CCN1 expression in hepatocytes contributes to macrophage infiltration in nonalcoholic fatty liver disease in mice

Our objective was to investigate the potential roles of CCN1 in the inflammation and macrophage infiltration of nonalcoholic fatty liver disease (NAFLD). The regulation of hepatic CCN1 expression was investigated in vitro with murine primary hepatocytes treated with free fatty acids or lipopolysaccharide (LPS) and in vivo with high-fat (HF) diet-fed mice or ob/ob mice. CCN1 protein and a liver-specific CCN1 expression plasmid were administered to mice fed a normal diet (ND) or HF diet. Myeloid-derived macrophages and RAW264.7 cells were also treated with CCN1 in vitro to determine the chemotactic effects of CCN1 on macrophages. LPS treatment significantly increased hepatic CCN1 expression in HF diet-fed mice and ob/ob mice. LPS and FFAs induced CCN1 expression in primary murine hepatocytes in vitro through the TLR4/MyD88/AP-1 pathway. CCN1 protein and overexpression of CCN1 in the liver induced more severe hepatic inflammation and macrophage infiltrates in HF mice than in ND mice. CCN1 recruited macrophages through activation of the Mek/Erk signaling pathway in myeloid-derived macrophages and RAW264.7 cells in vitro. Endotoxin and FFA-induced CCN1 expression in hepatocytes is involved in the hepatic proinflammatory response and macrophage infiltration in murine NAFLD. Our objective was to investigate the potential roles of CCN1 in the inflammation and macrophage infiltration of nonalcoholic fatty liver disease (NAFLD). The regulation of hepatic CCN1 expression was investigated in vitro with murine primary hepatocytes treated with free fatty acids or lipopolysaccharide (LPS) and in vivo with high-fat (HF) diet-fed mice or ob/ob mice. CCN1 protein and a liver-specific CCN1 expression plasmid were administered to mice fed a normal diet (ND) or HF diet. Myeloid-derived macrophages and RAW264.7 cells were also treated with CCN1 in vitro to determine the chemotactic effects of CCN1 on macrophages. LPS treatment significantly increased hepatic CCN1 expression in HF diet-fed mice and ob/ob mice. LPS and FFAs induced CCN1 expression in primary murine hepatocytes in vitro through the TLR4/MyD88/AP-1 pathway. CCN1 protein and overexpression of CCN1 in the liver induced more severe hepatic inflammation and macrophage infiltrates in HF mice than in ND mice. CCN1 recruited macrophages through activation of the Mek/Erk signaling pathway in myeloid-derived macrophages and RAW264.7 cells in vitro. Endotoxin and FFA-induced CCN1 expression in hepatocytes is involved in the hepatic proinflammatory response and macrophage infiltration in murine NAFLD. alanine aminotransferase activating protein-1 aspartate aminotransferase cysteine-rich protein 61, Cyr61 high-fat interleukin lipopolysaccharide monocyte chemotactic protein 1 macrophage inflammatory protein 1 α myeloid differentiation factor 88 nonalcoholic fatty liver disease nonalcoholic steatohepatitis normal diet toll-like receptor tumor necrosis factor α terminal deoxynucleotidyl transferase dUTP nick end labeling wild-type Nonalcoholic fatty liver disease (NAFLD) begins with the aberrant accumulation of triglycerides in the liver (simple steatosis), which in some individuals elicits an inflammatory response (nonalcoholic steatohepatitis, NASH) that can progress to cirrhosis and liver cancer (1Cohen J.C. Horton J.D. Hobbs H.H. Human fatty liver disease: old questions and new insights.Science. 2011; 332: 1519-1523Crossref PubMed Scopus (1566) Google Scholar). NASH is characterized by hepatocellular ballooning, lobular inflammation, and fibrosis in histology (2Farrell G.C. Larter C.Z. Nonalcoholic fatty liver disease: from steatosis to cirrhosis.Hepatology. 