Regulatory Pathways in Osteoclast Formation and Function

描述（由申请人提供）：类风湿性关节炎、牙周病和一些肿瘤相关骨转移的过度骨质流失，主要是由于免疫系统的异常激活导致破骨细胞（OCs）的过度刺激和激活[1-3]。识别影响骨免疫系统的常见信号分子及其对正常和病理性骨质流失的影响可能为未来治疗或影响骨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）作为RANKL （NFkappaB配体受体激活因子）诱导的破骨细胞发生的中心介质的新作用，独立于PLCgamma1。OCs是主要的骨吸收细胞，由骨髓巨噬细胞（BMMs）发育而来，主要受两大调节因子的影响：巨噬细胞集落刺激因子（M-CSF）和NFkappaB配体受体激活因子（RANKL），以及鲜为人知的共刺激因子，这些共刺激因子通过含有免疫受体酪氨酸激活基元（ITAM）的受体作用于OC前体细胞[2,4,5]。我们的数据表明，在小鼠中靶向删除PLCgamma2会导致骨质疏松表型。plcgamma2缺陷OC前体由于AP1和NFkappaB激活缺陷以及NFATc1（破骨细胞发生的主要转录因子）上调失败而无法分化。虽然PLCgamma2的磷酸化及其催化活性是ITAM依赖性NFATc1上调所必需的，但JNK和NFkappaB途径的激活可能依赖于PLCgamma2结合和激活GAB2的能力，GAB2是一种适配器蛋白，在rankl介导的破骨细胞发生[6]中发挥重要作用，并调节GAB2向RANK信号复合物的募集。

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.