Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide.
Two forms of cyclooxygenase are known to be present in eukaryotic organisms: a cyclooxygenase (COX-1) first purified from ram seminal vesicles encoded by a 2.8-kilobase mRNA, and a newly discovered mitogen-inducible cyclooxygenase (COX-2) encoded by a 4-kilobase mRNA. Expression of these two forms of the enzyme in rat alveolar macrophages stimulated with lipopolysaccharide was investigated by 1) determining the activity of newly synthesized enzyme after inactivating the endogenous enzyme with aspirin; 2) comparing levels of newly synthesized enzyme proteins in cells treated with or without lipopolysaccharide; and 3) assessing the expression of the mRNAs encoding COX-1 and COX-2. Levels of enzyme proteins were assessed by Western blot analysis and immunoprecipitation of 35S-labeled enzyme using two different antibodies, one specific for COX-2 and the other recognizing both forms of the enzyme but preferentially recognizing COX-1. We report here that the enhanced cyclooxygenase activity induced by the bacterial lipopolysaccharide in rat alveolar macrophages is caused by selective expression of the COX-2. Expression of COX-2 in macrophages stimulated by lipopolysaccharide was completely inhibited by dexamethasone, whereas COX-1 was unaffected. In resting unstimulated macrophages, only COX-1 but not COX-2 was detected. Levels of mRNA for the COX-2 in macrophages were increased, but those of the COX-1 were not affected by lipopolysaccharide as assessed by reverse transcription coupled with polymerase chain reaction. These results indicate that increased synthesis of prostaglandins and thromboxanes in lipopolysaccharide-stimulated macrophages results from selective expression of COX-2.
Lee
S H
SH
Pennington Biomedical Research Center, Louisiana State University, Baton Rouge 70808.
Soyoola
E
E
Chanmugam
P
P
Hart
S
S
Sun
W
W
Zhong
H
H
Liou
S
S
Simmons
D
D
Hwang
D
D
D000818:Animals; D001241:Aspirin / Q000494:pharmacology*; D001483:Base Sequence; D015153:Blotting, Western; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D015232:Dinoprostone / Q000378:metabolism; D004790:Enzyme Induction; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D016676:Macrophages, Alveolar / Q000187:drug effects* / Q000201:enzymology*; D008934:Mitogens / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction / Q000379:methods; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic
D007527:Isoenzymes; D008070:Lipopolysaccharides; D008934:Mitogens; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D003609:Dactinomycin; D003907:Dexamethasone; D003513:Cycloheximide; D011451:Prostaglandin-Endoperoxide Synthases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D015232:Dinoprostone; D001241:Aspirin