Induction of cyclooxygenase-2 by platelet-derived growth factor (PDGF) and its inhibition by dexamethasone are independent of NF-kappaB/IkappaB transcription factors.
Glucocorticoids are potent inhibitors of cyclooxygenase-2 (prostaglandin G/H synthase-2, COX-2) expression. The focus of this work was to investigate the molecular mechanisms, by which glucocorticoids interfere with platelet-derived growth factor (PDGF)-mediated induction of COX-2 with special emphasis on the role of the transcription factors NF-kappaB/IkappaB alpha. In rat renal mesangial cells, PDGF induced a rapid and transient increase of COX-2 mRNA and protein, which reached maximal levels after 1-2 and 4 h, respectively. The in vivo half-life of COX-2 mRNA, which was estimated to be less than 1 h, was reduced by dexamethasone. Kinetic studies and COX-2 promoter activity assays indicated that dexamethasone also interfered with COX-2 transcription. Inhibition of COX-2 induction by dexamethasone was abrogated by cycloheximide, an inhibitor of translation, indicating dependence on de novo protein synthesis. As a possible mediator of dexamethasone action, the NF-kappaB/IkappaB alpha system of transcription factors was investigated. Dexamethasone doubled IkappaB alpha protein levels within 1 h and reduced complex formation of nuclear NF-kappaB proteins with DNA. Newly synthesized IkappaB alpha may thus bind to NF-kappaB and interfere with gene activation. PDGF-induced signalling, however, barely affected the NF-kappaB/IkappaB alpha system: IkappaB alpha protein remained unaltered for 30 min after treatment of mesangial cells with PDGF and was only reduced by 30% after 1 h. Concomitantly, binding of NF-kappaB proteins to DNA, detected by electrophoretic mobility shift assays, was slightly increased by 30%. Furthermore, stably transfected COX-2 promoter constructs with and without the NF-KB binding site were comparably activated by PDGF (2.5-fold increase of luciferase activity). Taken together, these data indicate that although dexamethasone interferes with the NF-kappaB/IkappaB alpha system of transcription factors, this mechanism is not essential for the inhibition of PDGF-induced COX-2 expression.
Goppelt-Struebe
M
M
Medizinische Klinik IV, Universität Erlangen-Nürnberg, Erlangen, Germany. Goppelt-Struebe@rzmail.uni-erlangen.de
Rehm
M
M
Schaefers
H J
HJ
D000818:Animals; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D051546:Cyclooxygenase 2; D004247:DNA / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology*; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D007678:Kidney Glomerulus / Q000166:cytology; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015533:Transcriptional Activation
D020672:I-kappa B Proteins; D007527:Isoenzymes; D016328:NF-kappa B; D019384:Nucleic Acid Synthesis Inhibitors; D010982:Platelet-Derived Growth Factor; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D003609:Dactinomycin; D003907:Dexamethasone; D004247:DNA; D003513:Cycloheximide; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases