Both differentiation of osteoclasts (OCs) from their myeloid precursors and important aspects of their function depend on activation by M-CSF of c-Fms, the sole receptor for this cytokine. This event prompts phosphorylation of tyrosine residues in the c-Fms cytoplasmic tail, with consequent down-stream signaling. Given that M-CSF is key to OC formation and function, the components of c-Fms that mediate intracellular signaling events eventuating in osteoclastogenesis present themselves as potential anti-osteoporosis targets. Unfortunately, data regarding the elements of c-Fms that are central to its osteoclastogenic properties are lacking. This paucity of relevant information reflects the fact that studies addressing structure/function of c-Fms have been performed exclusively in cells, such as fibroblasts, which do not normally express the receptor, or in transformed myeloid lines, neither of which differentiate into OCs. With this in mind, we have developed an experimental system that permits us to study c-Fms signaling in authentic OCs and their bone marrow macrophage precursors (BMMs). With the realization that they express endogenous c-Fms, we transduce authentic OC precursors with a retrovirus expressing a chimera comprising the external domain of the Epo receptor (EpoR) linked to the transmembrane and intracellular domains of murine c-Fms. Treatment of EpoR transduced BMMS with Epo, as an M-CSF surrogate, plus RANK ligand (RANKL), generates OCs indistinguishable from those obtained by treating the same cells with M-CSF and RANKL. By expressing chimeric Epo/c-Fms receptors containing tyrosine to phenylalanine point mutations in their cytoplasmic tail, we can activate various c-Fms signals in authentic OC precursors, in the absence of endogenous c-Fms occupancy. We are therefore positioned to delineate the role specific components of the c-Fms cytoplasmic domain in OC biology. M-CSF signals, transmitted by phosphorylation of specific c-Fms cytoplasmic tail tyrosine residues, a) are critical for the proliferation of OC precursors, b) stimulate expression of RANK, the receptor for the key osteoclastogenic cytokine RANKL and c) regulate the function and morphology of mature OCs. Given these facts, we hypothesize that: specific amino acid residues in the c-Fms cytoplasmic tail mediate M-CSF-stimulated proliferation, RANK-expression and cytoskeletal-dependent function of OCs and/or their precursors. Thus, our Specific Aims are to identify: 1. the mechanisms by which c-Fms activation mediates proliferation of osteoclast precursors. 2. the mechanisms by which c-Fms activation stimulates expression of RANK, the receptor for the key osteoclastogenic cytokine RANKL. 3. the mechanisms by which c-Fms activation mediates re-organization of the cytoskeleton of mature osteoclasts.
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