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

创建

• 批准号: 8283524
• 负责人: Vicki Rosen
• 金额: $ 22.82万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8283524
• 关键词: Activins; Adult; Age; Age-Related Bone Loss; Aging; Biosensor; Calvaria; Cells; Chondrocytes; Data; Disease; Elderly; Environment; Equilibrium; Fluorescence; Generations; Health; In Vitro; Individual; Link; Maps; Mus; Nature; Osteoblasts; Osteogenesis; Pathway interactions; RNA Interference; Reagent; Reporter; Research Personnel; Risk; S100A8 gene; Signal Pathway; Signal Transduction; Skeleton; System; Techniques; Testing; Time; Validation; Variant; activin B; bone; bone cell; bone mass; extracellular; in vivo; innovation; novel; osteoblast differentiation; research study; skeletal; small hairpin RNA

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.

描述（由申请人提供）：bmp、TGF-？和激活素已被证明对成人骨量有深远的影响，大多数研究人员发现抑制典型TGF-？/activin信号促进骨形成，而抑制典型BMP信号则降低骨形成。然而，越来越多的研究提供证据表明，典型的BMP、TGF-？激活素信号对成骨细胞的分化和功能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，骨骼靶细胞很可能同时接受TGF-？/activin和BMP经典信号通路同时存在。在大多数用TGF-？/activin或bmp是对两种典型信号通路之间可能发生的潜在细胞内拮抗作用的检查。研究集中在软骨细胞上，这是另一种对TGF-？这些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种典型途径的信号。我们假设典型的BMP和TGF-?之间存在双向的细胞内拮抗作用。这种相互作用产生的综合信号调节了成人骨骼的骨量。作为BMP和TGF-？/activin信号只给出各通路独立激活的信息，需要确定各通路之间的串扰，我们提出构建新的BMP/TGF-？/激活素生物传感器系统，利用两个独立的报告通过相互RNA干扰连接。短发夹RNA的包含将两个报告基因连接起来，因此，通过相互拮抗，在平衡信号处，任何一个报告基因都不会发出荧光，而当通过一个途径的转导占主导地位时，则会发出荧光信号。一旦在体外得到验证，这种生物传感器将用于绘制BMP与TGF-？/激活素信号在骨发育和体内成人骨骼中的作用。如果成功，我们预计这种生物传感器