Levels of tyrosinase and its mRNA in coat-color mutants of C57BL/10J congenic mice: Effects of genic substitution at the agouti, brown, albino, dilute, and pink-eyed dilution loci
Abstract
The levels of tyrosinase activities, tyrosinase cross-reacting material (TY-CRM), and tyrosinase mRNA were determined for skins from five congenic strains of mice (Mus musculus) with different coat colors. The dopa oxidase activity and melanin formation activity were directly proportional to the abundance of TY-CRM in all of the mutants. The levels of the enzyme activities and TY-CRM were increased in brown (b/b) and dilute (d/d) mice, while they were reduced in agouti (A/A) and pink-eyed dilution (p/p) animals. In albino (c/c) mice, the tyrosinase activities and melanin formation activity were scarcely detectable, and no TY-CRM was observed.
Contrary to these, the levels of tyrosinase mRNA in agouti and albino mutants were almost the same as that in black mice, and those in brown and dilution mutants were 20–30% higher than that in black mice. These results indicate that the mutations at the brown and dilute loci exert their influence on the tyrosinase mRNA level, presumably by affecting the transcription of the tyrosinase gene, and that the mutations at the agouti and albino loci exert their influence on the TY-CRM level by regulating translation of tyrosinase mRNA and/or post-translation modification of the enzyme.
... The PMEL gene codes for a transmembrane glycoprotein that is regulated by multiple proteolytic processes resulting in an amyloid fibrillar matrix, which enables the deposition of melanin pigments and potentially prevents the toxic effect of melanin derivatives in the melanosome (Bissig et al., 2016). The OCA2 gene, also known as the pink-eyed dilution protein or p-gene in human, plays an important role in the biogenesis of melanosomes and regulation of eumelanin content in melanocytes through the processing and trafficking of the tyrosinase enzyme (Hirobe et al., 2002;Orlow and Brilliant, 1999;Ozeki et al., 1995;Tamate et al., 1989). We did not include the expression levels of MC2R gene in our analyses because the expression of this gene was at the limit of detection, even after preamplification. ...
Glucocorticoid hormones are important intermediates between an organism and its environment. They enable an organism to adjust its behavioural and physiological processes in response to environmental changes by binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) expressed in many tissues, including the integument. The regulation of glucocorticoids co-varies with melanin-based colouration in numerous species, an association that might result from pleiotropic effects of genes in the melanocortin system and evolve within a signalling context. Most studies have focused on the circulating levels of glucocorticoids disregarding the receptors that mediate their action, and that might partly account for the covariation between the regulation of stress and melanin-based colouration. We investigated the association of the expression levels of GR and MR genes with melanin-based colouration in the growing feathers of nestling barn owls (Tyto alba). We also explored the association between GR and MR expression levels and the expression of genes related to the melanocortin system and melanogenesis to better understand the origin of the link between the expression of receptors to which corticosterone binds and melanin-based colouration. Nestling barn owls displaying larger eumelanic black feather spots expressed GR and MR at lower levels than smaller-spotted individuals. However, we found that the expression of the GR and MR genes was positively rather than negatively correlated with the expression of genes involved in the deposition of melanin pigments at the time we sampled the nestlings. This provides mixed evidence of the association between melanin-based traits and MR and GR gene expression. The finding that the expression of GR and MR was positively associated with the expression of the PCSK2 gene (encoding one of the protein convertase responsible for the production of hormone peptide ACTH and α-MSH) suggests that the melanocortin system may be implicated in the establishment of the covariation between melanin-based colour and the expression of receptors to which glucocorticoids bind. However, further studies investigating the expression of melanin-based traits with stress-related endpoints at different time points of feather development will be necessary to understand better the proximate mechanism linking melanin-based traits with stress.
... The PMEL gene codes for a transmembrane glycoprotein that is regulated by multiple proteolytic processes resulting in an amyloid fibrillar matrix, which enables the deposition of melanin pigments and potentially prevents the toxic effect of melanin derivatives in the melanosome (Bissig et al., 2016). The OCA2 gene, also known as the pink-eyed dilution protein or p-gene in human, plays an important role in the biogenesis of melanosomes and regulation of eumelanin content in melanocytes through the processing and trafficking of the tyrosinase enzyme (Hirobe et al., 2002;Orlow and Brilliant, 1999;Ozeki et al., 1995;Tamate et al., 1989). We did not include the expression levels of MC2R gene in our analyses because the expression of this gene was at the limit of detection, even after preamplification. ...