2006; 43: S99-S112Crossref PubMed Scopus (1961) Google Scholar–4Brunt E.M. Nonalcoholic steatohepatitis.Semin. Liver Dis. 2004; 24: 3-20Crossref PubMed Scopus (456) Google Scholar). A good model to explain the pathogenesis of NASH is the “multiple parallel hits” hypothesis proposed by Tilg and Moschen, which states that various parallel processes, especially gut-derived and adipose tissue-derived factors, may be conducive to the development of liver inflammation in NAFLD (5Tilg H. Moschen A.R. Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis.Hepatology. 2010; 52: 1836-1846Crossref PubMed Scopus (1604) Google Scholar). Members of the CCN (Cyr61/CTGF/NOV) family have emerged as dynamically expressed, extracellular matrix-associated proteins that play critical roles in regulating cell adhesion, migration, proliferation, differentiation, apoptosis, and survival (6Chen C.C. Lau L.F. Functions and mechanisms of action of CCN matricellular proteins.Int. J. Biochem. Cell Biol. 2009; 41: 771-783Crossref PubMed Scopus (449) Google Scholar, 7Perbal B. CCN proteins: multifunctional signalling regulators.Lancet. 2004; 363: 62-64Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar). CCN1 (also known as cysteine-rich protein 61, Cyr61) is normally expressed at a low level in most tissues but is increased as a result of inflammation or tissue repair. It has been shown that CCN1 activates the NF-κB signaling pathway in macrophages, leading to the expression of multiple proinflammatory cytokines and chemokines characteristic of classical activated M1 macrophages (8Bai T. Chen C.C. Lau L.F. Matricellular protein CCN1 activates a proinflammatory genetic program in murine macrophages.J. Immunol. 2010; 184: 3223-3232Crossref PubMed Scopus (122) Google Scholar). The definitive role of CCN1 in liver diseases remains unclear, although it has been shown to be a tumor suppressor in hepatocarcinogenesis (9Feng P. Wang B. Ren E.C. Cyr61/CCN1 is a tumor suppressor in human hepatocellular carcinoma and involved in DNA damage response.Int. J. Biochem. Cell Biol. 2008; 40: 98-109Crossref PubMed Scopus (39) Google Scholar) and is involved in the oxidative DNA damage response in ConA-treated livers (10Chen C.C. Young J.L. Monzon R.I. Chen N. Todorovic V. Lau L.F. Cytotoxicity of TNFalpha is regulated by integrin-mediated matrix signaling.EMBO J. 2007; 26: 1257-1267Crossref PubMed Scopus (127) Google Scholar). In this study, we showed for the first time that induction of CCN1 by FFAs and LPS in hepatocytes resulted in macrophage infiltration and inflammation in the liver in hepatic steatosis. Male C57BL/6 mice (6–8 weeks of age) were purchased from the Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China) and housed under pathogen-free conditions in the animal facility of the Shanghai Jiao Tong University School of Medicine. Mice were fed either a high-fat (HF) diet (SLAC Laboratory Animal Co. Ltd) providing 59% of calories from fat, 25% from carbohydrates, and 16% from protein for eight weeks or an isocaloric normal diet (ND) containing less fat (12% fat, 59% total carbohydrate. and 29% protein). Male C57BL/10ScN toll-like receptor 4 (TLR4) knockout mice, male C57BL/6 myeloid differentiation factor 88 (MyD88) knockout mice, their control littermates, and male C57BL/6 ob/ob mice (6–8 weeks of age) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). All mice were maintained in a temperature- and light-controlled facility with ad libitum access to food and water. Escherichia coli LPS (50 μg/mouse, Sigma, St. Louis, MO) was injected intraperitoneally, and mice were euthanized 6 h later. The CCN1 protein displays a remarkable degree of evolutionary conservation, with 92.8% identity between mouse and human CCN1 (11Jay P. Berge-Lefranc J.L. Marsollier C. Mejean C. Taviaux S. Berta P. The human growth factor-inducible immediate early gene, CYR61, maps to chromosome 1p.Oncogene. 