Francois Rouzaud

Mesenchymal–epithelial interactions in the skin: increased expression of dickkopf1 by palmoplantar fibroblasts inhibits melanocyte growth and differentiation

We investigated whether or not the topographic regulation of melanocyte differentiation is determined by mesenchymal–epithelial interactions via fibroblast-derived factors. The melanocyte density in palmoplantar human skin (i.e., skin on the palms and the soles) is five times lower than that found in nonpalmoplantar sites. Palmoplantar fibroblasts significantly suppressed the growth and pigmentation of melanocytes compared with nonpalmoplantar fibroblasts. Using cDNA microarray analysis, fibroblasts derived from palmoplantar skin expressed high levels of dickkopf 1 (DKK1; an inhibitor of the canonical Wnt signaling pathway), whereas nonpalmoplantar fibroblasts expressed higher levels of DKK3. Transfection studies revealed that DKK1 decreased melanocyte function, probably through β-catenin–mediated regulation of microphthalmia-associated transcription factor activity, which in turn modulates the growth and differentiation of melanocytes. Thus, our results provide a basis to explain why skin on the palms and the soles is generally hypopigmented compared with other areas of the body, and might explain why melanocytes stop migrating in the palmoplantar area during human embryogenesis.

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)

The repeat domain of the melanosomal matrix protein PMEL17/GP100 is required for the formation of organellar fibers

Over 125 pigmentation-related genes have been identified to date. Of those, PMEL17/GP100 has been widely studied as a melanoma-specific antigen as well as a protein required for the formation of fibrils in melanosomes. PMEL17 is synthesized, glycosylated, processed, and delivered to melanosomes, allowing them to mature from amorphous round vesicles to elongated fibrillar structures. In contrast to other melanosomal proteins such as TYR and TYRP1, the processing and sorting of PMEL17 is highly complex. Monoclonal antibody HMB45 is commonly used for melanoma detection, but has the added advantage that it specifically reacts with sialylated PMEL17 in the fibrillar matrix in melanosomes. In this study, we generated mutant forms of PMEL17 to clarify the subdomain of PMEL17 required for formation of the fibrillar matrix, a process critical to pigmentation. The internal proline/serine/threonine-rich repeat domain (called the RPT domain) of PMEL17 undergoes variable proteolytic cleavage. Deletion of the RPT domain abolished its recognition by HMB45 and its capacity to form fibrils. Truncation of the C-terminal domain did not significantly affect the processing or trafficking of PMEL17, but, in contrast, deletion of the N-terminal domain abrogated both. We conclude that the RPT domain is essential for its function in generating the fibrillar matrix of melanosomes and that the luminal domain is necessary for its correct processing and trafficking to those organelles.

Voluntary exercise prevents the obese and diabetic metabolic syndrome of the melanocortin-4 receptor knockout mouse

Exercise is a mechanism for maintenance of body weight in humans. Morbidly obese human patients have been shown to possess single nucleotide polymorphisms in the melanocortin‐4 receptor (MC4R). MC4R knockout mice have been well characterized as a genetic model that possesses phenotypic metabolic disorders, including obesity, hyperphagia, hyperinsulinemia, and hyperleptinemia, similar to those observed in humans possessing dysfunctional hMC4Rs. Using this model, we examined the effect of voluntary exercise of MC4R knockout mice that were allowed access to a running wheel for a duration of 8 wk. Physiological parameters that were measured included body weight, body composition of fat and lean mass, food consumption, body length, and blood levels of cholesterol and nonfasted glucose, insulin, and leptin. At the termination of the experiment, hypothalamic mRNA expression levels of neuropeptide Y (NPY), agouti‐related protein (AGRP), proopiomelanocortin (POMC), cocaine‐and amphetamine‐regulated transcript (CART), orexin, brain‐derived neurotropic factor (BDNF), phosphatase with tensin homology (Pten), melanocortin‐3 receptor (MC3R), and NPY‐Y1R were determined. In addition, islet cell distribution and function in the pancreas were examined. In the exercising MC4R knockout mice, the pancreatic islet cell morphology and other physiological parameters resembled those observed in the wild‐type littermate controls. Gene expression profiles identified exercise as having a significant effect on hypothalamic POMC, orexin, and MC3R levels. Genotype had a significant effect on AGRP, POMC, CART, and NPY‐Y1R, with an exercise and genotype interaction effect on NPY gene expression. These data support the hypothesis that voluntary exercise can prevent the genetic predisposition of melanocortin‐4 receptor‐associated obesity and diabetes.—Haskell‐Luevano, C., Schaub, J. W., Andreasen, A., Haskell, K. R., Moore, M. C., Koerper, L. M., Rouzaud, F., Baker, H. V., Millard, W. J., Walter, G., Litherland, S. A., Xiang, Z. Voluntary exercise prevents the obese and diabetic metabolic syndrome of the melanocortin‐4 receptor knockout mouse. FASEB J. 23, 642–655 (2009)

