Cooperative effects of interferon-γ on the induction of NADPH oxidase by retinoic acid or 1,25(OH)2-vitamin D3 in monocytic U937 cells

Abstract The effect of retinoic acid (RA), 1,25-dihydroxyvitamin D 3 (1,25-D 3 ) or human recombinant interferon-γ (IFN-γ) on the induction of NADPH oxidase was studied in premonocytic U937 cells. Differentiation with the combination of either RA (1 μM) or 1,25-D 3 (10 nM) with IFN-γ (100 IU/ml) induced NADPH oxidase activity as demonstrated by increased superoxide anion (O 2 − ) generation in response to stimulation with phorbol myristate acetate (PMA, 100 nM). Induction of NADPH oxidase activity was preceded by increases in mRNA levels of p47-phox, p67-phox and gp91-phox, which encode three subunits of the enzyme, and immunoblot analysis of the p47-phox and p67-phox proteins revealed that the increases in mRNA levels were equally reflected by increases in protein levels. In contrast, RA, 1,25-D 3 or IFN-γ alone did not induce NADPH oxidase activity which correlated with their failure to increase p67-phox and gp91-phox mRNA levels. The mRNA of p21 racl , a GTP-binding protein that regulates NADPH oxidase activity in macrophages, was constitutively expressed in undifferentiated cells and was not affected by differentiation. These data indicate that induction of a functional NADPH oxidase in premonocytic U937 cells requires the cooperative actions of IFN-γ plus RA or 1,25-D 3 and is reflected in the increased expression of p67-phox and gp91-phox.