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

Primary HSC were obtained by in situ enzymatic perfusion of rat liver. NF-κB activation was assessed by immunoblotting for IκBα phosphorylation and degradation and by NF-κB p50 or p65 nuclear accumulation. NF-κB DNA-binding activity was determined by gel mobility shift assay while NF-κB 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.CCN2 induced IκBα phosphorylation and degradation as well as nuclear accumulation of NF-κB. Activated NF-κB 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-κB luciferase reporter. CCN2 promoted survival of serum-starved HSC and protected the cells from death induced by blocking the NF-κB signaling pathway using Bay-11-7082, a specific inhibitor of IκBα phosphorylation.CCN2 contributes to the survival of primary HSC through the NF-κB pathway.Hepatic stellate cells (HSC) are the primary targets of fibrogenic stimuli in the injured liver. During the development of fibrosis, HSC undergo a transition from resting vitamin A-rich cells to an activated myofibroblastic phenotype characterized by loss of vitamin A, expression of α-smooth muscle actin, enhanced proliferation and increased production of various extracellular matrix components [1-4]. Activation of HSC has been identified as a central event in hepatic fibrosis and is regulated by a wide variety of molecules including cytokines, cell-surface receptors, signal transduction molecules and factors that regulate HSC gene expression at the transcriptional and post-transcriptional levels [3-6].Connective tissue growth factor (CCN2, also known as CTGF) is a cysteine-rich matricellular protein that regulates cell adhesion, migration, proliferation, survival, and differentiation [7