients with a frequency of 26%, while patients with systemic lupus erythematosus, those with dermatomyositis, those with keloid and healthy subjects did not have anti-HSP47 antibodies. IgG1 and IgG2 were the major Ig isotypes of the autoantibodies. Patients positive for anti-HSP47 had a significantly shorter duration of disease than those who were negative. Anti-HSP47 autoantibodies were also positive in 79% of TSK/+ mice. Thus, autoantibodies to HSP47 were present in the sera from SSc patients as well as those from TSK mice, and may be associated with the pathogenesis of SSc.
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Affiliation(s)
- M Fujimoto
- Department of Regenerative Medicine, Research Institute, International Medical Center of Japan, Tokyo, Japan.
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27
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van de Bovenkamp M, Groothuis GMM, Draaisma AL, Merema MT, Bezuijen JI, van Gils MJ, Meijer DKF, Friedman SL, Olinga P. Precision-cut liver slices as a new model to study toxicity-induced hepatic stellate cell activation in a physiologic milieu. Toxicol Sci 2005; 85:632-8. [PMID: 15728706 DOI: 10.1093/toxsci/kfi127] [Citation(s) in RCA: 71] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Hepatic stellate cell (HSC) activation is a key event in the natural process of wound healing as well as in fibrosis development in liver. Current in vitro models for HSC activation contribute significantly to the understanding of HSC biology and fibrogenesis but still fall far short of recapitulating in vivo intercellular functional and anatomic relationships. In addition, when cultured on uncoated plastic, HSC spontaneously activate, which makes HSC activation difficult to regulate or analyze. We have examined whether the use of precision-cut liver slices might overcome these limitations. Liver slices (8 mm diameter, 250 microm thickness) were generated from normal rat liver and incubated for 3 or 16 h with increasing doses of carbon tetrachloride (CCl4). Rat liver slices remained viable during incubation, as shown by minimal enzyme leakage. Expression of markers for HSC activation and the onset of fibrogenesis in the liver slices was studied using real-time PCR and Western blotting. In unstimulated liver slices, mRNA and protein levels of desmin, heat shock protein 47, and alpha B-crystallin remained constant, indicating quiescence of HSC, whereas Krüppel-like factor 6 expression was increased. In contrast, incubation with CCl4 led to a time- and dose-dependent increase in mRNA expression of all markers and an increased alpha B-crystallin protein expression. In conclusion, we have developed a technique to induce activation of quiescent HSC in rat liver slices. This model permits the study of toxicity-induced HSC activation within a physiological milieu, not only in animal but ultimately also in human tissue, and could contribute to the reduction of animal experiments.
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Affiliation(s)
- Marja van de Bovenkamp
- Department of Pharmacokinetics and Drug Delivery, University Center for Pharmacy, Groningen, The Netherlands.
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28
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Park DH, Lee MS, Kim HJ, Kim HS, Lee YL, Kwon MS, Jang JJ, Lee MJ. Chronic Hepatotoxicity of Carbon Tetrachloride in HSP-70 Knock Out Mice. Exp Anim 2004; 53:27-30. [PMID: 14993737 DOI: 10.1538/expanim.53.27] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022] Open
Abstract
The chronic hepatotoxic effects of carbon tetrachloride (CCl(4)) in heat-shock protein (HSP) 70 knock out (HSP70-/-) mice were examined. After repeated intraperitoneal injections of CCl(4) for six weeks, the level of ALT and weight ratio of the liver to body were lower in HSP70-/- mice than in the control (WT) mice. The levels of HSP25 and HSP47 were lowered in HSP70-/- mice as compared with WT mice. The grades of hepatic necrosis and neutrophil infiltration were not significantly different between HSP70-/- and WT mice. The collagen content was not affected significantly by CCl(4) treatment.
