Effects of Coccoloba uvifera L. on UV‐stimulated melanocytes

Background: Exposure to ultraviolet (UV) radiation induces generation of reactive oxygen species, production of proinflammatory cytokines and melanocyte‐stimulating hormone (MSH) as well as increase in tyrosinase activity. The potential photoprotective effects of Coccoloba uvifera extract (CUE) were evaluated in UV‐stimulated melanocytes. Methods: Human epidermal melanocytes were used as an in vitro model to evaluate the effects of CUE on the production interleukin‐1α (IL‐1α), tumor necrosis factor α (TNF‐α), and α‐MSH under basal and UV‐stimulated conditions. Antioxidant and anti‐tyrosinase activities were also evaluated in membrane lipid peroxidation and mushroom tyrosinase assay, respectively. Results: Coccoloba uvifera L. showed antioxidant and anti‐tyrosinase activities and also inhibited the production of IL‐1α, TNF‐α and α‐MSH in melanocytes subjected to UV radiation (P<0.01). Moreover, CUE inhibited the activity of tyrosine kinase in cell culture under basal and UV radiation conditions (P<0.001), corroborating the findings of the mushroom tyrosinase assay. Conclusion: This study supports the photoprotective potential of CUE.     

Prostaglandin D2 production in FM55 melanoma cells is regulated by α‐melanocyte‐stimulating hormone and is not related to melanin production

Please cite this paper as: Prostaglandin D₂ production in FM55 melanoma cells is regulated by α‐melanocyte‐stimulating hormone and is not related to melanin production. Experimental Dermatology 2010; 19 : 751–753. Abstract: This study shows that prostaglandins in human FM55 melanoma cells and epidermal melanocytes are produced by COX‐1. Prostaglandin production in FM55 melanoma cells was unrelated to that of melanin suggesting that the two processes can occur independently. α‐Melanocyte‐stimulating hormone, which had no effect on melanin production in FM55 cells, stimulated PGD₂ production in these cells without affecting PGE₂. While cAMP pathways may be involved in regulating PGD₂ production, our results suggest that α‐MSH acts independently of cAMP, possibly by regulating the activity of lipocalin‐type PGD synthase. This α‐MSH‐mediated effect may be associated with its role as an immune modulator.     

Increased alpha‐melanocyte‐stimulating hormone (α‐MSH) levels and melanocortin receptors expression associated with pigmentation in an NC/Nga mouse model of atopic dermatitis

Please cite this paper as: Increased alpha‐melanocyte‐stimulating hormone (α‐MSH) levels and melanocortin receptors expression associated with pigmentation in an NC/Nga mouse model of atopic dermatitis. Experimental Dermatology 2010; 19: 132–136. Abstract: Patients with a specific subtype of atopic dermatitis (AD) display particular patterns of pigmentation, such as ripple pattern pigmentation on the neck, pigmented macules on the lip and diffuse pigmentation. However, the mechanism underlying these patterns has not been determined. The purpose of our research is to investigate the factors influencing this type of pigmentation in AD. We observed that AD model mice (NC/Nga mice) displayed an increase in the number of 3, 4‐dihydroxyphenylalanine (Dopa)‐positive melanocytes in the epidermis and intestine (jejunum and colon) while in the inflammatory state. The plasma levels of alpha‐melanocyte‐stimulating hormone (α‐MSH) and adrenocoticotropin (ACTH) also increased in NC/Nga mice with dermatitis. Furthermore, the expression of melanocortin receptor 5 and melanocortin receptor 1 (MC1R) increased in the skin, and melanocortin receptor 3 (MC3R) expression increased in the intestine. However, the changes in the Dopa‐positive cells of conventional NC/Nga mice were not induced by treatment with either agouti (an MC1R antagonist) or agouti‐related protein (an MC3R antagonist). These results indicate that the pigmentation of AD is related to increased levels of α‐MSH, MC1R (in the skin) and MC3R (in the intestines).     

