M-CSF Signaling in Osteoclast Formation and Function

描述（由申请人提供）：破骨细胞（OCs）从髓系前体的分化及其功能的重要方面都依赖于c-Fms的M-CSF的激活，c-Fms是该细胞因子的唯一受体。这一事件促使c-Fms细胞质尾部酪氨酸残基磷酸化，并随之产生下游信号。鉴于M-CSF是OC形成和功能的关键，在破骨细胞形成过程中介导细胞内信号事件的c-Fms成分作为潜在的抗骨质疏松靶点出现。不幸的是，关于c-Fms中对其破骨细胞特性至关重要的成分的数据缺乏。相关信息的缺乏反映了这样一个事实，即针对c-Fms结构/功能的研究仅在细胞中进行，例如通常不表达受体的成纤维细胞，或转化的髓系，两者都不能分化为OCs。考虑到这一点，我们开发了一个实验系统，使我们能够研究真正的OCs及其骨髓巨噬细胞前体（BMMs）中的c-Fms信号。认识到它们表达内源性c-Fms，我们用表达嵌合体的逆转录病毒转导真正的OC前体，嵌合体包括与小鼠c-Fms的跨膜和胞内结构域连接的Epo受体（EpoR）的外部结构域。用Epo作为M-CSF替代物，加上RANK配体（RANKL）处理EpoR转导的BMMS，产生的OCs与用M-CSF和RANKL处理相同细胞获得的OCs没有区别。通过将含有酪氨酸的嵌合Epo/c-Fms受体表达到其细胞质尾部的苯丙氨酸点突变中，我们可以在没有内源性c-Fms占据的情况下激活真实OC前体中的各种c-Fms信号。因此，我们定位于描绘c-Fms细胞质结构域在OC生物学中的作用特异性成分。M-CSF信号通过特定c- fms细胞质尾部酪氨酸残基的磷酸化传递，a)对OC前体的增殖至关重要，b)刺激关键破骨细胞因子RANKL受体RANK的表达，c)调节成熟OC的功能和形态。鉴于这些事实，我们假设：c-Fms细胞质尾部的特定氨基酸残基介导m - csf刺激的OCs和/或其前体的增殖、rank表达和细胞骨架依赖功能。因此，我们的具体目标是确定：1。c-Fms活化介导破骨细胞前体增殖的机制。2. c-Fms激活刺激RANK（关键破骨细胞因子RANKL的受体）表达的机制。3. c-Fms活化介导成熟破骨细胞骨架重组的机制。

DESCRIPTION (provided by applicant): 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.