Sorting of Pmel17 to melanosomes through the plasma membrane by AP1 and AP2: evidence for the polarized nature of melanocytes

Adaptor proteins (AP) play important roles in the sorting of proteins from the trans-Golgi network, but how they function in the sorting of various melanosome-specific proteins such as Pmel17, an essential structural component of melanosomes, in melanocytes is unknown. We characterized the processing and trafficking of Pmel17 via adaptor protein complexes within melanocytic cells. Proteomics analysis detected Pmel17, AP1 and AP2, but not AP3 or AP4 in early melanosomes. Real-time PCR, immunolabeling and tissue in-situ hybridization confirmed the coexpression of AP1 isoforms μ1A and μ1B (expressed only in polarized cells) in melanocytes and keratinocytes, but expression of μ1B is missing in some melanoma cell lines. Transfection with AP1 isoforms (μ1A or μ1B) showed two distinct distribution patterns that involved Pmel17, and only μ1B was able to restore the sorting of Pmel17 to the plasma membrane in cells lacking μ1B expression. Finally, we established that expression of μ1B is regulated physiologically in melanocytes by UV radiation or DKK1. These results show that Pmel17 is sorted to melanosomes by various intracellular routes, directly or indirectly through the plasma membrane, and the presence of basolateral elements in melanocytes suggests their polarized nature.

Mutations in dopachrome tautomerase (Dct) affect eumelanin/pheomelanin synthesis, but do not affect intracellular trafficking of the mutant protein.

Dopachrome tautomerase (Dct) is a type I membrane protein and an important regulatory enzyme that plays a pivotal role in the biosynthesis of melanin and in the rapid metabolism of its toxic intermediates. Dct-mutant melanocytes carrying the slaty or slaty light mutations were derived from the skin of newborn congenic C57BL/6J non-agouti black mice and were used to study the effect(s) of these mutations on the intracellular trafficking of Dct and on the pigmentation of the cells. Dct activity is 3-fold lower in slaty cells compared with non-agouti black melanocytes, whereas slaty light melanocytes have a surprisingly 28-fold lower Dct activity. Homology modelling of the active site of Dct suggests that the slaty mutation [R194Q (Arg194-->Gln)] is located in the active site and may alter the ability of the enzyme to transform the substrate. Transmembrane prediction methods indicate that the slaty light mutation [G486R (Gly486-->Arg)] may result in the sliding of the transmembrane domain towards the N-terminus, thus interfering with Dct function. Chemical analysis showed that both Dct mutations increase pheomelanin and reduce eumelanin produced by melanocytes in culture. Thus the enzymatic activity of Dct may play a role in determining whether the eumelanin or pheomelanin pathway is preferred for pigment biosynthesis.

Regulation of melanocortin 1 receptor expression at the mRNA and protein levels by its natural agonist and antagonist

Five melanocortin receptors, which form a subfamily of G protein‐coupled receptors, are expressed in mammalian tissues and regulate such diverse physiological processes as pigmentation, adrenal function, energy homeostasis, feeding efficiency, and sebaceous gland lipid production, as well as immune and sexual function. Pigmentation in mammals is stimulated by α‐melanocyte stimulating hormone (MSH), which binds to the melanocortin 1 receptor (Mc1r) and induces an activation of melanogenic enzymes through stimulation of adenylate cyclase and protein kinase A. The antagonist agouti signal protein (ASP) interacts with the Mc1r and blocks its stimulation by MSH. We examined the influence of ASP or MSH on Mc1r gene expression, and we report that both ligands influence the Mc1r 5′ promoter structure in distinct manners. Our study further shows that MSH regulates Mc1r function at both the mRNA and protein levels, whereas ASP acts only on its translation.

