Thierry Passeron

What are melanocytes really doing all day long...

Abstract:  Everyone knows and seems to agree that melanocytes are there to generate melanin – an intriguing, but underestimated multipurpose molecule that is capable of doing far more than providing pigment and UV protection to skin ( 1 ). What about the cell that generates melanin, then? Is this dendritic, neural crest‐derived cell still serving useful (or even important) functions when no‐one looks at the pigmentation of our skin and its appendages and when there is essentially no UV exposure? In other words, what do epidermal and hair follicle melanocytes do in their spare time – at night, under your bedcover? How much of the full portfolio of physiological melanocyte functions in mammalian skin has really been elucidated already? Does the presence or absence of melanoctyes matter for normal epidermal and/or hair follicle functions (beyond pigmentation and UV protection), and for skin immune responses? Do melanocytes even deserve as much credit for UV protection as conventional wisdom attributes to them? In which interactions do these promiscuous cells engage with their immediate epithelial environment and who is controlling whom? What lessons might be distilled from looking at lower vertebrate melanophores and at extracutaneous melanocytes in the endeavour to reveal the ‘secret identity’ of melanocytes? The current Controversies feature explores these far too infrequently posed, biologically and clinically important questions. Complementing a companion viewpoint essay on malignant melanocytes ( 2 ), this critical re‐examination of melanocyte biology provides a cornucopia of old, but under‐appreciated concepts and novel ideas on the slowly emerging complexity of physiological melanocyte functions, and delineates important, thought‐provoking questions that remain to be definitively answered by future research.

Dickkopf 1 (DKK1) regulates skin pigmentation and thickness by affecting Wnt/β-catenin signaling in keratinocytes

The epidermis (containing primarily ker‐atinocytes and melanocytes) overlies the dermis (containing primarily fibroblasts) of human skin. We previously reported that dickkopf 1 (DKK1) secreted by fibroblasts in the dermis elicits the hypopigmented phenotype of palmoplantar skin due to suppression of melanocyte function and growth via the regulation of two important signaling factors, microphthalmia‐associ‐ated transcription factor (MITF) and β‐catenin. We now report that treatment of keratinocytes with DKK1 increases their proliferation and decreases their uptake of melanin and that treatment of reconstructed skin with DKK1 induces a thicker and less pigmented epidermis. DNA microarray analysis revealed many genes regulated by DKK1, and several with critical expression patterns were validated by reverse transcriptase‐poly‐merase chain reaction and Western blotting. DKK1 induced the expression of keratin 9 and α‐Kelch‐like ECT2 interacting protein (αKLEIP) but down‐regulated the expression of β‐catenin, glycogen synthase kinase 3β, protein kinase C, and proteinase‐activated recep‐tor‐2 (PAR‐2), which is consistent with the expression patterns of those proteins in human palmoplantar skin. Treatment of reconstructed skin with DKK1 repro‐duced the expression patterns of those key proteins observed in palmoplantar skin. These findings further elucidate why human skin is thicker and paler on the palms and soles than on the trunk through topographical and site‐specific differences in the secretion of DKK1 by dermal fibroblasts that affects the overlying epidermis. Yamaguchi Y., Passeron, T., Hoashi, T., Watabe, H., Rouzaud, F., Yasumoto, K., Hara, T., Tohyama, C., Katayama, I., Miki, T., Hearing V. J. Dickkopf 1 (DKK1) regulates skin pigmentation and thickness by affecting Wnt/β‐catenin signaling in kera‐tinocytes. FASEB J. 22, 1009–1020 (2008)

SOX9 is a key player in ultraviolet B-induced melanocyte differentiation and pigmentation

SOX (SRY type HMG box) proteins are transcription factors that are predominantly known for their roles during development. During melanocyte development from the neural crest, SOX10 regulates microphthalmia-associated transcription factor, which controls a set of genes critical for pigment cell development and pigmentation, including dopachrome tautomerase and tyrosinase. We report here that another SOX factor, SOX9, is expressed by melanocytes in neonatal and adult human skin and is up-regulated by UVB exposure. We demonstrate that this regulation is mediated by cAMP and protein kinase. We also show that agouti signal protein, a secreted factor known to decrease pigmentation, down-regulates SOX9 expression. In adult and neonatal melanocytes, SOX9 regulates microphthalmia-associated transcription factor, dopachrome tautomerase, and tyrosinase promoters, leading to an increase in the expression of these key melanogenic proteins and finally to a stimulation of pigmentation. SOX9 completes the complex and tightly regulated process leading to the production of melanin by acting at a very upstream level. This role of SOX9 in pigmentation emphasizes the poorly understood impact of SOX proteins in adult tissues.

