interleukin-8 and hymenialdisine

The nuclear factor-kappaB (NF-kappaB) family of transcription factors have been implicated in the inducible expression of genes involved in inflammatory and immune responses. As such, a specific inhibitor of NF-kappaB would be a useful therapeutic agent in a variety of inflammatory disorders. The marine natural product hymenialdisine was evaluated as an inhibitor of NF-kappaB in U937 cells. U937 cells were transfected with either a luciferase reporter plasmid containing the human immunodeficiency virus long terminal repeat or the interleukin-8 (IL-8) core promoter, both of which are activated by NF-kappaB. Hymenialdisine caused a concentration-dependent decrease in luciferase production from both reporters when the cells were stimulated with tumor necrosis factor-alpha, lipopolysaccharide or phorbol myristate acetate. An electrophoretic mobility shift assay confirmed its activity by inhibiting DNA binding of NF-kappaB. Hymenialdisine was shown to be a selective inhibitor of NF-kappaB in that it had no effect on the binding of other transcription factors to their DNA concensus motifs; these included activator protein-1, CCAAT/enhancer binding protein and Sp1. Functional studies showed hymenialdisine to be an inhibitor of IL-8 production and IL-8 mRNA formation in the U937 cell. Investigation into the mechanism of action of hymenialdisine showed that it was not due to inhibition of protein kinase C because the selective protein kinase C inhibitor RO 32-0432 was inactive against tumor necrosis factor-alpha-stimulated luciferase and IL-8 production. The compound also had no effect on IkappaB alpha or IkappaB beta phosphorylation and degradation. Thus, hymenialdisine is a potent inhibitor of NF-kappaB and IL-8 production in U937 cells.

Topics: Azepines; Humans; Interleukin-8; Luciferases; NF-kappa B; Proto-Oncogene Proteins; Pyrroles; RNA, Messenger; Transcription Factor RelB; Transcription Factors; Tumor Cells, Cultured

Exposure of human rheumatoid synovial fibroblasts (RSF) to interleukin 1beta (IL-1beta) results in the coordinate up-regulation of 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX II) and subsequent biosynthesis of prostaglandin E2 (PGE2). We have recently demonstrated, through the use of oligonucleotide decoys and antisense, the participation of the proinflammatory transcription factor, nuclear factor kappaB (NFkappaB), in the regulation of the prostanoid-metabolizing enzymes. Hymenialdisine, a marine natural product has recently been characterized as an inhibitor of NFkappaB activation and exposure of IL-1-stimulated RSF-inhibited PGE2 production in a concentration-dependent manner (IC50 approximately 1 microM). Alternatively, both an analog, aldisine, and the protein kinase C inhibitor, RO 32-0432, were without affect. Direct action of hymenialdisine on IL-1-induced NFkappaB activation was demonstrated by a significant reduction (approximately 80%) in NFkappaB binding to the classical kappaB consensus motif (as assessed by electrophoretic mobility shift assay) and inhibition of stimulated p65 migration from the cytosol of treated cells (as assessed by Western analysis). Consistent with the role of NFkappaB in the transcriptional regulation of COX II and 85-kDa PLA2, hymenialdisine-treated RSF did not transcribe the respective mRNAs in response to IL-1. This led to reductions in their respective protein levels and subsequent reductions in the ability to produce PGE2. Specificity of action is suggested as IL-1-stimulated interleukin-8 (IL-8) production, which is known to be an NFkappaB-regulated event, was also inhibited by hymenialdisine, whereas IL-1-induced production of vascular endothelial growth factor, a non-NFkappaB-regulated gene, was not affected by exposure to hymenialdisine. Taken together, hymenialdisine inhibits IL-1-stimulated-RSF PGE2 formation acting predominately through modulation of NFkappaB activation and offers an interesting novel tool to evaluate the role of NFkappaB in inflammatory disease.

Topics: Adult; Arthritis, Rheumatoid; Azepines; Cells, Cultured; Dinoprostone; Endothelial Growth Factors; Humans; Interleukin-1; Interleukin-8; Lymphokines; NF-kappa B; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Pyrroles; Synovial Membrane; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors