Original Article
Copyright ©2014 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Sep 28, 2014; 20(36): 13088-13104
Published online Sep 28, 2014. doi: 10.3748/wjg.v20.i36.13088
Osteopontin is an important mediator of alcoholic liver disease via hepatic stellate cell activation
Devanshi Seth, Alastair Duly, Paul C Kuo, Geoffrey W McCaughan, Paul S Haber
Devanshi Seth, Paul S Haber, Drug Health Services, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia
Alastair Duly, Garvan Institute, Darlinghurst, NSW 2010, Australia
Devanshi Seth, Paul S Haber, Central Clinical School of Medicine, the University of Sydney, Sydney, NSW 2050, Australia
Paul C Kuo, Department of Surgery, Loyola University Medical Centre, Maywood, IL 60153, United States
Geoffrey W McCaughan, AW Morrow Gastroenterology and Liver Centre, RPAH, Camperdown, NSW 2050, Australia
Author contributions: Seth D wrote and revised the manuscript, designed, performed and supervised human, cell culture; Animal experiments performed by Duly A; Kuo PC supplied reagents and expert advice for OPN aptamer experiments; McCaughan GW provided critical review of the manuscript; Haber PS assisted with human sample collection, clinical expertise and critical review of the manuscript.
Supported by Philanthropic Anonymous Source; the University of Sydney Bridging Support Grant, in part for Honours Project; and by the National Health and Medical Research Council, No. NHMRC Practitioner Research Fellowship for PH support
Correspondence to: Devanshi Seth, Principal Scientist, Drug Health Services, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia. d.seth@sydney.edu.au
Telephone: +61-2-95157201 Fax: +61-2-95158970
Received: January 3, 2014
Revised: March 11, 2014
Accepted: April 30, 2014
Published online: September 28, 2014
Abstract

AIM: To investigate over-expression of Osteopontin (OPN) pathway expression and mechanisms of action in human alcoholic liver disease (ALD), in vivo and in vitro acute alcohol models.

METHODS: OPN pathway was evaluated in livers from patients with progressive stages of human ALD and serum from drinkers with and without liver cirrhosis. In vitro stellate LX2 cells exposed to acute alcohol and in vivo in acute alcoholic steatosis mouse models were also investigated for OPN pathway expression and function. WT and OPN-/- mice were administered an acute dose of alcohol and extent of liver injury was examined by histopathology and liver biochemistry after 16-24 h. The causative role of OPN was studied in OPN knockout animals and in vitro in stellate LX2 cells, utilizing siRNA, aptamer and neutralizing antibodies to block OPN and OPN pathway. OPN pathway expression and downstream functional consequences were measured for signaling by Western blotting, plasmin activation by spectrophotometric assays and cell migration by confocal imaging and quantitation.

RESULTS: OPN expression positively correlated with disease severity in patients with progressive stages of ALD. In vivo, associated with alcoholic steatosis, a single dose of acute alcohol significantly increased hepatic OPN mRNA and protein, and a cleaved OPN form in a dose dependent manner. OPN mRNA and secreted OPN also increased in parallel with activation of LX2 stellate cells within 4 h of a single dose of alcohol. Expression of OPN receptors, αvβ3-integrin and CD44, increased in human ALD, and in vivo and in vitro with alcohol administration. This was accompanied by downstream phosphorylation of Akt and Erk, increased mRNA expression of several fibrogenesis, fibrinolysis and extracellular matrix pathway genes, plasmin activation and hepatic stellate cell (HSC) migration. Inhibition of OPN and OPN-receptor mediated signaling partially inhibited alcohol-induced HSC activation, plasmin activity and cell migration.

CONCLUSION: OPN is a key mediator of the alcohol-induced effects on hepatic stellate cell functions and liver fibrogenesis.

Keywords: Hepatic stellate cells, Liver cirrhosis, Plasmin, Steatosis, Fibrogenesis, Transforming growth factor β, Osteopontin isoform

Core tip: The present study confirms a novel hypothesis that alcohol induced plasmin is mediated via Osteopontin (OPN) in hepatic stellate cell (HSC). We show that OPN has a key role in alcohol-induced HSC functions such as signalling, cell migration and activation of fibrinolysis, extracellular matrix and fibrogenic pathways. Identification of transcriptional isoform OPN-C in patients with alcoholic cirrhosis and LX2, and proteolytically cleaved cOPN in mice with a single dose of alcohol is novel. Importantly, we have defined novel mechanisms of OPN action in alcohol-induced liver injury that have a broader significance in other forms of liver injury.