Tae-Jin Yoon

Inhibitory Effects of Arbutin on Melanin Biosynthesis of α-Melanocyte Stimulating Hormone-induced Hyperpigmentation in Cultured Brownish Guinea Pig Skin Tissues

Arbutin has been used as a whitening agent in cosmetic products. Melanin, the major pigment that gives color to skin, may be over-produced with sun exposure or in conditions such as melasma or hyperpigmentary diseases. Tyrosinase is a key enzyme that catalyzes melanin synthesis in melanocytes; therefore, inhibitors of the tyrosinase enzyme could be used for cosmetic skin whitening. A recent study has reported that arbutin decreases melanin biosynthesis through the inhibition of tyrosinase activity. However, this inhibitory mechanism of arbutin was not sufficiently demonstrated in skin tissue models. We found that arbutin both inhibits melanin production in B16 cells induced with α-MSH and decreases tyrosinase activity in a cell-free system. Furthermore, the hyperpigmentation effects of α-MSH were abrogated by the addition of arbutin to brownish guinea pig and human skin tissues. These results suggest that arbutin may be a useful agent for skin whitening.

Involvement of pigment globules containing multiple melanosomes in the transfer of melanosomes from melanocytes to keratinocytes

The mechanism of melanosome transfer from melanocytes to keratinocytes has not been fully clarified. We now show a route of melanosome transfer using co-cultures of normal human melanocytes and keratinocytes. Substantial levels of melanosome transfer were elicited in co-cultures of melanocytes and keratinocytes separated by a microporous membrane filter. The melanocyte dendrites penetrated into the keratinocyte layer through the filter and many pigment globules were observed in keratinocytes. Electron microscopic observations revealed that melanosomes incorporated in keratinocytes were packed in clusters enclosed by a double membrane. Numerous pigment globules budded off from melanocyte dendrites and were released into the culture medium. Those pigment globules were filled with multiple melanosomes and a few mitochondria but no nuclei. When those globules were added to the culture medium of keratinocytes, they were incorporated and showed double membrane-enclosed melano-phagolysosomes consistent with the structures obtained from the co-culture system. In contrast, when individual naked melanosomes isolated from melanocytes were added to keratinocytes, they were also phagocytosed by keratinocytes but were enclosed by a single membrane in a manner distinct from the co-culture system. These results suggest a novel mechanism of melanosome transfer, wherein melanosomes are packed in membrane globules that bud off from melanocyte dendrites, where they are released into the extracellular space and then phagocytosed by keratinocytes.

Pitx2, a β-catenin-regulated transcription factor, regulates the differentiation of outer root sheath cells cultured in vitro

BACKGROUND Beta-catenin exerts its crucial role in hair follicle development and hair growth cycle. Although the importance of Wnt/beta-catenin is well recognized, the downstream effectors of beta-catenin have not been clearly elucidated yet. OBJECTIVE The aim of this study is to identify the beta-catenin-regulated genes in cultured human hair outer root sheath (ORS) cells. METHODS We transduced ORS cells with adenovirus harboring the expression cassette for constitutive active form of beta-catenin, then performed cDNA microarray. RESULTS Overexpression of beta-catenin led to the upregulation of hair cell differentiation markers such as keratin 16 and 17. In addition, the expression of Pitx2, a bicoid-type homeodomain transcription factor, was also increased by overexpression of beta-catenin in ORS cells cultured in vitro. To investigate the potential role of Pitx2, we made the recombinant adenovirus expressing Pitx2, then transduced into the cultured ORS cells. Interestingly, Pitx2 induced the expression of keratin 16 and 17, indicating that Pitx2 activates ORS cells towards the follicular differentiation pathway preferentially. CONCLUSION Our results implicate the potential importance of Pitx2 as a beta-catenin downstream modulator in hair growth control.

