• Archives of Pharmacal Research
• /
• v.24 no.4
• /
• pp.307-311
• /
• 2001

The activation of NF-$\kappa$B induced by kojic Acid, an inhibitor of tyrosinase for biosynthesis of melanin in melanocytes, was investigated in human transfectant HaCaT and SCC-13 cells. These two keratinocyte cell lines transfected with pNF-$\kappa$B-SEAP-NPT plasmid were used to determine the activation of NF-$\kappa$B. Transfectant cells release the secretory alkaline phosphatase (SEAP) as a transcription reporter in response to the NF-$\kappa$B activity and contain the neomycin phosphotransferase (NPT) gene for the dominant selective marker of geneticin resistance. NF-$\kappa$B activation was measured in the SEAP reporter gene assay using a fluorescence detection method. Kojic Acid showed the inhibition of cellular NF-$\kappa$B activity in both human keratinocyte transfectants. It could also downregulate the ultraviolet ray (UVR)-induced activation of NF-$\kappa$B expression in transfectant HaCaT cells. Moreover, the inhibitory activity of kojic Acid in transfectant HaCaT cells was found to be more potent than known antioxidants, e.g., vitamin C and N~acetyl-L-cysteine. These results indicate that kojic Acid is a potential inhibitor of NF-$\kappa$B activation in human keratinocytes, and suggest the hypothesis that NF-$\kappa$B activation may be involved in kojic Acid induced anti-melanogenic effect.

• Proceedings of the SCSK Conference
• /
• 2003.09a
• /
• pp.780-803
• /
• 2003

Exposure of skin cells, particularly keratinocytes to various nuclear factor-kappaB ($\textrm{NF}_{-{\kappa}}\textrm{B}$) activators [e.g. tumor necrosis factor-$\alpha$, interleukin-1, lipopolysaccharides, and ultraviolet light] leads to phosphorylation and degradation of the inhibitory protein, $\textrm{I}_{{\kappa}}\textrm{B}$. Liberated $\textrm{NF}_{-{\kappa}}\textrm{B}$ 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. In order to demonstrate the possible role of $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation on the synthesis of melanotrophic factors from the keratinocytes, the activities of $\textrm{NF}_{-{\kappa}}\textrm{B}$ induced by melanogenic inhibitors (MIs) were determined in human HaCaT keratinocytes transfected with $\textrm{pNF}_{-{\kappa}}\textrm{B}$-SEAP-NPT plasmid. Transfectant cells released the secretory alkaline phosphatase (SEAP) as a transcription reporter in response to the $\textrm{NF}_{-{\kappa}}\textrm{B}$ activity and contain the neomycin phosphotransferase (NPT) gene for the dominant selection marker for geneticin resistance. 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. $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation was measured with the SEAP reporter gene assay using a fluorescence detection method. Of the Mis tested, kojic acid ($IC_{50}$/ = 60 $\mu$M) was found to be the most potent inhibitor of UVB-upregulating $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation in transfectant HaCaT cells, which is followed by niacinamide ($IC_{50}$/= 540 $\mu$M). Pretreatment of the transfectant HaCaT cells with the Mis, especially kojic acid and niacinamide, effectively lowered $\textrm{NF}_{-{\kappa}}\textrm{B}$ 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. 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.

• Journal of the Society of Cosmetic Scientists of Korea
• /
• v.27 no.1
• /
• pp.17-27
• /
• 2001

A cell-based assay system for monitoring NF-$textsc{k}$B activity was developed to determine the influence of activated NF-$textsc{k}$B in human HaCaT cells. The pNF-$textsc{k}$B-SEAP-NPT plasmid that permits expression of the secreted alkaline phosphatase (SEAP) reported gene in response to the NF-$textsc{k}$B activity and contains neomycin phosphotransferase (NPT) gene for the geneticin resistance in host cells was constructed and transfected into human keratinocyte cell line HaCaT. Human HaCaT transfectant cells secreted the SEAP enzyme into the culture medium in a time-dependent manner until 72h. NF-$textsc{k}$B activities were measured in the SEAP reporter gene assay using a fluorescent detection method. The treatment of HaCaT cell transfectants with known antioxidants [e.g., N-acetyl-L-cysteine and vitamin C] showed inhibition of NF-$textsc{k}$B activity in a time-and concentration-dependent manner. The phorbol 12-myristate 13-acetate (PMA) known as a stimulator of NF-$textsc{k}$B expression demonstrated that it increased NF-$textsc{k}$B activity in a time- and concentration-dependent manner. This assay system could be used to determine the quantitative measurement of NF-$textsc{k}$B activity in the human skin and allow the screening of anti-inflammatory agents from various synthetic chemicals and natural products for dermatological purpose. Abbrevitions used: NF-$textsc{k}$B, nuclear factor kappa B; I-$textsc{k}$B, Inhibitory kappa B; SEAP, secreted alkaline phosphatase; NPT, neomycin phosphotransferease; PCR, polymerase chain reaction: dNTP, deoxynucleoside triphosphates; DMEM, dulbecco’s modified eagle medium; FBS, fetal bovine serum; PBs, phosphate-buffered saline; MUP, 4-methylumbellifery phosphate; NAC, N-acetyl-L-cysteine; DMSO, dimethyl sulfoxide; PMA, phorbol 12-myristate 13-acetate.

• Proceedings of the PSK Conference
• /
• 2002.10a
• /
• pp.390.3-391
• /
• 2002

Nuclear-kappa B(NF-$\kappa$B) plays a role in the regulation of genes responsible for inflammatory and immune responses as well as growth control of cells. A cell-based assay system for guiding NF-KB activity was developed to determine the influence of activated NF-KB in human keratinocytes. It suggested that this system could be used to determine the quantitative measurement of NF-$\kappa$B activity in the human skin and allow the monitoring of anti-inflammatory agent for dermatological means from various environmental stimuli. (omitted)