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Affiliation(s)
- Dae-Hun Park
- Laboratory of Immunopharmacology, Department of Veterinary Medicine, Kangwon National University, Korea
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29
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Nakatani K, Okuyama H, Shimahara Y, Saeki S, Kim DH, Nakajima Y, Seki S, Kawada N, Yoshizato K. Cytoglobin/STAP, its unique localization in splanchnic fibroblast-like cells and function in organ fibrogenesis. J Transl Med 2004; 84:91-101. [PMID: 14647402 DOI: 10.1038/labinvest.3700013] [Citation(s) in RCA: 100] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
Cytoglobin/stellate cell activation-associated protein (Cygb/STAP) consists of a new class of hexacoordinate globin superfamily, which was recently discovered by a proteome analysis on the rat hepatic stellate cells. Unlike haemoglobin, myoglobin, and neuroglobin, Cygb/STAP is ubiquitously expressed in several organs, although its detailed localization has not been clarified. Immunohistochemistry and immunoelectron microscopy revealed that Cygb/STAP is uniquely localized in fibroblast-like cells in splanchnic organs, namely the vitamin A-storing cell lineage, but neither in epithelial cells, endothelial cells, muscle cells, blood cells, macrophages, nor dermal fibroblasts. The expression of Cygb/STAP was upregulated in fibrotic lesions of the pancreas and kidney in which activated fibroblast-like cells or myofibroblasts are known to increase in number. In cultured hepatic stellate cells, Cygb/STAP expression was augmented by the stimulation with sera, platelet-derived growth factor-BB, and transforming growth factor-beta 1. Overexpression of Cygb/STAP in NIH 3T3 cells induced the cells to lessen migratory activities and increase the expression of collagen alpha1(I) mRNA. These results indicate that Cygb/STAP is a tissue globin uniquely localized in splanchnic fibroblastic cell lineage and may play a role in fibrotic organ disorder.
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Affiliation(s)
- Kazuki Nakatani
- Department of Anatomy, Graduate School of Medicine, Osaka City University, Japan
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30
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Kamada Y, Tamura S, Kiso S, Matsumoto H, Saji Y, Yoshida Y, Fukui K, Maeda N, Nishizawa H, Nagaretani H, Okamoto Y, Kihara S, Miyagawa JI, Shinomura Y, Funahashi T, Matsuzawa Y. Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin. Gastroenterology 2003; 125:1796-807. [PMID: 14724832 DOI: 10.1053/j.gastro.2003.08.029] [Citation(s) in RCA: 354] [Impact Index Per Article: 16.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
BACKGROUND & AIMS Obesity is one of the risk factors for liver fibrosis, in which plasma adiponectin, an adipocytokine, levels are decreased. Hepatic stellate cells play central roles in liver fibrosis. When they are activated, they undergo transformation to myofibroblast-like cells. Adiponectin suppresses the proliferation and migration of vascular smooth muscle cells, whose characteristics are similar to those of hepatic stellate cells. Adiponectin could have biological significances in liver fibrosis. METHODS The role of adiponectin on liver fibrosis induced by the administration of carbon tetrachloride twice a week for 12 weeks was tested by using adiponectin-knockout mice and an adenovirus-mediated adiponectin-expression system. We also investigated the effect of adiponectin in activated hepatic stellate cells. RESULTS When mice were administered carbon tetrachloride (300 microL/kg body weight) twice a week for 12 weeks, knockout mice showed extensive liver fibrosis with an enhanced expression of transforming growth factor-beta 1 and connective tissue growth factor compared with wild-type mice (P < 0.05). Injection of adenovirus producing adiponectin (AdADN) before carbon tetrachloride (1000 microL/kg body weight) treatment prevented liver fibrosis in wild-type mice (P < 0.001). Injection of AdADN at 6 weeks attenuated liver fibrosis even though carbon tetrachloride was given for an additional 6 weeks (total of 12 weeks). In cultured hepatic stellate cells, adiponectin suppressed platelet-derived growth factor-induced proliferation and migration and attenuated the effect of transforming growth factor-beta 1 on the gene expression of transforming growth factor-beta 1 and connective tissue growth factor and on nuclear translocation of Smad2. CONCLUSIONS The findings indicate that adiponectin attenuates liver fibrosis and could be a novel approach in its prevention.
