Casein Kinase II-mediated Phosphorylation of NF-κB 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- Kappa B (NF- Kappa B). In the present study, we further analyzed the role of NF- Kappa B 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)-1 beta 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- Kappa B in EMT-6J cells. In response to IL-1 beta , the kinetics of degradation of NF- Kappa B inhibitors I Kappa B- alpha and I Kappa B- 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-1 beta -induced phosphorylation of serine residues in NF- Kappa B p65 subunit was observed in EMT-6J, but not in EMT-6H, cells. This IL-1 beta -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- Kappa B transcriptional activity and NOSII gene transcription in IL-1 beta 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- Kappa B 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-1 beta or LPS.