Melanin concentrating hormone. I. Influence of nerves and hormones on the control of trout melanophores

When melanophores on trout scales are cultured in vitro they show a transitory melanin concentration, which can be prevented by addition to the medium of the alpha adrenergic blocker, phentolamine. This indicates the release of endogenous nor-epinephrine from local nerve terminals. This initial phase of melanin aggregation is followed by redispersion and then by a second, more gradual melanin concentration over several days, which is not antagonized by phentolamine. A final melanophore index of between 2-2.5 is attained which may be the resting state of trout melanophores. Using short-term cultured melanophores which have passed the phase of endogenous nor-epinephrine release, it is shown that exogenous nor-epinephrine will interact synergistically with the melanin-concentrating hormone to achieve full melanin concentration. Evidence is discussed for believing that in the trout, such synergy is necessary to achieve maximum pallor in vivo.     

Control of melanin synthesis and secretion by B16/C3 melanoma cells

In culture, B16/C3 murine melanoma cells grown in the presence of serum undergo melanogenesis at a specific time after plating. At this time, melanin is synthesized intracellularly and then secreted into the extracellular culture fluid. We have found that melanin secretion is dependent on the presence of serum in the growth medium. When confluent cultures are deprived of serum, that is, refed with serum-free medium, cells remain viable but do not undergo melanogenesis. Addition of serum-free medium supplemented with either melanocyte-stimulating hormone (MSH) or dibutyryl cAMP induced melanogenesis in these cells but did not result in melanin secretion. Furthermore, when B16/C3 cells are grown in serum-free, hormone-supplemented medium, they also undergo melanogenesis but fail to release melanin. The addition of serum, however, to B16/C3 cells induced to undergo melanogenesis with MSH, dibutyryl cAMP, or hormone-supplemented medium promotes melanin secretion. Fractionation studies hence revealed that serum contains specific factors capable of inducing melanin secretion. These results demonstrate that factors that regulate melanin synthesis are distinct from those that induce cells to release melanin into their extracellular environment. Furthermore, the ability to induce melanogenesis with single factors will permit us to study the precise sequence of events leading to differentiation in B16/C3 cells under chemically defined conditions.     

Mechanism of skin pigmentation

Melanin is a pigment that plays an important role in providing coloration and protecting human skin from the harmful effects of UV light radiation. Human skin color is determined by the type and amount of melanins that are synthesized and deposited within the melanosomes. In addition, the transfer of these specialized membrane-bound organelles from melanocytes to surrounding keratinocytes also plays a role in dictating human skin color. In order to investigate the principle features of skin pigmentation, the origin, function, and production ability of melanin should be highly understood in terms of biological and pathophysiological aspects. Furthermore, a deep understanding of melanin synthesis will also contribute to cosmetics and drugs development. In this review, the processes of melanin biosynthesis, such as survival, proliferation, and differentiation of melanin cells, as well as the biological regulation of human pigmentation were described.     

Melanosomal Proteins – Role in Melanin Polymerization

Melanin, the major determinant of skin colour, is a tyrosine‐based heteropolymer of indeterminate molecular weight. In vivo, melanin synthesis occurs within highly specialized organelles called melanosomes. Coated vesicles encapsulating the enzyme tyrosinase and tyrosinase related proteins, fuse with premelanosomes that contain structural proteins to form mature melanosomes. Coated vesicles and premelanosomes have been shown to have only melanin monomers but not the polymer. Our earlier results have clearly shown that the presence of proteins other than tyrosinase are critical for the post‐tyrosinase steps of melanin polymerization at acidic pH. Proteins in melanosomes are difficult to purify because of their firm association with melanin. Thus, with progressive melanization, melanoproteins become progressively insoluble. In this paper, we discuss the isolation and purification of melanosomal proteins and their role in melanin polymerization. We have hypothesized that the initiation of polymerization and the binding of melanin to proteins are two discrete events and we have developed assays to quantify these events. Purified melanosomal proteins differ in their ability to polymerize melanin monomers. Further, we have also shown that two polypeptides (28 and 45 kDa) purified from melanosomes inhibit melanin polymerization but can bind preformed melanin. In conclusion, melanosomal proteins regulate melanin polymerization and differ in their ability to bind melanin. Polymerization and binding abilities of melanosomal proteins are specific to each protein and melanin–protein interaction is not nonspecific.     

Regulation of eumelanin/pheomelanin synthesis and visible pigmentation in melanocytes by ligands of the melanocortin 1 receptor

The production of melanin in the hair and skin is tightly regulated by the melanocortin 1 receptor (MC1R) whose activation is controlled by two secreted ligands, alpha-melanocyte stimulating hormone (alphaMSH) and agouti signal protein (ASP). As melanin is extremely stable, lasting years in biological tissues, the mechanism underlying the relatively rapid decrease in visible pigmentation elicited by ASP is of obvious interest. In this study, the effects of ASP and alphaMSH on the regulation of melanin synthesis and on visible pigmentation were assessed in normal murine melanocytes and were compared with the quick depigmenting effect of the tyrosinase inhibitor, phenylthiourea (PTU). alphaMSH increased pheomelanin levels prior to increasing eumelanin content over 4 days of treatment. Conversely, ASP switched off the pigment synthesis pathway, reducing eu- and pheo-melanin synthesis within 1 day of treatment that was proportional to the decrease in tyrosinase protein level and activity. These results demonstrate that the visible depigmentation of melanocytes induced by ASP does not require the degradation of existing melanin but rather is due to the dilution of existing melanin by melanocyte turnover, which emphasizes the importance of pigment distribution to visible color.     

Melanosomal proteins--role in melanin polymerization

Melanin, the major determinant of skin colour, is a tyrosine-based heteropolymer of indeterminate molecular weight. In vivo, melanin synthesis occurs within highly specialized organelles called melanosomes. Coated vesicles encapsulating the enzyme tyrosinase and tyrosinase related proteins, fuse with premelanosomes that contain structural proteins to form mature melanosomes. Coated vesicles and premelanosomes have been shown to have only melanin monomers but not the polymer. Our earlier results have clearly shown that the presence of proteins other than tyrosinase are critical for the post-tyrosinase steps of melanin polymerization at acidic pH. Proteins in melanosomes are difficult to purify because of their firm association with melanin. Thus, with progressive melanization, melanoproteins become progressively insoluble. In this paper, we discuss the isolation and purification of melanosomal proteins and their role in melanin polymerization. We have hypothesized that the initiation of polymerization and the binding of melanin to proteins are two discrete events and we have developed assays to quantify these events. Purified melanosomal proteins differ in their ability to polymerize melanin monomers. Further, we have also shown that two polypeptides (28 and 45 kDa) purified from melanosomes inhibit melanin polymerization but can bind preformed melanin. In conclusion, melanosomal proteins regulate melanin polymerization and differ in their ability to bind melanin. Polymerization and binding abilities of melanosomal proteins are specific to each protein and melanin-protein interaction is not nonspecific.     

Melanin concentrating hormone (MCH) effects on teleost (Chrysiptera cyanea) melanophores

The in vitro biological actions of synthetic chum salmon melanin concentrating hormone (MCH) on melanophores of the blue damselfish (a teleost), Chrysiptera cyanea, were studied. This cyclic heptadecapeptide stimulated melanosome (melanin granule) aggregation (centripetal migration) within melanophores at a threshold concentration of about 10(-10) M. The action of this putative hormone was not blocked by alpha- or beta-adrenoceptor antagonists. It was concluded that the effects of MCH were direct and were not mediated indirectly through the actions of adrenergic neurotransmitters released from nerve terminals. Further evidence for this view comes from the observation that, unlike the case of neurotransmitter release, melanosome aggregation in response to MCH proceeded in the absence of calcium. The possible role of MCH in the control of color change of teleost fishes is discussed.     

