STIM1 but not STIM2 is an essential regulator of Ca2+ influx-mediated NADPH oxidase activity in neutrophil-like HL-60 cells

• Published in: Biochemical pharmacology Vol. 78; no. 5; pp. 504 - 513
• Main Authors: Bréchard, S., Plançon, S., Melchior, C., Tschirhart, E.J.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 01-09-2009; Elsevier
• Subjects: Biological and medical sciences; Blotting, Western; Calcium - metabolism; Caspase 3 - metabolism; Caspase 8 - metabolism; Cell Adhesion Molecules - genetics; Cell Adhesion Molecules - physiology; Chemoattractant; Enzyme Activation; Gene Knockdown Techniques; HL-60 Cells; Humans; Ion Transport; Medical sciences; Membrane Proteins - genetics; Membrane Proteins - physiology; NADPH oxidase; NADPH Oxidases - metabolism; Neoplasm Proteins - genetics; Neoplasm Proteins - physiology; Neutrophils; Neutrophils - enzymology; Pharmacology. Drug treatments; Polymerase Chain Reaction; STIM1; STIM2; Store-operated Ca2+ entry; Stromal Interaction Molecule 1; Stromal Interaction Molecule 2; [Ca2+]c; Store-operated Ca2+ entry; STIM; Neutrophils; siRNA; SOCs; 2-APB; fMLF; Chemoattractant; STIM1; STIM2; NADPH oxidase; SOCE; Calcium; Enzyme; Granulocyte; Store-operated Ca; Regulator; Inorganic element; Chemotaxis; Biological activity; HL60 cell line; NAD(P)H oxidase; entry; Neutrophil; Oxidoreductases
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2009.05.006

STIM1, not STIM2, regulates 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.