Creation & Validation of a BMP/TGF-B/Activin Biosensor System

描述(由申请人提供)：BMPS，转化生长因子-？激活素已被证明对成年人的骨量有深远的影响，大多数研究人员发现，抑制典型的转化生长因子-β/激活素信号促进骨形成，而抑制典型的BMP信号减少骨形成。然而，越来越多的研究提供了证据，表明典型的BMP、转化生长因子？激活素信号对成骨细胞的分化和功能既有积极作用，也有消极作用。由于这些分子经常同时存在于细胞外环境中，骨骼靶细胞很可能同时接受来自转化生长因子-β/激活素和骨形态发生蛋白信号通路的输入。在用转化生长因子-β/激活素或骨形态发生蛋白处理骨细胞的大多数实验中，基本上没有对这两条典型信号通路之间可能发生的潜在的细胞内拮抗作用进行检查。对软骨细胞的研究提供了有趣的数据，表明这些细胞在决定细胞命运时整合了来自两条典型途径的信号。软骨细胞是另一种对转化生长因子-β/激活素和骨形态发生蛋白有反应的靶细胞。我们假设在骨细胞中典型的BMP和转化生长因子-β/激活素信号通路之间存在双向的细胞内拮抗作用，这种相互作用产生的整合信号调节成人骨骼中的骨量。鉴于现有的BMP和转化生长因子-β/激活素信号转导系统只提供了有关每个信号通路独立激活的信息，而我们需要确定信号通路之间的串扰，我们建议构建一种新型的BMP/转化生长因子-β/激活素生物传感器系统，该系统利用两个独立的记者通过相互的RNA干扰连接起来。短发夹状RNA的包含将两个报告连接在一起，因此，通过相互拮抗，两个报告中的任何一个都不会在平衡信号下出现荧光，但当通过一条途径转导占主导地位时，将出现荧光信号。一旦在体外得到验证，这种生物传感器将被用来在体内定位BMP与转化生长因子-β/激活素信号在发育中的骨骼和成人骨骼中的关系。如果成功，我们预计这种生物传感器 对于需要了解BMP和转化生长因子-β/激活素信号之间的平衡的多个领域的许多研究人员来说，这将是有用的。因此，我们认为，这种理解规范信号的创新方法的影响值得在开发这种新试剂时固有的风险。 公共卫生相关性：与老龄化相关的疾病对所有美国公民的健康和福祉有巨大影响。这一点对于骨骼来说尤其如此，老年人的骨骼缺失威胁着他们的独立性。为了更好地了解如何随着年龄的增长保持骨量，我们建议开发一种新型的生物传感器，使我们能够评估调控骨形成的BMP/转化生长因子-β/激活素水平的变化是否与年龄相关的骨丢失有关。

DESCRIPTION (provided by applicant): BMPs, TGF-? and activin have been shown to have profound effects on bone mass in adults where most investigators have found that inhibiting canonical TGF-?/activin signaling enhances bone formation while inhibiting canonical BMP signaling decreases bone formation. However, an increasing number of studies have provided evidence to suggest that canonical BMP, TGF-? and activin signaling can have both positive and negative effects on osteoblast differentiation and function. As these molecules are often present in the extracellular environment at the same time, skeletal target cells are likely to receive inputs from both the TGF-?/activin and BMP canonical signaling pathways concurrently. Largely absent from most experiments where bone cells are treated with TGF-?/activin or BMPs is an examination of the potential intracellular antagonism that may occur between the two canonical signaling pathways. Studies focused on chondrocytes, another target cell that responds to TGF-?/activin and BMPs, provide intriguing data to suggest that these cells integrate signals from the two canonical pathways when making cell fate decisions. We hypothesize that there is bidirectional intracellular antagonism between the canonical BMP and TGF-?/activin signaling pathways in bone cells, and that the integrated signal that results from this interaction regulates bone mass in the adult skeleton. As existing reporters for BMP and TGF-?/activin signaling only give information about the independent activation of each pathway, and we need to determine the crosstalk between the pathways, we propose to construct a novel BMP/TGF-?/activin biosensor system that utilizes two independent reporters linked through reciprocal RNA interference. The inclusion of short hairpin RNA links the two reporters so that, through mutual antagonism, no fluorescence from either reporter will occur at a balanced signal but a fluorescent signal will occur when transduction through one pathway is dominant. Once validated in vitro, this biosensor will be used to map BMP vs. TGF-?/activin signaling in developing bone and in the adult skeleton in vivo. If successful, we anticipate that this biosensor will be useful to many investigators in multiple fields where an understanding of the balance between BMP and TGF-?/activin signaling is required. As such we believe that the impact of this innovative approach to understanding canonical signaling warrants the risks inherent in developing this novel reagent. PUBLIC HEALTH RELEVANCE: Diseases associated with aging have a tremendous impact on the health and wellbeing of all US citizens. This is particularly true for the skeleton where loss f bone in older adults threatens their independence. To better understand how to maintain bone mass as we age, we propose to develop a novel biosensor that allows us to assess if variation in levels of BMP/TGF-?/activin, signals that regulate bone formation, are responsible for age-related bone loss.