Signal Transduction In Mast Cells

肥大细胞中的信号转导

• 批准号: 7967078
• 负责人: Reuben P. Siraganian
• 金额: $ 160.9万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967078
• 关键词: Affinity; Amino Acids; Antibodies; Antigens; Binding; Binding Sites; Biological; Biological Assay; Calcineurin B; Calcium; Catalytic Domain; Cell Degranulation; Cell Line; Cell Nucleus; Cell physiology; Cells; Cloning; Complementary DNA; Cytokine Gene; Cytoplasmic Protein; Cytoplasmic Tail; Disease; Docking; Dose; Enzymes; Event; Extrinsic asthma; Family; GD1b ganglioside; Galactose; Galactose Binding Lectin; Galactosyltransferases; Genes; Genetic Transcription; Glycolipids; Green Fluorescent Proteins; Housekeeping; ITAM; IgE; IgE Receptors; Immune system; Immunologic Receptors; In Vitro; Inflammatory; Knockout Mice; Laboratories; Lipids; Mediating; Mediator of activation protein; Membrane Microdomains; Minor; Mitogen-Activated Protein Kinases; Molecular; Molecular Conformation; Molecular Weight; Monoclonal Antibodies; Mus; Mutate; Mutation; NF-kappa B; PPP2CA gene; Pathway interactions; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Plasmids; Play; Protein Tyrosine Kinase; Protein phosphatase; Proteins; Proto-Oncogene Proteins c-fyn; RNA Interference; RNA library; Reaction; Relative (related person); Reporter; Role; Serine; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Sorting - Cell Movement; Sucrose; System; T-Cell Activation; Testing; Thin Layer Chromatography; Transfection; Tyrosine; Tyrosine Phosphorylation; Variant; cDNA Library; calcineurin phosphatase; cytokine; design; enhanced green fluorescent protein; expression cloning; inhibitor/antagonist; knock-down; mast cell; member; mitogen-activated protein kinase p38; mutant; nuclear factors of activated T-cells; protein function; protein kinase Cmu; receptor; receptor-mediated signaling; research study; response; scaffold; src-Family Kinases; tool; transcription factor

The activation of the high affinity IgE receptor (FceRI) results in rapid protein tyrosine phosphorylation and activation of the cytoplasmic protein tyrosine kinase Syk which is essential for this receptor-mediated signaling in mast cells. The binding of Syk to the tyrosine phosphorylated ITAM of the beta and gamma subunits of FceRI results in a conformational change in Syk, with an increase in its enzymatic activity that leads to tyrosine phosphorylation of several proteins and the downstream propagation of signals. The conformational change of Syk exposes its carboxy-terminal region to binding by an antibody. Syk has three Tyr residues located four amino acids from its carboxy terminal. To characterize the role of these Tyr residues in mast cell signaling, mutant Syk with these three Tyr mutated to Phe was expressed in a Syk-deficient variant of the RBL-2H3 mast cells. Compared with wild-type Syk, mutation of the three Tyr residues resulted in a dramatic decrease of FceRI-stimulated mast cell degranulation and signaling to NFAT and NF-kB activation with a parallel decrease in activation of the Erk and p38 MAP kinases. In vitro this mutated Syk had decreased kinase activity but when expressed in cells it was constitutively tyrosine phosphorylated with an increase in phosphorylation of the negative regulatory Y317, but decreased phosphorylation of the activation loop tyrosines. Mutation of each of the three Tyr residues separately showed that the last two and especially the third was the most important for Syk function. These results suggest that phosphorylation of these Tyr residues contribute to the activate state by keeping the molecule in an extended conformation. These Tyr residues may also contribute to signaling in the cell by providing docking sites after phosphorylation for other molecules. Studies with mast cells from knockout mice have suggested that the tyrosine kinase Fyn and its downstream substrate Gab2 may play a role in FceRI-mediated mast cell activation. To examine the relative role of these two molecules and compare them to that of Syk, we transiently transfected mouse mast cells with small interference RNA (siRNA) to specifically decrease the expression of Fyn, Gab2 or Syk.. There was decreased activation of phosphoinositide-3-kinase (PI3K) pathway as indicated by a change in Akt phosphorylation after suppression of Gab2 but not Fyn demonstrating that Gab2 but not Fyn regulates this pathway. The decreased expression of Gab2 slightly enhanced degranulation whereas decreased Fyn levels did not have any effect. There were some minor changes in NFAT or NFkB activation in cells with decreased expression of Fyn or Gab2. Decreased Gab2 but not Fyn reduced the FceRI-induced activation of the Erk, Jnk and p38 MAP kinases and the release of TNFa. In contrast, decreased expression of Syk dramatically reduced FceRI-induced degranulation, activation of NFAT and