Identification of DEAD-box RNA helicase 6 (DDX6) as a cellular modulator of vascular endothelial growth factor expression under hypoxia
| Autor: | Jürgen Bernhagen, Henning Urlaub, Sebastian de Vries, Antje Ostareck-Lederer, Isabel S. Naarmann-de Vries, Dirk H. Ostareck, Hongqi Lue |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Vascular Endothelial Growth Factor A
Cytoplasm Angiogenesis Biology Biochemistry Gene Expression Regulation Enzymologic Mass Spectrometry DEAD-box RNA Helicases 03 medical and health sciences chemistry.chemical_compound Proto-Oncogene Proteins Translational regulation Human Umbilical Vein Endothelial Cells Humans Micrococcal Nuclease RNA Messenger RNA Processing Post-Transcriptional Hypoxia Molecular Biology Post-transcriptional regulation 3' Untranslated Regions In Situ Hybridization Fluorescence 030304 developmental biology 0303 health sciences Gene knockdown Neovascularization Pathologic 030302 biochemistry & molecular biology Translation (biology) Cell Biology Molecular biology Recombinant Proteins Cell biology Vascular endothelial growth factor Gene Expression Regulation Neoplastic Vascular endothelial growth factor A Internal ribosome entry site chemistry MCF-7 Cells RNA RNA Interference 5' Untranslated Regions |
| Zdroj: | Journal of Biological Chemistry |
| Popis: | Vascular endothelial growth factor A (VEGF) is a crucial proangiogenic factor, which regulates blood vessel supply under physiologic and pathologic conditions. The VEGF mRNA 5′-untranslated region (5′-UTR) bears internal ribosome entry sites (IRES), which confer sustained VEGF mRNA translation under hypoxia when 5′-cap-dependent mRNA translation is inhibited. VEGF IRES-mediated initiation of translation requires the modulated interaction of trans-acting factors. To identify trans-acting factors that control VEGF mRNA translation under hypoxic conditions we established an in vitro translation system based on human adenocarcinoma cells (MCF-7). Cytoplasmic extracts of MCF-7 cells grown under hypoxia (1% oxygen) recapitulate VEGF IRES-mediated reporter mRNA translation. Employing the VEGF mRNA 5′-UTR and 3′-UTR in an RNA affinity approach we isolated interacting proteins from translational active MCF-7 extract prepared from cells grown under normoxia or hypoxia. Interestingly, mass spectrometry analysis identified the DEAD-box RNA helicase 6 (DDX6) that interacts with the VEGF mRNA 5′-UTR. Recombinant DDX6 inhibits VEGF IRES-mediated translation in normoxic MCF-7 extract. Under hypoxia the level of DDX6 declines, and its interaction with VEGF mRNA is diminished in vivo. Depletion of DDX6 by RNAi further promotes VEGF expression in MCF-7 cells. Increased secretion of VEGF from DDX6 knockdown cells positively affects vascular tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Our results indicate that the decrease of DDX6 under hypoxia contributes to the activation of VEGF expression and promotes its proangiogenic function. Background: We explore post-transcriptional control of VEGF expression. Results: DDX6 specifically binds to the VEGF mRNA 5′-UTR and regulates VEGF mRNA translation in vitro and in vivo. Decline of DDX6 under hypoxia leads to enhanced VEGF secretion, which increases angiogenesis in vitro. Conclusion: In MCF-7 breast cancer cells DDX6 modulates VEGF translation in response to oxygen supply. Significance: Hypoxia-induced DDX6 reduction positively affects proangiogenic VEGF expression. |
| Databáze: | OpenAIRE |
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