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 Ca2+. To study signal integration in macrophages, we screened RAW264.7 cells and bone marrow-derived macrophages (BMDM) for their Ca2+ 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 G alpha(i)-dependent, whereas the UDP, PAF, and LPA responses were G alpha(q)-dependent. Synergy between C5a and UDP, mediated by the C5a and P2Y6 receptors, required dual receptor occupancy, and affected the initial release of Ca2+ from intracellular stores as well as sustained Ca2+ levels. C5a and UDP synergized in generating inositol 1,4,5-trisphosphate, suggesting synergy in activating phospholipase C (PLC) beta. Macrophages expressed transcripts for three PLC beta isoforms (PLC beta 2, PLC beta 3, and PLC beta 4), but GPCR ligands selectively used these isoforms in Ca2+ signaling. C5a predominantly used PLC beta 3, whereas UDP used PLC beta 3 but also PLC beta 4. Neither ligand required PLC beta 2. Synergy between C5a and UDP likewise depended primarily on PLC beta 3. Importantly, the Ca2+ signaling deficiency observed in PLC beta 3-deficient BMDM was reversed by re-constitution with PLC beta 3. Neither phosphatidylinositol (PI) 3-kinase nor protein kinase C was required for synergy. In contrast to Ca2+, PI 3-kinase activation by C5a was inhibited by UDP, as was macropinocytosis, which depends on PI 3-kinase. PLC beta 3 may thus provide a selective target for inhibiting Ca2+ responses to mediators of inflammation, including C5a, UDP, PAF, and LPA.