M-CSF Signaling in Osteoclast Formation and Function

描述（由申请人提供）：破骨细胞（OC）从其髓样前体细胞的分化及其功能的重要方面都依赖于M-CSF对c-Fms（该细胞因子的唯一受体）的激活。该事件促使c-Fms胞质尾中酪氨酸残基的磷酸化，随之产生下游信号传导。鉴于M-CSF是OC形成和功能的关键，介导破骨细胞生成中发生的细胞内信号传导事件的c-Fms组分本身是潜在的抗骨质疏松症靶点。不幸的是，缺乏关于c-Fms的元素的数据，这些元素是其破骨细胞生成特性的核心。这种相关信息的缺乏反映了这样的事实，即针对c-Fms的结构/功能的研究仅在细胞中进行，例如成纤维细胞，其通常不表达受体，或在转化的髓系中进行，两者都不分化成OC。考虑到这一点，我们已经开发了一个实验系统，使我们能够研究真正的OC和他们的骨髓巨噬细胞前体细胞（BMPs）中的c-Fms信号。随着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纯化了真正的OC前体，所述嵌合体包含连接到鼠c-Fms的跨膜和胞内结构域的Epo受体（EpoR）的外部结构域。用Epo（作为M-CSF替代物）加RANK配体（RANKL）处理EpoR转导的BMMS，产生的OC与用M-CSF和RANKL处理相同细胞获得的OC无法区分。通过表达嵌合Epo/c-Fms受体，在其胞质尾区中含有酪氨酸至苯丙氨酸的点突变，我们可以在不存在内源性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，我们定位于描绘的作用特定的组件的c-Fms细胞质结构域在OC生物学。通过特定c-Fms胞质尾酪氨酸残基的磷酸化传递的M-CSF信号，a）对于OC前体的增殖至关重要，B）刺激RANK（关键破骨细胞生成细胞因子RANKL的受体）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胞质尾中的特定氨基酸残基介导M-CSF刺激的增殖、RANK表达和OC和/或其前体的细胞毒性依赖性功能。因此，我们的具体目标是确定：1。c-Fms激活介导破骨细胞前体增殖的机制。2. c-Fms激活刺激RANK（关键破骨细胞生成细胞因子RANKL的受体）表达的机制。3. c-Fms激活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.