Pigment-type Switching Research Articles

Elucidation of Melanogenesis Cascade for Identifying Pathophysiology and Therapeutic Approach of Pigmentary Disorders and Melanoma.

Melanogenesis is the biological and biochemical process of melanin and melanosome biosynthesis. Melanin is formed by enzymic reactions of tyrosinase family proteins that convert tyrosine to form brown-black eumelanin and yellow-red pheomelanin within melanosomal compartments in melanocytes, following the cascades of events interacting with a series of autocrine and paracrine signals. Fully melanized melanosomes are delivered to keratinocytes of the skin and hair. The symbiotic relation of a melanocyte and an associated pool of keratinocytes is called epidermal melanin unit (EMU). Microphthalmia-associated transcription factor (MITF) plays a vital role in melanocyte development and differentiation. MITF regulates expression of numerous pigmentation genes for promoting melanocyte differentiation, as well as fundamental genes for maintaining cell homeostasis. Diseases involving alterations of EMU show various forms of pigmentation phenotypes. This review introduces four major topics of melanogenesis cascade that include (1) melanocyte development and differentiation, (2) melanogenesis and intracellular trafficking for melanosome biosynthesis, (3) melanin pigmentation and pigment-type switching, and (4) development of a novel therapeutic approach for malignant melanoma by elucidation of melanogenesis cascade.

The Serine Protease Activity of Corin Is Required for Normal Pigment Type Switching

The Serine Protease Activity of Corin Is Required for Normal Pigment Type Switching

MGRN1‐dependent pigment‐type switching requires its ubiquitination activity but not its interaction with TSG101 or NEDD4

Mice lacking the E3 ubiquitin ligase mahogunin ring finger-1 (MGRN1) have a pleiotropic phenotype that includes spongiform neurodegeneration, embryonic patterning defects, and dark fur due to a defect in pigment-type switching. The only MGRN1 ubiquitination target identified to date is tumor susceptibility gene 101 (TSG101), a component of the endosomal trafficking machinery. Here, we show that MGRN1 also interacts with but does not ubiquitinate NEDD4, a HECT-domain ubiquitin ligase involved in endosomal trafficking. Using transgenesis in mice, we demonstrate that pigment-type switching likely requires MGRN1's ubiquitin ligase activity but not its ability to bind TSG101 or NEDD4. This indicates that MGRN1-dependent ubiquitination of an as-yet unidentified target protein is required for agouti-mediated melanocortin signaling.

Characterization of different 5′‐untranslated exons of the ASIP gene in black‐and‐tan Doberman Pinscher and brindle Boxer dogs

Differential expression of the ASIP gene and its interaction with MC1R have provided basic insight into pigment-type switching in mammals. Here, we report the characterization of a specific red-haired skin transcript and a specific black-haired skin transcript in the ASIP gene in the black-and-tan Doberman Pinscher. It is also shown that the brindle-haired skin of the Boxer exhibits a deregulated expression resulting in various 5'-untranslated exons. Comparative sequence analysis revealed a short interspersed element and a poly(A) stretch inserted within the promoter region of the ASIP in the Boxer. Genotyping studies have shown that both insertions are also present in brindle and fawn animals of the Boxer and Great Dane breeds. Furthermore, we genotyped MC1R and K loci for their known variants that affect coat color in dogs. As expected, all animals were homozygotes (E(M) /E(M) ) for the mask mutation, and fawn animals were k(y) /k(y) . Unexpectedly, we found that all brindle animals were heterozygotes k(B) /k(y) . Our results suggest that differential expression of ASIP determine pigment-type switching in a MC1R and K allele-dependent manner in dogs.

P-REX1, a Rac guanine exchange factor, links melanocyte development and melanoma progression

P-REX1, a Rac guanine exchange factor, links melanocyte development and melanoma progression

Mahoganoid and Mahogany Mutations Rectify the Obesity of the Yellow Mouse by Effects on Endosomal Traffic of MC4R Protein

