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Marañón P, Isaza SC, Rey E, Rada P, García-García Y, Dear JW, García-Monzón C, Valverde ÁM, Egea J, González-Rodríguez Á. BMP6 participates in the molecular mechanisms involved in APAP hepatotoxicity. Arch Toxicol 2025; 99:1187-1202. [PMID: 39827450 PMCID: PMC11821676 DOI: 10.1007/s00204-024-03954-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2024] [Accepted: 12/23/2024] [Indexed: 01/22/2025]
Abstract
Given the lack of accurate diagnostic methods of acetaminophen (APAP)-induced acute liver failure (ALF), the search for new biomarkers for its diagnosis is an urgent need. The aim of this study was to evaluate the role of bone morphogenetic protein 6 (BMP6) in APAP-induced ALF progression and its potential value as a biomarker of ALF. Hepatic and circulating BMP6 expression was assessed in APAP-treated mice and in serum samples from patients with APAP overdose. In addition, BMP6 expression and release was evaluated in hepatocytes after APAP exposure. BMP6 gene was silenced in Huh7 cells prior to APAP treatment and the culture medium (CM) was added to THP1 cells to evaluate the paracrine effects of hepatocyte BMP6 on APAP toxicity. Hepatic and serum BMP6 levels were increased in mice after APAP-induced ALF. In addition, a positive correlation was observed between circulating BMP6 and ALT activity in patients exposed to APAP overdose. Moreover, hepatocytes expressed and released BMP6 to the CM after APAP treatment. Indeed, the CM from APAP-treated Huh7 cells upregulated M1 and M2 markers in THP1 monocytes. The CM from BMP6-silenced Huh7, which was depleted of BMP6, reduced the expression of M2 markers in THP1 cells. In fact, expression of M2 markers was increased in THP1 cells exposed to BMP6. This study reveals that hepatic BMP6 expression is increased in APAP-induced acute liver injury, positioning it as a potential new biomarker of liver damage severity. Moreover, our data indicate that BMP6 might play a role in the hepatocyte-macrophage crosstalk during APAP-induced hepatotoxicity.
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Affiliation(s)
- Patricia Marañón
- Unidad de Investigación, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa (IIS-P), Madrid, Spain.
| | - Stephania C Isaza
- Unidad de Investigación, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa (IIS-P), Madrid, Spain
| | - Esther Rey
- Unidad de Investigación, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa (IIS-P), Madrid, Spain
| | - Patricia Rada
- Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain
- Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain
| | - Yaiza García-García
- Unidad de Investigación, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa (IIS-P), Madrid, Spain
| | - James W Dear
- Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, Scotland, UK
| | - Carmelo García-Monzón
- Unidad de Investigación, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa (IIS-P), Madrid, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Madrid, Spain
| | - Ángela M Valverde
- Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain
- Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain
| | - Javier Egea
- Unidad de Investigación, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa (IIS-P), Madrid, Spain
| | - Águeda González-Rodríguez
- Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain.
- Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain.
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2
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Bloomer SA, Brown KE. Hepcidin and Iron Metabolism in Experimental Liver Injury. THE AMERICAN JOURNAL OF PATHOLOGY 2021; 191:1165-1179. [PMID: 33891874 DOI: 10.1016/j.ajpath.2021.04.005] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/08/2020] [Revised: 02/25/2021] [Accepted: 04/06/2021] [Indexed: 11/18/2022]
Abstract
The liver plays a pivotal role in the regulation of iron metabolism through its ability to sense and respond to iron stores by release of the hormone hepcidin. Under physiologic conditions, regulation of hepcidin expression in response to iron status maintains iron homeostasis. In response to tissue injury, hepcidin expression can be modulated by other factors, such as inflammation and oxidative stress. The resulting dysregulation of hepcidin is proposed to account for alterations in iron homeostasis that are sometimes observed in patients with liver disease. This review describes the effects of experimental forms of liver injury on iron metabolism and hepcidin expression. In general, models of acute liver injury demonstrate increases in hepcidin mRNA and hypoferremia, consistent with hepcidin's role as an acute-phase reactant. Conversely, diverse models of chronic liver injury are associated with decreased hepcidin mRNA but with variable effects on iron status. Elucidating the reasons for the disparate impact of different chronic injuries on iron metabolism is an important research priority, as is a deeper understanding of the interplay among various stimuli, both positive and negative, on hepcidin regulation. Future studies should provide a clearer picture of how dysregulation of hepcidin expression and altered iron homeostasis impact the progression of liver diseases and whether they are a cause or consequence of these pathologies.
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Affiliation(s)
- Steven A Bloomer
- Division of Science and Engineering, Penn State Abington, Abington, Pennsylvania
| | - Kyle E Brown
- Iowa City Veterans Administration Medical Center, Iowa City, Iowa; Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, Iowa; Program in Free Radical and Radiation Biology, Department of Radiation Oncology, University of Iowa Carver College of Medicine, Iowa City, Iowa.
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Radhakrishnan K, Kim YH, Jung YS, Kim J, Kim DK, Cho SJ, Lee IK, Dooley S, Lee CH, Choi HS. Orphan Nuclear Receptor ERRγ Is a Novel Transcriptional Regulator of IL-6 Mediated Hepatic BMP6 Gene Expression in Mice. Int J Mol Sci 2020; 21:7148. [PMID: 32998264 PMCID: PMC7582774 DOI: 10.3390/ijms21197148] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2020] [Revised: 09/24/2020] [Accepted: 09/24/2020] [Indexed: 12/23/2022] Open
Abstract
Bone morphogenetic protein 6 (BMP6) is a multifunctional growth factor involved in organ development and homeostasis. BMP6 controls expression of the liver hormone, hepcidin, and thereby plays a crucial role in regulating iron homeostasis. BMP6 gene transcriptional regulation in liver is largely unknown, but would be of great help to externally modulate iron load in pathologic conditions. Here, we describe a detailed molecular mechanism of hepatic BMP6 gene expression by an orphan nuclear receptor, estrogen-related receptor γ (ERRγ), in response to the pro-inflammatory cytokine interleukin 6 (IL-6). Recombinant IL-6 treatment increases hepatic ERRγ and BMP6 expression. Overexpression of ERRγ is sufficient to increase BMP6 gene expression in hepatocytes, suggesting that IL-6 is upstream of ERRγ. In line, knock-down of ERRγ in cell lines or a hepatocyte specific knock-out of ERRγ in mice significantly decreases IL-6 mediated BMP6 expression. Promoter studies show that ERRγ directly binds to the ERR response element (ERRE) in the mouse BMP6 gene promoter and positively regulates BMP6 gene transcription in IL-6 treatment conditions, which is further confirmed by ERRE mutated mBMP6-luciferase reporter assays. Finally, an inverse agonist of ERRγ, GSK5182, markedly inhibits IL-6 induced hepatic BMP6 expression in vitro and in vivo. Taken together, these results reveal a novel molecular mechanism on ERRγ mediated transcriptional regulation of hepatic BMP6 gene expression in response to IL-6.
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Affiliation(s)
- Kamalakannan Radhakrishnan
- School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Korea; (K.R.); (Y.S.J.)
| | - Yong-Hoon Kim
- Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea;
- Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon 34141, Korea
| | - Yoon Seok Jung
- School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Korea; (K.R.); (Y.S.J.)
| | - Jina Kim
- New Drug Development Center, Daegu Gyeongbuk Medical Innovation Foundation, Daegu 41061, Korea; (J.K.); (S.J.C.)
| | - Don-Kyu Kim
- Department of Integrative Food, Bioscience and Biotechnology, Chonnam National University, Gwangju 61186, Korea;
| | - Sung Jin Cho
- New Drug Development Center, Daegu Gyeongbuk Medical Innovation Foundation, Daegu 41061, Korea; (J.K.); (S.J.C.)
- Leading-Edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University Hospital, Daegu 41404, Korea;
| | - In-Kyu Lee
- Leading-Edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University Hospital, Daegu 41404, Korea;
- Department of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, Daegu 41944, Korea
| | - Steven Dooley
- Department of Medicine II, Medical Faculty Mannheim, Heidelberg University, 68167 Mannheim, Germany;
| | - Chul-Ho Lee
- Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea;
- Department of Functional Genomics, KRIBB School of Bioscience, University of Science and Technology (UST), Daejeon 34141, Korea
| | - Hueng-Sik Choi
- School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Korea; (K.R.); (Y.S.J.)
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Xu F, Yu Y, Wang F, Sun W, Li P, Wu HF, Bian ZP, Chen XJ, Dong-Jie X. Analysis of gene expression profiling of amyloidogenic immunoglobulin light-chains on cultured rat cardiomyocytes. Exp Ther Med 2020; 19:3767-3777. [PMID: 32346441 PMCID: PMC7185198 DOI: 10.3892/etm.2020.8610] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2019] [Accepted: 02/25/2020] [Indexed: 11/20/2022] Open
Abstract
The present study aimed to investigate the toxic effects of different amyloidogenic light-chains (LCs) on cardiomyocytes, and demonstrate the differentially expressed genes (DEGs) and signaling pathways that participate in this process. Cultured cardiomyocytes were treated with recombinant κ LC peptide (AL-09) or with serum from a patient diagnosed with multiple myeloma (λ LC) with cardiac involvement. The 6xHis peptide or serum from healthy patients was used as peptide control or serum control, respectively. Cell viability was determined using CCK-8 assay and apoptosis was analyzed by flow cytometry. The DEGs were detected by RNA sequencing (RNA-Seq), followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Changes in gene expression levels were confirmed by reverse transcription-quantitative PCR. The cell viability in the AL-09 peptide-treated (0.2 mg/ml) and patient serum-treated (1:10 dilution) cardiomyocytes decreased to 42 and -72% of the corresponding control groups. The extent of cell apoptosis increased in AL-09-treated cardiomyocytes compared with the control group. RNA-Seq showed 256 DEGs co-existed in the two paired groups, including 127 upregulated and 88 downregulated genes. The KEGG pathways for upregulated expressed genes included the ‘TGF-β signaling pathway’, the ‘Hedgehog signaling pathway’, the ‘ErbB signaling pathway’ and ‘lysine degradation’. The higher mRNA expression of bone morphogenetic protein (Bmp) 4, Bmp6, prostaglandin G/H synthase (Ptgs)1, Ptgs2, epiregulin, Tgfa and procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 were confirmed. The KEGG pathways of downregulated expressed genes included genes involved with the ‘p53 signaling pathway’ and the ‘cell cycle’. The mRNA expression levels of E3 ubiquitin-protein ligase CCNB1IP1 showed significant downregulation in the AL-09 peptide group compared with those in the 6xHis peptide group. In conclusion, cardiomyocytes treated with amyloidogenic λ and κ LCs presented with decreased cell viability compared with controls. Cell apoptosis increased in κ LC-treated cells compared with controls. The gene expression profiles associated with transforming growth factor-β-bone morphogenetic protein, the receptor tyrosine-protein kinase erbB-2 signaling pathways, prostaglandins, collagen production, the p53 signaling pathway and the cell cycle were altered in light-chain-treated cardiomyocytes.
