TUMOR-NECROSIS-FACTOR DECREASES THROMBIN RECEPTOR EXPRESSION IN ENDOTHELIAL-CELLS

We examined the effects of the proinflammatory cytokine, tumor necrosi s factor-alpha (TNFalpha), on the expression of proteolytically activa ted thrombin receptor (PATR) in human umbilical vein endothelial cells (HUVEC). PATR mRNA and protein levels were measured in confluent HUVE C monolayers after challenge with TNFalpha. Northern analysis indicate d that TNFalpha treatment resulted in 2- to 3-fold decrease in PATR mR NA in a time- and dose-dependent manner. PATR mRNA level returned to t he control level within 6 hr. The nuclear run-on assay indicated that the decreased mRNA signal was due to reduction in the transcription ra te. Immunoblotting experiments indicated that the decrease in expressi on of PATR protein followed in time the decrease in mRNA; the lowest l evel of protein expression was achieved at 22 hr after TNFalpha treatm ent. PATR protein returned to basal value with in 40 hr after TNFalpha challenge. To assess alterations in endothelial cell function after T NFalpha treatment, we measured thrombin-induced increase in cytosolic Ca2+ ([Ca2+](i)) and the cell shape change (measured by decrease in el ectrical impedance of endothelial cell monolayer). In HUVEC treated wi th TNFalpha (100 U/ml for 22 hr), the rise in [Ca2+](i) after thrombin challenge was similar to 2-fold less than in control thrombin-treated cells. The decrease in electrical impedance of HUVEC monolayers in re sponse to thrombin after TNFalpha treatment was also significantly red uced. However, the rise in [Ca2+](i) in response to histamine was not altered by TNFalpha pretreatment. In conclusion, TNFalpha exposure of endothelial cells decreased both mRNA and protein expression of PATR, which explain the decreased activation of thrombin generated signals a fter the TNFalpha exposure. (C) 1996 Wiley-Liss, Inc.