PHOSPHATIDYLINOSITOL KINASE, M-CSF AND OSTEOCLASTOGENESIS

描述(申请人提供)：Ibeta型磷脂酰肌醇-4-磷酸-激酶(PIP5Ka)最初被鉴定为M-CSF受体的伙伴。随后，PIP5Kβ被证明被募集到M-CSF受体和EGF受体上。未能招募PIP5Kβ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对M-CSF受体的激活是如何与I型PIP5K家族的激活相耦合的；(Ii)M-CSFR与PIP5K的相互作用如何整合激活的受体在巨噬细胞和破骨细胞中的信号和内吞运输；(Iii)PIP5K和M-CSF对巨噬细胞和破骨细胞内小体结构和组成的影响。巨噬细胞集落刺激因子(c-FMS)是调节单核/巨噬细胞存活、增殖和分化的重要细胞因子之一。此外，单核/巨噬细胞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受体信号和转运之间的关系可以揭示新的治疗靶点，并对我们理解包括骨质疏松在内的许多M-CSF依赖的病理生理过程具有深远的影响。本研究的目的是研究PIP5Kβ与M-CSF受体的相互作用，并确定其在破骨细胞M-CSF信号转导中的作用，破骨细胞M-CSF活性的主要部位以及巨噬细胞/破骨细胞内体结构和功能中PIP5Kβ的作用。(1)研究PIP5K与骨髓巨噬细胞和c-FMS嵌合受体表达的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和截断突变。(2)初步数据表明，PIP5K是RTK信号转导的重要早期决定因素，通过MAP和PKB/AKT信号转导途径。我们将通过表达骨髓巨噬细胞的M-CSFR和EPO/c-FMS来确定PIP5Kbeta作为MCSF信号转导的媒介的作用。我们的假设是，PIP5Kβ启动受体内在化，这可能有利于一个或多个信号转导途径。(3)研究PIP5K和M-CSF对巨噬细胞和破骨细胞内小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。破骨细胞内体在调节和调节细胞表面成分和功能(如骨吸收)以及从M-CSF和其他破骨细胞生长因子产生信号方面都被认为是重要的细胞器。膜筏也可能在受体信号转导中发挥关键作用。利用已建立的破骨细胞梯度沉淀和分级程序，我们建议利用质谱学和蛋白质组学方法，探索在M-CSF和RANKL刺激后，PIP5K对破骨细胞内体和筏的结构和组成的影响。

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.