1997; 14: 1753-1757Crossref PubMed Scopus (61) Google Scholar). Recombinant human CCN1 (5 μg/mouse, R and D Systems, Minneapolis, MN) was administered intravenously through the tail vein, and mice were euthanized 24 h later. The neutralizing anti-integrin αM antibody (0.1 mg/mouse, BD Pharmingen, San Diego, CA) or Z-VAD-FMK (200 μg/mouse, Sigma), which inhibits induction of apoptosis, was injected through the tail vein 2 h before administration of CCN1. Control animals were injected with vehicle only. The plasmid encoding mouse CCN1 cDNA or control vector (50 μg/mouse) was dissolved in PBS with a volume of 2 ml or 3 ml and then delivered to ND and HF mice by hydrodynamic tail vein injection, respectively (12Le Saux O. Fulop K. Yamaguchi Y. Ilias A. Szabo Z. Brampton C.N. Pomozi V. Huszár K. Arányi T. Váradi A. Expression and in vivo rescue of human ABCC6 disease-causing mutants in mouse liver.PLoS ONE. 2011; 6: e24738Crossref PubMed Scopus (59) Google Scholar). All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine and were conducted in accordance with the National Research Council Guide for Care and Use of Laboratory Animals. The construction of the liver-specific CCN1 expression plasmid was carried out as previously described (12Le Saux O. Fulop K. Yamaguchi Y. Ilias A. Szabo Z. Brampton C.N. Pomozi V. Huszár K. Arányi T. Váradi A. Expression and in vivo rescue of human ABCC6 disease-causing mutants in mouse liver.PLoS ONE. 2011; 6: e24738Crossref PubMed Scopus (59) Google Scholar), using the Enh1mTTR (ET) promoter generated by fusing a synthetic hepatocyte-specific enhancer to the murine transthyretin promoter (13Mátrai J. Cantore A. Bartholomae C.C. Annoni A. Wang W. Acosta-Sanchez A. Samara-Kuko E. De Waele L. Ma L. Genovese P. et al.Hepatocyte-targeted expression by integrase-defective lentiviral vectors induces antigen-specific tolerance in mice with low genotoxic risk.Hepatology. 2011; 53: 1696-1707Crossref PubMed Scopus (111) Google Scholar). The CCN1 cDNA inserted into the vector was expressed under the control of ET promoter. The control plasmid contained the GFP gene under control of the same promoter. Primary murine hepatocytes were isolated from mice by in situ collagenase liver perfusion (14Nyagode B.A. Lee C.M. Morgan E.T. Modulation of hepatic cytochrome P450s by Citrobacter rodentium infection in interleukin-6- and interferon-{gamma}-null mice.J. Pharmacol. Exp. Ther. 2010; 335: 480-488Crossref PubMed Scopus (27) Google Scholar) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 mg/ml streptomycin, and 100 U/ml penicillin (Invitrogen, Carlsbad, CA). Primary murine macrophages were obtained as previously described with minor modifications (15Gersuk G.M. Razai L.W. Marr K.A. Methods of in vitro macrophage maturation confer variable inflammatory responses in association with altered expression of cell surface dectin-1.J. Immunol. Methods. 2008; 329: 157-166Crossref PubMed Scopus (35) Google Scholar) and cultured in DMEM supplemented with 10% FBS, 20 ng/ml macrophage colony-stimulating factor (M-CSF; R and D Systems), 100 mg/ml streptomycin, and 100 U/ml penicillin. The macrophage RAW264.7 cell line was purchased from the Shanghai Institute of Cell Biology (Chinese Academy of Sciences, Shanghai, China) and cultured with DMEM plus 10% FBS. Cells were precultured with JNK inhibitor II (SP600125, 10 μM), IKK-2 inhibitor V (IMD0354, 1 μM), U0126 (10 μM), PD98059 (10 μM) (Merck, Darmstadt, Germany), or anti-integrin αM (5 μg/ml, BD Pharmingen) for 2 h before stimulation with LPS, OP 2:1 (mixture of oleic acid and palmitic acid at a ratio of 2:1, Sigma) or CCN1, respectively. RAW264.7 cells or murine myeloid-derived macrophages were precultured with or without neutralizing anti-integrin αM (5 μg/ml), U0126 (10 μM) for 30 min, and then 1 × 105 cells