NFkB. These results suggest that Syk is the major regulator of FceRI-mediated reactions while Fyn has minor if any effects and Gab2 regulates primarily late events including MAP kinase activation and release of cytokines. The mAb BD6 is a monoclonal antibody that inhibits the binding of IgE to FceRI, without directly reacting with this receptor. By expression cloning, mAb BD6 identified the alpha1,3-galactosyltransferase gene. Both galactose and melibiose decreased the binding of mAb BD6 in a dose dependent manner on RBL-2H3 cells and abolished its binding on alpha1,3-galactosyltransferase transfected PEAK cells. There was also partial competition between mAb BD6 and IB4, a galactose binding lectin. MAb BD6 recognized a low molecular weight lipid separated by thin layer chromatography. By sucrose gradient analysis mAb BD6 bound to the lipid rafts fractions. By repeated sorting and cloning, RBL-2H3 variants were isolated deficient in mAb BD6 binding; these cells lacked the GD1b ganglioside and had low expression of GM1. However, they still had normal FceRI induced degranulation. These results suggest that mAb BD6 inhibits IgE binding by reacting with a galactose containing glycolipid present in lipid rafts. FceRI stimulation results in an increase in intracellular calcium that activates the serine phosphatase calcineurin, which then dephosphorylates the nuclear factor of activated T cells (NFAT). The dephosphorylated NFAT rapidly translocates into the nucleus and induces the transcription of various cytokine genes in cells. Therefore, NFAT was used as readout for mast cell activation. A plasmid containing three tandem NFAT binding sites fused to the cDNA of enhanced green fluorescent protein (GFP) was transfected into the RBL-2H3 cells and a cell line was selected that became strongly GFP-postive only after FceRI stimulation. Transient transfection of a plasmid containing the cDNA for the NH2-half of Syk that lacks the enzymatic domain (Syk-SH2) inhibits this GFP response. Transient transfection of these cells with plasmids from an RBL-2H3 cDNA library were used to screen for molecules that could inhibit or enhance the receptor-induced GFP response. In a screen of 300 plasmids, there were a number of positives; among these are genes that have effects on cellular housekeeping and several others that are similar to signaling molecules. The pathways leading from FceRI aggregation to cellular responses depend on protein phosphorylations regulated by both kinases and phosphatases. To gain an understanding on the functions played by phosphatases in IgE-mediated mast cell activation, a siRNA library that targets all mouse phosphatase genes was screened. Following each target siRNA transfection, IgE-antigen induced mast cell degranulation was assayed for three days as a functional readout of targeted protein knock-down. Out of 198 phosphatases, 10 enhanced and 7 inhibited FceRI-induced mast cell mediator release. For 7 of the strongest hits, four different siRNAs per target were tested, which defined three unambiguous hits, Pten, Mtmr4 and Ppp3r1 (calcineurin B). Furthermore, the mechanism of the inhibition of mast cell degranulation due to calcineurin B deficiency was investigated. Calcineurin B deficiency reduced the phosphorylation of MAP kinases and the phosphorylation of PKD/PKCmu and PKCdelta, which are involved in FceRI signaling. Therefore, this siRNA screen identified several new molecules that are critical for FceRI-induced degranulation. Blocking the function of these proteins may be potential targets for the treatment of asthma and allergic diseases. Compared to the studies using knock-out mice in which the targeted protein is absent, the results with siRNA transfection probably reproduce better what would occur with an inhibitor of a molecule in this signaling pathway and therefore is more useful for the design of pharmacological inhibitors. Immune receptor stimulated synthesis of cytokines depends on NFAT and NFkB transcription factors. To study FceRI induced activation of these pathways,mast cell lines that have NFAT or NFkB reporter systems were screened with a siRNA library that targets phosphatases. Among the 198 phosphatases, 31 enhanced or inhibited FceRI-initiated NFAT or NFkB activation. Among the positive hits, the siRNA for PPP2CA (catalytic subunit of Ser/Thr phosphatase 2A) slightly reduced FceRI-initiated NFAT activation, but dramatically enhanced NFkB activity. While the siRNA of PPP1CA (catalytic subunit of the Ser/Thr phosphatase 1A) slightly enhanced NFAT, but had no effect on NFkB activation. These results suggest that Ser/Thr phosphatases are involved in FceRI signaling with protein phosphatase 1 and 2 having divergent roles in mast cell functions contrary to what had been concluded from experiments with inhibitors that indiscriminately block both enzymes.

