Introduction: In human skin, melanocytes are located in the dermal-epidermal junction. Keratinocytes, fibroblasts and melanocytes are communicated with each other via interactive factors to regulate the function and the phenotype of the skin color. It is well known that keratinocyte-derived paracrine factors, for example Endothelin-1, significantly contribute to UV-induced pigmentation. Recently, there is a great interest in regulation of melanin production in melanocytes by fibroblast-derived paracrine factors. In previous studies, several fibroblast-derived paracrine factors such as Dickkopf1 (DKK1) and Stromal Derived Factor-1 and others have been identified as inhibitory regulators of melanin production, while Neuregulin-1 and Stem Cell Factor and others have been identified as promotive regulators. The amount of these pigmentation-related fibroblast-derived paracrine factors are altered in sun-exposed areas, suggesting that sun-exposed keratinocytes might contribute to the secretion of fibroblast-derived paracrine factors. However, the involvement of keratinocytes in interaction between fibroblast-derived paracrine factors and UV-induced pigmentation has not yet been confirmed.
Objective: The purpose of this study was to investigate the possibility that UV-exposed keratinocyte-derived paracrine factors modulate fibroblast-derived paracrine factors involved in melanin production.
Methods: All experiments were conducted with normal human cultured cells. First, normal human epidermal keratinocytes were pre-treated with or without an active ingredient (Zinc Glycinate) for 24 hours and then were exposed to UVB and were further incubated for 24 hours. After 24 hours incubation, the conditioned medium of keratinocytes (UVB-exposed Keratinocytes CM) was collected and was added to normal human dermal fibroblasts. Next, the cultured fibroblasts were incubated with UVB-exposed Keratinocytes CM for 24 hours, and then the conditioned medium (UVB-KC-Fibroblasts CM) was collected. Finally, normal human epidermal melanocytes were cultured in melanocyte growth medium with UVB-KC-Fibroblasts CM and were incubated for 72 hours. The amount of intracellular melanin contents was measured using a method of alkaline solubilization. The melanocytes with non-UVB-irradiated conditioned medium (KC-Fibroblasts CM) were used as a control. Interleukin-1α (IL-1α; a keratinocyte-derived inflammatory factor) and DKK1 (a pigmentation related fibroblast-derived factor) were quantified by ELISA. In addition, we confirmed the effect of Zinc-Glycinate on the UV-induced pigmentation. Zinc-Glycinate was reported as a suitable agent to suppress the release of inflammatory cytokines regarding the crosstalk between keratinocytes and fibroblasts in our previous study.
Results and Discussions: To investigate the crosstalk among keratinocytes, fibroblasts and melanocytes, we observed the melanin production in melanocytes which were treated with each conditioned medium of fibroblasts. The amount of melanin in melanocytes treated with KC-Fibroblasts CM was markedly reduced compared to normal melanocytes as previously reported in vitro. In contrast, the amount of melanin in UVB-KC-Fibroblasts CM treated melanocytes was comparable with normal melanocytes. Then, IL-1α protein production was increased in UVB-exposed Keratinocytes CM, and DKK1 protein production was significantly reduced in UVB-KC-Fibroblasts CM. We also found that a reduction in DKK1 was induced in IL-1α-treated fibroblasts as well as in UVB-Keratinocytes CM-treated fibroblasts. These results suggest that UV-induced keratinocyte-derived paracrine factor, such as IL-1α, suppresses the ability of fibroblast-derived paracrine factor DKK1 to regulate melanin production. This finding may be a novel mechanism supporting the promotion of epidermal inflammation-induced pigmentation. In addition, we found that Zinc-Glycinate suppressed the increase in IL-1α secretion in UVB-exposed Keratinocytes CM and recovered DKK1 expression in UVB-exposed Keratinocytes CM-treated fibroblasts and maintained the ability of fibroblasts to suppress melanin production in melanocytes.
Conclusion: This is a novel finding suggesting that the secretion of paracrine factors of fibroblasts involved in pigmentation is regulated by keratinocytes. In conclusion, the approach about interaction between keratinocytes and fibroblasts may be useful in the prevention of UVB-induced pigmentation.