were placed on a 0.8 μm membrane (Millicell Hanging Cell Culture Insert, PET, Millipore, Billerica, MA) in DMEM with 10% FBS. The cells were allowed to migrate toward CCN1 in the medium below the membrane at the indicated concentrations (250 ng/ml, 500 ng/ml, 1,000 ng/ml) for 90 min and 24 h, respectively. Migration was quantified as the number of crystal violet-stained cells observed on the underside of the membrane by light microscopy (10 randomly chosen fields per membrane and three replicate wells per treatment). Immunoblotting analyses were carried out using either cell extracts or whole-liver extracts, as described previously (16Ma X. Hua J. Li Z. Probiotics improve high fat diet-induced hepatic steatosis and insulin resistance by increasing hepatic NKT cells.J. Hepatol. 2008; 49: 821-830Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). Immunoblots were probed with antibodies to CCN1 (Abcam, Cambridge, UK), phosphorylated and total Mek1/2, Erk1/2, C-jun, and IκB-α (Cell Signaling Technology, Boston, MA). Immunohistochemistry was performed on formalin-fixed, paraffin-embedded liver tissues using antibodies against F4/80 (1:100, Genetex, Irvine, CA) and CCN1 (1:100). The procedures were performed as previously described with minor modifications (17Birjandi S.Z. Ippolito J.A. Ramadorai A.K. Witte P.L. Alterations in marginal zone macrophages and marginal zone B cells in old mice.J. Immunol. 2011; 186: 3441-3451Crossref PubMed Scopus (65) Google Scholar). Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), using the TUNEL FITC kit (ShanghaRuian bioTechnologies Co., Ltd, Shanghai, China) according to the manufacturer's instructions. Data are expressed as the mean ± SD. The group means were compared using ANOVA. All statistical analysis was performed using the SPSS statistical software version 16.0 (SPSS Inc., Chicago, IL). P values < 0.05 were considered statistically significant. Our previous studies demonstrated that mice fed HF diets became obese and developed hepatic steatosis (18Li Z. Soloski M.J. Diehl A.M. Dietary factors alter hepatic innate immune system in mice with nonalcoholic fatty liver disease.Hepatology. 2005; 42: 880-885Crossref PubMed Scopus (253) Google Scholar, 19Ma X. Hua J. Mohamood A.R. Hamad A.R. Ravi R. Li Z. A high-fat diet and regulatory T cells influence susceptibility to endotoxin-induced liver injury.Hepatology. 2007; 46: 1519-1529Crossref PubMed Scopus (135) Google Scholar). In the present study, HF mice also developed significant hepatic steatosis characterized by the presence of macrovesicular and microvesicular lipid droplets. Immunohistochemical analysis showed that LPS administration significantly increased hepatic CCN1 expression in HF mice compared with HF mice without LPS administration and LPS-treated ND mice that hepatic CCN1 was increased in steatosis ob/ob mice were treated with or without hepatic CCN1 expression was increased in ob/ob mice compared with mice. LPS treatment increased hepatic CCN1 expression in ob/ob mice analysis showed that HF and LPS treatment increased hepatic expression of CCN1 in ND mice. LPS-treated HF mice significantly hepatic CCN1 expression compared with the LPS-treated ND mice The same was also observed in ob/ob mice analysis we that CCN1 was expressed in analysis showed that the hepatocytes significantly CCN1 expression than the that the of CCN1 protein in the liver is hepatocytes we hepatocytes as cells for experiments to the regulation of CCN1 LPS at the concentrations of 1 and 2 induced CCN1 expression by in primary murine hepatocytes in vitro In CCN1 expression was at h and a at h LPS treatment at 2 Primary murine hepatocytes were with a of fatty acids to a steatosis model A. H. hepatic steatosis to liver in mice.J. Hepatol. Full Text Full Text PDF PubMed Scopus Google Scholar). FFAs