Glucocorticoid-mediated gene suppression of rat cytokine-induced neutrophil chemoattractant CINC/gro, a member of the interleukin-8 family, through impairment of NF-κB activation

Abstract

The glucocorticoid dexamethasone inhibited the production of the rat cytokine-induced neutrophil chemoattractant CINC/gro, a counterpart of human melanoma growth-stimulating activity that belongs to the interleukin-8 (IL- 8) family, in the normal rat kidney epithelial cell line NRK-52E stimulated with interleukin-1β (IL-1β), lipopolysaccharide, or tumor necrosis factor α. The accumulation of CINC/gro mRNA induced by these activators was also decreased comparably by dexamethasone. A nuclear run-on assay revealed that dexamethasone decreased the IL-1β-induced transcription of the CINC/gro gene. The half-life of CINC/gro mRNA transcripts did not change significantly after exposure to dexamethasone, suggesting that this glucocorticoid acts mainly at the transcriptional level. Transfection with luciferase expression vectors containing 5'-deleted and mutated CINC/gro gene sequences demonstrated that the 5'-flanking region containing the NF-κB binding site is involved in the IL-1β- and dexamethasone-induced activation and repression of the CINC/gro gene expression, respectively. Furthermore, a tandem repeat of the NF-κB sequence in the CINC/gro gene conferred the inducibility by IL-1β and suppression of luciferase activity by dexamethasone. In an electrophoretic mobility shift assay, dexamethasone diminished the IL-1β-induced formation of NF-κB complexes, which consisted of p65 and p50. Western blotting revealed that dexamethasone inhibited the IL-1β-induced translocation of p65 from the cytoplasm into the nucleus, while the nuclear level of NF-κB p50 remained almost unchanged. In addition, the degradation of IκB-α induced by IL-1β was not inhibited by dexamethasone. These results indicated that the suppression of the CINC/gro gene transcription by glucocorticoid occurs through the impairment of NF-κB activation, possibly by interference with the translocation of NF-κB p65 from the cytoplasm into the nucleus, thereby suppressing transactivation of the CINC/gro gene.