The ubiquitous overexpression of agouti-signaling protein (ASP), a paracrine-signaling molecule that regulates pigment-type switching in the hair follicle of the mouse, is responsible for the obesity and yellow pelage of the Yellow mouse (A(y)). Mahogany (Attractin, Atrn/mg) and mahoganoid (Mahogunin Ring Finger-1, Mgrn1/md) are mutations epistatic to A(y). These mutations have been described as suppressors of ASP action, blocking its antagonizing effects on the melanocortin 1 and 4 receptors (MC1R and MC4R) in the skin and the brain, respectively, via unknown mechanisms. Here, we describe the molecular bases for the md- and mg-dependent rescue of the A(y) phenotype at the MC4R. We show that overexpression of ASP inhibits the rise in cAMP levels in response to α-melanocyte-stimulating hormone, an MC4R agonist, by blocking ligand binding and by directing MC4R trafficking to the lysosome. Loss-of-function of either attractin or MGRN1 blocks ASP-dependent MC4R degradation and promotes increased trafficking of internalized MC4R to the cell surface, but it does not restore α-melanocyte-stimulating hormone-dependent cAMP signaling. We propose that MGRN1 and attractin are components of an evolutionarily conserved receptor trafficking pathway and that the md and mg mutations rescue the A(y) phenotypes by a primarily cAMP-independent mechanism promoting trafficking of MC4R and likely MC1R away from the lysosome toward the cell surface.

How are proliferation and differentiation of melanocytes regulated?

Coat colors are determined by melanin (eumelanin and pheomelanin). Melanin is synthesized in melanocytes and accumulates in special organelles, melanosomes, which upon maturation are transferred to keratinocytes. Melanocytes differentiate from undifferentiated precursors, called melanoblasts, which are derived from neural crest cells. Melanoblast/melanocyte proliferation and differentiation are regulated by the tissue environment, especially by keratinocytes, which synthesize endothelins, steel factor, hepatocyte growth factor, leukemia inhibitory factor and granulocyte-macrophage colony-stimulating factor. Melanocyte differentiation is also stimulated by alpha-melanocyte stimulating hormone; in the mouse, however, this hormone is likely carried through the bloodstream and not produced locally in the skin. Melanoblast migration, proliferation and differentiation are also regulated by many coat color genes otherwise known for their ability to regulate melanosome formation and maturation, pigment type switching and melanosome distribution and transfer. Thus, melanocyte proliferation and differentiation are not only regulated by genes encoding typical growth factors and their receptors but also by genes classically known for their role in pigment formation.

Mice in Camouflage

Natural variation in vertebrate color pattern is one of the most conspicuous traits related to fitness. Using genetic and functional analyses in natural populations of Peromyscus mice, Manceau et al. (p. [1062][1]) uncover the developmental mechanism for the establishment and subsequent evolution of color patterns, which function in camouflage from visual predators. The Agouti protein, known to be involved in pigment-type switching in adult skin, establishes a molecular prepattern during embryonic development to generate the adult color pattern. These changes in the time, place, and level of embryonic expression of Agouti affect adult color pattern and explain the difference in coloration between locally camouflaged mouse populations. [1]: /lookup/doi/10.1126/science.1200684

β-Catenin activity in the dermal papilla of the hair follicle regulates pigment-type switching

The switch between black and yellow pigment is mediated by the interaction between Melanocortin receptor 1 (Mc1r) and its antagonist Agouti, but the genetic and developmental mechanisms that modify this interaction to obtain different coat color in distinct environments are poorly understood. Here, the role of Wnt/β-catenin signaling in the regulation of pigment-type switching was studied. Loss and gain of function of β-catenin in the dermal papilla (DP) of the hair follicle results in yellow and black animals, respectively. β-Catenin activity in the DP suppresses Agouti expression and activates Corin, a negative regulator of Agouti activity. In addition, β-catenin activity in the DP regulates melanocyte activity by a mechanism that is independent of both Agouti and Corin. The coordinate and inverse regulation of Agouti and Corin renders pelage pigmentation sensitive to changes in β-catenin activity in the DP that do not alter pelage structure. As a result, the signals that specify two biologically distinct quantitative traits are partially uncoupled despite their common regulation by the β-catenin pathway in the same cells.

Shades of meaning: the pigment‐type switching system as a tool for discovery

The pigment-type switching system, which controls whether melanocytes produce black/brown eumelanin or yellow/red pheomelanin, is responsible for many familiar coat coloration patterns in both domestic and wild mammals. In conjunction with the accessory proteins attractin and mahogunin ring finger 1, endogenous agonists and antagonists modulate signaling by the melanocortin 1 receptor to determine pigment type. Mutations in pigment-type switching genes can cause a variety of pleiotropic phenotypes, and these are often similar between mutants at different loci because the proteins encoded by these genes act together as part of conserved molecular pathways that are deployed in multiple biological contexts. When this is the case, pigment-type switching provides a powerful model system for elucidating the shared molecular mechanisms underlying the pigmentary and non-pigmentary phenotypes. This review outlines the current understanding of the pigment-type switching pathway and discusses the opportunities that exist for exploring the molecular basis of pleiotropic phenotypes using this model system.

