Molecular Control of RANKL Gene Expression

RANKL 基因表达的分子控制

• 批准号: 8090418
• 负责人: CHARLES A O'BRIEN
• 金额: $ 30.32万
• 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 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8090418
• 关键词: Ablation; Address; Alleles; Binding; Bone Resorption; Bone remodeling; Cells; Commit; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Distal; Distant; Enhancers; Equilibrium; Family; Family member; Funding; Gene Expression; Genes; Genetic Transcription; Goals; Green Fluorescent Proteins; Health; Hormones; In Vitro; Knockout Mice; Knowledge; LacZ Genes; Ligands; Mesenchymal; Mesenchymal Stem Cells; Molecular; Mouse Strains; Mus; Osteoblasts; Osteoclasts; Osteocytes; Parathyroid gland; Pathway interactions; Play; Population; Process; Production; Protein Binding; Regulatory Element; Reporter; Risk; Role; Source; Stromal Cells; Supporting Cell; TNFSF11 gene; Tamoxifen; Therapeutic Intervention; Transgenic Mice; Tumor necrosis factor receptor 11b; base; bone; bone mass; bone strength; cell type; cytokine; design; expectation; in vivo; loss of function; member; osteoblast differentiation; osteoclastogenesis; osteoporosis with pathological fracture; progenitor; protein expression; receptor; receptor expression; recombinase; small hairpin RNA; therapy development; transcription factor

DESCRIPTION (provided by applicant): Osteoclast differentiation depends on support provided by stromal cells. Although indirect evidence suggests that these stromal cells may be of the osteoblast lineage, the identity of these cells and their relationship to matrix-synthesizing osteoblasts remains unknown. One of the key mechanisms by which stromal cells support osteoclast differentiation is expression of receptor activator of NF(B ligand (RANKL). Hormones that stimulate osteoclast formation, such as PTH, do so by stimulating RANKL expression in stromal cells. However, the mechanisms controlling RANKL expression in these cells are only partially understood. The goals of this application are to elucidate the molecular mechanisms that control RANKL expression in stromal cells and determine the relationship, if any, of these cells to matrix-synthesizing osteoblasts. During the previous funding cycle, we demonstrated that PTH controls RANKL expression in stromal cells via a distant transcriptional enhancer that we have designated the RANKL distal control region (DCR). DCR knockout mice have reduced RANKL expression and increased bone mass and strength. The transcription factor Runx2 binds to the DCR, suggesting a linkage to osteoblast differentiation; however, stimulation of RANKL was not altered in cells from Runx2-deficient mice. Therefore, the molecular basis of DCR action and RANKL cell type-specific expression remains unclear. We also determined that ablation of matrix-synthesizing osteoblasts and their immediate precursors did not alter RANKL expression or osteoclast formation in bone, highlighting the contention that the identity of stromal cells remains unknown. Based on these findings, we hypothesize that matrix-synthesizing osteoblasts and the stromal cells that support osteoclastogenesis are derived from a common mesenchymal precursor, which bifurcates early in its specification into these two distinct lineages. Aim 1 will identify the molecular mechanisms by which the DCR functions by determining whether other members of the Runx family of transcription factors are required for RANKL expression in the absence of Runx2. In addition, other proteins that bind the DCR will be identified by footprinting and their role in DCR action determined by gain- and loss-of-function studies. Aim 2 will identify osteoclast support cells using two complementary approaches: (1) by localizing RANKL-expressing cells and their descendants in bone and (2) by determining whether RANKL expression in genetically defined cell populations is required for osteoclast formation. PUBLIC HEALTH RELEVANCE: The proposed studies seek to identify stromal cells that support production of bone-degrading cells. This will be accomplished by studying a gene expressed in stromal cells that is essential for production of bone-degrading cells. Increased understanding of the mechanisms that control bone resorption will provide essential information for the development of therapies to maintain or increase bone mass and strength, thereby reducing the risk of osteoporotic fractures.

描述(申请人提供)：破骨细胞分化依赖于基质细胞提供的支持。虽然间接证据表明这些基质细胞可能属于成骨细胞系，但这些细胞的特性及其与基质合成成骨细胞的关系仍不清楚。基质细胞支持破骨细胞分化的关键机制之一是表达核因子受体激活剂B配体(RANKL)。刺激破骨细胞形成的激素，如甲状旁腺素，是通过刺激基质细胞中RANKL的表达来实现的。然而，控制RANKL在这些细胞中表达的机制只被部分了解。这项应用的目的是阐明控制基质细胞RANKL表达的分子机制，并确定这些细胞与基质合成成骨细胞的关系。在之前的资金周期中，我们证明了PTH通过一个我们命名为RANKL远端控制区(DCR)的远程转录增强子来控制基质细胞中RANKL的表达。DCR基因敲除小鼠降低了RANKL的表达，增加了骨量和强度。转录因子Runx2与DCR结合，提示与成骨细胞分化有关；然而，在Runx2缺陷小鼠的细胞中，RANKL的刺激没有改变。因此，DCR作用和RANKL细胞类型特异性表达的分子基础仍不清楚。我们还确定，基质合成成骨细胞及其直接前体的消融不会改变骨中RANKL的表达或破骨细胞的形成，这突显了基质细胞的特性仍然未知的论点。基于这些发现，我们假设合成基质的成骨细胞和支持破骨细胞形成的基质细胞来自共同的间充质前体，这种前体在其指定的早期分成这两个不同的谱系。目的1通过确定在缺少Runx2的情况下，RUNX转录因子家族的其他成员是否需要RANKL的表达，来确定DCR发挥作用的分子机制。此外，结合DCR的其他蛋白质将通过足迹和它们在DCR作用中的作用来确定，这是通过功能获得和丧失研究确定的。目的2将通过两种互补的方法鉴定破骨细胞支持细胞：(1)通过定位RANKL表达的细胞及其后代在骨中的定位；(2)通过确定RANKL在基因定义的细胞群中的表达是否是形成破骨细胞所必需的。公共卫生相关性：拟议的研究试图确定支持骨降解细胞产生的基质细胞。这将通过研究在基质细胞中表达的一种基因来实现，该基因对产生骨降解细胞是必不可少的。增加对骨吸收控制机制的了解将为开发保持或增加骨量和强度的治疗方法提供必要的信息，从而降低骨质疏松性骨折的风险。