Keratinocytes in culture accumulate phagocytosed melanosomes in the perinuclear area

There are many techniques for evaluating melanosome transfer to keratinocytes but the spectrophotometric quantification of melanosomes incorporated by keratinocyte phagocytosis has not been previously reported. Here we describe a new method that allows the spectrophotometric visualization of melanosome uptake by normal human keratinocytes in culture. Fontana‐Masson staining of keratinocytes incubated with isolated melanosomes showed the accumulation of incorporated melanosomes in the perinuclear areas of keratinocytes within 48 h. Electron microscopic observations of melanosomes ingested by keratinocytes revealed that many phagosomes containing clusters of melanosomes or their fragments were localized in the perinuclear area. A known inhibitor of keratinocyte phagocytosis which inhibits protease‐activated receptor‐2, i.e., soybean trypsin inhibitor, decreased melanosome uptake by keratinocytes in a dose‐dependent manner. These data suggest that our method is a useful model to quantitate keratinocyte phagocytosis of melanosomes visually in vitro.

Induction of Melanogenesis by Rapamycin in Human MNT-1 Melanoma Cells

Background Melanogenesis is one of the characteristic parameters of differentiation in melanocytes and melanoma cells. Specific inhibitors of phosphatidylinositol 3-kinase (PI3K), such as wortmannin and LY294002, stimulate melanin production in mouse and in human melanoma cells, suggesting that PI3K and mammalian target of rapamycin (mTOR) might be involved in the regulation of melanogenesis. Objective The involvement of the mTOR pathway in regulating melanogenesis was examined using human MNT-1 melanoma cells, and the effects of the potent inhibitor of mTOR, rapamycin, in the presence or absence of α-melanocyte-stimulating hormone (α-MSH) were evaluated. Methods In cells treated with rapamycin, cell viability, melanin content, and tyrosinase (TYR) activity were measured and compared with untreated controls. Protein levels of TYR, tyrosinase-related protein (TYRP)-1, TYRP-2, and microphthalmia-associated transcription factor (MITF) were also analyzed by Western blot. Results In rapamycin-treated cells, the melanin content increased concomitantly with an elevation in TYR activity, which plays a major role in melanogenesis. There was also an up-regulation of TYR, TYRP-1, and MITF proteins. Combined treatment with rapamycin or wortmannin and α-MSH increased melanogenesis more strongly than α-MSH alone. Conclusion Rapamycin-induced melanin formation may be mediated through the up-regulation of TYR protein and activity. Furthermore, rapamycin and wortmannin, inhibitors of mTOR and PI3K, respectively, have co-stimulatory effects with α-MSH in enhancing melanogenesis in melanocyte cells.

Melanosome uptake is associated with the proliferation and differentiation of keratinocytes

Melanosomes are synthesized in melanocytes and transferred to neighboring keratinocytes. However, the associations of melanosome uptake with the proliferation and differentiation of keratinocytes are not fully understood. We examined the associations of melanosome uptake with keratinocyte differentiation and proliferation. SV40T-transformed human epidermal keratinocytes (SV-HEKs) were treated with isolated melanosomes. The effects of melanosome uptake on the proliferation and differentiation of the keratinocytes were analyzed by Western blotting and flow cytometry. The relationship between melanosome uptake and keratinocyte differentiation status was verified by determining the melanin content in the cells. Melanosomes reduced the proliferation of SV-HEKs in a dose-dependent manner, but did not induce differentiation. Melanosome uptake was higher in differentiating keratinocytes compared to non-differentiating keratinocytes, and inhibited significantly by PAR-2 inhibitor. Melanosomes inhibit keratinocyte proliferation. Moreover, melanosome uptake is influenced by keratinocyte differentiation status, being highest in mid-stage differentiating keratinocytes in a PAR-2 dependent manner.

