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) g ene in human chondrocytes was examined. Okadaic acid (OKA), an inhibit or of protein phosphatases 1 (PP-1) and 2A (PP-2A), induced a delayed, time-dependent increase in the rate of COX-2 gene transcription (runo ff assay) resulting in increased steady-state mRNA levels and enzyme s ynthesis. The latter response was dose dependent over a narrow range o f 1-30 nmol/L with declining Expression and synthesis of COX-2 at high er concentrations due to cell toxicity. The delayed increase in COX-2 mRNA expression was accompanied by the induction of the proto-oncogene s c-jun, junB, junD, and c-fos (but not FosB or Fra-1). Increased phos phorylation of CREB-1/ATF-1 transcription factors was observed beginni ng at 4 h and reached a zenith at 8 h. Gel-shift analysis confirmed th e up-regulation of AP-l and CRE nuclear binding proteins, though there was little or no OKA-induced nuclear protein binding to SP-1, AP-2, N F-kappa B or NF-IL-6 regulatory elements. OKA-induced nuclear protein binding to P-32-CRE oligonucleotides was abrogated by a pharmacologica l inhibitor of protein kinase A (PKA), KT-5720; the latter compound al so inhibited OKA-induced COX-2 enzyme synthesis. Calphostin C (CalC), an inhibitor of PKC isoenzymes, had little effect in this regard. Inhi bition of P-32-CRE binding was also observed in the presence of an ant ibody to CREB-binding protein (265-kDa CBP), an integrator and coactiv ator of cAMP-responsive genes. The binding to P-32-CRE was unaffected in the presence of excess radioinert AP-1 and COX-2 NF-IL-6 oligonucle otides, although a COX-2 CRE-oligo competed very efficiently. P-32-AP- 1 consensus sequence binding was unaffected by incubation of chondrocy tes with KT-5720 or CalC, but was dramatically diminished by excess ra dioinert AP-1 and CRE-COX-2 oligos. Supershift analysis in the presenc e of antibodies to c-Jun, c-Fos, JunD, and JunB suggested that AP-1 co mplexes were composed oi c-Fos, JunB, and possibly c-Jun. OKA has no e ffect on total cellular PKC activity but caused a delayed time-depende nt increase in total PKA activity and synthesis. OKA suppressed the ac tivity of the MAP kinases, ERK1/2 in a time-dependent fashion,suggesti ng that the Raf-1/MEKK1/MEK1/ERK1,2 cascade was compromised by OKA tre atment. By contrast, OKA caused a dramatic increase in SAPK/JNK expres sion and activity, indicative of an activation of MEKK1/JNKK/SAPK/JNK pathway. OKA stimulated a dose-dependent activation of CAT activity us ing transfected promoter-CAT constructs harboring the regulatory eleme nts AP-1 (c-jun promoter) and CRE (CRE-tkCAT). We conclude that in pri mary phenotypically stable human chondrocytes, COX-2 gene expression m ay be controlled by critical phosphatases that interact with phosphory lation dependent (e.g., MAP kinases:AP-1, PKA:CREB/ATF) signaling path ways. AP-1 and CREB/ATF families of transcription factors may be impor tant substrates for PP-1/PP-2A in human chondrocytes. (C) 1998 Wiley-L iss, Inc.