The human phospholipase D1 (hPLD1) has recently been cloned. Although recent data have implicated PLD in receptor-stimulated secretion, the regulation of the activity of PLD enzymes remains to be clarified. Purified hPLD1 is activated by several cytosolic cofactors among which are protein kinase C␣, ARF, and RhoA. In human granulocytes, a strong correlation between tyrosine phosphorylation of proteins and PLD activity has been established. In this study, the presence of hPLD1 in HL-60 granulocytes and its phosphorylation on tyrosine residues have been studied. We generated antipeptide antibodies (Abs) specific for hPLD1 but not PLD2 as shown by Western blotting (WB) of recombinant PLD1 and PLD2. These Abs identified the presence of hPLD1 in HL-60 cells with the bulk of it being detected in the membranes and only a minor fraction in the cytosol. The hPLD1 Abs detected a major band at 120 kDa (PLD1a) and a minor band at 115 kDa (PLD1b). The specificity of the Abs was confirmed using PLD antisera neutralized with the immunizing peptides. The two forms of hPLD1 were consistently detected by immunoprecipitation under nondenaturing and denaturing conditions following a WB analysis with hPLD1 Abs. Following exposure of HL-60 cells to peroxides of vanadate (V 4؉ -OOH), an inhibitor of tyrosine phosphatases, hPLD1 was immunoprecipitated under nondenaturing conditions from HL-60 cell lysates and assayed for tyrosine phosphorylation by WB. hPLD1 comigrated with a 120-kDa tyrosine phosphorylated protein by gel electrophoresis. Other tyrosine-phosphorylated peptides of 160, 140, 135, 90, and 75-80 kDa were also detected in hPLD1 immune complexes. hPLD1 and the associated tyrosine-phosphorylated proteins were not immunoprecipitated by neutralized hPLD1 Abs. Using denaturing conditions, the PLD immunoprecipitates were sequentially immunoblotted with anti-PLD1 and anti-phosphotyrosine Abs. PLD1a and PLD1b were detected, and the major PLD1a protein was superimposable with a major tyrosine-phosphorylated protein detected at 120 kDa. Conversely, PLD1a and PLD1b were recovered, at least in part, in the anti-phosphotyrosine immunoprecipitates. These results provide evidence that two PLD1 forms are expressed in human granulocytes. Furthermore, in response to stimulation by V 4؉ -OOH, PLD1 was tyrosinephosphorylated and associated with several, presently undefined, tyrosine-phosphorylated proteins.
Four types of adenosine receptors have been identified in different tissues and cell types, namely, A1, A2A, A2B, and A3 receptors. We report that A2AR but not A2BR mRNA in freshly isolated polymorphonuclear neutrophils (PMN) is maximally up-regulated after 4 h stimulation with lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-alpha) and to a lesser extent, with interleukin (IL)-1beta. These effects were maintained up to 21 h. Consistent with changes in A2AR mRNA expression, up-regulation of A2AR protein was also detected after 4 h of LPS or TNF-alpha exposure. Up-regulation of A2AR protein expression was transient and returned to near basal levels after 12 h or 16 h stimulation with TNF-alpha or LPS, respectively. Conversely, IL-1beta failed to promote A2AR protein expression. Suppression of thapsigargin-induced leukotriene synthesis by the selective A2AR agonist CGS-21680 was found to be more pronounced when PMN were cultured for 4 h with LPS or TNF-alpha. In contrast, the up-regulation of A2AR has no impact on CGS-21680-induced inhibition of phospholipase D activation and superoxide production in response to formyl-Met-Leu-Phe. These results demonstrate that the A2AR is up-regulated by specific T helper cell type 1 cytokines and LPS. Although this could represent a potential feedback mechanism to control inflammation, the effect of A2AR up-regulation varied depending on the stimulus used to stimulate PMN functional responses after their incubation with proinflammatory mediators.
