Pre-osteoclast fusion

描述(申请人提供)：破骨细胞(OCs)的骨吸收超过成骨细胞(OBS)的形成是一个生物学问题，在广泛且代价高昂的疾病中起着核心作用，包括骨质疏松症、骨性和类风湿性关节炎、牙周病以及假体/植入物松动。由于正常的骨吸收需要融合，因此描述单核前体细胞与多核OCs融合的机制和调控是很重要的。在我们对OC分化过程中基因表达变化的研究中，我们发现了OC-STAMP(破骨细胞刺激跨膜蛋白)，并注意到与其他人显示的对融合至关重要的一系列惊人的相似之处，称为DC-STAMP。相似之处包括：1)RANKL刺激OC前体时上调mRNA和蛋白质；2)预测跨膜(TM)螺旋的拓扑结构；3)OC-STAMP的C末端存在DC-STAMP家族共识序列；4)通过敲除、敲除或抗体抑制体内和体外多核破骨细胞的形成；5)在过度表达时刺激融合。这两种蛋白质在陆生脊椎动物中都高度保守。在骨质疏松症大鼠的急性诱导OC分化模型中，我们提出了初步证据，表明抗OC-STAMP抗体抑制体内破骨细胞的形成。[最近的一份报告证实了我们的假设，即OC-STAMP-/-小鼠和DC-STAMP-/-小鼠一样，会有单核OCS。]本方案将在3个特定目标下研究OC前融合，以阐明其在OC细胞生物学和体内活性中的作用，作为控制骨丢失的潜在治疗靶点，以及与DC-STAMP作为融合因子的关系。SA1将建立OC-STAMP和DC-STAMP的膜拓扑(DC-STAMP已被报道为7-TM超家族成员，但不同的算法预测不同的拓扑)。所获得的信息应有助于针对已证实的Fusigen胞外环的“下一代”抗体和/或抑制性多肽，并为关于活性和调节的进一步假说提供基础。根据SA2，将生产OC-STAMP和DC-STAMP基因敲除小鼠(现有线路的请求被拒绝)。我们将在手中确认这两个菌株的骨骼和OC表型，并将使用它们衍生的细胞进行融合实验，以确定OC-STAMP和DC-STAMP之间的相互作用或相互依赖进行融合。将进行研究，以确定OC-STAMP和DC-STAMP的潜在配体和调节因子。在SA3下，我们将扩大我们对具有抗C-STAMP抗体的tl/tl大鼠的急性体内OC抑制的初步研究规模。我们还将研究抗[Fusigens]抗体抑制在已证实的去卵巢大鼠骨质疏松模型中的作用。总之，这些研究将为OC-STAMP和DC-STAMP的拓扑学和细胞生物学、OC-STAMP前融合、OC-STAMP丢失对骨骼的表型影响以及原则上抑制OC-STAMP前融合是否是阻止体内骨丢失的有效方法提供重要的新信息。

DESCRIPTION (provided by applicant): Bone resorption by osteoclasts (OCs) exceeding formation by osteoblasts (OBs) is a biological problem with a central role in widespread and costly disorders, including osteoporosis, osteo- and rheumatoid arthritis, periodontal disease, and prosthesis/implant loosening. Delineation of mechanisms and regulation of the fusion of mononuclear precursors into multinucleated OCs is important since fusion is needed for normal bone resorption. In our studies of gene expression changes during OC differentiation, we discovered OC-STAMP (osteoclast-stimulatory transmembrane protein) and noted a series of striking similarities to a factor shown by others to be essential to fusion, called DC-STAMP. Similarities include 1) up- regulation of mRNA and protein upon stimulation of OC precursors by RANKL; 2) predicted topology of transmembrane (TM) helices; 3) presence of a DC-STAMP family consensus sequence in the C-terminal half of OC-STAMP; 4) suppression of formation of multinucleated osteoclasts in vivo and in vitro by either knockout, knockdown, or antibody; 5) stimulation of fusion upon overexpression. Both proteins are highly conserved in terrestrial vertebrates. In a model of acute, induced OC differentiation in an osteopetrotic rat model, we present preliminary evidence that anti-OC-STAMP antibody suppresses osteoclastogenesis in vivo. [A recent report validated our hypothesis that OC-STAMP -/- mice would, like DC-STAMP -/- mice, have mononuclear OCs.] This proposal will investigate pre-OC fusion under 3 Specific Aims to clarify its role in OC cell biology and activity in vivo, as a potential therapeutic targetto control bone loss, and in relation to DC- STAMP as a fusion factor. SA1 will establish the membrane topology of both OC- and DC-STAMP (DC- STAMP has been reported to be a 7-TM superfamily member, yet different algorithms predict different topologies). Information gained should facilitate "next-generation" antibodies and/or inhibitory peptides directed at proven extracellular loops of these fusigens and provide a basis for further hypotheses about activities and regulation. Under SA2, OC- and DC-STAMP knockout mice will be produced (requests for existing lines were refused). We will confirm the skeletal and OC phenotypes of both strains in our hands, and we will use cells derived from them to perform fusion experiments to determine interaction or interdependence of OC- and DC- STAMP for fusion. Studies will be done to identify potential ligands and regulatory factors for OC- and DC- STAMP. Under SA3, we will increase the size of our preliminary study of acute, in vivo OC inhibition in tl/tl rats with anti-C-STAMP antibody. We will also investigate the effects of anti-[fusigens] antibody inhibition in the proven, ovariectomized rat model of osteoporosis using athymic rats. Together, these studies should provide significant new information about the topology and cell biology of both OC-STAMP and DC-STAMP, about pre- OC fusion, about the phenotypic impact of loss of OC-STAMP on the skeleton, and whether in principle, inhibition of pre-OC fusion is a valid approach to stemming bone loss in vivo.