Impact of NAD(P)H:Quinone Oxidoreductase-1 on Pigmentation

We obtained metastasized melanoma tissue from a primary acral lentiginous melanoma (ALM) patient and established a melanoma cell line named primary culture of melanoma cell derived from lymph node (PML)-1. PML-1 cells had a light brown color and decreased the expression of melanogenesis markers, including tyrosinase (TYR), microphthalmia-associated transcription factor, and tyrosinase-related protein-1. To identify genes differentially regulated in PML-1 melanoma cells, we performed DNA microarray and two-dimensional matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. Among the candidate genes identified, we chose NAD(P)H:quinone oxidoreductase-1 (NQO1) for further study. Reverse transcription-PCR and western blot analyses showed that NQO1 was markedly decreased in PML-1 cells and in several amelanotic melanoma cell lines. To investigate whether NQO1 affects the melanogenesis, we treated the cultured normal human melanocytes (NHMC) and zebrafish with NQO1 inhibitors, ES936 and dicoumarol. Interestingly, melanogenesis was significantly decreased by the addition of NQO1 inhibitors in both NHMC and zebrafish models. In contrast, overexpression of NQO1 using a recombinant adenovirus clearly induced melanogenesis, concomitantly with an increase of TYR protein level. These results suggest that NQO1 is a positive regulator of the pigmentation process.

Nrf2 negatively regulates melanogenesis by modulating PI3K/Akt signaling.

Nrf2 plays a role in protection of cells against oxidative stress and xenobiotic damage by regulating cytoprotective genes. In this study, we investigated the effect of Nrf2 on melanogenesis in normal human melanocytes (NHMCs). When NHMCs were transduced with a recombinant adenovirus expressing Nrf2, melanin synthesis was significantly decreased. Consistent with this result, overexpression of Nrf2 decreased the expression of tyrosinase and tyrosinase-related protein 1. The inhibitory effect of Nrf2 was reversed by overexpression of Keap1, an intracellular regulator of Nrf2. Interestingly, Nrf2 overexpression resulted in marked activation of PI3K/Akt signaling. Conversely, inhibition of PI3K activity by treatment with wortmannin reversed the depigmentary effects of Nrf2. Taken together, these results strongly suggest that Nrf2 negatively regulates melanogenesis by modulating the PI3K/Akt signaling pathway.

Hair greying is associated with active hair growth

Background  Hair greying is an obvious sign of ageing in humans. White (nonpigmented) hair is thicker than black (pigmented) hair. The growth rate of white hair is also significantly higher than that of black hair. However, the mechanism underlying this is largely unknown.

protective effect of tetracycline against dermal toxicity induced by Jellyfish venom.

Background Previously, we have reported that most, if not all, of the Scyphozoan jellyfish venoms contain multiple components of metalloproteinases, which apparently linked to the venom toxicity. Further, it is also well known that there is a positive correlation between the inflammatory reaction of dermal tissues and their tissue metalloproteinase activity. Based on these, the use of metalloproteinase inhibitors appears to be a promising therapeutic alternative for the treatment of jellyfish envenomation. Methodology and Principal Findings Tetracycline (a metalloproteinase inhibitor) has been examined for its activity to reduce or prevent the dermal toxicity induced by Nemopilema nomurai (Scyphozoa: Rhizostomeae) jellyfish venom (NnV) using in vitro and in vivo models. HaCaT (human keratinocyte) and NIH3T3 (mouse fibroblast) incubated with NnV showed decreases in cell viability, which is associated with the inductions of metalloproteinase-2 and -9. This result suggests that the use of metalloproteinase inhibitors, such as tetracycline, may prevent the jellyfish venom-mediated local tissue damage. In vivo experiments showed that comparing with NnV-alone treatment, tetracycline pre-mixed NnV demonstrated a significantly reduced progression of dermal toxicity upon the inoculation onto rabbit skin. Conclusions/Significance It is believed that there has been no previous report on the therapeutic agent of synthetic chemical origin for the treatment of jellyfish venom-induced dermonecrosis based on understanding its mechanism of action except the use of antivenom treatment. Furthermore, the current study, for the first time, has proposed a novel mechanism-based therapeutic intervention for skin damages caused by jellyfish stings.