Short communication Extracellular nucleotides induce COX-2 up-regulation and prostaglandin E 2 production in human A549 alveolar type II epithelial cells Brice Marcet a, ⁎ , Frédérick Libert a , Jean-Marie Boeynaems a,b , Didier Communi a a Institute of Interdisciplinary Research, IRIBHM, Université Libre de Bruxelles, Bât C5-110, Route de Lennik, 808, 1070 Brussels, Belgium b Department of Medical Chemistry, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium Received 9 January 2007; received in revised form 27 March 2007; accepted 1 April 2007 Available online 14 April 2007 Abstract Extracellular nucleotides regulate ion transport, mucociliary clearance as well as inflammatory properties of the airway epithelium by acting on P2 receptors. Cyclooxygenase-2 (COX-2) is a key enzyme involved in the synthesis of prostaglandins during inflammation. In this study, using calcium imaging, DNA microarray experiments, real-time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and prostaglandin E 2 (PGE 2 ) measurement, we show for the first time that ATP, UTP or INS365 compound (P2Y 2 receptor agonists) up-regulate COX-2 expression by ∼ 3-fold and enhance the release of PGE 2 in human A549 airway epithelial cells. Our data suggest that P2Y receptors may represent putative targets in airway inflammatory diseases. © 2007 Elsevier B.V. All rights reserved. Keywords: P2Y receptor; Airway epithelium; COX-2; Prostaglandin E 2 ; Cystic fibrosis; Extracellular nucleotide 1. Introduction Nucleotides are released across the membrane of airway epithelial cells into the extracellular compartment in response to different stresses such as mechanical stimulations (Marcet and Boeynaems, 2006). They interact at the cell surface with metabotropic P2Y receptors and thereby regulate the integrated airway epithelial functions like mucociliary clearance, airway defense responses and airway immune function (Marcet and Boeynaems, 2006; Marcet et al., 2007). Cyclooxygenases (COX-1 and COX-2), enzymes involved in prostaglandin synthesis, are key modulators of airway inflammation (Mitchell and Evans, 1998). The human airway type II alveolar epithelial carcinoma cell line A549 (Lieber et al., 1976), widely used to examine airway inflammation, does not express COX-1 but constitutively expresses COX-2 (Mitchell et al., 1994; Watkins et al., 1999). In addition, nucleotides stimulated the release of arachidonic acid, a rate- limiting step in prostaglandin E 2 (PGE 2 ) synthesis, and up- regulated inducible nitric oxide synthase expression in A549 cells (Laubinger et al., 2006). Thus, COX-2 and extracellular nucleotides both play a key role in airway inflammation but it remained unknown whether extracellular nucleotides may affect COX-2 expression. Here, we showed that COX-2 was a target gene of P2Y 2 receptor agonists using microarray technology and real-time RT-PCR and that PGE 2 release was induced by extracellular nucleotides in A549 airway epithelial cells. 2. Materials and methods 2.1. Cell culture A549 cells, cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal calf serum, antibiotics and amphotericin at 37 °C with 5% CO 2 /95% air in 6-well plates to 80% confluency, were then stimulated in serum-free medium by nucleotides. ATP, ATPγS, UTP, UDP, NS398, actinomycin D, European Journal of Pharmacology 566 (2007) 167 – 171 www.elsevier.com/locate/ejphar ⁎ Corresponding author. Tel.: +32 33 4 93 95 77 90; fax: +32 33 4 93 95 77 08. E-mail addresses: marcet@ipmc.cnrs.fr, brice_marcet@hotmail.com (B. Marcet). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.04.003