Inhibitory effect of Elephantopus mollis H.B. and K. extract on melanogenesis in B16 murine melanoma cells by downregulating microphthalmia-associated transcription factor expression.

In this study, the inhibitory effect of Elephantopus mollis H.B. and K. extract on melanogenesis in B16 murine melanoma cells was examined and possible mechanisms were elucidated. The melanin content in B16 cells decreased when they were treated with E. mollis extract. Inhibition was accompanied by reduced expression of tyrosinase (TYR) and tyrosinase-related protein 1 (TRP1). Furthermore, the expression level of microphthalmia-associated transcription factor (MITF), a major transcriptional regulator of genes encoding melanogenic enzymes such as Tyr and Trp1, decreased as assessed by western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR). These results suggest that E. mollis extract reduces melanogenesis by downregulating Mitf expression, leading to reduced expression of Tyr and Trp1. In addition, melanocortin-1 receptor (MC1R) expression was downregulated by E. mollis extract, suggesting desensitization to α-melanocyte-stimulating hormone (α-MSH) of cells treated with the extract.

Effects of fibroblast-derived factors on the proliferation and differentiation of human melanocytes in culture.

BACKGROUND: Although keratinocyte-derived factors are known to promote the proliferation and differentiation of human epidermal melanocytes, it is not fully understood whether fibroblast-derived factors work in a similar way. OBJECTIVE: The aim of this study is to clarify whether fibroblast-derived factors are involved in regulating the proliferation and differentiation of human melanocytes with or without keratinocytes using serum-free culture system. METHODS: Human epidermal melanoblasts and melanocytes were cultured in a serum-free growth medium supplemented with fibroblast-derived factors such as keratinocyte growth factor (KGF) with or without keratinocytes, and the effects of KGF on the proliferation and differentiation of melanocytes were studied. RESULTS: KGF stimulated the proliferation of melanoblasts in the presence of dibutyryl cAMP (DBcAMP), basic fibroblast growth factor (bFGF), transferrin (Tf), and endothelin-1 (ET-1). Although KGF stimulated the differentiation, melanogenesis, and dendritogenesis in the presence of DBcAMP, Tf, and ET-1 without keratinocytes, KGF required the presence of keratinocytes for the stimulation of melanocyte proliferation. CONCLUSION: These results suggest that fibroblast-derived KGF stimulates the proliferation of human melanoblasts in synergy with cAMP, bFGF, Tf, and ET-1, the differentiation of melanocytes in synergy with cAMP, Tf, and ET-1, and the proliferation of melanocytes in synergy with cAMP, Tf, ET-1, and undefined keratinocyte-derived factors.

Immunohistochemical survey of the distribution of epidermal melanoblasts and melanocytes during the development of UVB-induced pigmented spots.

BACKGROUND: Repeated exposures to ultraviolet B radiation (UVB) induce pigmented spots on dorsal skin of (HR-1 x HR/De) F(1) hairless mouse. We showed previously that this mouse is suitable for studies of melanocyte function. OBJECTIVE: To clarify the mechanism of development of pigmented spots induced by chronic UVB exposure. METHODS: We used light and fluorescence microscopy to quantify changes in the numbers of differentiated melanocytes containing melanin pigments (MM) and melanoblasts/melanocytes immunohistochemically positive for tyrosinase-related protein (TRP)-1, TRP-2 (dopachrome tautomerase), and c-kit in epidermis during the development of pigmented spots in hairless mice chronically exposed to UVB (99 mJ/cm(2), 3 times/week, 8 weeks). RESULTS: The change in the number of TRP-1-positive cells during chronic UVB exposure was similar to that of MM: both increased dramatically during the stage of acute pigmentation, then decreased sharply after cessation of UVB, concomitantly with depigmentation; subsequently they increased gradually with the development of pigmented spots. In contrast, after two UVB exposures, no c-kit-positive cells were detected, then the number gradually increased during UVB irradiation, and continued to increase after cessation of irradiation; TRP-2-positive cells showed a rather similar pattern, except that they did not disappear initially. CONCLUSION: Our results indicate that chronic UVB irradiation induces differentiation and proliferation of melanoblasts, followed by an increase of differentiated melanocytes, leading to the development of pigmented spots. The sequence of expression of markers appeared to be c-kit, TRP-2, TRP-1, and finally melanin, as it is during normal melanocyte differentiation.