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Affiliation(s)
- Fei Xu
- Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
| | - Yue Yu
- Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
| | - Fang Wang
- Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
| | - Wei Sun
- Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
| | - Peng Li
- Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
| | - Heng-Fang Wu
- Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
| | - Zhi-Ping Bian
- Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
| | - Xiang-Jian Chen
- Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
| | - Xu Dong-Jie
- Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
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Kleven MD, Enns CA, Zhang AS. Bone Morphogenetic Protein-6 Mutations Take Their Place in Iron Overload Diseases. Gastroenterology 2016; 150:556-9. [PMID: 26820052 DOI: 10.1053/j.gastro.2016.01.016] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Affiliation(s)
- Mark D Kleven
- Department of Cell, Developmental and Cancer Biology, Oregon Health & Science University, Portland, Oregon
| | - Caroline A Enns
- Department of Cell, Developmental and Cancer Biology, Oregon Health & Science University, Portland, Oregon.
| | - An-Sheng Zhang
- Department of Cell, Developmental and Cancer Biology, Oregon Health & Science University, Portland, Oregon
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Pietrangelo A. Genetics, Genetic Testing, and Management of Hemochromatosis: 15 Years Since Hepcidin. Gastroenterology 2015; 149:1240-1251.e4. [PMID: 26164493 DOI: 10.1053/j.gastro.2015.06.045] [Citation(s) in RCA: 104] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/30/2015] [Revised: 06/19/2015] [Accepted: 06/30/2015] [Indexed: 12/13/2022]
Abstract
The discovery of hepcidin in 2000 and the subsequent unprecedented explosion of research and discoveries in the iron field have dramatically changed our understanding of human disorders of iron metabolism. Today, hereditary hemochromatosis, the paradigmatic iron-loading disorder, is recognized as an endocrine disease due to the genetic loss of hepcidin, the iron hormone produced by the liver. This syndrome is due to unchecked transfer of iron into the bloodstream in the absence of increased erythropoietic needs and its toxic effects in parenchymatous organs. It is caused by mutations that affect any of the proteins that help hepcidin to monitor serum iron, including HFE and, in rarer instances, transferrin-receptor 2 and hemojuvelin, or make its receptor ferroportin, resistant to the hormone. In Caucasians, C282Y HFE homozygotes are numerous, but they are only predisposed to hemochromatosis; complete organ disease develops in a minority, due to alcohol abuse or concurrent genetic modifiers that are now being identified. HFE gene testing can be used to diagnose hemochromatosis in symptomatic patients, but analyses of liver histology and full gene sequencing are required to identify patients with rare, non-HFE forms of the disease. Due to the central pathogenic role of hepcidin, it is anticipated that nongenetic causes of hepcidin loss (eg, end-stage liver disease) can cause acquired forms of hemochromatosis. The mainstay of hemochromatosis management is still removal of iron by phlebotomy, first introduced in 1950s, but identification of hepcidin has not only shed new light on the pathogenesis of the disease and the approach to diagnosis, but etiologic therapeutic applications from these advances are now foreseen.
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Affiliation(s)
- Antonello Pietrangelo
- Unit of Internal Medicine 2 and Centre for Hemochromatosis, University Hospital of Modena, Modena, Italy.
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Rausa M, Pagani A, Nai A, Campanella A, Gilberti ME, Apostoli P, Camaschella C, Silvestri L. Bmp6 expression in murine liver non parenchymal cells: a mechanism to control their high iron exporter activity and protect hepatocytes from iron overload? PLoS One 2015; 10:e0122696. [PMID: 25860887 PMCID: PMC4393274 DOI: 10.1371/journal.pone.0122696] [Citation(s) in RCA: 57] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2014] [Accepted: 02/12/2015] [Indexed: 02/06/2023] Open
Abstract
Bmp6 is the main activator of hepcidin, the liver hormone that negatively regulates plasma iron influx by degrading the sole iron exporter ferroportin in enterocytes and macrophages. Bmp6 expression is modulated by iron but the molecular mechanisms are unknown. Although hepcidin is expressed almost exclusively by hepatocytes (HCs), Bmp6 is produced also by non-parenchymal cells (NPCs), mainly sinusoidal endothelial cells (LSECs). To investigate the regulation of Bmp6 in HCs and NPCs, liver cells were isolated from adult wild type mice whose diet was modified in iron content in acute or chronic manner and in disease models of iron deficiency (Tmprss6 KO mouse) and overload (Hjv KO mouse). With manipulation of dietary iron in wild-type mice, Bmp6 and Tfr1 expression in both HCs and NPCs was inversely related, as expected. When hepcidin expression is abnormal in murine models of iron overload (Hjv KO mice) and deficiency (Tmprss6 KO mice), Bmp6 expression in NPCs was not related to Tfr1. Despite the low Bmp6 in NPCs from Tmprss6 KO mice, Tfr1 mRNA was also low. Conversely, despite body iron overload and high expression of Bmp6 in NPCs from Hjv KO mice, Tfr1 mRNA and protein were increased. However, in the same cells ferritin L was only slightly increased, but the iron content was not, suggesting that Bmp6 in these cells reflects the high intracellular iron import and export. We propose that NPCs, sensing the iron flux, not only increase hepcidin through Bmp6 with a paracrine mechanism to control systemic iron homeostasis but, controlling hepcidin, they regulate their own ferroportin, inducing iron retention or release and further modulating Bmp6 production in an autocrine manner. This mechanism, that contributes to protect HC from iron loading or deficiency, is lost in disease models of hepcidin production.
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MESH Headings
- Anemia, Iron-Deficiency/metabolism
- Anemia, Iron-Deficiency/pathology
- Animals
- Apoferritins/metabolism
- Bone Morphogenetic Protein 6/genetics
- Bone Morphogenetic Protein 6/metabolism
- Cells, Cultured
- Disease Models, Animal
- GPI-Linked Proteins
- Hemochromatosis Protein
- Hepatocytes/cytology
- Hepatocytes/drug effects
- Hepatocytes/metabolism
- Hepcidins/metabolism
- Iron/metabolism
- Iron Deficiencies
- Iron Overload/metabolism
- Iron Overload/pathology
- Iron, Dietary/pharmacology
- Male
- Membrane Proteins/deficiency
- Membrane Proteins/genetics
- Mice
- Mice, Inbred C57BL
- Mice, Knockout
- RNA, Messenger/metabolism
- Receptors, Transferrin/genetics
- Receptors, Transferrin/metabolism
- Serine Endopeptidases/deficiency
- Serine Endopeptidases/genetics
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Affiliation(s)
- Marco Rausa
- Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy
- Vita Salute University, Milan, Italy
| | - Alessia Pagani
- Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy
- Vita Salute University, Milan, Italy
| | - Antonella Nai
- Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy
- Vita Salute University, Milan, Italy
| | - Alessandro Campanella
- Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy
- Vita Salute University, Milan, Italy
| | - Maria Enrica Gilberti
- Unit of Occupational Health and Industrial Hygiene, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia, Brescia, Italy
| | - Pietro Apostoli
- Unit of Occupational Health and Industrial Hygiene, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia, Brescia, Italy
| | - Clara Camaschella
- Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy
- Vita Salute University, Milan, Italy
- * E-mail: (LS); (CC)
| | - Laura Silvestri
- Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy
- Vita Salute University, Milan, Italy
- * E-mail: (LS); (CC)
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Pauk M, Grgurevic L, Brkljacic J, Kufner V, Bordukalo-Niksic T, Grabusic K, Razdorov G, Rogic D, Zuvic M, Oppermann H, Babitt JL, Lin HY, Volarevic S, Vukicevic S. Exogenous BMP7 corrects plasma iron overload and bone loss in Bmp6-/- mice. INTERNATIONAL ORTHOPAEDICS 2014; 39:161-72. [PMID: 25300398 DOI: 10.1007/s00264-014-2550-4] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/06/2014] [Accepted: 09/22/2014] [Indexed: 02/07/2023]
Abstract
PURPOSE Iron overload accelerates bone loss in mice lacking the bone morphogenetic protein 6 (Bmp6) gene, which is the key endogenous regulator of hepcidin, iron homeostasis gene. We investigated involvement of other BMPs in preventing haemochromatosis and subsequent osteopenia in Bmp6-/- mice. METHODS Iron-treated wild-type (WT) and Bmp6-/- mice were analysed for hepcidin messenger RNA (mRNA) and tissue and blood BMP levels by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry, Western blot, enzyme-linked immunosorbent assay (ELISA) and proximity extension assay. BMPs labeled with technetium-99m were used in pharmacokinetic studies. RESULTS In WT mice, 4 h following iron challenge, liver Bmp6 and hepcidin expression were increased, while expression of other Bmps was not affected. In parallel, we provided the first evidence that BMP6 circulates in WT mice and that iron increased the BMP6 serum level and the specific liver uptake of (99m)Tc-BMP6. In Bmp6-/- mice, iron challenge led to blunted activation of liver Smad signaling and hepcidin expression with a delay of 24 h, associated with increased Bmp5 and Bmp7 expression and increased Bmp2, 4, 5 and 9 expression in the duodenum. Liver Bmp7 expression and increased circulating BMP9 eventually contributed to the late hepcidin response. This was further supported by exogenous BMP7 therapy resulting in an effective hepcidin expression followed by a rapid normalisation of plasma iron values and restored osteopenia in Bmp6-/- mice. CONCLUSION In Bmp6-/- mice, iron activated endogenous compensatory mechanisms of other BMPs that were not sufficient for preventing hemochromatosis and bone loss. Administration of exogenous BMP7 was effective in correcting the plasma iron level and bone loss, indicating that BMP6 is an essential but not exclusive in vivo regulator of iron homeostasis.
