| Calcium oscillation occurs in many cells, play essential in many physiologic and pathological response. Asthma is a chronic allergic airway inflammatory disease, many inflammatory cells and cytokines are involved in its pathogenesis. Bronchial epithelial cells are the main target cells for airway inflammation, involved in the existence, chemotaxis and activation of inflammation cells. Interleukin-13 (IL-13) and interleukin-4(IL-4) derived from Th2 cells, play a key role in the pathogenesis of asthma. IL-13 mediates epithelial hyperplasia and mucus excessive secretion, IL-4 involved in most process in the pathogenesis of asthma. Tumor necrosis factor(TNF-α)is the most important initiator in asthma inflammation,induce many cytokines. In this study, we treat human bronchial epithelial cells with these three cytokines, to investigate their effect on histamine induced cellular calcium oscillation. Lipid rafts are a membrane microdomain enriched with sphingolipid, cholesterol, acylated protein, forming relative stable and functional rafts floating in mobile lipid membrane, play a crucial role in cellular signal transduction, maybe involved in calcium signal transduction. FKBP12.6(FK506 binding protein of 12.6kDa)is a member of immunophilin superfamily, it is the accessory protein of Ryanodine2 Ca2+ releasing receptor (RyR2) on sarcoplasmic reticulum, binding of FKBP12.6 to RyR2 stabilize RyR2 . Calcium sensing receptor (CaSR) is a G-protein-coupled receptor, playing a key role in maintaining Ca2+ homeostasis. So, we hypothesized that the interaction of lipid rafts with FKBP12.6 or CaSR regulate cytokines induced cellular calcium oscillation. Objective The aim of this study is to investigate the effect of lipid rafts andits interaction with FKBP12.6 or CaSR on cytokines induced cellular calcium oscillation, to illustrate the role of lipid rafts in the pathogenesis of asthma, provide new strategy for the prevention and treatment of asthma.Methods1. Human bronchial epithelial cells were treated with 10ng/ml IL-4,IL-13,TNF-αfor 15 min, respectively, incubated with calcium-sensitive fluorophore FLUO-4, [Ca2+]i alteration induced by 10μm/ml histamine were monitored by laser confocal microscopy.2. Fixed cells were stained with CaSR or FKBP12.6 polyclonal antibody, respectively, followed by incubation with Alexa Fluor488 conjugated Cholera Toxin Subunit B (CTX-B), the colocalization of lipid rafts with FKBP12.6 or CaSR were observed and recorded with laser confocal microscopy.3. Treated HBE with FilipinⅢto inhibit lipid rafts, followed by monitor histamine induced [Ca2+]i changes.Results1. After pretreat with 10ng/ml IL-4 in human epithelial cells, the frequency of [Ca2+]i oscillations during histamine stimulation increased, the amplititude decreased; Lipid rafts aggregated significantly, were found to colocalize with FKBP12.6 /CaSR; Intranuclear CaSR protein level decreased, lower than that in endochylema and membrane. Under control normal condition, FKBP12.6 is expressed in endochylema, addition of IL-4 produced a significant increase of FKBP12.6 in nuclear.2. 10ng/ml IL-13 stimulated [Ca2+]i oscillations in human bronchial epithelial cells, followed by addition of histamine did not trigger [Ca2+]i oscillations. No obvious lipid rafts aggregation were found; Intranuclear CaSR protein level decreased, lower than that in endochylema and membrane. Intracellular FKBP12.6 level remains unchanged.3. After pretreatment with 10ng/ml TNF-αin human epithelial cells, Histamine did not trigger [Ca2+]i oscillations; Lipid rafts aggregated significantly, were found to colocalize with FKBP12.6 /CaSR; Intracellular CaSR level remain unchanged, intranuclear FKBP12.6 level increased significantly.4. Pretreating with FilipinⅢinhibited lipid rafts aggregation, histamine did not trigger [Ca2+]i oscillations with or without IL-4 / TNF-α.ConclusionAddition of IL-4 in human epithelial cells increased the frequency of [Ca2+]i oscillations during histamine stimulation, It is possible result from lipid rafts aggregation and its interaction with CaSR. IL-13 stimulated [Ca2+]i oscillations possiblely depend on the translocation of CaSR, independent of lipid rafts aggregation... |
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