Molecular Control of RANKL Gene Expression

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，这些基质细胞可能是成骨细胞谱系，这些细胞的身份和他们的关系，基质合成成骨细胞仍然未知。基质细胞支持破骨细胞分化的关键机制之一是表达NF（B配体）受体激活因子（RANKL）。刺激破骨细胞形成的激素，如PTH，通过刺激基质细胞中RANKL的表达来实现。然而，在这些细胞中控制RANKL表达的机制仅部分了解。本申请的目的是阐明控制基质细胞中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（如有）。在之前的资助周期中，我们证明了PTH通过我们指定为RANKL远端控制区（DCR）的远端转录增强子控制基质细胞中的RANKL表达。DCR敲除小鼠RANKL表达减少，骨量和强度增加。转录因子Runx 2与DCR结合，表明与成骨细胞分化有关;然而，在Runx 2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。我们还确定了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，突出了基质细胞的身份仍然未知的论点。基于这些发现，我们假设基质合成成骨细胞和支持破骨细胞生成的基质细胞来自于共同的间充质前体，其在其规范的早期分叉成这两个不同的谱系。目的1将通过确定Runx家族转录因子的其他成员是否在Runx 2不存在的情况下是RANKL表达所必需的，来确定DCR发挥功能的分子机制。此外，结合DCR的其他蛋白质将通过足迹法鉴定，并通过功能获得和丧失研究确定其在DCR作用中的作用。目的2将使用两种互补的方法鉴定破骨细胞支持细胞：（1）通过定位RANKL表达细胞及其在骨中的后代和（2）通过确定破骨细胞形成是否需要遗传定义的细胞群中的RANKL表达。公共卫生相关性：拟议的研究旨在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基因来实现，该基因对骨降解细胞的产生至关重要。增加对控制骨吸收机制的了解将为开发维持或增加骨量和强度的治疗方法提供重要信息，从而降低骨质疏松性骨折的风险。

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.