Bone morphogenetic protein 9 as a key regulator of liver progenitor cells in DDC-induced cholestatic liver injury
Annalisa Addante
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorCesáreo Roncero
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorLaura Almalé
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorNerea Lazcanoiturburu
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorMaría García-Álvaro
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorMargarita Fernández
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorJulián Sanz
Department Pathology, Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorSeddik Hammad
Medical Faculty Mannheim, Department Medicine II, Heidelberg University, Manhheim, Germany
Search for more papers by this authorZeribe C. Nwosu
Medical Faculty Mannheim, Department Medicine II, Heidelberg University, Manhheim, Germany
Search for more papers by this authorSe-Jin Lee
Department Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Search for more papers by this authorIsabel Fabregat
Bellvitge Biomedical Research Institute, L’Hospitalet de Llobregat, Barcelona, Spain
Search for more papers by this authorSteven Dooley
Medical Faculty Mannheim, Department Medicine II, Heidelberg University, Manhheim, Germany
Search for more papers by this authorPeter ten Dijke
Department Cell and Chemical Biology and Oncode Institute, Leiden University Medical Center, RC, Leiden, The Netherlands
Search for more papers by this authorBlanca Herrera
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorCorresponding Author
Aránzazu Sánchez
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Correspondence
Aránzazu Sánchez, Faculty of Pharmacy, Department of Biochemistry and Molecular Biology, Complutense University of Madrid, Madrid, Spain.
Email: [email protected]
Search for more papers by this authorAnnalisa Addante
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorCesáreo Roncero
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorLaura Almalé
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorNerea Lazcanoiturburu
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorMaría García-Álvaro
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorMargarita Fernández
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorJulián Sanz
Department Pathology, Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorSeddik Hammad
Medical Faculty Mannheim, Department Medicine II, Heidelberg University, Manhheim, Germany
Search for more papers by this authorZeribe C. Nwosu
Medical Faculty Mannheim, Department Medicine II, Heidelberg University, Manhheim, Germany
Search for more papers by this authorSe-Jin Lee
Department Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Search for more papers by this authorIsabel Fabregat
Bellvitge Biomedical Research Institute, L’Hospitalet de Llobregat, Barcelona, Spain
Search for more papers by this authorSteven Dooley
Medical Faculty Mannheim, Department Medicine II, Heidelberg University, Manhheim, Germany
Search for more papers by this authorPeter ten Dijke
Department Cell and Chemical Biology and Oncode Institute, Leiden University Medical Center, RC, Leiden, The Netherlands
Search for more papers by this authorBlanca Herrera
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Search for more papers by this authorCorresponding Author
Aránzazu Sánchez
Faculty of Pharmacy, Department Biochemistry and Molecular Biology, Complutense University of Madrid, Health Research Institute of the Hospital Clínico San Carlos, Madrid, Spain
Correspondence
Aránzazu Sánchez, Faculty of Pharmacy, Department of Biochemistry and Molecular Biology, Complutense University of Madrid, Madrid, Spain.
Email: [email protected]
Search for more papers by this authorFunding Information
This work was supported by a Marie Curie Action of FP7-2012 (Grant #PITN-GA-2012-316549; IT-LIVER); Ministry of Economy and Competitiveness, Spain (Grant #SAF2015-69145-R); Health Research Fund-Institute of Health Carlos III, Spain (Grant #PI10/00274); and General Direction of Universities and Research of the Autonomous Community of Madrid, Spain (Grant #S2010/BMD-2402).
Abstract
Background & Aims
Bone morphogenetic protein 9 (BMP9) interferes with liver regeneration upon acute injury, while promoting fibrosis upon carbon tetrachloride-induced chronic injury. We have now addressed the role of BMP9 in 3,5 diethoxicarbonyl-1,4 dihydrocollidine (DDC)-induced cholestatic liver injury, a model of liver regeneration mediated by hepatic progenitor cell (known as oval cell), exemplified as ductular reaction and oval cell expansion.
Methods
WT and BMP9KO mice were submitted to DDC diet. Livers were examined for liver injury, fibrosis, inflammation and oval cell expansion by serum biochemistry, histology, RT-qPCR and western blot. BMP9 signalling and effects in oval cells were studied in vitro using western blot and transcriptional assays, plus functional assays of DNA synthesis, cell viability and apoptosis. Crosslinking assays and short hairpin RNA approaches were used to identify the receptors mediating BMP9 effects.
Results
Deletion of BMP9 reduces liver damage and fibrosis, but enhances inflammation upon DDC feeding. Molecularly, absence of BMP9 results in overactivation of PI3K/AKT, ERK-MAPKs and c-Met signalling pathways, which together with an enhanced ductular reaction and oval cell expansion evidence an improved regenerative response and decreased damage in response to DDC feeding. Importantly, BMP9 directly targets oval cells, it activates SMAD1,5,8, decreases cell growth and promotes apoptosis, effects that are mediated by Activin Receptor-Like Kinase 2 (ALK2) type I receptor.
