Regulatory Pathways in Osteoclast Formation and Function

破骨细胞形成和功能的调节途径

• 批准号: 7672245
• 负责人: Roberta Faccio
• 金额: $ 28.64万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-20 至 2010-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7672245
• 关键词: Affect; Binding; Bone Marrow; Cells; Chimeric Proteins; Complex; Data; Disease; Failure; Future; GAB2 gene; Genetic Transcription; ITAM; Immune system; Inflammatory; Ligands; MAPK8 gene; Macrophage Colony-Stimulating Factor; Macrophage Colony-Stimulating Factor Receptor; Mediating; Mediator of activation protein; Metastatic Neoplasm to the Bone; Mus; Mutate; NF-kappa B; Osteoclasts; Osteolysis; Osteolytic; PLCgamma2; Pathway interactions; Periodontal Diseases; Phenotype; Phospholipase; Phosphorylation; Proteins; Regulation; Regulatory Pathway; Rheumatoid Arthritis; Role; Signal Transduction; Signaling Molecule; Testing; Transcription Factor AP-1; Up-Regulation; adapter protein; base; bone; bone loss; in vivo; macrophage; mutant; novel; osteoclastogenesis; precursor cell; receptor; response; transcription factor; tumor

DESCRIPTION (provided by applicant): Excessive bone loss in rheumatoid arthritis, periodontal disease and some tumor- associated bone metastasis is mostly due to an abnormal activation of the immune system leading to hyper-stimulation and activation of osteoclasts (OCs) [1-3]. Identifying common signaling molecules affecting the osteo-immune system and their impact on normal and pathological bone loss may lay the groundwork for future therapies or the variety of diseases that affect both bone and the immune system. We have identified a novel role for the immunomodulatory protein phospholipase Cgamma2 (PLCgamma2) as central mediator of RANKL (Receptor Activator of NFkappaB Ligand)-induced osteoclastogenesis, independent of PLCgamma1. OCs, the principal bone resorbing cells, develop from bone marrow macrophages (BMMs) primarily under the influence of two major regulators: the macrophage colony stimulating factor (M-CSF) and receptor activator of NFkappaB ligand (RANKL), and less understood costimulatory factors that act via immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors on OC precursor cells [2,4, 5]. Our data indicate that targeted deletion of PLCgamma2 in mice leads to an osteopetrotic phenotype. PLCgamma2-deficient OC precursors fail to differentiate due to defective activation of AP1 and NFkappaB and a failure in upregulation of NFATc1, a master transcription actor for osteoclastogenesis. While PLCgamma2 phosphorylation and its catalytic activity are required for ITAM - dependent upregulation of NFATc1, activation of JNK and NFkappaB pathways may be dependent on the capacity of PLCgamma2 to bind and activate GAB2, an adapter protein shown to be important in RANKL-mediated osteoclastogenesis [6], and modulate GAB2 recruitment to the RANK signaling complex. The non-redundant role of PLCgamma2 in OC differentiation suggests that identifying the structural domains which make PLCgamma2 unique in its capacity to promote OC differentiation/function may unveil novel OC regulatory mechanisms and provide the basis for new antiresorptive therapies. Based on observations presented under preliminary data we hypothesize that: 1) the catalytic activity of PLCgamma2 is required for OC differentiation by modulating NFATc1 upregulation, but not JNK or NFkappaB 2) the adapter function of PLCgamma2 is required for osteoclastogenesis by modulating GAB2 activation and subsequent activation of JNK and NFkappaB 3) PLCgamma2 mediates osteoclastogenesis and osteolytic responses in vivo, at least in part, through the JNK/NFkappaB axis and the NFATc1 pathway. In this proposal we seek to: Specific Aim 1: a) generate PLCgamma2 mutants to clarify the role of the catalytic activity and the adapter function of PLCgamma2 during regulation of OC differentiation, and b) generate TAT-fusion-proteins expressing unique regions of PLCgamma2 required for osteoclastogenesis with the intent to identify novel anti-resorptive approaches. Specific Aim 2: a) determine the role of PLCgamma2 in OC differentiation and inflammatory osteolysis, in vivo, and b) test the in vivo osteolytic response in mice harboring mutated forms of PLCgamma2 and the anti-resorptive effects of TAT-fusion proteins encompassing functional domains of PLCgamma2.

描述（由申请人提供）：类风湿关节炎，牙周疾病和某些肿瘤相关的骨转移的过度骨质流失主要是由于免疫系统异常激活导致过度刺激和激活破骨细胞（OCS）[1-3]。鉴定影响骨免疫系统的常见信号分子及其对正常和病理骨质流失的影响可能为未来疗法或影响骨骼和免疫系统的各种疾病奠定基础。我们已经确定了免疫调节蛋白磷脂酶CGAMMA2（PLCGAMMA2）的新作用，是RANKL（NFKAPPAB配体的受体激活剂）诱导的骨质核组织发生的中心介体，独立于PLCGAMMAMMA1。 OCs, the principal bone resorbing cells, develop from bone marrow macrophages (BMMs) primarily under the influence of two major regulators: the macrophage colony stimulating factor (M-CSF) and receptor activator of NFkappaB ligand (RANKL), and less understood costimulatory factors that act via immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors on OC precursor cells [2,4，5]。我们的数据表明，针对小鼠的PLCGAMMA2的靶向缺失会导致骨质术表型。由于AP1和NFKappab的有缺陷激活以及NFATC1的上调（用于骨质碎裂的主转录参与者）上调，缺乏PLCGAMMA2缺陷的OC前体无法区分。虽然ITAM的依赖性上调NFATC1需要PLCGAMMA2磷酸化及其催化活性，但JNK和NFKAPPAB途径的激活可能取决于PLCGAMMA2结合和激活GAB2的能力，激活GAB2，这是一种在Rankl介导的构造构造中所显示的适应性蛋白，对rankl介导的构造构成型GAB [6] [6] [6]] [6]]。 PLCGAMMA2在OC分化中的非冗余作用表明，识别使Plcgamma2在促进OC分化/功能的能力上独特的结构结构域可能会揭示新型的OC调节机制，并为新的抗疾病疗法提供基础。根据初步数据下提出的观察结果，我们假设： 1）PLCGAMMA2的催化活性是通过调节NFATC1上调的OC分化所必需的，而不是JNK或NFKAPPAB 2）PLCGAMMA2的适配器函数是通过调节GAB2激活和随后的JNK和NFKAPPAB的激活来进行破骨细胞生成所必需的 3）PLCGAMMA2至少部分通过JNK/NFKAPPAB轴和NFATC1途径介导了体内的破骨细胞生成和骨化反应。 在此提案中，我们寻求： 具体目的1：a）生成PLCGAMMA2突变体，以阐明PLCAMMA2在OC分化调节过程中的催化活性和适配器功能，并且b）生成Tat-Fusion-proteins表达plcgamma2独特区域所需的plcgamma2所需的独特区域，以识别出新型抗骨化抗肿瘤生成的方法，以识别新型的抗抗抗流抗抗抗性方法。 具体目的2：a）确定plcgamma2在体内的OC分化和炎症性溶解中的作用，b）在具有突变形式的PLCAMMA2突变形式的小鼠中测试体内溶性反应，以及PLCAMMA2的突变形式和抗抑制蛋白包含PLCGAMMA2的tat-Fusion蛋白包含功能域的抗敏感性。