SPECIFIC OBJECTIVES COMPLETED THIS PAST YEAR: 1) With respect to PLD, previous studies revealed that PLD2 co-localized with components of specialized lipid microdomains in the plasma membrane sometimes """"""""lipid rafts"""""""" (unpublished data). We have now investigated whether PLD itself is required for the functional integrity of """"""""lipid rafts"""""""" because localized production of phosphatidic acid and diacylglycerol through PLD could have a substantial impact on membrane dynamics (Ref. 1). Suppression of PLD function, either with primary butanol, which subverts production of phosphatidic acid to the inert phosphatidylbutanol, or siRNA against PLD2 and to a much lesser extent PLD1 results in dispersal of lipid raft components including LAT, Thy1, and GPI. Stimulated translocation of FceRI and its associated tyrosine kinases, Lyn and Syk, into lipid rafts as well as downstream phosphorylation events and degranulation are also blocked. PLD2 also colocalizes with lipid raft constituents, a process that is prevented by the lipid raft dispersing agents. Consistent results were obtained when cells were examined by use of fluorescent tagged molecules and confocal microscopy or by membrane fractionation techniques. These and earlier studies suggest that PLD2 activation is not only dependent on lipid raft integrity but also ensures functionality of lipid rafts. 2) With respect to calcium signaling and as noted in the Introduction, we believed that CRAC was not the only mechanism for the entry of Ca2+ because Sr2+ and other divalent cations also permeate and support degranulation in stimulated mast cells. The transient receptor potential cannonical (TRPC) channels were originally considered as candidates for CRAC but their electrophysiological properties did not fit with those of CRAC and some TRPCs were known to conduct Sr2+ and other divalent metal ions. We found, however, that TRPCs do interact with the recently identified CRAC components, Orai1 and its Ca2+ sensor STIM1 (J. Immunol. 180:2233, 2008). We have since observed similar scenarios in both tumor and primary mast cell lines of rodent and human origin and have confirmed the requirement for TRPC channels in BMMC from TRPC -/- mice. These mice exhibit attenuated allergic reactions and BMMC derived therefrom exhibit defective a calcium signal, degranulation and much reduced production of inflammatory cytokines (studies in collaboration with Alasdair M. Gilfillan, LAD, NIAID). Most recently, it has become apparent that initiation of the calcium signal is absolutely dependent of TRPC channels and that this occurs in discrete regions (cell protuberances) where regional flickers merge into propagating calcium waves (Ref. 2). 3) Finally we examined the regulation of calcium mobilization by SK. In last year's report we described our failure to demonstrate a PLD/SK/Ca2+ pathway as reported by Melendez and coworkers. However, we have found by use of SK deficient BMMC an impairment of migration of the Ca2+-sensor STIM1 to the cell periphery and its presumed interaction with Orai1 and STIM 1 with modest impairment of Ca2+ influx (unpublished data). Our conclusion at this stage of our studies is that SK deficiency results in disfunctional membrane trafficking (the best studied role of SK)and that the effects on calcium signaling are secondary. The above findings tie seemingly paradoxical observations of non-selective uptake of divalent metal ions by stimulated mast cells (early sudies by us) and highly selective uptake of Ca2+ through CRAC/Orai1 (recent studies by others) (reviewed in refs 3 and 4). Collectively our studies on calcium and PKC signaling over the past 25 years have revealed much about the contribution of of these signals to mast cell activation and potential therapeutic targets (reviewed in ref. 4). There is still more to be learnt about specific details of the contributions of PLD. The specific details of role of SK, although clearly involved in allergic reactions (reviewed in Ref. 5), remain obscure.
| Cohen, Roy; Torres, Alexis; Ma, Hong-Tao et al. (2009) Ca2+ waves initiate antigen-stimulated Ca2+ responses in mast cells. J Immunol 183:6478-88 |
| Park, Seung-Kiel; Qiao, Huihong; Beaven, Michael A (2009) Src-like adaptor protein (SLAP) is upregulated in antigen-stimulated mast cells and acts as a negative regulator. Mol Immunol 46:2133-9 |
| Lisboa, Felipe A; Peng, Ze; Combs, Christian A et al. (2009) Phospholipase d promotes lipid microdomain-associated signaling events in mast cells. J Immunol 183:5104-12 |
| Ma, Hong-Tao; Beaven, Michael A (2009) Regulation of Ca2+ signaling with particular focus on mast cells. Crit Rev Immunol 29:155-86 |
| Beaven, Michael A (2009) Our perception of the mast cell from Paul Ehrlich to now. Eur J Immunol 39:11-25 |
