alsterpaullone and pyrazolanthrone

Inducible prostaglandin synthase (cyclooxygenase-2, COX-2) is highly expressed in inflammation. The signaling mechanisms involved in the up-regulation of COX-2 are not known in detail. In the present study we investigated the role of c-Jun NH2-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family in COX-2 expression and prostaglandin (PG) E2 production in murine J774 macrophages activated by bacterial lipopolysaccharide (LPS). LPS caused a transient activation of JNK which was followed by increased COX-2 expression. Anthra(1,9-cd)pyrazol-6(2H)-one (SP600125), an inhibitor of JNK, inhibited phosphorylation of c-Jun with an IC50 of 5-10 microM. At the same concentrations SP600125 suppressed also LPS-induced COX-2 protein levels and PGE2 production. SP600125 did not alter LPS-induced COX-2 mRNA levels when measured 3 h after addition of LPS, whereas mRNA levels were significantly reduced in SP600125-treated cells when measured 24 h after addition of LPS. LPS-induced COX-2 mRNA levels reduced faster in cells treated with SP600125 than in control cells. Cycloheximide (that is known to activate JNK) enhanced COX-2 expression and its effect was inhibited by SP600125. The present results suggest that JNK pathway is involved in the up-regulation of COX-2 expression possibly by a mechanism related to the stability of COX-2 mRNA.

Topics: Animals; Anthracenes; Benzazepines; Cell Line; Cyclin-Dependent Kinase 2; Cycloheximide; Cyclooxygenase 2; Dactinomycin; Dinoprostone; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Indoles; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Macrophages; Mice; Oximes; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-jun; RNA, Messenger; Transcription, Genetic

We have previously examined the specificities of 28 commercially available compounds, reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases [Davies, Reddy, Caivano and Cohen (2000) Biochem. J. 351, 95-105]. In the present study, we have extended this analysis to a further 14 compounds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a number of protein kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more protein kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiological roles of cyclin-dependent protein kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiological roles of glycogen synthase kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 protein kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated kinase 1A (DYRK1A; IC(50)=0.33 microM) and p38-regulated/activated kinase (PRAK; IC(50)=1.0 microM).

Topics: Alkaloids; Anthracenes; Azepines; Benzazepines; Carbazoles; Catechin; Cyclin-Dependent Kinases; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Indoles; JNK Mitogen-Activated Protein Kinases; Kinetin; Mitogen-Activated Protein Kinases; Naphthalenes; Oximes; Protein Kinase Inhibitors; Purines; Pyrazoles; Pyrimidines; Signal Transduction; src-Family Kinases; Substrate Specificity