Phagocyte cell migration is mediated by phospholipases PLD1 and PLD2.
We have investigated whether the signaling protein phospholipase D is implicated in leukocyte cell motility. Treating differentiated HL-60 cells with small interfering RNAs (siRNAs), to deplete endogenous expression of the PLD1 isoform, led to an abolishment of basal chemokinesis that could not be rescued with chemoattractants ENA-78, FMLP, and IL-8. Transient overexpression of PLD1 increased both chemokinesis and chemotaxis toward IL-8 and FMLP but not toward ENA-78. Chemokinesis was not mediated by the enzymatic activity of PLD1, but the chemotactic response was, because a lipase-inactive mutant (PLD1-K830R) negated all chemokine-induced potentiating actions and because IL-8 and FMLP increased activity in vitro. Gene expression silencing of the other mammalian isoform, PLD2, also led to cell migration arrest, whereas ENA-78 selectively increased endogenous PLD2 activity and chemotaxis of HL-60 cells overexpressing a myc-pcDNA-PLD2 construct. Thus, PLD1 is differentially activated by CXCR-1, whereas CXCR-2 (and possibly CXCR-1) mediates PLD2 activation. Finally, immunofluorescence microscopy showed that both isoforms were associated with cell polarity and directionality concomitantly with adhesion and F-actin polymerization in response to IL-8. These data represent the first demonstration of the involvement of PLD and its enzymatic activity toward chemokines in the key physiologic process of leukocyte migration.
Lehman
Nicholas
N
Department of Cell Biology and Physiology, Wright State University School of Medicine, Dayton, OH 45435, USA.
Di Fulvio
Mauricio
M
McCray
Nicholas
N
Campos
Isabel
I
Tabatabaian
Farnaz
F
Gomez-Cambronero
Julian
J
D002448:Cell Adhesion; D002465:Cell Movement / Q000187:drug effects; D016764:Cell Polarity; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D016209:Interleukin-8 / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000502:physiology; D010586:Phagocytes / Q000502:physiology*; D010739:Phospholipase D / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D023062:Receptors, Interleukin-8A / Q000502:physiology; D023063:Receptors, Interleukin-8B / Q000502:physiology
D016209:Interleukin-8; D034741:RNA, Small Interfering; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D009240:N-Formylmethionine Leucyl-Phenylalanine; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1