Open AccessTranscriptional regulation of the tissue‐type plasminogen‐activator gene in human endothelial cells : identification of nuclear factors that recognise functional elements in the tissue‐type plasminogen‐activator gene promoter
Author(s) -
Costa Magdaline,
Shen Yang,
Maurer Fabienne,
Medcalf Robert L.
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2580123.x
Subject(s) - microbiology and biotechnology , biology , reporter gene , transfection , promoter , gene , activator (genetics) , transcription factor , plasminogen activator , transcriptional regulation , response element , creb , gene expression , genetics
The gene encoding human tissue‐type plasminogen activator (t‐PA) is regulated in a cell‐type‐specific manner. Previous studies in non‐endothelial cells have indicated that basal and phorbol ester mediated induction is controlled by a cAMP response element (CRE) referred to as the tPACRE, and an activating protein 2 (AP‐2)‐like site. The classification of the AP‐2‐like site was assigned on the basis of its sequence homology, but has been shown in some cell systems to be recognised by promoter‐specific transcription factor‐1 (Sp‐1). Here, we have investigated the transcriptional regulation of the t‐PA gene in endothelial cells and addressed the functional roles of the tPACRE and the Sp‐1/AP‐2‐like sites. 5′‐RACE experiments indicate that the t‐PA gene uses two transcription initiation sites in these cells with the downstream site being preferred. Functional analyses of the t‐PA promoter using reporter‐gene constructs transfected into C11STH endothelial cells demonstrate that the first 410 bp of the t‐PA promoter confers an increase in reporter‐gene activity on treatment with 4β‐phorbol 12‐myristate 13‐acetate (PMA). Mutagenesis of either the tPACRE or the Sp‐1/AP‐2 site weakens both basal and inducible expression, while disruption of both sites renders the promoter completely unresponsive. Using supershift assays, we identify the predominant tPACRE‐binding proteins in nuclear extracts prepared from both C11STH cells and primary umbilical vein endothelial cells (HUVECs) as activating transcription factor 2, CREB (cAMP‐responsive‐element‐binding protein), CREM (cAMP response element modulator) and c‐jun. Treatment of cells with PMA results in a selective recruitment of jun‐D to the tPACRE, while Sp‐1 was identified as the major transcription factor that recognises the AP‐2‐like site. Based on this data and previous reports, we have reassigned this as a Sp‐1‐binding site. Finally, the identification of specific endothelial‐derived t‐PACRE‐binding proteins suggests an integral role for these factors in the regulation of t‐PA gene expression in human endothelial cells.