title: Expression of the Robo4 receptor in endothelial cells is regulated by two AP-1 protein complexes.
abstract: Roundabout4 (Robo4) is an endothelial cell-specific gene that plays an important role in endothelial cell stability. We previously identified a 3-kb Robo4 promoter and demonstrated the importance of its proximal region in regulating Robo4 gene expression. To investigate the role of the upstream promoter in Robo4 gene regulation, we searched evolutionarily conserved promoter regions by phylogenetic footprinting and identified three conserved promoter regions. The most upstream region included a conserved AP-1 binding motif at position -2875. A mutation in the AP-1 motif significantly decreased Robo4 promoter activity in a transient reporter assay. An electrophoretic mobility shift assay and a chromatin immunoprecipitation assay demonstrated binding of a c-Jun/c-Jun complex and a c-Jun/Fra-1 complex to the AP-1 motif. Knockdown experiments using siRNA revealed that both c-Jun/c-Jun and c-Jun/Fra-1 complexes regulate Robo4 gene expression, and that the c-Jun/c-Jun complex is essential for maximum promoter activation. Collectively, these results indicate that AP-1 complexes regulate Robo4 gene expression in endothelial cells.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan. Electronic address: okadabos@phs.osaka-u.ac.jp.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Okada
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Naruse
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Funahashi
authors.affiliation: Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA.
authors.forename: Erzsébet Ravasz
authors.initials: ER
authors.lastname: Regan
authors.affiliation: Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA.
authors.forename: Kazuma
authors.initials: K
authors.lastname: Yamakawa
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
authors.forename: Nobumasa
authors.initials: N
authors.lastname: Hino
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Ishimoto
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
authors.forename: Takefumi
authors.initials: T
authors.lastname: Doi
authors.affiliation: Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA.
authors.forename: William C
authors.initials: WC
authors.lastname: Aird
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D011401:Promoter Regions, Genetic; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D018808:Transcription Factor AP-1 / Q000378:metabolism / Q000502:physiology*; D000097905:Roundabout Proteins
chemical_list: D011956:Receptors, Cell Surface; D018808:Transcription Factor AP-1; C462951:ROBO4 protein, human; D000097905:Roundabout Proteins
keywords: AP-1; Endothelial cell; Fra-1; Robo4; c-Jun