Inhibition of IL-6 signaling by a mechanism involving mTOR inactivation

通过 mTOR 失活机制抑制 IL-6 信号传导

• 批准号: 8148336
• 负责人: MICHEL BERNIER
• 金额: $ 39.28万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148336
• 关键词: 70-kDa Ribosomal Protein S6 Kinases; Acute-Phase Proteins; Amino Acid Metabolism Pathway; Biochemical; Biological; Chronic; Disease; Fibrinogen; Gene Expression; Genes; Growth; Hepatocarcinogenesis; Human; Immediate-Early Genes; Immunoblot Analysis; Interleukin-6; Pathway interactions; Pharmacotherapy; STAT3 gene; Signal Transduction; Transcript; Ubiquitination; base; human FRAP1 protein; in vitro Model; liver metabolism; multicatalytic endopeptidase complex; pyrrolidine dithiocarbamate

A global comparison of mRNA from PDTC versus IL-6 identified a highly significant difference of dysregulated gene expression in HepG2 cells. The human HepG2 hepatoma cell line was used because it secretes a variety of major acute phase proteins (e.g., fibrinogen, plasminogen, α2-macroglobulin) and will respond to stimulation with IL-6. HepG2 cells are also used as a suitable in vitro model system for studies of liver metabolism, hepatocarcinogenesis, and for drug targeting studies. Here, an unbiased pathway analysis using the Parameterized Analysis of Gene Set Enrichment method detected the mammalian target of rapamycin (mTOR) pathway, amino acid metabolism and mitochondrial oxidative function as the most differentially regulated pathways between PDTC and IL-6 treatments. Recent studies have indicated the role of mTOR signaling in limiting mammalian lifespan through the control of mitochondrial oxidative function. Moreover, many human disorders occur as a result of dysregulated cellular signal transduction through mTOR, the key player that coordinates proper cell growth and proliferation by regulating ribosomal biogenesis and protein translation. Interestingly, we identified DDIT4 as an early-immediate gene whose transcript, termed REDD1, was drastically up- and down-regulated by PDTC and IL-6 in HepG2 cells, respectively. The REDD1 tumor suppressor protein tightly controls mTOR. To assess the relevance of these observations, we performed immunoblot analysis and found that PDTC inhibits whereas IL-6 augments p70 S6 kinase phosphorylation at Ser 371, a known target of mTOR kinase activity. These and other results identify dysregulated mTOR activity as an important component of PDTC-mediated inhibition of IL-6 signaling through the control of DDIT4 (REDD1) expression. Little is known about the signaling pathways and post-translational modifications that regulate REDD1 expression and function. We are currently investigating the role of phosphorylation and ubiquitination in proteasome-mediated degradation of REDD1 in cells exposed to PDTC and/or IL-6. Genetic, biochemical and pharmacological approaches are also being used to study the effects of the JAK/STAT pathway in controlling DDIT4 (REDD1) expression at transcriptional and post-transcriptional levels. Taken together, our findings thus suggest that the degradation of REDD1 we detect with IL-6 may trigger proliferative and survival signals in cancer cells through up-regulation of the mTOR pathway. REDD1 and its signaling cascade may represent novel potential therapeutic targets for human diseases.

对来自PDTC和IL-6的mRNA的全球比较发现，HepG2细胞中基因表达异常存在高度显著差异。之所以使用人HepG2肝癌细胞系，是因为它分泌多种主要的急性期蛋白（如纤维蛋白原、纤溶酶原、2-巨球蛋白），并对IL-6的刺激有反应。HepG2细胞也被用作肝脏代谢、肝癌发生和药物靶向研究的合适体外模型系统。本研究采用参数化基因集富集方法进行无偏途径分析，发现哺乳动物雷帕霉素靶蛋白（mTOR）途径、氨基酸代谢和线粒体氧化功能是PDTC和IL-6处理之间差异最大的调控途径。最近的研究表明，mTOR信号通过控制线粒体氧化功能来限制哺乳动物的寿命。此外，许多人类疾病的发生是由于通过mTOR的细胞信号转导失调的结果，mTOR是通过调节核糖体生物发生和蛋白质翻译来协调细胞正常生长和增殖的关键角色。有趣的是，我们发现DDIT4是一个早期直接基因，其转录物被称为REDD1，在HepG2细胞中分别被PDTC和IL-6大幅上调和下调。REDD1肿瘤抑制蛋白严格控制mTOR。为了评估这些观察结果的相关性，我们进行了免疫印迹分析，发现PDTC抑制而IL-6增强p70 S6激酶Ser 371位点的磷酸化，Ser 371是mTOR激酶活性的已知靶点。这些结果和其他结果表明，mTOR活性失调是pdtc通过控制DDIT4 （REDD1）表达介导的IL-6信号抑制的重要组成部分。