UVB‐inhibited H19 activates melanogenesis by paracrine effects

The long noncoding RNA H19 was reported to associate with melanogenesis. However, it remains unknown whether H19 expression will be changed by UVB irradiation and whether H19 will regulate melanocytes melanogenesis by paracrine effects. Here, we analysed the expression changes of H19 irradiated by UVB in keratinocytes and explored the mechanism of melanogenesis stimulated by H19 through paracrine effects. First, after keratinocytes were exposed to UVB irradiation, expression of H19 and pro‐opiomelanocortin (POMC) was measured by qRT‐PCR. Also, α‐melanocyte‐stimulating hormone (α‐MSH) contents in cells supernatant were measured by ELISA. Then, H19 siRNAs were designed and transfected into keratinocytes by liposome. The expression changes of H19, POMC and α‐MSH were detected. Besides, expression of p53 was detected by Western blot. After that, supernatant of keratinocytes with H19 siRNAs or negative control siRNA was cocultured with immortalized melanocyte line PIG1. Expression levels of MiTF, TYR, Rab27A, TYRP2, FSCN1 and MYO5A in PIG1 cells were detected by Western blot and qRT‐PCR. We found that H19 expression of keratinocytes cells decreased after UVB irradiation. However, the levels of POMC, α‐MSH and p53 were upregulated in UVB‐irradiated cells. Compared with the negative control, H19 siRNAs could significantly increase the expression of POMC, α‐MSH and p53. After supernatant of keratinocytes transfected with H19 siRNAs was cocultured with PIG1 cells, the levels of MiTF, TYR and Rab27A were upregulated in PIG1 cells. In conclusion, UVB‐inhibited H19 may promote α‐MSH secretion by p53 in keratinocytes and then regulate melanocytes melanogenesis through paracrine effects.     

Melanocyte‐protective effect of afzelin is mediated by the Nrf2‐ARE signalling pathway via GSK‐3β inactivation

Vitiligo is an acquired condition characterized by depigmented, cutaneous lesions that result from the death of pigment‐producing cells, melanocytes. The occurrence of oxidative stress has been proposed as a pathogenetic mechanism for melanocyte degeneration in vitiligo. Therefore, in this study, we investigated the cytoprotective effects of afzelin against oxidative stress and its mechanism of action in human epidermal melanocytes. We found that afzelin significantly inhibited hydrogen peroxide‐induced cell death, cellular reactive oxygen species production and lipid peroxidation in melanocytes. In an antioxidant response element (ARE)‐luciferase reporter assay, afzelin increased ARE‐luciferase reporter activity in a concentration‐dependent manner. Consistently, the expression of antioxidant genes, including NF‐E2‐related factor 2 (Nrf2), haem oxygenase‐1 (HO‐1) and catalase, was enhanced by afzelin treatment. Nuclear translocation of Nrf2 also increased in response to afzelin treatment. In addition, the phosphorylation of glycogen synthase kinase‐3β (GSK‐3β) was induced by afzelin treatment. The enhancement of HO‐1 gene expression by afzelin treatment was reduced by Nrf2‐siRNA expression. Furthermore, we found that the expression of Nrf2‐siRNA significantly attenuated the cytoprotective effect of afzelin against hydrogen peroxide. These data suggest that the cytoprotective effects of afzelin against hydrogen peroxide may be mediated by Nrf2‐ARE signalling via GSK‐3β inactivation. Our data suggest the novel use of afzelin for the prevention of oxidative stress‐induced damage in melanocytes and its potential as a therapeutic agent for vitiligo.     