Glucocorticoid hormones, such as corticosterone, are important intermediates between an organism and its environment. They enable an organism to adjust his behavioural and physiological processes in response to environmental changes by binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) expressed in many tissues, including the integument. The regulation of glucocorticoids covaries with melanin-based colouration in numerous species, an association that may result from the melanocortin system. Most studies have focused on the circulating levels of glucocorticoids disregarding the receptors that mediate their action and the proximate mechanisms underlying the covariation with melanin-based coloration. We investigated in nestling barn owls (Tyto alba) the association between melanin-based coloration and the expression levels of GR and MR in growing feathers. We also considered the expression levels of genes related to the melanocortin system and melanogenesis to better understand the origin of the link between the expression of receptors to which corticosterone binds and melanin-based coloration. Nestling barn owls displaying larger black feather spots expressed GR and MR at lower levels than did smaller-spotted individuals. Because the expression of the GR and MR genes were positively, rather than negatively, correlated with the expression of melanogenic genes, we suggest that the link between melanin-based coloration, GR and MR is not directly associated to melanogenesis. The finding that the expression of GR and MR was positively associated with the PCSK2 gene that encodes the protein convertase responsible for post-translational modification of the proopiomelanocortin hormone, suggests that the melanocortin system may be implicated in the establishment of the covariation between melanism and the expression of receptors to which glucocorticoids bind. Together with previous studies, our results suggest that coloration is not only related to the production of glucocorticoids but also to the sensitivity of the integument to glucocorticoids.
... [22][23][24] Mice with TYRP1 mutations have 30-40% less eumelanin in their brown fur than those with wild-type black fur, and if the same is true in cats that segregate for these polymorphisms, this decreased requirement for pigmentation may be associated with decreased risk of hyperthyroidism. 25 British shorthairs are accepted in a range of colors including colorpoint by the GCCF, but this breed is probably most well known for its classic British blue phenotype. The majority of BSH in this study were a dilute color, usually blue, but an effect of color dilution was not found on risk of hyperthyroidism in nonpurebreds. ...
Background: Hyperthyroidism is very common in older cats, but the etiopathogenesis is poorly understood. Decreased risk of hyperthyroidism has been reported in certain colorpoint breeds, and this observation previously has been hypothesized to result from relatively greater tyrosine availability for thyroid hormone production because of limited ability to convert tyr- osine to melanin pigment. However, studies investigating a potential link between coat pigmentation and risk of hyperthy- roidism are limited.
Objective: To identify associations between coat phenotype and hyperthyroidism by investigation of breed, coat color, and hair length as risk factors for the disease.
Animals: Data were used from 4,705 cats aged ≥10 years, referred to a single veterinary teaching hospital (2006–2014) in the United Kingdom.
Methods: Retrospective, epidemiological, cross-sectional study using Bayesian multivariable logistic regression to assess risk factors for hyperthyroidism.
Results: Burmese (odds ratio [OR], 0.01; 0.00–0.23; P = .004), Tonkinese (OR, 0.05; 0.00–0.95; P = .046), Persian (OR, 0.21; 0.10–0.44; P < .001), Siamese (OR, 0.27; 0.12–0.61; P = .002), Abyssinian (OR, 0.04; 0.00–0.74; P = .031), and British shorthair (OR, 0.47; 0.28–0.79; P = .004) breeds had decreased risk of hyperthyroidism compared to domestic shorthairs. Longhaired, nonpurebred cats (OR, 1.30; 1.03–1.64; P = .028) were at increased risk of hyperthyroidism. Coat color/pattern was not associated with hyperthyroidism in nonpurebred cats.
Conclusions and Clinical Importance: We identified decreased risk of hyperthyroidism in the Tonkinese, Abyssinian, and British shorthair breeds, identified an association between risk of hyperthyroidism and hair length, and confirmed decreased risk in Burmese, Siamese, and Persian breeds. Additional studies are warranted to further investigate these findings.
... More than 80 alleles have so far been reported [5]. The mouse pink-eyed dilution (p/Oca2 p , oculocutaneous albinism II) locus is one of the classical coat color loci and the locus controls tyrosinase activity, melanin synthesis, and melanosome development [6][7][8][9][10][11]. ...
Background:
The mouse pink-eyed dilution (oculocutaneous albinism II; p/Oca2(p)) locus is known to control tyrosinase activity, melanin content, and melanosome development in melanocytes. Pink-eyed dilution castaneus (p(cas)/Oca2(p-cas)) is a novel mutant allele on mouse chromosome 7 that arose spontaneously in Indonesian wild mice, Mus musculus castaneus. Mice homozygous for Oca2(p-cas) usually exhibit pink eyes and beige-colored coat on nonagouti C57BL/6 (B6) background. Recently, a novel spontaneous mutation occurred in the progeny between this mutant and B6 mice. The eyes of this novel mutant progressively become black from pink and the coat becomes dark gray from beige with aging.