Upregulation of SOX9 inhibits the growth of human and mouse melanomas and restores their sensitivity to retinoic acid.

Treatments for primary and metastatic melanomas are rarely effective. Even therapeutics such as retinoic acid (RA) that are successfully used to treat several other forms of cancer are ineffective. Recent evidence indicates that the antiproliferative effects of RA are mediated by the transcription factor SOX9 in human cancer cell lines. As we have previously shown that SOX9 is expressed in normal melanocytes, here we investigated SOX9 expression and function in human melanomas. Although SOX9 was expressed in normal human skin, it was increasingly downregulated as melanocytes progressed to the premalignant and then the malignant and metastatic states. Overexpression of SOX9 in both human and mouse melanoma cell lines induced cell cycle arrest by increasing p21 transcription and restored sensitivity to RA by downregulating expression of PRAME, a melanoma antigen. Furthermore, SOX9 overexpression in melanoma cell lines inhibited tumorigenicity both in mice and in a human ex vivo model of melanoma. Treatment of melanoma cell lines with PGD2 increased SOX9 expression and restored sensitivity to RA. Thus, combined treatment with PGD2 and RA substantially decreased tumor growth in human ex vivo and mouse in vivo models of melanoma. The results of our experiments targeting SOX9 provide insight into the pathophysiology of melanoma. Further, the effects of SOX9 on melanoma cell proliferation and RA sensitivity suggest the encouraging possibility of a noncytotoxic approach to the treatment of melanoma.

The deceptive nature of UVA tanning versus the modest protective effects of UVB tanning on human skin.

The relationship between human skin pigmentation and protection from ultraviolet (UV) radiation is an important element underlying differences in skin carcinogenesis rates. The association between UV damage and the risk of skin cancer is clear, yet a strategic balance in exposure to UV needs to be met. Dark skin is protected from UV‐induced DNA damage significantly more than light skin owing to the constitutively higher pigmentation, but an as yet unresolved and important question is what photoprotective benefit, if any, is afforded by facultative pigmentation (i.e. a tan induced by UV exposure). To address that and to compare the effects of various wavelengths of UV, we repetitively exposed human skin to suberythemal doses of UVA and/or UVB over 2 weeks after which a challenge dose of UVA and UVB was given. Although visual skin pigmentation (tanning) elicited by different UV exposure protocols was similar, the melanin content and UV‐protective effects against DNA damage in UVB‐tanned skin (but not in UVA‐tanned skin) were significantly higher. UVA‐induced tans seem to result from the photooxidation of existing melanin and its precursors with some redistribution of pigment granules, while UVB stimulates melanocytes to up‐regulate melanin synthesis and increases pigmentation coverage, effects that are synergistically stimulated in UVA and UVB‐exposed skin. Thus, UVA tanning contributes essentially no photoprotection, although all types of UV‐induced tanning result in DNA and cellular damage, which can eventually lead to photocarcinogenesis.

Glycoprotein nonmetastatic melanoma protein b, a melanocytic cell marker, is a melanosome-specific and proteolytically released protein