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Affiliation(s)
- Yoshihiro Kamada
- Department of Internal Medicine and Molecular Science, Osaka University, Graduate School of Medicine, Suita, Japan
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31
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Wang JF, Olson ME, Winkfein RJ, Kulyk WM, Wright JB, Hart DA. Molecular and cell biology of porcine HSP47 during wound healing: complete cDNA sequence and regulation of gene expression. Wound Repair Regen 2002; 10:230-40. [PMID: 12191005 DOI: 10.1046/j.1524-475x.2002.10406.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Heat shock protein (HSP) 47 is a major stress-inducible protein that is localized to the endoplasmic reticulum of avian and mammalian cells and is thought to act as a molecular chaperone specific for the processing of procollagen. However, limited information is available regarding the regulation of HSP47 during wound healing. Using a polymerase chain reaction strategy, screening of a cDNA library, and RACE-polymerase chain reaction approaches, the sequence of a full-length porcine HSP47 cDNA has been identified. The cDNA contained 2096 bp that encodes for an 18 amino acid signal peptide and a mature protein coding region consisting of 401 amino acid residues. It also included 108 bp of the 5' noncoding region and a 731-bp 3' noncoding region. The deduced amino acid is 83% identical to chicken, 87% identical to mouse, 88% identical to rat, and 91% identical to human HSP47. It also shares between 26% and 30% identity with different members of the serine protease inhibitor superfamily. The protein contains a RDEL endoplasmic reticulum retention signal, and two potential glycosylation sites. All of these features are characteristic of HSP47 in higher vertebrates. Heat shock treatment of porcine fibroblasts led to up-regulation of HSP47 at both the transcriptional and translational levels. HSP47 protein levels were also up-regulated during skin wound healing in a pig model. Moreover, a higher molecular weight complex at approximately 140 Kda containing HSP47 was detected at the stage of healing that was coincident with the maximal transcriptional expression of HSP47 during wound healing in this animal model. Further investigation of how HSP47 is regulated during normal and abnormal skin wound healing may lead to new therapeutic approaches to improve the healing process.
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Affiliation(s)
- Jian Fei Wang
- McCaig Center for Joint Injury and Arthritis Research, University of Calgary, Calgary, Alberta, Canada
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Liu D, Razzaque MS, Nazneen A, Naito T, Taguchi T. Role of heat shock protein 47 on tubulointerstitium in experimental radiation nephropathy. Pathol Int 2002; 52:340-7. [PMID: 12100516 DOI: 10.1046/j.1440-1827.2002.01362.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
The molecular mechanisms of fibrosis in radiation nephropathy have received scant attention. Heat shock protein 47 (HSP47), a collagen-binding stress protein, helps in the intracellular processing of procollagen molecules during collagen synthesis. We investigated the role of HSP47 in the progression of radiation nephropathy using experimental radiation nephropathy. Experimental rat groups were as follows: (i) group I, sham operated (n = 12); (ii) group II, single doses of irradiation, either 7, 15 or 25 Gy to left kidney (n = 60); and (iii) group III, a similar irradiation procedure as group II after right nephrectomy (n = 60). The rats were followed up until 9 months after renal exposure to radiation. Renal dysfunction (as determined by serum creatinine and blood urea nitrogen) and hypertension were noted in group III rats, along with inflammatory cell infiltration and interstitial fibrosis (as determined by increased deposition of collagens). Compared to control rat kidneys, an increased expression of HSP47 was noted in kidneys obtained from irradiated rats. By double immunostaining, HSP47-expressing cells were identified as alpha-smooth muscle actin-positive myofibroblasts and vimentin-positive tubular epithelial cells. Increased expression of HSP47 was closely associated with increased deposition of collagens in the widened interstitium of irradiated rats. Overexpression of HSP47 by phenotypically altered tubulointerstitial cells might play a role in excessive assembly/synthesis of collagens and could contribute to tubulointerstitial fibrosis in radiation nephropathy.
