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‐κB 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 32P‐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. J. Cell. Biochem. 69:392–413, 1998. © 1998 Wiley‐Liss, Inc.