Mutant alleles at the brown locus encoding tyrosinase-related protein-1 (TRP-1) affect proliferation of mouse melanocytes in culture

Tyrosinase related protein-1 (TRP-1) is a melanocyte-specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP-1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1b (brown) and Tyrp1b-cj (cordovan) compared with wild type Tyrp1B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP-1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP-1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.     

A dominant mutation in tyrp1A leads to melanophore death in zebrafish

Melanin biosynthesis in vertebrates depends on the function of three enzymes of the tyrosinase family, tyrosinase (Tyr), tyrosinase‐related protein 1 (Tyrp1), and dopachrome tautomerase (Dct or Tyrp2). Tyrp1 might play an additional role in the survival and proliferation of melanocytes. Here, we describe a mutation in tyrp1A, one of the two tyrp1 paralogs in zebrafish, which causes melanophore death leading to a semi‐dominant phenotype. The mutation, an Arg‐>Cys change in the amino‐terminal part of the protein, is similar to mutations in humans and mice where they lead to blond hair (in melanesians) or dark hair with white bases, respectively. We demonstrate that the phenotype in zebrafish depends on the presence of the mutant protein and on melanin synthesis. Ultrastructural analysis shows that the melanosome morphology and pigment content are altered in the mutants. These structural changes might be the underlying cause for the observed cell death, which, surprisingly, does not result in patterning defects.     

Tyrp1 and Oculocutaneous Albinism Type 3

Tyrosinase‐related protein 1 (Tyrp1) is a melanocyte‐specific gene product involved in eumelanin synthesis. Mutations in the mouse Tyrp1 gene are associated with brown pelage, and in the human TYRP1 gene with oculocutaneous albinism type 3 (OCA3). In the murine system, Tyrp1 expresses significant dihydroxyindole carboxylic acid oxidase (i.e. DHICA oxidase) activity. However, in humans, TYRP1 is enigmatic in that despite extensive efforts focused on the study of its function, its actual role in the human melanocyte is still unclear. There is mounting evidence demonstrating that in addition to its role in eumelanin synthesis, Tyrp1 is involved in maintaining stability of tyrosinase protein and modulating its catalytic activity. Tyrp1 is also involved in maintenance of melanosome ultrastructure and affects melanocyte proliferation and melanocyte cell death. The current review is an attempt to consolidate our understanding of the role of Tyrp1 in the melanocyte.     

Keratinocytes Suppress TRP‐1 Expression and Reduce Cell Number of Co‐cultured Melanocytes – Implications For Grafting of Patients with Vitiligo

A pilot study for grafting of patients with vitiligo using cultured epithelial autografts containing melanocytes gave disappointing clinical results, with pigmentation achieved in only one out of five patients. Irrespective of the fate of melanocytes grafted back onto the patients, we experienced problems in identifying melanocytes within these well‐integrated keratinocyte sheets. This led us to explore the fate of these cells within these sheets in vitro and to seek to improve their number and function within the sheets. We report that the introduction of a fibroblast feeder layer can improve melanocyte number within melanocyte/keratinocyte co‐cultures initially, but at very high keratinocyte density, there is a marked loss of melanocytes (as detected by staining for S100). Additionally, we found that keratinocytes not only down‐regulate melanocyte number, but also pigmentary function; thus, it was possible to identify melanocytes that were S100 positive but tyrosinase‐related protein‐1 (TRP‐1) negative in confluent well‐integrated keratinocyte sheets. In summary, our data suggest that keratinocytes at high density initially suppress melanocyte pigmentation (as evidenced by a lack of TRP‐1 expression) and then cause a physical loss of melanocytes. The introduction of a fibroblast feeder layer can help maintain melanocyte number while keratinocytes are subconfluent, but fails to oppose the inhibitory influence of the keratinocytes on melanocyte TRP‐1 expression.     

A premature stop codon in the TYRP1 gene is associated with brown coat colour in the European rabbit (Oryctolagus cuniculus)

Classical genetic studies in European rabbits (Oryctolagus cuniculus) suggested the presence of two alleles at the brown coat colour locus: a wild‐type B allele that gives dense black pigment throughout the coat and a recessive b allele that in the homozygous condition (b/b genotype) produces brown rabbits that are unable to develop black pigmentation. In several other species, this locus is determined by mutations in the tyrosinase‐related protein 1 (TYRP1) gene, encoding a melanocyte enzyme needed for the production of dark eumelanin. In this study, we investigated the rabbit TYRP1 gene as a strong candidate for the rabbit brown coat colour locus. A total of 3846 bp of the TYRP1 gene were sequenced in eight rabbits of different breeds and identified 23 single nucleotide polymorphisms (SNPs; 12 in intronic regions, five in exons and six in the 3′‐untranslated region) and an insertion/deletion of 13 bp, in the 3′‐untranslated region, organised in a few haplotypes. A mutation in exon 2 (g.41360196G>A) leads to a premature stop codon at position 190 of the deduced amino acid sequence (p.Trp190ter). Therefore, translation predicts a truncated TYRP1 protein lacking almost completely the tyrosinase domain. Genotyping 203 rabbits of 32 different breeds identified this mutation only in brown Havana rabbits. Its potential functional relevance in disrupting the TYRP1 protein and its presence only in brown animals strongly argue for this non‐sense mutation being a causative mutation for the recessive b allele at the brown locus in Oryctolagus cuniculus.     

