Quantitative Imaging at Subcellular Levels in Chondrocyte Mechanotransduction

软骨细胞机械转导中亚细胞水平的定量成像

• 批准号: 8773662
• 负责人: Sungsoo Na
• 金额: $ 7.8万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-03 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8773662
• 关键词: Address; Attenuated; Biosensor; Cartilage; Cell Culture Techniques; Cells; Chondrocytes; Complex; Cytokine Signaling; Degenerative Disorder; Degenerative polyarthritis; Family; Fluorescence Resonance Energy Transfer; Focal Adhesion Kinase 1; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Image; Individual; Inflammatory; Integrins; Intercellular Fluid; Interleukins; Life; Link; Maintenance; Mechanics; Mediating; Mitogen-Activated Protein Kinases; Modeling; Molecular; Osteoarthrosis Deformans; Outcome; Patients; Pattern; Peptide Hydrolases; Physiological; Play; Role; Signal Transduction; Signaling Molecule; Stimulus; Testing; Tumor Necrosis Factor-alpha; base; cell type; cytokine; fluid flow; insight; novel; response; rho; rho GTP-Binding Proteins; spatiotemporal

DESCRIPTION (provided by applicant): The long-term objective of the proposed project is to determine the effects of mechanical loading on osteoarthritic (OA) cartilage. Integrin-mediated Src/FAK signaling as well as Rho family GTPase such as RhoA is known to play an important role in cartilage maintenance and degradation. Accumulating evidence suggests that in OA cartilage inflammatory cytokines such as interleukin 1β (IL1β) and tumor necrosis factor α (TNFα) elevate catabolic activities, whereas mechanical loading may act as a beneficial regulator depending on the loading magnitude. Despite previous studies, it remains unclear whether mechanical loading could attenuate cytokine-induced activation of Src/FAK and Rho GTPases that is linked to cartilage degradation. Furthermore, little has been known about spatiotemporal dynamics of intracellular signaling of Src/FAK and GTPases at the sub-cellular level in response to cytokines and interstitial fluid flow. We address a question whether normal and OA chondrocytes have differential baseline activities of Src, FAK, and Rho family GTPases. We hypothesize that interstitial fluid flow generates spatiotemporal patterns of these signaling molecules distinctively different in normal and OA/cytokine-treated chondrocytes, and it suppresses cytokine-induced and/or OA-induced signaling activities. To test our hypotheses, we propose three specific aims using normal chondrocytes, cytokine-treated chondrocytes, and chondrocytes isolated from OA patients under varying magnitudes of interstitial fluid flow. " Aim 1: Determine the spatiotemporal patterns of activities of Src and FAK " Aim 2: Determine the spatiotemporal patterns of activities of Rho family GTPases (RhoA, Rac1, etc.)" Aim 3: Examine interactions of Src and FAK to Rho family GTPases In this proposal, fluorescence resonance energy transfer (FRET)-based biosensors together with a three- dimensional (3D) culture model will allow us to visualize the activities of signaling molecules at the sub- cellular level and evaluate the responses to fluid flow in the presence and absence of inflammatory cytokines. We expect that this project will advance our understanding of the molecular mechanism of mechanotransduction involved in cartilage degradation, and it will contribute to treatment of degenerative joint diseases such as osteoarthritis.

描述（由申请人提供）：拟议项目的长期目标是确定机械负荷对骨关节炎（OA）软骨的影响。已知整合素介导的Src/FAK信号传导以及Rho家族GT3（如RhoA）在软骨维持和降解中起重要作用。越来越多的证据表明，在OA软骨炎性细胞因子，如白细胞介素1β（IL 1 β）和肿瘤坏死因子α（TNFα）提高分解代谢活动，而机械负荷可能作为一个有益的调节器取决于负荷的大小。尽管以前的研究，它仍然不清楚是否机械负荷可以减弱姜黄素诱导的Src/FAK和Rho GTP酶的激活，这是与软骨降解。此外，很少有人知道Src/FAK和GTP酶的细胞内信号转导在亚细胞水平的时空动态响应细胞因子和间质液流。我们解决了一个问题，是否正常和OA软骨细胞有不同的基线活动的Src，FAK，和Rho家族GTP酶。我们假设，间质液流产生的时空模式，这些信号分子明显不同的正常和OA/精氨酸处理的软骨细胞，它抑制精氨酸诱导和/或OA诱导的信号活动。为了验证我们的假设，我们提出了三个具体的目标，使用正常的软骨细胞，姜黄素处理的软骨细胞，软骨细胞分离的OA患者在不同程度的间质液流量。“目标1：确定Src和FAK的活性的时空模式“目的2：确定Rho家族GTP酶（RhoA、Rac 1等）的活性的时空模式。“目标3：检查Src和FAK与Rho家族GTP酶的相互作用在该提议中，基于荧光共振能量转移（FRET）的生物传感器与三维（3D）培养模型一起将允许我们在亚细胞水平上可视化信号分子的活性，并评估在存在和不存在炎性细胞因子的情况下对流体流动的响应。我们期望该项目将促进我们对软骨降解中所涉及的机械传导的分子机制的理解，并且它将有助于退行性关节疾病如骨关节炎的治疗。