Alternative NF-KB as a negative regulator of osteogenesis

 描述(申请人提供)：骨量调节是一个高度复杂的过程，涉及成骨细胞(OBS)、破骨细胞和骨细胞的协调作用。异常信号在骨质疏松症、骨折和癌症等多种疾病状态中都很明显。这项建议将首次研究替代的核因子-κB在基础骨内稳和应激条件下的OB谱系中的直接作用。转基因小鼠模型将被用来调节OB血统中替代的NF-κB，使用OSX-Cre。在诱导替代的NF-κB时，Nik使IKA磷酸化。由于目前尚不存在NIKF/f模型，将使用OSX-Cre；IKKaf/f动物(CKO)来检测OB谱系中替代的NF-κB的抑制情况。NIKdeltaT3(NT3)是Nik的一个结构性活性等位基因，位于有牙线的终止盒的下游。与OSX-Cre的重组将激活OB谱系中替代的NF-κB(CACT)。在目标1a中，通过对对照组、CKO和CACT小鼠的显微CT、组织形态计量学和机械强度的分析，将确定OB谱系中替代的NF-κB途径的丢失或激活如何影响基础骨表型。在目标1b中，将CKO和CACT培养的体外OB表型与对照进行比较。添加已知的成骨激活剂(WNT3a、BMP2)将描述替代的NF-κB可能的阶段特异性效应以及与各种成骨信号的相互作用。在目标2中，对病理机械刺激或激素合成代谢刺激的反应将在与目标1相同的动物中进行评估。目标2a将使用体内加载方案来监测OB谱系细胞对机械应激的反应。在AIM 2b中，将通过间歇性甲状旁腺激素治疗动物来评估OB血统细胞对激素刺激的反应能力，甲状旁腺激素是目前使用的一种骨质疏松疗法。骨形成率和骨量将在AIMS 2a和2b中进行量化，以评估替代的NF-κB途径的丢失或激活如何影响OB谱系细胞对这些合成代谢刺激的反应能力。因此，体内和体外实验都将检验最重要的假设，即替代的NF-κB是OB谱系分化和/或功能的负面调节因子。这一建议的结果将为成骨的分子机制提供有价值的见解，这可能有助于指导未来治疗骨相关病理的干预措施。

 DESCRIPTION (provided by applicant): Regulation of bone mass is a highly complex process involving the coordinated actions of osteoblasts (OBs), osteoclasts, and osteocytes. Aberrant signaling is manifest in a host of disease states such as osteoporosis, fracture, and cancer. This proposal will examine for the first time the direct role of alternative NF-κB in the OB lineage during basal bone homeostasis and under stress conditions. Transgenic mouse models will be used to modulate alternative NF-κB in the OB lineage using Osx-Cre. Upon induction of alternative NF-κB, NIK phosphorylates IKKa. As a NIKf/f model does not currently exist, Osx-Cre;IKKaf/f animals (cKO) will be used to examine inhibition of alternative NF-κB in the OB lineage. NIKdeltaT3 (NT3) is a constitutively active allele of NIK placed downstream of a floxed STOP cassette. Recombination with Osx-Cre will activate alternative NF-κB in the OB lineage (cACT). In Aim 1a, microCT, histomorphometric and mechanical strength analyses of control, cKO, and cACT mice will establish how loss or activation of the alternative NF-κB pathway in the OB lineage impacts basal bone phenotype. In Aim 1b, the in vitro OB phenotype of cKO and cACT cultures will be compared to control. Addition of known activators (Wnt3a, BMP2) of osteogenesis will delineate possible stage-specific effects of alternative NF-κB and interactions with various osteogenic signals. In Aim 2, the response to either pathological mechanical or hormonal anabolic stimuli will be evaluated in the same animals used in Aim 1. Aim 2a will use an in vivo loading protocol to monitor OB lineage cell response to mechanical stress. In Aim 2b, the ability of OB lineage cells to respond to hormonal stimulus will be evaluated by treating animals with intermittent parathyroid hormone, a currently used osteoporosis therapy. Bone formation rate and bone mass will be quantified in both Aims 2a and 2b to assess how loss or activation of the alternative NF-κB pathway influences the ability of OB lineage cells to respond to these anabolic stimuli. Thus, both in vivo and in vitro experiments will test the overarching hypothesis that alternative NF-κB is a negative regulator of OB lineage differentiation and/or function. The results of this proposal will provide valuable insight into the molecular mechanisms of osteogenesis, which may help guide future interventions to treat bone-related pathologies.