PI3KC2 Beta in Ca2+ Signaling via Sphingosine Kinase

PI3KC2 Beta 通过鞘氨醇激酶参与 Ca2 信号传导

• 批准号: 6620543
• 负责人: OKSOON H CHOI
• 金额: $ 2.7万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-05 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6620543
• 关键词: alcohol phosphotransferase; antibody receptor; calcium flux; cell line; endoplasmic reticulum; flow cytometry; immunoglobulin E; inositol phosphates; laboratory rabbit; leukocyte activation /transformation; mast cell; phosphates; phosphatidylinositol 3 kinase; phospholipase C; protein protein interaction; protein tyrosine kinase; sphingosine

Stimulation of mast cells via the high affinity receptor for IgE (FcepsilonRI) leads to the release of inflammatory mediators that are responsible for allergic inflammatory diseases, such as asthma and allergic dermatitis. The release of inflammatory mediators is dependent on the increase in cytosolic free Ca2+ ([Ca2+]i), which is initiated by the mobilization of Ca2+ from the endoplasmic reticulum and followed by influx of Ca2+ from outside of the cell. It is generally believed that inositol 1,4,5-trisphosphate (IP3) is responsible for mobilizing Ca2+ from the intracellular stores. However, we have shown that FcepsilonRI-mediated production of IP3 is not sufficient to account for the increase in [Ca2+]i. This suggests an alternative mechanism for Ca2+ mobilization via sphingosine 1- phosphate, a product of sphingosine kinase. Furthermore, the increase in sphingosine 1-phosphate production and Ca2+ mobilization has been observed in an antigen-stimulated mast cell line, RBL-2H3 cells that has been overexpressed with the beta isoform of Class II phosphoinositide 3-kinase (PI3KC2beta). Our long-range goal is to understand the mechanisms underlying [Ca2+]i increase in mast cells. The objective of this particular application is to understand the role of Class II PI3K in sphingosine kinase activation and Ca2+ mobilization. The central hypothesis for the proposed research is that activation of a Class II PI3K via FcepsilonRI increases production of sphingosine 1-phosphate, which causes Ca2+ mobilization in conjunction with IP3 in RBL-2H3 Cells. We formulated this hypothesis based on strong preliminary findings, which demonstrate that overexpression of PI3KC2beta increases FcepsilonRI-mediated production of sphingosine 1-phosphate and Ca2+ mobilization. In addition, increased production of sphingosine 1-phosphate leads to increased mobilization of Ca2+ only in the presence of IP3. The rationale for the proposed research is that, once the alternative pathway of Ca2+ mobilization is elucidated, it can be targeted as a new approach to treat allergic inflammatory diseases. The central hypothesis will be tested by pursuing three specific aims: 1) Identify initiating biochemical events for FcepsilonRI-mediated Ca2+ mobilization. 2) Identify intermediate signaling molecule(s) in the alternative pathway for FcepsilonRI-mediated Ca2+ mobilization. 3) Determine how FcepsilonRI-mediated Ca2+ mobilization is regulated by sphingosine kinase and phospholipase C pathways in RBL-2H3 cells. It is our expectation that the resultant approach will delineate an alternative Ca2+ signaling pathway that involves the activation of PI3KC2beta, sphingosine kinase and phospholipase C. The research proposed in this application is significant, because the outcomes are expected to provide new targets for therapeutic interventions for the prevention and treatment of allergic inflammatory diseases. In addition, they are expected to offer a better fundamental understanding of how Ca2+ is mobilized in mast cells.

通过高亲和力受体刺激肥大细胞（Fcepsilonri）导致释放炎症介质，这些炎症介质导致了导致过敏性炎症性疾病（例如哮喘和过敏性皮炎）。 炎症介质的释放取决于胞质游离Ca2+（[Ca2+] i）的增加，这是由内质网的Ca2+动员引发的，然后从细胞外部从细胞外部涌入Ca2+。 通常认为，肌醇1,4,5-三磷酸（IP3）负责从细胞内存储中动员Ca2+。 但是，我们已经表明，fcepsilonri介导的IP3产生不足以说明[Ca2+] i的增加。 这表明了通过1-磷酸盐（鞘氨醇激酶产物）通过鞘氨醇1-磷酸盐动员的替代机制。 此外，已经在抗原刺激的肥大细胞系中观察到鞘氨醇1-磷酸盐产生和Ca2+动员的增加，RBL-2H3细胞已被II类磷酸辛酶硫代酶3-激酶（PI3KC2BETA）过表达的RBL-2H3细胞。 我们的远程目标是了解[Ca2+] I在肥大细胞中增加的机制。 该特定应用的目的是了解II类PI3K在鞘氨酸激酶激活和Ca2+动员中的作用。 拟议研究的中心假设是，通过Fcepsilonri激活II类PI3K会增加1-磷酸鞘氨醇的产生，从而导致RBL-2H3细胞中的IP3导致Ca2+动员。 我们基于强烈的初步发现提出了这一假设，该发现表明PI3KC2BETA的过表达增加了Fcepsilonri介导的1-磷酸盐和Ca2+动员的产生。 另外，仅在IP3存在下，鞘氨醇1-磷酸盐的产生才会导致Ca2+的动员增加。拟议的研究的理由是，一旦阐明了CA2+动员的替代途径，就可以将其作为治疗过敏性炎症性疾病的新方法。 中心假设将通过追求三个具体目的来检验：1）确定用于Fcepsilonri介导的Ca2+动员的启动生化事件。 2）在fcepsilonri介导的Ca2+动员的替代途径中确定中间信号分子。 3）确定RBL-2H3细胞中的鞘氨酸激酶和磷脂酶C途径如何调节Fcepsilonri介导的Ca2+动员。我们期望由此产生的方法将描绘出涉及PI3KC2BETA，鞘氨酸激酶和磷脂酶C的激活的替代性CA2+信号传导途径C。在此应用中提出的研究很重要，因为预计结果有望为预防性和治疗过敏性疾病提供新的目标。 此外，预计他们将对肥大细胞中的Ca2+如何动员提供更好的基本了解。