Study of osteoblast regulation in TNF-mediated bone loss

描述（由申请人提供）：慢性炎症性疾病患者，如类风湿关节炎（RA），经常患有严重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TNF转基因（TNF- tg）小鼠（RA模型）的间充质干细胞（MSCs）显著降低成骨细胞（obl）分化。在RA中介导抑制obl功能的分子是未知的，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TNF-Tg小鼠和WT幼崽纯化MSCs的RNA测序数据进行了途径分析。我们发现Notch通路在TNF-Tg MSCs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MSCs中Notch升高。我们发现间歇性抑制Notch可防止TNF- tg小鼠骨质流失，TNF通过非典型NF-？B p52&RelB蛋白。基于这些新发现，我们假设在RA MSCs中，TNF通过非典型NF-？B p52和relb，导致obl分化受阻。间歇性Notch抑制可防止TNF-Tg小鼠的骨质流失，而持续的Notch抑制则会因MSC池耗尽而加剧骨质流失。这些假设将在三个具体目标中进行检验。在目的1中，我们将确定来自Hes1-GFP小鼠的CD45-Hes1+细胞是否具有notch报告细胞的功能。我们将检测不同年龄Hes1- gfp /TNF-Tg小鼠Hes1+MSCs在RA发展过程中功能和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NF-？B p52和relb，以及典型NF-？bp50和rela介导TNF对Notch激活的影响，如果这是通过典型的Notch信号通路。在目标3中，我们将确定是否是间歇性的，而不是持续性的，

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-?B p52&RelB protein. Based on these new findings, we hypothesize that in RA MSCs, TNF activates Notch signaling through non-canonical NF-?B 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-?B p52&RelB, as well as canonical NF-?B 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-?B 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.