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Affiliation(s)
- Diange Liu
- Second Department of Pathology, Nagasaki University School of Medicine, Sakamoto, Nagasaki, Japan
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33
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Razzaque MS, Ahmed AR. Collagens, collagen-binding heat shock protein 47 and transforming growth factor-beta 1 are induced in cicatricial pemphigoid: possible role(s) in dermal fibrosis. Cytokine 2002; 17:311-6. [PMID: 12061838 DOI: 10.1006/cyto.2002.1020] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Cicatricial pemphigoid (CP) is an autoimmune mucocutaneous blistering disease associated with scarring. Heat shock protein 47 (HSP47) is thought to play an important role in fibrogenesis, but its role in skin lesions of cicatricial pemphigoid is not yet known. In the present study, we examined the role of HSP47 in dermal fibrosis in cutaneous lesions of a CP patient. Skin biopsies from a patient with CP, and from normal subjects were studied for the expression of HSP47, and interstitial collagens (type I and type III collagens) by immunohistochemistry. Dermal fibroblasts isolated from skin of normal individuals and from fibrotic skin of a CP patient were used to study the expression of HSP47, transforming growth factor beta 1 (TGF-beta 1), type I and type III collagens. Compared to the control skin sections, an increased expression of HSP47 was associated with an increased deposition of interstitial collagens in the fibrotic skin section of the CP patient. Similarly, in contrast to control dermal fibroblasts, the fibroblasts isolated and cultured from fibrotic skin of the CP patient, and grown in vitro, exhibited increased expression of HSP47, type I and type III collagens. Furthermore, compared to the normal control fibroblasts, an increased expression of TGF-beta 1 was detected in the dermal fibroblasts isolated from fibrotic skin of the CP patient. When dermal fibroblasts were treated with various concentrations of TGF-beta 1 (6.25, 12.5, 25, 50 and 100 ng/ml for 24 h), it induced the expression of both type I collagen and HSP47, as determined by quantitative real-time PCR. In conclusion, the expression of TGF-beta 1, HSP47, type I collagen and type III collagen was up-regulated in the fibrotic skin of CP patient, and a complex interaction of these molecules may initiate and propagate the fibrotic cascade in the skin of CP patients.
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Affiliation(s)
- M S Razzaque
- Department of Dermatology, Harvard Medical School, Boston, MA, USA
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Hattori T, Kubota S, Yutani Y, Fujisawa T, Nakanishi T, Takahashi K, Takigawa M. Change in cellular localization of a rheumatoid arthritis-related antigen (RA-A47) with downregulation upon stimulation by inflammatory cytokines in chondrocytes. J Cell Physiol 2001; 186:268-281. [PMID: 11169453 DOI: 10.1002/1097-4652(200002)186:2<168::aid-jcp1022>3.0.co;2-m] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
We previously isolated a rheumatoid arthritis-related antigen (RA-A47) protein that had reactivity with RA sera from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8. Sequencing analysis of ra-a47 cDNA revealed RA-A47 as a product of the colligin-2 gene, which is also known as the human heat shock protein (HSP) 47 gene. Expression of hsp47 has been shown to be cooperatively altered with that of collagen genes upon stimulation. In this study, it was confirmed that the mRNA expression of ra-a47 and COL2A1, a type II collagen gene, was upregulated on stimulation with transforming growth factor (TGF) beta in chondrocytes. However, in contrast, inflammatory cytokines such as tumor necrosis factor (TNF) alpha, interferon (IFN) beta, and interleukin (IL)-6 downregulated the expression of ra-a47 mRNA, whereas the expression of COL2A1 mRNA was not repressed, or even upregulated, in HCS-2/8 cells. Of note, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP)-9 mRNAs were strongly stimulated by TNFalpha. We also found that cell-surface type II collagen disappeared upon such a stimulation, suggesting that decrement of RA-A47 may inhibit the secretion of type II collagen and lead to its accumulation inside the cells. RA-A47 was detected in the cultured medium of TNFalpha-treated HCS-2/8 cells and of IL-1-treated rabbit chondrocytes by Western blot analysis. Under the same conditions, RA-A47 was detected on the cell surface by immunofluorescence staining. These findings demonstrate that the RA-A47 chaperone protein is specifically downregulated, causing the intracellular accumulation of unsecretable type II collagen, while the extracellular matrix (ECM) is degraded by MMPs and iNOS through the stimulation of chondrocytes by TNFalpha. The altered localization of RA-A47 to the surface or outside of cells may represent the mechanism for the recognition of RA-A47 as an autoantigen during rheumatoid arthritis.
