Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47phox, a major cytosolic component of this oxidase. Protein kinase C ζ (PKC ζ), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC ζ in p47phox phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47phox with recombinant PKC ζ induced a time- and concentration-dependent phosphorylation of p47phox with an apparent Km value of 2 μM. Phosphopeptide mapping analysis of p47phox showed that PKC ζ phosphorylated fewer selective sites in comparison to “conventional” PKCs. Serine 303/304 and serine 315 were identified as targets of PKC ζ by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC ζ that correlated to that of p47phox. A cell-permeant-specific peptide antagonist of PKC ζ inhibited both fMLP-induced phosphorylation of p47phox and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC50 of 10 μM), but not that induced by PMA. The inhibition of PKC ζ expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 μM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47phox is a substrate for PKC ζ and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to superoxide anion (O 2 . ) at the expense of NADPH. This multicomponent enzyme is dormant in resting cells but is activated on exposure to an appropriate stimulus. The phosphorylation-dependent mechanisms regulating the activation of the respiratory burst oxidase are unclear, particularly the phosphorylation status of the cytosolic component p67 phox. In this study, we found that activation of human neutrophils with formyl-methionylleucyl-phenylalanine (fMLP), a chemotactic peptide, or phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), resulted in the phosphorylation of p67 phox . Using an anti-p67 phox antibody or an antip47 phox antibody, we showed that phosphorylated p67 phox and p47 phox form a complex. Phosphoamino acid analysis of the phosphorylated p67 phox revealed only 32 P-labeled serine residues. Two-dimensional tryptic peptide mapping analysis showed that p67 phox is phosphorylated at the same peptide whether fMLP or PMA is used as a stimulus. In addition, PKC induced the phosphorylation of recombinant GST-p67 phox in vitro, at the same peptide as that phosphorylated in intact cells. PMA-induced phosphorylation of p67 phox was strongly inhibited by the PKC inhibitor GF109203X. In contrast, fMLP-induced phosphorylation was minimally affected by this PKC inhibitor. Taken together, these results show that p67 phox is phosphorylated in human neutrophils by different pathways, one of which involves protein kinase C.

Vascular endothelial growth factor (VEGF ), an endothelial cell mitogen, is a potent angiogenic factor produced by several cell types. Whether human neutrophils are potential producers of VEGF has not yet been described. The present work shows that phorbol-12-myristate 13-acetate (PMA), fMet-Leu-Phe, and tumor necrosis factor-α (TNF-α) triggered a time-dependent secretion of VEGF by human neutrophils. Cells incubated with 50 ng/mL of PMA released significant amounts of VEGF after 15 minutes. Because the extracellular content of VEGF in human neutrophils supernatants remained constant over a period of 2 to 24 hours and because PMA is a potent inducer of human neutrophil degranulation, the PMA-induced secretion of VEGF may be due to a pre-existing intracellular pool of this molecule. This hypothesis was reinforced by the absence of cycloheximide effect on the PMA-induced secretion of VEGF. The existence of an intracellular pool of VEGF was confirmed by measuring the intracellular content of VEGF in resting neutrophils. A dosedependent inhibition of PMA-induced VEGF secretion was observed when the cells were incubated in the presence of pentoxifylline, a methylxanthine known to inhibit neutrophil degranulation. To confirm the implication of neutrophil degranulation in VEGF release, the effects of two inducers of physiologic degranulation, fMet-Leu-Phe and TNF-α, were determined. Both agonists induced a release of VEGF in the absence of cytochalasin B, confirming the involvement of neutrophil degranulation and suggesting the intracellular localization of VEGF in the specific granule fraction. In addition, the kinetics of fMet-Leu-Phe– and TNF-α–induced secretion of lactoferrin were similar to those of VEGF release induced by these two both agonists. The subcellular fractionation of human neutrophils showed a granule-specific distribution of the intracellular pool of VEGF in resting neutrophils. The finding that human neutrophils contain an intracellular pool of VEGF, secreted in the extracellular space under PMA-, fMet-Leu-Phe–, and TNF-α–induced degranulation, suggests a role for human neutrophils as cellular effectors of physiologic as well as pathologic angiogenesis.

Tissue factor (TF) assembled with activated factor VII (FVIIa) initiates the coagulation cascade. We recently showed that TF was essential for FVIIa-induced vascular endothelial growth factor (VEGF) production by human fibroblasts. We investigated whether this production resulted from TF activation by its binding to FVIIa or from the production of clotting factors activated downstream. Incubation of fibroblasts with a plasma-derived FVIIa concentrate induced the generation of activated factor X (FXa) and thrombin and the secretion of VEGF, which was inhibited by hirudin and FXa inhibitors. By contrast, the addition of recombinant FVIIa to fibroblasts did not induce VEGF secretion unless factor X was present. Moreover, thrombin and FXa induced VEGF secretion and VEGF mRNA accumulation, which were blocked by hirudin and FXa inhibitors, respectively. The effect of thrombin was mediated by its specific receptor, protease-activated receptor-1; in contrast, the effect of FXa did not appear to involve effector cell protease receptor-1, because it was not affected by an anti-effector cell protease receptor-1 antibody. An increase in intracellular calcium with the calcium ionophore A23187 or intracellular calcium chelation by BAPTA-AM had no effect on either basal or FXa-induced VEGF secretion, suggesting that the calcium signaling pathway was not sufficient to induce VEGF secretion. Finally, FVIIa, by itself, had no effect on mitogen-activated protein (MAP) kinase activation, contrary to thrombin and FXa, which activate the p44/p42 MAP kinase pathway, as shown by the blocking effect of PD 98059 and by Western blotting of activated MAP kinases. These findings indicate that FVIIa protease induction of VEGF expression is mediated by thrombin and FXa generated in response to FVIIa binding to TF-expressing fibroblasts; they also exclude a direct signaling involving MAP kinase activation via the intracellular domain of TF when expressed by these cells. (Arterioscler Thromb Vasc Biol. 2000;20:1374-1381.) Key Words: vascular endothelial growth factor tissue factor activated factor VII fibroblasts proteases T issue factor (TF), the cell-surface receptor for activated factor VII (FVIIa), is the primary regulator of blood coagulation. The TF-FVIIa complex cleaves and activates factors IX and X into factors IXa and factor Xa (FXa), respectively, which lead to thrombin generation. 1 Beyond its role as a procoagulant activator, TF participates in other cellular processes, including metastasis, 2 tumor-associated angiogenesis, 3 and embryogenesis. 4 Accordingly, several cell functions are modified in response to FVIIa binding to TF, its receptor. These include intracellular signaling, 5 activation of the p44/42 mitogen-activated protein (MAP) kinase pathway , 6 induction of tyrosine phosphorylation in monocytes, 7 upregulation of poly(A) polymerase, 8 cell spreading and phosphorylation of focal adhesion kinase, 9 enhanced expression of urokinase receptor by pancreatic cancer cell lines, 10 and vascular endothelial growth factor (VEGF)...