Arsenic inhibits NF-kappaB-mediated gene transcription by blocking IkappaB kinase activity and IkappaBalpha phosphorylation and degradation.
The inflammatory cytokine, TNF-alpha, induces IL-8 gene transcription via a mechanism involving proteasome-mediated IkappaBalpha degradation and NF-kappaB activation. Here, we investigated whether arsenic, which has been shown to inhibit the ubiquitin-proteasome pathway, could inhibit TNF-alpha-mediated increases in IL-8 expression. Using RT-PCR, we show that the addition of TNF-alpha to human bronchial epithelial (BEAS 2B) or embryonic kidney (HEK293) cells resulted in increased steady-state levels of IL-8 mRNA. This was preceded by a rapid decrease in cellular IkappaBalpha levels, as demonstrated by Western analysis, and an increase in nuclear levels of NF-kappaB, as demonstrated by gel shift analysis. Further demonstrating the activation of NF-kappaB, TNF-alpha induced the transcription of a NF-kappaB-dependent reporter gene. Exposing the cells to 500 microM arsenite, prior to adding TNF-alpha, completely inhibited IkappaBalpha degradation, NF-kappaB translocation, NF-kappaB-dependent gene transcription, and transcription of the endogenous gene for IL-8. In comparison with the proteasome inhibitor MG-132, which does not affect the phosphorylation and ubiquitination of IkappaBalpha, arsenite inhibited the phosphorylation of IkappaBalpha. Furthermore, arsenite directly blocked the activity of IKK, the kinase responsible for IkappaBalpha phosphorylation. These studies demonstrate that high levels of arsenic may inhibit NF-kappaB-mediated gene transcription by specifically blocking IKK activity, thereby limiting the phosphorylation and subsequent degradation of the NF-kappaB inhibitor, IkappaBalpha.
Roussel
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RR
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Barchowsky
A
A
D000841:Anisomycin / Q000494:pharmacology; D018053:Arsenites / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D016209:Interleukin-8 / Q000235:genetics; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000502:physiology; D014158:Transcription, Genetic / Q000187:drug effects*; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
D018053:Arsenites; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016209:Interleukin-8; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D019000:Phosphotyrosine; D000841:Anisomycin; D004247:DNA; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C015001:arsenite