also induced CCN1 expression in a at FFAs were shown to the CCN1 expression level at 24 h demonstrated that LPS and FFAs induced CCN1 expression of hepatocytes in a and In LPS administration the hepatic CCN1 expression in significant in CCN1 expression was detected between treatment with LPS and treatment without LPS in LPS and FFAs CCN1 expression in hepatocytes isolated from that LPS and FFA-induced CCN1 expression is on the signaling pathway. the signaling pathway of involved in this for the and NF-κB were and significantly the of of and respectively the expression of CCN1 in primary hepatocytes with LPS or FFAs that LPS and FFAs CCN1 expression in hepatocytes through activation of the TLR4/MyD88/AP-1 signaling pathway. CCN1 protein induced to hepatic inflammation in ND mice, it induced severe hepatic inflammation in HF mice CCN1 treatment increased the of hepatic inflammatory in ND and HF mice compared with ND and HF mice without respectively. CCN1 induced more hepatic inflammatory in HF mice compared with ND mice treated with CCN1 CCN1 treatment increased serum and in ND and HF mice with the HF mice than ND mice that the HF mice were more to hepatic inflammation and liver CCN1 induces macrophage and activation through (8Bai T. Chen C.C. Lau L.F. Matricellular protein CCN1 activates a proinflammatory genetic program in murine macrophages.J. Immunol. 2010; 184: 3223-3232Crossref PubMed Scopus (122) Google Scholar, Chen N. Lau L.F. of as an receptor on for Cyr61 and tissue growth factor gene expressed in PubMed Scopus Google Scholar), a neutralizing antibody against αM was to the between CCN1 and macrophages. of αM hepatic inflammation in ND and HF mice, indicated by less hepatic inflammatory infiltration in and serum and compared with ND and HF mice treated with CCN1 that CCN1 induced monocyte infiltration through to αM on the surface of CCN1 also induced hepatic inflammation in ob/ob mice but to the in HF mice of a liver-specific CCN1 to CCN1 induced more severe hepatic inflammation in HF mice than in ND mice we macrophage infiltration in CCN1 hepatic inflammatory in ND and HF mice by using F4/80 F4/80 cells were detected that macrophages are the in liver CCN1 induced more macrophage infiltrates in HF mice compared with ND mice treated with CCN1 of αM hepatic macrophage infiltrates in ND and HF mice analysis showed that the of F4/80 cells in the liver expressed αM that αM may macrophage infiltration in hepatic with the of CCN1 protein the expression of CCN1 also induced macrophage in ND and HF mice It was that CCN1 apoptosis by and in vivo and vitro (10Chen C.C. Young J.L. Monzon R.I. Chen N. Todorovic V. Lau L.F. Cytotoxicity of TNFalpha is regulated by integrin-mediated matrix signaling.EMBO J. 2007; 26: 1257-1267Crossref PubMed Scopus (127) Google Scholar, V. Chen C.C. Lau L.F. apoptosis is regulated by the extracellular matrix protein CCN1 in vitro and in Biol. 2009; PubMed Scopus (61) Google Scholar). we observed apoptosis a critical role in hepatic inflammation and macrophage infiltration induced by CCN1. CCN1 induced apoptosis in the livers of ND mice but also of HF mice, and a this Z-VAD-FMK significantly the hepatic inflammation induced by CCN1 in either ND or HF mice In macrophage infiltration was significantly by Z-VAD-FMK that the role of CCN1 on hepatic inflammation and macrophage infiltration is of apoptosis induced by CCN1. CCN1 in increased the of and in the RAW264.7 cell line CCN1 recruited primary murine myeloid-derived macrophages in a from to 1,000 ng/ml at 90 min and 24 with the neutralizing antibody against αM or an inhibitor for significantly the level of macrophage in response to CCN1 were also in the murine macrophage RAW264.7 cell line that CCN1 macrophages through activation of the signaling pathway. In this study, we demonstrated for the first time that FFAs and LPS induced CCN1 expression in hepatocytes through the TLR4/MyD88/AP-1 signaling pathway in vitro and in CCN1 severe hepatic inflammation and apoptosis in mice with and CCN1 was shown to macrophage through the signaling pathway. CCN1 may play roles in the inflammatory response and macrophage infiltration in NAFLD. that innate is involved in insulin resistance and a critical role in the pathogenesis of NASH P. J.C. insulin innate in nonalcoholic 2008; PubMed Scopus Google Scholar). innate and fatty insulin resistance H. K. H. innate and fatty insulin 2006; PubMed Scopus Google Scholar, cells in fatty liver disease: the Hepatol. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). is a receptor for LPS, a of the of and is considered a in the pathogenesis of NAFLD A. X. C. X. E. C. et LPS signaling in and in PubMed Scopus Google Scholar). LPS has been shown to of in macrophages, apoptosis in a murine NASH model H. T. Y. K. W. T. apoptosis in a murine steatohepatitis Hepatol. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). In an role in proinflammatory effects of FFAs may as the that to activation in the of inflammation, and insulin 2010; PubMed Scopus Google Scholar). FFAs may and from steatosis to NASH mechanisms J. Diehl A.M. of disease in nonalcoholic fatty liver Liver Dis. 2008; PubMed Scopus Google Scholar). acid can to leading to activation of NF-κB and as and in macrophages and mice fed a HF are against development of insulin to inflammatory gene expression in liver and fat tissues H. K. H. innate and fatty insulin 2006; PubMed Scopus Google Scholar). FFAs also of proinflammatory cytokines A. R. fatty acids hepatic by expression a 2004; 40: PubMed Scopus Google Scholar) and S. K. L. P. acid induces of proinflammatory from 2007; 46: PubMed Scopus Google Scholar) from to hepatic inflammation and liver and showed that the palmitic acid the which into and induces to LPS for in hepatocytes T. J. K. A. Szabo acid and in mouse hepatocytes that to immune 2011; PubMed Scopus Google Scholar). Our showed that LPS and FFAs induced of CCN1 in which may be inflammatory factor in the and studies that hepatocytes are but that also are involved in responses to in are of innate in NAFLD. In adipose macrophages are a of proinflammatory which can in a and an to insulin The that activation of hepatic macrophages at the is an in the pathogenesis of NAFLD cells in fatty liver disease: the Hepatol. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). It was shown that CCN1 can a proinflammatory genetic program in macrophages, as monocyte chemotactic protein 1 and macrophage inflammatory protein 1 α (8Bai T. Chen C.C. Lau L.F. Matricellular protein CCN1 activates a proinflammatory genetic program in murine macrophages.J. Immunol. 2010; 184: 3223-3232Crossref PubMed Scopus (122) Google Scholar). In study, we showed that CCN1 macrophages through to αM on the surface of macrophages, which at the macrophage infiltrates in murine liver CCN1 of CCN1 by hepatocytes treatment with LPS and FFAs may and macrophages into the activated hepatic macrophages then various as which in macrophages, a that the number of macrophages in the liver and the inflammatory inflammation, and insulin 2010; PubMed Scopus Google Scholar). In the proinflammatory is in NAFLD by induction of macrophage infiltration into the liver through and by In we for the first time that and FFAs expression of the matricellular signaling CCN1 in hepatocytes through activation of the TLR4/MyD88/AP-1 signaling pathway in mice. CCN1 then the of macrophages into the the inflammatory and macrophage The potential role of CCN1 in the of from steatosis to NASH in be in CCN1 may a potential to disease to in NAFLD. The Animal Research Center of Nanjing for with of

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.