Signaling and Cross-talk by C5a and UDP in Macrophages

Studies in fibroblasts, neurons, and platelets have demonstrated the integration of signals from different G proteincoupled receptors (GPCRs) in raising intracellular freeCa2 . 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 i-dependent, whereas the UDP, PAF, and LPA responseswereG q-dependent. SynergybetweenC5a andUDP, mediated by theC5a andP2Y6 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) . Macrophages expressed transcripts for three PLC isoforms (PLC 2, PLC 3, and PLC 4), but GPCR ligands selectively used these isoforms in Ca2 signaling. C5a predominantly used PLC 3, whereas UDP used PLC 3 but also PLC 4. Neither ligand required PLC 2. Synergy between C5a and UDP likewise depended primarily on PLC 3. Importantly, the Ca2 signaling deficiency observed in PLC 3-deficient BMDMwas reversed by re-constitution with PLC 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 3 may thus provide a selective target for inhibiting Ca2 responses to mediators of inflammation, including C5a, UDP, PAF, and LPA. Calcium is an important messenger involved in the regulation of multiple cellular processes, and levels of intracellular free calcium ([Ca2 ]i) are precisely regulated (1–3). Increases in intracellular [Ca2 ]i are initiated by the phospholipase C (PLC) family of enzymes, which hydrolyze membrane-associated phosphatidylinositol 4,5-diphosphate (PIP2) to produce inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (4). IP3 triggers the release of Ca2 from stores in the endoplasmic reticulum, whereas diacylglycerol activates members of the protein kinase C (PKC) family. Following activation of stored Ca2 by IP3, influx of extracellular Ca2 across the plasma membrane may further contribute to an increase in [Ca2 ]i, which is regulated by several Ca2 pumps and buffers (1). The net level and duration of these Ca2 signals regulate cellular responses, including transcription, apoptosis, endocytosis, chemotaxis, and metabolism (3). Simultaneous stimulation of twoGPCRs coupled to different G subunits, often G i or G s in combination with G q, has been shown to yield synergistic Ca2 responses in several model systems (reviewed in Ref. 5). Limited studies have demonstrated this synergy in primary cells, including neurons and platelets, but the mechanisms of synergy vary and are not well defined (6, 7). Synergistic Ca2 responses resulting from heterologous GPCR ligation have been little studied in macrophages, where members of the GPCR superfamily can stimulate an increase in [Ca2 ]i by activating members of the PLC family (4). As part of a systematic screen of RAW264.7macrophage cells, C5a and UDP demonstrated synergy in producing a rise in [Ca2 ]i (8)). C5a is an important inflammatory mediator for macrophages and UDP, which is released following cell damage, is also present at sites of injury or infection (9, 10). Both ligands signal through GPCRs; C5a signals through C5aR (11), and UDP signals through P2Y6 receptors (12). To examineGPCR cross-talk by these ligands in mouse macrophages we studied both * This work was supported, in whole or in part, by National Institutes of Health Grant GM 62114. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4. 1 Both authors contributed equally to this manuscript. 2 To whom correspondence may be addressed: VAMC 111R, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-750-2104; Fax: 415-750-6920; E-mail: [email protected]. 3 To whom correspondence may be addressed. E-mail: [email protected]. 4 To whom correspondence may be addressed. E-mail: bseaman@medicine. ucsf.edu. 5 The abbreviations used are: [Ca ]i, intracellular free calcium; BMDM, bone marrow-derived macrophages; C5a, complement component 5a; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; IP3, inositol 1,4,5-trisphosphate; LPA, lysophosphatidic acid; PAF, platelet activating factor; PIP2, phosphatidylinositol 4,5-diphosphate; PKC, protein kinase C; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; PTX, pertussis toxin; ERK, extracellular signal-regulated kinase; WT, wild type; YFP, yellow fluorescent protein; RNAi, RNA interfering; shRNA, short hairpin RNA. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 25, pp. 17351–17361, June 20, 2008 Printed in the U.S.A.