Recently, a novel peptide (Trp-Lys-Tyr-Met-Val-D-Met, WKYMVm) has been shown to induce superoxide generation in human monocytes. The peptide stimulated phospholipase A2 (PLA2) activity in a concentration- and time-dependent manner. Superoxide generation as well as arachidonic acid (AA) release evoked by treatment with WKYMVm could be almost completely blocked by pretreatment of the cells with cytosolic PLA2 (cPLA2)-specific inhibitors. The involvement of cPLA2 in the peptide-induced AA release was further supported by translocation of cPLA2 to the nuclear membrane of monocytes incubated with WKYMVm. WKYMVm-induced phosphatidylbutanol formation was completely abolished by pretreatment with PKC inhibitors. Immunoblot showed that monocytes express phospholipase D1 (PLD1), but not PLD2. GF109203X as well as butan-1-ol inhibited peptide-induced superoxide generation in monocytes. Furthermore, the interrelationship between the two phospholipases, cPLA2 and PLD1, and upstream signaling molecules involved in WKYMVm-dependent activation was investigated. The inhibition of cPLA2 did not blunt peptide-stimulated PLD1 activation or vice versa. Intracellular Ca2+ mobilization was indispensable for the activation of PLD1 as well as cPLA2. The WKYMVm-dependent stimulation of cPLA2 activity was partially dependent on the activation of PKC and mitogen-activated protein kinase, while PKC activation, but not mitogen-activated protein kinase activation, was an essential prerequisite for stimulation of PLD1. Taken together, activation of the two phospholipases, which are absolutely required for superoxide generation, takes place through independent signaling pathways that diverge from a common pathway at a point downstream of Ca2+.

Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) is a synthetic peptide that stimulates phosphoinositide (PI) hydrolysis in human leukocytes. The peptide binds to a unique cell surface receptor(s). Recently we had demonstrated that human neutrophils, monocytes, and B lymphocytes express this peptide-specific receptor and that stimulation of human leukocytes with the peptide leads to activation of the oxidative respiratory system and the bactericidal activity of neutrophils or monocytes. In this study we showed that the peptide induces chemotaxis of phagocytic leukocytes and studied the signaling pathway leading to chemotaxis in human monocytes. The peptide-induced monocyte chemotaxis is pertussis toxin (PTX)-sensitive. This fact correlates with the peptide's stimulation of PI hydrolysis and intracellular Ca2+ ([Ca2+]i) release, which is also PTX-sensitive. We demonstrate that the peptide-specific receptor is different from receptor(s) for monocyte chemoattractant protein-1 (MCP-1). We also show that intracellular signaling of WKYMVm leading to monocyte chemotaxis is different from that of MCP-1. The peptide-mediated monocyte chemotaxis is insensitive to protein kinase C (PKC) inhibitor (GF109203X) and butan-1-ol, ruling out PKC and phospholipase D participation in this process. On the other hand, a tyrosine kinase inhibitor (genistein) and RhoA inhibitor (C3 transferase) curtailed the peptide-induced chemotaxis in a concentration-dependent manner, implying the involvement of tyrosine kinase and RhoA, respectively. Treatment of human monocytes with the peptide stimulates tyrosine phosphorylation of several cellular proteins, including p125FAK and Pyk2 and translocation of RhoA from the cytosol to the membrane. We conclude that WKYMVm induces chemotaxis of human phagocytic leukocytes via unique receptors and signaling.