BACKGROUND: Exposure of skin cells, particularly keratinocytes to various nuclear factor-kappaB (NF-kappaB) activators (e.g. tumor necrosis factor-alpha, interleukin-1, lipopolysaccharides, and ultraviolet light) leads to phosphorylation and degradation of the inhibitory protein, IkappaB. Liberated NF-kappaB 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-kappaB activation on the synthesis of melanotrophic factors from the keratinocytes, the activities of NF-kappaB induced by melanogenic inhibitors (MIs) were determined in human HaCaT keratinocytes transfected with pNF-kappaB-SEAP-NPT plasmid. Transfectant cells released the secretory alkaline phosphatase (SEAP) as a transcription reporter in response to the NF-kappaB 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-kappaB activation was measured with the SEAP reporter gene assay using a fluorescence detection method. RESULTS: Of the MIs tested, kojic acid (IC(50)=60 microM) was found to be the most potent inhibitor of UVB-upregulating NF-kappaB activation in transfectant HaCaT cells, which is followed by niacinamide (IC(50)=540 microM). Pretreatment of the transfectant HaCaT cells with the MIs, especially kojic acid and niacinamide, effectively lowered NF-kappaB 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.
BACKGROUND: Exposure of skin cells, particularly keratinocytes to various nuclear factor-kappaB (NF-kappaB) activators (e.g. tumor necrosis factor-alpha, interleukin-1, lipopolysaccharides, and ultraviolet light) leads to phosphorylation and degradation of the inhibitory protein, IkappaB. Liberated NF-kappaB 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-kappaB activation on the synthesis of melanotrophic factors from the keratinocytes, the activities of NF-kappaB induced by melanogenic inhibitors (MIs) were determined in human HaCaT keratinocytes transfected with pNF-kappaB-SEAP-NPT plasmid. Transfectant cells released the secretory alkaline phosphatase (SEAP) as a transcription reporter in response to the NF-kappaB 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-kappaB activation was measured with the SEAP reporter gene assay using a fluorescence detection method. RESULTS: Of the MIs tested, kojic acid (IC(50)=60 microM) was found to be the most potent inhibitor of UVB-upregulating NF-kappaB activation in transfectant HaCaT cells, which is followed by niacinamide (IC(50)=540 microM). Pretreatment of the transfectant HaCaT cells with the MIs, especially kojic acid and niacinamide, effectively lowered NF-kappaB 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.