endothelin-1 and astaxanthine

Topics: Endothelin-1; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Skin; Skin Aging; Stem Cell Factor; Xanthophylls

Paracrine interactions between keratinocytes and melanocytes via cytokines play an essential role in regulating pigmentation in epidermal hyperpigmentary disorders. There is an urgent need for a human epidermal model in which melanogenic paracrine interactions between UVB-exposed keratinocytes and melanocytes can be precisely evaluated because human epidermal equivalents consisting of multilayered keratinocytes and melanocytes have significant limitations in this respect.. To resolve this challenge, we established a co-culture system with cell inserts using human keratinocytes and human melanocytes that serves as an appropriate new model for UVB-induced hyperpigmentation. Using that new model, we examined the blocking effects of two natural chemicals, astaxanthin and withaferin A, on paracrine cytokine interactions between UVB-exposed keratinocytes and melanocytes and characterized their mechanisms of action.. RT-PCR analysis showed that co-culture of human keratinocytes that had been exposed to UVB significantly stimulated human melanocytes to increase their expression of genes encoding microphthalmia-associated transcription factor, tyrosinase and tyrosinase-related protein 1. The catalytic activity of tyrosinase was also increased. ELISA assays revealed that UVB significantly increased the secretion of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1 but not α-melanocyte stimulating hormone. The addition of an endothelin-1 neutralizing antibody significantly abrogated the increase of tyrosinase activity. Post-irradiation treatment with astaxanthin or withaferin A significantly abolished the up-regulation of tyrosinase activity induced by UVB. Treatment with astaxanthin or withaferin A significantly reduced the increased levels of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1. Withaferin A but not astaxanthin also significantly abrogated the endothelin-1-stimulated activity of tyrosinase in melanocytes. Western blot analysis of intracellular signaling factors revealed that withaferin A but not astaxanthin significantly abolished the endothelin-1-stimulated phosphorylation of Raf-1, MEK, ERK, MITF and CREB in human melanocytes.. These results demonstrate that this co-culture system is an appropriate model to characterize melanogenic paracrine interactions and that astaxanthin and withaferin A serve as potent inhibitors of those interactions. Their effects are caused not only by down-regulating the increased secretion of an intrinsic melanogenic cytokine, endothelin-1, by UVB-exposed human keratinocytes, but also by interrupting the endothelin-1-triggered downstream intracellular signaling between protein kinase C and Raf-1 in human melanocytes (only for withaferin A).

Topics: Antibodies; Calcium; Cell Line, Tumor; Coculture Techniques; Cytokines; Dithiothreitol; Endothelin-1; Gene Expression Regulation; Humans; Intracellular Space; Keratinocytes; Melanocytes; Melanoma; Monophenol Monooxygenase; Paracrine Communication; Phosphorylation; Signal Transduction; Ultraviolet Rays; Withanolides; Xanthophylls

We established a model for the stem cell factor (SCF)-associated stimulation of human epidermal equivalent (HEE) pigmentation. The addition of SCF (at 5 nM) gradually stimulated the visible pigmentation of HEEs over 14 days of treatment. A time course study using real-time RT-PCR and western blotting analysis demonstrated that the expression of all melanocyte-specific genes and proteins examined was gradually up-regulated over 7-10 days of treatment with SCF. The addition of astaxanthin (Ax) at concentrations of 1, 4, or 8 μM markedly abolished the SCF- but not the endothelin (EDN)1-elicited increase in visible pigmentation over 14 days in a dose-dependent manner, with almost complete inhibition at 8 μM. While no degeneration of the epidermal tissue was visible at day 14 by HE staining, melanin deposition throughout the epidermis was markedly reduced in the Ax-treated HEEs at day 14 compared to untreated controls. Ax significantly reduced the eumelanin content of HEEs to the non-SCF-stimulated level at concentrations of 4 or 8 μM compared with untreated controls. Real-time RT-PCR and western blotting of Ax-treated HEEs revealed that the SCF-stimulated expression of tyrosinase (TYR), TYR-related protein-1 (TYRP1), and Pmel17, as well as microphthalmia-associated transcription factor (MITF), is significantly suppressed by Ax at the transcriptional and translational levels. Studies using cultured normal human melanocytes revealed that pre-treatment with Ax interrupts the SCF- but not the EDN1-induced stimulation of TYR activity, and there was no direct inhibitory effect of Ax on TYR activity in vitro. These findings indicate that Ax attenuates SCF-stimulated pigmentation by directly interrupting SCF-associated intracellular signaling linkages through increased expression of MITF, which leads to the stimulated expression of melanogenic genes and proteins in a reactive oxygen species depletion-independent mechanism.

Topics: Cells, Cultured; Endothelin-1; Enzyme Activation; Epidermis; Gene Expression Regulation; gp100 Melanoma Antigen; Humans; Hyperpigmentation; Melanocytes; Membrane Glycoproteins; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Oxidoreductases; Skin Pigmentation; Stem Cell Factor; Xanthophylls