ROS and MAPK signal cascades in corneal myofibroblast genesis

角膜肌成纤维细胞发生中的ROS和MAPK信号级联

• 批准号: 9788093
• 负责人: JOSE MARIO WOLOSIN
• 金额: $ 42.38万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9788093
• 关键词: Ablation; Address; Alkalies; Bone Marrow; Calcium; Cell Culture Techniques; Cells; Chemicals; Collagen; Complement; Cornea; Distant; Enzymes; Event; Extracellular Matrix; Eye; Family suidae; Feedback; Fibroblasts; Fibrosis; Gel; Generations; Human; Hydrogen Peroxide; Injury; Japanese Population; Knockout Mice; Laboratories; Location; MADH2 gene; MAP Kinase Gene; MAPK14 gene; Mediating; Mediator of activation protein; Mitogen-Activated Protein Kinase Kinases; Modeling; Monocular Blindness; Mus; Myofibroblast; NADPH Oxidase; Organ Culture Techniques; Participant; Pathway interactions; Peptide Signal Sequences; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Process; Production; Proteins; Proteomics; Publishing; Reactive Oxygen Species; Recurrence; Reporting; Research; Research Personnel; Role; Signal Transduction; Smooth Muscle; Stress Fibers; Superoxides; TRPV1 gene; Testing; Time; Transforming Growth Factor beta; Vision; Wound Healing; base; corneal scar; inhibitor/antagonist; knowledge base; novel; outcome prediction; prevent; receptor; response; sensor; transmission process; voltage; wound

Project Summary Following stromal wounds, intra-stromal cells (resident and bone marrow-derived) change or `transform' into myofibroblasts (MFs). This change involves the synthesis α-SMA containing stress fibers and secretion of extracellular matrix components. Persistence of the myofibroblast phenotype brings about fibrosis, i.e., the formation of dense, disorganized opaque collagenous material that blocks and distorts vision. MF genesis is controlled by TGFβ. Exposure of the mouse eye to alkali induces the de novo expression of α-SMA and complete corneal opacification. Japanese researcher demonstrated that these events do not occur in Transient Receptor Protein Voltage activated channel one knockout mice (TRPV1-/- mice), indicating a critical role of this channel in the fibrotic process. Following their groundbreaking studies, a collaborative study in corneal fibroblasts between three laboratories demonstrated that, a) myofibroblast formation in the wounded pig cornea is also dependent on TRPV1 activity; b) the phenotype change is underpinned by a positive feedback process that starts when the activated TGFβ receptor induces SMAD2 activation and the concurrent generation of ROS through a process that is NADPH oxidase (NOX)-dependent; c) in turn, ROS activates TRPV1 leading to a [Ca] increased) this [Ca] rise is instrumental in the activation of p38 (p-p38), and d) in turn, p-p38 directly or indirectly increases pSMAD2 levels establishing thereby recurrent cycles of .pSMAD2->ROS->TRPV1->p- p38->pSMAD2 soon after TGFβR activation. This recurring positive feedback loop is essential to generate the accumulation of the high levels of pSMAD that are necessary to drive maximal fibroblast to myofibroblast conversion (FMC). We seek now to identify and characterize the full complement of proteins and transduction events that underpin the described cycle. In Specific Aim 1 using genetically encoded fluorescent sensors we determine the temporal relationships between the start of ROS generation, pSMAD2 activity and p38 phosphorylation and identify the NOX subtype(s) involved and it (their) location(s). In Specific Aim 2, we address the involvement of intracellular kinases of the MAPK cascades upstream from p38 and test a novel hypothesis for the mechanisms involved in the p.p38 enhancements of SMAD2 activation and use phosphor- proteomic approaches to identify undiscovered mediators of the feedback loop. In Specific Aim 3, we test the hypothesis that the cell culture results are accurate predictors of outcome in an organ culture pig model that appears to be relevant to physical and chemically induced human corneal fibrosis and test the impact of NOX4 in corneal fibrosis in NOX4-/- mice. The spatial and temporal information gathered in the studies above on ROS production, protein phosphorylation changes and other activated entities that will be critical to elucidate the sequence of signal transduction cause and effect in the induction of the myofibroblast phenotype from corneal keratocytes.

项目摘要 基质创伤后，基质内细胞（固有细胞和骨髓来源细胞）发生变化或“转化” 转化成肌成纤维细胞（MF）。这种变化涉及含α-SMA的应力纤维的合成和分泌， 细胞外基质成分。肌成纤维细胞表型的持续存在导致纤维化，即，的 形成致密、无组织的不透明胶原物质，阻碍和扭曲视力。MF起源是 由TGFβ控制。将小鼠眼暴露于碱诱导α-SMA的重新表达， 角膜完全混浊。日本研究人员证明，这些事件不会发生在瞬态 受体蛋白电压激活通道1基因敲除小鼠（TRPV 1-/-小鼠），表明这一关键作用。 在纤维化过程中的通道。在他们的开创性研究之后，一项关于角膜的合作研究 三个实验室之间的成纤维细胞表明，a）受伤猪中的肌成纤维细胞形成 角膜也依赖于TRPV 1活性; B）表型变化由正反馈支持 当活化的TGFβ受体诱导SMAD 2活化时开始的过程， ROS通过NADPH氧化酶（NOX）依赖性过程; c）反过来，ROS激活TRPV 1， [Ca]升高）这种[Ca]升高有助于p38（p-p38）的激活，以及d）反过来，p-p38直接 或间接地增加pSMAD 2水平，从而建立pSMAD 2->ROS-> TRPV 1->p- TGFβR活化后p38 → pSMAD 2。这种反复出现的正反馈循环对于产生 高水平pSMAD的积累，其是驱动最大成纤维细胞到肌成纤维细胞所必需的 转换（FMC）。我们现在寻求确定和表征蛋白质和转导的完整补充 这些事件支撑了所描述的周期。在特定目标1中，使用基因编码的荧光传感器， 确定ROS产生开始、pSMAD 2活性和p38之间的时间关系 本发明的目的在于检测NOX的磷酸化并鉴定所涉及的NOX亚型及其位置。在Aim Specific 2中，我们 解决了MAPK级联上游p38的细胞内激酶的参与，并测试了一种新的 p.p38增强SMAD 2激活的机制的假设，并使用磷- 蛋白质组学的方法来确定未发现的介质的反馈回路。在具体目标3中，我们测试了 假设细胞培养结果是器官培养猪模型结果的准确预测因子， 似乎与物理和化学诱导的人角膜纤维化有关，并测试了NOX 4 NOX 4-/-小鼠的角膜纤维化。在上述研究中收集的空间和时间信息 活性氧的产生、蛋白质磷酸化的变化和其他活化的实体，这对阐明 信号转导的顺序在诱导肌成纤维细胞表型中的因果关系， 角膜基质细胞