Secretion Of Granules From Mast Cells

肥大细胞分泌颗粒

• 批准号: 7594370
• 负责人: Michael A Beaven
• 金额: $ 95.62万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 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/7594370
• 关键词: 1,2-diacylglycerol; 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one; 76-kDa SH2 domain-containing leukocyte protein; Alcohols; Allergens; Antigens; Ascaridil; Binding; Calcium; Calcium Channel; Calcium Signaling; Cell Degranulation; Cell membrane; Cells; Co-Immunoprecipitations; Condition; Confocal Microscopy; Cytoplasmic Granules; Data; Diglycerides; Disruption; Divalent Cations; Enzyme Activators; Enzymes; Event; Fractionation; Generations; Genetic; Golgi Apparatus; Hand; Histamine; IgE; IgE Receptors; Inflammatory; Inflammatory Response; Investigation; LCP2 gene; Lead; Link; Lipids; Localized; Location; Manuscripts; Mediating; Membrane; Membrane Lipids; Membrane Microdomains; Natural Immunity; Pathologic; Pathway interactions; Phase; Phosphatidic Acid; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase; Phospholipase D; Phosphorylation; Phosphotransferases; Physiological; Play; Population; Process; Production; Protein Isoforms; Protein Kinase C; Protein Overexpression; Protein Tyrosine Kinase; Proteins; Regulation; Reporting; Research; Role; STIM1 gene; SYK gene; Series; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; Source; Sphingosine; Techniques; Therapeutic Agents; Vesicle; antigen challenge; cytokine; genetic manipulation; genetic regulatory protein; human STIM1 protein; in vivo; interest; kinase inhibitor; knock-down; mast cell; methyl-beta-cyclodextrin; mutant; phosphatidylinositol 4-phosphate; prevent; receptor; release of sequestered calcium ion into cytoplasm; response; sphingosine 1-phosphate; sphingosine kinase; src-Family Kinases

INTRODUCTION: We have shown that mast cell degranulation is dependent on a rise in intracellular calcium (calcium signal)in conjunction with activation of protein kinase (PK) C and phospholipase(PL)D. We have focussed on PLD in recent years because least is known about this enzyme. PLD is activated in mast cells by antigen and other stimulants and is the major source of biologically lipids such as phosphatidic acid and diglycerides. PLD appears to play an essential role in degranulation because PLD activation is closely correlated with degranulation under a variety of experimental conditions which include pharmacological disruption (Chadi et al., Mol. Immunol. 38:1269, 2001)and knock down of PLD with iRNAs (Peng and Beaven, J. Immunol.174:5201,2005). Furthermore, the two known isoforms of PLD, PLD1 and PLD2, reside in different locations within the cell. PLD1 is associated with Golgi and granule membranes and PLD2 with the plasma membrane (Choi et al., J. Immunol. 278:12039,2002). Both isoforms regulate different phases of degranulation in mast cells, granule-associated PLD1 in the translocation of granules to the plasma membrane and plasma membrane-associated PLD2 in the fusion of granules with the plasma membrane. However, the mechanisms by which PLD is activated and the downstream PLD-dependent signaling events remain largely undefined. These include possible links to entry of Ca2+ via phosphatidylinositol 5-kinase (PI5K) and sphingosine kinase (SK). OBJECTIVES: We are pusuing two lines of investigation in regard to PLD: the first is to elucidate the mechanisms of activation of PLD and the second is to identify downstream events that are regulated by PLD. PLD2 is of particular interest because its location at the plasma membrane makes this isoform accessible to FcepsilonRI-associated Src kinases. As reported last year, we found that PLD2, but not PLD1, is phosphorylated by the Src kinases, Fyn and Fgr, and that this phosphorylation regulates PLD2 activation and degranulation (Choi et al., Mol. Cell. Biol. 24:6980,2004). These earlier studies also revealed that PLD2 co-localized with "lipid rafts" and that phosphorylation of PLD2 was dependent on integrity of these "lipid rafts" (unpublished data). In the current period we have investigated whether PLD itself is required for the functional integrity of "lipid rafts" and the activation of downstream kinases such as Syk which plays a critical role in initiating several signaling cascades in mast cells. Also, following our observation that diglyceride-dependent isoforms of PKC are highly regulated by PLD (Peng and Beaven, J. Immunol. 