STIM1 but not STIM2 is an essential regulator of Ca2+ influx-mediated NADPH oxidase activity in neutrophil-like HL-60 cells.
Extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has been linked to various inflammatory diseases, regulatory mechanisms that control Ca2+ influx-induced NOX2 activation are poorly understood in SOCE. The role of STIM1, a Ca2+ sensor that transduces the store depletion signal to the plasma membrane, seems well established and supported by numerous studies in non-phagocytic cells. Here, in neutrophil-like HL-60 cells we used a siRNA approach to delineate the effect of STIM1 knock-down on NOX2 activity regulated by Ca2+ influx. Because the function of the STIM1 homolog, STIM2, is still unclear, we determined the consequence of STIM2 knock-down on Ca2+ and NOX2. STIM1 and STIM2 knock-down was effective and isoform specific when assayed by real-time PCR and Western blotting. Consistent with a unique role of STIM1 in the regulation of SOCE, STIM1, but not STIM2, siRNA significantly decreased Ca2+ influx induced by fMLF or the SERCA pump inhibitor thapsigargin. A redistribution of STIM1, originally localized intracellularly, near the plasma membrane was observed by confocal microscopy upon stimulation by fMLF. Inhibition of STIM1-induced SOCE led to a marked decrease in NOX2 activity while STIM2 siRNA had no effect. Thus, our results provide evidence for a role of STIM1 protein in the control of Ca2+ influx in neutrophils excluding a STIM2 involvement in this process. It also places STIM1 as a key modulator of NOX2 activity with a potential interest for anti-inflammatory pharmacological development.
Life Sciences Research Unit, Université du Luxembourg, 162A, Avenue de la Faïencerie, L-1511 Luxembourg, Luxembourg.
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Bréchard
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Plançon
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Melchior
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Tschirhart
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000502:physiology*; D004789:Enzyme Activation; D055785:Gene Knockdown Techniques; D018922:HL-60 Cells; D006801:Humans; D017136:Ion Transport; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D019255:NADPH Oxidases / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000502:physiology*; D009504:Neutrophils / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D000071737:Stromal Interaction Molecule 1; D000071738:Stromal Interaction Molecule 2
D015815:Cell Adhesion Molecules; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C103530:STIM1 protein, human; C513279:STIM2 protein, human; D000071737:Stromal Interaction Molecule 1; D000071738:Stromal Interaction Molecule 2; D019255:NADPH Oxidases; D053148:Caspase 3; D053181:Caspase 8; D002118:Calcium