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Affiliation(s)
- T Hattori
- Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, Okayama, Japan
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35
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Yoneyama H, Matsuno K, Zhang Y, Murai M, Itakura M, Ishikawa S, Hasegawa G, Naito M, Asakura H, Matsushima K. Regulation by chemokines of circulating dendritic cell precursors, and the formation of portal tract-associated lymphoid tissue, in a granulomatous liver disease. J Exp Med 2001; 193:35-49. [PMID: 11136819 PMCID: PMC2195882 DOI: 10.1084/jem.193.1.35] [Citation(s) in RCA: 161] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
We have studied the recruitment and roles of distinct dendritic cell (DC) precursors from the circulation into Propionibacterium acnes-induced granulomas in mouse liver. During infection, F4/80(-)B220(-)CD11c(+) DC precursors appeared in the circulation, migrated into the perisinusoidal space, and matured within newly formed granulomas. Recruited DCs later migrated to the portal area to interact with T cells in what we term "portal tract-associated lymphoid tissue" (PALT). Macrophage inflammatory protein 1alpha attracted blood DC precursors to the sinusoidal granuloma, whereas secondary lymphoid organ chemokine (SLC) attracted mature DCs to the newly identified PALT. Anti-SLC antibody diminished PALT expansion while exacerbating granuloma formation. Therefore, circulating DC precursors can migrate into a solid organ like liver, and participate in the granulomatous reaction in response to specific chemokines.
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Affiliation(s)
- Hiroyuki Yoneyama
- Department of Molecular Preventive Medicine, School of Medicine and Core Research for Evolutional Science and Technology (CREST), The University of Tokyo, Bunkyoku, Tokyo 113-0033, Japan
- Third Department of Internal Medicine, Niigata University School of Medicine, Niigata-shi, Niigata 951-8122, Japan
| | - Kenjiro Matsuno
- Department of Anatomy II, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan
| | - Yanyun Zhang
- Department of Molecular Preventive Medicine, School of Medicine and Core Research for Evolutional Science and Technology (CREST), The University of Tokyo, Bunkyoku, Tokyo 113-0033, Japan
| | - Masako Murai
- Department of Molecular Preventive Medicine, School of Medicine and Core Research for Evolutional Science and Technology (CREST), The University of Tokyo, Bunkyoku, Tokyo 113-0033, Japan
- Third Department of Internal Medicine, Niigata University School of Medicine, Niigata-shi, Niigata 951-8122, Japan
| | - Meiji Itakura
- Department of Molecular Preventive Medicine, School of Medicine and Core Research for Evolutional Science and Technology (CREST), The University of Tokyo, Bunkyoku, Tokyo 113-0033, Japan
| | - Sho Ishikawa
- Department of Molecular Preventive Medicine, School of Medicine and Core Research for Evolutional Science and Technology (CREST), The University of Tokyo, Bunkyoku, Tokyo 113-0033, Japan
| | - Go Hasegawa
- Second Department of Pathology, Niigata University School of Medicine, Niigata-shi, Niigata 951-8122, Japan
| | - Makoto Naito
- Second Department of Pathology, Niigata University School of Medicine, Niigata-shi, Niigata 951-8122, Japan
| | - Hitoshi Asakura
- Third Department of Internal Medicine, Niigata University School of Medicine, Niigata-shi, Niigata 951-8122, Japan
| | - Kouji Matsushima
- Department of Molecular Preventive Medicine, School of Medicine and Core Research for Evolutional Science and Technology (CREST), The University of Tokyo, Bunkyoku, Tokyo 113-0033, Japan
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Razzaque MS, Taguchi T. Role of glomerular epithelial cell-derived heat shock protein 47 in experimental lipid nephropathy. KIDNEY INTERNATIONAL. SUPPLEMENT 1999; 71:S256-9. [PMID: 10412793 DOI: 10.1046/j.1523-1755.1999.07169.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
