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）的激活导致快速蛋白酪氨酸磷酸化和细胞质蛋白酪氨酸激酶SYK的激活，这对于肥大细胞中该受体介导的信号传导至关重要。 SYK与酪氨酸的结合使FCERI的β和伽马亚基的ITAM磷酸化导致SYK的构象变化，其酶促活性的增加，导致了几种蛋白质的酪氨酸磷酸化以及信号的下游传播。 SYK的构象变化使其羧基末端区域暴露于抗体结合。 Syk的三个Tyr残留物位于其羧基末端的四个氨基酸。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缺陷型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信号转导向NFAT和NF-KB激活，而ERK和p38 MAP激酶的激活却降低。在体外，这种突变的SYK的激酶活性降低，但是当细胞中表达时，酪氨酸在阴性调节Y317的磷酸化增加时被组成性酪氨酸磷酸化，但激活环酪氨酸的磷酸化降低。三个Tyr残基中每个残基的突变分别表明，最后两个，尤其是第三个是对Syk功能最重要的。这些结果表明，这些Tyr残基的磷酸化通过使分子保持扩展构象来促进激活状态。这些Tyr残基也可能通过在其他分子后提供对接位点来导致细胞中的信号传导。 对敲除小鼠的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起作用。 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那个GAB2但没有Fyn调节这一途径。 GAB2表达降低略有增强的脱粒化，而FYN水平降低没有任何作用。在FYN或GAB2表达降低的细胞中，NFAT或NFKB激活发生了一些较小的变化。 降低的GAB2但没有FYN降低了FCERI诱导的ERK，JNK和P38 MAP激酶的激活以及TNFA的释放。 相反，SYK表达降低，大幅度降低了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剂，而FYN如果任何作用和GAB2的调节，则主要是较低的事件，主要是较晚的事件，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，而无需直接与该受体反应。通过表达克隆，MAB BD6鉴定出α1,3-乳糖基转移酶基因。半乳糖和麦芽糖都以剂量依赖于RBL-2H3细胞的剂量降低了MAB BD6的结合，并消除了其对α1,3-乳糖基转移酶转染的峰细胞的结合。 MAB BD6和IB4（一种半乳糖结合凝集素）之间也有部分竞争。 MAB BD6识别出薄层色谱分离的低分子量脂质。 通过蔗糖梯度分析MAB BD6与脂质筏的分数结合。通过重复分类和克隆，RBL-2H3变体在MAB BD6结合中被分离出来。这些细胞缺乏GD1B神经节苷脂，GM1表达较低。但是，它们仍然具有正常的FCERI诱导的脱粒。这些结果表明，MAB BD6通过与含有脂质筏中存在的糖脂的半乳糖反应来抑制IgE结合。 FCERI刺激导致细胞内钙的增加，从而激活丝氨酸磷酸酶钙调蛋白，然后将活化T细胞（NFAT）的核因子脱磷酸化。 去磷酸化的NFAT迅速转移到细胞核中，并诱导细胞中各种细胞因子基因的转录。因此，NFAT用作肥大细胞激活的读数。含有三个串联NFAT结合位点的质粒与增强的绿色荧光蛋白（GFP）融合的质粒被转染到RBL-2H3细胞中，并选择了仅在FCERI刺激后才能强烈GFP稳定性的细胞系。缺乏酶促结构域（SYK-SH2）的SYK NH2半级cDNA的质粒的瞬时转染抑制了这种GFP响应。 用来自RBL-2H3 cDNA文库的质粒将这些细胞的瞬时转染用于筛选可以抑制或增强受体诱导的GFP反应的分子。在300个质粒的屏幕中，有许多阳性。其中是对细胞家政的影响以及其他几个类似于信号分子的基因。 从FCER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所发挥的功能，筛选了针对所有小鼠磷酸酶基因的siRNA库。在每个靶标siRNA转染之后，测定IgE抗原诱导的肥大细胞脱粒化三天，作为靶向蛋白质敲低的功能读数。在198个磷酸酶中，有10种增强和7种抑制了FCERI诱导的肥大细胞介质释放。对于7个最强的命中，测试了每个目标四个不同的siRNA，这定义了三个明确的命中PTEN，MTMR4和PPP3R1（钙调神经素B）。此外，研究了钙调神经酶B缺乏引起的肥大细胞脱粒的机制。钙调神经素B缺乏降低了与FCERI信号传导有关的MAP激酶的磷酸化和PKD/PKCMU和PKCDELTA的磷酸化。因此，该siRNA屏幕确定了几个对于FCERI诱导的脱粒至关重要的新分子。阻止这些蛋白质的功能可能是治疗哮喘和过敏性疾病的潜在靶标。与使用缺乏靶向蛋白的敲除小鼠的研究相比，siRNA转染的结果可能会更好地再现该信号传导途径中分子抑制剂会发生的结果，因此对于药理抑制剂的设计更有用。 免疫受体刺激的细胞因子合成取决于NFAT和NFKB转录因子。为了研究FCERI诱导的这些途径的激活，用靶向磷酸酶的siRNA库筛选了具有NFAT或NFKB报告基因系统的肥大细胞系。在198个磷酸酶中，有31种增强或抑制了FCERI引起的NFAT或NFKB激活。在正命中中，PPP2CA的siRNA（Ser/Thr磷酸酶2A的催化亚基）略微降低了FCERI引起的NFAT激活，但急剧增强了NFKB活性。而PPP1CA的siRNA（Ser/Thr磷酸酶1A的催化亚基）略微增强了NFAT，但对NFKB激活没有影响。这些结果表明，Ser/Thr磷酸酶与蛋白质磷酸酶1和2的FCERI信号传导在肥大细胞功能中具有不同作用，与与不可分解的抑制剂实验相反，这些抑制剂不可分割地阻止了这两种酶。