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

• 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
• 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

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(申请人提供)：我们将研究香烟烟雾中的氧化剂和其他亲电体如何激活上皮-间充质转化(EMT)之前的Src激酶。我们的初步结果表明，香烟烟雾提取物(CSE)激活Src并导致EMT，这两者都被抗氧化剂和谷胱甘肽前体N-乙酰半胱氨酸(NAC)或PP2抑制，在人类非小细胞肺癌细胞系H358中。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 通过激活多个细胞内信号通路，由不同的刺激触发。吸烟与特发性肺纤维化和肺癌有关。CSE含有数千种化合物，其中许多可能是诱导EMT的原因。因此，理清EMT启动的机制似乎是一项艰巨的任务。幸运的是，至少有一个共同的因素，大多数生理激动剂和CSE的无数成分似乎都是通过这个共同因素发挥作用的。这是NAC抑制EMT所提示的氧化还原机制的共同参与。此外，被大多数EMT诱导剂激活的Src可能是这种氧化还原调节的共同焦点。受生长因子受体结合刺激的SRC激活似乎需要Tyr529的去磷酸化，然后是Tyr418的自动磷酸化，从而产生活性酶。但CSE如何激活Src在很大程度上仍未解决。我们的初步结果支持CSE通过氧化或烷基化激活Src的机制，而不是Try529去磷酸化。目的1验证CSE和两个CSE组分过氧化氢(H_2O_2)和丙烯醛通过调节半胱氨酸残基的氧化或烷基化来启动Src激活的假说。为了验证这一点，我们将构建一个His标记的-Src质粒，并在H358和HBE1细胞中表达。在确认Src激活后，将分离出Src蛋白，并进行半胱氨酸氧化和/或烷基化的质谱分析。预计低剂量的CSE暴露会激活Src 修饰Src上九个半胱氨酸残基中的一些，而其他的将保持还原形式(-SH)。然后，AIM 2将识别和确认其氧化还原修饰对氧化Src激活至关重要的单个半胱氨酸残基。在AIM 1中鉴定的半胱氨酸残基将单独或成对突变为亮氨酸，以模拟烷基化反应，突变的质粒将在细胞中表达。然后将通过测量Tyr418处的Src磷酸化和Src活性来确定CSE对SRC的激活。目的3是确定与通过经典途径激活相比，CSE、H_2O_2或丙烯醛对Src的激活是否延长。我们将检查CSE、H_2O_2或丙烯醛激活的Src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。 与公共卫生相关：SRC是一种酶，对于将正常的上皮细胞转化为与纤维化和转移性癌症相关的其他类型的细胞至关重要。吸烟可以引起这种转化，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 导致Src活性延长。