ROS and MAPK signal cascades in corneal myofibroblast genesis

项目摘要 间质损伤后，间质内细胞(常驻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(MFS)。这种变化涉及含有应力纤维的α-SMA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会导致纤维化，即 形成致密的、散乱的、不透明的胶原质，阻碍和扭曲视力。多发性骨髓瘤的起源是 由转化生长因子β控制。碱暴露诱导小鼠眼部α-SMA的从头表达 完全的角膜混浊。一名日本研究人员证明，这些事件不是瞬时发生的 受体蛋白电压激活的通道1基因敲除小鼠(TRPV1-/-小鼠)，表明这一关键作用 经络在纤维化过程中的作用。在他们开创性的研究之后，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)在受伤的猪中形成了肌成纤维细胞 角膜也依赖于TRPV1的活性；b)表型的改变是由正反馈支持的 当激活的转化生长因子β受体诱导Smad2激活和同时产生 通过依赖于NADPH氧化酶(NOX)的过程来激活ROS；c)反过来，ROS激活TRPV1前导 到[Ca]增加)这种[Ca]升高有助于激活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-&gt；ROS-&gt；TRPV1-&gt；p- P38--转化生长因子β受体激活后不久的pSMAD2。这种循环的正反馈循环对于生成 促进成纤维细胞向肌成纤维细胞转化所必需的高水平的pSMAD积聚 转换(FMC)。我们现在寻求鉴定和表征蛋白质和转导的全部补充。 作为所述循环的基础的事件。在具体目标1中，我们使用基因编码的荧光传感器 确定ROS产生开始、pSMAD2活性和p38之间的时间关系 并确定参与其中的氮氧化物亚型(S)及其位置(S)。在具体目标2中，我们 解决从p38上游MAPK级联的细胞内激酶的参与，并测试一种新的 关于p.p38增强Smad2激活和使用磷的机制的假说- 用蛋白质组学方法确定反馈环未被发现的介体。在特定目标3中，我们测试 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 在NOX4-/-小鼠角膜纤维化中的作用。在上述研究中收集的时空信息 ROS的产生，蛋白质磷酸化的变化和其他对阐明至关重要的激活实体 肌成纤维细胞表型诱导过程中的信号转导因果序列 角膜基质细胞。

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.