Phosphorylation of iκbα and nf-κb activation are required for macrophage-differentiation of U937 cells

NF-κB is a heterodimer comprised of a DNA-binding subunit (p50) and a transactivator (p65). It is sequestered in the cytoplasm by association with an inhibitory protein, known as IκBα. The role of IκBα phosphorylation and activity of NF-κB during phorbol 12-myristate 13-acetate (PMA)-induced differentiation of U937 cells was investigated. Treatment of U937 cells with PMA (10−6M) for 24 hr resulted in macrophage-like differentiation by light microscopy. PMA rapidly induced activation of NF-κB, as demonstrated by translocation of p65 subunit into the nucleus (by Western blotting). By 2 hr p65 subunit was predominantly localized in the nucleus, with little residual cytoplasmic p65. Specific inhibitors of NF-κB activation, CAPE and SN50, completely blocked the activation of NF—κB and partially (40%) inhibited macrophage-like differentiation. PMA also rapidly induced phosphorylation of IκBα, transient loss (for about 1 hr) of IκBα followed by a marked increase of phosphorylated IκBα from 2 to 24 h. In contrast, IκBβ protein levels remained constant for 24 h after initiating PMA treatment. The IκBα phosphorylation inhibitor Bay 11-7085 completely inhibited the phosphorylation of IκBα and macrophage differentiation of the cells, without affecting IκBβ levels, These results suggest that both phosphorylation of IκBα and NF-κB activation are required for macrophage-like differentiation of U937 cells; The prolonged phosphorylation of IκBα has more profound effect on differentiation, which suggests that the IκBα may regulate other molecules that are also required for macrophage-like differentiation of U937 cells.