174:5201,2005), we are now investigating the role of PLD in the activation of PI5K and SK because PLD products are potential activators of these enzymes. As both PI5K and SK have been implicated in the generation of a calcium signal, this topic has become an important component of our recent research. PLD AND LIPID RAFT FUNCTION: Suppression of PLD function, either with primary alcohols or siRNAs, results in dispersal of lipid raft components including LAT, Thy1, and GPI and suppression of the translocation of the IgE receptor (FceRI) and its associated tyrosine kinases, Lyn and Syk, into lipid rafts following antigen stimulation. Downstream phosphorylation events are also blocked. Consistent results were obtained whether cells were examined by use of fluorescent tagged molecules and confocal microscopy or by classical membrane fractionation techniques. These techniques indicate that tagged PLD2 also colocalizes with lipid raft constituents, a process that is prevented by the lipid raft dispersing agent, methyl beta-cyclodextrin. These observations suggest that not only is PLD2 activation dependent on lipid raft integrity but that this activation contributes to the functional organization of lipid rafts (unpublished data). PLD2 INTERACTS WITH SYK AND REGULATES SYK ACTIVITY AND DOWNSTREAM EVENTS: PLD2 associates with and promotes activation of Syk which, as noted above is a key enzyme in mast cell activation. Antigen stimulation results in co-localization of Syk with PLD2 within membrane lipid rafts as indicated by co-immunoprecipitation and confocal microscopy. This association is dependent on the initial phosphorylation of Syk by Lyn and binding of PLD2 to Syk via its Phox homology (PX) domain to induce further phosphorylation and activation of Syk and its downstream targets, LAT and SLP76. Paradoxically, expression studies indicated that a catalytically inactive mutant of PLD2 was equally active in this regard. Overexpression of PLD2, or the PLD2 mutant, enhanced antigen-induced phosphorylations of Syk and downnstream targets while expression of a PLD2 siRNA blocked these phosphorylations. Similar genetic manipulations suggested that the interaction of PLD2 with Syk was necessary for degranulation. These findings indicate a dual role for PLD2 as as a signaling molecule. It can act in a catalytic manner to produce biologically active lipids such as phosphatidic acid and diglycerides and as an adaptor molecule to facilitate signal transduction via Syk (Ref. 1). REGULATION OF PHOSPHATIDYLINOSITOL 5-KINASE, SPHINGOSINE KINASE, CALCIUM MOBILIZATION, AND DEGRANULATION BY PLD: As reported last year, the production of phosphatidic acid by PLD is the primary source of diacylglycerides in mast cells and is essential for sustained activation of diglyceride-dependent isoforms of PKC and degranulation. However, additional PLD-dependent processes appear to be necessary for antigen-mediated degranulation. One such process could be the activation of Syk as described above(Peng and Beaven, J. Immunol. 174:5201,2005). Pharmacological and genetic studies now show that PLD is linked to the activation of PI5K and SK in addition to PKC. PI5K catalyzes the conversion of phosphatidylinositol 4-phosphate (PIP) to phosphatidylinositol 4,5-bisphosphate (PIP2). SK kinase phosphorylates sphingosine to form sphingosine 1-phosphate (S1P) (ref. 2). Reports indicate that PIP2 regulates translocation of the TRPC5 Ca2+ channel protein from proximal vesicles to the plasma membrane a. S1P acting in conjunction with IP3 promotes release of Ca2+ from intracellular stores and subsequently influx of external Ca2+. In our hands, knockdown of PLD2 or PI5K blocks entry of Ca2+, as do SK inhibitors, with minimal effect on release of Ca2+ from intracellular stores in stimulated mast cells. These studies clearly demonstrate that PI5K and SK regulate Ca2+ influx rather than Ca2+ release. We are currently defining the mechanisms inolved in studies with inhibitory RNAs and genetically deficient mast cells (see ref.3). IDENTIFICATION OF CALCIUM CHANNELS AND REGULATORY PROTEINS FOR CALCIUM ENTRY Degranulation of mast cells is dependent on emptying of intracellular stores of Ca2+ and the ensuing influx of external Ca2+, also referred to as store-operated calcium entry (SOCE). The well characterized calcium release-activated calcium current (CRAC) has been identified as a primary candidate for mediating SOCE. We were sceptical that CRAC was the sole mechanism for the entry of Ca2+ because Sr2+ and other divalent cations also permeate and support degranulation in stimulated mast cells (see previous reports in this series). We find that overexpression of STIM1 and Orai1, which were recently shown to be essential components of CRAC, allows entry of Ca2+ but not Sr2+ in stimulated mast cells. However, influx of Ca2+ and Sr2+ as well as degranulation are dependent on the presence of TRPC5 (see previous section), in addition to STIM1 and Orai1, as demonstrated by siRNA knock down of each of these proteins. Our observations suggest that TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca2+ or Sr+ to generate a signal for degranulation (manuscript submitted).