Of patches, blotches, speckles and stripes

From flags to fashion, people love patterns of colors and shapes and use them to convey meaning. Nature is also replete with colorful patterns and employs them to send signals, but how are such patterns generated without our doing? By information or self-organization? John Maynard Smith pointedly asks in his book entitled ‘Shaping Life’. A good question, as the debate has not yet been entirely settled. Proponents of self-organization believe that most if not all repetitive coat patterns can be explained by mathematical models originally based on the reaction-diffusion mechanism proposed by Turing in 1952. Suppose a factor exists in the skin that causes melanocytes to go black, induces an inhibitor that causes them to go white, and is not precisely at equal concentrations at every point on a body surface. Suppose also that the inhibitor diffuses faster than the activator and needs to act before the activator steps in, and – bingo!– there will be black spots surrounded by white areas. One need only tinker with the parameters of an otherwise common algorithm to generate virtually every pattern in nature on a computer screen. Conceptually, then, genes would set the parameters (body size, concentrations, diffusion rates, etc.) and patterns would inevitably and logically emerge. But then there are those who hold that this view underplays the importance of genetic programming, which, after all, underlies all adaptive evolution. They would argue that the pigment cell is not just pushed around by extrinsic factors but is actively engaged in the communication with the local environment and uses its own color genes to draw patterns. As is often the case in such debates, the truth may lie somewhere in between. There is evidence suggesting that patterning genes conceivably influencing reaction-diffusion processes in the integument may have appeared early in evolution and been preserved, and that pigmentation genes interpreting these pattern genes may have evolved later and independently on multiple occasions. This and other questions concerning pigment patterns are discussed in this issue in the commentary by Kaelin and Barsh on the genetics of pigmentation in the cat, by Walker and Gunn in a review on pigment type switching, and by myself in a News and Views piece on sex-specific coloration in fish. Hellström and colleagues from Leif Anderson’s group in Uppsala, Sweden, investigate the genetics of sex-linked barring in chickens and find, intriguingly, that the cell cycle and tumor suppressor gene CDKN2A is involved. Harris and colleagues from the Pavan laboratory at NIH in the US review the various roles SOX proteins play in the development of pigmentation and its regulation in the adult, and they also discuss the involvement of these transcription factors in malignant transformation. Alas, this brings us to pathology, the flip side of all the joy we get from the beauty in animal coloration. In fact, PCMR devotes a substantial number of pages to pathology, for the analysis and treatment of hypo- and hyperpigmentations or pigment cell-associated malignancies. There are several papers in this issue that deal with one or the other factor that is dysregulated in melanoma. Ryan and colleagues, for instance, use comparative genome hybridization to show that the gene encoding Topoisomerase-1 is amplified in aggressive melanoma, giving a poor prognosis. Wang and colleagues show that poor melanoma prognosis is also predicted by aberrant expression of alpha-1 antichymotrypsin. Chen and colleagues show increased expression of Cullin1, which is part of a large ubiquitin E3 ligase complex, in early stage melanoma. VanBrocklin and colleagues use a promising system in mice that makes it possible to evaluate the role of individual genes in melanoma formation alone and in combination. Needless to say, there are many more interesting papers in this issue that should be highlighted, and I encourage you, the reader, to leaf through the pages and mark (and cite!) all the compelling studies that pique your interest.

Piecing together the pigment-type switching puzzle

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Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with Gαs