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Affiliation(s)
- Martina Pauk
- Center for Translational and Clinical Research, University of Zagreb School of Medicine, Salata 11, 10000, Zagreb, Croatia,
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Abstract
Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-β) superfamily of signaling molecules. In addition to protean roles in embryonic development, germ-line specification, and cellular differentiation, a central role in iron homeostasis has recently been demonstrated for certain BMPs. Specifically, BMP6 serves to relate hepatic iron stores to the hepatocellular expression of the iron-regulatory hormone hepcidin. This regulation occurs via cellular SMAD-signaling molecules and is strongly modulated by the BMP coreceptor hemojuvelin (HJV). Mutations in certain genes influencing signaling to hepcidin via the BMP/SMAD pathway are associated with human disorders of iron metabolism, such as hereditary hemochromatosis and iron-refractory iron-deficiency anemia. Evidence suggests that signals in addition to iron stores influence hepcidin expression via the BMP/SMAD pathway. This review summarizes the details of BMP/SMAD signaling, with a particular focus on its role in iron homeostasis and iron-related diseases.
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Affiliation(s)
- Nermi L Parrow
- Division of Molecular and Clinical Nutrition, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892
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Worthen CA, Enns CA. The role of hepatic transferrin receptor 2 in the regulation of iron homeostasis in the body. Front Pharmacol 2014; 5:34. [PMID: 24639653 PMCID: PMC3944196 DOI: 10.3389/fphar.2014.00034] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2014] [Accepted: 02/18/2014] [Indexed: 12/22/2022] Open
Abstract
Fine-tuning of body iron is required to prevent diseases such as iron-overload and anemia. The putative iron sensor, transferrin receptor 2 (TfR2), is expressed in the liver and mutations in this protein result in the iron-overload disease Type III hereditary hemochromatosis (HH). With the loss of functional TfR2, the liver produces about 2-fold less of the peptide hormone hepcidin, which is responsible for negatively regulating iron uptake from the diet. This reduction in hepcidin expression leads to the slow accumulation of iron in the liver, heart, joints, and pancreas and subsequent cirrhosis, heart disease, arthritis, and diabetes. TfR2 can bind iron-loaded transferrin (Tf) in the bloodstream, and hepatocytes treated with Tf respond with a 2-fold increase in hepcidin expression through stimulation of the bone morphogenetic protein (BMP)-signaling pathway. Loss of functional TfR2 or its binding partner, the original HH protein, results in a loss of this transferrin-sensitivity. While much is known about the trafficking and regulation of TfR2, the mechanism of its transferrin-sensitivity through the BMP-signaling pathway is still not known.
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Affiliation(s)
- Christal A Worthen
- Department of Cell and Developmental Biology, Oregon Health and Science University Portland, OR, USA
| | - Caroline A Enns
- Department of Cell and Developmental Biology, Oregon Health and Science University Portland, OR, USA
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11
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Abstract
A classic Girl Scout song says, "Make new friends/but keep the old/One is silver/and the other gold." This review focuses on the past decade of discovery in the field of iron homeostasis, which has identified "new friends" or key modifiers of the critical systemic iron regulator, hepcidin antimicrobial peptide. The foundation for these discoveries has been the identification of mutated genes in well-characterized cohorts of patients with inherited hemochromatosis from across the globe. Transgenic mouse models of iron overload and iron-restricted anemia have also contributed to understanding molecular pathophysiology in ways that could never be accomplished in human subjects alone. The majority of these newly discovered molecules coordinate signaling through the bone morphogenetic protein pathway of ligands, receptors and coreceptors, intracellular signaling and transcription. The discovery of these proteins and their interactions with "old friends," such as the 1st known hereditary hemochromatosis gene product, HFE and transferrin receptor, has opened the field of iron homeostasis to include regulatory networks involving signal transduction pathways, in particular, the mitogen-activated protein kinase and Smad pathways. These newly discovered partnerships have also made way for opportunities to develop novel therapeutics for the treatment of iron regulatory disorders, including hemochromatosis.
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Enns CA, Ahmed R, Wang J, Ueno A, Worthen C, Tsukamoto H, Zhang AS. Increased iron loading induces Bmp6 expression in the non-parenchymal cells of the liver independent of the BMP-signaling pathway. PLoS One 2013; 8:e60534. [PMID: 23565256 PMCID: PMC3615098 DOI: 10.1371/journal.pone.0060534] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2012] [Accepted: 02/27/2013] [Indexed: 02/07/2023] Open
Abstract
Bone morphogenetic protein 6 (BMP6) is an essential cytokine for the expression of hepcidin, an iron regulatory hormone secreted predominantly by hepatocytes. Bmp6 expression is upregulated by increased iron-levels in the liver. Both hepatocytes and non-parenchymal liver cells have detectable Bmp6 mRNA. Here we showed that induction of hepcidin expression in hepatocytes by dietary iron is associated with an elevation of Bmp6 mRNA in the non-parenchymal cells of the liver. Consistently, incubation with iron-saturated transferrin induces Bmp6 mRNA expression in isolated hepatic stellate cells, but not in hepatocytes. These observations suggest an important role of the non-parenchymal liver cells in regulating iron-homeostasis by acting as a source of Bmp6.
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Affiliation(s)
- Caroline A. Enns
- Department of Cell and Developmental Biology, Oregon Health & Science University, Portland, Oregon, United States of America
- * E-mail: (CAE); (A-SZ)
| | - Riffat Ahmed
- Department of Cell and Developmental Biology, Oregon Health & Science University, Portland, Oregon, United States of America
| | - Jiaohong Wang
- Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States of America
| | - Akiko Ueno
- Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States of America
| | - Christal Worthen
- Department of Cell and Developmental Biology, Oregon Health & Science University, Portland, Oregon, United States of America
| | - Hidekazu Tsukamoto
- Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States of America
- Department of Veteran Affairs, Greater Los Angeles Healthcare System, Los Angeles, California, United States of America
| | - An-Sheng Zhang
- Department of Cell and Developmental Biology, Oregon Health & Science University, Portland, Oregon, United States of America
- * E-mail: (CAE); (A-SZ)
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Feng Q, Migas MC, Waheed A, Britton RS, Fleming RE. Ferritin upregulates hepatic expression of bone morphogenetic protein 6 and hepcidin in mice. Am J Physiol Gastrointest Liver Physiol 2012; 302:G1397-404. [PMID: 22517766 PMCID: PMC3378091 DOI: 10.1152/ajpgi.00020.2012] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Hepcidin is a hepatocellular hormone that inhibits the release of iron from certain cell populations, including enterocytes and reticuloendothelial cells. The regulation of hepcidin (HAMP) gene expression by iron status is mediated in part by the signaling molecule bone morphogenetic protein 6 (BMP6). We took advantage of the low iron status of juvenile mice to characterize the regulation of Bmp6 and Hamp1 expression by iron administered in three forms: 1) ferri-transferrin (Fe-Tf), 2) ferric ammonium citrate (FAC), and 3) liver ferritin. Each of these forms of iron enters cells by distinct mechanisms and chemical forms. Iron was parenterally administered to 10-day-old mice, and hepatic expression of Bmp6 and Hamp1 mRNAs was measured 6 h later. We observed that hepatic Bmp6 expression increased in response to ferritin but was unchanged by Fe-Tf or FAC. Hepatic Hamp1 expression likewise increased in response to ferritin and Fe-Tf but was decreased by FAC. Exogenous ferritin increased Bmp6 and Hamp1 expression in older mice as well. Removing iron from ferritin markedly decreased its effect on Bmp6 expression. Exogenously administered ferritin and the derived iron localized in the liver primarily to sinusoidal lining cells. Moreover, expression of Bmp6 mRNA in isolated adult rodent liver cells was much higher in sinusoidal lining cells than hepatocytes (endothelial >> stellate > Kupffer). We conclude that exogenous iron-containing ferritin upregulates hepatic Bmp6 expression, and we speculate that liver ferritin contributes to regulation of Bmp6 and, thus, Hamp1 genes.
