Casein kinase II-mediated phosphorylation of NF-kappaB p65 subunit enhances inducible nitric-oxide synthase gene transcription in vivo.
Nitric oxide (NO) produced by inducible nitric-oxide synthase (NOSII) is mainly regulated at the transcriptional level by the nuclear factor-kappaB (NF-kappaB). In the present study, we further analyzed the role of NF-kappaB in the in vivo transcriptional regulation of NOSII gene by comparing two clones isolated from the EMT-6 mouse mammary cancer cell line. In response to interleukin (IL)-1beta or lipopolysaccharide (LPS), EMT-6 clone J (EMT-6J) cells produce 3-fold more NO than EMT-6 clone H (EMT-6H) cells, an effect correlated with enhanced activation of NF-kappaB in EMT-6J cells. In response to IL-1beta, the kinetics of degradation of NF-kappaB inhibitors IkappaB-alpha and IkappaB-beta, the nucleo-cytoplasmic shuttling of the transcription factor and its binding to a specific DNA sequence were similar in both clones. In contrast, an IL-1beta-induced phosphorylation of serine residues in NF-kappaB p65 subunit was observed in EMT-6J, but not in EMT-6H, cells. This IL-1beta-induced phosphorylation of p65 was specifically prevented by pretreatment of EMT-6J cells with the casein kinase II inhibitor DRB. Small interfering RNA-mediated depletion of casein kinase II-alpha subunit also decreased NF-kappaB transcriptional activity and NOSII gene transcription in IL-1beta and LPS-stimulated EMT-6J cells to the levels observed in EMT-6H cells treated in the same conditions. Altogether, these data indicate that casein kinase II-mediated phosphorylation of p65 subunit can enhance the transcriptional activity of NF-kappaB in vivo. This post-translational modification of the transcription factor can be responsible for increased NOSII gene transcription and NO production in tumor cells exposed to either IL-1beta or LPS.
Chantôme
Aurélie
A
Cancer Immunotherapy Laboratory, Ecole Pratique des Hautes Etudes, INSERM U517, Faculty of Medicine, Dijon, France.
Pance
Alena
A
Gauthier
Nolwenn
N
Vandroux
David
D
Chenu
Julie
J
Solary
Eric
E
Jeannin
Jean-François
JF
Reveneau
Sylvie
S
D000818:Animals; D015152:Blotting, Northern; D047390:Casein Kinase II; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D017930:Genes, Reporter; D015151:Immunoblotting; D007375:Interleukin-1 / Q000378:metabolism; D007700:Kinetics; D008070:Lipopolysaccharides / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D051996:Transcription Factor RelA; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D007375:Interleukin-1; D008070:Lipopolysaccharides; D016328:NF-kappa B; D034741:RNA, Small Interfering; D051996:Transcription Factor RelA; D009569:Nitric Oxide; D012694:Serine; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases