Effects of Glucocorticoids on Tumor Necrosis Factor α-Dependent Activation of Nuclear Factor κB and Expression of the Intercellular Adhesion Molecule 1 Gene in Osteoblast-Like ROS17/2.8 Cells

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Abstract
Recently, we showed that tumor necrosis factor α (TNF‐α) stimulates expression of the intercellular adhesion molecule 1 (ICAM‐1) and interleukin‐6 (IL‐6) genes through activation of p65‐p50 heterodimer nuclear factor κB (NF‐κB) in rat osteoblast‐like ROS17/2.8 cells. In the present study, we investigated effects of a synthetic glucocorticoid, dexamethasone (Dex), on TNF‐α‐dependent activation of NF‐κB and expression of the ICAM‐1 gene. ROS17/2.8 cells were pretreated with Dex for 6 h and then exposed to TNF‐α. Electrophoretic mobility shift assay (EMSA) revealed that TNF‐α‐dependent activation of NF‐κB was almost completely suppressed by Dex treatment. Increase in ICAM‐1 messenger RNA (mRNA) level by TNF‐α also was markedly suppressed by Dex. Western blot and immunocytochemical analyses showed that Dex attenuated the TNF‐α‐induced nuclear translocation of p65. Treatment with protein synthesis inhibitor cycloheximide (CHX) reversed the Dex effect, indicating that Dex requires de novo protein synthesis for its action. Northern blot analysis revealed that Dex increased IκB‐α mRNA level synergistically with TNF‐α, whereas it decreased p65 mRNA level. The p105 and IκB‐β mRNA levels were not altered by Dex. Consistent with the mRNA level, Dex increased the amount of IκB‐α protein in the cytoplasm in either the presence or the absence of TNF‐α. Considering a role of IκB to sequester NF‐κB in the cytoplasm, it was suggested that an increase in IκB‐α protein and the concomitant decrease in p65 synthesis account for the Dex‐induced suppression of NF‐κB activation in osteoblastic cells.