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

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Abstract

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-α (TNFα) 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. Human endothelial cells isolated from umbilical vein (HUVEC) were exposed to PMA and TNFα 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 F (6-keto-PGF ) 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. Data show that TNFα 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 TNFα. Of interest, intracellular ROS generation and consequent GSH/GSSG ratio reduction represents a common step through which PMA and TNFα 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. 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.

Original languageEnglish
Pages (from-to)683-693
Number of pages11
JournalCardiovascular Research
Volume65
Issue number3
DOIs
Publication statusPublished - Feb 15 2005

Keywords

  • Cyclooxygenase
  • Endothelial function
  • Infection/inflammation
  • Prostaglandins
  • Redox signaling

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

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