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

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).

Immortalized mouse HSC lines JS1, JS2, and JS3 that were either TLR4(+/+), TLR4(-/-), or MyD88(-/-) were transfected with NF-κB 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-κB 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 μg/ml TGF-β1 and LPS or HMGB1 and collected for assessment of fibrogenic mRNA and protein expression.

HMGB1 stimulation markedly up-regulated MCP-1 mRNA expression and protein secretion, and enhanced TGF-β1-stimulated collagen α2(I) and α-SMA expression in JS1 cells. This was associated with enhanced activation of NF-κB 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.

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-β1 to stimulate fibrogenic protein expression, which is likely to be TLR4 dependent.