PHOSPHATIDYLINOSITOL KINASE, M-CSF AND OSTEOCLASTOGENESIS

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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.