Granulocyte-macrophage colony-stimulating factor is a keratinocyte-derived factor involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture.

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and a melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with granulocyte-macrophage colony-stimulating factor (GMCSF) from 14 days (keratinocyte depletion). GMCSF stimulated the number of melanoblasts/melanocytes as well as the percentage of differentiated melanocytes in keratinocyte-depleted cultures. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and G(2)/M phases of the cell cycle were increased by the treatment with GMCSF. Moreover, anti-GMCSF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts/melanocytes as well as the differentiation of melanocytes. Enzyme-linked immunosorbent assay of culture media revealed that GMCSF was secreted from keratinocytes, but not from melanocytes. These results suggest that GMCSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanoblasts/melanocytes in culture in cooperation with cAMP elevator and bFGF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the proliferation and differentiation of mouse epidermal melanocytes from pigmented spots induced by ultraviolet radiation B.

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB-induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor-free medium supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF induced the proliferation and differentiation of melanocytes in those keratinocyte-depleted cultures. Moreover, an antibody to GM-CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV-irradiated mice, but not from control mice. Further, the GM-CSF antibody inhibited the proliferation and differentiation of melanocytes co-cultured with keratinocytes derived from UV-irradiated mice, but not from control mice. The quantity of GM-CSF secreted from keratinocytes derived from the pigmented spots of UV-irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM-CSF in keratinocytes derived from the pigmented spots of skin in UV-irradiated mice, but not from normal skin in control mice. These results suggest that GM-CSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB-induced pigmented spots.

Changes in the proliferative activity of epidermal melanocytes in serum-free primary culture during the development of ultraviolet radiation B-induced pigmented spots in hairless mice.

Long-term exposure to ultraviolet radiation B (UVB) induced pigmented spots in the dorsal skin of hairless mice of strain (HR-1 X HR/De)F1. To clarify the cellular mechanism for the development of these UVB-induced pigmented spots, we investigated changes in the proliferative activity of epidermal melanoblasts and melanocytes in the dorsal skin at various weeks after UVB irradiation. Epidermal cell suspensions from the dorsal skin of hairless mice were cultured in a serum-free medium supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF). The suspensions were prepared from dorsal skins of mice exposed to UVB for 4 weeks (the stage of hyperpigmentation). Suspensions were also prepared from mice at 3 (the stage of depigmentation), 8 (the stage of appearance of pigmented spots), 20 (the stage of development of small-sized pigmented spots) and 37 (the stage of development of medium-sized pigmented spots) weeks after the cessation of 8-week UVB exposure. At the stage of hyperpigmentation the proliferative activity of melanoblasts and melanocytes was suppressed. With the development of pigmented spots, the proliferative activity of undifferentiated melanoblasts gradually increased, and then followed the increase in the proliferative activity of differentiated melanocytes. These results suggest that the proliferative activity of epidermal melanoblasts and melanocytes in UVB-irradiated skin increases with the development of pigmented spots.

Keratinocytes control the proliferation and differentiation of cultured epidermal melanocytes from ultraviolet radiation B-induced pigmented spots in the dorsal skin of hairless mice.

Long-term exposure of ultraviolet radiation B (UVB)-induced pigmented spots in the dorsal skin of hairless mice of Hos:(HR-1 X HR//De) F1. Previous study showed that the proliferative and differentiative activities of cultured epidermal melanoblasts/melanocytes from UVB-induced pigmented spots increased with the development of the pigmented spots. To determine whether the increase in the proliferative and differentiative activities of epidermal melanoblasts/melanocytes was brought about by direct changes in melanocytes, or by indirect changes in surrounding keratinocytes, pure cultured melanoblasts/melanocytes and keratinocytes were prepared and co-cultured in combination with control and irradiated mice in a serum-free culture medium. Keratinocytes from irradiated mice stimulated the proliferation and differentiation of both neonatal and adult non-irradiated melanoblasts/melanocytes more greatly than those from non-irradiated mice. In contrast, both non-irradiated and irradiated adult melanocytes proliferated and differentiated similarly when they were co-cultured with irradiated adult keratinocytes. These results suggest that the increased proliferative and differentiative activities of mouse epidermal melanocytes from UVB-induced pigmented spots are regulated by keratinocytes, rather than melanocytes.