Novel three‐way complex rearrangement of TRPM1‐PUM1‐LCK in a case of agminated Spitz nevi arising in a giant congenital hyperpigmented macule

The genetic anomalies associated with the agminated variant of Spitz nevus have so far been limited to HRAS G13R mutations, especially when arising within a nevus spilus. A previous report exposed the case of a man with a giant pigmented macule involving his upper right limb and trunk. Since childhood, Spitz nevi have been periodically arising, within the pigmented area. The histopathology of several lesions displayed the usual criteria of junctional, compound, or intradermal Spitz nevi with a diversity of cytomorphological and architectural features. Some lesions spontaneously regressed. Genetic studies confirmed in three lesions an identical translocation involving TRPM1, PUM1, and LCK. No mutations in HRAS, NRAS, BRAF, or other known fusion genes linked to Spitz nevus were detected. LCK break‐apart fluorescence in situ hybridization confirmed the rearrangement was present not only in the melanocytic proliferation but also in the surrounding non‐spitzoid melanocytes. This report expands the list of genetic alterations involved both in giant congenital macules and in agminated Spitz nevi, and also extends the concept of mosaicism in melanocytes to gene translocations.     

Mutational Analysis of the Modulation of Tyrosinase by Tyrosinase‐Related Proteins 1 and 2 In Vitro

The albino (tyrosinase, Tyr^c), brown (tyrosinase‐related protein 1, Tyrp1^b) and slaty (tyrosinase‐related protein 2, tyrp2^slt) loci are all involved in the regulation of melanogenesis. Phenotypes of inbred mice mutant at two or more of these loci are not always explicable by simple summation of the established or suspected catalytic functions of the gene products. These phenotypes suggest that relationships among the proteins extend beyond the obvious fact that they catalyze different steps in the same melanogenic pathway, and that they may also interact intimately in such a way that a mutation in one impacts the function of the other(s). Previous studies have attributed catalytic activities to each member of this trio; however, it has been difficult to study the proteins individually, either in vivo or in tissues or cells. Therefore, we undertook to transfect the genes, in revealing combinations, into COS‐7 cells (which have no melanogenic apparatus of their own) to clarify the interacting functions of their encoded proteins. Specifically, we attempted to evaluate the effects of Tyrp1 and Tyrp2 proteins on tyrosinase protein. We report evidence that Tyrp1 stabilizes tyrosinase, confirming previous observations, and, in addition, demonstrate that Tyrp1 decreases tyrosinase activity. By contrast, Tyrp2 increases tyrosinase activity by stabilizing the protein. We conclude that both Tyrp1 and Tyrp2, in addition to other catalytic functions they may possess, act together to modulate tyrosinase activity.     

Generation of albino Xenopus tropicalis using zinc‐finger nucleases

To generate albino lines of Xenopus tropicalis, we injected fertilized eggs with mRNAs encoding zinc‐finger nucleases (ZFNs) targeting the tyrosinase coding region. Surprisingly, vitiligo was observed on the skin of F0 frogs that had been injected with ZFN mRNAs, indicating that both tyrosinase genes in the genome were disrupted in all melanocytes within the vitiligo patches. Mutation analysis using genomic DNA from the skin revealed that two mosaic F0 frogs underwent spatially complex tyrosinase gene mutations. The data implies that the ZFN‐induced tyrosinase gene ablations occurred randomly over space and time throughout the entire body, possibly until the young tadpole stage, and that melanocyte precursors lacking functional tyrosinase proliferated and formed vitiligo patches. Several albino X. tropicalis, which are compound heterozygotes for biallelic tyrosinase mutations, were obtained by mating the mosaic F0 frogs. To our knowledge, this is the first report of the albino vertebrates generated by the targeted gene knockout.     

Pleiotrophin inhibits melanogenesis via Erk1/2‐MITF signaling in normal human melanocytes

Pleiotrophin (PTN) is a secreted heparin‐binding protein that is involved in various biological functions of cell growth and differentiation. Little is known about the effects of PTN on the melanocyte function and skin pigmentation. In this study, we investigated whether PTN would affect melanogenesis. PTN was expressed in melanocytes and fibroblasts of human skin. Transfection studies revealed that PTN decreased melanogenesis, probably through MITF degradation via Erk1/2 activation in melanocytes. The inhibitory action of PTN in pigmentation was further confirmed in ex vivo cultured skin and in the melanocytes cocultured with fibroblasts. These findings suggest that PTN is a crucial factor for the regulation of melanogenesis in the skin.     

