1α,25‐Dihydroxyvitamin D 3 and a Variety of its Natural Metabolites Transcriptionally Repress Nuclear‐Factor‐κB‐Mediated Interleukin‐8 Gene Expression

Regulation of interleukin‐8 (IL‐8) gene transcription occurs mainly through the sequences ‐94 to ‐71 of the 5′‐flanking region of the IL‐8 gene, involving the transcription factors nuclear factor for interleukin‐6 (NF‐IL‐6) and nuclear factor κB (NF‐κB). The human melanoma cell line A3 was derived from G‐361 cells by stable transfection with an IL‐8 promoter–luciferase construct containing these sequences. 1α,25‐Dihydroxyvitamin D3 (calcitriol) repressed IL‐8 promoter activity induced by tumor necrosis factor‐α (TNF‐α) by 50%, compared to 30% inhibition using dexamethasone, an effect consistent with its effect on TNF‐α‐induced IL‐8 release and IL‐8 mRNA levels. A variety of vitamin D metabolites caused the same repressive effect on IL‐8 promoter activation as calcitriol. However, only those metabolites which were able to transactivate a classical vitamin D response element had the ability to repress IL‐8 promoter activation, suggesting that this repression is mediated via vitamin D receptor (VDR). Furthermore, overexpression of VDR in the parental G‐361 cell line enhanced the repressive effect of calcitriol on activation of the IL‐8 promoter by either TNF‐α stimulation or overexpression of the NF‐κB subunit p65. Electrophoretic mobility shift assays using nuclear extracts from A3 cells showed that calcitriol decreased the abundance of nuclear factors bound to the NF‐κB binding site of the IL‐8 promoter and this reduced binding of NF‐κB proteins presumably contributes to its inhibitory action.