Paper
British Journal of Pharmacology (2002) 136, 558–567; doi:10.1038/sj.bjp.0704777
YC-1 increases cyclo-oxygenase-2 expression through protein kinase G- and p44/42 mitogen-activated protein kinase-dependent pathways in A549 cells
Ming-Shyan Chang1, Wen-Sen Lee1,2, Che-Ming Teng3, Horng-Mo Lee4, Joen-Rong Sheu1, George Hsiao1 and Chien-Huang Lin4,5
- 1Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan
- 2Department of Physiology, School of Medicine, Taipei Medical University, Taipei, Taiwan
- 3Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
- 4Graduate Institute of Biomedical Technology, Taipei Medical University, Taipei, Taiwan
- 5School of Respiratory Therapy, Taipei Medical University, Taipei, Taiwan
Correspondence: Chien-Huang Lin, Graduate Institute of Biomedical Technology, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. E-mail: chlin@tmu.edu.tw
Received 22 October 2001; Revised 19 April 2002; Accepted 23 April 2002.
Abstract
- YC-1, an activator of soluble guanylate cyclase (sGC), has been shown to increase the intracellular cGMP concentration. This study was designed to investigate the signaling pathway involved in the YC-1-induced COX-2 expression in A549 cells.
- YC-1 caused a concentration- and time-dependent increase in COX activity and COX-2 expression in A549 cells. Pretreatment of the cells with the sGC inhibitor (ODQ), the protein kinase G (PKG) inhibitor (KT-5823), and the PKC inhibitors (Go 6976 and GF10923X), attenuated the YC-1-induced increase in COX activity and COX-2 expression.
- Exposure of A549 cells to YC-1 caused an increase in PKC activity; this effect was inhibited by ODQ, KT-5823 or Go 6976. Western blot analyses showed that PKC-
, -
, -
, -
and -
isoforms were detected in A549 cells. Treatment of A549 cells with YC-1 or PMA caused a translocation of PKC-, but not other isoforms, from the cytosol to the membrane fraction. Long-term (24 h) treatment of A549 cells with PMA down-regulated the PKC-. - The MEK inhibitor, PD 98059 (10–50 M), concentration-dependently attenuated the YC-1-induced increases in COX activity and COX-2 expression. Treatment of A549 cells with YC-1 caused an activation of p44/42 MAPK; this effect was inhibited by KT-5823, Go 6976, long-term (24 h) PMA treatment or PD98059, but not the p38 MAPK inhibitor, SB 203580.
- These results indicate that in human pulmonary epithelial cells, YC-1 might activate PKG through an upstream sGC/cGMP pathway to elicit PKC- activation, which in turn, initiates p44/42 MAPK activation, and finally induces COX-2 expression.
Keywords:
YC-1, guanylate cyclase, cyclo-oxygenase-2, protein kinase C, p44/42 MAPK, A549 cells
Abbreviations:
BCIP, 5-bromo-4-chloro-3-indolyl-phosphate; cGMP, 3',5'-cyclic guanosine monophosphate; COX, cyclo-oxygenase; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol bis (
-aminoethyl ether)-N,N,N',N'-tetraacetic acid; ERK, extracellular signal-regulated protein kinase; FCS, foetal calf serum; HEPES, 4-(2-hydroxy-ethyl)-1-piperazineethanesulphonic acid; iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; NBT, 4-nitro blue tetrazolium; NO, nitric oxide; NP-40, nonident P-40; ODQ, 1H-(1,2,4)oxadiazolo[4,3-a]quinozalin-1-one; YC-1, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; PBS, phosphate buffer saline; PGE2, prostaglandin E2; PGs, prostaglandins; PKC, protein kinase C; PKG, protein kinase G; PMA, phorbol-12-myristate-13-acetate; PMSF, phenylmethylsulphonyl fluoride; SDS, sodium dodecyl sulphate; sGC, soluble guanylate cyclase; TNF-, tumour necrosis factor-