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Affiliation(s)
- Qi Feng
- 1Department of Pediatrics, Saint Louis University School of Medicine, St. Louis, Missouri;
| | - Mary C. Migas
- 1Department of Pediatrics, Saint Louis University School of Medicine, St. Louis, Missouri;
| | - Abdul Waheed
- 3Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri
| | - Robert S. Britton
- 2Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, Missouri; and
| | - Robert E. Fleming
- 1Department of Pediatrics, Saint Louis University School of Medicine, St. Louis, Missouri; ,3Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri
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Chen J, Enns CA. Hereditary hemochromatosis and transferrin receptor 2. Biochim Biophys Acta Gen Subj 2011; 1820:256-63. [PMID: 21864651 DOI: 10.1016/j.bbagen.2011.07.015] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2011] [Revised: 07/26/2011] [Accepted: 07/29/2011] [Indexed: 02/08/2023]
Abstract
BACKGROUND Multicellular organisms regulate the uptake of calories, trace elements, and other nutrients by complex feedback mechanisms. In the case of iron, the body senses internal iron stores, iron requirements for hematopoiesis, and inflammatory status, and regulates iron uptake by modulating the uptake of dietary iron from the intestine. Both the liver and the intestine participate in the coordination of iron uptake and distribution in the body. The liver senses inflammatory signals and iron status of the organism and secretes a peptide hormone, hepcidin. Under high iron or inflammatory conditions hepcidin levels increase. Hepcidin binds to the iron transport protein, ferroportin (FPN), promoting FPN internalization and degradation. Decreased FPN levels reduce iron efflux out of intestinal epithelial cells and macrophages into the circulation. Derangements in iron metabolism result in either the abnormal accumulation of iron in the body, or in anemias. The identification of the mutations that cause the iron overload disease, hereditary hemochromatosis (HH), or iron-refractory iron-deficiency anemia has revealed many of the proteins used to regulate iron uptake. SCOPE OF THE REVIEW In this review we discuss recent data concerning the regulation of iron homeostasis in the body by the liver and how transferrin receptor 2 (TfR2) affects this process. MAJOR CONCLUSIONS TfR2 plays a key role in regulating iron homeostasis in the body. GENERAL SIGNIFICANCE The regulation of iron homeostasis is important. One third of the people in the world are anemic. HH is the most common inherited disease in people of Northern European origin and can lead to severe health complications if left untreated. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.
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Affiliation(s)
- Juxing Chen
- Department of Cell and Developmental Biology L215, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., Portland, OR 97239, USA
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Suppression of hepatic hepcidin expression in response to acute iron deprivation is associated with an increase of matriptase-2 protein. Blood 2010; 117:1687-99. [PMID: 21115976 DOI: 10.1182/blood-2010-06-287292] [Citation(s) in RCA: 88] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Recent studies demonstrate a pivotal role for bone morphogenic protein-6 (BMP6) and matriptase-2, a protein encoded by the TMPRSS6 gene, in the induction and suppression of hepatic hepcidin expression, respectively. We examined their expression profiles in the liver and showed a predominant localization of BMP6 mRNA in nonparenchymal cells and exclusive expression of TMPRSS6 mRNA in hepatocytes. In rats fed an iron-deficient (ID) diet for 24 hours, the rapid decrease of transferrin saturation from 71% to 24% (control vs ID diet) was associated with a 100-fold decrease in hepcidin mRNA compared with the corresponding controls. These results indicated a close correlation of low transferrin saturation with decreased hepcidin mRNA. The lower phosphorylated Smad1/5/8 detected in the ID rat livers suggests that the suppressed hepcidin expression results from the inhibition of BMP signaling. Quantitative real-time reverse transcription polymerase chain reaction analysis revealed no significant change in either BMP6 or TMPRSS6 mRNA in the liver. However, an increase in matriptase-2 protein in the liver from ID rats was detected, suggesting that suppression of hepcidin expression in response to acute iron deprivation is mediated by an increase in matriptase-2 protein levels.
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Abstract
Iron-loading disorders (haemochromatosis) represent an important class of human diseases. Primary iron loading results from inherited disturbances in the mechanisms regulating intestinal iron absorption, such that excess iron is taken up from the diet. Body iron load can also be increased by repeated blood transfusions (secondary iron loading), usually as part of the treatment for various haematological disorders. In these syndromes, an element of enhanced iron absorption is also often involved. The central regulator of body iron trafficking is the liver-derived peptide hepcidin. Hepcidin limits iron entry into the plasma from macrophages, intestinal enterocytes and other cells by binding to the sole iron-export protein ferroportin, and facilitating its removal from the plasma membrane. Mutations in hepcidin or its upstream regulators (HFE, TFR2, HFE2 and BMP6) lead to reduced or absent hepcidin expression and a concomitant increase in iron absorption. Mutations in ferroportin that prevent hepcidin binding produce a similar result. Increased ineffective erythropoiesis, which often characterises erythrocyte disorders, also leads to reduced hepcidin expression and increased absorption. Recent advances in our understanding of hepcidin and body iron homeostasis provide the potential for a range of new diagnostic and therapeutic tools for haemochromatosis and related conditions.
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17
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Piscaglia F, Dudás J, Knittel T, Di Rocco P, Kobold D, Saile B, Zocco MA, Timpl R, Ramadori G. Expression of ECM proteins fibulin-1 and -2 in acute and chronic liver disease and in cultured rat liver cells. Cell Tissue Res 2009; 337:449-462. [PMID: 19609566 PMCID: PMC2728066 DOI: 10.1007/s00441-009-0823-9] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2009] [Accepted: 05/25/2009] [Indexed: 01/10/2023]
Abstract
Fibulin-2 has previously been considered as a marker to distinguish rat liver myofibroblasts from hepatic stellate cells. The function of other fibulins in acute or chronic liver damage has not yet been investigated. The aim of this study has been to evaluate the expression of fibulin-1 and -2 in models of rat liver injury and in human liver cirrhosis. Their cellular sources have also been investigated. In normal rat liver, fibulin-1 and -2 were both mainly present in the portal field. Fibulin-1-coding transcripts were detected in total RNA of normal rat liver, whereas fibulin-2 mRNA was only detected by sensitive, real-time quantitative polymerase chain reaction. In acute liver injury, the expression of fibulin-1 was significantly increased (17.23-fold after 48 h), whereas that of fibulin-2 was not modified. The expression of both fibulin-1 and -2 was increased in experimental rat liver cirrhosis (19.16- and 26.47-fold, respectively). At the cellular level, fibulin-1 was detectable in hepatocytes, "activated" hepatic stellate cells, and liver myofibroblasts (2.71-, 122.65-, and 469.48-fold over the expression in normal rat liver), whereas fibulin-2 was restricted to liver myofibroblasts and was regulated by transforming growth factor beta-1 (TGF-beta1) in 2-day-old hepatocyte cultures and in liver myofibroblasts. Thus, fibulin-1 and -2 respond differentially to single and repeated damaging noxae, and their expression is differently present in liver cells. Expression of the fibulin-2 gene is regulated by TGF-beta1 in liver myofibroblasts.
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Affiliation(s)
- Fabio Piscaglia
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
- Present Address: Divisione di Medicina Interna – Bolondi, University of Bologna, Via Albertoni 15, 40138 Bologna, Italy
| | - József Dudás
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
- Present Address: Department of Otorhinolaryngology, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria
| | - Thomas Knittel
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| | - Paola Di Rocco
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| | - Dominik Kobold
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| | - Bernhard Saile
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| | - Maria Assunta Zocco
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
| | - Rupert Timpl
- Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany
| | - Giuliano Ramadori
- Department of Internal Medicine and Gastroenterology, University of Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
- Department of Gastroenterology and Endocrinology, Robert-Koch-Strasse 40, 37075 Göttingen, Germany
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18
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BMP/Smad signaling is not enhanced in Hfe-deficient mice despite increased Bmp6 expression. Blood 2009; 114:2515-20. [PMID: 19622835 DOI: 10.1182/blood-2009-02-206771] [Citation(s) in RCA: 96] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Impaired regulation of hepcidin expression in response to iron loading appears to be the pathogenic mechanism for hereditary hemochromatosis. Iron normally induces expression of the BMP6 ligand, which, in turn, activates the BMP/Smad signaling cascade directing hepcidin expression. The molecular function of the HFE protein, involved in the most common form of hereditary hemochromatosis, is still unknown. We have used Hfe-deficient mice of different genetic backgrounds to test whether HFE has a role in the signaling cascade induced by BMP6. At 7 weeks of age, these mice have accumulated iron in their liver and have increased Bmp6 mRNA and protein. However, in contrast to mice with secondary iron overload, levels of phosphorylated Smads 1/5/8 and of Id1 mRNA, both indicators of BMP signaling, are not significantly higher in the liver of these mice than in wild-type livers. As a consequence, hepcidin mRNA levels in Hfe-deficient mice are similar or marginally reduced, compared with 7-week-old wild-type mice. The inappropriately low levels of Id1 and hepcidin mRNA observed at weaning further suggest that Hfe deficiency triggers iron overload by impairing hepatic Bmp/Smad signaling. HFE therefore appears to facilitate signal transduction induced by the BMP6 ligand.
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19
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Anderson GJ. Things that go BMP in the liver: bone morphogenetic protein 6 and the control of body iron homeostasis. Hepatology 2009; 50:316-9. [PMID: 19554550 DOI: 10.1002/hep.23106] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/07/2022]
Affiliation(s)
- Gregory J Anderson
- Iron Metabolism Laboratory, Queensland Institute of Medical Research, Brisbane, Australia
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20
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Ji WM, Xu CP. Expression of BMP2 mRNA in regenerated liver tissue of rats and its significance. Shijie Huaren Xiaohua Zazhi 2008; 16:2453-2457. [DOI: 10.11569/wcjd.v16.i22.2453] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To analyze the dynamic changes of BMP2 mRNA in liver regeneration process, and to investigate the effect of BMP2 mRNA on liver regeneration in combination with the characteristics of nuclear factor kappa B (NF-κB) expression.
METHODS: Fifty-four healthy adult male Wister rats were randomly divided into 3 groups: normal control group (NC, n = 6), sham operation group (SO, n = 24), partial hepatectomy group (PH, n = 24). Using in situ hybridization and immunohistochemistry method, we measured the expression of BMP2 mRNA and NF-κB in regenerated liver tissue, separately.
RESULTS: BMP2 mRNA was significantly different between the SO and PH group at 6, 12 and 24 (gray values: 99.74 ± 6.85 vs 114.41 ± 5.12, 130.59 ± 6.74, 113.74 ± 7.32; all P < 0.05). There was no significance between the SO group and NC group. NF-κB was negative in the NC group, and there was no specific staining at various postoperative time points in the SO group. NF-κB was weakly positive at the initial stage in the PH group, and it was increased at 6, 12 and 24 h (gray values: 96.22 ± 3.12, 89.59 ± 3.24, 83.72 ± 4.32) (P < 0.05).