Tropisetron via α7 nicotinic acetylcholine receptor suppresses tumor necrosis factor‐α‐mediated cell responses of human keratinocytes

Tropisetron is a serotonin receptor (5‐HT‐R)‐modulating agent and approved as an antiemetic for patients undergoing chemotherapy. In the gut, it acts via specific serotonin receptors, 5‐HT₃‐R, to elicit its beneficial effects against nausea. We investigated whether tropisetron can affect inflammatory cell responses of human primary epidermal keratinocytes (NHK) which are key cells in the regulation of skin homoeostasis. Tropisetron significantly and dose‐dependently suppressed tumor necrosis factor (TNF)‐α‐mediated mRNA expression and protein secretion of interleukin (IL)‐6 and IL‐8 in these cells. This effect of tropisetron was independent of p65/NF‐κB as shown by various NF‐κB signal transduction read‐outs. Importantly, the anti‐inflammatory tropisetron effect on NHK was neither mediated by 5‐HT₃‐R nor 5‐HT₄‐R since these receptors were absent in NHK. In contrast, NHK expressed α7 nicotinic acetylcholine receptors (α7nAchR) which previously were found to bind tropisetron. The α7nAchR antagonist α‐bungarotoxin neutralized, whereas AR‐R17779, a specific α7nAchR agonist, mimicked the suppressive effect of tropisetron on TNF‐α‐mediated IL‐6 and IL‐8 expression in NHK. Our findings suggest that tropisetron and probably other α7nAchR‐activating agents could be useful for the future therapy of inflammatory skin diseases.     

Role of epidermal γδ T‐cell‐derived interleukin 13 in the skin‐whitening effect of Ginsenoside F1

Ginsenoside F1 (GF1) is a metabolite of ginsenoside Rg1. Although GF1 has several benefits for skin physiology, the effect of GF1 on skin pigmentation has not been reported. We found that a cream containing 0.1% GF1 showed a significant whitening effect on artificially tanned human skin after 8 weeks of application. However, GF1 did not inhibit mRNA expression of tyrosinase or dopachrome tautomerase (DCT) in normal human epidermal melanocytes (NHEMs) or cocultured NHEMs/normal human epidermal keratinocytes. Interestingly, GF1 enhanced production of interleukin 13 (IL‐13) from human epidermal γδ T cells. IL‐13 significantly reduced the mRNA expression and protein amount of both tyrosinase and DCT and reduced melanin synthesis activities in NHEMs, resulting in visible brightening of NHEM pellet. These results suggest that enhancement of IL‐13 production by GF1 from epidermal γδ T cells might play a role in the skin‐whitening effect of GF1 via the suppression of tyrosinase and DCT.     

Effective induction of melanoma‐antigen‐specific CD8+ T cells via Vγ9γδT cell expansion by CD56high+ Interferon‐α‐induced dendritic cells

Dendritic cells (DCs) can be differentiated from CD14⁺ monocytes in the presence of interferon‐α (IFNα) and granulocyte/macrophage‐colony stimulating factor (GM‐CSF) in vitro and are known as IFN‐DCs. Circulating blood CD56⁺ cells expressing high levels of CD14, HLA‐DR and CD86 have been shown to spontaneously differentiate into DC‐like cells in vitro after their isolation from blood. We show here that IFN‐DCs expressing high levels of CD56 (hereafter, CD56^high+ IFN‐DCs) can be differentiated in vitro from monocytes obtained as adherent cells from healthy donors and patients with metastatic melanoma. These cells expressed high levels of CD14, HLA‐DR and CD86 and possessed many pseudopodia. These CD56^high+ IFN‐DCs may be an in vitro counterpart of the circulating CD56⁺ CD14⁺ CD86⁺ HLA‐DR⁺ cells in blood. Conventional mature DCs differentiated from monocytes as adherent cells in the presence of GM‐CSF, IL‐4 and TNF‐α (hereafter, mIL‐4DCs) did not express CD56 or CD14. In contrast to mIL‐4DCs, the CD56^high+ IFN‐DCs exhibited a stronger capacity to stimulate autologous CD56⁺ Vγ9γδT cells highly producing IFNγ in the presence of zoledronate and IL‐2. The CD56^high+ IFN‐DCs possessing HLA‐A*0201 effectively induced Mart‐1‐modified melanoma peptide (A27L)‐specific CD8⁺ T cells through preferential expansion of CD56⁺ Vγ9γδT cells in the presence of A27L, zoledronate and IL‐2. Vaccination with CD56^high+ IFN‐DCs copulsed with tumor antigens and zoledronate may orchestrate the induction of various CD56⁺ immune cells possessing high effector functions, resulting in strong immunological responses against tumor cells. This study may be relevant to the design of future clinical trials of CD56^high+ IFN‐DCs‐based immunotherapies for patients with melanoma.     

