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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Pereira ER

PROVIDER: S-ECPF-GEOD-21979 | biostudies-other | 2010

REPOSITORIES: biostudies-other

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Project description: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.

Project description:The Unfolded Protein Response (UPR) is a cascade of intracellular stress signaling from the endoplasmic reticulum (ER) that protect the cells from the stress caused by accumulation of unfolded or misfolded proteins in the ER. Activating transcription factor 6 (ATF6) is one of primary UPR transducers that remodels the stressed cells through transcriptional regulation. Although the activation mechanism and biological roles of ATF6 have been well studied, the understanding of the negative or feedback regulation of ATF6 remains elusive. In this report, we showed that ATF6 protein can be modified by small ubiquitin-like modification (SUMOylation) and that the transcriptional activity of ATF6 is negatively regulated by SUMOylation. We identified that SUMOylation of ATF6 is significantly increased in the cells expressing misfolded cystic fibrosis transmembrane conductance regulator (CFTR) encoded by the mutant human CFTR gene (dF508CFTR). Further analyses revealed two highly conserved SUMOylation motifs within the trans-activation domain of ATF6 protein of human, mouse, or rat specie. The human ATF6 protein can be SUMOylated mediated through the small ubiquitin-like modifier protein 1 (SUMO-1) and E3 SUMO-protein ligase 1 (PIAS1) at the conserved sumoylation residue Lys149 that is located at the N-terminal of the activated form of ATF6 protein. Bimolecular fluorescence complementation (BiFC) analysis confirmed that the activated ATF6 protein can be SUMOylated and that the ATF6 sumoylation occurs in the nuclei. Moreover, trans-activation reporter analysis demonstrated that SUMOylation of the ATF6 protein at the conserved residue Lys149 represses the transcriptional activity of ATF6. In summary, our study revealed a negative regulation of the UPR transducer ATF6 through post-translational SUMOylation. The information from this study will not only increase our understanding of the fine-tuning regulation of the UPR signaling but will also be informative to the modulation of the UPR for therapeutic benefits.

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

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