Transcriptional induction of cyclooxygenase-2 gene by okadaic acid inhibition of phosphatase activity in human chondrocytes: co-stimulation of AP-1 and CRE nuclear binding proteins.
The involvement of serine/threonine protein phosphatases in signaling pathways that control the expression of the cyclooxygenase-2 (COX-2) gene in human chondrocytes was examined. Okadaic acid (OKA), an inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A), induced a delayed, time-dependent increase in the rate of COX-2 gene transcription (runoff assay) resulting in increased steady-state mRNA levels and enzyme synthesis. The latter response was dose dependent over a narrow range of 1-30 nmol/L with declining expression and synthesis of COX-2 at higher concentrations due to cell toxicity. The delayed increase in COX-2 mRNA expression was accompanied by the induction of the proto-oncogenes c-jun, junB, junD, and c-fos (but not FosB or Fra-1). Increased phosphorylation of CREB-1/ATF-1 transcription factors was observed beginning at 4 h and reached a zenith at 8 h. Gel-shift analysis confirmed the up-regulation of AP-1 and CRE nuclear binding proteins, though there was little or no OKA-induced nuclear protein binding to SP-1, AP-2, NF-kappaB or NF-IL-6 regulatory elements. OKA-induced nuclear protein binding to 32P-CRE oligonucleotides was abrogated by a pharmacological inhibitor of protein kinase A (PKA), KT-5720; the latter compound also inhibited OKA-induced COX-2 enzyme synthesis. Calphostin C (CalC), an inhibitor of PKC isoenzymes, had little effect in this regard. Inhibition of 12P-CRE binding was also observed in the presence of an antibody to CREB-binding protein (265-kDa CBP), an integrator and coactivator of cAMP-responsive genes. The binding to 32P-CRE was unaffected in the presence of excess radioinert AP-1 and COX-2 NF-IL-6 oligonucleotides, although a COX-2 CRE-oligo competed very efficiently. 32P-AP-1 consensus sequence binding was unaffected by incubation of chondrocytes with KT-5720 or CalC, but was dramatically diminished by excess radioinert AP-1 and CRE-COX-2 oligos. Supershift analysis in the presence of antibodies to c-Jun, c-Fos, JunD, and JunB suggested that AP-1 complexes were composed of c-Fos, JunB, and possibly c-Jun. OKA has no effect on total cellular PKC activity but caused a delayed time-dependent increase in total PKA activity and synthesis. OKA suppressed the activity of the MAP kinases, ERK1/2 in a time-dependent fashion, suggesting that the Raf-1/MEKK1/MEK1/ERK1,2 cascade was compromised by OKA treatment. By contrast, OKA caused a dramatic increase in SAPK/JNK expression and activity, indicative of an activation of MEKK1/JNKK/SAPK/JNK pathway. OKA stimulated a dose-dependent activation of CAT activity using transfected promoter-CAT constructs harboring the regulatory elements AP-1 (c-jun promoter) and CRE (CRE-tkCAT). We conclude that in primary phenotypically stable human chondrocytes, COX-2 gene expression may be controlled by critical phosphatases that interact with phosphorylation dependent (e.g., MAP kinases:AP-1, PKA:CREB/ATF) signaling pathways. AP-1 and CREB/ATF families of transcription factors may be important substrates for PP-1/PP-2A in human chondrocytes.
Miller
C
C
Department of Medicine, University of Montreal, Quebec, Canada.
Zhang
M
M
He
Y
Y
Zhao
J
J
Pelletier
J P
JP
Martel-Pelletier
J
J
Di Battista
J A
JA
D000975:Antioxidants / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051546:Cyclooxygenase 2; D004247:DNA / Q000378:metabolism; D004790:Enzyme Induction / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D008565:Membrane Proteins; D019319:Okadaic Acid / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000502:physiology*; D010766:Phosphorylation; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000235:genetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011519:Proto-Oncogenes / Q000235:genetics; D011759:Pyrrolidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000502:physiology; D013859:Thiocarbamates / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D015533:Transcriptional Activation
D000975:Antioxidants; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D011759:Pyrrolidines; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013859:Thiocarbamates; D018808:Transcription Factor AP-1; D019319:Okadaic Acid; C020972:pyrrolidine dithiocarbamic acid; D004247:DNA; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases