Study of osteoblast regulation in TNF-mediated bone loss

描述（由申请人提供）：患有慢性炎症性疾病（如类风湿性关节炎（RA））的患者通常患有严重的骨质疏松症，骨折风险增加，部分原因是成骨细胞骨形成减少。我们报道了间充质干细胞（MSC）的TNF转基因（TNF-Tg）小鼠，RA模型，有显着降低成骨细胞（obl）分化。在RA中介导obl功能抑制的分子是未知的，并且没有有效的治疗。为了鉴定MSC中负责obl抑制的新分子，我们使用来自TNF-Tg小鼠和WT同窝小鼠的纯化MSC中的RNA测序的数据进行了途径分析。我们发现，参与Notch的途径在TNF-Tg MSC中显著上调。在我们的初步研究中，我们证实了TNF-Tg RA小鼠和RA患者的MSC中Notch增加。我们发现间歇性Notch抑制可防止TNF-Tg小鼠的骨丢失，并且TNF通过非经典NF-κB p52和RelB蛋白激活Notch。基于这些新的发现，我们假设在RA MSC中，TNF通过非经典NF-κB p52 & RelB激活Notch信号传导，导致obl分化的阻断。间歇性Notch抑制可防止TNF-Tg小鼠的骨丢失，而连续性Notch抑制可由于MSC池的消耗而加剧骨丢失。这些假设将在3个具体目标中进行检验。 在目标1中，我们将确定来自Hes 1-GFP小鼠的CD 45-Hes 1+细胞是否作为Notch报告基因MSC起作用。我们将研究在RA发展过程中，来自不同年龄的Hes 1-GFP/TNF-Tg小鼠的Hes 1 + MSC的功能和Notch激活状态的变化。 目的2：利用生物化学和分子生物学方法研究经典NF-κB p52和RelB以及经典NF-κB p50和RelA介导TNF对Notch激活的作用机制，以及这是否通过经典Notch信号通路。 在目标3中，我们将确定是否是间歇性的，但不是持续的， 施用Notch抑制剂挽救了TNF-Tg小鼠的骨损失表型，并且如果使用TNF抑制剂通过TNF介导了TNF-Tg小鼠MSC中Notch活化的增加和obl的抑制。 我们的研究将揭示Notch在介导RA相关骨丢失中的新作用以及非经典NF-κB和Notch之间的相互作用，并产生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疗法的临床前信息。

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.