The role of p62 in the mechanism of Cr(VI) carcinogenesis

尽管活性氧在Cr（VI）诱导的恶性细胞中起重要作用， 转化，其潜在机制仍有待研究。最近的研究表明，ROS 在诱导自噬和随后上调p62中很重要。我们的初步结果表明 人支气管上皮（BEAS-2B）细胞慢性暴露于Cr（VI）上调p62， 动物暴露于Cr（VI）颗粒上调了肺中的这种蛋白质。BEAS中p62的强制表达- 2B细胞引起这些细胞的恶性转化，并在异种移植模型中产生肿瘤。照经上所 在BEAS-2B细胞中，ROS可能上调p62及其下游，导致细胞转化。我们 初步研究表明，在Cr（VI）转化的细胞中，p62被上调。在六个主要 p62的结构域/区域，我们将研究（a）TRAF 6结合结构域，其结合TRAF 6并导致其 磷酸化，导致NF-κB活化，和（B）Keap相互作用区，其结合Keap 1 (Nrf2抑制剂）并引起Nrf 2的组成性激活。核心假设是在正常细胞中Cr（VI） 激活NADPH氧化酶，产生ROS，然后上调p62，导致恶性细胞转化 在Cr（VI）转化细胞中，p62通过TRAF 6结合结构域激活NF-κB， 通过Keap相互作用区域的组成性Nrf 2激活，随后上调其下游抗- 炎症，抗氧化剂和抗凋亡蛋白，导致增加的生存和肿瘤发生 这些转化的细胞目的1将检验在正常细胞中Cr（VI）激活NADPH氧化酶的假设， 产生ROS并上调p62，导致恶性细胞转化。我们会进行以下工作 问题研究(a)由Cr（VI）产生的ROS上调p62。我们将抑制ROS以显示p62的减少。(b)我们 将抑制ROS或p62以证明对Cr（VI）诱导的恶性细胞转化的抑制。(c)我们将 使用全转录组（RNA Seq）分析来鉴定负责Cr（VI）诱导的p62下游基因。 恶性转化目的2将检验p62通过TRAF 6 - 1上调NF-κB的假设。 p62通过其Keap相互作用区引起组成性Nrf 2激活，导致 有利于Cr（VI）转化细胞存活的微环境的产生以及随后的 肿瘤发生我们将展示（a）TRAF 6结合结构域在NF-κB活化中的作用;（B）Keap-κ B在NF-κB活化中的作用。 在组成性Nrf 2激活和上调其下游炎症，抗氧化， 和抗凋亡蛋白;和（c）p62及其下游NF-κB和Nrf 2途径在 Cr（VI）转化细胞的肿瘤发生。目的3探讨p62在Cr（VI）作用机制中的作用 使用p62野生型和敲除小鼠的致癌作用。我们将测量p62及其下游蛋白， 慢性暴露于Cr（VI）的动物和职业暴露于Cr（VI）的工人的肺部问题， 加强细胞和动物Cr（VI）暴露研究。

Although reactive oxygen species (ROS) are considered important in Cr(VI)-induced malignant cell transformation, the underlying mechanism remains to be investigated. Recent studies show that ROS are important in the induction of autophagy and subsequently upregulation of p62. Our preliminary results show that chronic exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) upregulated p62 and that chronic exposure of animals to Cr(VI) particles upregulated this protein in the lung. Forced expression of p62 in BEAS- 2B cells caused malignant transformation of these cells and generated tumor in xenograft model. Thus, it is likely that in BEAS-2B cells ROS upregulate p62 and its downstream, leading to cell transformation. Our preliminary studies show that in Cr(VI)-transformed cells p62 was upregulated. Among six main domains/regions of p62, we will study (a) the TRAF6-binding domain, which binds to TRAF6 and causes its phosphorylation, leading to activation of NF-κB, and (b) the Keap-interacting region, which binds to Keap1 (Nrf2 inhibitor) and causes constitutive activation of Nrf2. The central hypothesis is that in normal cells Cr(VI) activates NADPH oxidase, generates ROS, and then upregulates p62, leading to malignant cell transformation and that in Cr(VI)-transformed cells p62 activates NF-κB through TRAF6-binding domain and causes constitutive Nrf2 activation through Keap-interacting region and subsequently upregulates its downstream anti- inflammatory, antioxidant, and anti-apoptotic proteins, resulting in increased survival and tumorigenesis of these transformed cells. Aim 1 will test the hypothesis that in normal cells Cr(VI) activates NADPH oxidase, generates ROS, and upregulates p62, leading to malignant cell transformation. We will carry out the following studies. (a) ROS generated by Cr(VI) upregulate p62. We will inhibit ROS to show the decrease of p62. (b) We will inhibit ROS or p62 to demonstrate the inhibition of Cr(VI)-induced malignant cell transformation. (c) We will use whole transcriptome (RNA Seq) analysis to identify p62 downstream genes responsible for Cr(VI)-induced malignantly transformation. Aim 2 will test the hypothesis that p62 upregulates NF-κB through its TRAF6- binding domain and that p62 causes constitutive Nrf2 activation through its Keap-interacting region, resulting in the generation of microenvironment favorable for survival of Cr(VI)-transformed cells and subsequent tumorigenesis. We will show (a) the role of TRAF6-binding domain in NF-κB activation; (b) the role of Keap- interacting region in constitutive Nrf2 activation and upregulation of its downstream inflammatory, antioxidant, and anti-apoptotic proteins; and (c) the role of p62 and its downstream NF-κB and Nrf2 pathways in tumorigenesis of Cr(VI)-transformed cells. Aim 3 will investigate the role of p62 in the mechanism of Cr(VI) carcinogenesis using p62 wildtype and knockout mice. We will measure p62 and its downstream proteins in lung issues of animals chronically exposed to Cr(VI) and of workers occupationally exposed to Cr(VI) to strengthen the cellular and animal Cr(VI) exposure studies.