Inhibition of NFκB in activated rat hepatic stellate cells by proteasome inhibitors and an IκB super‐repressor

Abstract The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. Cytokines induce NFκB activity in activated but not in quiescent HSCs with subsequent expression of NFκB‐responsive genes, such as intercellular adhesion molecule (ICAM)‐1 and interleukin (IL)‐6. We investigated the effect of proteasome inhibitors and an IκB super‐repressor on the cytokine mediated activation of NFκB, ICAM‐1, and IL‐6 in activated HSCs. Culture‐activated HSCs were stimulated with IL‐1β or tumor necrosis factor α (TNFα) in the presence or absence of proteasome inhibitors, ALLN or MG‐132, or after infection with an adenovirus expressing the IκB super‐repressor (Ad5IκB) or β‐galactosidase (Ad5LacZ) as a control. NFκB activity was evaluated by immunofluorescence and by electrophoretic mobility shift assay. The steady state level of cytoplasmic IκB protein was measured by Western Blot. ICAM‐1 and IL‐6 expression was measured by reverse transcriptase‐polymerase chain reaction and enzyme‐linked immunosorbant assay. Proteasome inhibitors, which block the degradation of IκB, and the Ad5IκB, which provides an exogenous nondegradable IκB, block the stimulation of NFκB activity by TNFα and IL‐1β in activated HSCs. These reagents block the subsequent nuclear translocation of p65 NFκB and induction of ICAM‐1 and IL‐6 by cytokines. The specificities of the proteasome inhibitors and the IκB super‐repressor are demonstrated by their failure to block c‐Jun N‐terminal kinase induction by cytokines. Cytokine‐induced stimulation of NFκB, ICAM‐1, and IL‐6 is blocked by proteasome inhibitors and Ad5IκB in activated HSCs. Inhibition of IκBα degradation is a potential target for anti‐inflammatory therapy in the liver and might influence the activation process of HSCs following fibrotic stimuli.