Identification and Characterization of Signaling Pathways and Mediators Regulating Mast Cell-Related Disorders

调节肥大细胞相关疾病的信号通路和介质的鉴定和表征

• 批准号: 10272217
• 负责人: Dean D Metcalfe
• 金额: $ 156.05万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/10272217
• 关键词: 11q12; Adhesions; Affect; Affinity; Alcohols; Aldehydes; Allergic; Allergic Disease; Allergic inflammation; Area; Asthma; Automobile Driving; Awareness; Biological; Biological Process; Blood Vessels; Bone Diseases; Cell Surface Receptors; Cells; Chromosomes; Code; Collaborations; Complex; Data; Differentiation and Growth; Disease; Disease Management; Disease Marker; Dissociation; Effector Cell; Environment; Event; Family; Flushing; Foundations; Friction; G-Protein-Coupled Receptors; Gene Cluster; Genes; Genetic; Genetic Polymorphism; Genotype; Goals; Hepatic Stellate Cell; Homeostasis; Human; Hypersensitivity; IgE; IgE Receptors; In Vitro; Incidence; Inflammation; Inflammatory; Inherited; Intercept; Interleukin-6; Investigation; Knowledge; Lead; Ligands; Light; Link; Liver; Liver Fibrosis; MAPK3 gene; MS4A1 gene; Mechanics; Mediating; Mediator of activation protein; Membrane; Membrane Microdomains; Missense Mutation; Mitochondria; Mus; Mutation; Oncogenic; Osteoblasts; Osteopenia; Osteoporosis; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Phenotype; Phosphatidylinositols; Phospholipase C; Phosphorylation; Phosphotransferases; Play; Predisposition; Process; Production; Prostaglandin D2; Protein Kinase C; Protein Tyrosine Phosphatase; Pruritus; Reaction; Reactive Oxygen Species; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Regulation; Role; Serum; Severity of illness; Signal Pathway; Signal Transduction; Signaling Protein; Skin; Source; Stat5 protein; Stem Cell Factor; Stimulus; Swelling; Systemic Mastocytosis; Tissues; Tryptase; Up-Regulation; Urticaria; Variant; aldehyde dehydrogenases; alternative treatment; antigen binding; asthma exacerbation; bone; cell behavior; disorder risk; extracellular vesicles; genetic linkage; genetic variant; genomic locus; in vivo; inflammatory milieu; insight; intercellular communication; mast cell; mastocytosis; mechanical force; neoplastic; neoplastic cell; oxidative damage; patient response; patient subsets; receptor; receptor-mediated signaling; release of sequestered calcium ion into cytoplasm; response; stellate cell; therapeutic target; vibration

