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

性状（由申请方提供）：BMP、TGF-？和激活素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TGF-β/激活素信号传导增强骨形成，而抑制典型BMP信号传导降低骨形成。然而，越来越多的研究提供的证据表明，典型的BMP，TGF-？激活素信号传导对成骨细胞的分化和功能具有积极和消极的影响。由于这些分子通常同时存在于细胞外环境中，骨骼靶细胞可能接受来自TGF-β/TGF-β受体的输入。激活素和BMP经典信号通路同时进行。在大多数实验中，骨细胞用TGF-β处理，激活素或BMP是对可能发生在两种典型信号传导途径之间的潜在细胞内拮抗作用的检查。研究集中在软骨细胞，另一种对TGF-？/激活素和骨形成蛋白提供了有趣的数据，表明这些细胞在做出细胞命运决定时整合了来自两个典型途径的信号。我们假设在典型的BMP和TGF-β之间存在双向的细胞内拮抗作用。骨细胞中的激活素信号通路，并且由这种相互作用产生的整合信号调节成人骨骼中的骨量。作为BMP和TGF-？/激活素信号仅提供关于每个通路的独立激活的信息，并且我们需要确定通路之间的串扰，我们建议构建新的BMP/TGF-？/激活素生物传感器系统，其利用通过相互RNA干扰连接的两个独立报告子。短发夹RNA的包含连接两个报告子，使得通过相互拮抗作用，在平衡信号下不会发生来自任一报告子的荧光，但当通过一个途径的转导占主导地位时会发生荧光信号。一旦在体外验证，该生物传感器将用于映射BMP与TGF-？/激活素信号在发育中的骨和在体内的成人骨骼。如果成功，我们预计这种生物传感器 将是有用的，许多研究人员在多个领域，了解BMP和TGF-？/需要激活素信号传导。因此，我们认为这种创新方法对理解经典信号传导的影响保证了开发这种新型试剂的固有风险。 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产生巨大影响。这对于骨骼尤其如此，老年人的骨骼损失威胁到他们的独立性。为了更好地了解如何保持骨量，因为我们的年龄，我们建议开发一种新的生物传感器，使我们能够评估，如果变化的BMP/TGF-？/激活素是调节骨形成的信号，是与年龄相关的骨丢失的原因。

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.