Hmgb1 recruits hepatic stellate cells and liver endothelial cells to sites of ethanol-induced parenchymal cell injury

Hepatic stellate cells (HSC) and liver endothelial cells (LEC) migrate to sites of injury and perpetuate alcohol-induced liver injury. Highmobility group box 1 (HMGB1) is a protein released from the nucleus of injured cells that has been implicated as a proinflammatory mediator. We hypothesized that HMGB1 may be released from ethanolstimulated liver parenchymal cells and contribute to HSC and LEC recruitment. Ethanol stimulation of rat hepatocytes and HepG2 cells resulted in translocation of HMGB1 from the nucleus as assessed by Western blot. HMGB1 protein levels were increased in the supernatant of ethanol-treated hepatocytes compared with vehicle-treated cells. Migration of both HSC and LEC was increased in response to conditioned medium for ethanol-stimulated hepatocytes (CME_tOH) compared with vehicle-stimulated hepatocytes (CM_VEH) (P< 0.05). However, the effect of CME_tOH on migration was almost entirely reversed by treatment with HMGB1-neutralizing antibody or when HepG2 cells were pretransfected with HMGB1-siRNA compared with control siRNA-transfected HepG2 cells (P< 0.05). Recombinant HMGB1 (100 ng/ml) also stimulated migration of HSC and LEC compared with vehicle stimulation (P< 0.05 for both HSC and LEC). HMGB1 stimulation of HSC increased the phosphorylation of Src and Erk and HMGB1-induced HSC migration was blocked by the Src inhibitor PP2 and the Erk inhibitor U0126. Hepatocytes release HMGB1 in response to ethanol with subsequent recruitment of HSC and LEC. This pathway has implications for HSC and LEC recruitment to sites of ethanol-induced liver injury.