Mast cells are effector and regulatory cells involved in the pathogenesis of allergic inflammation. These responses are generally, but not exclusively, mediated through canonical activation of the high affinity IgE receptor. Binding of an antigen to IgE/IgE receptor initiates complex intracellular signaling events that drive mast cell responses, including the secretion of vascular mediators and inflammatory substances. In addition, signaling by other cell surface receptors activated by signals in the tissue environment play critical roles in enhancing or inhibiting allergic processes or other mast cell-driven disorders. An example is the tyrosine kinase receptor KIT which mediates important biological processes in mast cells such as differentiation, growth, survival and activation. Moreover, mutations that result in constitutive activation of KIT are associated with mastocytosis, a mast cell-related disorder characterized by abnormal accumulation of mast cells in tissues. Despite the increased incidence and awareness of allergic diseases and other disorders involving mast cells, the strategies to intercept mast cell activation remain limited. A deeper understanding of receptors that activate or inhibit mast cell responses, their biological roles and signaling mechanisms gives important insights into the regulation of allergic and non-allergic hypersensitivity reactions, and treatment options for these human illnesses. Relating to objective 1: In FY2020, in collaboration with Dr. Cruse, we found that Membrane Spanning 4- Domains A4A (MS4A4A) stimulates IgE receptor-mediated phospholipase C (PLC) signaling and enhances Orai1-mediated calcium flux, processes required for IgE-mediated mast cell activation. MS4A4A promoted the translocation of the IgE receptor and signaling proteins to lipid rafts thus facilitating the formation of signaling complexes and phosphorylation cascades. MS4A4A belongs to the same family as the gene coding for the beta chain of the IgE receptor (MS4A2). These genes are clustered around chromosome 11q12-q13, a region linked to allergy and asthma susceptibility. Thus, our data advances the understanding of canonical activation of mast cells and supports the potential for MS4A4A as a candidate for the linkage of the genetic loci to allergy, a hypothesis that requires further investigation. In FY2020, we also found evidence for a role of mast cell mitochondrial aldehyde dehydrogenase (ALDH2) in KIT-mediated responses. ALDH2 metabolizes endogenous and exogenous aldehydes and protects cells against oxidative injury. Inactivating genetic polymorphisms in humans are common and associate with alcohol flush reactions and exacerbations of asthma, conditions often linked to mast cell activation. Our data indicated that mouse mast cells with a genetic deletion in Aldh2 have increased proliferation and IL-6 production after stimulation with stem cell factor (SCF), the ligand for Kit, and when co-stimulated with SCF and IgE/Ag, show enhanced mediator release. We further demonstrated that these effects were associated with an increase in reactive oxygen species levels in Aldh2 -deficient mast cells and a reduction in the activity of a tyrosine phosphatase (Shp-1), that negatively regulates Kit activity. Our findings reveal a functional role for mast cell intrinsic Aldh2 in the control of Kit activation and Kit-mediated responses, which may lead to a better understanding of mast cell reactivity in conditions related to ALDH2 polymorphisms in humans. In systemic mastocytosis (SM), neoplastic accumulation of mast cells associates with genetic variants, particularly the missense mutation D816V in KIT that causes ligand-independent activation, oncogenic signaling and abnormal mast cell responses. In FY2018 through FY2020, we demonstrated that mast cells with D816V-KIT are a significant source for IL-6 in SM and that D816V-KIT-mediated STAT5 hyperactivation drives persistent IL-6 production. This study provided insights into mechanisms leading to IL-6 upregulation in mastocytosis, a parameter associated with disease severity and risk of progression. An example of mast cell activation driving disease independently of IgE-mediated activation is found in patients with vibratory urticaria, where mast cells respond excessively to a mechanical stimulus, such as friction of the skin resulting in itching, swelling and hives on affected areas, and often, systemic manifestations. In previous years, we found that a missense mutation in the adhesion G-protein coupled receptor E2 (ADGRE2) abundantly expressed in human mast cells, associates with disease presentation in a subset of patients with vibratory urticaria. In FY2020 we have characterized the responses and intracellular signals elicited by mechanical activation in human mast cells expressing this variant (p.C492Y-ADGRE2). We found that the p.C492Y variant enhances the sensitivity to vibration and assembles functionally distinct signaling events leading to degranulation and the release of prostaglandin D2 (PGD2), respectively. PLC activation and calcium mobilization were upstream of the activation of phosphoinositide 3-kinase (PI3K)- and extracellular-signal-regulated kinase (ERK) 1/2-pathways, and key to both degranulation and PGD2 release. However, while degranulation was also dependent on protein kinase C (PKC) and partly dependent on PI3K activation, release of PGD2, also found increased in serum of patients with vibratory urticaria after vibration, was dependent of ERK1/2 pathways. The data identify the signals causing the pathologic presentation of vibratory urticaria and thus provide potential therapeutic targets for treatment; and contribute to the understanding of adhesion G-protein coupled receptor (aGPCR)-mediated signaling elicited by mechanical forces. Our prior studies on ADGRE2 also suggested that dissociation of the alpha and beta subunits are the first steps triggering ADGRE2 activation and that p.C492Y variant destabilizes the interaction between the alpha and beta subunits, rendering the receptor more prone to activation by physical forces. In fiscal years FY2019 and FY2020, in collaboration with Drs. Schwartz, Lyons, and Milner it was unveiled that separation of the subunits of ADGRE2 facilitated by proteolytic cleavage via an uncommon heterotetramer of mast cell tryptase present in patients with hereditary alpha-tryptasemia, also sensitizes ADGRE2 to a vibratory stimulus leading to mast cell activation. This mechanism links their genotype with the higher responsiveness of these patients to a vibratory stimuli in the skin. Relating to objective 2: In previous years, we found that serum from patients with SM contains elevated concentrations of extracellular vesicles (EVs) with a mast cell signature and associate with markers of disease severity. Furthermore, EVs from these patients (SM-EVs) induce a fibrotic phenotype in hepatic stellate cells (a recognized as a step in the pathogenesis of liver fibrosis) in vitro and in vivo by transferring functionally active KIT, thus linking SM-EVs to liver pathology in SM. The identification of EVs from neoplastic cells has opened the possibility that their specific cargos can be shuttled into cells other than stellate cells and potentially contribute to some aspects of the pathogenesis of mast cell-related diseases. During FY2019 and FY2020, we examined the role of SM-EVs on the bone environment where mast cells pathologically accumulate. Bone disease, including osteopenia and osteoporosis, is one of the most frequent and debilitating complications in patients with SM. However, the pathogenesis of bone disease in SM is not understood. Our investigations may shed light into the contributions of EVs to bone homeostasis by affecting osteoblast functions and the pathogenesis of bone disease in SM.

肥大细胞是参与过敏性炎症发病机制的效应细胞和调节细胞。这些反应一般，但不完全是通过高亲和力IgE受体的典型激活介导的。抗原与IgE/IgE受体结合启动复杂的细胞内信号事件，驱动肥大细胞反应，包括血管介质和炎症物质的分泌。此外，组织环境中信号激活的其他细胞表面受体的信号在增强或抑制过敏过程或其他肥大细胞驱动的疾病中起着关键作用。一个例子是酪氨酸激酶受体KIT，它介导肥大细胞的重要生物过程，如分化、生长、存活和激活。此外，导致KIT组成性激活的突变与肥大细胞增多症有关，肥大细胞增多症是一种肥大细胞相关疾病，其特征是肥大细胞在组织中异常积聚。