简介：我们已经表明，肥大细胞脱粒取决于蛋白激酶（PK）C和磷脂酶（PL）d的激活以及蛋白激酶（PK）C的激活，取决于细胞内钙（钙信号）的升高。近年来，我们专注于PLD，因为对这种酶的了解最少。 PLD通过抗原和其他刺激剂在肥大细胞中激活，是生物学上脂质（例如磷脂酸和甘油三酸酯）的主要来源。 PLD似乎在脱粒中起着至关重要的作用，因为PLD激活与各种实验条件下的脱粒密切相关，包括药理学破坏（Chadi等，Mol。38：1269，2001），并用IRNAS敲击PLD（Peng and Beaven，J.Immunol.174：5201,5201,2005）。此外，PLD，PLD1和PLD2的两个已知同工型都位于细胞内的不同位置。 PLD1与质膜与高尔基体和颗粒膜以及PLD2相关（Choi等，J。Immunol。278：12039,2002）。两种同工型都调节了肥大细胞中脱粒的不同阶段，颗粒相关的PLD1在颗粒转移到质膜和质膜相关的PLD2中与颗粒与质膜融合中的脱位相关。但是，激活PLD并下游PLD依赖性信号事件的机制在很大程度上不确定。其中包括通过磷脂酰肌醇5-激酶（PI5K）和鞘氨醇激酶（SK）进入Ca2+的可能链接。 目的：我们正在对PLD进行两条研究：第一个是阐明PLD激活的机制，第二个是识别受PLD调节的下游事件。 PLD2特别有趣，因为它在质膜上的位置使得与Fcepsilonri相关的SRC激酶可以使用的同工型。如去年所报道的那样，我们发现PLD2而不是PLD1被SRC激酶Fyn和FGR磷酸化，并且这种磷酸化调节PLD2激活和脱粒（Choi等，Mol。Cell。Biol。Biol。24：6980,2004）。这些早期的研究还表明，PLD2与“脂质筏”共定位，并且PLD2的磷酸化取决于这些“脂质筏”的完整性（未发表的数据）。在当前时期，我们研究了“脂质筏”的功能完整性以及下游激酶（例如SYK）的激活（例如SYK）在启动肥大细胞中的多个信号级联反应中起关键作用的激活是否需要PLD本身。同样，在我们观察到，PKC的二酸酯依赖性同工型受PLD高度调节（Peng和Beaven，J。Immunol。174：5201,2005），我们现在正在研究PLD在PI5K和SK激活中的作用，因为PLD产品是PLD产品的潜在激活剂。由于PI5K和SK都与钙信号的产生有关，因此该主题已成为我们最近研究的重要组成部分。 PLD和脂质RAFT功能：抑制PLD功能，无论是用醇还是siRNA，都会散布脂质筏成分，包括LAT，THY1和GPI，以及抑制IgE受体（FCERI）的易位（FCERI）及其相关的酪氨酸激酶，Lyn和Syk，lyn和Syk，lyn lyn和Syk，lipids lipids suft taintigeNAntigeN AntigeN刺激。下游磷酸化事件也被阻塞。无论是通过使用荧光标记的分子和共聚焦显微镜还是经典的膜分馏技术检查细胞的一致结果。这些技术表明，标记的PLD2还与脂质筏成分共定位，这一过程由脂质筏分散剂甲基β-蛋白丝脱蛋白所阻止。这些观察结果表明，不仅PLD2激活取决于脂质筏完整性，而且这种激活有助于脂质筏的功能组织（未发表的数据）。 