CONCLUSION: BMP2 is expressed in normal liver tissue of rats, and it displays a trend of increase after an initial decrease in hepatic regeneration. NF-κB is not expressed in normal liver tissue, but its expression gradually increases in the process of liver regeneration. Therefore, BMP2 may inhibit liver regeneration.
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21
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Neuman MG, Sha K, Esguerra R, Zakhari S, Winkler RE, Hilzenrat N, Wyse J, Cooper CL, Seth D, Gorrell MD, Haber PS, McCaughan GW, Leo MA, Lieber CS, Voiculescu M, Buzatu E, Ionescu C, Dudas J, Saile B, Ramadori G. Inflammation and repair in viral hepatitis C. Dig Dis Sci 2008; 53:1468-1487. [PMID: 17994278 DOI: 10.1007/s10620-007-0047-3] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/11/2007] [Accepted: 09/26/2007] [Indexed: 02/07/2023]
Abstract
Hepatitis C viral infection (HCV) results in liver damage leading to inflammation and fibrosis of the liver and increasing rates of hepatic decompensation and hepatocellular carcinoma (HCC). However, the host's immune response and viral determinants of liver disease progression are poorly understood. This review will address the determinants of liver injury in chronic HCV infection and the risk factors leading to rapid disease progression. We aim to better understand the factors that distinguish a relatively benign course of HCV from one with progression to cirrhosis. We will accomplish this task by discussion of three topics: (1) the role of cytokines in the adaptive immune response against the HCV infection; (2) the progression of fibrosis; and (3) the risk factors of co-morbidity with alcohol and human immunodeficiency virus (HIV) in HCV-infected individuals. Despite recent improvements in treating HCV infection using pegylated interferon alpha (PEGIFN-alpha) and ribavirin, about half of individuals infected with some genotypes, for example genotypes 1 and 4, will not respond to treatment or cannot be treated because of contraindications. This review will also aim to describe the importance of IFN-alpha-based therapies in HCV infection, ways of monitoring them, and associated complications.
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Affiliation(s)
- Manuela G Neuman
- In Vitro Drug Safety and Biotechnology, Department of Pharmacology, Biophysics and Global Health, Institute of Drug Research, University of Toronto, Toronto, ON, Canada.
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22
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Effect of cholinergic denervation on hepatic fibrosis induced by carbon tetrachloride in rats. Neurosci Lett 2008; 438:90-5. [PMID: 18472332 DOI: 10.1016/j.neulet.2008.04.048] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2008] [Revised: 04/15/2008] [Accepted: 04/15/2008] [Indexed: 11/21/2022]
Abstract
Various factors involved in the development of liver fibrosis, including hepatic stellate cells (HSCs), cholinergic nervous activity and fibrogenetic cytokines. The present study aims to investigate the role of cholinergic regulation in the promoting of liver fibrogenesis relating to bone morphogenetic protein-6 (BMP-6) and/or transforming growth factor-beta1 (TGFbeta1). We treated carbon tetrachloride (CCl(4)) into rats for eight weeks to induce liver fibrosis and arranged these rats for cholinergic denervation, hepatic branch vagotomy or atropine administration. Acetylcholinesterase (AChE) staining showed the distribution of cholinergic nerve around fibrosis scaring septa. The immunohistochemical staining for alpha smooth muscle actin (alphaSMA) indicated the less HSCs in CCl(4) treated rat liver with cholinergic denervation as compared to the sham-operated CCl(4) treated rats. It seems that cholinergic nerve not only innervates around the fibrosis area but also promotes HSCs. We also detected TGFbeta1 and BMP-6 expressions using RT-PCR and immunohistochemistry. The obtained results show that cholinergic denerveration decreases BMP-6 and TGF-beta1 expressions in CCl(4) induced liver fibrosis of rats. In conclusion, cholinergic nerve may influence HSCs in addition to the lowering of BMP-6 and TGF-beta1 gene expressions to modify liver fibrosis.
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23
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Du J, Yang S, Wang Z, Zhai C, Yuan W, Lei R, Zhang J, Zhu T. Bone morphogenetic protein 6 inhibit stress-induced breast cancer cells apoptosis via both smad and P38 pathways. J Cell Biochem 2008; 103:1584-97. [PMID: 17879955 DOI: 10.1002/jcb.21547] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Breast carcinoma is one of the most common malignant tumors and has become a more common cancer in women. BMP6 was abnormally expressed in breast cancer specimens and cell lines. However, the contribution of BMP6 in promoting breast cancer progression remains unknown. The purpose of our study was to establish whether expression of BMP6 in breast cancer cells affect their proliferation or apoptosis and the mechanism. We found that BMP6 inhibited proliferation of MDA-MB-231 cells and blocked cell cycle at G(0)/G(1) stage. BMP6 also inhibited serum deprivation induced apoptosis in MDA-MB-231 cells. At the 4 days of serum starvation, BMP6 reduced the percentage of caspase-3 positive cells from 49% to 21%, BMP6 also reduced sub-G(1) peak induced by serum starvation. In contrast, BMP6 significantly enhanced survivin expression both at mRNA and protein levels. Dominant negative-survivin and Antisense-survivin impaired BMP6 induced antiapoptotic effect. BMP6 enhanced survivin expression at the transcription level in a Smad-dependent manner. BMP6 also played its antiapoptotic effect through activation p38 MAPK signal pathway, independent of smad/survivin pathway. These results suggested that BMP6 induced cell cycle arrest in estrogen-insensitive breast cancer cells. BMP6 inhibits stress-induced apoptosis via both Smad and p38 signal pathways.
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Affiliation(s)
- Jun Du
- Medical College of Nankai University, Tianjin, China
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24
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Babitt JL, Huang FW, Xia Y, Sidis Y, Andrews NC, Lin HY. Modulation of bone morphogenetic protein signaling in vivo regulates systemic iron balance. J Clin Invest 2007; 117:1933-9. [PMID: 17607365 PMCID: PMC1904317 DOI: 10.1172/jci31342] [Citation(s) in RCA: 346] [Impact Index Per Article: 19.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2006] [Accepted: 04/10/2007] [Indexed: 11/17/2022] Open
Abstract
Systemic iron balance is regulated by hepcidin, a peptide hormone secreted by the liver. By decreasing cell surface expression of the iron exporter ferroportin, hepcidin decreases iron absorption from the intestine and iron release from reticuloendothelial stores. Hepcidin excess has been implicated in the pathogenesis of anemia of chronic disease, while hepcidin deficiency has a key role in the pathogenesis of the iron overload disorder hemochromatosis. We have recently shown that hemojuvelin is a coreceptor for bone morphogenetic protein (BMP) signaling and that BMP signaling positively regulates hepcidin expression in liver cells in vitro. Here we show that BMP-2 administration increases hepcidin expression and decreases serum iron levels in vivo. We also show that soluble hemojuvelin (HJV.Fc) selectively inhibits BMP induction of hepcidin expression in vitro and that administration of HJV.Fc decreases hepcidin expression, increases ferroportin expression, mobilizes splenic iron stores, and increases serum iron levels in vivo. These data support a role for modulators of the BMP signaling pathway in treating diseases of iron overload and anemia of chronic disease.
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Affiliation(s)
- Jodie L Babitt
- Program in Membrane Biology and Nephrology Division, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
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25
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Xu CP, Ji WM, van den Brink GR, Peppelenbosch MP. Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver. World J Gastroenterol 2006; 12:7621-5. [PMID: 17171790 PMCID: PMC4088043 DOI: 10.3748/wjg.v12.i47.7621] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [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 characterize the expression and dynamic changes of bone morphogenetic protein (BMP)-2 in hepatocytes in the regenerating liver in rats after partial hepatectomy (PH), and examine the effects of BMP-2 on proliferation of human Huh7 hepatoma cells.
METHODS: Fifty-four adult male Wistar rats were randomly divided into three groups: A normal control (NC) group, a partial hepatectomized (PH) group and a sham operated (SO) group. To study the effect of liver regeneration on BMP-2 expression, rats were sacrificed before and at different time points after PH or the sham intervention (6, 12, 24 and 48 h). For each time point, six rats were used in parallel. Expression and distribution of BMP-2 protein were determined in regenerating liver tissue by Western blot analysis and immunohistochemistry. Effects of BMP-2 on cell proliferation of human Huh7 hepatoma cell line were assessed using an MTT assay.
RESULTS: In the normal liver strong BMP-2 expression was observed around the central and portal veins. The expression of BMP-2 decreased rapidly as measured by both immunohistochemistry and Western blot analysis. This decrease was at a maximum of 3.22 fold after 12 h and returned to normal levels at 48 h after PH. No significant changes in BMP-2 immunoreactivity were observed in the SO group. BMP-2 inhibited serum induced Huh7 cell proliferation.
CONCLUSION: BMP-2 is expressed in normal adult rat liver and negatively regulates hepatocyte proliferation. The observed down regulation of BMP-2 following partial hepatectomy suggests that such down regulation may be necessary for hepatocyte proliferation.
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Affiliation(s)
- Cui-Ping Xu
- Department of Digestive Diseases, First Clinical College, Shanxi Medical University, 85 Jiefang Nanlu, Taiyuan 030001, Shanxi Province, China.
