The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. Cytokines induce NFB activity in activated but not in quiescent HSCs with subsequent expression of NFBresponsive genes, such as intercellular adhesion molecule (ICAM)-1 and interleukin (IL)-6. We investigated the effect of proteasome inhibitors and an IB super-repressor on the cytokine mediated activation of NFB, ICAM-1, and IL-6 in activated HSCs. Culture-activated HSCs were stimulated with IL-1 or tumor necrosis factor ␣ (TNF␣) in the presence or absence of proteasome inhibitors, ALLN or MG-132, or after infection with an adenovirus expressing the IB super-repressor (Ad5IB) or -galactosidase (Ad5LacZ) as a control. NFB activity was evaluated by immunofluorescence and by electrophoretic mobility shift assay. The steady state level of cytoplasmic IB 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 IB, and the Ad5IB, which provides an exogenous nondegradable IB, block the stimulation of NFB activity by TNF␣ and IL-1 in activated HSCs. These reagents block the subsequent nuclear translocation of p65 NFB and induction of ICAM-1 and IL-6 by cytokines. The specificities of the proteasome inhibitors and the IB super-repressor are demonstrated by their failure to block c-Jun N-terminal kinase induction by cytokines. Cytokine-induced stimulation of NFB, ICAM-1, and IL-6 is blocked by proteasome inhibitors and Ad5IB in activated HSCs. Inhibition of IB␣ degradation is a potential target for anti-inflammatory therapy in the liver and might influence the activation process of HSCs following fibrotic stimuli. (HEPATOLOGY 1998;27:1285-1295.)
Following a fibrogenic stimulus, the hepatic stellate cell (HSC; also called Ito cell, lipocyte, or fat-storing cell) changes from a quiescent to an activated cell, 1,2 which includes increased proliferation, a phenotypic transformation to a myofibroblast-like cell, increased synthesis of extracellular matrix proteins, and contractility. [3][4][5][6] The activated HSC is the predominant source of the increased extracellular matrix proteins, which characterize hepatic fibrosis and cirrhosis. Many of the changes demonstrated in HSC activation during fibrogenesis in vivo are also observed in HSCs grown in culture on plastic. 2,7,8 Furthermore, HSCs have been implicated in hepatic inflammation through their ability to secrete cytokines 9 and chemokines [10][11][12] and express adhesion molecules. 13 Although many aspects of the molecular regulation of the activation of HSCs are unknown, a role for the transcription factor NFB is evolving. NFB is responsible for the rapid induction of many cytokines and adhesion molecules implicated in the immune and inflammatory responses. [14][15][16] Classic NFB is a heterodimer of p65 (Rel A) and p50 (NFB-1), but NFB may consist of a variety of homodimers and heterodimers. 17 NFB is retained in an inactive form in the cytoplasm through association with an IB inhibitory protein, such as IB␣. 18 Following cellular stimulation, IB␣ is phosphorylated at serines 32 and 36 by a specific kinase IKK, 19,20 ubiquinated, and undergoes proteolysis in proteasomes, enabling NFB to translocate to the nucleus in which it binds to NFB DNA binding sites and stimulates transcription of many cytokines, chemokines, and adhesion molecules. 21 NFB is induced upon the transformation of quiescent to activated HSCs. 22 Using differential display, we demonstrated the expression of ICAM-1, an NFB-responsive gene, in activated but not in quiescent HSCs. 13 Furthermore, treatment of activated, but not quiescent, HSCs with the cytokines TNF␣ or interleukin (IL)-1 resulted in IB␣ degradation, NFB nuclear translocation, increased NFB DNA binding activity, and induction of NFB-responsive genes (Hellerbrand et al., manuscript submitted October 1997).
The signaling pathway leading to the induction of NFBresponsive genes provides opportunities to specifically block this response. A super-repressor form of IB␣, containing serine-to-alanine mutations at amino acids 32 and 36, inhibits signal-induced phosphorylation and subsequent degradation of IB␣. 23,24 The proteasome complex is required for degradation of IB␣. Proteasome inhibitors, such as ALLN and MG-132, block the induction of NFB activity in cultured cells 25,26 and prevent inflammation in vivo. 27 This study uses two different approaches to block the NFB pathway in activated HSCs, as follows: 1