Melanosomes are organelles specialized for the production of melanin pigment and are specifically produced by melanocytic cells. More than 150 pigmentation‐related genes have been identified, including glycoprotein nonmetastatic melanoma protein b (GPNMB). A recent proteomics analysis revealed that GPNMB is localized in melanosomes, and GPNMB is a membrane‐bound glycoprotein that shows high homology with a well‐known melanosomal structural protein, Pmel17/gp100. In this study, we show that GPNMB is expressed in melanocytes of normal human skin, as well as in human melanoma cells. GPNMB is heavily glycosylated and is enriched in mature (stage III and IV) melanosomes in contrast to MART‐1 and Pmel17, which are abundant in early (stage I and II) melanosomes. MART‐1 and Pmel17 play critical roles in the maturation of early melanosomes; thus, we speculate that GPNMB might be important in the functions of late melanosomes, possibly their transport and/or transfer to keratinocytes. We also demonstrate that a secreted form of GPNMB is released by ectodomain shedding from the largely Golgi‐modified form of GPNMB and that the PKC and Ca2+ intracellular signaling pathways regulate that shedding. We conclude that GPNMB is a melanosomal protein that is released by proteolytic ectodomain shedding and might be a useful and specific histological marker of melanocytic cells.—Hoashi, T., Sato, S., Yamaguchi, Y., Passeron, T., Tamaki, K., Hearing, V. J. Glycoprotein nonmetastatic melanoma protein b, a melanocytic cell marker, is a melanosome‐specific and proteolytically released protein. FASEB J. 24, 1616–1629 (2010). www.fasebj.org

In vivo reflectance confocal microscopy detects pigmentary changes in melasma at a cellular level resolution

Please cite this paper as: In vivo reflectance confocal microscopy detects pigmentary changes in melasma at a cellular level resolution. Experimental Dermatology 2010; 19: e228–e233.

Microarray analysis sheds light on the dedifferentiating role of agouti signal protein in murine melanocytes via the Mc1r

The melanocortin-1 receptor (MC1R) is a key regulator of pigmentation in mammals and is tightly linked to an increased risk of skin cancers, including melanoma, in humans. Physiologically activated by α-melanocyte stimulating hormone (αMSH), MC1R function can be antagonized by a secreted factor, agouti signal protein (ASP), which is responsible for the lighter phenotypes in mammals (including humans), and is also associated with increased risk of skin cancer. It is therefore of great interest to characterize the molecular effects elicited by those MC1R ligands. In this study, we determined the gene expression profiles of murine melan-a melanocytes treated with ASP or αMSH over a 4-day time course using genome-wide oligonucleotide microarrays. As expected, there were significant reductions in expression of numerous melanogenic proteins elicited by ASP, which correlates with its inhibition of pigmentation. ASP also unexpectedly modulated the expression of genes involved in various other cellular pathways, including glutathione synthesis and redox metabolism. Many genes up-regulated by ASP are involved in morphogenesis (especially in nervous system development), cell adhesion, and extracellular matrix-receptor interactions. Concomitantly, ASP enhanced the migratory potential and the invasiveness of melanocytic cells in vitro. These results demonstrate the role of ASP in the dedifferentiation of melanocytes, identify pigment-related genes targeted by ASP and by αMSH, and provide insights into the pleiotropic molecular effects of MC1R signaling that may function during development and may affect skin cancer risk.

Melasma Treatment With Pulsed-Dye Laser and Triple Combination Cream: A Prospective, Randomized, Single-Blind, Split-Face Study

R ecent data show that melasma lesions have, in addition to increased pigmentation, more elastosis and vascularization than perilesional skin. The stabilized formulation of Kligman preparation has shown significant improvements in the treatment of melasma. However, most of the treatments only target the pigmentation, and none of them has been demonstrated so far to prevent the frequent relapses. Pulsed-dye laser treatment (PDL) is considered the gold standard therapy for vascular lesions. By targeting not only melanin but also vascularization and at least in part elastosis, PDL might provide, in combination with blanching cream, an effective and complete therapeutic approach for melasma. The objective of this pilot study was to evaluate the dual treatment of fixed triple combination cream (TCC) and PDL in the treatment of melasma.

Forskolin protects keratinocytes from UVB-induced apoptosis and increases DNA repair independent of its effects on melanogenesis.

Melanin pigments provide efficient protection against ultraviolet B (UVB) radiation but DNA repair also plays a key role in eliminating UV-induced damage and preventing the development of skin cancers. In this study, we demonstrate that forskolin (FSK), an agent that increases intracellular levels of cAMP, protects keratinocytes from UVB-induced apoptosis independently from the amount of melanin in the skin. FSK enhances the removal of the two major types of UVB-induced DNA damage, cyclobutane pyrimidine dimers and 6,4-photoproducts, by facilitating DNA repair. These findings suggest new preventive approaches with topical formulations of FSK or other bioactive agents that could be applied to the skin before sun exposure to increase its ability to repair DNA damage.