| Autor: |
Kelher, Marguerite R., McLaughlin, Nathan J. D., Banerjee, Anirban, Elzi, David J., Gamboni, Fabia, Khan, Samina Y., Meng, Xianzhong, Mitra, Sanchayita, Silliman, Christopher C. |
| Zdroj: |
Journal of Leukocyte Biology; January 2017, Vol. 101 Issue: 1 p261-273, 13p |
| Abstrakt: |
Lysophosphatidylcholines (lysoPCs) are effective polymorphonuclear neutrophil (PMN) priming agents implicated in transfusion-related acute lung injury (TRALI). LysoPCs cause ligation of the G2A receptor, cytosolic Ca2+flux, and activation of Hck. We hypothesize that lysoPCs induce Hck-dependent activation of protein kinase C (PKC), resulting in phosphorylation and membrane translocation of 47 kDa phagocyte oxidase protein (p47phox). PMNs, human or murine, were primed with lysoPCs and were smeared onto slides and examined by digital microscopy or separated into subcellular fractions or whole-cell lysates. Proteins were immunoprecipitated or separated by polyacrylamide gel electrophoresis and immunoblotted for proteins of interest. Wild-type (WT) and PKC? knockout (KO) mice were used in a 2-event model of TRALI. LysoPCs induced Hck coprecipitation with PKCd and PKC? and the PKCd:PKC? complex also had a fluorescence resonance energy transfer (FRET)+interaction with lipid rafts and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2 (WAVE2). PKC? then coprecipitated with p47phox. Immunoblotting, immunoprecipitation (IP), specific inhibitors, intracellular depletion of PKC isoforms, and PMNs from PKC? KO mice demonstrated that Hck elicited activation/Tyr phosphorylation (Tyr311 and Tyr525) of PKCd, which became Thr phosphorylated (Thr507). Activated PKCd then caused activation of PKC?, both by Tyr phosphorylation (?yr514) and Ser phosphorylation, which induced phosphorylation and membrane translocation of p47phox. In PKC? KO PMNs, lysoPCs induced Hck translocation but did not evidence a FRET+interaction between PKCd and PKC? nor prime PMNs. In WT mice, lysoPCs served as the second event in a 2-event in vivo model of TRALI but did not induce TRALI in PKC? KO mice. We conclude that lysoPCs prime PMNs through Hck-dependent activation of PKCd, which stimulates PKC?, resulting in translocation of phosphorylated p47phox. LysoPCs elicit Hck translocation, causing nPKC (PKCd) to activate cPKC (PKC?), which results in priming of neutrophil NADPH oxidase. |
| Databáze: |
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