BACKGROUND Heat shock protein 47 (hsp47) is a collagen-specific stress protein and is shown to be involved in the synthesis/assembly of various collagens as a molecular chaperone. This study was undertaken to investigate the possible role of hsp47 in dietary-induced hypercholesterolemic rat kidneys, which showed glomerular hypercellularity with expansion of mesangial matrix. METHODS Dietary-induced hypercholesterolemia was induced in male Wistar rats by giving 2% cholesterol diet for four months. Immunohistochemistry was used for localization of protein products for collagens (types I, III, and IV). alpha-smooth muscle actin, vimentin, desmin, and ED-1, a macrophage/monocyte marker, and hsp47 in control and hypercholesterolemic rat kidneys. RESULTS Compared with the control, increased accumulation of collagens was accompanied with increased expression of hsp47 in hypercholesterolemic rat kidneys, with predominant expression in the glomeruli. By double immunostaining, desmin-positive glomerular epithelial cells were found to be the main source of hsp47 in hypercholesterolemic rat kidneys. CONCLUSION From these results, it is concluded that induced expression of hsp47 by phenotypically altered glomerular epithelial cells might play a role in the excessive assembly/synthesis of collagens and could thereby contribute to the glomerulosclerosis found in dietary-induced hypercholesterolemic rat kidneys.
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Affiliation(s)
- M S Razzaque
- Second Department of Pathology, Nagasaki University School of Medicine, Japan
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Razzaque MS, Shimokawa I, Nazneen A, Liu D, Naito T, Higami Y, Taguchi T. Life-long dietary restriction modulates the expression of collagens and collagen-binding heat shock protein 47 in aged Fischer 344 rat kidney. THE HISTOCHEMICAL JOURNAL 1999; 31:123-32. [PMID: 10416684 DOI: 10.1023/a:1003578928487] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
It has been shown that the expression of HSP47 and collagens is substantially increased in the sclerotic/fibrotic process in various organs, including kidney. However, the factors regulating the increased expression of HSP47 are not yet clear. In this study, we examined the effect of dietary restriction for the expression of collagens and collagen-binding HSP47 in the kidneys of 6- and 24-month-old male Fischer 344 (F 344) rats fed ad libitum or 30% diet-restricted. No significant histological alteration was found in the kidneys of 6-month-old fed or diet-restricted rats. Kidneys obtained from 24-month-old freely fed rats showed glomerulosclerosis with marked tubulointerstitial damage including interstitial fibrosis, while in the kidneys of 24-month-old diet-restricted rats, renal damage was remarkably less than those noted in 24-month-old freely fed rat kidneys. Immunohistochemical analysis showed an increased accumulation of type I, type III and type IV collagens in areas of glomerulosclerosis and interstitial fibrosis in old rat kidneys. Dietary restriction significantly reduces renal accumulation of collagens in old age. Aging enhanced expression of HSP47 in 24-month-old freely fed rat kidneys whereas dietary restriction suppressed its expression in 24-month-old diet-restricted rat kidneys. Also, phenotypic alterations of mesangial cells and interstitial cells (immunopositive for alpha-smooth muscle actin), glomerular epithelial cells (immunopositive for desmin) and tubular epithelial cells (immunopositive for vimentin) were seen in 24-month-old freely fed rat kidneys and found to express HSP47. Dietary restriction significantly diminished phenotypically altered renal cells in 24-month-old rat kidneys. Our results suggest that increased expression of HSP47 is associated with age-related renal damage and that diet-restricted alteration of its expression is associated with the modulation of age-associated renal sclerosis/fibrosis.
