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

Background: Moderate alcohol consumption has been correlated to reduced coronary artery disease (CAD) risk and mortality. This alcohol effect may be mediated in part by an increased endothelial cell (EC) fibrinolysis. ECs synthesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase type plasminogen activator (u-PA), and plasminogen activator inhibitor type-1(PAI-l). In addition, they synthesize and regulate receptors for fibrinolytic proteins, namely (t-PA and plasminogen receptor) Annexin II and u-PA 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-localized 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 fibrinolytic activity, will be expected to reduce the risk for thrombosis, CAD, and myocardial infarction (MI). We have previously shown that low ethanol up-regulates t-PA and u-PA gene transcription, while it down-regulates PAI-1, hence resulting in increased (sustained, 24 hr) surface-localized EC fibrinolytic activity. The current studies were carried out to determine whether low ethanol increased u-PAR expression in cultured human umbilical cord vein ECs (HUVECs). Methods: Cultured HUVECs were preincubated (1 hr) in the absence/presence of 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 then measured after 0-24 hr. To determine whether the ethanol-induced changes in the u-PAR expression were transcriptional, transient transfection studies 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 vector]. Results: uPAR mRNA levels increased 2- to 3-fold and antigen levels (western 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/luc construct resulted in a 2- to 3-fold increase in promoter activity in ethanol-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 further identify/define the contribution and role of another fibrinolytic protein in the overall ethanol-induced increase in surface-localized EC fibrinolysis that may underlie and contribute, in part, to the cardioprotection attributed to moderate alcohol consumption.