title: Dependence of Proximal GC Boxes and Binding Transcription Factors in the Regulation of Basal and Valproic Acid-Induced Expression of t-PA.
abstract: Objective. Endothelial tissue-type plasminogen activator (t-PA) release is a pivotal response to protect the circulation from occluding thrombosis. We have shown that the t-PA gene is epigenetically regulated and greatly induced by the histone deacetylase (HDAC) inhibitor valproic acid (VPA). We now investigated involvement of known t-PA promoter regulatory elements and evaluated dependence of potential interacting transcription factors/cofactors. Methods. A reporter vector with an insert, separately mutated at either the t-PA promoter CRE or GC box II or GC box III elements, was transfected into HT-1080 and HUVECs and challenged with VPA. HUVECs were targeted with siRNA against histone acetyl transferases (HAT) and selected transcription factors from the Sp/KLF family. Results. An intact VPA-response was observed with CRE mutated constructs, whereas mutation of GC boxes II and III reduced the magnitude of the induction by 54 and 79% in HT-1080 and 49 and 50% in HUVECs, respectively. An attenuated induction of t-PA mRNA was observed after Sp2, Sp4, and KLF5 depletion. KLF2 and p300 (HAT) were identified as positive regulators of basal t-PA expression and Sp4 and KLF9 as repressors. Conclusion. VPA-induced t-PA expression is dependent on the proximal GC boxes in the t-PA promoter and may involve interactions with Sp2, Sp4, and KLF5.
authors.affiliation: The Wallenberg Laboratory for Cardiovascular Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, 413 45 Gothenburg, Sweden.
authors.forename: Erik
authors.identifier: 0000-0002-6326-5175
authors.initials: E
authors.lastname: Ulfhammer
authors.affiliation: The Wallenberg Laboratory for Cardiovascular Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, 413 45 Gothenburg, Sweden.
authors.forename: Pia
authors.initials: P
authors.lastname: Larsson
authors.affiliation: The Wallenberg Laboratory for Cardiovascular Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, 413 45 Gothenburg, Sweden.
authors.forename: Mia
authors.initials: M
authors.lastname: Magnusson
authors.affiliation: The Wallenberg Laboratory for Cardiovascular Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, 413 45 Gothenburg, Sweden.
authors.forename: Lena
authors.initials: L
authors.lastname: Karlsson
authors.affiliation: The Wallenberg Laboratory for Cardiovascular Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, 413 45 Gothenburg, Sweden.
authors.forename: Niklas
authors.initials: N
authors.lastname: Bergh
authors.affiliation: The Wallenberg Laboratory for Cardiovascular Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, 413 45 Gothenburg, Sweden.
authors.forename: Sverker
authors.initials: S
authors.lastname: Jern