Ethanol-induced up-regulation of the urokinase receptor in cultured human endothelial cells

Background: Moderate alcohol consumption has been correlated to reduced cor onary artery disease (CAD) risk and mortality. This alcohol effect may be m ediated in part by an increased endothelial cell (EC) fibrinolysis. ECs syn thesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokina se type plasminogen activator (u-PA), and plasminogen activator inhibitor t ype-1(PAI-l). In addition, they synthesize and regulate receptors for fibri nolytic proteins, namely (t-PA and plasminogen receptor) Annexin II and u-P A receptor (u-PAR). These receptors play an important role in the regulated expression of receptor-bound plasminogen activator conversion of receptor- bound plasminogen to receptor-bound plasmin on the EC surface (surface-loca lized fibrinolytic activity). Therefore, systemic factors, such as ethanol, that affect the level, or activity or interaction of one or more of these components, resulting in the increased expression of surface-localized EC f ibrinolytic activity, will be expected to reduce the risk for thrombosis, C AD, and myocardial infarction (MI). We have previously shown that low ethan ol up-regulates t-PA and u-PA gene transcription, while it down-regulates P AI-1, hence resulting in increased (sustained, 24 hr) surface-localized EC fibrinolytic activity. The current studies were carried out to determine wh ether low ethanol increased u-PAR expression in cultured human umbilical co rd vein ECs (HUVECs). Methods: Cultured HUVECs were preincubated (1 hr) in the absence/presence o f ethanol (0.025- 0.2%, v/v); u-PAR mRNA (RT-PCR), antigen (western blot), and activity (I-125-u-PA ligand binding/Scatchard analysis) levels were the n measured after 0-24 hr. To determine whether the ethanol-induced changes in the u-PAR expression were transcriptional, transient transfection studie s were carried out using a u-PAR/ luciferase promoter construct (pu-PAR120/ luc [1.2-kb u-PAR promoter fragment ligated to a promoterless luciferase ve ctor]. Results: uPAR mRNA levels increased 2- to 3-fold and antigen levels (wester n blot) increased 2- to 4-fold while u-PA binding activity increased 36% (1 .25 vs. 1.7 x 10(5) sites/cell, B-max) without significantly affecting the K-d (1-2 nM). Transient transfection of cultured HUVECs with a pu-PAR120/lu c construct resulted in a 2- to 3-fold increase in promoter activity in eth anol-induced cultures, compared with controls. Conclusion: These combined results demonstrate that low ethanol (less than or equal to0.1%, v/v) induces the upregulation of u-PAR gene transcription, resulting in increased u-PAR ligand binding activity. These results also f urther identify/define the contribution and role of another fibrinolytic pr otein in the overall ethanol-induced increase in surface-localized EC fibri nolysis that may underlie and contribute, in part, to the cardioprotection attributed to moderate alcohol consumption.