Objective:
The aim of this study is to clarify whatever differences exist in melanocyte proliferation and differentiation between the ordinary (pink-eyed) and novel (black-eyed) mutant using serum-free primary culture system.
Methods:
The characteristics of melanocyte proliferation and differentiation were investigated by serum-free primary culture system using melanocyte-proliferation medium (MDMD).
Results:
The proliferation of melanoblasts in MDMD did not differ between the two mice. However, when the epidermal cell suspensions were cultured with MDMD supplemented with l-tyrosine (Tyr), the differentiation of black-eyed melanocytes was greatly induced in a concentration-dependent manner compared with pink-eyed melanocytes. Immunocytochemistry demonstrated that the expression of tyrosinase and tyrosinase-related protein-1 (Tyrp1) was greatly induced or stimulated both in pink-eyed and black-eyed melanocytes, whereas the expression of microphthalmia-associated transcription factor (Mitf) was stimulated only in black-eyed melanocytes.
Conclusion:
These results suggest that the age-related coat darkening in black-eyed mutant may be caused by the increased ability of melanocyte differentiation dependent on l-Tyr through the upregulation of tyrosinase, Tyrp1, and Mitf. This mutant mouse may be useful for animal model to clarify the mechanisms of age-related pigmentation in human skin, such as melasma and solar lentigines.
... However, the siRNA was not affected the cellular tyrosinase activity as well as the expression in melan-p1 cells (Figs. 7c, 9c). Previous studies on p-mutant also reported the diminution in tyrosinase expression [25][26][27]. The siRNA had no effect on expression of TRP-1 and TRP-2 in melan-a, B16F10 and melan-p1 cells. ...
The pink-eyed dilution protein (P-protein) plays a critical role in melanin synthesis in melanocytes and retinal pigment epithelium cells. Mutation in this protein may cause complete or partial albinism. Role of the P-protein ranges in melanin synthesis to maturation and trafficking of the melanosomes. The aim of the present study was to evaluate the effect of P-protein inhibition on melanosome biology by comparing the shape, size, count, and types of melanosomes in melan-a melanocytes. The cells were extensively examined by the transmission electron microscopy. The P-protein inhibition was carried by P-protein-siRNA transfection to melan-a melanocytes, B16F10 mouse melanoma, and melan-p1 cells. Measurement of melanin contents, cellular tyrosinase, and different tyrosinase related proteins were also determined to investigate the effect of P-protein siRNA transfection on melanocytes. Results suggested that the inhibition of P-protein can significantly change the melanosomal morphology, types and their respective numbers, and provided a novel strategy for the control of melanin synthesis.
... Levels of tyrosinase, the rate-limiting enzyme in melanin synthesis, are greatly decreased in the skin of p/p mice (Tamate et al., 1989) and in cultured epidermal melanocytes of newborn p/p mice (Hirobe et al., 1998). The product of the p gene is an integral membrane protein that localizes in melanosomes (Rosemblat et al., 1994); its predicted secondary structure is a 12-transmembrane domain protein similar to a channel or transporter (Gardner et al., 1992;Rinchik et al., 1993). ...
... Levels of tyrosinase, the rate-limiting enzyme in melanin synthesis, are greatly decreased in the skin of p/p mice (Tamate et al., 1989) and in cultured epidermal melanocytes of newborn p/p mice (Hirobe et al., 1998). The product of the p gene is an integral membrane protein that localizes in melanosomes (Rosemblat et al., 1994); its predicted secondary structure is a 12-transmembrane domain protein similar to a channel or transporter (Gardner et al., 1992;Rinchik et al., 1993). ...
It is known that melanin pigments composed of black to dark brown eumelanin (EM) and yellow to reddish-brown pheomelanin (PM) are widely distributed in vertebrates. Melanin pigments in vertebrate are produced in melanocytes which are distributed in the epidermis, hair follicles, choroid, iris, inner ear, and other tissues. The diversity of the color in the internal and external tissues in vertebrates is mainly attributed to the quantity and ratio of EM and PM. Melanin pigments are highly oxidized and complex pigments. It is thus important to analyze these two types of melanin pigments in studies of the biochemical and genetic bases of pigmentation in vertebrates. Two microanalytical methods to perform the simultaneous measurement of eumelanin and pheomelanin were established to characterize melanin and melanogenesis. In this chapter, we explain microanalytical application with the chemical degradation of melanin and focus on internal and external melanins produced by pigment cells in vertebrates: mammals, birds, fish, reptiles, and amphibians. By using these methods for the evaluation of the “chemical phenotype,” the mutual triangular relationship among “chemical phenotype,” “visual phenotype,” and “genotype” is reviewed.
