Activation of NFkappaB is necessary for IL-1beta-induced cyclooxygenase-2 (COX-2) expression in human gingival fibroblasts.
The immediate-early cyclooxygenase-2 (COX-2) gene encodes an inducible prostaglandin synthase enzyme which is implicated in inflammatory and proliferative diseases. COX-2 is highly induced during cell activation by various factors, including mitogens, hormones and cytokines. Since pro-inflammatory cytokine IL-1beta has been shown to induce prostaglandin E2 (PGE2) release in human gingival fibroblasts (HGF), here we analyzed the effect of IL-1beta on the expression of COX-2 and the activation of NFkappaB in HGF. Northern hybridization analysis revealed that IL-1beta (200 pg/ml) increased the expression of COX-2 mRNA in HGF. The effect of IL-1beta was abrogated by herbimycin A, a protein tyrosine kinase inhibitor, and enhanced by orthovanadate, a protein tyrosine phosphatase inhibitor. IL-1beta-induced PGE2 release was blocked by the tyrosine kinase inhibitor and increased by the tyrosine phosphatase inhibitor. The results of transient transfection assays using chimeric constructs of the human COX-2 promoter (nt -1432 approximately +59) ligated to a luciferase reporter gene indicated that IL-1beta stimulated the transcriptional activity approximately 1.5-fold. Gel mobility shift assays with a radiolabelled COX-2-NFkappaB oligonucleotide (nts-223 to-214) revealed an increase in the binding of nuclear proteins from IL-1beta-stimulated HGF. This increase of DNA-protein complex formation induced by IL-1beta was blocked by herbimycin A and another tyrosine kinase inhibitor, genistein. These results suggest that NFkappaB is an important transcription factor for IL-1beta-induced COX-2 gene expression, and is involved in inducing COX-2 gene transcription through tyrosine phosphorylation in HGF.
Nakao
S
S
Department of Pharmacology, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Ogata
Y
Y
Shimizu-Sasaki
E
E
Yamazaki
M
M
Furuyama
S
S
Sugiya
H
H
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D005881:Gingiva / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D007527:Isoenzymes / Q000235:genetics; D008565:Membrane Proteins; D016328:NF-kappa B / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology
D007375:Interleukin-1; D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014638:Vanadates; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D017027:Protein Tyrosine Phosphatases; D015232:Dinoprostone