Abstract
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.