Interleukin-17 Stimulates STAT3-Mediated Endothelial Cell Activation for Neutrophil Recruitment
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Background/Aims: Interleukin-17 (IL-17) is a major pro-inflammatory cytokine that initiates and maintains inflammation. However, the molecular mechanisms as to how IL-17 influences endothelial cells to promote neutrophil recruitment are not fully understood. Methods: Human endothelial cells (HMECs) were stimulated with IL-17, and investigated for proliferation, migration, and tubule formation activities. Transwell chemotaxis and adhesion assays were performed to assess neutrophil recruitment. Cytokine production was measured by Cytokine Array Chip and ELISA. Western blotting and immunofluorescent analysis were used to detect the phosphorylation and translocation of STAT3. Specific inhibitors, small interfering RNA, and phosphorylation mutants were used to confirm that IL-17 induced STAT3 activation via IL-17RA signaling. Results: Activation of HMECs with IL-17 induced STAT3 phosphorylation and nuclear translocation, which were associated with induction of GRO-α, GM-CSF and IL-8, and neutrophil recruitment. Phosphorylation of STAT3 was identified mainly at the tyrosine in position 705 (Y705), and the Y705F mutants attenuated IL-17-mediated STAT3 activation. Moreover, specific inhibitors, FLLL31, or siRNA silencing of STAT3 attenuated HMECs activation, resulting in inhibition of GRO-α, GM-CSF, IL-8 production, and neutrophil recruitment. Furthermore, phosphorylation of STAT3 was identified as downstream of IL-17RA signaling. Conclusions: IL-17 induced STAT3 activation as a necessary step in endothelial cell activation and neutrophil recruitment.Cite
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Objective STAT3 protein is an important member of STAT family and it is activated mainly by the phosphorylation of tyrosine.This study is to analyse the expression of STAT3 in Promyelocytic leukemia cells.Methods Promyelocytic leukemia cell line HL-60 are cultured and stimulate with human IL(hIL) 5 in different concentration(0,1.0,10,100 ng/ml) then are detected with immunoprecipitation,SDS-PAGE and Western Blot.Result HL-60 cell line expresses tyrosine(Y705) phosphorylated STAT3α and STAT3β in different concentration.Conclusion hIL-5 induces tyrosine(Y705) phosphorylation of STAT3α and STAT3β in HL-60 cell line in a certain range of concentration and the tyrosine phosphorylation of STAT3 αand STAT3β induced by hIL-5 is dose depended.
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Background: Signal Transducer and Activator of Transcription 3 (STAT3) is an intracellular signalling molecule, which is used by several cytokines, including leukemia inhibitory factor (LIF), epithelial growth factor (EGF), and interleukin‐6 (IL‐6). It induces a variety of gene transcripts and cell functions. In trophoblast cells and in tumor cells, its tyrosine phosphorylation is directly linked to their invasiveness. The regulation and function of STAT3 serine phosphorylation is still widely unclear. Material and Methods: Jeg‐3 choriocarcinoma cells were stimulated with different concentrations of EGF, IL‐6 and LIF. STAT3 serine (727) and tyrosine (705) phosphorylation were analyzed 5–60 min after stimulation by SDS‐PAGE electrophoresis followed by Western blotting. Results: Jeg‐3 cells display spontaneous STAT3 serine phosphorylation. 100 ng/mL EGF induces a time‐dependent reduction starting 15 min after stimulation. Tyrosine phosphorylation does not occur spontaneously, but is strongly induced by EGF at all analyzed time points. LIF induces tyrosine phosphorylation, but affects serine phosphorylation only very slightly. IL‐6 did not influence neither serine phosphorylation nor tyrosine phosphorylation. Discussion: The EGF induced STAT3 tyrosine phosphorylation may be responsible for its invasion triggering capacities. The parallel reduction of serine phosphorylation may enhance this effect. LIF was formerly shown to enhance trophoblast invasion via STAT3 tyrosine phosphorylation. IL‐6 displays very little effects on STAT3 and seems to use other pathways for signalling.
Phosphorylation cascade
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Phosphoproteomics
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UVradiation isknowntoinduce lymphocyte nonresponsiveness bothinvitro andinvivo. Wehavefoundthat UVradiation rapidly induced tyrosine phosphorylation andcalcium signaling innormal humanperipheral blood lymphocytes. Intheleukemic Tcell line Jurkat andtheBurkitt's lymphoma cell line Ramos, UVrapidly induced tyrosine phosphorylation inawavelength-dependent manner, giving strong signals after UVBandUVC,butnot UVA,irradiation. Similarly, inJurkat cells UV-induced calcium signals weredependent on thedoseofUVBorUVCirradiation overarange of150-1200 J/m2, butonlyasmall signal wasobserved forUVA atadoseof1200J/m2. TheUV-induced calcium signals were blocked bythetyrosine kinase inhibitor herbimycin A,indicating that they weredependent ontyrosine phosphorylation. Phospholipase C(PLC)oylwastyrosine phosphorylated in response toUV irradiation buttoalesser extent thanobserved after CD3cross-linking. However, PLCy1-associated proteins demonstrated tobindtothePLCY1SH2domain weretyrosine phosphorylated strongly after UVirradiation. A similar doseresponse was observed fortheinhibition byherbimycin AofUV-induced calcium signals andUV-induced tyrosine phosphorylation ofPLCy1 andassociated proteins. Wepropose that incontrast toCD3/Tistimulation, UV aberrantly triggers lymphocyte signal transduction pathways byamechanism that bypasses normal receptor control.
Jurkat cells
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