Mahogunin ring finger-1 (MGRN1) is a RING domain-containing ubiquitin ligase mutated in mahoganoid, a mouse mutation causing coat color darkening, congenital heart defects, high embryonic lethality, and spongiform neurodegeneration. The melanocortin hormones regulate pigmentation, cortisol production, food intake, and body weight by signaling through five G protein-coupled receptors positively coupled to the cAMP pathway (MC1R-MC5R). Genetic analysis has shown that mouse Mgrn1 is an accessory protein for melanocortin signaling that may inhibit MC1R and MC4R by unknown mechanisms. These melanocortin receptors (MCRs) regulate pigmentation and body weight, respectively. We show that human melanoma cells express 4 MGRN1 isoforms differing in the C-terminal exon 17 and in usage of exon 12. This exon contains nuclear localization signals. MGRN1 isoforms decreased MC1R and MC4R signaling to cAMP, without effect on beta(2)-adrenergic receptor. Inhibition was independent on receptor plasma membrane expression, ubiquitylation, internalization, or stability and occurred upstream of Galpha(s) binding to/activation of adenylyl cyclase. MGRN1 co-immunoprecipitated with MCRs, suggesting a physical interaction of the proteins. Significantly, overexpression of Galpha(s) abolished the inhibitory effect of MGRN1 and decreased co-immunoprecipitation with MCRs, suggesting competition between MGRN1 and Galpha(s) for binding to MCRs. Although all MGRN1s were located in the cytosol in the absence of MCRs, exon 12-containing isoforms accumulated in the nuclei upon co-expression with the receptors. Therefore, MGRN1 inhibits MCR signaling by a new mechanism involving displacement of Galpha(s), thus accounting for key features of the mahoganoid phenotype. Moreover, MGRN1 might provide a novel pathway for melanocortin signaling from the cell surface to the nucleus.

Genetics ofSex-linked yellowin the Syrian Hamster

Alternating patches of black and yellow pigment are a ubiquitous feature of mammalian color variation that contributes to camouflage, species recognition, and morphologic diversity. X-linked determinants of this pattern--recognized by variegation in females but not in males--have been described in the domestic cat as Orange, and in the Syrian hamster as Sex-linked yellow (Sly), but are curiously absent from other vertebrate species. Using a comparative genomic approach, we develop molecular markers and a linkage map for the euchromatic region of the Syrian hamster X chromosome that places Sly in a region homologous to the centromere-proximal region of human Xp. Comparison to analogous work carried out for Orange in domestic cats indicates, surprisingly, that the cat and hamster mutations lie in nonhomologous regions of the X chromosome. We also identify the molecular cause of recessively inherited black coat color in hamsters (historically referred to as nonagouti) as a Cys115Tyr mutation in the Agouti gene. Animals doubly mutant for Sly and nonagouti exhibit a Sly phenotype. Our results indicate that Sly represents a melanocortin pathway component that acts similarly to, but is genetically distinct from, Mc1r and that has implications for understanding both the evolutionary history and the mutational mechanisms of pigment-type switching.

β‐Defensin 3: a novel and unexpected key that unlocks the melanocortin 1 receptor

β‐Defensin 3: a novel and unexpected key that unlocks the melanocortin 1 receptor

Linkage and Segregation Analysis of Black and Brindle Coat Color in Domestic Dogs

Mutations of pigment type switching have provided basic insight into melanocortin physiology and evolutionary adaptation. In all vertebrates that have been studied to date, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls the switch between synthesis of red-yellow pheomelanin vs. black-brown eumelanin. However, in domestic dogs, historical studies based on pedigree and segregation analysis have suggested that the pigment type-switching system is more complicated and fundamentally different from other mammals. Using a genomewide linkage scan on a Labrador x greyhound cross segregating for black, yellow, and brindle coat colors, we demonstrate that pigment type switching is controlled by an additional gene, the K locus. Our results reveal three alleles with a dominance order of black (K(B)) > brindle (k(br)) > yellow (k(y)), whose genetic map position on dog chromosome 16 is distinct from the predicted location of other pigmentation genes. Interaction studies reveal that Mc1r is epistatic to variation at Agouti or K and that the epistatic relationship between Agouti and K depends on the alleles being tested. These findings suggest a molecular model for a new component of the melanocortin signaling pathway and reveal how coat-color patterns and pigmentary diversity have been shaped by recent selection.

Mice with mutations in Mahogunin ring finger‐1 (Mgrn1) exhibit abnormal patterning of the left–right axis

