Interleukin-1h-induced cyclooxygenase-2 expression is mediated through activation of p42/44 and p38 MAPKS, and NF-nB pathways in canine tracheal smooth muscle cells Chuen-Mao Yang a, * , Chin-Sung Chien a , Li-Der Hsiao a , Shu-Fen Luo b , Chuan-Chwan Wang b a Department of Physiology and Pharmacology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan b Department of Internal Medicine, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan Received 6 February 2002; accepted 8 April 2002 Abstract Interleukin-h (IL-1h) was found to induce inflammatory responses in the airways, which exerted a potent stimulus for PG synthesis. This study was to determine the mechanisms of IL-1h-enhanced cyclooxygenase (COX)-2 expression associated with PGE 2 synthesis in tracheal smooth muscle cells (TSMCs). IL-1h markedly increased COX-2 expression and PGE 2 formation in a time- and concentration-dependent manner in TSMCs. Both COX-2 expression and PGE 2 formation in response to IL-1h were attenuated by a tyrosine kinase inhibitor, genistein, a phosphatidylcholine-phospholipase C inhibitor, D609, a phosphatidylinositol-phospholipase C inhibitor, U73122, protein kinase C inhibitors, GF109203X and staurosporine, removal of Ca 2+ by addition of BAPTA/AM plus EGTA, and phosphatidylinositol 3-kinase (PI3-K) inhibitors, LY294002 and wortmannin. IL-1h-induced activation of NF-nB correlated with the degradation of InB-a in TSMCs. IL- 1h-induced NF-nB activation, COX-2 expression, and PGE 2 synthesis were inhibited by the dominant negative mutants of NIK and IKK-a, but not by IKK-h. IL-1h-induced COX-2 expression and PGE 2 synthesis were completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 inhibitor), but these two inhibitors had no effect on IL-1h-induced NF-nB activation, indicating that activation of p42/44 and p38 MAPK and NF-nB signalling pathways were independently required for these responses. These findings suggest that the increased expression of COX-2 correlates with the release of PGE 2 from IL-1h-challenged TSMCs, at least in part, independently mediated through MAPKs and NF-nB signalling pathways in canine TSMCs. IL-1h-mediated responses were modulated by PLC, Ca 2+ , PKC, tyrosine kinase, and PI3-K in these cells. D 2002 Elsevier Science Inc. All rights reserved. Keywords: COX-2; IL-1h; MAPK; NF-nB; Tracheal smooth muscle cells 1. Introduction Cytokines are potent immunoregulatory and pro-inflam- matory mediators secreted by a variety of cells’ response to infection, activated by lymphocyte products, microbial tox- ins, and other stimuli [1]. Elevated levels of pro-inflamma- tory cytokines including interleukin-1h (IL-1h) in the bronchoalveolar lavage fluid have been detected in allergic asthmatic patients [2,3]. IL-1h exerted a potent stimulus for prostaglandin (PG) and thromboxane synthesis, which is implicated in inflammatory responses in the airways. The enzyme cyclooxygenase (COX) is responsible for conver- sion of arachidonic acid to PGH 2 that can be further metabolized to prostanoids and can modulate various airway functions [4,5]. It has been demonstrated that COX exists in at least two isoforms. The COX-1 expressed constitutively in most tissues appears to support the levels of prostanoid biosynthesis required for maintaining normal physiological homeostasis [6]. In contrast, the COX-2 is recognized to mediate inflammatory responses and is highly restricted 0898-6568/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII:S0898-6568(02)00037-2 Abbreviations: BCA, bicinchoninic acid; COX, cyclooxygenase; DMEM, Dulbecco’s modified Eagle’s medium; EC 50 , concentration required for half-maximal stimulation; ECL, enhanced chemiluminescence; F-12, Ham’s nutrient mixture F-12; FBS, fetal bovine serum; IKK, InB kinase; IL-1h, interleukin-1h; MAPK, mitogen-activated protein kinase; MEK1/2, MAPK kinase; NF-nB, nuclear factor-nB; PG, prostaglandin; PC- PLC, phosphatidylcholine-phospholipase C; PKC, protein kinase C; PTX, pertussis toxin; PI3-K, phosphatidylinositol 3-kinase; PI-PLC, phosphati- dylinositol-phospholipase C; TSMCs, tracheal smooth muscle cells. * Corresponding author. Department of Pharmacology, College of Medicine, Chang Gung University, 259 Wen-Hwa 1 Road, Kwei-San, Tao- Yuan, Taiwan. Tel.: +886-3-328-3016x5123; fax: +886-3-328-2365. E-mail address: chuenmao@mail.cgu.edu.tw (C.-M. Yang). www.elsevier.com/locate/cellsig Cellular Signalling 14 (2002) 899– 911