Cysteine S-conjugates activate transcription factor NF-kappa B in cultured renal epithelial cells.
Activation of NF-kappa B by the nephrotoxic and cytotoxic cysteine S-conjugate S-(1,2-dichlorovinyl)-L-cysteine (DCVC) was investigated in porcine kidney-derived LLC-PK1 cells. DCVC induced binding of nuclear proteins to an NF-kappa B consensus oligonucleotide from the immunoglobulin kappa-light chain enhancer region, as determined by electrophoretic mobility shift assays, and the activated proteins were identified as the p50/RelA heterodimeric complex of NF-kappa B. Transient transfection experiments with a kappa B-controlled luciferase reporter construct showed that the NF-kappa B complex activated by DCVC was transcriptionally active. NF-kappa B transactivation was blocked by inhibition of DCVC bioactivation with the cysteine conjugate beta-lyase inhibitor (aminooxy)acetic acid, by the antioxidants N,N'-diphenyl-p-phenylenediamine and N-acetyl-L-cysteine, and by the protein kinase inhibitor staurosporine. The cysteine S-conjugates S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine also activated NF-kappa B in LLC-PK1 cells. These results demonstrate that the NF-kappa B pathway is present in LLC-PK1 cells and is induced by cysteine S-conjugates. Inhibition of DCVC-induced transactivation of NF-kappa B by staurosporine and by antioxidants indicate roles for protein kinases and oxidative stress in the NF-kappa B pathway.
Otieno
M A
MA
University of Rochester, School of Medicine and Dentistry, Department of Pharmacology and Physiology, New York 14642, USA.
Anders
M W
MW
D000111:Acetylcysteine / Q000494:pharmacology*; D000625:Aminooxyacetic Acid / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D016384:Consensus Sequence; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D017930:Genes, Reporter; D007668:Kidney / Q000378:metabolism*; D008156:Luciferases / Q000096:biosynthesis; D046911:Macromolecular Substances; D016328:NF-kappa B / Q000096:biosynthesis* / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013552:Swine; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000975:Antioxidants; D046911:Macromolecular Substances; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D000625:Aminooxyacetic Acid; C039961:S-(1,2-dichlorovinyl)cysteine; D008156:Luciferases; D003545:Cysteine; D000111:Acetylcysteine