Differential effect of thalidomide and dexamethasone on the transcription factor NF-kappa B

Thalidomide was initially used as a sedative during pregnancy but was withd rawn from the market due to its teratogenic effects. In vitro studies have shown that thalidomide inhibits tumour necrosis factor alpha (TNF-alpha) mR NA expression and protein production by mitogen-stimulated macrophages and activated T cells. Even at the highest concentration (10(-1) mM) tested, ho wever, TNF-alpha levels are inhibited only partially and the mechanism of a ction is unknown. In the present investigations, we have examined the influ ence of thalidomide on nuclear levels of NF-kappaB in human peripheral bloo d mononuclear cells (PBMC) following activation with mitogen or phorbol myr istate acetate (PMA)/ionophore. Dexamethasone was used as a positive contro l due to its well-characterised mechanism of action and NF-kappaB-mediated effects on TNF-alpha expression. PBMC from healthy human volunteers were st imulated optimally with phytohemagglutinin (PHA) or PMA/ionophore in the pr esence of 10(-1)-10(-5) mM thalidomide or dexamethasone, concentrations tha t displayed a range of inhibitory effects on TNF-alpha production. Cells we re harvested at varying time points and nuclear extracts prepared. Nuclear levels of NF-kappaB were measured using electrophoretic mobility shift assa ys (EMSA) with a radiolabelled DNA probe specific for NF-kappaB. Results we re analysed using optical densitometry. Nuclear levels of NF-kappaB were fo und to be unaffected by thalidomide at all concentrations tested, including concentrations (10(-1)-10(-3) mM) that exhibited significant inhibition of TNF-alpha protein and mRNA expression. In concurrent experiments, dexameth asone was found to reduce NF-kappaB expression in a dose-dependent manner w ith maximal inhibition at the highest dose tested (10(-1) mM). TNF-alpha ge ne expression is controlled by at least three separate transcription factor s that are involved in binding to the promoter region. These observations s uggest that thalidomide does nut act directly on NF-KB and therefore inhibi ts TNF-alpha production through another independent mechanism. (C) 2001 Els evier Science B.V. All rights reserved.