Molecular Mechanisms of RANKL Activation in Osteoblasts

描述(申请人提供)：破骨细胞是大的，多核细胞，在骨吸收中起中心作用。它们来源于对RANKL反应的造血祖细胞。RANKL对破骨细胞的形成既是必要的，也是充分的，尽管M-CSF、TGFb、炎性细胞因子和前列腺素等因素在这一过程中也是重要的。RANKL在多种细胞类型中合成，包括成骨细胞系和活化的T细胞。体外和体内研究表明，RANKL基因的表达受关键的骨重建激素如甲状旁腺素和1，25(OH)2D3以及细胞因子如IL-1、TNFa、IL-6和前列腺素PGE2的生理调节，但其在T细胞亚群中的表达主要是由于T细胞的激活。RANKL的过度表达、骨吸收增强、溶骨性或骨质疏松性疾病与上述任何一种调节因子或细胞类型的异常产生和/或表达有关。尽管最近我们对成骨细胞系细胞中RANKL表达的分子机制的了解有所增加，但几乎对这些与T细胞相关的机制知之甚少。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与T细胞活化相关的骨吸收中的关键作用，我们建议在分子水平上确定在T细胞中RANKL基因表达的关键机制。目的：研究RANKL在小鼠和人T细胞模型及人和鼠原代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类T细胞模型，以探索RANKL的激活过程，并从mRNA和蛋白水平研究RANKL的诱导。目的：用芯片和芯片分析技术研究RANKL诱导小鼠和人T细胞分化的分子机制。我们计划利用一种无偏见的方法，使用芯片分析来识别RANKL基因间隔区分析中的T细胞特异性增强子，以通过T细胞激活诱导的RNA PolII招募和T细胞激活诱导的组蛋白4(H4)乙酰化(H4ac)来识别该基因间隔区的T细胞特异性增强子，然后识别和表征参与激活过程的转录因子。目的：鉴定RANKL基因座在体内对T细胞表达RANKL至关重要的关键成分。我们将制备一个含有CTCF绝缘子结合的RANKL基因的小鼠BAC克隆，将荧光素酶报告基因导入3‘非编码区，然后评估该克隆在转基因小鼠品系中的表达。这些研究将为体内和体外调节T细胞RANKL表达的机制提供重要的细节。 公共卫生相关性：RANKL是一种分子，它在正常的骨转换以及与衰老、更年期、自身免疫性疾病和癌症相关的骨丢失方面都发挥着重要作用。这里的研究试图确定该因子表达的基础机制，以便能够创造出更好、更具选择性的药物。

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.