高亲和力 IgE 受体 (FceRI) 的激活导致蛋白质酪氨酸快速磷酸化和细胞质蛋白酪氨酸激酶 Syk 的激活，这对于肥大细胞中受体介导的信号传导至关重要。 Syk 与 FceRI β 和 γ 亚基的酪氨酸磷酸化 ITAM 的结合导致 Syk 的构象变化，其酶活性增加，导致多种蛋白质的酪氨酸磷酸化和信号的下游传播。 Syk 的构象变化使其羧基末端区域暴露于抗体的结合。 Syk 具有三个酪氨酸残基，位于距其羧基末端四个氨基酸处。为了表征这些 Tyr 残基在肥大细胞信号传导中的作用，在 RBL-2H3 肥大细胞的 Syk 缺陷变体中表达了这三个 Tyr 突变为 Phe 的突变体 Syk。与野生型 Syk 相比，三个 Tyr 残基的突变导致 FceRI 刺激的肥大细胞脱粒和 NFAT 和 NF-kB 激活信号传导显着减少，同时 Erk 和 p38 MAP 激酶的激活也相应减少。在体外，这种突变的 Syk 激酶活性降低，但当在细胞中表达时，它会被组成型酪氨酸磷酸化，负调节 Y317 的磷酸化增加，但激活环酪氨酸的磷酸化减少。三个 Tyr 残基中每一个的突变表明最后两个，尤其是第三个对于 Syk 功能最重要。这些结果表明这些 Tyr 残基的磷酸化通过保持分子处于延伸构象而有助于激活状态。这些酪氨酸残基还可能通过在磷酸化后为其他分子提供对接位点来促进细胞中的信号传导。 对基因敲除小鼠肥大细胞的研究表明，酪氨酸激酶 Fyn 及其下游底物 Gab2 可能在 FceRI 介导的肥大细胞激活中发挥作用。为了检查这两种分子的相对作用并将其与 Syk 进行比较，我们用小干扰 RNA (siRNA) 瞬时转染小鼠肥大细胞，以特异性降低 Fyn、Gab2 或 Syk 的表达。抑制 Gab2 而不是 Fyn 后 Akt 磷酸化的变化表明了 3-激酶 (PI3K) 途径，表明 Gab2 而不是 Fyn 调节该途径。 Gab2 表达的减少略微增强了脱粒作用，而 Fyn 水平的降低则没有任何影响。 Fyn 或 Gab2 表达降低的细胞中 NFAT 或 NFkB 激活存在一些微小变化。 Gab2 的减少而非 Fyn 的减少会减少 FceRI 诱导的 Erk、Jnk 和 p38 MAP 激酶的激活以及 TNFa 的释放。 相反，Syk 表达的减少显着减少了 FceRI 诱导的脱粒、NFAT 和 NFkB 的激活。这些结果表明 Syk 是 FceRI 介导的反应的主要调节因子，而 Fyn 的影响较小（如果有的话），而 Gab2 主要调节晚期事件，包括 MAP 激酶激活和细胞因子的释放。 mAb BD6 是一种单克隆抗体，可抑制 IgE 与 FceRI 的结合，但不直接与该受体发生反应。通过表达克隆，mAb BD6 鉴定了 α1,3-半乳糖基转移酶基因。半乳糖和蜜二糖均以剂量依赖性方式降低 mAb BD6 对 RBL-2H3 细胞的结合，并消除其对 α1,3-半乳糖基转移酶转染 PEAK 细胞的结合。 mAb BD6 和 IB4（一种半乳糖结合凝集素）之间也存在部分竞争。 MAb BD6 识别通过薄层色谱分离的低分子量脂质。 通过蔗糖梯度分析，mAb BD6 与脂筏组分结合。通过重复分选和克隆，分离出缺乏 mAb BD6 结合的 RBL-2H3 变体；这些细胞缺乏 GD1b 神经节苷脂，并且 GM1 表达较低。