PLD2与SYK相互作用并调节SYK活性和下游事件：PLD2与SYK的激活相关并促进，如上所述，这是肥大细胞激活中的关键酶。抗原刺激导致SYK与PLD2在膜脂质筏中共定位，如共免疫沉淀和共聚焦显微镜所示。该关联取决于SYK通过LYN对SYK的初始磷酸化以及PLD2通过其Phox同源（PX）结构域与SYK与SYK的结合，以诱导SYK及其下游靶标的进一步磷酸化和激活，LAT和SLP76。矛盾的是，表达研究表明，在这方面，PLD2的催化无效突变体同样活跃。 PLD2或PLD2突变体的过表达增强了抗原诱导的SYK和Downnstream靶标的磷酸化，而PLD2 siRNA的表达阻止了这些磷酸化。类似的遗传操作表明，PLD2与SYK的相互作用是脱粒所必需的。这些发现表明PLD2作为信号分子的双重作用。它可以以催化方式作用，以产生具有生物活性的脂质，例如磷脂酸和甘油酸，以及作为衔接子分子，以促进通过SYK的信号转导（参考文献1）。 PLD的磷脂酰肌醇5-激酶，鞘氨酸激酶，钙动员和脱粒的调节：如去年所报道，PLD通过PLD的磷脂酸的产生是肥大细胞中二酰基甘油酸酯的主要来源，并且对于依赖于Diglyceride的PKC和Degrans of Degran和Degrans的激活是必不可少的。但是，对于抗原介导的脱粒似乎是必需的其他PLD依赖性过程。这样的过程可以是如上所述的SYK激活（Peng and Beaven，J。Immunol。174：5201,2005）。 药理学和遗传研究现在表明，除PKC外，PLD与PI5K和SK的激活有关。 PI5K催化磷脂酰肌醇4-磷酸（PIP）转化为磷脂酰肌醇4,5-双磷酸（PIP2）。 SK激酶磷酸化鞘氨醇形成1-磷酸盐（S1P）（参考文献2）。报告表明，PIP2调节TRPC5 Ca2+通道蛋白从近端囊泡转移到质膜a。与IP3结合起作用的S1P促进了细胞内存储中Ca2+的释放，然后促进了外部Ca2+的涌入。在我们的手中，与SK抑制剂一样，PLD2或PI5K阻滞了Ca2+的进入，对刺激肥大细胞中细胞内存储的Ca2+释放的影响很小。这些研究清楚地表明，PI5K和SK调节Ca2+流入而不是Ca2+释放。目前，我们正在定义抑制性RNA和遗传缺陷肥大细胞的研究中的机制（请参见参考文献3）。 识别钙进入钙的钙通道和调节蛋白 肥大细胞的脱粒取决于Ca2+的细胞内存储和随之而来的外部Ca2+的流入，也称为商店繁殖的钙进入（SOCE）。表征良好的钙释放激活的钙电流（CRAC）已被确定为介导SOCE的主要候选者。我们对CRAC是进入Ca2+的唯一机制，因为SR2+和其他二价阳离子也渗透并支持刺激的肥大细胞中的脱粒（请参阅本系列的先前报告）。我们发现，最近被证明是CRAC所必需的组成部分的STIM1和ORAI1的过表达允许进入Ca2+但不能进入刺激的肥大细胞中SR2+。然而，除了sirna敲低这些蛋白质的sirna证明，Ca2+和Sr2+的流入以及脱粒取决于TRPC5的存在（请参阅上一节）。我们的观察结果表明，TRPC5以化学计量的方式与STIM1和ORAI1相关联，以增强Ca2+或SR+的进入以生成脱粒信号（手稿提交）。