Experimental study on cross‐reactivity of α‐arbutin toward p‐phenylenediamine and hydroquinone in guinea pigs

Hydroquinone (HQ) has been used as a skin‐lightening cosmetic ingredient, while it has been known that HQ shows sensitizing potential and cross‐reactivity toward a strong sensitizer, p‐phenylenediamine (PPD). α‐Arbutin, a glycoside of HQ (4‐hydroxyphenyl α‐D‐glucopyranoside), is used worldwide as a skin‐lightening agent. The aim of this study was to evaluate the cross‐reactivity of α‐arbutin toward PPD and HQ. All tests were performed using the guinea pig maximization test. In experiments on the cross‐reactivity of α‐arbutin or HQ to PPD, six animals in each group were induced with PPD at 0.1% by i.d. injection and at 1.0% by topical application. The animals were challenged with α‐arbutin, HQ or PPD (as a positive control) at concentrations of 0.01%, 0.05% and 0.1%. In experiments on the cross‐reactivity of α‐arbutin to HQ, four animals in each group were induced with HQ at 2% by i.d. injection and at 1% by topical application. The animals were challenged with α‐arbutin or HQ (as a positive control) at concentrations of 0.2%, 2% and 20%. The cross‐reactivity toward PPD was observed with HQ (4/6) only at 0.1% challenge. However, α‐arbutin showed no apparent cross‐reactivity to either PPD or HQ even at their highest challenge concentrations. Potent sensitization was observed with PPD (6/6) even at 0.01% challenge and with HQ (3/4) at 0.2%. In conclusion, glycosylation of HQ remarkably reduced the sensitization potency of HQ and the cross‐reactivity of HQ to PPD.     

Autophagy induced by resveratrol suppresses α‐MSH‐induced melanogenesis

Autophagy degrades cellular components and organelles through a cooperative process involving autophagosomes and lysosomes. Although autophagy is known to mainly regulate the turnover of cellular components, the role of autophagy in melanogenesis has not been well addressed. Here, we show that inhibition of autophagy suppresses the antimelanogenesis activity of resveratrol (RSV), a well‐known antimelanogenic agent. RSV strongly increased autophagy in melanocytes. However, the depletion of ATG5 significantly suppressed RSV‐mediated antimelanogenesis as well as RSV‐induced autophagy in melanocytes. Moreover, suppression of ATG5 retrieved the RSV‐mediated downregulation of tyrosinase and TRP1 in α‐MSH‐treated cells. Most importantly, electron microscopy analysis revealed that autophagosomes engulfed melanin or melanosomes after combined treatment of α‐MSH and RSV. Taken together, these results suggest that RSV‐mediated autophagy regulates melanogenesis.     

Mannosylerythritol lipids inhibit melanogenesis via suppressing ERK‐CREB‐MiTF‐tyrosinase signalling in normal human melanocytes and a three‐dimensional human skin equivalent

