title: The PNPLA3 I148M variant modulates the fibrogenic phenotype of human hepatic stellate cells.
abstract: The genetic polymorphism I148M of patatin-like phospholipase domain-containing 3 (PNPLA3) is robustly associated with hepatic steatosis and its progression to steatohepatitis, fibrosis, and cancer. Hepatic stellate cells (HSCs) are key players in the development of liver fibrosis, but the role of PNPLA3 and its variant I148M in this process is poorly understood. Here we analyzed the expression of PNPLA3 during human HSC activation and thereby explored how a PNPLA3 variant impacts hepatic fibrogenesis. We show that expression of PNPLA3 gene and protein increases during the early phases of activation and remains elevated in fully activated HSCs (P < 0.01). Knockdown of PNPLA3 significantly decreases the profibrogenic protein alpha-smooth muscle actin (P < 0.05). Primary human I148M HSCs displayed significantly higher expression and release of proinflammatory cytokines, such as chemokine (C-C motif) ligand 5 (P < 0.01) and granulocyte-macrophage colony-stimulating factor (P < 0.001), thus contributing to migration of immune cells (P < 0.05). Primary I148M HSCs showed reduced retinol (P < 0.001) but higher lipid droplet content (P < 0.001). In line with this, LX-2 cells stably overexpressing I148M showed augmented proliferation and migration, lower retinol, and abolished retinoid X receptor/retinoid A receptor transcriptional activities but more lipid droplets. Knockdown of I148M PNPLA3 (P < 0.001) also reduces chemokine (C-C motif) ligand 5 and collagen1α1 expression (P < 0.05). Notably, I148M cells display reduced peroxisome proliferator-activated receptor gamma transcriptional activity, and this effect was attributed to increased c-Jun N-terminal kinase, thereby inhibiting peroxisome proliferator-activated receptor gamma through serine 84 phosphorylation and promoting activator protein 1 transcription. Conversely, the c-Jun N-terminal kinase inhibitor SP600125 and the peroxisome proliferator-activated receptor gamma agonist rosiglitazone decreased activator protein 1 promoter activity.
  
  
  
  These data indicate that PNPLA3 is required for HSC activation and that its genetic variant I148M potentiates the profibrogenic features of HSCs, providing a molecular mechanism for the higher risk of progression and severity of liver diseases conferred to patients carrying the I148M variant. (Hepatology 2017;65:1875-1890).
authors.affiliation: Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology & Hepatology, Medical University of Vienna, Vienna, Austria.
authors.forename: Francesca Virginia
authors.initials: FV
authors.lastname: Bruschi
authors.affiliation: Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology & Hepatology, Medical University of Vienna, Vienna, Austria.
authors.forename: Thierry
authors.initials: T
authors.lastname: Claudel
authors.affiliation: Christian Doppler-Laboratory for Cardio-Metabolic Immunotherapy and Clinical Division of Endocrinology and Metabolism, Internal Medicine III, Medical University of Vienna, Vienna, Austria.
authors.forename: Matteo
authors.initials: M
authors.lastname: Tardelli
authors.affiliation: Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Caligiuri
authors.affiliation: Christian Doppler-Laboratory for Cardio-Metabolic Immunotherapy and Clinical Division of Endocrinology and Metabolism, Internal Medicine III, Medical University of Vienna, Vienna, Austria.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Stulnig
authors.affiliation: Christian Doppler-Laboratory for Cardio-Metabolic Immunotherapy and Clinical Division of Endocrinology and Metabolism, Internal Medicine III, Medical University of Vienna, Vienna, Austria.
authors.forename: Fabio
authors.initials: F
authors.lastname: Marra
authors.affiliation: Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology & Hepatology, Medical University of Vienna, Vienna, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Trauner
mesh_terms: D015153:Blotting, Western; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D002478:Cells, Cultured; D002849:Chromatography, Gas / Q000379:methods; D016207:Cytokines / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D005786:Gene Expression Regulation; D020022:Genetic Predisposition to Disease; D055166:Hepatic Stellate Cells / Q000378:metabolism*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008049:Lipase / Q000235:genetics*; D010641:Phenotype; D011110:Polymorphism, Genetic; D060888:Real-Time Polymerase Chain Reaction; D012016:Reference Values; D000217:Acyltransferases
chemical_list: D016207:Cytokines; D048031:JNK Mitogen-Activated Protein Kinases; D008049:Lipase; C091246:PNPLA4 protein, human; D000217:Acyltransferases