Oxidative activation of Src in smoke-induced epithelial mesenchymal transition

烟雾诱导的上皮间质转化中 Src 的氧化激活

基本信息

批准号：
8383384
负责人：
HENRY Jay FORMAN
金额：
$ 24.6万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8383384
关键词：
Acetylcysteine Acrolein Affect Agonist Alkylation Amino Acids Antioxidants Biochemical Cell Line Cells Cysteine Cytoskeleton Data Development Disseminated Malignant Neoplasm Disulfides Dose E-Cadherin EGF gene Enzymes Epithelial Epithelial Cells Event Exposure to Family member Fibrosis Glutathione Growth Factor Hamman-Rich syndrome Human Hydrogen Peroxide In Vitro Individual Leucine Lung Malignant Epithelial Cell Malignant neoplasm of lung Mass Spectrum Analysis Measures Mediating Mesenchymal Methods Modeling Modification Molecular Molecular Conformation Mutate Neoplasm Metastasis Non-Small-Cell Lung Carcinoma Oxidants Oxidation-Reduction Oxidative Stress Pathway interactions Phenotype Phosphorylation Physiological Plasmids Platelet-Derived Growth Factor Protein Dephosphorylation Protein Kinase Protein Tyrosine Kinase Proteins Regulation Relative (related person)Reporting Rest Signal Pathway Signal Transduction Site Smoke Sorting - Cell Movement Stimulus Testing Tetrachlorodibenzodioxin Vimentin Zinc Zinc deficiency base cancer cell cell transformation cell type cigarette smoke-induced cigarette smoking cigarette smoking in vivo inhibitor/antagonist oxidation prevent receptor receptor binding src-Family Kinases

项目摘要

DESCRIPTION (provided by applicant): We will investigate how oxidants and other electrophiles in cigarette smoke activate Src kinase, which precedes epithelial-mesenchymal transition (EMT). Our preliminary results show that cigarette smoke extract (CSE) activates Src and causes EMT and that both are inhibited by N-acetylcysteine (NAC), an antioxidant and glutathione precursor, or by PP2, a Src inhibitor in the human non-small cell lung carcinoma cell line, H358. Receptor-mediated Src activation appears to require Tyr529 dephosphorylation; however, we observed that Src activation occurs without Tyr529 dephosphorylation, which may affect duration of activity. EMT is implicated in both lung fibrosis and cancer metastasis. It can be triggered by diverse stimuli through activation of multiple intracellular signaling pathways. Cigarette smoking is implicated in idiopathic pulmonary fibrosis and lung cancer. CSE contains thousands of compounds, many of which could be responsible for inducing EMT. Thus, sorting out the mechanism for EMT initiation would seem a monumental task. Fortunately, there is at least one common factor through which most physiological agonists and the myriad components of CSE, appear to act. This is the common involvement of redox mechanisms suggested by NAC inhibition of EMT. Also, Src, which is activated by most if not all EMT inducers may be a common focal point for this redox regulation. Src activation stimulated by receptor binding of growth factors appears to require dephosphorylation of Tyr529 followed by autophosphorylation at Tyr418 that produces the active enzyme. But how CSE activates Src remains largely unresolved. Our preliminary results support a mechanism for Src activation by CSE through oxidation or alkylation without Try529 dephosphorylation. Aim 1 is to test the hypothesis that CSE and two CSE components, hydrogen peroxide (H2O2) and acrolein, initiate Src activation through oxidation or alkylation of regulatory cysteine residues. To test this, a his-tagged- Src plasmid will be constructed and expressed in H358 and HBE1 cells. After confirming Src activation, Src protein will be isolated and analyzed with mass spectrometry for cysteine oxidation and/or alkylation. It is expected that a low dose exposure to CSE that activates Src will modify some of the nine cysteine residues on Src while the others will remain in the reduced form (-SH). Aim 2 will then identify and confirm individual cysteine residue whose redox modification is critical for oxidative Src activation. Cysteine residues identified in Aim 1 will b mutated individually or in pairs to leucine to mimic alkylation and the mutated plasmid will be expressed in cells. Src activation by CSE will then be determined by measuring Src phosphorylation at Tyr418 and Src activity. Aim 3 is to determine if Src activation by CSE, H2O2 or acrolein is prolonged in comparison with its activation through the classical pathway. We will examine whether Src activation by CSE, H2O2 or acrolein lasts longer than Src activated by agonists that act through Tyr529 dephosphorylation. PUBLIC HEALTH RELEVANCE: Src is an enzyme that is critical to the transformation of normal epithelial cells into other cell types that are associated with fibrosis and metastatic cancer. Cigarette smoking can cause this transformation and our results suggest that oxidizing and alkylating compounds in smoke are the cause of Src activation and cell transformation. We will investigate how Src is activated at a molecular level and whether this kind of activation also causes prolonged Src activity.

描述（由申请人提供）：我们将研究香烟烟雾中的氧化剂和其他亲电子试剂如何激活 Src 激酶，该激酶发生在上皮间质转化 (EMT) 之前。我们的初步结果表明，香烟烟雾提取物 (CSE) 激活 Src 并引起 EMT，并且两者均受到 N-乙酰半胱氨酸 (NAC)（一种抗氧化剂和谷胱甘肽前体）或 PP2（人非小细胞肺中的 Src 抑制剂）的抑制癌细胞系，H358。受体介导的 Src 激活似乎需要 Tyr529 去磷酸化；然而，我们观察到 Src 激活发生时没有 Tyr529 去磷酸化，这可能会影响活性持续时间。 EMT 与肺纤维化和癌症转移有关。它可以通过激活多种细胞内信号通路，由多种刺激触发。吸烟与特发性肺纤维化和肺癌有关。 CSE 含有数千种化合物，其中许多可能负责诱导 EMT。因此，理清 EMT 启动机制似乎是一项艰巨的任务。幸运的是，大多数生理激动剂和 CSE 的无数成分似乎都通过至少一个共同因素发挥作用。这是 NAC 抑制 EMT 所提示的氧化还原机制的共同参与。此外，Src 被大多数（如果不是全部）EMT 诱导剂激活，可能是这种氧化还原调节的共同焦点。生长因子受体结合刺激的 Src 激活似乎需要 Tyr529 去磷酸化，然后在 Tyr418 处进行自磷酸化，从而产生活性酶。但 CSE 如何激活 Src 很大程度上仍未解决。我们的初步结果支持 CSE 通过氧化或烷基化而不进行 Try529 去磷酸化来激活 Src 的机制。目标 1 是检验以下假设：CSE 和两种 CSE 成分（过氧化氢 (H2O2) 和丙烯醛）通过调节性半胱氨酸残基的氧化或烷基化启动 Src 激活。为了测试这一点，将构建带有 his 标签的 Src 质粒并在 H358 和 HBE1 细胞中表达。确认 Src 激活后，将分离 Src 蛋白并用质谱分析半胱氨酸氧化和/或烷基化。预计低剂量接触 CSE 会激活 Src 修改 Src 上九个半胱氨酸残基中的一些，而其他残基将保持还原形式 (-SH)。然后，目标 2 将识别并确认单个半胱氨酸残基，其氧化还原修饰对于氧化 Src 激活至关重要。目标 1 中鉴定的半胱氨酸残基将单独或成对突变为亮氨酸以模拟烷基化，并且突变的质粒将在细胞中表达。然后通过测量 Tyr418 处的 Src 磷酸化和 Src 活性来确定 CSE 的 Src 激活。目标 3 是确定与通过经典途径激活相比，CSE、H2O2 或丙烯醛激活 Src 是否会延长。我们将检查 CSE、H2O2 或丙烯醛激活 Src 的持续时间是否比通过 Tyr529 去磷酸化作用的激动剂激活的 Src 持续时间更长。公共健康相关性：Src 是一种酶，对于正常上皮细胞转化为与纤维化和转移性癌症相关的其他细胞类型至关重要。吸烟会导致这种转化，我们的结果表明烟雾中的氧化和烷基化化合物是 Src 激活和细胞转化的原因。我们将研究Src是如何在分子水平上被激活的，以及这种激活是否也能在分子水平上被激活。导致 Src 活性延长。