TRANSCRIPTIONAL REGULATION OF THE TISSUE-TYPE PLASMINOGEN-ACTIVATOR GENE IN HUMAN ENDOTHELIAL-CELLS - IDENTIFICATION OF NUCLEAR FACTORS THAT RECOGNIZE FUNCTIONAL ELEMENTS IN THE TISSUE-TYPE PLASMINOGEN-ACTIVATOR GENE PROMOTER

The gene encoding human tissue-type plasminogen activator (t-PA) is re gulated in a cell-type-specific manner. Previous studies in non-endoth elial cells have indicated that basal and phorbol ester mediated induc tion is controlled by a cAMP response element (CRE) referred to as the tPACRE, and an activating protein 2 (AP-2)-like site. The classificat ion of the AP-2-like site was assigned on the basis of its sequence ho mology, but has been shown in some cell systems to be recognised by pr omoter-specific transcription factor-1 (Sp-1). Here, we have investiga ted the transcriptional regulation of the t-PA gene in endothelial cel ls 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 t ranscription initiation sites in these cells with the downstream site being preferred. Functional analyses of the t-PA promoter using report er-gene constructs transfected into C11STH endothelial cells demonstra te that the first 410 bp of the t-PA promoter confers an increase in r eporter-gene activity on treatment with 4 beta-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 e ndothelial 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 selecti ve recruitment of jun-D to the tPACRE, while Sp-l was identified as th e major transcription factor that recognises the AP-2-like site. Based on this data and previous reports, we have reassigned this as a Sp-l- binding site. Finally, the identification of specific endothelial-deri ved t-PACRE-binding proteins suggests an integral role for these facto rs in the regulation of t-PA gene expression in human endothelial cell s.