Conclusions
We identify BMP9 as a negative regulator of oval cell expansion in cholestatic injury, its deletion enhancing liver regeneration. Likewise, our work further supports BMP9 as an attractive therapeutic target for chronic liver diseases.
CONFLICT OF INTEREST
The authors do not have any disclosures to report.
Supporting Information
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| liv13879-sup-0003-TableS2.docxWord document, 18.7 KB | |
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REFERENCES
- 1Kohn-Gaone J, Gogoi-Tiwari J, Ramm GA, Olynyk JK, Tirnitz-Parker JE. The role of liver progenitor cells during liver regeneration, fibrogenesis, and carcinogenesis. Am J Physiol Gastrointest Liver Physiol. 2016; 310: G143-G154.
- 2Alison MR, Lin WR. Regenerative medicine: hepatic progenitor cells up their game in the therapeutic stakes. Nat Rev Gastroenterol Hepatol. 2015; 12: 610-611.
- 3Lu WY, Bird TG, Boulter L, et al. Hepatic progenitor cells of biliary origin with liver repopulation capacity. Nat Cell Biol. 2015; 17: 971-983.
- 4Chen J, Chen L, Zern MA, et al. The diversity and plasticity of adult hepatic progenitor cells and their niche. Liver Int 2017; 37: 1260-1271.
- 5Giannelli G, Mikulits W, Dooley S, et al. The rationale for targeting TGF-beta in chronic liver diseases. Eur J Clin Invest. 2016; 46: 349-361.
- 6Herrera B, Addante A, Sanchez A. BMP signalling at the crossroad of liver fibrosis and regeneration. Int J Mol Sci 2017; 19:pii: E39.
- 7Bidart M, Ricard N, Levet S, et al. BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain. Cell Mol Life Sci 2012; 69: 313-324.
- 8Miller AF, Harvey SA, Thies RS, Olson MS. Bone morphogenetic protein-9. An autocrine/paracrine cytokine in the liver. J Biol Chem. 2000; 275: 17937-17945.
- 9Herrera B, Garcia-Alvaro M, Cruz S, et al. BMP9 is a proliferative and survival factor for human hepatocellular carcinoma cells. PLoS ONE. 2013; 8: e69535.
- 10Li Q, Gu X, Weng H, et al. Bone morphogenetic protein-9 (BMP-9) induces epithelial to mesenchymal transition (EMT) in hepatocellular carcinoma cells. Cancer Sci. 2013; 104: 398-408.
- 11Li P, Li Y, Zhu L, et al. Targeting secreted cytokine BMP9 gates the attenuation of hepatic fibrosis. Biochim Biophys Acta. 2017; 1864: 709-720.
- 12Breitkopf-Heinlein K, Meyer C, Konig C, et al. BMP-9 interferes with liver regeneration and promotes liver fibrosis. Gut. 2017; 66: 939-954.
- 13Fickert P, Stoger U, Fuchsbichler A, et al. A new xenobiotic-induced mouse model of sclerosing cholangitis and biliary fibrosis. Am J Pathol. 2007; 171: 525-536.
- 14Jakubowski A, Ambrose C, Parr M, et al. TWEAK induces liver progenitor cell proliferation. J Clin Invest. 2005; 115: 2330-2340.
- 15Ricard N, Ciais D, Levet S, et al. BMP9 and BMP10 are critical for postnatal retinal vascular remodeling. Blood. 2012; 119: 6162-6171.
- 16Preisegger KH, Factor VM, Fuchsbichler A, et al. Atypical ductular proliferation and its inhibition by transforming growth factor beta1 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model for chronic alcoholic liver disease. Lab Invest. 1999; 79: 103-109.
- 17del Castillo G, Factor VM, Fernandez M, et al. Deletion of the Met tyrosine kinase in liver progenitor oval cells increases sensitivity to apoptosis in vitro. Am J Pathol. 2008; 172: 1238-1247.
- 18Knodell RG, Ishak KG, Black WC, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology. 1981; 1: 431-435.
- 19Pollheimer MJ, Fickert P, Stieger B. Chronic cholestatic liver diseases: clues from histopathology for pathogenesis. Mol Aspects Med. 2014; 37: 35-56.
- 20Yamazaki Y, Moore R, Negishi M. Nuclear receptor CAR (NR1I3) is essential for DDC-induced liver injury and oval cell proliferation in mouse liver. Lab Invest. 2011; 91: 1624-1633.
- 21Kitade M, Factor VM, Andersen JB, et al. Specific fate decisions in adult hepatic progenitor cells driven by MET and EGFR signaling. Genes Dev. 2013; 27: 1706-1717.
- 22Morales-Ruiz M, Santel A, Ribera J, Jimenez W. The Role of Akt in Chronic Liver Disease and Liver Regeneration. Semin Liver Dis. 2017; 37: 11-16.
- 23Gomez-Quiroz LE, Seo D, Lee YH, et al. Loss of c-Met signaling sensitizes hepatocytes to lipotoxicity and induces cholestatic liver damage by aggravating oxidative stress. Toxicology. 2016; 361–362: 39-48.
- 24Huh CG, Factor VM, Sanchez A, et al. Hepatocyte growth factor/c-met signaling pathway is required for efficient liver regeneration and repair. Proc Natl Acad Sci U S A. 2004; 101: 4477-4482.
- 25Ishikawa T, Factor VM, Marquardt JU, et al. Hepatocyte growth factor/c-met signaling is required for stem-cell-mediated liver regeneration in mice. Hepatology. 2012; 55: 1215-1226.
- 26Thompson MD, Wickline ED, Bowen WB, et al. Spontaneous repopulation of beta-catenin null livers with beta-catenin-positive hepatocytes after chronic murine liver injury. Hepatology. 2011; 54: 1333-1343.
- 27Garcia-Alvaro M, Addante A, Roncero C, et al. BMP9-Induced Survival Effect in Liver Tumor Cells Requires p38MAPK Activation. Int J Mol Sci. 2015; 16: 20431-20448.
- 28Brown MA, Zhao Q, Baker KA, et al. Crystal structure of BMP-9 and functional interactions with pro-region and receptors. J Biol Chem. 2005; 280: 25111-25118.
- 29Herrera B, van Dinther M, Ten Dijke P, Inman GJ. Autocrine bone morphogenetic protein-9 signals through activin receptor-like kinase-2/Smad1/Smad4 to promote ovarian cancer cell proliferation. Cancer Res. 2009; 69: 9254-9262.
- 30Scharpfenecker M, van Dinther M, Liu Z, et al. BMP-9 signals via ALK1 and inhibits bFGF-induced endothelial cell proliferation and VEGF-stimulated angiogenesis. J Cell Sci. 2007; 120: 964-972.
- 31Papp V, Rokusz A, Dezso K, et al. Expansion of hepatic stem cell compartment boosts liver regeneration. Stem Cells Dev. 2014; 23: 56-65.
- 32Strick-Marchand H, Weiss MC. Embryonic liver cells and permanent lines as models for hepatocyte and bile duct cell differentiation. Mech Dev. 2003; 120: 89-98.
- 33Wang X, Foster M, Al-Dhalimy M, et al. The origin and liver repopulating capacity of murine oval cells. Proc Natl Acad Sci U S A. 2003; 100(Suppl 1): 11881-11888.
- 34Clouston AD, Powell EE, Walsh MJ, et al. Fibrosis correlates with a ductular reaction in hepatitis C: roles of impaired replication, progenitor cells and steatosis. Hepatology. 2005; 41: 809-818.
- 35Pi L, Robinson PM, Jorgensen M, et al. Connective tissue growth factor and integrin alphavbeta6: a new pair of regulators critical for ductular reaction and biliary fibrosis in mice. Hepatology. 2015; 61: 678-691.
- 36Weng HL, Feng DC, Radaeva S, et al. IFN-gamma inhibits liver progenitor cell proliferation in HBV-infected patients and in 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet-fed mice. J Hepatol. 2013; 59: 738-745.
- 37Xiang S, Dong HH, Liang HF, et al. Oval cell response is attenuated by depletion of liver resident macrophages in the 2-AAF/partial hepatectomy rat. PLoS ONE. 2012; 7: e35180.
- 38Young K, Tweedie E, Conley B, et al. BMP9 Crosstalk with the Hippo Pathway Regulates Endothelial Cell Matricellular and Chemokine Responses. PLoS ONE. 2015; 10: e0122892.
- 39Zhao WM, Qin YL, Niu ZP, et al. Branches of the NF-kappaB signaling pathway regulate proliferation of oval cells in rat liver regeneration. Genet Mol Res 2016; 15: gmr7750.
- 40Giebeler A, Boekschoten MV, Klein C, et al. c-Met confers protection against chronic liver tissue damage and fibrosis progression after bile duct ligation in mice. Gastroenterology 2009; 137: 297-308. 308, e291-294.
- 41Factor VM, Seo D, Ishikawa T, et al. Loss of c-Met disrupts gene expression program required for G2/M progression during liver regeneration in mice. PLoS ONE 2010; 5: e12739.
- 42Martinez-Palacian A, del Castillo G, Suarez-Causado A, et al. Mouse hepatic oval cells require Met-dependent PI3K to impair TGF-beta-induced oxidative stress and apoptosis. PLoS ONE. 2013; 8: e53108.
- 43Wagner M, Trauner M. Recent advances in understanding and managing cholestasis. F1000Res. 2016; 5: 705.
10.12688/f1000research.8012.1 Google Scholar
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