High mobility group box 1 activates toll like receptor 4 signaling in hepatic stellate cells

Aims: The aim of the present study was to investigate the effect of high mobility group box 1 (HMGB1), a damage pattern molecule that signals the presence of necrosis, on TLR4 signaling in hepatic stellate cells (HSC). Main methods: Immortalized mouse HSC lines JS1, JS2, and JS3 that were either TLR4(+/+), TLR4(-/-), or MyD88(-/-) were transfected with NF-kappa B or AP-1 responsive luciferase reporter plasmids, followed by stimulation with 100 ng/ml lipopolysacchride (the exogenous TLR4 ligand) or 100 ng/ml HMGB1. The activation of NF-kappa B or AP-1 activities was determined by a dual-luciferase reporter assay system. The cells were also stimulated with LPS or HMGB1 and collected for the determination of chemotactic cytokine MCP-1 mRNA or proteins secretion. In a separate experiment, the cells were co-stimulated with 10 mu g/ml TGF-beta 1 and LPS or HMGB1 and collected for assessment of fibrogenic mRNA and protein expression. Key findings: HMGB1 stimulation markedly up-regulated MCP-1 mRNA expression and protein secretion, and enhanced TGF-beta 1-stimulated collagen alpha 2(I) and alpha-SMA expression in JS1 cells. This was associated with enhanced activation of NF-kappa B and AP-1 responsive luciferase reporters. On the contrary, JS2 and JS3 cells were hyporesponsive to both LPS and HGMB1 stimulation compared to JS1 cells. Significance: As an endogenous ligand of TLR4, HMGB1 activates TLR4 signaling in HSCs to enhance their inflammatory phenotype, indicating that TLR4 signaling need not rely solely on gut-derived LPS for activation during liver injury. HMGB1 also has a synergistic effect with TGF-beta 1 to stimulate fibrogenic protein expression, which is likely to be TLR4 dependent. (c) 2012 Elsevier Inc. All rights reserved.