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。它们是由造血祖细胞在多种调节因子的作用下产生的，其中最重要的是RANKL。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与这一过程相关的事件中也是重要的。RANKL在包括成骨细胞系在内的多种细胞类型中合成。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。任何一种调节因子的异常产生和/或表达都与RANKL的过度表达、增强的骨吸收和溶骨性或骨质疏松性疾病有关。尽管有相当多的生理和病理认识，但对控制RANKL从支持细胞转录输出的分子机制知之甚少。然而，初步研究表明，造成这一现象的机制可能非常复杂。鉴于RANKL在骨吸收中的重要作用，我们建议在分子水平上确定RANKL基因表达的关键机制。目的1：研究位于小鼠RANKL转录起始点(TSS)上游76kb的RANKL远端控制区(RL-DCR)上与VDR、CREB和STATS激活RANKL基因相关的分子事件。这个站点的相关性将使用转基因小鼠模型进行确认。目的：研究位于小鼠RANKL基因TSS上游-16、-22、-60和-69kb的4个额外调控结构域的作用。这些位点对RANKL基因表达调控的贡献将通过制备一个独特的小鼠模型来检验。目的：研究RANKL基因上游区域转录因子占位对染色质组织、修饰和结构的影响，以及核糖核酸聚合酶II的功能。这些研究将为促钙激素和细胞因子调节RANKL表达的机制提供重要的细节，RANKL基因的产物是骨吸收的核心。

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 a number of regulatory factors, the most important of which is RANKL. RANKL is both necessary and sufficient for osteoclastogenesis, although factors such as M-CSF, TGFb, inflammatory cytokines and prostaglandins are also important in the events associated with this process. RANKL is synthesized in a number of cell types, including those of the osteoblast lineage. Studies both in vitro and in vivo indicate that 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. Aberrant production and/or expression of any one these modulators has been implicated in RANKL overexpression, enhanced bone resorption and osteolytic or osteoporotic disease. Despite considerable physiologic and pathologic insight, little is known of the molecular mechanisms that control the transcriptional output of RANKL from support cells. Initial studies, however, suggest that the mechanisms responsible are likely to be highly complex. In view of the critical impact of RANKL on bone resorption, we propose to define key mechanisms instrumental to RANKL gene expression at the molecular level. Aim 1: To define the molecular events associated with activation of the RANKL gene by VDR, CREB, and STATS at the RANKL distal control region (RL- DCR) located 76 kb upstream of the mouse RankL transcriptional start site (TSS). The relevance of this site will be confirmed using a genetically altered mouse model. Aim 2: To evaluate the contribution of four additional regulatory domains located upstream of the mouse RankL gene TSS at -16, -22, -60 and -69 kb. The contribution of these sites to the regulation of RankL gene expression will be examined through preparation of a unique mouse model. Aim 3: To assess the consequence of transcription factor occupancy within the RankL gene upstream region on chromatin organization, modification and structure, and to determine the function of RNA polymerase II which appears specifically at these sites in response to activation. These studies will provide important detail into the mechanism by which calciotropic hormones and cytokines regulate RANKL expression, a gene whose product is central to bone resorption.