Hair bulb melanocytes involved in the expression of the coat colors of mice are derived from epidermal melanocytes. The biological study of epidermal melanocytes is important to understand the expression of the coat color. Analysis of the melanocyte and melanoblast-melanocyte population in the mouse epidermis has shown marked strain differences, suggesting that the two populations are regulated by genetic factors. From the results of genetic crosses between C57BL/1OJ and C3H/He mice semidominant genes were shown to be involved in regulating the two populations. The two populations in C57BL/1OJ-A/A congenic mouse did not differ from those in C57BL/1OJ. The distributions of the two populations in C3H/He were gradually transferred to those in C57BL/1OJ by repeated backcrossing.The semidominant genes are thought to affect the melanocyte proliferation, melanocyte differentiation, or induction of tyrosinase activity by MSH. Such effects may change the pigmentation in the hair bulb and produce a variety of the hair color intensity of mice, which may in turn be influenced by selection, mutation, random mating, and environment.
Three different cDNA clones (pmcTyr1, pmcTyr2 and pmcTyr3) representing mRNAs originating by alternative splicing of the primary transcript of mouse tyrosinase gene, were identified and characterized by sequence analysis and by a functional assay. These cDNAs were subcloned into the newly constructed expression vector pHD. After electroporation of these hybrid clones into tyrosinase negative cells, protein extracts were prepared and tested for tyrosinase enzyme activity. Only the cDNA insert of pmcTyr1 was able to confer tyrosinase enzyme activity. This cDNA encodes a protein 533 amino acid residues in length containing a putative leader peptide of 18 amino acids and six putative glycosylation sites. Comparisons of the deduced amino acid sequence of the cDNA clone pmcTyr1 with the protein sequence of tyrosinases from man, Streptomyces, Neurospora and with haemocyanin subunits from a spider showed two regions of sequence conservation. One of these regions is known to be involved in copper binding. Since this gene with the coding capacity for tyrosinase is absent in all studied c-locus lethal deletion mutant mice, we have evidence that albinism in mice is caused by mutations of the tyrosinase gene.
This chapter discusses the method of guanidine isothiocyanate preparation of total RNA, which is a versatile and efficient way to extract intact RNA from most tissues and cultured cells, even if the endogenous level of RNase is high. The cells are lysed in guanidine isothiocyanate using a tissue homogenizer. The lysate is layered onto a CsCl gradient and spun in an ultracentrifuge. Proteins remain in the aqueous guanidine portion, DNA bands in the CsCl, and RNA pellets in the bottom of the tube. The RNA is recovered by redissolving the pellet. The recovery of RNA is usually excellent if the capacity of the gradient is not exceeded. The method can also be used to isolate RNA from tissue or to isolate both RNA and DNA from cells. The time taken to follow this method is two days.
Tyrosinase, the enzyme that controls the synthesis of melanin, is a unique product of melanocytes. Normal and malignant human melanocytes grown in culture were used to study the factors that regulate the expression of tyrosinase. Immunoprecipitation experiments showed that newly synthesized tyrosinase appeared as a protein with an apparent molecular weight of 70,000 that was processed to a protein with an apparent molecular weight of 80,000. Neither tunicamycin nor 2-deoxy-D-glucose inhibited this conversion, suggesting that O-glycosylation is the major biochemical event in the posttranslational modification of tyrosinase. Agents that stimulated the proliferation of normal melanocytes also stimulated tyrosinase activity. Melanocytes with low levels of tyrosinase activity synthesized less tyrosinase, processed the enzyme more slowly, and degraded it more rapidly than melanocytes with high levels of tyrosinase activity. We conclude that tyrosinase activity in cultures of human melanocytes derived from different donors is determined predominantly by its abundance.
A cDNA library was constructed from poly (A)+ mRNA from mouse melanocytes and screened using anti-tyrosinase antiserum and oligonucleotide probes corresponding to amino acid sequence of tyrosinase. Sequencing of some cDNA clones positive in these screenings gave a nucleotide sequence of 1838 nucleotides including a open reading frame of 1344 nucleotides.
1) In the mouse, melanoblasts positive to the dopa-oxidase reaction appear in the basal layer of the epidermis at neo-natal stages, though they can not be detected in the epidermis of adult mice.2) There was a significant difference in the number of the dopa-positive melanoblasts per unit area of the epidermis at the neo-natal stages between the two strains examined, C3H/HeNSa and C57BL/6.3) The difference seems to be attributable to the difference in dopa-oxidase activity of the epidermal melanoblasts from the two strains as determined microspectrophotometrically.4) The F1 from the reciprocal crosses between the two strains exhibited an intermediate value in both the number of dopa-positive cells and the enzyme activity of the cells.5) The F2 showed a considerably wider distribution in the number of dopa-positive cells.6) These results seem to suggest an inhibitory factor is involved in the strain difference in the dopa-oxidase activity and is controlled by more than two loci.