Colocalization and interaction of cyclooxygenase-2 with caveolin-1 in human fibroblasts

Results from our previous study suggest that cyclooxygenase-2 (COX-2) induc ed by phorbol 12-myristate 13-acetate (PMA) may be localized to caveolae-li ke structures (Liou, J.-Y., Shyue, S.-K., Tsai, M.-J., Chung, C.-L., Chu, K .-Y., and Wu, K. K. (2000) J. Biol. Chem. 275, 15314-15320). In this study, we determined subcellular localization of COX-2 and caveolin-1 by confocal microscopy. COX-2 in human foreskin fibroblasts stimulated by PMA (100 mM) or interleukin-1 beta (1 ng/ml) for 6 h was localized to plasma membrane i n addition to endoplasmic reticulum and nuclear envelope. Caveolin-1 was lo calized to plasma membrane, and image overlay showed colocalization of COX- 2 with caveolin-1. This was confirmed by the presence of COX-2 and caveolin -1 in the detergent-insoluble membrane fraction of cells stimulated by PMA. Immunoprecipitation showed complex formation of COX-2 with caveolin-1 in a time-dependent manner. A larger quantity of COX-2 was complexed with caveo lin-1 in PMA-treated than in interleukin-Ig-treated cells. Purified COX-2 c omplexed with glutathione S-transferase-fused caveolin-1, which was not inh ibited by the scaffolding domain peptide. Caveolin-1-bound COX-2 was cataly tically active, and its activity was not inhibited by the scaffolding domai n peptide. These results suggest that COX-2 induced by PMA and interleukin- 10 is colocalized with caveolin-1 in the segregated caveolae compartment. B ecause caveolae, are rich in signaling molecules, this COX-2 compartment ma y play an important role in diverse pathophysiological processes.