Natural,semisynthetic and synthetic tyrosinase inhibitors

Tyrosinase plays a pivotal role in the synthesis of melanin pigment synthesis on skin utilizing tyrosine as a substrate. Melanin is responsible for the protection against harmful ultraviolet irradiation, which can cause significant pathological conditions, such as skin cancers. However, it can also create esthetic problems when accumulated as hyperpigmented spots. Various skin-whitening ingredients which inhibit tyrosinase activity have been identified. Some of them, especially ones with natural product origins, possess phenolic moiety and have been employed in cosmetic products. Semi-synthetic and synthetic inhibitors have also been developed under inspiration of the natural inhibitors yet some of which have no phenolic groups. In this review, tyrosinase inhibitors with natural, semi-synthetic and synthetic origins are listed up with their structures, activities and characteristics. Further, a recent report on the adverse effect of a natural melanin synthesis inhibitor which was included in skin-whitening cosmetics is also briefly discussed.     

Aminopiperidine indazoles as orally efficacious melanin concentrating hormone receptor-1 antagonists

The synthesis and biological evaluation of novel 3-amino indazole melanin concentrating hormone receptor-1 antagonists are reported, several of which demonstrated functional activity of less than 100nM. Compounds 19 and 28, two of the more potent compounds identified in this study, were characterized by high exposure in the brain and demonstrated robust efficacy when dosed in diet-induced obese mice.     

A hormone-sensitive stage in the development of the retinal pigment epithelium in Hunter rats with hereditary retinal dystrophy

The sensitivity of the retinal pigment epithelium (RPE) to the melanotropic effects of alpha-MSH and dbcAMP was assayed in an organ culture of the eye scleral part in the Hunter rats with inherited retinal dystrophy. The melanin synthesis was estimated by liquid scintillation on the RPE isolated enzymatically after 48-h cultivation. One eye from every animal was cultivated in a medium without natural components and with the hormone or dbcAMP (experiment), while the other in a hormone-free medium (control). The melanin synthesis was estimated by 14C-thiouracil incorporation. The result was expressed as a cpm/microgram DNA (experiment) to cpm/microgram DNA (control) ratio. In addition, the index of labelled nuclei was determined using 3H-thymidine autoradiography in the central RPE zone of the eyes from young rats of the same litter. The experiments with alpha-MSH confirmed the earlier data according to which the RPE of the 3 day old Hunter rats were insensitive to melanotropic hormones. This was not due to defects in the cytoplasmic melanin-synthesizing system, since under the influence of dbcAMP the melanin synthesis in the RPE increases more than four-fold as compared with the control; dbcAMP stimulates also the total protein synthesis, as estimated by 3H-leucine incorporation. The RPE of the 4 day old rats proved to be sensitive to alpha-MSH: the melanin synthesis increases more than twice suggesting the healthy state of the RPE membrane melanotropic receptors. alpha-MSH also stimulates the total protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular and biochemical mechanisms of human iris color: A comprehensive review

Eye color is determined as a polymorphism and polygenic trait. Brown is the most common eye color in the world, accounting for about 79%, blue eye color for about 8–10%, hazel for 5%, and green for 2%. Rare-colored eyes include gray and red/violet. Different factors are involved in determining eye color. The two most important factors are the iris pigment and the way light is scattered from the iris. Gene expression determines the iris pigmentation and how much melanin is present in the eye, which is the number of melanin subunits that identify eye color. The genes involved in the pigmentation of single-nucleotide polymorphism (SNP) have a significant role; and even some genes are included only in the eye color through SNP. MicroRNAs also affect melanocyte synthesis, which is usually affected by the downregulation of essential genes involved in pigmentation. In this study, we assess the biochemical pathways of melanin synthesis, and the role of each gene in this pathway also has been examined in the signaling pathway that stimulates melanin synthesis.     

PDE5 inhibitor promotes melanin synthesis through the PKG pathway in B16 melanoma cells

PDE inhibitors could increase cellular cGMP levels and are used to treat erectile dysfunction as well as pulmonary arterial hypertension. cGMP production was reported to be necessary for UVB-induced melanin synthesis, however, the effect of PDE5 inhibitor on melanin synthesis has not been examined. We found that PDE5 inhibitor (sildenafil or vardenafil) and the cGMP analog 8-CPT-cGMP stimulated CREB phosphorylation, leading to increased tyrosinase expression and melanin synthesis, which was counteracted by KT5823, a selective cGMP-dependent protein kinase (PKG) inhibitor. However, KT5823 did not affect cAMP-elevating agent-mediated melanin synthesis, indicating that KT5823 selectively inhibited cGMP-induced melanin synthesis. This is the first study to find that PDE5 inhibitor can promote melanin synthesis and reveal that PKG-dependent CREB phosphorylation and tyrosinase expression is involved in cGMP-induced melanin synthesis. Our results suggest that PDE5 inhibitor may be beneficial for the treatment of hypopigmentation diseases.     

