title: Overexpression of truncated IkappaBalpha induces TNF-alpha-dependent apoptosis in human vascular smooth muscle cells.
abstract: Dysregulation of apoptosis is one of the likely underlying mechanisms of neointimal thickening, a disorder in which proinflammatory cytokines may influence the function of vascular smooth muscle cells (VSMCs) and contribute to atherogenesis. One of these cytokines, tumor necrosis factor-alpha (TNF-alpha), induces 2 possibly conflicting pathways, 1 leading to the activation of nuclear factor-kappaB (NF-kappaB) and the other leading to caspase-mediated apoptosis. We investigated whether specific inhibition of NF-kappaB affects TNF-alpha-dependent apoptosis in human VSMCs. To inhibit NF-kappaB activation specifically, we constructed a recombinant adenovirus vector expressing a truncated form of the inhibitor protein IkappaBalpha (AdexIkappaBDeltaN) that lacks the phosphorylation sites essential for activation of NF-kappaB. The IkappaBDeltaN was overexpressed by adenoviral infection and was resistant to stimulus-dependent degradation. Electromobility gel shift and luciferase assays demonstrated that overexpression of IkappaBDeltaN inhibited NF-kappaB activation induced by TNF-alpha or interleukin-1beta (IL-1beta). In cells overexpressing IkappaBDeltaN, TNF-alpha dramatically induced apoptosis, whereas IL-1beta had no effect. The induction was suppressed by treatment with a selective inhibitor of the caspase-3 family, Z-DEVD-fmk, and the overexpression of IkappaBDeltaN induced TNF-alpha-mediated caspase-3 and caspase-2 activity. These results indicate that overexpression of IkappaBDeltaN induces TNF-alpha-dependent apoptosis by efficient and specific suppression of NF-kappaB and upregulation of caspase-3 and caspase-2 activity in human VSMCs. Our findings suggest that adenovirus-mediated IkappaBDeltaN gene transfer may be useful in the treatment of disorders associated with inflammatory conditions, such as the response to vascular injury and atherosclerosis.
authors.affiliation: Department of Surgery, Keio University School of Medicine, Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Obara
authors.forename: A
authors.initials: A
authors.lastname: Takayanagi
authors.forename: J
authors.initials: J
authors.lastname: Hirahashi
authors.forename: K
authors.initials: K
authors.lastname: Tanaka
authors.forename: G
authors.initials: G
authors.lastname: Wakabayashi
authors.forename: K
authors.initials: K
authors.lastname: Matsumoto
authors.forename: M
authors.initials: M
authors.lastname: Shimazu
authors.forename: N
authors.initials: N
authors.lastname: Shimizu
authors.forename: M
authors.initials: M
authors.lastname: Kitajima
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D001011:Aorta / Q000378:metabolism; D017209:Apoptosis; D001161:Arteriosclerosis / Q000517:prevention & control; D015153:Blotting, Western; D053143:Caspase 2; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D053938:DNA Fragmentation; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D004586:Electrophoresis; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D006801:Humans; D020672:I-kappa B Proteins; D007375:Interleukin-1 / Q000378:metabolism / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D007375:Interleukin-1; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D012097:Repressor Proteins; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D008156:Luciferases; C505407:CASP3 protein, human; D053143:Caspase 2; D053148:Caspase 3; D020169:Caspases