A Mechanism for the Up-regulation of the IL-8 Gene Expression in Keratinocytes by All-trans Retinoic Acid

Background: Retinoic acid (RA) has been reported to induce the up-regulation of inflammatory cytokines such as IL-1, TNF- and IL-8 in dermal fibroblasts and keratinocytes. There is no evidence to support a direct interaction between the RA-mediated transcriptional machinery and IL-8 gene transcription. Objective: The aim of this study is to clarify the mechanism of the up-regulation of IL-8 in keratinocytes by RA. Methods: The IL-1, IL-8, TNF- and MCP-1 mRNA expressions in HaCaT cells stimulated by RA were measured by quantitative RT-PCR. The effects of a NF-B inhibitor and IL-1 receptor antagonist (ra) on the IL-8 mRNA expression were measured by quantitative RT-PCR. Electrophoretic motility shift assay (EMSA) was conducted on the RA-stimulated HaCaT cells that were or were not treated with NF-B inhibitor to measure the NF-B binding activity in each group. The phospho-IB activity in the HaCaT cells after stimulation with RA was also measured by Western blotting. Results: An up-regulation of the IL-8 gene expression by RA was demonstrated in the HaCaT cells. The inhibition assay revealed the involvement of the NF-B binding site of the IL-8 gene in the RA-enhanced promoter activity. EMSA demonstrated that RA enhanced the formation of the DNA-NF-B complex. There was no evidence to support IL-1 as an intermediate stimulus between the RA-mediated transcriptional machinery and IL-8 gene transcription. Western blot analysis revealed increased phospho-IB activity in the HaCaT cells after stimulation with RA. Conclusion: Our result suggested that the IL-8 gene expression of HaCaT cells after RA stimulation is caused by the activation of IKK and the dissociation of IB from NF-B and the transcription of NF-B in the nucleus.