title: Identification of DEAD-box RNA helicase 6 (DDX6) as a cellular modulator of vascular endothelial growth factor expression under hypoxia.
abstract: 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.
authors.affiliation: Experimental Research Unit, Department of Intensive Care and Intermediate Care, University Hospital, RWTH Aachen University, 52074 Aachen, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: de Vries
authors.forename: Isabel S
authors.initials: IS
authors.lastname: Naarmann-de Vries
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Hongqi
authors.initials: H
authors.lastname: Lue
authors.forename: Jürgen
authors.initials: J
authors.lastname: Bernhagen
authors.forename: Dirk H
authors.initials: DH
authors.lastname: Ostareck
authors.forename: Antje
authors.initials: A
authors.lastname: Ostareck-Lederer
mesh_terms: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D003593:Cytoplasm / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D000860:Hypoxia; D017404:In Situ Hybridization, Fluorescence; D061986:MCF-7 Cells; D013058:Mass Spectrometry / Q000379:methods; D008836:Micrococcal Nuclease / Q000378:metabolism; D009389:Neovascularization, Pathologic; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000502:physiology*; D034622:RNA Interference; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis*
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D008836:Micrococcal Nuclease; C094683:DDX6 protein, human; D053487:DEAD-box RNA Helicases