Ca2+-dependent regulation of NOX2 activity via MRP proteins in HL-60 granulocytes

Recently, two proteins of the S100 protein family, the myeloid-related calcium-binding proteins MRP-8 and MRP-14 have been implicated in the Ca2+-induced activation of the neutrophil NADPH oxidase (NOX2) but the mechanism underlying this process remains unclear. In this study, the role of MRP-8/14 in the Ca2+-dependent regulation of NOX2 activity was characterized in neutrophil-like HL-60 cells using small interfering RNAs (siRNAs) to knock-down endogenous MRP-8 and/or MRP-14 expression. Real-time PCR and Western blot revealed that MRP-8 and MRP-14 expression was 20 times higher in dimethylsulfoxide-differentiated neutrophil-like HL-60 cells compared to quiescent HL-60 cells. Knock-down of MRP-8 and MRP-14 in differentiated HL-60 cells decreased protein levels by 30 and 45% respectively. The impact of the reduced MRP-8/14 protein expression on NOX2 activity was investigated by measuring fMLF-induced H2O2 production. In cells simultaneously transfected with MRP-8 and MRP14 siRNAs, H2O2 production was reduced by 50%, suggesting that both MRP-8 and MRP-14 are required for NOX2 activity; single knock-downs were inefficient. To elucidate the role of Ca2+ in MRP8/14, and consequently in NOX2 activation, siRNA-transfected cells were treated with the Ca2+ ionophore ionomycin prior to stimulation with PMA, a Ca2+-independent protein kinase C activator. PMA-induced H2O2 production was enhanced by ionomycin. This amplification of NOX2 activity was abolished by MRP8/14 knock-down, indicating that both MRP-8 and MRP-14 are necessary to regulate Ca2+-induced NOX2 activation. Taken together, our results suggest that the mechanism of MRPs activation is highly dependent on the increase of intracellular Ca2+ level for a full activation of NOX2.