Sialylated Core 1 O-Glycans Influence the Sorting of Pmel17/gp100 and Determine Its Capacity to Form Fibrils

Pmel17 is a melanocyte/melanoma-specific protein that is essential for the maturation of melanosomes to form mature, fibrillar, and pigmented organelles. Recently, we reported that the less glycosylated form of Pmel17 (termed iPmel17) is sorted via the plasma membrane in a manner distinct from mature Pmel17 (termed mPmel17), which is sorted directly to melanosomes. To clarify the mechanism(s) underlying the distinct processing and sorting of Pmel17, we generated a highly specific antibody (termed αPEP25h) against an epitope within the repeat domain of Pmel17 that is sensitive to changes in O-glycosylation. αPEP25h recognizes only iPmel17 and allows analysis of the processing and sorting of iPmel17 when compared with αPEP13h, an antibody that recognizes both iPmel17 and mPmel17. Our novel findings using αPEP25h demonstrate that iPmel17 differs from mPmel17 not only in its sensitivity to endoglycosidase H, but also in the content of core 1 O-glycans modified with sialic acid. This evidence reveals that iPmel17 is glycosylated differently in the Golgi and that it is sorted through the secretory pathway. Analysis of Pmel17 processing in glycosylation-deficient mutant cells reveals that Pmel17 lacking the correct addition of sialic acid and galactose loses the ability to form fibrils. Furthermore, we show that addition of sialic acid affects the stability and sorting of Pmel17 and reduces pigmentation. Alterations in sialyltransferase activity and substrates differ between normal and transformed melanocytes and may represent a critical change during malignant transformation.

Regulation of constitutive and UVR-induced skin pigmentation by melanocortin 1 receptor isoforms

Melanin synthesized by epidermal melanocytes protects the skin against UVR‐induced DNA damage and skin cancer. Exposure to UVR increases the synthesis of the photoprotective eumelanin on activation of MC1R, a melanoma susceptibility gene. We studied the expression of MC1R under UVR and α‐MSH stimulation in skin of different ethnic origins and in melanocytes of various pigmentary levels. This study identifies and characterizes a novel MC1R isoform (MC1R350) generated by alternative splicing of the classically known MC1R (MC1R317). We demonstrate that the melanin content of melanocytes shows a significant positive correlation with MC1R317 levels but correlates inversely with the amount of MC1R350, suggesting that this latter isoform could act as a negative regulator of melanin synthesis. We confirmed that hypothesis by showing that while MC1R317 signaling significantly increases the expression of MITF and tyrosinase, two key factors in the melanin synthesis pathway, MC1R350 dramatically hampers their expression. In the skin, we show that UVR does not increase MC1R350 expression but does significantly increase MC1R317. Taken together, our results strongly suggest that MC1R350 acts as a negative regulator of skin pigmentation and demonstrate for the first time that MC1R isoform‐specific expression is closely related to skin pigmentation and photoprotection.—Rouzaud, F., Costin, G‐E., Yamaguchi, Y., Valencia, J. C., Berens, W. F., Chen, K. G., Hoashi, T., Böhm, M., Abdel‐Malek, Z. A., Hearing, V. J. Regulation of constitutive and UVR‐induced skin pigmentation by melanocortin 1 receptor isoforms. FASEB J. 20, E1338–E1350 (2006)

Regulatory elements of the melanocortin 1 receptor

Among more than 120 genes that are now known to regulate mammalian pigmentation, one of the key genes is MC1R, which encodes the melanocortin 1 receptor, a seven transmembrane G protein-coupled receptor expressed on the surface of melanocytes. Since the monoexonic sequence of the gene was cloned and characterized more than a decade ago, tremendous efforts have been dedicated to the extensive genotyping of mostly red-haired populations all around the world, thus providing allelic variants that may or may not account for melanoma susceptibility in the presence or absence of ultraviolet (UV) exposure. Soluble factors, such as proopiomelanocortin (POMC) derivatives, agouti signal protein (ASP) and others, regulate MC1R expression, leading to improved photoprotection via increased eumelanin synthesis or in contrast, inducing the switch to pheomelanin. However, there is an obvious lack of knowledge regarding the numerous and complex regulatory mechanisms that govern the expression of MC1R at the intra-cellular level, from gene transcription in response to an external stimulus to the expression of the mature receptor on the melanocyte surface.