Polymorphonuclear neutrophil (PMN) stimulation with fMLP stimulates small G proteins such as ADP-ribosylation factors (Arfs) Arf1 and Arf6, leading to phospholipase D (PLD) activation and functions such as degranulation and the oxidative burst. However, the molecular links between fMLF receptors and PLD remain unclear. PMNs express cytohesin-1, an Arf-guanine exchange factor that activates Arfs, and its expression is strongly induced during the acquisition of the neutrophilic phenotype by neutrophil-like cells. The role of cytohesin-1 in the activation of the fMLF-Arf-PLD signaling axis, and the accomplishment of superoxide anion production, and degranulation was investigated in PMNs using the selective inhibitor of cytohesin, Sec 7 inhibitor H3 (secinH3). Cytohesin-1 inhibition with secinH3 leads to Arf6 but not Arf1 inhibition, demonstrating the specificity for Arf6, and fMLF-mediated activation of PLD and of the oxidative burst as well. We observed a decrease in fMLF-mediated protein secretion and expression of cell surface markers corresponding to primary (CD63/myeloperoxidase), secondary (CD66/lactoferrin), and tertiary (matrix metalloproteinase-9) granules in PMNs incubated with secinH3. Similarly, silencing cytohesin-1 or Arf6 in PLB-985 cells negatively affected fMLF-induced activation of PLD, superoxide production, and expression of granule markers on the cell surface. In contrast, stable overexpression of cytohesin-1 in PLB-985 cells enhanced fMLF-induced activation of Arf6, PLD, and NADPH oxidase. The results of this study provide evidence for an involvement of cytohesin-1 in the regulation of the functional responses of human PMNs and link these events, in part at least, to the activation of Arf6.
Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors in both the plasma membrane and the cytosol. HL-60 cytosol was fractionated by ammonium sulfate and gel-permeation chromatography. Two cytosolic protein fractions were found to reconstitute the GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-stimulated PLD in a reconstitution assay consisting of 3H-labeled HL-60 membranes and eluted column fractions. The major peak of reconstituting activity was in the region of 50 kDa, and a second discrete peak of PLD reconstitution activity was observed in the region of 18 kDa. Rho GDP/GTP exchange inhibitor, Rho GDI, comigrated with Rac2 and RhoA, but not Rac1. RhoA and Rac2 were entirely complexed with Rho GDI and eluted with an apparent molecular mass of 43 kDa by gel filtration chromatography. The partial overlap between cytosolic Rac2 and RhoA with the 50-kDa peak of reconstituting activity was not consistent with the participation of cytosolic Rho-related GTPases in the activation of PLD by guanine nucleotides. However, recombinant Rho GDI, which inhibits nucleotide exchange on the Rho family of small GTP-binding proteins, reduced GTP gamma S-stimulated PLD activity in HL-60 homogenates. The stimulatory exchange factor, Smg GDS, which is active on Rho and Rac, could be partially separated from the PLD-stimulating factor(s) by gel-permeation chromatography. Moreover, recombinant Smg GDS failed to stimulate GTP-dependent PLD activity. Cytosolic ADP-ribosylation factor (ARF) was exclusively located in the 18-kDa peak of reconstitution activity. Faint amounts of membrane-bound ARF were also detected using the monoclonal antibody 1D9. The effects of the 50-kDa and 18-kDa PLD-inducing factors on the salt-extracted PLD activity were synergistic. The weak stimulatory effect of ARF alone suggested that the GTP gamma S-stimulated PLD activity is dependent on the presence of another protein(s), presumably ARF-regulatory proteins. We propose that a membrane-bound GTP-binding protein, possibly ARF, may be involved in the activation of PLD when combined with the component(s) of the 50-kDa fraction.
Adenosine (Ado) is an important autocrine modulator of neutrophil functions. In this study, we determined the effects of endogenous Ado on fMet-Leu-Phe (fMLP)–induced phospholipase D (PLD) activity in neutrophils. The removal of extracellular Ado by Ado deaminase (ADA) or the blockade of its action by the A2a receptor antagonists 8-(3-chlorostyryl) caffeine (CSC) or CGS15943 markedly increased fMLP-induced PLD activation. The concentration-dependent stimulatory effects of CSC and CGS15943 were abolished by a pretreatment of neutrophil suspensionswith ADA. In contrast, the selective A2a receptor agonist CGS21680 suppressed fMLP-induced PLD activation. Furthermore, inhibition by CGS21680 of fMLP-induced PLD activity was reversed by CSC or CGS15943. The removal of Ado by ADA or the blockade of its action by CSC or CGS15943, markedly increased the membrane recruitment of cytosolic protein kinase C (PKC), RhoA, and ADP-ribosylation factor (ARF) in response to fMLP. As shown for PLD activity, the stimulatory effect of Ado receptor antagonists on PLD cofactors translocation was abolished by a pretreatment of the cells with ADA. Moreover, the membrane translocation of both PKC, RhoA, and ARF in response to fMLP was attenuated by CGS21680 and this effect of the A2a receptor agonist was antagonized by CSC or CGS15943. These data demonstrate that Ado released by neutrophils in the extracellular milieu inhibits PLD activation by blocking membrane association of ARF, RhoA, and PKC through Ado A2a receptor occupancy.
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