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

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.

Figure 1

Effect of CCN2 on stimulation of IκBα phophorylation and NF-κB translocation. Freshly isolated rat HSC were cultured for 24 h in 5% FBS DMEM and for another 48 h in serum-free medium. The cells were harvested following incubating the cells for 30 min in the absence or presence of 100 ng/ml CCN2, and nuclear extracts were prepared. Western blot analysis shows that CCN2 induces IκB phosphorylation and degradation (A) and translocation of the NF-κB subunits p65/p50 from cytoplasm to nucleus (B).

Figure 2

Modulation of NF-κB DNA binding activity by CCN2. HSC were harvested at the desired time points after treatment with or without 100 ng/ml CCN2. 6 μg nuclear protein extract were used in 20 μl reactions, containing 0.2 ng ³²P-labeled double strand NF-κB oligonucleotides. Reactions were fractioned through a nondenaturing 4% polyacrylamide gel. (A) Complex 1 (p65/p50) and complex 2 were enhanced after stimulation with CCN2. "Ctrl" represents a reaction lacking nuclear extract. (B) Bay11-7082 inhibited complex formation when added prior to CCN2 treatment ("Bay 11 + CCN2") but not when added subsequent to a 1 hour pretreatment with CCN2 ("CCN2 + Bay11"). (C) A supershift assay was performed by incubating pre-assembled gel shift assay complexes containing 8 μg nuclear extract with either 2 μg normal rabbit IgG or 2 μg anti-NF-κB antibody prior to separation through 8% polyacrylamide gel, showing that CCN2 stimulates the formation of an anti-p65/p65/anti-p50/p50/NF-κB oligonucleotide (S1) and an anti-p65/p65/NF-κB oligonucleotide (S2) supershift complexes. (D) A gel shift assay was performed following pre-incubating the nuclear extracts with either ³²P-labeled p50/p65 site mutant oligonucleotides or CBF-1 site mutant oligonucleotides.

Figure 3

Effect of CCN2 on expression of NF-κB response genes. (A) Freshly isolated rat HSC were placed in 12-well plates, transfected with 1 μg pTA-Luc or pNF-κB-Luc, and then incubated for another 24 h in the absence or presence of 100 ng/ml CCN2. (B) Transfected cells were pre-treated with 10 μM Bay11-7082 for 30 min and cultured for another 24 h in the absence or presence of 100 ng/ml CCN2. **P < 0.01 vs. pNF-κB-Luc; ^##P < 0.01 vs. pNF-κB-Luc + CCN2.

Figure 4

Effect of CCN2 in sustaining HSC survival. Freshly isolated rat HSC were cultured in 6-well plates in 5% FBS DMEM for 24 h, followed by serum deprivation for 48 h. The cells were cultured for another 24 h in the absence or presence of 100 ng/ml CCN2 ("CCN2"). In the CCN2 protection assay, the cells were incubated with 10 μM Bay11-7082 for 24 h alone ("Bay11") or following pre-treatment of the cells with CCN2 for 1 h ("CCN2+Bay11"). For the Bay11 blocking assay, the cells were pre-treated with Bay11-7082 for 30 min and cultured for another 24 h in the presence of 100 ng/ml CCN2 ("Bay11+CCN2"). At the end of incubation time period, (A) cells were trypsinized and survival was determined by Trypan blue exclusion, or (B) cell viability was also quantified by measurement of the fluorescence intensity using CellTiter-Glo™ reagent, or (C) cell apoptosis was assessed by measurement of caspase-3 activity at 405 nm using a luminescence assay kit. *P < 0.05 vs. control; ^#P < 0.05 vs. CCN2 group; **P < 0.01 and * P < 0.05 vs. Bay11-7082 group.