Diversity and similarity in signaling events leading to rapid Cox-2 induction by tumor necrosis factor-α and phorbol ester in human endothelial cells

Objective: This study examines whether cyclooxygenase 2 (Cox-2) synthesis in human endothelial cells involves different signaling pathways when induced by the proinflammatory cytokine tumor necrosis factor-alpha (TNFalpha) or by the tumor and angiogenic promoter phorbol ester (PMA). Moreover, the hypothesis that reactive oxygen species (ROS) and an altered redox status within the cell are fundamental steps for Cox-2 synthesis is verified. Methods: Human endothelial cells isolated from umbilical vein (HUVEC) were exposed to PMA and TNFa and Cox-2 protein and mRNA levels were evaluated by Western blot and Real-Time Quantitative Reverse Transcription-PCR analysis. Prostaglandin E-2 (PGE(2)) and 6-keto prostaglandin F-1alpha (6-keto-PGF(1alpha)) levels were measured in cell medium as an index of Cox-2 activity. Intracellular ROS formation was detected by flow cytometry in HUVEC loaded with the oxidant-sensitive 2',7'-dichlorofluorescein diacetate (DCFH-DA) and by nitroblue tetrazolium (NBT) reduction. Reduced and oxidized glutathione (GSH and GSSG) were measured by HPLC. Results: Data show that TNFa and PMA signal for early Cox-2 induction through distinct pathways. PMA-induced Cox-2 expression involves a small GTPase-dependent pathway acting via tyrosine kinase, activation of protein kinase C (PKC) and of the mitogen-activated protein kinase (MAPK) ERK1/2. Conversely, MAPK p38 is critical for Cox-2 induction by TNFa. Of interest, intracellular ROS generation and consequent GSH/GSSG ratio reduction represents a common step through which PMA and TNFalpha signal for early Cox-2 induction. In addition, we provide evidence that phosphatidylinositol 3 (P13)-kinase activation plays a regulatory role for Cox-2 synthesis in HUVEC. Conclusion: Cox-2 represents a critical link among vascular homeostasis, inflammatory response, angiogenesis and tumor growth. The finding that two independent pathways and an overlapping upstream event signal for Cox-2 induction in HUVEC may be of relevance to develop strategies aimed at selectively interfering with Cox-2 regulating pathways. (C) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.