Elucidation of Shear-Induced Signaling Networks

剪切诱导信号网络的阐明

• 批准号: 7483197
• 负责人: Konstantinos Konstantopoulos
• 金额: $ 34.09万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-18 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7483197
• 关键词: Antioxidants; Apoptosis; Apoptotic; Arthritis; BCL2 gene; Binding Sites; Bioinformatics; Biological Assay; Biology; Bioreactors; Cartilage; Cells; Chondrocytes; Comparative Study; Condition; Coupled; Cyclic AMP-Dependent Protein Kinases; Data; Disease; Dominant-Negative Mutation; Drug Metabolic Detoxication; Elements; Engineering; Enzymes; Family; Gel; Gene Expression; Gene Targeting; Genes; Human; Individual; Inflammation; Inflammatory; JUN gene; Knowledge; Liquid substance; Literature; MAP Kinase Kinase Kinase; Maps; Mechanics; Mediating; Methodology; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Molecular Target; Mutation; N-terminal; Nitric Oxide Synthase; Numbers; Osteoarthrosis Deformans; Oxidation-Reduction; Pathogenesis; Pathway interactions; Peroxisome Proliferator-Activated Receptors; Phase; Process; Prostaglandin-Endoperoxide Synthase; Prostaglandins; Protein Isoforms; Protein Kinase C; Protein Overexpression; Proteins; RNA Interference; Rate; Regulation; Regulator Genes; Reporter; Research Personnel; Role; Series; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Small Interfering RNA; Stimulus; Technology; Testing; Tissue Engineering; Tissues; Trans-Activators; base; cDNA Arrays; cyclooxygenase 1; cyclooxygenase 2; design; novel; novel therapeutics; programs; promoter; receptor; repaired; research study; response; rho GTP-Binding Proteins; shear stress; tool; transcription factor

DESCRIPTION (provided by applicant): Overexpression of cyclooxygenase-2 (COX-2) in articular tissues is an earmark of arthritis associated with increased chondrocyte apoptosis. Although high fluid shear induces chondrocyte apoptosis, the intracellular signaling pathways regulating this process remain largely unknown. In this application, we will test the hypothesis supported by compelling data that COX-2 activity is responsible for the decreased antioxidant capacity of chondrocytes and their apoptosis in response to shear stimulation. An extensive characterization of the signaling network regulating chondrocyte apoptosis may offer novel avenues for its control. We believe that the most judicious approach is to characterize the signaling mechanisms regulating COX-2 expression, and identify downstream targets of COX-2 activity which in turn regulate the activity of phase 2 antioxidant enzymes, and key pro-/anti- apoptotic genes. As has been argued in the literature, the signaling mechanisms are species-, cell- and stimulus- specific. Thus, Aim 1a will identify the cis-elements and their cognate trans- acting factors of shear-induced COX-2 expression in human chondrocytes. Using bioinformatics tools and cDNA microarrays coupled with selective, individual gene knockdowns via dominant negative/siRNA techno- logy, we will delineate the upstream signaling molecules of COX-2 expression (Aim 1b). This methodology will also enable us to define the downstream targets of COX-2 activity as well as other intermediate gene targets responsible for the differential expression of phase 2 genes (Aim 2). Aim 3 will provide a mechanistic interpretation for the shear-induced chondrocyte apoptosis by identifying the key pro- and anti- apoptotic genes of the Bcl-2 family as well as their upstream regulatory genes. Comparative studies of the effects of fluid shear and cyclic strain on chondrocyte function will also be performed. This application integrates engineering principles with quantitative biology to reconstruct gene networks in shear-activated chondrocytes. The knowledge gained from these studies will provide a basis for the design of novel cell- based approaches for cartilage repair, and defining ideal bioreactor operating conditions for culturing artificial cartilage. Moreover, this approach will be vital for developing novel therapeutic strategies targeting molecular pathways relevant to arthritic pathogenesis and progression.

描述（由申请人提供）：关节组织中环氧合酶2（COX-2）的过表达是与软骨细胞凋亡增加有关的关节炎的指定。尽管高流体剪切会诱导软骨细胞凋亡，但调节该过程的细胞内信号通路仍在很大程度上未知。在此应用程序中，我们将通过令人信服的数据来检验COX-2活性造成软骨细胞抗氧化能力及其对剪切刺激的抗氧化能力的降低及其凋亡的原因。调节软骨细胞凋亡的信号网络的广泛表征可能为其控制提供新的途径。我们认为，最明智的方法是表征调节COX-2表达的信号传导机制，并确定COX-2活性的下游靶标，这反过来又调节了2期抗氧化剂酶的活性以及关键的促/抗/抗凋亡基因。正如文献中所论述的那样，信号传导机制是物种，细胞和刺激特异性的。因此，AIM 1a将识别人类软骨细胞中剪切诱导的COX-2表达的顺式元素及其同源型转移因子。使用生物信息学工具和cDNA微阵列以及通过主导/siRNA技术的选择性，单个基因敲低的结合，我们将描述Cox-2表达的上游信号分子（AIM 1B）。该方法还将使我们能够定义COX-2活性的下游靶标以及其他负责2阶段基因差异表达的中间基因靶标（AIM 2）。 AIM 3将通过鉴定Bcl-2家族的关键促促促凋亡基因及其上游调节基因来为剪切诱导的软骨细胞凋亡提供一种机械解释。还将对流体剪切和环状应变对软骨细胞功能的影响进行比较研究。该应用程序将工程原理与定量生物学集成在一起，以在剪切激活的软骨细胞中重建基因网络。从这些研究中获得的知识将为设计新型细胞修复方法设计提供基础，并定义理想的生物反应器工作条件以培养人造软骨。此外，这种方法对于制定针对与关节炎发病机理和进展相关的分子途径的新型治疗策略至关重要。