Mahogunin Ring Finger 1 (Mgrn1) encodes a RING-containing protein with ubiquitin ligase activity that has been implicated in pigment-type switching. In addition to having dark fur, mice lacking MGRN1 develop adult-onset spongy degeneration of the central nervous system and have reduced embryonic viability. Observation of complete situs inversus in a small proportion of adult Mgrn1 mutant mice suggested that embryonic lethality resulted from congenital heart defects due to defective establishment and/or maintenance of the left-right (LR) axis. Here we report that Mgrn1 is expressed in a pattern consistent with a role in LR patterning during early development and that many Mgrn1 mutant embryos show abnormal expression of asymmetrically expressed genes involved in LR patterning. A range of complex heart defects was observed in 20-25% of mid-to-late gestation Mgrn1 mutant embryos and another 20% were dead. This finding was consistent with 46-60% mortality of mutants by weaning age. Our results indicate that Mgrn1 acts early in the LR signaling cascade and is likely to provide new insight into this developmental process as Nodal expression was uncoupled from expression of other Nodal-responsive genes in Mgrn1 mutant embryos. Our work identifies a novel role for MGRN1 in embryonic patterning and suggests that the ubiquitination of MGRN1 target genes is essential for the proper establishment and/or maintenance of the LR axis.

Characterization of Mahogunin Ring Finger‐1 expression in mice

Mutations in mouse Mahogunin Ring Finger-1 (Mgrn1) were first recognized for their effect on agouti-mediated pigment-type switching. Mgrn1 null mutants are completely black and develop spongiform degeneration of the brain. Mgrn1 hypomorphs have dark fur but do not develop neurodegeneration. We characterized a new Mgrn1 hypomorphic allele caused by a gene-trap insertion. Mice homozygous for this mutation are slightly darker than non-mutant animals. They show reduced overall expression of Mgrn1 and two of the four normal Mgrn1 isoforms are replaced by beta-GEO fusion proteins that differ from the normal proteins at their carboxy termini. To investigate the role of different Mgrn1 isoforms in pigment-type switching, we used quantitative relative reverse transcriptase polymerase chain reaction to examine their expression in the skin of Mgrn1 mutant and control mice. Most Mgrn1 mutants produce little or no normal Mgrn1 in the skin. Mgrn1 null mutant mice overexpressing isoform I or III, which are normally absent or weakly expressed in adult skin, had normal agouti-banded hairs. Our results indicate that reduced levels of MGRN1 cause the pigmentation phenotypes of Mgrn1 mutant mice and that there are no significant differences in the function of the four MGRN1 isoforms in pigment-type switching.

Characterization of the dog Agouti gene and a nonagoutimutation in German Shepherd Dogs

The interaction between two genes, Agouti and Melanocortin-1 receptor ( Mc1r), produces diverse pigment patterns in mammals by regulating the type, amount, and distribution pattern of the two pigment types found in mammalian hair: eumelanin (brown/black) and pheomelanin (yellow/red). In domestic dogs ( Canis familiaris), there is a tremendous variation in coat color patterns between and within breeds; however, previous studies suggest that the molecular genetics of pigment-type switching in dogs may differ from that of other mammals. Here we report the identification and characterization of the Agouti gene from domestic dogs, predicted to encode a 131-amino-acid secreted protein 98% identical to the fox homolog, and which maps to chromosome CFA24 in a region of conserved linkage. Comparative analysis of the Doberman Pinscher Agouti cDNA, the fox cDNA, and 180 kb of Doberman Pinscher genomic DNA suggests that, as with laboratory mice, different pigment-type-switching patterns in the canine family are controlled by alternative usage of different promoters and untranslated first exons. A small survey of Labrador Retrievers, Greyhounds, Australian Shepherds, and German Shepherd Dogs did not uncover any polymorphisms, but we identified a single nucleotide variant in black German Shepherd Dogs predicted to cause an Arg-to-Cys substitution at codon 96, which is likely to account for recessive inheritance of a uniform black coat.

The color loci of mice--a genetic century.

Color loci in mammals are those genetic loci in which mutations can affect pigmentation of the hair, skin, and/or eyes. In the mouse, over 800 phenotypic alleles are now known, at 127 identified color loci. As the number of color loci passed 100 only recently, we celebrate this 'century' with an overview of these loci, especially the 59 that have been cloned and sequenced. These fall into a number of functional groups representing melanocyte development and differentiation, melanosomal components, organelle biogenesis, organelle transport, control of pigment-type switching, and some systemic effects. A human ortholog has been identified in all cases, and the majority of these human genes are found to be loci for human disorders, often affecting other body systems as well as pigmentation. We expect that a significant number of color loci remain to be identified. Nonetheless, the large number known already provide a treasury of resources for reconstruction of the mechanisms, at the subcellular, cellular and tissue levels, that produce a functional pigmentary system and contribute to the normal development and functioning of many other organ systems. The mutant mice also provide valuable models for the study of human disease.