Store-operated Ca2+ channels formed by TRPC1, TRPC6 and Orai1 and non-store-operated channels formed by TRPC3 are involved in the regulation of NADPH oxidase in HL-60 granulocytes.
Ca(2+) influx has been shown to be essential for NADPH oxidase activity which is involved in the inflammatory process. Ca(2+) conditions underlying the oxidative response are clearly delineated. Here, we show that store-operated Ca(2+) entry (SOCE) is required at the beginning of NADPH oxidase activation in response to fMLF (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) in neutrophil-like HL-60 cells. When extracellular Ca(2+) is initially removed, early addition of Ca(2+) after stimulation causes a complete restoration of Ca(2+) entry and H(2)O(2) production. Both Ca(2+) entry and H(2)O(2) production are decreased by purported SOCE blockers, 2-aminoethoxydiphenyl borane (2-APB) and SK&F 96365. Endogenously expressed TRPC (transient receptor potential canonical) homologues and Orai1 were investigated for their role in supporting store-operated Ca(2+) channels activity. TRPC1, TRPC6 and Orai1 knock-out by siRNA resulted in the inhibition of Ca(2+) influx and H(2)O(2) production in response to fMLF and thapsigargin while suppression of TRPC3 had no effect on thapsigargin induced-SOCE. 2-APB and SK&F 96365 were able to amplify the reduction of fMLF-stimulated Ca(2+) entry and H(2)O(2) production observed in cells transfected by TRPC3 siRNA. In summary, Ca(2+) influx in HL-60 cells relies on different membrane TRPC channels and Orai1 for allowing NADPH oxidase activation. TRPC3 primarily mediates SOCE-independent pathways and TRPC1, TRPC6 and Orai1 exclusively contribute to SOCE.
Bréchard
S
S
Life Sciences Research Unit, Université du Luxembourg, 162A, Avenue de la Faïencerie, L-1511 Luxembourg, Luxembourg.
Melchior
C
C
Plançon
S
S
Schenten
V
V
Tschirhart
E J
EJ
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D006098:Granulocytes / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D022423:Myeloid Cells / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism; D000071740:ORAI1 Protein; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D000074287:TRPC6 Cation Channel; D019284:Thapsigargin / Q000378:metabolism
D015220:Calcium Channels; D004791:Enzyme Inhibitors; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D034741:RNA, Small Interfering; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C096351:transient receptor potential cation channel, subfamily C, member 1; D008345:Manganese; D019284:Thapsigargin; D009532:Nickel; D019255:NADPH Oxidases; D002118:Calcium