然而，它们仍然具有正常的 FceRI 诱导的脱颗粒。这些结果表明 mAb BD6 通过与脂筏中存在的含有半乳糖的糖脂反应来抑制 IgE 结合。 FceRI 刺激导致细胞内钙增加，从而激活丝氨酸磷酸酶钙调神经磷酸酶，然后使活化 T 细胞的核因子 (NFAT) 去磷酸化。 去磷酸化的NFAT迅速转入细胞核并诱导细胞内各种细胞因子基因的转录。因此，NFAT 被用作肥大​​细胞激活的读数。将含有与增强型绿色荧光蛋白（GFP）的 cDNA 融合的三个串联 NFAT 结合位点的质粒转染至 RBL-2H3 细胞中，并选择仅在 FceRI 刺激后才变为强 GFP 阳性的细胞系。瞬时转染含有缺少酶结构域的 Syk NH2-half (Syk-SH2) cDNA 的质粒会抑制这种 GFP 反应。 用来自 RBL-2H3 cDNA 文库的质粒瞬时转染这些细胞，用于筛选可以抑制或增强受体诱导的 GFP 反应的分子。在300个质粒的筛选中，出现了许多阳性；其中包括对细胞管家有影响的基因以及其他一些类似于信号分子的基因。 从 FceRI 聚集到细胞反应的途径取决于激酶和磷酸酶调节的蛋白质磷酸化。为了了解磷酸酶在 IgE 介导的肥大细胞激活中发挥的功能，筛选了针对所有小鼠磷酸酶基因的 siRNA 文库。每次靶标 siRNA 转染后，对 IgE 抗原诱导的肥大细胞脱粒进行三天的测定，作为靶标蛋白敲低的功能读数。在 198 种磷酸酶中，10 种增强，7 种抑制 FceRI 诱导的肥大细胞介质释放。对于 7 个最强的命中，测试了每个靶标的四种不同 siRNA，这定义了三个明确的命中：Pten、Mtmr4 和 Ppp3r1（钙调神经磷酸酶 B）。此外，还研究了由于钙调神经磷酸酶B缺乏而抑制肥大细胞脱颗粒的机制。钙调神经磷酸酶 B 缺乏会降低 MAP 激酶的磷酸化以及参与 FceRI 信号传导的 PKD/PKCmu 和 PKCdelta 的磷酸化。因此，该 siRNA 筛选鉴定了几种对 FceRI 诱导的脱颗粒至关重要的新分子。阻断这些蛋白质的功能可能是治疗哮喘和过敏性疾病的潜在目标。与使用缺失靶蛋白的基因敲除小鼠进行的研究相比，siRNA转染的结果可能更好地再现了该信号通路中分子抑制剂所发生的情况，因此对于药理学抑制剂的设计更有用。 免疫受体刺激的细胞因子合成依赖于 NFAT 和 NFkB 转录因子。为了研究 FceRI 诱导的这些途径的激活，使用靶向磷酸酶的 siRNA 文库筛选了具有 NFAT 或 NFkB 报告系统的肥大细胞系。在 198 种磷酸酶中，有 31 种增强或抑制 FceRI 启动的 NFAT 或 NFkB 激活。在积极的结果中，PPP2CA（Ser/Thr 磷酸酶 2A 催化亚基）的 siRNA 略微降低了 FceRI 引发的 NFAT 激活，但显着增强了 NFkB 活性。而PPP1CA（Ser/Thr磷酸酶1A的催化亚基）的siRNA略微增强了NFAT，但对NFkB激活没有影响。这些结果表明，Ser/Thr 磷酸酶参与 FceRI 信号传导，而蛋白磷酸酶 1 和 2 在肥大细胞功能中具有不同的作用，这与使用不加区别地阻断两种酶的抑制剂实验得出的结论相反。