Transcriptional and post-transcriptional regulation of VEGF by the unfolded protein response

Inadequate extracellular conditions can adversely affect the environment of the ER and impinge on the maturation of nascent proteins. The resultant accumulation of unfolded proteins activates a signal transduction pathway, known as the unfolded protein response, which serves primarily to protect the cell during stress and helps restore homeostasis to the ER. Microarray analysis of the unfolded protein response in a human medulloblastoma cell line treated with thapsigargin revealed that, in addition to known targets, a large number of proangiogenic factors were up-regulated. Real-time PCR analyses confirmed that four of these factors, VEGF, FGF2, angiogenin and IL-8, were transcriptionally up-regulated in multiple cell lines by various ER stress inducers. Our studies on VEGF regulation revealed that XBP-1(S), a UPR-inducible transcription factor, bound to two regions on the VEGF promoter, and analysis of XBP-1 null mouse embryonic fibroblasts revealed that it contributes to VEGF expression in response to ER stress. ATF4, another UPR-inducible transcription factor, also binds to the VEGF gene, although its contribution to VEGF transcription appeared to be fairly modest. We also found that VEGF mRNA stability is increased in response to UPR activation, via activation of the AMP and p38MAP kinases, demonstrating that increased mRNA levels occur at two regulatory points. In keeping with the mRNA levels, we found that VEGF protein is secreted at levels as high as or higher than that achieved in response to hypoxia. Our results indicate that the UPR plays a significant role in inducing positive regulators of angiogenesis. It also regulates VEGF expression at multiple levels and is likely to have widespread implications for promoting angiogenesis in response to normal physiological cues as well as in pathological conditions like cancer. We used microarrays to perform genome wide expression analysis in Daoy medulloblastoma cells to identify gene profiles that change in response to thapsigargin treatment which causes induction of the unfolded protein response.

细胞外微环境的不足可对内质网（Endoplasmic Reticulum, ER）的稳态造成不利影响，并干扰新生蛋白质的成熟过程。未折叠蛋白的异常蓄积会激活一条信号转导通路，即未折叠蛋白反应（unfolded protein response, UPR），该通路主要在应激状态下保护细胞，并帮助恢复内质网的稳态。对经毒胡萝卜素（thapsigargin）处理的人髓母细胞瘤细胞系开展未折叠蛋白反应的微阵列分析结果显示，除已知靶基因外，大量促血管生成因子（proangiogenic factors）的表达水平显著上调。实时定量PCR（real-time PCR）分析证实，在内质网应激诱导剂处理的多种细胞系中，血管内皮生长因子（vascular endothelial growth factor, VEGF）、成纤维细胞生长因子2（fibroblast growth factor 2, FGF2）、血管生成素（angiogenin）以及白细胞介素8（interleukin 8, IL-8）这四种因子的转录水平均出现上调。针对血管内皮生长因子调控机制的研究显示，未折叠蛋白反应诱导的转录因子X盒结合蛋白1剪接体（X-box binding protein 1 spliced, XBP-1(S)）可结合至血管内皮生长因子启动子的两个区域；而对XBP-1敲除小鼠胚胎成纤维细胞的分析表明，该因子在内质网应激诱导的血管内皮生长因子表达中发挥了重要作用。另一种未折叠蛋白反应诱导的转录因子激活转录因子4（activating transcription factor 4, ATF4）同样可结合至血管内皮生长因子基因，尽管其对血管内皮生长因子转录的调控作用相对较弱。本研究还发现，未折叠蛋白反应激活可通过激活AMP激酶与p38丝裂原活化蛋白激酶（p38MAP kinases）提升血管内皮生长因子mRNA的稳定性，这表明血管内皮生长因子的mRNA水平升高可通过两个调控环节实现。与mRNA水平的变化一致，研究人员检测到血管内皮生长因子蛋白的分泌水平至少与缺氧诱导的分泌水平相当，甚至更高。本研究结果表明，未折叠蛋白反应在诱导血管生成正向调控因子的过程中发挥了重要作用。该反应可通过多个层面调控血管内皮生长因子的表达，这对于机体在正常生理信号刺激下以及癌症等病理状态下的血管生成过程均具有广泛的潜在影响。本研究利用微阵列技术，对Daoy髓母细胞瘤细胞开展全基因组表达分析，以筛选出经可诱导未折叠蛋白反应的毒胡萝卜素处理后，表达水平发生改变的基因谱。