Role of COX-1 and COX-2 in the release of prostanoids in murine lung and isolated lung fibroblasts

Abstract

Cyclooxygenase (COX) is the first enzyme in the conversion of arachidonic acid to prostanoids. There are two isoforms of COX; COX-1, which is constitutively expressed with a homeostatic role in most tissues, and COX-2, which while constitutively expressed in some discreet sites is generally inducible by growth factors and during inflammation. In the current study, we have used tissues and cells from knock-out mice to investigate the relative contributions of COX-1 and COX-2 to PGE2 production by lung tissue ex vivo and by proliferating lung fibroblasts in vitro. Lung tissues from WT (C57Bl6), COX-1-/- and COX-2-/- mice were immediately dissected (<15 min after death) and incubated (37 °C) for 30 min in DMEM containing 50 µM calcium ionophore (A23187). Release of PGE2 was determined by competitive immunoassay. In parallel studies, murine lung fibroblasts from COX-1-/- and COX-2-/- mice were explanted and cultured before being seeded in 96-well plates at sub-confluence (5000-8000/well) and incubated for 24-48 hours in the presence of 10% FCS. Accumulated release of PGE2 was then measured as above. Over 30 min PGE2 was released by lung pieces from wild type (1117 ± 55 pg/ml) and COX-2-/- (2013 ± 255 pg/ml) but not from COX-1-/- (<61pg/ml) mice (n=4). In contrast, proliferating lung fibroblasts from COX-1-/- (4978.9 ± 1392 pg/ml) mice released higher levels of PGE2 than cells from COX-2-/- (1194 ± 617 ng/ml) mice (n=4 using cells from 2-3 separate mice for each genotype). These results show that COX-1 activity underpins the stimulated release of PGE2 in healthy mouse lung tissue. Conversely, COX-2 activity predominates in proliferating lung fibroblasts, which may be important as COX-derived PGE2 mediates proliferation of lung fibroblasts (Trends Immunol.2004;25(1):40-6). Our results suggest a switch in COX isoform in lung cells during proliferation which could be relevant to our understanding of conditions such as idiopathic pulmonary fibrosis.