Regulatory Factors of Pheo- and Eumelanogenesis in Melanogenic Compartments

Melanogenesis, i.e., synthesis of melanin and melanosomes, is a “cascade'’of event which is channelled by internal and external regulatory factors. The recognition and selection of this information and subsequent differentiation of melanogenesis (melanin type and melanosomal development) would be regulated significantly by melanosomal membrane. The melangenesis type could be switched relatively easily by UV light, hormone, and availability of tyrosinase substrate. The role of sulphydryl compounds as a regulatory factor in melangenesis type (in particular for pheomelanogenesis) may not be tied to its absolute presence or absence, but rather, to the effective concentration within the melanocyte at a given time. It is, therefore, probable that the morphogenesis of melanosomes may not follow immediately in response to melanogenesis-type changes, hence the melanocyte revealing more often mosaic forms of melanosomes in nature after exposure to non-genetic factors. The switch of melanogenesis would be significantly controlled by structural and functional availability of vesiculoglobular bodies which are encoded or associated with HMSA-5 (69 kDa) glycoprotein. This HMSA-5 protein shares a significant homology with gp75 “b-locus'’protein. However, because of our hypothesis that vesiculoglobular bodies carry post-(and pre-) tyrosinase regulatory factors involving in both pheo- and eumelanogenesis, the term “b-protein'’which focuses only on eumelanogenesis may not be applied to HMSA-5.     

The role of melanocytes in protection against photooxidative stress Krystyna Stepień

Excessive exposure to solar ultraviolet radiation is an essential etiological factor for skin cancer. UV radiation, directly or indirectly through the generation of reactive oxygen species (ROS), causes damage to DNA, proteins and lipids, and induces inflammation and immunosuppression. Cutaneous pigmentation afforded by melanocytes is the main photoprotective mechanism in human skin. In response to UV, melanocytes produce melanin pigments and transfer them to adjacent keratinocytes. This review describes: (i) the photoprotective action of melanin; (ii) the regulation of UV-induced melanogenesis and the role of p53 in this process; (iii) the relation between melanogenic and antioxidant activities in melanocytes. The possible involvement of UV-induced ROS in the stimulation of melanin synthesis is also discussed.     

BmPAH Catalyzes the Initial Melanin Biosynthetic Step in Bombyx mori

Pigmentation during insect development is a primal adaptive requirement. In the silkworm, melanin is the primary component of larval pigments. The rate limiting substrate in melanin synthesis is tyrosine, which is converted from phenylalanine by the rate-limiting enzyme phenylalanine hydroxylase (PAH). While the role of tyrosine, derived from phenylalanine, in the synthesis of fiber proteins has long been known, the role of PAH in melanin synthesis is still unknown in silkworm. To define the importance of PAH, we cloned the cDNA sequence of BmPAH and expressed its complete coding sequence using the Bac-to-Bac baculovirus expression system. Purified recombinant protein had high PAH activity, some tryptophan hydroxylase activity, but no tyrosine hydroxylase activity, which are typical properties of PAH in invertebrates. Because melanin synthesis is most robust during the embryonic stage and larval integument recoloring stage, we injected BmPAH dsRNA into silkworm eggs and observed that decreasing BmPAH mRNA reduced neonatal larval tyrosine and caused insect coloration to fail. In vitro cultures and injection of 4^th instar larval integuments with PAH inhibitor revealed that PAH activity was essential for larval marking coloration. These data show that BmPAH is necessary for melanin synthesis and we propose that conversion of phenylalanine to tyrosine by PAH is the first step in the melanin biosynthetic pathway in the silkworm.