NF-kappaB induction by bisperoxovanadium compounds requires CD45, p36(LAT), PKC, and IKK activity and exhibits kinetics of activation comparable to those of TCR/CD28 coengagement.
We have previously shown that bisperoxovanadium (bpV) phosphotyrosyl phosphatase inhibitors can potently activate NF-kappaB. We have already determined that p56(lck), ZAP-70, SLP-76, capacitative entry of calcium, and calcium-regulated effectors are important in bpV-induced NF-kappaB activation. In this study, we evaluated whether other signal transducers previously reported in NF-kappaB induction by T cell activating stimuli are also activated by bpV compounds. Nuclear translocation of NF-kappaB was evaluated in cell lines deficient for either CD45 or p36(LAT) to assess the role of these signal transducers in bpV-mediated NF-kappaB activation. A deficiency of either protein greatly reduced the extent of NF-kappaB nuclear translocation following bpV treatment. Isoform-specific PKC inhibitors were then used to show that bpV compounds activate NF-kappaB through both calcium-sensitive and -insensitive PKC isoforms. The implication of the IkappaB-kinase complex was then investigated through the use of an IkappaBalpha-specific kinase assay and plasmids expressing catalytically inactive forms of IKKalpha and IKKbeta. Upstream kinases involved in IKK complex activation such as TPL-2/COT, NIK, and IKKepsilon were also shown to play an important role in bpV-mediated NF-kappaB activation. Finally, reporter gene transcriptional assays and gel shift assays were performed to compare the kinetics of activation of NF-kappaB by bpV with those of antigenic and TNFalpha stimulation. We demonstrate, both in Jurkat cells and in primary T cells, that bpV-mediated NF-kappaB activation kinetics are comparable to those of an antigenic stimulation but occur much slower than the kinetics seen upon TNFalpha treatment.
Ouellet
Michel
M
Centre de Recherche en Infectiologie, Hôpital CHUL, Centre Hospitalier Universitaire de Québec, and Département de Biologie Médicale, Faculté de Médecine, Université Laval, Ste-Foy, Quebéc, Canada.
Roy
Jocelyn
J
Barbeau
Benoit
B
Geleziunas
Romas
R
Tremblay
Michel J
MJ
D018106:CD28 Antigens / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D017493:Leukocyte Common Antigens / Q000378:metabolism*; D016328:NF-kappa B / Q000096:biosynthesis*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D017968:Vanadium Compounds / Q000494:pharmacology*
D018106:CD28 Antigens; D016328:NF-kappa B; D011948:Receptors, Antigen, T-Cell; D017968:Vanadium Compounds; C472777:bisperoxovanadium; D014443:Tyrosine; D011493:Protein Kinase C; D017493:Leukocyte Common Antigens