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26
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Sammons J, Ahmed N, El-Sheemy M, Hassan HT. The role of BMP-6, IL-6, and BMP-4 in mesenchymal stem cell-dependent bone development: effects on osteoblastic differentiation induced by parathyroid hormone and vitamin D(3). Stem Cells Dev 2006; 13:273-80. [PMID: 15186723 DOI: 10.1089/154732804323099208] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Human bone marrow-derived mesenchymal stem cells (MSCs) represent an ideal source for cell therapy for inherited and degenerative diseases, bone and cartilage repair, and as target for gene therapy. The role of the combination of human parathyroid hormone (PTH) and vitamin D(3) in bone formation and mineralization has been established in several osteoblast cell culture studies. The aim of the present study was to evaluate the role of this hormonal combination alone and in the presence of bone morphogenetic protein-4 (BMP-4) or-6 (BMP-6) in inducing osteogenic differentiation of human MSC. Human MSC derived from adult normal bone marrow that are positive for CD29, CD44, CD105, and CD166 and negative for CD14, CD34, and CD45, were treated with the PTH and 1,25-dihydroxyvitamin D(3) in the presence and absence of recombinant human BMP-4 or BMP6. PTH and vitamin D(3) induced high levels of expression of two key markers of bone formation: osteocalcin and alkaline phosphatase by MSCs. BMP-6 but not BMP-4 increased osteocalcin expression induced by PTH and vitamin D(3). Both BMPs enhanced calcium formation in MSC cultures and this response was potentiated by PTH and vitamin D(3). The present results revealed a novel potent effect of PTH and vitamin D(3) plus BMPs in inducing bone development by human MSCs. These results may facilitate therapeutic utility of MSCs for bone disease and help clarify mechanisms involved in stem cell-mediated bone development.
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Affiliation(s)
- J Sammons
- Biomedical Science Division, University of Wolverhampton, Wolverhampton WV1 1ST, UK
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Babitt JL, Huang FW, Wrighting DM, Xia Y, Sidis Y, Samad TA, Campagna JA, Chung RT, Schneyer AL, Woolf CJ, Andrews NC, Lin HY. Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression. Nat Genet 2006; 38:531-9. [PMID: 16604073 DOI: 10.1038/ng1777] [Citation(s) in RCA: 779] [Impact Index Per Article: 41.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2005] [Accepted: 03/07/2006] [Indexed: 02/07/2023]
Abstract
Hepcidin is a key regulator of systemic iron homeostasis. Hepcidin deficiency induces iron overload, whereas hepcidin excess induces anemia. Mutations in the gene encoding hemojuvelin (HFE2, also known as HJV) cause severe iron overload and correlate with low hepcidin levels, suggesting that hemojuvelin positively regulates hepcidin expression. Hemojuvelin is a member of the repulsive guidance molecule (RGM) family, which also includes the bone morphogenetic protein (BMP) coreceptors RGMA and DRAGON (RGMB). Here, we report that hemojuvelin is a BMP coreceptor and that hemojuvelin mutants associated with hemochromatosis have impaired BMP signaling ability. Furthermore, BMP upregulates hepatocyte hepcidin expression, a process enhanced by hemojuvelin and blunted in Hfe2-/- hepatocytes. Our data suggest a mechanism by which HFE2 mutations cause hemochromatosis: hemojuvelin dysfunction decreases BMP signaling, thereby lowering hepcidin expression.
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Affiliation(s)
- Jodie L Babitt
- Program in Membrane Biology and Nephrology Division, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
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28
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Zavala-Góngora R, Kroner A, Bernthaler P, Knaus P, Brehm K. A member of the transforming growth factor-beta receptor family from Echinococcus multilocularis is activated by human bone morphogenetic protein 2. Mol Biochem Parasitol 2006; 146:265-71. [PMID: 16434111 DOI: 10.1016/j.molbiopara.2005.12.011] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2005] [Revised: 12/16/2005] [Accepted: 12/19/2005] [Indexed: 10/25/2022]
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Li H, Li JZ, Pittman DD, Amalfitano A, Hankins GR, Helm GA. Comparison of osteogenic potentials of human rat BMP4 and BMP6 gene therapy using [E1-] and [E1-,E2b-] adenoviral vectors. Int J Med Sci 2006; 3:97-105. [PMID: 16761078 PMCID: PMC1475427 DOI: 10.7150/ijms.3.97] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/02/2006] [Accepted: 05/31/2006] [Indexed: 11/15/2022] Open
Abstract
Osteogenic potentials of some recombinant human bone morphogenetic protein (BMP) first-generation adenoviral vectors (ADhBMPs) are significantly limited in immunocompetent animals. It is unclear what role expression of viral proteins and foreign proteins transduced by adenoviral vectors play in the host immune response and in ectopic bone formation. In this study two sets of experiments were designed and performed. First, rat BMP6 cDNA were amplified, sequenced, and recombined in first-generation adenoviral vector (ADrBMP6). A comparison of human and rat BMP6 adenoviral vectors demonstrated identical osteogenic activities in both immunodeficient and immunocompetent rats. Second, the activities of recombinant human BMP6 in E1- (ADhBMP6) and [E1-,E2b-] ( [E1-,E2b-]ADGFP&hBMP6, and [E1-,E2b-]ADhBMP6) adenoviral vectors were compared in both in vitro and in vivo models. Similar activities of these two generations of BMP adenoviral vectors were found in all models. These results indicate that the amount of viral gene expression and the source of the BMP cDNA are not major factors in the interruption of osteogenic potentials of recombinant BMP6 adenoviral vectors in immunocompetent animals.
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Affiliation(s)
- Hongwei Li
- Department of Neurological Surgery, University of Virginia Health System, Charlottesville, Virginia 22908, USA
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30
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Abstract
The main non-parenchymal cells of the liver, Kupffer cells, sinusoidal endothelial cells and stellate cells, participate in liver growth with respect to both their own proliferation, and effects on hepatocyte proliferation. In the well-characterised paradigm of 70% partial hepatectomy, they undergo DNA synthesis and cell division 20-24h later than the hepatocyte population. They exert both positive and negative influences on hepatocyte proliferation, including provision of an extracellular matrix-bound reservoir of hepatocyte growth factor that is activated after damage; priming of hepatocytes for DNA synthesis through rapid generation of TNF-alpha and IL-6; and generation of factors at later time points that curb hepatocyte DNA synthesis (IL-1, TGF-beta) and initiate reconstruction and reformation of matrix proteins.
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Affiliation(s)
- Raza Malik
- Centre for Hepatology, Royal Free and University College Medical School, Rowland Hill Street, Hampstead, NW3 2PF, London, UK
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31
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Saile B, Matthes N, Neubauer K, Eisenbach C, El-Armouche H, Dudas J, Ramadori G. Rat liver myofibroblasts and hepatic stellate cells differ in CD95-mediated apoptosis and response to TNF-alpha. Am J Physiol Gastrointest Liver Physiol 2002; 283:G435-44. [PMID: 12121892 DOI: 10.1152/ajpgi.00441.2001] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Hepatic stellate cells (HSC), particularly activated HSC, are thought to be the principle matrix-producing cell of the diseased liver. However, other cell types of the fibroblast lineage, especially the rat liver myofibroblasts (rMF), also have fibrogenic potential. A major difference between the two cell types is the different life span under culture conditions. Although nearly no spontaneous apoptosis could be shown in rMF cultures, 18 +/- 2% of the activated HSC (day 7) were apoptotic. Compared with activated HSC, CD95R was expressed in 70% higher amounts in rMF. CD95L could only be detected in activated HSC. Stimulation of the CD95 system by agonistic antibodies (1 ng/ml) led to apoptosis of all rMF within 2 h, whereas activated HSC were more resistant (5.3 h/ 40% of total cells). Although transforming growth factor-beta downregulated apoptosis in both activated HSC and rMF, tumor necrosis factor-alpha (TNF-alpha) upregulated apoptosis in rMF. Lack of spontaneous apoptosis and CD95L expression in rMF and the different reaction on TNF-alpha stimulation reveal that activated HSC and rMF belong to different cell populations.
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Affiliation(s)
- Bernhard Saile
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, University of Göttingen, Germany
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32
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Simon M, Feliers D, Arar M, Bhandari B, Abboud HE. Cloning of the 5'-flanking region of the murine bone morphogenetic protein-7 gene. Mol Cell Biochem 2002; 233:31-7. [PMID: 12083377 DOI: 10.1023/a:1015546615027] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
BMP-7, a member of the bone morphogenetic protein subfamily of the TGFbeta-superfamily is highly expressed in the murine kidney. BMP-7 is involved in fetal nephron development and mesenchymal to epithelial cell differentiation. Constitutive BMP-7 expression is found in tubular and glomerular epithelial cells of the adult kidney. BMP-7 may play a role in physiology and pathophysiology of the adult kidney since BMP-7 gene expression in acute renal ischemia is diminished and injection of recombinant BMP-7 into rats with ischemic acute renal failure preserves renal function. In order to investigate the transcriptional regulation of BMP-7, this study was undertaken to clone and characterize the promoter of the murine BMP-7 gene. A 1394 bp sequence of the 5'-flanking region of the BMP-7 gene was isolated and subcloned. No TATA and CAAT box consensus motifs could be identified as shown for promoters of other BMPs. Using in vitro transfection assays, the 5'-flanking region revealed moderate to strong basal promoter activity. PMA increased basal BMP-7 promoter activity. Thus BMP-7 gene transcription might involve at least in part a PKC-dependent pathway. The cloning of a 5'-flanking region of the BMP-7 gene should provide a useful tool for future studies on the transcriptional regulation of BMP-7 gene expression.
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Affiliation(s)
- Matthias Simon
- South Texas Veterans Health Care System, Department of Medicine, University of Texas Health Science Center, San Antonio, USA.
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33
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Azari K, Doll BA, Sfeir C, Mu Y, Hollinger JO. Therapeutic potential of bone morphogenetic proteins. Expert Opin Investig Drugs 2001; 10:1677-86. [PMID: 11772277 DOI: 10.1517/13543784.10.9.1677] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Recently, there has been substantial progress in the area of bone morphogenetic protein (BMP) research. This review serves as an up-to-date summary of the history of BMPs, the mechanisms of BMP signalling and the role of BMPs in adipose, kidney, liver, bone and nervous system. The potential of BMPs as therapeutic agents will also be discussed.
