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Clostridium difficile toxin B differentially affects GPCR-stimulated Ca^(2+) responses in macrophages: independent roles for Rho and PLA_2

Rebres, Robert A. and Moon, Christina and DeCamp, Dianne and Lin, Keng-Mean and Fraser, Iain D. and Milne, Stephen B. and Roach, Tamara I. A. and Brown, H. Alex and Seaman, William E. (2010) Clostridium difficile toxin B differentially affects GPCR-stimulated Ca^(2+) responses in macrophages: independent roles for Rho and PLA_2. Journal of Leukocyte Biology, 87 (6). pp. 1041-1057. ISSN 0741-5400.

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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 Ca^(2+) responses to Gαi-linked receptors, including the C5aR, but reduced responses to Gαq-linked receptors, including the UDP receptors. Other Rho inhibitors also reduced UDP Ca^(2+) 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 PLCβ isoform-deficient BMDM, we found that ToxB inhibited Ca^(2+) signaling through PLCβ4 but enhanced signaling through PLCβ3. Effects of ToxB on GPCR Ca^(2+) responses correlated with GPCR use of PLCβ3 versus PLCβ4. ToxB inhibited UDP Ca^(2+) signaling without reducing InsP3 production or the sensitivity of cellular Ca^(2+) stores to exogenous InsP3, suggesting that ToxB impairs UDP signaling at the level of InsP3/Ca^(2+) coupling. In contrast, ToxB elevated InsP3 production by C5a, and the enhancement of Ca^(2+) 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 Ca^(2+) signaling by different GPCR-linked PLCβ isoforms in macrophages.

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Additional Information:© 2010 Society for Leukocyte Biology. Received November 20, 2008; Revised January 13, 2010; Accepted January 16, 2010. This work was supported by National Institutes of Health grant GM 62114. We thank Amanda Norton (San Francisco Veterans Mfairs Medical Center) and Jamie Liu, Leah Santat, and Lucas Cheadle (Caltech) for excellent technical assistance. Authorship: Robert A. Rebres: project design, statistical analyses, authorship, supervision, cell culture, expression constructs and transfections, population and single-cell Ca2 + assays, Western blots, Rho assays, and cytoskeletal assessments; Christina Moon: cell culture, transfections, population Ca2+ assays, Western blots, InsP3 analyses, and phosphatidylinositol analyses; Dianne DeCamp: InsP3 analyses; Keng-Mean Lin: expression constructs and InsP3 analyses; lain D. Fraser: project design, cell culture, and expression constructs; Stephen B. Milne: phosphatidylinositol analyses; Tamara 1. A. Roach: authorship and supervision; H. Alex Brown: project design and supervision; and William E. Seaman: project design, authorship, and supervision.
Funding AgencyGrant Number
NIHGM 62114
Subject Keywords:phospholipase C; lipoxygenase; complement
Issue or Number:6
Record Number:CaltechAUTHORS:20100810-093926145
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:19367
Deposited By: Tony Diaz
Deposited On:10 Aug 2010 21:49
Last Modified:03 Oct 2019 01:55

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