Hyperpigmentation is caused by excessive production of melanin in melanocytes. Mannosylerythritol lipids (MELs) are glycolipid biosurfactants that are abundantly produced by yeasts and used commercially in cosmetics. However, the potential depigmenting efficacy of MELs has not been evaluated. In this study, the depigmentary effect of MELs was tested in primary normal human melanocytes (NHMs), α‐melanocyte‐stimulating hormone (MSH)‐stimulated B16 cells (murine melanoma cells) and a human skin equivalent (MelanoDerm) using photography, Fontana‐Masson (F&M) staining and two‐photon microscopy. Mannosylerythritol lipids significantly decreased the melanin contents in NHMs and α‐MSH‐stimulated B16 cells. Consistent with these findings, MELs treatment had a clear whitening effect in a human skin equivalent, brightening the tissue colour and reducing the melanin content. The molecular mechanism underlying the anti‐melanogenic effect of MELs treatment was examined by real‐time PCR and Western blotting. Mechanistically, MELs clearly suppressed the gene expression levels of representative melanogenic enzymes, including tyrosinase, Tyrp‐1 and Tyrp‐2, by inhibiting the ERK/CREB/MiTF signalling pathway in NHMs. This work demonstrates for the first time that MELs exert whitening effects on human melanocytes and skin equivalent. Thus, we suggest that MELs could be developed as a potent anti‐melanogenic agent for effective whitening, beyond their use as a biosurfactant in cosmetics.     

Melanotropic peptides: more than just ‘Barbie drugs’ and ‘sun‐tan jabs’?

While ultraviolet radiation (UVR) is a major cause of skin ageing and carcinogenesis, public pursuit of a novel tanning strategy circumventing the need for UVR is increasingly reported in the media and scientific press. This involves the subcutaneous self‐administration of unregulated products labelled as melanotan I and/or II, synthetic analogues of α‐melanocyte stimulating hormone (α‐MSH), as obtained via the internet, tanning salons and gyms. The Medicines and Healthcare products Regulatory Authority has recently raised awareness of the public health risk of transmission of blood‐borne viruses from the needle sharing that may occur, and of the potential impurity of these products. Dermatologists should also be aware that these agents can complicate the clinical presentation of patients with pigmented lesions; their use may be suspected in unexpectedly tanned individuals with rapidly pigmenting naevi. Meanwhile, the regulated α‐MSH analogue afamelanotide (Clinuvel Pharmaceuticals Ltd, Melbourne, Australia) is showing promise for its photoprotective potential, and is undergoing phase II and III clinical trials in people with photosensitivity disorders and those prone to nonmelanoma skin cancer. The photoprotective and other biological effects of α‐MSH analogues await full determination.     

Staphylococcal α‐toxin induces a functional upregulation of TLR‐2 on human peripheral blood monocytes

Resistance to bacterial skin infections, for example with Staphylococcus aureus (S. aureus), is based on the function of intact innate immune mechanisms. Toll‐like receptor (TLR)‐2 recognizes components of S. aureus and is known to be expressed on monocytes. Staphylococcal exotoxins such as staphylococcal enterotoxin B (SEB) or α‐toxin are produced by many S. aureus strains. To investigate TLR‐2 regulation and function on human monocytes upon stimulation with staphylococcal exotoxins to elucidate a putative feedback loop between different staphylococcal components. Monocytes were stimulated with α‐toxin or SEB, respectively. TLR‐2 expression and regulation as well as functional effects of TLR‐2 stimulation with Pam3Cys (TLR‐2/TLR‐1), lipoteichoic acid (LTA) (TLR‐2/TLR‐6) and peptidoglycan (PGN) (TLR‐2 and Nod) were then investigated both at the mRNA and protein level and compared to monocytes from patients with psoriasis. α‐toxin significantly upregulated TLR‐2 expression. TLR‐2 mediated IL‐1β, IL‐6 and IL‐8 secretion was significantly augmented after upregulation with staphylococcal exotoxins. CD36 expression was significantly more downregulated after TLR‐2 upregulation with SEB and consecutive LTA stimulation and TLR‐2 upregulation with α‐toxin following LTA and PGN stimulation, respectively. PGN enhanced CD54 expression after upregulation of the receptor with α‐toxin. Expression of HLA‐DR was unaffected. However, no differences were observed in monocytes from psoriasis patients compared to healthy controls. Together, our findings provide a new link between staphylococcal α‐toxin and TLR‐2 signalling in monocytes which may have implications for skin diseases where skin colonization with S. aureus and dysregulation of TLR‐2 have been described.