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Affiliation(s)
- M S Razzaque
- Second Department of Pathology, Nagasaki University School of Medicine, Japan
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Moriyama T, Kawada N, Ando A, Yamauchi A, Horio M, Nagata K, Imai E, Hori M. Up-regulation of HSP47 in the mouse kidneys with unilateral ureteral obstruction. Kidney Int 1998; 54:110-9. [PMID: 9648069 DOI: 10.1046/j.1523-1755.1998.00964.x] [Citation(s) in RCA: 72] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
BACKGROUND Unilateral ureteral obstruction (UUO) is a well established experimental model of renal injury leading to interstitial fibrosis. The molecular and cellular mechanism(s) of interstitial fibrosis in UUO are beginning to be elucidated. In the progression of interstitial fibrosis in UUO, up-regulation of collagen synthesis is commonly observed. HSP47 is a collagen-binding stress protein and is thought to be a collagen-specific molecular chaperone, which plays a pivotal role during the biosynthesis and secretion of collagen molecules in the endoplasmic reticulum. The synthesis of HSP47 has been demonstrated to always parallel that of collagen in physiological and pathophysiological conditions. It is well recognized that renin-angiotensin system (RAS) is enhanced in the setting of UUO and that enhanced RAS has been implicated in the pathogenesis of interstitial fibrosis in the obstructed kidneys. METHODS To investigate the role of HSP47 in the progression of interstitial fibrosis in mouse UUO, the expression of HSP47 was examined by Northern blotting, immunohistochemistry and in situ hybridization in the obstructed kidneys. To test the possible involvement of enhanced RAS on the HSP47 expression, we examined the effects of lisinopril, an angiotensin converting enzyme inhibitor, on interstitial fibrosis. HSP47 and type I collagen mRNA expression. RESULTS By Northern blot analysis, HSP47 mRNA was significantly up-regulated at 12 hours (about twice that of sham operated kidneys) after the onset of ureteral obstruction, further increased and stayed at the increased level until seven days (about 8 times that of sham operated kidneys). HSP47 mRNA and protein expression were observed in the periglomerular and peritubular interstitial regions of the obstructed kidneys. Distribution of smooth muscle alpha actin and type I collagen immunoreactivity were similar to the HSP47 distribution pattern, suggesting that HSP47 was up-regulated in the myofibroblasts. Lisinopril ameliorated the expansion of cortical interstitium in the obstructed kidneys at four and seven days after ureteral obstruction. HSP47 mRNA expression was suppressed at four and seven days, whereas type I collagen mRNA was suppressed only at seven days after the onset of ureteral obstruction. CONCLUSIONS These results demonstrate the early and persistent up-regulation of HSP47 during the progression of interstitial fibrosis in mouse UUO kidneys, and further suggest the potential role of HSP47 in the pathogenesis of interstitial fibrosis in the obstructed kidneys. Partial suppression of HSP47 mRNA expression by lisinopril at day 4 and day 7 after ureteral obstruction suggests that there are other immediate trigger(s) that induce the HSP47 mRNA expression. Identification of the molecular mechanism of HSP47 induction during UUO may give an insight into the novel aspects of the molecular pathophysiology of interstitial fibrosis in obstructive nephropathy.
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Affiliation(s)
- T Moriyama
- First Department of Medicine, Osaka University School of Medicine, Faculty of Health and Sport Sciences, Japan.
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