The involvement of NF-kappaB in the constitutive overexpression of cyclooxygenase-2 in cyclooxygenase-1 null cells.
We recently reported that there was enhanced cyclooxygenase (COX)-2 expression and prostaglandin E(2) biosynthesis in COX-1 deficient (COX-1(-/-)) cells. We also observed that the growth of COX-1(-/-) cells was significantly retarded compared to wild-type (WT) and COX-2 deficient (COX-2(-/-)) cells. In this study, COX-2 expression and its promoter activity were compared in immortalized, nontransformed fibroblasts from WT, COX-1(-/-) or COX-2(-/-) mice in the context of the role of COX-2 as a growth regulator. When compared with WT cells expressing both COX isoenzymes, constitutive COX-2 protein and promoter activity were significantly higher in COX-1(-/-) cells as determined by Western blotting and luciferase assays using a 5'-flanking promoter construct of the murine COX-2 gene. The luciferase assay using a series of luciferase-linked COX-2 promoter deletions transfected into COX-1(-/-) cells indicated that a region involving NF-kappaB plays a significant role in regulating constitutive COX-2 expression. Data from electrophoretic mobility shift assays showed that COX-1(-/-) cells contained higher levels of activated NF-kappaB than either WT or COX-2(-/-) cells. Furthermore, COX-2 promoter activity was significantly inhibited by the oligonucleotides (ODNs) containing the NF-kappaB element (NF-kappaB decoy ODNs) but not by the scrambled control ODNs, as examined by the luciferase assay. These findings indicate that constitutive COX-2 promoter activity and protein expression are enhanced in COX-1(-/-) fibroblasts and that signaling via the NF-kappaB pathway is involved in the transcriptional control of constitutive COX-2 expression.
Kanekura
Takuro
T
Department of Dermatology, Kagoshima University Faculty of Medicine, Japan. takurok@m2.kufm.kagoshima-u.ac.jp
Goorha
Sarita
S
Kirtikara
Kanyawim
K
Ballou
Leslie R
LR
D000818:Animals; D002453:Cell Cycle; D002455:Cell Division; D002478:Cells, Cultured; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000172:deficiency* / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D008565:Membrane Proteins; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000494:pharmacology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000172:deficiency* / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D009841:Oligonucleotides; D008156:Luciferases; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; C496512:Ptgs1 protein, mouse