Nuclear factor κB in proliferation, activation, and apoptosis in rat hepatic stellate cells

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Abstract

Background/Aims: Activation of the transcription factor NFκB has been demonstrated in activated hepatic stellate cells (HSCs). We investigated the role of NFκB in proliferation, in activation, and in TNFα-induced apoptosis of HSCs.

Methods: NFκB activation was inhibited using an adenovirus expressing an IκB dominant negative protein (Ad5IκB) in both quiescent and activated HSCs. Quiescent HSCs were infected with Ad5IκB or an adenovirus expressing β-galactosidase (Ad5LacZ). The cells were cultured for 7 days. HSCs activation was determined by cell morphology, smooth muscle α-actin (α-sma) expression, and steady-state mRNA levels of α1(I) collagen as assessed by Western blot and RNase protection assay, respectively. Proliferation was determined in culture-activated HSCs by 3H-thy-midine incorporation and direct cell counting. Apoptosis was analyzed by infecting quiescent or activated HSCs with Ad5IκB or Ad5LacZ, and then treating with TNFα. Apoptosis was demonstrated by determining cell number, assessing nuclear morphology, TUNEL assay and caspase 3 activity.

Results: After 7 days in culture no differences were noted between the Ad5IκB- and the Ad5LacZ-in-fected cells in the morphology, α-sma expression or in α1(I) collagen mRNA levels. Ad5IκB infection did not modify proliferation in activated HSCs. TNFα induced apoptosis only in Ad5IκB-infected activated, but not quiescent HSCs. Apoptosis was initially demonstrated 12 h after exposure to TNFα. Twenty-four h after the TNFα treatment, 60% of the activated HSCs were apoptotic.

Conclusion: NFκB activity is not required for proliferation or activation of HSCs; however, NFκB protects activated HSCs against TNFα-induced apoptosis.

Section snippets

HSC isolation and culture

HSCs were isolated from Sprague-Dawley retired breeder rats (>400 g) by sequential digestion of the liver with pronase and collagenase, followed by arabinogalactan gradient centrifugation, as previously described (10). HSC purity was assessed microscopically and by using the autofluorescence property of the stored retinoids in the HSC. Cell viability was determined using Trypan blue exclusion staining. HSC populations were between 90–99% pure and 95% viable. HSCs were cultured on plastic using

Infection of HSCs with adenovirus

To evaluate adenoviral infection of quiescent HSCs, cells were infected with either Ad5LacZ or Ad5IκB at day 1 after isolation (day 1), fixed on day 3 and either stained for β-galactosidase for the control AdLacZ or stained for the HA epitope of the Ad5IκB. At a moi of 500, 90% of the cells were infected by both viruses (Fig. 1A,C). To evaluate protein expression of genes delivered by adenoviral infection 1 week after infection, cells were infected on day 1, cultured and then stained 7 days

Discussion

Following liver injury the HSC undergoes a transformation from a quiescent cell to an activated proliferating myofibroblast-like cell, which secretes cytokines and extracellular matrix proteins. NFκB is activated following the transformation of quiescent to activated HSCs 13., 14., 25., 26., and NFκB responsive genes are induced in the activated HSCs (10). To determine the relationship between NFκB and HSC activation, NFκB activity was blocked in quiescent HSCs, and this inhibition was

Acknowledgements

This work was supported by National Institute of Health grants AA-10459, DK-34987, GM-41804 and AA-11605.

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