Endothelin‐1 is a transcriptional target of p53 in epidermal keratinocytes and regulates ultraviolet‐induced melanocyte homeostasis

Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here, we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV‐induced melanocyte homeostasis. Selective ablation of endothelin‐1 (EDN1) in murine epidermis (EDN1^ep?/?) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non‐cell autonomous manner controls melanocyte proliferation, migration, DNA damage, and apoptosis after ultraviolet B (UVB) irradiation. Expression of other keratinocyte‐derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV‐induced melanocyte activation and recapitulated the phenotype seen in EDN1^ep?/? mice. Altogether, the present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV‐induced melanocyte homeostasis in vivo.     

The TYRP1‐mediated protection of human tyrosinase activity does not involve stable interactions of tyrosinase domains

Tyrosinases are melanocyte‐specific enzymes involved in melanin biosynthesis. Mutations in their genes cause oculocutaneous albinism associated with reduced or altered pigmentation of skin, hair, and eyes. Here, the recombinant human intra‐melanosomal domains of tyrosinase, TYRtr (19–469), and tyrosinase‐related protein 1, TYRP1tr (25–472), were studied in vitro to define their functional relationship. Proteins were expressed or coexpressed in whole Trichoplusia ni larvae and purified. Their associations were studied using gel filtration and sedimentation equilibrium methods. Protection of TYRtr was studied by measuring the kinetics of tyrosinase diphenol oxidase activity in the presence (1:1 and 1:20 molar ratios) or the absence of TYRP1tr for 10 hr under conditions mimicking melanosomal and ER pH values. Our data indicate that TYRtr incubation with excess TYRP1tr protects TYR, increasing its stability over time. However, this mechanism does not appear to involve the formation of stable hetero‐oligomeric complexes to maintain the protective function.     

Metabotropic glutamate receptor 6 signaling enhances TRPM1 calcium channel function and increases melanin content in human melanocytes

Mutations in TRPM1, a calcium channel expressed in retinal bipolar cells and epidermal melanocytes, cause complete congenital stationary night blindness with no discernible skin phenotype. In the retina, TRPM1 activity is negatively coupled to metabotropic glutamate receptor 6 (mGluR6) signaling through Gα_o and TRPM1 mutations result in the loss of responsiveness of TRPM1 to mGluR6 signaling. Here, we show that human melanocytes express mGluR6, and treatment of melanocytes with L‐AP4, a type III mGluR‐selective agonist, enhances Ca²⁺ uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L‐AP4‐induced Ca²⁺ influx and TRPM1 currents, showing that TRPM1 activity in melanocytes is positively coupled to mGluR6 signaling. Gα_o protein is absent in melanocytes. However, forced expression of Gα_o restored negative coupling of TRPM1 to mGluR6 signaling, but treatment with pertussis toxin, an inhibitor of G_i/G_o proteins, did not affect basal or mGluR6‐induced Ca²⁺ uptake. Additionally, chronic stimulation of mGluR6 altered melanocyte morphology and increased melanin content. These data suggest differences in coupling of TRPM1 function to mGluR6 signaling explain different cellular responses to glutamate in the retina and the skin.     

Nucleocytoplasmic trafficking is essential for BAK1‐ and BKK1‐mediated cell‐death control

BRI1‐ASSOCIATED KINASE 1 (BAK1) was initially identified as a co‐receptor of the brassinosteroid (BR) receptor BRI1. Genetic analyses also revealed that BAK1 and its closest homolog BAK1‐LIKE 1 (BKK1) regulate a BR‐independent cell‐death control pathway. The double null mutant bak1 bkk1 displays a salicylic acid‐ and light‐dependent cell‐death phenotype even without pathogen invasion. Molecular mechanisms of the spontaneous cell death mediated by BAK1 and BKK1 remain unknown. Here we report our identification of a suppressor of bak1 bkk1 (sbb1–1). Genetic analyses indicated that cell‐death symptoms in a weak double mutant, bak1–3 bkk1–1, were completely suppressed by the loss‐of‐function mutation in SBB1, which encodes a nucleoporin (NUP) 85‐like protein. Genetic analyses also demonstrated that individually knocking out three other nucleoporin genes from the SBB1‐located sub‐complex was also able to rescue the cell‐death phenotype of bak1–3 bkk1–1. In addition, a DEAD‐box RNA helicase, DRH1, was identified in the same protein complex as SBB1 via a proteomic approach. The drh1 mutation also rescues the cell‐death symptoms of bak1–3 bkk1–1. Further analyses indicated that export of poly(A)⁺ RNA was greatly blocked in the nup and drh1 mutants, resulting in accumulation of significant levels of mRNAs in the nuclei. Over‐expression of a bacterial NahG gene to inactivate salicylic acid also rescues the cell‐death phenotype of bak1–3 bkk1–1. Mutants suppressing cell‐death symptoms always showed greatly reduced salicylic acid contents. These results suggest that nucleocytoplasmic trafficking, especially of molecules directly or indirectly involved in endogenous salicylic acid accumulation, is critical in BAK1‐ and BKK1‐mediated cell‐death control.