Downregulation of NF-κB activation in human keratinocytes by melanogenic inhibitors

Abstract

Background: Exposure of skin cells, particularly keratinocytes to various nuclear factor-kappaB (NF-κB) activators (e.g. tumor necrosis factor-α, interleukin-1, lipopolysaccharides, and ultraviolet light) leads to phosphorylation and degradation of the inhibitory protein, IκB. Liberated NF-κB is translocated into the nucleus where it can change or alter expression of target genes, resulting in the secretion of extracellular signaling molecules including melanotrophic factors affecting melanocyte . Objective: In order to demonstrate the possible role of NF-κB activation on the synthesis of melanotrophic factors from the keratinocytes, the activities of NF-κB induced by melanogenic inhibitors (MIs) were determined in human HaCaT keratinocytes transfected with pNF-κB–SEAP–NPT plasmid. Transfectant cells released the secretory alkaline phosphatase (SEAP) as a transcription reporter in response to the NF-κB activity and contain the neomycin phosphotransferase (NPT) gene for the dominant selection marker for geneticin resistance. Methods: MIs such as niacinamide, kojic acid, hydroquinone, resorcinol, arbutin, and glycolic acid were preincubated with transfectant HaCaT cells for 3 h and then ultraviolet B (UVB) was irradiated. NF-κB activation was measured with the SEAP reporter gene assay using a fluorescence detection method. Results: Of the MIs tested, kojic acid (IC 50=60 μM) was found to be the most potent inhibitor of UVB-upregulating NF-κB activation in transfectant HaCaT cells, which is followed by niacinamide (IC 50=540 μM). Pretreatment of the transfectant HaCaT cells with the MIs, especially kojic acid and niacinamide, effectively lowered NF-κB binding measured by electrophoretic mobility shift assay. Furthermore, these two inhibitors remarkably reduced the secretion level of IL-6, one of melanotrophic factors, triggered by UV-radiation of the HaCaT cells. Conclusion: These observations suggest that MIs working at the in vivo level might act partially through the modulation of the synthesis of melanotrophic factors in keratinocyte.