Colocalization and interaction of cyclooxygenase-2 with caveolin-1 in human fibroblasts.
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.
Liou
J Y
JY
Vascular Biology Research Center and Division of Hematology, University of Texas-Houston Medical School, Houston, Texas 77030, USA.
Deng
W G
WG
Gilroy
D W
DW
Shyue
S K
SK
Wu
K K
KK
D015153:Blotting, Western; D021941:Caveolae / Q000737:chemistry; D051242:Caveolin 1; D022461:Caveolins / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D007527:Isoenzymes / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D008565:Membrane Proteins; D018613:Microscopy, Confocal; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D007375:Interleukin-1; D007527:Isoenzymes; D008565:Membrane Proteins; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D013755:Tetradecanoylphorbol Acetate