Abstract
AIMS: Glucocorticoids (GCs) exert some of their anti-inflammatory actions by preventing the activation of the transcription factor nuclear factor (NF)-kappaB. The GC-dependent inhibition of NF-kappaB may occur at different levels, but the mechanisms involved are still incompletely understood. In this work, we investigated whether the synthetic GC, dexamethasone (Dex), modulates the activity of NF-kappaB in the lymphoblastic CCRF-CEM cell line. We also evaluated the ability of Dex to prevent the activation of NF-kappaB in response to the potent proinflammatory cytokine, interleukin (IL)-1beta. RESULTS: Exposure of the cells to Dex (1 microM) induced the rapid degradation of IkappaB-alpha, leading to the transient translocation of the NF-kappaB family members p65 and p50 from the cytoplasm to the nucleus, as evaluated by western blot. Electrophoretic mobility shift assays revealed that, in the nucleus, these NF-kappaB proteins formed protein-DNA complexes, indicating a transient activation of NF-kappaB. Additionally, Dex also induced de novo synthesis of IkappaB-alpha, following its degradation. Finally, when the cells were exposed to Dex (1 microM) prior to stimulation with IL-1beta (20 ng/ml), Dex was efficient in preventing IL-1beta-induced NF-kappaB activation. The GC antagonist, RU 486 (10 microM), did not prevent any of the effects of Dex reported here. CONCLUSION: Our results indicate that, in CCRF-CEM cells, Dex prevents NF-kappaB activation, induced by IL-1beta, by a mechanism that involves the upregulation of IkappaB-alpha synthesis, and that depends on the early and transient activation of NF-kappaB.
Highlights
The transcription factor nuclear factor (NF)-kB is composed of several structurally related (Rel) proteins, which include p50, p52, p65 (RelA), c-Rel and RelB.1Á3 The Rel proteins can form either homodimers or heterodimers with each other, except RelB, which can only form heterodimers.[4]
To evaluate the ability of Dex to modulate the levels of IkB-a, western blot analysis was performed using cytoplasmic extracts obtained from CCRF-CEM cells treated with Dex in concentrations ranging from 50 nM to 1 mM, for 5 min Á/1 h
Pretreatment of the cells with CHX, for 1 h, completely abrogated the appearance of IkB-a in cytoplasmic extracts from cells treated with Dex, for 1 h, showing that de novo protein synthesis is required for the Dex-induced IkB-a accumulation in the cytoplasm that follows its initial degradation (Fig. 1)
Summary
The transcription factor nuclear factor (NF)-kB is composed of several structurally related (Rel) proteins, which include p50 (and its precursor p105), p52 (and its precursor p100), p65 (RelA), c-Rel and RelB.1Á3 The Rel proteins can form either homodimers or heterodimers with each other, except RelB, which can only form heterodimers.[4] Á 6. The activity of NF-kB is usually transient,[1,3] as a consequence of the rapid re-synthesis of IkB-a following its degradation. Synthesized IkB-a replenishes its cytoplasmic levels and simultaneously enters the nucleus, where it binds to the NF-kB dimers, thereby blocking their DNA binding ability and restoring the cytoplasmic pool of latent NF-kB.[1,2]
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