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

PDTC与IL-6的mRNA的总体比较鉴定了HepG 2细胞中失调基因表达的高度显著差异。使用人HepG 2肝细胞瘤细胞系是因为其分泌多种主要急性期蛋白（例如，纤维蛋白原、纤溶酶原、β 2-巨球蛋白），并将对IL-6的刺激作出反应。HepG 2细胞也被用作研究肝脏代谢、肝癌发生和药物靶向研究的合适的体外模型系统。在这里，使用基因集富集方法的参数化分析的无偏途径分析检测到雷帕霉素（mTOR）途径，氨基酸代谢和线粒体氧化功能作为PDTC和IL-6治疗之间最差异调节的途径的哺乳动物靶。最近的研究表明mTOR信号通过控制线粒体氧化功能在限制哺乳动物寿命中的作用。此外，许多人类疾病是由于通过mTOR的失调的细胞信号转导而发生的，mTOR是通过调节核糖体生物合成和蛋白质翻译来协调适当细胞生长和增殖的关键参与者。有趣的是，我们发现DDIT 4是一个早期即时基因，其转录本REDD 1在HepG 2细胞中分别被PDTC和IL-6显著上调和下调。REDD 1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激酶在Ser 371处的磷酸化，Ser 371是mTOR激酶活性的已知靶标。这些和其他结果鉴定了失调的mTOR活性作为通过控制DDIT 4（REDD 1）表达的PDTC介导的IL-6信号传导抑制的重要组分。 关于调节REDD 1表达和功能的信号通路和翻译后修饰知之甚少。我们目前正在研究磷酸化和泛素化在暴露于PDTC和/或IL-6的细胞中蛋白酶体介导的REDD 1降解中的作用。遗传、生物化学和药理学方法也被用于研究JAK/STAT途径在转录和转录后水平控制DDIT 4（REDD 1）表达的作用。综上所述，我们的研究结果表明，我们用IL-6检测到的REDD 1的降解可能通过上调mTOR通路触发癌细胞中的增殖和存活信号。REDD 1及其信号级联可能代表人类疾病的新的潜在治疗靶点。

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.