Signaling and Cross-talk by C5a and UDP in Macrophages Selectively Use PLCβ3 to Regulate Intracellular Free Calcium

Studies in fibroblasts, neurons, and platelets have demonstrated the integration of signals from different G protein-coupled receptors (GPCRs) in raising intracellular free Ca 2+ . To study signal integration in macrophages, we screened RAW264.7 cells and bone marrow-derived macrophages (BMDM) for their Ca 2+ response to GPCR ligands. We found a synergistic response to complement component 5a (C5a) in combination with uridine 5′-diphosphate (UDP), platelet activating factor (PAF), or lysophosphatidic acid (LPA). The C5a response was Gα i -dependent, whereas the UDP, PAF, and LPA responses were Gα q -dependent. Synergy between C5a and UDP, mediated by the C5a and P2Y6 receptors, required dual receptor occupancy, and affected the initial release of Ca 2+ from intracellular stores as well as sustained Ca 2+ levels. C5a and UDP synergized in generating inositol 1,4,5-trisphosphate, suggesting synergy in activating phospholipase C (PLC) β. Macrophages expressed transcripts for three PLCβ isoforms (PLCβ2, PLCβ3, and PLCβ4), but GPCR ligands selectively used these isoforms in Ca 2+ signaling. C5a predominantly used PLCβ3, whereas UDP used PLCβ3 but also PLCβ4. Neither ligand required PLCβ2. Synergy between C5a and UDP likewise depended primarily on PLCβ3. Importantly, the Ca 2+ signaling deficiency observed in PLCβ3-deficient BMDM was reversed by re-constitution with PLCβ3. Neither phosphatidylinositol (PI) 3-kinase nor protein kinase C was required for synergy. In contrast to Ca 2+ , PI 3-kinase activation by C5a was inhibited by UDP, as was macropinocytosis, which depends on PI 3-kinase. PLCβ3 may thus provide a selective target for inhibiting Ca 2+ responses to mediators of inflammation, including C5a, UDP, PAF, and LPA.