1,25-Dihydroxyvitamin D₃ induces biphasic NF-κB responses during HL-60 leukemia cells differentiation through protein induction and PI3K/Akt-dependent phosphorylation/degradation of IκB

1,25-Dihydroxyvitamin D₃ (VD₃) induces differentiation in a number of leukemia cell lines and under various conditions is able to either stimulate or inhibit nuclear factor kappa B (NF-κB) activity. Here we report a time-dependent biphasic regulation of NF-κB in VD₃-treated HL-60 leukemia cells. After VD₃ treatment there was an early ∼ 4 h suppression and a late 8-72 h prolonged reactivation of NF-κB. The reactivation of NF-κB was concomitant with increased IKK activities, IKK-mediated IκBα phosphorylation, p65 phosphorylation at residues S276 and S536, p65 nuclear translocation and p65 recruitment to the NF-κB/vitamin D responsive element promoters. In parallel with NF-κB stimulation, there was an up-regulation of NF-κB controlled inflammatory and anti-apoptotic genes such as TNFα, IL-1β and Bcl-xL. VD₃-triggered reactivation of NF-κB was associated with PI3K/Akt phosphorylation. PI3K/Akt antagonists suppressed VD₃-stimulated IκBα phosphorylation as well as NF-κB-controlled gene expression. The early ∼ 4 h VD₃-mediated NF-κB suppression coincided with a prolonged increase of IκBα protein which require de novo protein synthesis, lasted for as least 72 h and was insensitive to MAPK, IKK or PI3K/Akt inhibitors. Our data suggest a novel biphasic regulation of NF-κB in VD₃-treated leukemia cells and our results may have provided the first molecular explanation for the contradictory observations reported on VD₃-mediated immune-regulation.