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：
8538386
负责人：
HENRY Jay FORMAN
金额：
$ 20.1万
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 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8538386
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.

描述（由申请人提供）：我们将研究香烟中的氧化剂和其他电力烟雾激活SRC激酶，该激酶先于上皮 - 间质转变（EMT）。我们的初步结果表明，香烟烟雾提取物（CSE）激活SRC并引起EMT，并且两者都受到N-乙酰半胱氨酸（NAC）的抑制（NAC），一种抗氧化剂和谷胱甘肽前体，或PP2，或PP2（人类非小细胞肺癌细胞脑癌细胞中的SRC抑制剂），SRC抑制剂。受体介导的SRC激活似乎需要Tyr529去磷酸化。但是，我们观察到SRC激活发生在没有Tyr529的去磷酸化的情况下，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，可以通过多种刺激触发。吸烟与特发性肺纤维化和肺癌有关。 CSE包含数千种化合物，其中许多化合物可能导致诱导EMT。因此，整理EMT启动的机制似乎是一项艰巨的任务。幸运的是，至少有一个共同因素，大多数生理激动剂和CSE的无数组成部分似乎都在作用。这是NAC抑制EMT建议的氧化还原机制的普遍参与。同样，大多数（如果不是所有的EMT诱导剂）激活的SRC可能是该氧化还原调控的常见焦点。生长因子的受体结合刺激的SRC激活似乎需要在产生活性酶的Tyr418上的Tyr529，然后在Tyr418处进行自磷酸化。但是，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通过氧化或烷基化而无需TRY529去磷酸化而通过氧化或烷基化激活的机制。目的1是测试以下假设：CSE和两个CSE成分（H2O2）和丙烯醛蛋白通过调节性半胱氨酸残基的氧化或烷基化来启动SRC激活。为了测试这一点，将在H358和HBE1细胞中构建并表达His标记的SRC质粒。确认SRC激活后，将分离SRC蛋白，并用质谱法分析半胱氨酸氧化和/或烷基化。预计激活SRC的低剂量暴露于CSE会修改SRC上的九种半胱氨酸残基中的一些，而其他人将保持降低形式（-sh）。然后，AIM 2将识别并确认单个半胱氨酸残基的氧化还原修饰对于氧化SRC激活至关重要。在AIM 1中鉴定出的半胱氨酸残基将单独或成对与亮氨酸成对与模拟烷基化相结合，并且突变的质粒将在细胞中表达。然后，通过测量Tyr418和SRC活性下的SRC磷酸化来确定CSE的SRC激活。 AIM 3是确定与通过经典途径相比，CSE，H2O2或丙烯醛的SRC激活是否延长。我们将检查CSE，H2O2或丙烯醛的SRC激活是否比通过Tyr529脱磷酸化的激动剂激活的SRC持续时间更长。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Resveratrol protects SR-B1 levels in keratinocytes exposed to cigarette smoke.

白藜芦醇可保护暴露于香烟烟雾的角质形成细胞中的 SR-B1 水平。

DOI：
10.1016/j.freeradbiomed.2014.01.007
发表时间：
2014
期刊：
Free radical biology & medicine
影响因子：
7.4
作者：
Sticozzi,C;Belmonte,G;Cervellati,F;Muresan,XM;Pessina,F;Lim,Y;Forman,HJ;Valacchi,G
通讯作者：
Valacchi,G

Alteration of serum lipid profile, SRB1 loss, and impaired Nrf2 activation in CDKL5 disorder.

DOI：
10.1016/j.freeradbiomed.2015.05.010
发表时间：
2015-09
期刊：
Free radical biology & medicine
影响因子：
7.4
作者：
Pecorelli A;Belmonte G;Meloni I;Cervellati F;Gardi C;Sticozzi C;De Felice C;Signorini C;Cortelazzo A;Leoncini S;Ciccoli L;Renieri A;Jay Forman H;Hayek J;Valacchi G
通讯作者：
Valacchi G

4-Hydroxynonenal activates Src through a non-canonical pathway that involves EGFR/PTP1B.