Transcriptional 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.
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 4beta-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.
Costa
M
M
Monash University, Department of Medicine, Box Hill Hospital, Victoria, Australia.
Shen
Y
Y
Maurer
F
F
Medcalf
R L
RL
D001483:Base Sequence; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017931:DNA Primers; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D010959:Tissue Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate