Activation of nuclear factor kappa B (NF-kappaB) by connective tissue growth factor (CCN2) is involved in sustaining the survival of primary rat hepatic stellate cells.

BACKGROUND/AIMS: Connective tissue growth factor (CCN2) is a matricellular protein that plays a role in hepatic stellate cell (HSC)-mediated fibrogenesis. The aim of this study was to investigate the regulation by CCN2 of cell survival pathways in primary HSC.

METHODS

Primary HSC were obtained by in situ enzymatic perfusion of rat liver. NF-kappaB activation was assessed by immunoblotting for IkappaBalpha phosphorylation and degradation and by NF-kappaB p50 or p65 nuclear accumulation. NF-kappaB DNA-binding activity was determined by gel mobility shift assay while NF-kappaB response gene expression was evaluated using a luciferase reporter. Cell viability was assessed by Trypan blue staining or ATP luminescent assay while apoptosis was evaluated by caspase-3 activity.

RESULTS

CCN2 induced IkappaBalpha phosphorylation and degradation as well as nuclear accumulation of NF-kappaB. Activated NF-kappaB comprised three dimers, p65/p65, p65/p50 and p50/p50, that individually bound to DNA-binding sites and subsequently triggered transcriptional activity. This was confirmed by showing that CCN2 promoted activity of a NF-kappaB luciferase reporter. CCN2 promoted survival of serum-starved HSC and protected the cells from death induced by blocking the NF-kappaB signaling pathway using Bay-11-7082, a specific inhibitor of IkappaBalpha phosphorylation.

CONCLUSION

CCN2 contributes to the survival of primary HSC through the NF-kappaB pathway.