Molecular Mechanisms of RANKL Activation in Osteoblasts

描述（由申请人提供）：破骨细胞是大的，多核细胞，在骨吸收中起核心作用。它们来源于对RANKL反应的造血前体。尽管M-CSF、TGFb、炎症细胞因子和前列腺素等因子在破骨细胞生成过程中也很重要，但RANKL对于破骨细胞生成是必要的和充分的。RANKL在许多细胞类型中合成，包括成骨细胞谱系和活化的T细胞。体外和体内研究表明，尽管RANKL mRNA的表达受PTH和1,25(OH)2D3等关键骨重塑激素以及IL-1、TNFa、IL-6和前列腺素PGE2等细胞因子的生理调节，但其在T细胞亚群中的表达主要受T细胞活化的影响。这些调节因子或细胞类型中的任何一种的异常产生和/或表达都与RANKL过表达、骨吸收增强和溶骨或骨质疏松症有关。尽管最近我们对成骨细胞谱系中Rankl表达的分子机制的理解有所增加，但对这些与T细胞相关的机制几乎一无所知。鉴于RANKL在慢性炎症性疾病、自身免疫性疾病和转移性癌症中对骨吸收和T细胞活化的重要影响，我们建议从分子水平确定RANKL基因在T细胞中表达的关键机制。目的1：研究RankL在小鼠和人T细胞模型以及小鼠和人原代T细胞中的表达。RankL在初始T细胞中不存在，但在活化的CD4+和CD8+ T细胞群中显著诱导。我们计划建立小鼠和人T细胞模型来探索激活过程，并在mRNA和蛋白水平上研究RankL的诱导作用。目的2：利用ChIP和ChIP- ChIP分析表征RankL诱导小鼠和人T细胞的分子机制。我们计划利用ChIP-chip分析的无偏方法在RankL基因间区分析中鉴定T细胞特异性增强子，利用T细胞激活诱导的RNA pol II募集和T细胞激活诱导的组蛋白4 （H4）乙酰化（H4ac）鉴定该基因间区中的T细胞特异性增强子，然后鉴定和表征参与激活过程的转录因子。目的3：确定T细胞在体内表达Rankl所必需的Rankl位点的关键成分。我们将制备含有以CTCF绝缘子位点为结合的RankL位点的小鼠BAC克隆，在3' UTR中引入荧光素酶报告基因，然后在转基因小鼠品系中评价该克隆的表达。这些研究将提供重要的细节，通过在体外和体内调节T细胞中RANKL表达的机制。

DESCRIPTION (provided by applicant): Osteoclasts are large, multinucleated cells that play a central role in bone resorption. They are derived from hematopoietic precursors in response to RANKL. RANKL is both necessary and sufficient for osteoclastogenesis, although factors such as M-CSF, TGFb, inflammatory cytokines and prostaglandins are also important to this process. RANKL is synthesized in a number of cell types, including those of the osteoblast lineage and the activated T cell. Studies both in vitro and in vivo indicate that whereas RANKL mRNA expression is regulated physiologically by key bone remodeling hormones such as PTH and 1,25(OH)2D3 as well as by cytokines such as IL-1, TNFa, IL-6 and the prostaglandin PGE2, its expression in T cell subsets is largely due to T cell activation. Aberrant production and/or expression of any one these modulators or the cell types has been implicated in RANKL overexpression, enhanced bone resorption and osteolytic or osteoporotic disease. Despite a recent increase in our understanding of the molecular mechanisms of Rankl expression in osteoblast lineage cells, almost nothing is known of these mechanisms relative to T cells. In view of the critical impact of RANKL on bone resorption associated with activation of T cells in chronic inflammatory disease, autoimmune disease and metastatic cancer, we propose to define key mechanisms instrumental to RANKL gene expression at the molecular level in T cells. Aim 1: To investigate the expression of RankL in both mouse and human T cell models and in mouse and human primary T Cells. RankL is absent in naive T cells, but strikingly induced in activated CD4+ and CD8+ T cell populations. We plan to establish both mouse and human T cell models to explore the activation process and to study the induction of RankL at the mRNA and protein levels. Aim 2: To characterize the molecular mechanisms of RankL induction in mouse and human T cells using ChIP and ChIP-chip analysis. We plan to utilize an unbiased approach using ChIP-chip analysis to identify T cell specific enhancers in the RankL intergenic region analysis to identify T cell specific enhancers in this intergenic region using T cell activation-induced RNA pol II recruitment and T cell activation-induced histone 4 (H4) acetylation (H4ac) and then identify and characterize the transcription factors that are involved in the activation process. Aim 3: To identify key components of the Rankl locus essential for T cell expression of RankL in vivo. We will prepare a mouse BAC clone containing the RankL locus bounded by CTCF insulator sites, introduce a luciferase reporter into the 3' UTR, and then evaluate the expression of this clone in transgenic mouse strains. These studies will provide important detail into the mechanisms through which RANKL expression is regulated in T cells in vitro and in vivo. PUBLIC HEALTH RELEVANCE: RANKL is a molecule which plays a significant role in both normal bone turnover and the bone loss associated with ageing, menopause, autoimmune disease and cancer. The studies herein seek to identify the mechanisms that underlie the expression of this factor such that better and more selective medicines can be created.