Study of osteoblast regulation in TNF-mediated bone loss

描述(申请人提供)：患有慢性炎症性疾病的患者，如类风湿性关节炎(RA)，通常患有严重的骨质疏松症和骨折风险增加，部分原因是成骨细胞骨形成减少。我们报道了类风湿关节炎模型--肿瘤坏死因子转基因小鼠的间充质干细胞(MSCs)明显减少了成骨细胞(OBL)的分化。在RA中，调节OBL功能抑制的分子尚不清楚，也没有有效的治疗方法。为了确定MSCs中负责obl抑制的新分子，我们使用来自纯化的TNF-TG小鼠和WT仔鼠MSCs的RNA测序数据进行了路径分析。我们发现，在肿瘤坏死因子-甘油三酯-间充质干细胞中，Notch信号通路明显上调。在我们的初步研究中，我们证实了来自TNF-TGRA小鼠和RA患者的MSCs中Notch的增加。我们发现，间歇性抑制Notch可以防止肿瘤坏死因子-甘油三酯小鼠的骨丢失，而肿瘤坏死因子通过非规范的NF-κBp52和RelB蛋白激活Notch。基于这些新的发现，我们推测在RA MSCs中，肿瘤坏死因子通过非典型的NF-κB p52和RelB激活Notch信号，导致OBL分化的阻断。间歇性Notch抑制可以防止肿瘤坏死因子-甘油三酯小鼠的骨丢失，而持续的Notch抑制会加剧由于MSC池耗尽而造成的骨丢失。这些假设将在三个具体目标中得到检验。在目标1中，我们将确定来自Hes1-GFP小鼠的CD45-Hes1+细胞是否具有Notch报告MSCs的功能。我们将检测不同年龄的Hes1-GFP/TNF-TG小鼠Hes1+MSCs在RA形成过程中功能和Notch激活状态的变化。在目标2中，我们将使用生化和分子方法来研究典型的NF-κB p52和RelB以及典型的NF-κB p50和RelA介导肿瘤坏死因子对Notch激活的影响的机制，如果这是通过典型的Notch信号通路实现的。在目标3中，我们将确定是否间歇性，但不是持续的， 给予Notch抑制剂可挽救肿瘤坏死因子-甘油三酯小鼠的骨丢失表型，而在肿瘤坏死因子-甘油三酯诱导的小鼠骨髓间充质干细胞中Notch活性的增加和成骨细胞的抑制是通过使用肿瘤坏死因子抑制剂的肿瘤坏死因子介导的。我们的研究将揭示Notch在介导RA相关的骨丢失以及非规范的NF-κB和Notch之间的相互作用中的新作用，并产生关于Notch抑制剂作为治疗RA或其他慢性炎症相关骨质疏松症患者的新的潜在治疗方法的临床前信息。

DESCRIPTION (provided by applicant): Patients with chronic inflammatory disorders, such as rheumatoid arthritis (RA), often suffer from severe osteoporosis and increased risk of fracture, partially due to reduced osteoblastic bone formation. We reported that mesenchymal stem cells (MSCs) of TNF transgenic (TNF-Tg) mice, a model of RA, have significantly decreased osteoblast (obl) differentiation. The molecules that mediate inhibited obl function in RA are unknown and there is no effective therapy. To identify new molecules in MSCs that are responsible for obl inhibition, we performed a pathway analysis using data from RNA sequencing in purified MSCs of TNF-Tg mice and WT littermates. We found that the pathway involved in Notch is markedly up-regulated in TNF-Tg MSCs. In our preliminary study, we confirmed increased Notch in MSCs from TNF-Tg RA mice and RA patients. We found that intermittent Notch inhibition prevented bone loss in TNF-Tg mice and TNF activates Notch via non- canonical NF-κB p52&RelB protein. Based on these new findings, we hypothesize that in RA MSCs, TNF activates Notch signaling through non-canonical NF-κB p52&RelB, leading to a blockade of obl differentiation. Intermittent Notch inhibition prevents bone loss in TNF-Tg mice while continuous Notch inhibition exacerbates bone loss due to depletion of the MSC pools. These hypotheses will be tested in 3 specific aims. In aim 1, we will determine if CD45-Hes1+ cells from Hes1-GFP mice functions as Notch-reporter MSCs. We will examine changes of function and Notch activation status in Hes1+MSCs from different ages of Hes1-GFP/TNF-Tg mice during the course of RA development. In aim 2, we will use biochemical and molecular approaches to study mechanisms by which canonical NF-κB p52&RelB, as well as canonical NF-κB p50&RelA mediate the effect of TNF on Notch activation and if this is through the canonical Notch signaling pathway. In aim 3, we will determine if intermittent, but not continues, administration of Notch inhibitor rescues bone loss phenotype of TNF-Tg mice and if increased Notch activation and inhibited obls in TNF-Tg mouse MSCs are mediated by TNF using TNF inhibitor. Our study will reveal a novel role for Notch in mediating RA-associated bone loss and interaction between non-canonical NF-κB and Notch, and generate preclinical information on Notch inhibitors as a new potential therapy for treating patients with RA or other chronic inflammation-associated osteoporosis.