title: Inhibition of NFkappaB in activated rat hepatic stellate cells by proteasome inhibitors and an IkappaB super-repressor.
abstract: The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. Cytokines induce NFkappaB activity in activated but not in quiescent HSCs with subsequent expression of NFkappaB-responsive genes, such as intercellular adhesion molecule (ICAM)-1 and interleukin (IL)-6. We investigated the effect of proteasome inhibitors and an IkappaB super-repressor on the cytokine mediated activation of NFkappaB, ICAM-1, and IL-6 in activated HSCs. Culture-activated HSCs were stimulated with IL-1beta or tumor necrosis factor alpha (TNFalpha) in the presence or absence of proteasome inhibitors, ALLN or MG-132, or after infection with an adenovirus expressing the IkappaB super-repressor (Ad5IkappaB) or beta-galactosidase (Ad5LacZ) as a control. NFkappaB activity was evaluated by immunofluorescence and by electrophoretic mobility shift assay. The steady state level of cytoplasmic IkappaB 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 IkappaB, and the Ad5IkappaB, which provides an exogenous nondegradable IkappaB, block the stimulation of NFkappaB activity by TNFalpha and IL-1beta in activated HSCs. These reagents block the subsequent nuclear translocation of p65 NFkappaB and induction of ICAM-1 and IL-6 by cytokines. The specificities of the proteasome inhibitors and the IkappaB super-repressor are demonstrated by their failure to block c-Jun N-terminal kinase induction by cytokines. Cytokine-induced stimulation of NFkappaB, ICAM-1, and IL-6 is blocked by proteasome inhibitors and Ad5IkappaB in activated HSCs. Inhibition of IkappaBalpha degradation is a potential target for anti-inflammatory therapy in the liver and might influence the activation process of HSCs following fibrotic stimuli.
authors.affiliation: University of North Carolina at Chapel Hill, Department of Medicine, 27599-7080, USA.
authors.forename: C
authors.initials: C
authors.lastname: Hellerbrand
authors.forename: C
authors.initials: C
authors.lastname: Jobin
authors.forename: Y
authors.initials: Y
authors.lastname: Iimuro
authors.forename: L
authors.initials: L
authors.lastname: Licato
authors.forename: R B
authors.initials: RB
authors.lastname: Sartor
authors.forename: D A
authors.initials: DA
authors.lastname: Brenner
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008099:Liver / Q000166:cytology*; D008106:Liver Cirrhosis, Experimental / Q000473:pathology; D008297:Male; D009097:Multienzyme Complexes / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D051841:Transcription Factor RelB; D014157:Transcription Factors
chemical_list: D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D007976:Leupeptins; D009097:Multienzyme Complexes; D016328:NF-kappa B; D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C497595:Relb protein, rat; D014157:Transcription Factors; D018799:Intercellular Adhesion Molecule-1; D051841:Transcription Factor RelB; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde