Copyright © 2004, European Society of Cardiology
Diversity and similarity in signaling events leading to rapid Cox-2 induction by tumor necrosis factor-
and phorbol ester in human endothelial cells
aE. Grossi Paoletti Center, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy
bDepartment of Cardiac Surgery, Centro Cardiologico Fondazione Monzino I.R.C.C.S., University of Milan, Italy
* Corresponding author. Tel.: +39 02 50319913; fax: +39 02 50318250. Email address: Susanna.Colli{at}unimi.it
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-
(TNF) 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 TNF and Cox-2 protein and mRNA levels were evaluated by Western blot and Real-Time Quantitative Reverse Transcription–PCR analysis. Prostaglandin E2 (PGE2) and 6-keto prostaglandin F1 (6-keto-PGF1) 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 TNF 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 TNF. Of interest, intracellular ROS generation and consequent GSH/GSSG ratio reduction represents a common step through which PMA and TNF signal for early Cox-2 induction. In addition, we provide evidence that phosphatidylinositol 3 (PI3)-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.
KEYWORDS Endothelial function; Cyclooxygenase; Redox signaling; Infection/inflammation; Prostaglandins
Time for primary review 32 days
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