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Affiliation(s)
- K Azari
- Bone Tissue Engineering Center, Carnegie Mellon University, 125 Smith Hall, 5000 Forbes Avenue, Pittsburgh, PA 15213, USA.
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34
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Abstract
The transforming growth factor beta (TGF-beta) superfamily has profound effects on many aspects of animal development. In the last decade, our laboratory and others have performed in vivo functional studies on multiple components of the TGF-beta superfamily signal transduction pathway, including upstream ligands, transmembrane receptors, receptor-associated proteins and downstream Smad proteins. We have taken gene knockout approaches to generate null alleles of the genes of interest, as well as a gene knockin approach to replace the mature region of one TGF-beta superfamily ligand with another. We found that activin betaB, expressed in the spatiotemporal pattern of activin betaA, can function as a hypomorphic allele of activin betaA and rescue the craniofacial defects and neonatal lethal phenotype of activin betaA-deficient mice. With the knockout approach, we have shown that the expression pattern of a component in the TGF-beta superfamily signal transduction cascade does not necessarily predict its in vivo function. Two liver-specific activins, activin betaC and activin betaE are dispensable for liver development, regeneration and function, whereas ubiquitously expressed Smad5 has specific roles in the development of multiple embryonic and extraembryonic tissues.
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Affiliation(s)
- H Chang
- Department of Pathology, Baylor College of Medicine, One Baylor Plaza, 77030, Houston, TX, USA
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35
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Tamada H, Kitazawa R, Gohji K, Kitazawa S. Epigenetic regulation of human bone morphogenetic protein 6 gene expression in prostate cancer. J Bone Miner Res 2001; 16:487-96. [PMID: 11277266 DOI: 10.1359/jbmr.2001.16.3.487] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor-beta (TGF-beta) superfamily, are multifunctional molecules that regulate bone induction and organ development. Among BMPs, BMP-6 has been shown to be overexpressed in prostate cancer and is speculated to be associated with bone-forming skeletal metastasis. We investigated the regulatory mechanism of the BMP-6 gene expression in prostate cancer cell lines DU-145, LNCaP, PC-3, and PC-3M with regard to the methylation status of the CpG island in the 5' flanking region of the human BMP-6 gene. By sequence-specific analysis of methylated cytosines, we show here that the methylation status of the CpG loci around the Sp1 site of the BMP-6 promoter is related to its steady-state expression and an alternative splicing of messenger RNA (mRNA) in prostate cancer cell lines. Furthermore, a study of clinical cases of benign and malignant prostate lesion by in situ hybridization showed that BMP-6 expression was high at both primary and secondary sites in cases of advanced cancer with metastasis. Demethylation of the CpG loci around the Spl binding site was shown in cases with high BMP-6 expression by sequencing analysis of the methylated cytosine from paraffin-embedded materials. Our results suggested that during cancer progression, besides inactivation of tumor suppressor genes by hypermethylation, activation of certain genes like BMP-6 by selective demethylation was a common epigenetic event giving a variable character to the invading and metastasizing cancer cells.
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Affiliation(s)
- H Tamada
- Second Department of Pathology, University School of Medicine, Japan
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36
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Lau AL, Kumar TR, Nishimori K, Bonadio J, Matzuk MM. Activin betaC and betaE genes are not essential for mouse liver growth, differentiation, and regeneration. Mol Cell Biol 2000; 20:6127-37. [PMID: 10913194 PMCID: PMC86088 DOI: 10.1128/mcb.20.16.6127-6137.2000] [Citation(s) in RCA: 106] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2000] [Accepted: 05/16/2000] [Indexed: 11/20/2022] Open
Abstract
The liver is an essential organ that produces several serum proteins, stores vital nutrients, and detoxifies many carcinogenic and xenobiotic compounds. Various growth factors positively regulate liver growth, but only a few negative regulators are known. Among the latter are the transforming growth factor beta (TGF-beta) superfamily members TGF-beta1 and activin A. To study the function of novel activin family members, we have cloned and generated mice deficient in the activin betaC and betaE genes. Expression analyses demonstrated that these novel genes are liver specific in adult mice. Here, we show by RNase protection that activin betaC transcripts are present in the liver beginning at embryonic day 11.5 (E11.5) whereas activin betaE expression is detected starting from E17.5. Gene targeting in embryonic stem cells was used to generate mice with null mutations in either the individual activin betaC and betaE genes or both genes. In contrast to the structurally related activin betaA and betaB subunits, which are necessary for embryonic development and pituitary follicle-stimulating hormone homeostasis, mice deficient in activin betaC and betaE were viable, survived to adulthood, and demonstrated no reproductive abnormalities. Although activin betaC and betaE mRNAs are abundantly expressed in the liver of wild-type mice, the single and double mutants did not show any defects in liver development and function. Furthermore, in the homozygous mutant mice, liver regeneration after >70% partial hepatectomy was comparable to that in wild-type mice. Our results suggest that activin betaC and betaE are not essential for either embryonic development or liver function.
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Affiliation(s)
- A L Lau
- Departments of Pathology, Baylor College of Medicine, Houston, Texas 77030, USA
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37
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Miller AF, Harvey SA, Thies RS, Olson MS. Bone morphogenetic protein-9. An autocrine/paracrine cytokine in the liver. J Biol Chem 2000; 275:17937-45. [PMID: 10849432 DOI: 10.1074/jbc.275.24.17937] [Citation(s) in RCA: 134] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Bone morphogenetic proteins (BMPs) occupy important roles during development serving to direct cells through specific differentiation programs. While several BMPs are essential for embryonic viability, their significance in mediating intercellular communication in the context of adult organ systems remains largely unknown. In the adult rat we characterized the tissue- and cell-specific transcription and translation of BMP-9. Utilizing a ribonuclease protection assay, we determined that in the adult animal, BMP-9 expression occurs predominantly in the liver. Furthermore, we determined that the non-parenchymal cells of the liver, i.e. endothelial, Kupffer, and stellate cells, are the major sources of this message. Western analyses corroborate the ribonuclease protection assay results, confirming that LEC and KC contain an abundance of immunoreactive BMP-9. Using [(125)I]BMP-9, a receptor with specific binding affinity for BMP-9 was characterized in primary cultures of hepatic endothelial cells and Kupffer cells. BMP-9 binding to these cell types was observed to be fully reversible and highly specific for this ligand. Additionally, we demonstrate that BMP-9 is specifically internalized upon binding to its receptor. This may represent a novel BMP receptor and is the first to be characterized in primary cultures of mature liver non-parenchymal cells. Our results depict BMP-9 as a potential autocrine/paracrine mediator in the hepatic reticuloendothelial system.
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Affiliation(s)
- A F Miller
- Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7600, USA
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38
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Knittel T, Kobold D, Dudas J, Saile B, Ramadori G. Role of the Ets-1 transcription factor during activation of rat hepatic stellate cells in culture. THE AMERICAN JOURNAL OF PATHOLOGY 1999; 155:1841-8. [PMID: 10595913 PMCID: PMC1866949 DOI: 10.1016/s0002-9440(10)65502-2] [Citation(s) in RCA: 24] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
During liver tissue repair, hepatic stellate cells (HSCs), a pericyte-like nonparenchymal liver cell population, transform from a quiescent status (resting HSCs) into myofibroblast like cells (activated HSCs); the latter is the principal matrix-synthesizing cell of the liver. Although several factors have been shown to be involved in this important process, the molecular mechanisms regulating HSC activation are still under investigation. To identify key regulatory proteins involved in the HSC activation process, we used different mRNA display technologies, with cDNAs prepared from HSCs at different stages of in vitro activation. With the latter technique, the transcription factor Ets-1 was detected through its down-regulation during activation. As confirmed by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, mRNAs coding for Ets-1 were present in the highest amounts in freshly isolated HSCs and in HSCs 2 days after plating (classified as resting HSCs/early activated HSCs) and were diminished in HSCs 7 days after plating (activated cells). Ets-1 protein was present in HSC-lysates, as assessed by Western blot, and bound to an oligonucleotide containing the Ets-1 consensus cis-acting motif, as demonstrated by electrophoretic mobility shift assay. Ets-1 binding activity peaked in nuclear extracts prepared from resting/early activated cells and was diminished in extracts derived from fully activated cells. In contrast, binding activity of the transcription factors TFIID, AP-1, and SP-1 was highest in activated HSCs and only barely detectable in resting/early activated HSCs. By Northern blot and RT-PCR analysis, Ets-1-specific transcripts were present in parenchymal and other nonparenchymal liver cells too, illustrating that hepatic Ets-1 expression is not specific or restricted to HSCs. However, the unique pattern of Ets-1 binding activity present in resting versus activated HSCs and its known implications for cellular differentiation and tissue remodeling suggest that Ets-1 could be of crucial importance for HSC activation and hepatic tissue repair.
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Affiliation(s)
- T Knittel
- Department of Internal Medicine, Section of Gastroenterology and Endocrinology, University of Göttingen, Göttingen, Germany.
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Knittel T, Kobold D, Saile B, Grundmann A, Neubauer K, Piscaglia F, Ramadori G. Rat liver myofibroblasts and hepatic stellate cells: different cell populations of the fibroblast lineage with fibrogenic potential. Gastroenterology 1999; 117:1205-21. [PMID: 10535885 DOI: 10.1016/s0016-5085(99)70407-5] [Citation(s) in RCA: 282] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
BACKGROUND & AIMS Hepatic stellate cells (HSCs) are considered the principal matrix-producing cells of the damaged liver. However, other cell types of the fibroblast lineage that have not yet been characterized are also involved in liver tissue repair and fibrogenesis. METHODS We established cultures of cells of the fibroblast lineage, termed rat liver myofibroblasts, and analyzed their phenotypical and functional properties in comparison with HSCs. RESULTS HSCs and rat liver myofibroblasts were discernible by morphological criteria and growth behavior. Prolonged subcultivation of rat liver myofibroblasts was achieved, but HSCs were maintained in culture at maximum until second passage. HSCs were characterized by expression of glial fibrillary acidic protein, desmin, and vascular cell adhesion molecule 1, which were almost completely absent in rat liver myofibroblasts. For synthetic properties, HSCs and rat liver myofibroblasts displayed mostly overlapping properties with 4 striking differences. The complement-activating protease P100 and the protease inhibitor alpha(2)-macroglobulin were preferentially expressed by HSCs, whereas interleukin 6-coding messenger RNAs and the extracellular matrix protein fibulin 2 were almost exclusively detectable in rat liver myofibroblasts. CONCLUSIONS The data show that morphologically and functionally different fibroblastic populations, HSCs and rat liver myofibroblasts, can be derived from liver tissue. HSCs may not represent the single matrix-producing cell type of the fibroblast lineage in the liver.
