MOLECULAR CONTROL OF RANKL GENE EXPRESSION

RANKL 基因表达的分子控制

• 批准号: 7087002
• 负责人: CHARLES A O'BRIEN
• 金额: $ 24.82万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-29 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7087002
• 关键词: DNA footprinting; RNase protection assay; gel mobility shift assay; gene expression; genetic regulation; genetic regulatory element; genetic transcription; genetically modified animals; laboratory mouse; messenger RNA; nucleic acid sequence; osteoblasts; osteoprotegerin; parathyroid hormones; polymerase chain reaction; posttranscriptional RNA processing; site directed mutagenesis

DESCRIPTION (provided by applicant): The initiation of bone remodeling depends in part on the support of osteoclast differentiation by stromal cells that appear to be of the osteoblast lineage. The key mechanism by which these stromal/osteoblastic cells support osteoclastogenesis is expression of RANKL (receptor activator of NFkappaB ligand). Hormones that stimulate osteoclast formation, such as PTH, do so by stimulating RANKL expression in stromal/osteoblastic cells but not other cell types. However, strikingly little is known about how this is accomplished. The long-term goal of this project is to identify the mechanisms that control RANKL expression in stromal/osteoblastic cells. Preliminary studies have determined that PTH stimulates RANKL transcription via a PKA-CREB pathway and that PTH also stimulates RANKL mRNA stability. Promoter-reporter constructs containing up to 7 kb of RANKL 5'-flanking region are regulated weakly or not at all by osteoclastogenic agents, suggesting that regulation of this gene involves distant regulatory elements. Consistent with this, a 160-kb DNA fragment harboring the murine RANKL gene as well as extensive flanking regions contains sequences sufficient for appropriate regulation by PKA in stably transfected stromal/osteoblastic cells. In Aim 1, cis-acting elements required for transcriptional control of RANKL expression by PTH will be identified by deletion analysis of the 160 kb DNA fragment and DNase I hypersensitivity assays. Elements will be further localized by DNase I protection and gel shift assays. Site-directed mutagenesis of the full-length construct will confirm the significance of each site. In Aim 2, cis-acting sequences that mediate post-transcriptional control of RANKL mRNA by PTH will be identified. Tagged murine RANKL mRNA will be conditionally expressed in stromal/osteoblastic cells and sequences involved in destabilization/PTH-induced stabilization will be mapped by deletion analysis. Aim 3 will identify regions of the RANKL gene involved in tissue-specific expression. This will be accomplished by creating transgenic mice harboring luciferase reporter constructs containing different portions of the RANKL gene and flanking region. Information gained from these studies will lead to a better understanding of the cellular factors involved in the support of osteoclast formation and thereby identify novel targets for anti-resorptive therapy.

描述（由申请人提供）：骨重塑的启动部分取决于似乎属于成骨细胞谱系的基质细胞对破骨细胞分化的支持。这些基质/成骨细胞支持破骨细胞生成的关键机制是 RANKL（NFkappaB 配体受体激活剂）的表达。刺激破骨细胞形成的激素（例如 PTH）是通过刺激基质/成骨细胞（而非其他细胞类型）中的 RANKL 表达来实现这一目的。然而，人们对于如何实现这一点却知之甚少。该项目的长期目标是确定控制基质/成骨细胞中 RANKL 表达的机制。初步研究已确定 PTH 通过 PKA-CREB ​​途径刺激 RANKL 转录，并且 PTH 还刺激 RANKL mRNA 稳定性。含有高达 7 kb RANKL 5' 侧翼区域的启动子-报告基因构建体受破骨细胞因子的调节较弱或根本不调节，表明该基因的调节涉及远处的调节元件。与此一致的是，含有鼠 RANKL 基因以及广泛的侧翼区域的 160 kb DNA 片段包含足以在稳定转染的基质/成骨细胞中通过 PKA 进行适当调节的序列。在目标 1 中，将通过 160 kb DNA 片段的缺失分析和 DNase I 超敏试验来鉴定 PTH 转录控制 RANKL 表达所需的顺式作用元件。元素将通过 DNase I 保护和凝胶位移测定进一步定位。全长构建体的定点诱变将确认每个位点的重要性。在目标 2 中，将鉴定通过 PTH 介导 RANKL mRNA 转录后控制的顺式作用序列。标记的鼠 RANKL mRNA 将在基质细胞/成骨细胞中条件表达，并且通过缺失分析绘制参与不稳定/PTH 诱导稳定的序列。目标 3 将鉴定参与组织特异性表达的 RANKL 基因区域。这将通过创建转基因小鼠来实现，该小鼠携带含有 RANKL 基因和侧翼区域不同部分的荧光素酶报告基因构建体。从这些研究中获得的信息将有助于更好地了解支持破骨细胞形成的细胞因素，从而确定抗骨吸收治疗的新靶点。