Clostridium difficile toxin B differentially affects GPCR-stimulated Ca2+ responses in macrophages: independent roles for Rho and PLA2

Clostridium difficile toxins cause acute colitis by disrupting the enterocyte barrier and promoting inflammation. ToxB from C. difficile inactivates Rho family GTPases and causes release of cytokines and eicosanoids by macrophages. We studied the effects of ToxB on GPCR signaling in murine RAW264.7 macrophages and found that ToxB elevated Ca2+ responses to G alpha i-linked receptors, including the C5aR, but reduced responses to G alpha q-linked receptors, including the UDP receptors. Other Rho inhibitors also reduced UDP Ca2+ responses, but they did not affect C5a responses, suggesting that ToxB inhibited UDP responses by inhibiting Rho but enhanced C5a responses by other mechanisms. By using PLC beta isoform-deficient BMDM, we found that ToxB inhibited Ca2+ signaling through PLC beta 4 but enhanced signaling through PLC beta 3. Effects of ToxB on GPCR Ca2+ responses correlated with GPCR use of PLC beta 3 versus PLC beta 4. ToxB inhibited UDP Ca2+ signaling without reducing InsP3 production or the sensitivity of cellular Ca2+ stores to exogenous InsP3, suggesting that ToxB impairs UDP signaling at the level of InsP3/Ca2+ coupling. In contrast, ToxB elevated InsP3 production by C5a, and the enhancement of Ca2+ signaling by C5a was prevented by inhibition of PLA2 or 5-LOX but not COX, implicating LTs but not prostanoids in the mechanism. In sum, ToxB has opposing, independently regulated effects on Ca2+ signaling by different GPCR-linked PLC beta isoforms in macrophages. J. Leukoc. Biol. 87: 1041-1057; 2010.