PHOSPHATIDYLINOSITOL KINASE, M-CSF AND OSTEOCLASTOGENESIS

磷脂酰肌醇激酶、M-CSF 和破骨细胞生成

• 批准号: 6821853
• 负责人: PHILIP D STAHL
• 金额: $ 15.3万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6821853
• 关键词: alcohol phosphotransferase; biological signal transduction; cell component structure /function; cell differentiation; colony stimulating factor; cytokine; cytokine receptors; growth factor receptors; macrophage; mass spectrometry; molecular site; osteoclasts; protein isoforms; protein protein interaction; protein structure function; proteomics; receptor binding; receptor expression; receptor mediated endocytosis; tissue /cell culture; vesicle /vacuole

DESCRIPTION (provided by applicant): Type Ibeta phosphatidylinositol-4-phosphate-kinase (PIP5Ka) was initially identified as a partner of the M-CSF receptor. Subsequently, PIP5Kbeta was shown to be recruited to the M-CSF receptor and to the EGF receptor. Failure to recruit PIP5Kbeta prevented receptor internalization and altered signal transduction pathways. Our long term goal is to address three fundamental questions: (i) how M-CSF activation of the M-CSF receptor is coupled the activation of the type I PIP5K family of lipid modifying kinases, (ii) how M-CSFR interaction with PIP5K integrates signaling and endocytic trafficking of the activated receptor in macrophages and osteoclasts, (iii) what is the effect of PIP5K and M-CSF on macrophage and osteoclast endosome structure and composition. M-CSF (c-Fms) is one of the most important cytokines regulating monocyte/macrophage survival, proliferation and differentiation. Moreover, cells of the monocyte/macrophage lineage are precursors of the osteoclast, multinucleated cells essential for bone remodeling. Understanding the relationship between M-CSF receptor signaling and trafficking could reveal new therapeutic targets and have a profound effect on our understanding of a number of M-CSF dependent pathophysiological processes including osteoporosis. The goal of this proposal is to examine the interaction of PIP5Kbeta with the M-CSF receptor and to determine the role it plays in M-CSF signaling in osteoclasts, the major site of M-CSF activity and in macrophage/osteoclasts endosome structure and function. (1) We will study the interaction of the PIP5K with the MCSF receptor expressed in bone marrow macrophages and macrophages transfected with an Epo/c-fms chimeric receptor. We will take advantage of the many point and truncation mutants available in the Epo/c-fms chimeric receptor. (2) Preliminary data indicate that PIP5Ks are important early determinants of RTK signal transduction, both via the MAP kinase pathway and the PKB/akt pathway. We will establish the role of the PIP5Kbeta as a mediator of MCSF signal transduction using M-CSFR and Epo/c-fms expressing bone marrow macrophages. Our hypothesis is that PIP5Kbeta initiates receptor internalization which may favor one or more signal transduction pathways. (3) We will characterize the effect of PIP5K and M-CSF on macrophage and osteoclast endosome structure and composition. These studies will lay the groundwork for an osteoclast "endosome proteome" project. Osteoclast endosomes are predicted to be important organelles both in terms of regulating and modulating cell surface composition and function (e.g., bone resorption) and in generating signals from M-CSF and other osteoclastogenic growth factors. Membrane rafts may also play key roles in receptor signaling. Using established gradient sedimentation and fractionation procedures with cultured osteoclasts, we propose to explore the effect of PIP5K, following M-CSF and RANKL stimulation, on the structure and composition of the osteoclast endosomes and rafts using mass spectrometry and proteomics methodologies.

描述（由申请人提供）：I β型磷脂酰肌醇-4-磷酸激酶（PIP 5 Ka）最初被确定为M-CSF受体的伴侣。 随后，PIP 5 Kbeta被募集到M-CSF受体和EGF受体。不能募集PIP 5 K β阻止了受体内化并改变了信号转导途径。我们的长期目标是解决三个基本问题：（i）M-CSF受体的M-CSF活化如何与脂质修饰激酶的I型PIP 5 K家族的活化偶联，（ii）M-CSFR与PIP 5 K的相互作用如何整合巨噬细胞和破骨细胞中活化受体的信号传导和内吞运输，（iii）PIP 5 K和M-CSF对巨噬细胞和破骨细胞内体结构和组成的影响。M-CSF（c-Fms）是调节单核/巨噬细胞存活、增殖和分化的最重要的细胞因子之一。此外，单核细胞/巨噬细胞谱系的细胞是破骨细胞的前体，破骨细胞是骨重建所必需的多核细胞。 了解M-CSF受体信号传导和贩运之间的关系可以揭示新的治疗靶点，并对我们理解许多M-CSF依赖的病理生理过程（包括骨质疏松症）产生深远影响。本提案的目的是研究PIP 5 K β与M-CSF受体的相互作用，并确定其在破骨细胞（M-CSF活性的主要位点）中的M-CSF信号传导中以及在巨噬细胞/破骨细胞内体结构和功能中的作用。(1)我们将研究PIP 5 K与骨髓巨噬细胞和Epo/c-fms嵌合受体转染的巨噬细胞中表达的MCSF受体的相互作用。我们将利用Epo/c-fms嵌合受体中可用的许多点突变体和截短突变体。(2)初步数据表明，PIP 5 Ks是RTK信号转导的重要早期决定因素，通过MAP激酶途径和PKB/akt途径。我们将使用表达M-CSFR和Epo/c-fms的骨髓巨噬细胞来确定PIP 5 K β作为MCSF信号转导的介体的作用。我们的假设是，PIP 5 K β启动受体内化，这可能有利于一个或多个信号转导途径。(3)我们将表征PIP 5 K和M-CSF对巨噬细胞和破骨细胞内体结构和组成的影响。 这些研究将为破骨细胞“内体蛋白质组”项目奠定基础。破骨细胞内体被预测在调节和调节细胞表面组成和功能方面是重要的细胞器（例如，骨吸收）和从M-CSF和其它破骨细胞生长因子产生信号。膜筏也可能在受体信号传导中发挥关键作用。使用已建立的梯度沉降和分级程序与培养的破骨细胞，我们建议探索PIP 5 K的影响，M-CSF和RANKL刺激后，对破骨细胞内体和筏的结构和组成，使用质谱和蛋白质组学方法。