Colocalization and Interaction of Cyclooxygenase-2 with Caveolin-1 in Human Fibroblasts

Jun-Yang Liou,Wu-Guo Deng,Derek W Gilroy,Song-Kun Shyue,Kenneth K Wu

doi:10.1074/jbc.m105946200

Abstract

Results from our previous study suggest that cyclooxygenase-2 (COX-2) induced by phorbol 12-myristate 13-acetate (PMA) may be localized to caveolae-like 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 nm) or interleukin-1beta (1 ng/ml) for 6 h was localized to plasma membrane in addition to endoplasmic reticulum and nuclear envelope. Caveolin-1 was localized 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 caveolin-1 in PMA-treated than in interleukin-1beta-treated cells. Purified COX-2 complexed with glutathione S-transferase-fused caveolin-1, which was not inhibited by the scaffolding domain peptide. Caveolin-1-bound COX-2 was catalytically active, and its activity was not inhibited by the scaffolding domain peptide. These results suggest that COX-2 induced by PMA and interleukin-1beta is colocalized with caveolin-1 in the segregated caveolae compartment. Because caveolae are rich in signaling molecules, this COX-2 compartment may play an important role in diverse pathophysiological processes.

Highlights

Cyclooxygenase (COX,1 known as prostaglandin H synthase) catalyzes the oxygenation of arachidonate to form prostaglandin G2 (PGG2) and the reduction of PGG2 to PGH2 [1]
human foreskin fibroblasts (HFFs) were stimulated with phorbol 12-myristate 13-acetate (PMA) (100 nM) for 6 h and treated with anti-COX-2 and anti-Cav-1 antibodies followed by secondary antibodies conjugated to rhodamine and fluorescein isothiocyanate, respectively
To determine whether there is a direct interaction between COX-2 and Cav-1, lysates of HFFs stimulated with PMA (100 nM) or IL-1␤ (1 ng/ml) for 6 h were immunoprecipitated with an antibody directed against Cav-1, and COX-2 proteins in the precipitates were detected by Western blot analysis

Summary

Introduction

Cyclooxygenase (COX,1 known as prostaglandin H synthase) catalyzes the oxygenation of arachidonate to form prostaglandin G2 (PGG2) and the reduction of PGG2 to PGH2 [1]. These results suggest that COX-2 induced by PMA and interleukin-1␤ is colocalized with caveolin-1 in the segregated caveolae compartment. 1 The abbreviations used are: COX, cyclooxygenase; ER, endoplasmic reticulum; NE, nuclear envelope, IL-1␤, interleukin-1␤; PMA, phorbol 12-myristate 13-acetate; Cav-1, Caveolin-1; HFF, human foreskin fibroblasts; SD, scaffolding domain; DRM, detergent-resistant membrane fraction; PG, prostaglandin.

Results

Conclusion