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Affiliation(s)
- T Knittel
- Section of Gastroenterology, Department of Internal Medicine, University of Göttingen, Göttingen, Germany
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40
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Simon M, Maresh JG, Harris SE, Hernandez JD, Arar M, Olson MS, Abboud HE. Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney. THE AMERICAN JOURNAL OF PHYSIOLOGY 1999; 276:F382-9. [PMID: 10070161 DOI: 10.1152/ajprenal.1999.276.3.f382] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
BMP-7, a member of the bone morphogenic protein subfamily (BMPs) of the transforming growth factor-beta superfamily of secreted growth factors, is abundantly expressed in the fetal kidney. The precise role of this protein in renal physiology or pathology is unknown. A cDNA that encodes rat BMP-7 was cloned and used as a probe to localize BMP-7 mRNA expression by in situ hybridization in the adult rat kidney. The highest expression of BMP-7 mRNA could be seen in tubules of the outer medulla. In glomeruli, a few cells, mainly located at the periphery of the glomerular tuft, showed specific and strong signals. Also, high BMP-7 mRNA expression could be localized to the adventitia of renal arteries, as well as to the epithelial cell layer of the renal pelvis and the ureter. Preliminary evidence suggests that BMP-7 enhances recovery when infused into rats with ischemia-induced acute renal failure. We examined BMP-7 mRNA expression in kidneys with acute renal failure induced by unilateral renal artery clamping. BMP-7 mRNA abundance as analyzed by solution hybridization was reduced in ischemic kidneys after 6 and 16 h of reperfusion compared with the contralateral kidney. In situ hybridization in ischemic kidneys showed a marked decrease of BMP-7 mRNA in the outer medulla and in glomeruli. Utilizing rat metanephric mesenchymal cells in culture, we also demonstrate that BMP-7 induces epithelial cell differentiation. Taken together, these data suggest that BMP-7 is important in both stimulating and maintaining a healthy differentiated epithelial cell phenotype.
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Affiliation(s)
- M Simon
- Division of Nephrology, University of Texas Health Science Center, and Audie L. Murphy Memorial Veterans Affairs Hospital San Antonio, Texas 78284, USA
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41
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Clement JH, Sänger J, Höffken K. Expression of bone morphogenetic protein 6 in normal mammary tissue and breast cancer cell lines and its regulation by epidermal growth factor. Int J Cancer 1999; 80:250-6. [PMID: 9935207 DOI: 10.1002/(sici)1097-0215(19990118)80:2<250::aid-ijc14>3.0.co;2-d] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Bone morphogenetic proteins (BMPs) are multifunctional regulators of proliferation, differentiation and apoptosis. BMP-6 is involved in numerous developmental processes. We have demonstrated expression of BMP-6 in breast cancer cell lines by RT-PCR and immuno-histochemistry. The level of BMP-6 mRNA decreased upon serum starvation, whereas epidermal growth factor (EGF) treatment led to elevation of BMP-6 mRNA levels in a dose-dependent manner, with a maximum at 50 ng/ml EGF under serum-free conditions in hormone-sensitive (MCF-7) and in hormone-insensitive (SK-BR-3) breast cancer cell lines. The EGF-like growth factors transforming growth factor-alpha, amphiregulin and betacellulin were also able to elevate the BMP-6 mRNA level after 24 hr. Inhibition of EGF receptor tyrosine kinase with tyrphostine AG 1517 repressed the inductive effect of these growth factors, indicating an EGF receptor-mediated regulation of BMP-6 mRNA. In addition, BMP-6 mRNA was detected in tumor samples from breast carcinoma patients. However, levels were reduced in 18/44 samples compared with tumor-free resection margins. In 12 of these 18 patients, at least a 10-fold reduction of EGF receptor mRNA levels in tumor samples vs. tumor-free samples was observed. This suggests a putative relationship between EGF receptor and BMP-6 mRNA levels in breast cancer.
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Affiliation(s)
- J H Clement
- Department of Internal Medicine II, Friedrich Schiller University of Jena, Germany.
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42
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Knittel T, Mehde M, Kobold D, Saile B, Dinter C, Ramadori G. Expression patterns of matrix metalloproteinases and their inhibitors in parenchymal and non-parenchymal cells of rat liver: regulation by TNF-alpha and TGF-beta1. J Hepatol 1999; 30:48-60. [PMID: 9927150 DOI: 10.1016/s0168-8278(99)80007-5] [Citation(s) in RCA: 253] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
BACKGROUND/AIMS Although matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs) play an essential role in liver injury associated with tissue remodeling, the cellular origin of MMPs/TMPs within the liver remains to be clarified. METHODS Different liver cell populations were analysed with respect to their expression by reverse transcription-polymerase chain reaction, Northern blot analysis and zymography. RESULTS MMP and TIMP coding transcripts were detectable in all liver cell types by reverse transcription-polymerase chain reaction; however, the cellular expression levels were markedly different as assessed by Northern blot analysis. Gelatinase-B was predominantly expressed in Kupffer cells, gelatinase-A in hepatic stellate cells and rat liver myofibroblasts and stromelysins-1, -2 as well as collagenase in hepatic stellate cells. Membrane type-1 MMP (MMP-14) was found in significant amounts in all liver cells. TIMP-1 coding m-RNAs were present mainly in hepatic stellate cells and rat liver myofibroblasts, TIMP-2 additionally in Kupffer cells, while TIMP-3 expression was detectable only in hepatocytes. During in vitro activation of hepatic stellate cells, MMP expression was mostly downregulated, while TIMP expression was enhanced, thereby providing an explanation for matrix accumulation co-localised with these cells during chronic liver injury. In general, TNF-alpha stimulated both MMP and TIMP expression of hepatic stellate cells, while TGF-beta1 induced TIMP expression only. CONCLUSIONS Collectively these data demonstrate that all resident liver cells are involved in matrix degradation to some extent and that hepatic stellate cells play an important role in matrix breakdown in addition to matrix synthesis. The cytokine-specific regulation of MMP/TIMP expression in hepatic stellate cells suggests that the initial matrix breakdown following liver injury might be enhanced by TNF-alpha, while diminished matrix degradation during chronic tissue injury might be due to the action of TGF-beta1 through TIMP induction.
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Affiliation(s)
- T Knittel
- Department of Internal Medicine, University of Göttingen, Germany
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43
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Knittel T, Dinter C, Kobold D, Neubauer K, Mehde M, Eichhorst S, Ramadori G. Expression and regulation of cell adhesion molecules by hepatic stellate cells (HSC) of rat liver: involvement of HSC in recruitment of inflammatory cells during hepatic tissue repair. THE AMERICAN JOURNAL OF PATHOLOGY 1999; 154:153-67. [PMID: 9916930 PMCID: PMC1853435 DOI: 10.1016/s0002-9440(10)65262-5] [Citation(s) in RCA: 102] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Hepatic stellate cells (HSC), a pericyte-like nonparenchymal liver cell population, are regarded as the principal matrix-synthesizing cells of fibrotic liver. They might also play a role during liver inflammation. The present study analyzed (i) expression of cell adhesion molecules (CAMs) mediating cell infiltration, like intercellular adhesion molecule-1 (I-CAM-1) and vascular cell adhesion molecule-1 (V-CAM-1), by HSC, (ii) CAM regulation in HSC by growth factors and inflammatory cytokines, and (iii) CAM expression in situ during liver inflammation, using immunochemistry and Northern blot analysis. I-CAM-1 and V-CAM-1 expression was present in HSC in vitro and in cells located in the sinusoidal/perisinusoidal area of normal liver. Growth factors, eg, transforming growth factor-beta1, down-regulated I-CAM-1- and V-CAM-1-coding mRNAs and stimulated N-CAM expression of HSC. In contrast, inflammatory cytokines like tumor necrosis factor-alpha reduced N-CAM-coding mRNAs, whereas induction of I-CAM-1- and V-CAM-1-specific transcripts increased several fold. In situ, messengers specific for I-CAM-1 and V-CAM-1 were induced 3 hours after CCl4 treatment (thereby preceding mononuclear cell infiltration starting at 12 hours), were expressed at maximal levels 9-12 hours after CCl4 application, and decreased afterwards. I-CAM-1 and V-CAM-1 immunoreactivity increased in a linear fashion starting 3 hours after CCl4-induced liver injury, was detected in highest amounts at 24-48 hours characterized by maximal cell infiltration, and returned to baseline values at 96 hours. Interestingly, the induction/repression of CAM-specific messengers paralleled the time kinetics of tumor necrosis factor-alpha transforming growth factor-beta1 expression in injured liver. HSC might be important during the onset of hepatic tissue injury as proinflammatory elements and might interact with I-CAM-1 and V-CAM-1 ligand-bearing cells, namely lymphocyte function-associated antigen-1- or Mac-1/very late activation antigen-4-positive inflammatory cells, thereby modulating the recruitment and migration of mononuclear cells within the perisinusoidal space of diseased livers.
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Affiliation(s)
- T Knittel
- Department of Internal Medicine, University of Göttingen, Germany
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