Demonstration of an inducible cyclooxygenase in human endothelial cells using antibodies raised against the carboxyl-terminal region of the cyclooxygenase-2.
Cyclooxygenase (Cox) exists in two forms in human endothelial cells (HUVEC). We have raised antibodies that recognize the sequence of the carboxyl-terminal portion of the human Cox-2 (C)-NASSSRSGLD-DINPTVLLK. Cyclooxygenase activity of HUVEC challenged with interleukin 1 alpha or a phorbol ester increased in parallel with the mass of a protein doublet analyzed by Western blot using antibodies directed against the Cox-2 peptide; a monoclonal antibody directed against Cox-1 showed a small change in protein mass. A 35S-labeled protein doublet with a molecular mass of approximately 70,000 daltons was immunoprecipitated with the anti-Cox-2 antiserum in L-[35S] methionine-labeled cells stimulated with interleukin 1 alpha. This protein was not recovered by pretreating the antiserum with the Cox-2 peptide before immunoprecipitation. A minor variation in 35S-immunoprecipitated protein was obtained with the polyclonal anti-Cox-1 antibody. Both immunoprecipitated Cox-1 and Cox-2 possessed cyclooxygenase activity that was inhibited by flurbiprofen. Endoglycosidase H treatment of immunoprecipitated Cox-2 proteins caused a decline in the apparent molecular size similar to that observed with immunoprecipitated Cox-1 or sheep cyclooxygenase but did not suppress the doublet. These results show by direct protein measurement that HUVEC synthesize the novel Cox-2 under appropriate stimulation, with little changes of Cox-1.
Habib
A
A
Unité 348 Institut National de la Santé et de la Recherche Médicale, Hôpital Lariboisière, Paris, France.
Créminon
C
C
Frobert
Y
Y
Grassi
J
J
Pradelles
P
P
Maclouf
J
J
D000595:Amino Acid Sequence; D015153:Blotting, Western; D004730:Endothelium, Vascular / Q000201:enzymology*; D004790:Enzyme Induction; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007375:Interleukin-1; D010455:Peptides; D011451:Prostaglandin-Endoperoxide Synthases; D006596:Hexosaminidases; D013755:Tetradecanoylphorbol Acetate