Diversity and similarity in signaling events leading to rapid Cox-2 induction by tumor necrosis factor-alpha 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 (TNF alpha) 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 alpha 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(1 alpha) (6-keto-PGF(1 alpha)) 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 alpha 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 alpha. Of interest, intracellular ROS generation and consequent GSH/GSSG ratio reduction represents a common step through which PMA and TNF alpha 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.


CONCLUSIONS
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.
Eligini
Sonia
S
E. Grossi Paoletti Center, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy.
Barbieri
Silvia Stella
SS
Cavalca
Viviana
V
Camera
Marina
M
Brambilla
Marta
M
De Franceschi
Michela
M
Tremoli
Elena
E
Colli
Susanna
S
D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000502:physiology; D005978:Glutathione / Q000502:physiology; D019803:Glutathione Disulfide / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology
D008565:Membrane Proteins; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D048051:p38 Mitogen-Activated Protein Kinases; D020558:GTP Phosphohydrolases; D005978:Glutathione; D013755:Tetradecanoylphorbol Acetate; D019803:Glutathione Disulfide