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: 10179399
• 负责人: JOSE MARIO WOLOSIN
• 金额: $ 41.1万
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10179399
• 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; Mediating; Mediator of activation protein; Mitogen-Activated Protein Kinase Kinases; 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 models; base; corneal scar; inhibitor/antagonist; knowledge base; novel; outcome prediction; p38 Mitogen Activated Protein Kinase; porcine model; 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.

项目概要 基质伤口后，基质内细胞（驻留细胞和骨髓来源的细胞）发生变化或“转化” 转化为肌成纤维细胞（MF）。这种变化涉及含有应力纤维的α-SMA的合成和 细胞外基质成分。肌成纤维细胞表型的持续存在会导致纤维化，即 形成致密、无组织的不透明胶原材料，阻碍和扭曲视力。 MF 的起源是 由TGFβ控制。小鼠眼睛暴露于碱会诱导 α-SMA 的从头表达 角膜完全混浊。日本研究人员证明这些事件不会在瞬态中发生 受体蛋白电压激活通道一敲除小鼠（TRPV1-/- 小鼠），表明这一作用至关重要 纤维化过程中的通道。继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证明，a) 受伤猪中肌成纤维细胞的形成 角膜也依赖于 TRPV1 活性； b) 表型变化是由正反馈支撑的 当激活的 TGFβ 受体诱导 SMAD2 激活并同时生成时开始的过程 通过 NADPH 氧化酶 (NOX) 依赖的过程产生 ROS； c) 反过来，ROS 激活 TRPV1 引导 [Ca] 增加）这种 [Ca] 增加有助于激活 p38 (p-p38)，并且 d) 反过来，p-p38 直接 或间接增加 pSMAD2 水平，从而建立 .pSMAD2->ROS->TRPV1->p- 的循环周期 TGFβR 激活后不久，p38->pSMAD2。这种反复出现的正反馈循环对于产生 高水平 pSMAD 的积累是驱动最大成纤维细胞转变为肌成纤维细胞所必需的 转换（FMC）。我们现在寻求鉴定和表征完整的蛋白质和转导 支持所描述的周期的事件。在使用基因编码荧光传感器的具体目标 1 中，我们 确定 ROS 生成开始、pSMAD2 活性和 p38 之间的时间关系 磷酸化并识别所涉及的 NOX 亚型及其位置。在具体目标 2 中，我们 解决 p38 上游 MAPK 级联的细胞内激酶的参与并测试一种新的 涉及 p.p38 增强 SMAD2 激活的机制的假设并使用磷酸盐 蛋白质组学方法来识别反馈环路中未被发现的介质。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类角膜纤维化有关并测试 NOX4 的影响 NOX4-/- 小鼠的角膜纤维化。上述研究中收集的空间和时间信息 ROS 产生、蛋白质磷酸化变化和其他激活实体对于阐明至关重要 信号转导序列在诱导肌成纤维细胞表型中的因果关系 角膜角质细胞。