The Role Of Shp2 And NK1R In Mast Cell Activation And Its Molecular Mechanism

Part Ⅰ Study on the role of Shp2in FcεRI-mediated mast cell activationThe protein-tyrosine phosphatase (PTP) Shp2has been implicated in many immunoreceptor signaling pathways, but its role in immunoreceptor FcsRI signaling is still largely undefined. FcεRI is expressed abundantly on mast cells and blood basophils, with high affinity for immunoglobulin E (IgE). Aggregation of IgE-bearing FcεRI on mast cells induced by polyvalent antigen promotes assembly of following signal transductions, leading eventually to the activation of mast cells. In this study, Using Shp2knockdown RBL-2H3mast cells, we investigated the role of Shp2in FcεRI-evoked mast cell activation, and also explored its signaling regulatory mechanism in FcεRI signaling. Based on our data, we here reported that Shp2is required for the activation of RBL-2H3cells induced by FcεRI. FcεRI-evoked degranulation, calcium mobilization, and synthesis of cytokine transcripts (IL-1β, IL-10, and monocyte chemoattractant protein1(MCP-1)) were reduced in Shp2knockdown RBL-2H3cells. Signaling regulatory mechanism investigation using immunoblotting, immunoprecipitation, and GST pull-down assay reveals that the down-regulation of Shp2expression in RBL-2H3cells leads to decreased activities of many key signal molecules, including Fyn, Akt, JNK, p38MAPK, and Ras/Erkl/2, after FcεRI aggregation. Further study suggests that Paxillin phosphoryaltion was also impaired, but CBP/PAG phosphorylation was normal after FcεRI stimulation as a consequence of the inhibition of Shp2expression in RBL-2H3cells. Collectively, our data strongly indicate that Shp2is essential for the activation of RBL-2H3cells in response to FcεRI aggregation. Shp2regulates this process through Fyn and Ras with no involvement of CbP/PAG. In addition, we identify Paxillin as an indirect substrate of Shp2in FcεRI-initiated signaling of RBL cells. Part Ⅱ Study on the role of neurokinin1receptor (NK1R) in FcεRI-mediated mast cell activationMast cells are principal effectors in allergic diseases. In response to the specific antigen, mast cells are activated following the aggregation of the FcεRI, leading to the release of inflammatory mediators and thus contributing to allergic symptomology. Increasing numbers of reports reveal that there exist a close relationship between neurokinin-1receptor (NKIR) and various allergic diseases, indicating that NK1R may be involved in the activation of mast cells. In the current study, by transiently transfecting shRNAs against NKIR into RBL-2H3mast cells, we inhibited the expression of NKIR in RBL-2H3mast cells. This allowed us to study the role of NKIR in RBL-2H3mast cells after FcεRI cross-linking. We observed that MCP-1mRNA expression and calcium mobilization were reduced in NKIR knockdown RBL-2H3cells after FcεRI activation, indicating an essential role of NKIR in FceRI-evoked RBL-2H3mast cell activation. Investigation of the signaling regulatory mechanism suggest that NKIR is involved in the regulation of MAPK signaling pathways downstream of the FcεRI, as phosphorylaiton levels of MAPKs (Erkl/2, JNK, and p38) after DNP-BSA stimulation (via FcεRI) were all decreased in NKIR knockdown RBL-2H3mast cells compared with control RBL-2H3mast cells.Taken together, our data provide the first evidence for the functional roles of NKIR in FcεRI-evoked activation of RBL-2H3mast cells. NKIR exerts its functions in this process by promoting the synthesis of MCP-1mRNA and calcium mobilization, the mechanism behind which is involved with the regulation of MAPK signaling pathways.