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Department of Pathology, University of Tennessee, Memphis, Tennessee; Department of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, United Kingdom; Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai, Miyagi, Japan; and Department of Medicine, Southern Illinois University, Springfield, Illinois
ABSTRACT I. MELANIN PIGMENT A. Melanins: Chemical and Physical Properties B. Melanin Pigment in Skin Physiology and Pathology 1. Human skin A) SEXUAL DIFFERENCES IN SKIN MELANIZATION. B) MELANIN ROLE IN HUMAN PATHOLOGY. C) MELANIN METABOLISM. 2. Furry mammals (rodents) II. BIOCHEMISTRY AND CELLULAR AND MOLECULAR BIOLOGY OF MELANOGENESIS A. Melanocytes as Melanin-Producing Cells: Cell Biology and Ultrastructure B. Biochemistry of Melanogenesis 1. Introduction to melanogenesis 2. Tyrosinase, the main enzyme regulating melanin synthesis 3. TRP1 and TRP2 as modifiers of pathway velocity 4. Tetrahydropteridines, phenylalanine, and tyrosine hydroxylase as regulators of melanogenesis 5. Other melanogenesis-related proteins C. Extrapigmentary Functions of Melanogenesis-Related Proteins III. MECHANISMS OF REGULATION OF MELANOGENESIS A. Transcriptional Regulation B. Intracellular Signal Transduction Pathways C. Dual Function of L-Tyrosine and L-DOPA: Reaction Substrates and Bioregulators 1. Overview 2. L-Tyrosine and L-DOPA as positive regulators of subcellular apparatus of melanogenesis 3. L-Tyrosine promelanogenic effect and p locus 4. L-Tyrosine and L-DOPA regulate MSH receptor expression 5. L-Tyrosine and L-DOPA as ''hormonelike bioregulators'' D. Summary IV. HORMONAL STIMULATORS OF MELANOGENESIS AND THEIR RECEPTORS A. G Protein-Coupled Receptors and Ligands 1. Melanocortins, ACTH, and melanocortin receptors A) OVERVIEW OF MELANOCORTINS PHENOTYPIC EFFECTS. B) MOLECULAR CHARACTERIZATION OF MELANOCORTIN RECEPTORS. C) CELL BIOLOGY AND BIOCHEMISTRY OF MELANOCORTIN RECEPTORS. D) REGULATION OF MSH RECEPTORS EXPRESSION. E) PARA-, AUTO-, AND INTRACRINE MODES OF REGULATION OF MELANOCYTIC ACTIVITY. 2. {beta}-Endorphin and opioid receptors A) AN OVERVIEW. B) ROLE OF {beta}-ENDORPHIN IN SKIN BIOLOGY AND PIGMENTATION. 3. Endothelins and their receptors A) OVERVIEW. B) ENDOTHELINS AND MELANOCYTE DEVELOPMENT. C) ENDOTHELINS AS REGULATORS OF ADULT MELANOCYTES. D) ENDOTHELINS AND PATHOLOGY OF SKIN PIGMENTATION. 4. Histamine and its receptors A) OVERVIEW. B) CUTANEOUS HISTAMINE FUNCTION. C) HISTAMINE ACTION ON MELANOCYTES. D) HISTAMINE IN PIGMENTARY DISORDERS. 5. Eicosanoids and their receptors A) OVERVIEW. B) EICOSANOIDS AND MELANOCYTES. C) EICOSANOIDS AND PIGMENTARY DISORDERS. 6. Catecholamines and their receptors A) OVERVIEW. B) CATECHOLAMINE ACTION ON MELANOCYTES. C) CATECHOLAMINE IN PIGMENTARY DISORDERS. B. SCF and Its Receptor 1. Overview 2. SCF/c-kit in melanocytes 3. SCF/c-kit in pigmentary disorders C. Nuclear Receptors and Their Ligands 1. Estrogens and their receptors A) OVERVIEW. B) ESTROGENS AND MELANOCYTES. 2. Androgens and their receptors A) ANDROGEN FUNCTION AND SIGNALING. B) ANDROGENS AND MELANOCYTE BIOLOGY. C) ANDROGENS AND PIGMENTATION DISORDERS. 3. Vitamin D and its receptors A) OVERVIEW. B) VITAMIN D AND MELANOCYTE BIOLOGY. C) VITAMIN D AND PIGMENTARY DISORDERS. D. Other Positive Regulators of Melanogenesis: Bone Morphogenic Proteins 1. Bone morphogenic proteins and their receptors 2. Bone morphogenic proteins and melanogenesis E. Summary V. HORMONAL INHIBITORS OF MELANOGENESIS AND RECEPTORS A. G Protein-Coupled Receptors and Their Ligands 1. Serotonin and its receptors A) OVERVIEW. B) EFFECTS OF SEROTONIN AND ITS METABOLITES ON MELANOCYTES. 2. Melatonin and its receptors A) MELATONIN PRODUCTION. B) BIOLOGICAL FUNCTION OF MELATONIN AND ITS RECEPTORS. C) MELATONIN EFFECTS ON THE PIGMENTARY SYSTEM. 3. Dopamine and its receptors A) DOPAMINE AND MELANOGENESIS. B) DOPAMINE AND MELANOCYTE DISORDERS. 4. Acetylcholine and its receptors A) ACETYLCHOLINE AND MELANOGENESIS. B) ACETYLCHOLINE AND MELANOCYTE DISORDERS. B. Melanocortins Antagonists 1. Agouti protein A) AGOUTI-RELATED PROTEIN. 2. Agouti modifiers A) ATTRACTIN OR MAHOGANY. B) MAHOGANOID OR MAHOGUNIN. 3. Melanin concentrating hormone A) OVERVIEW. B) MCH SIGNALING SYSTEM AND MELANOCYTES. C. Cytokines, Growth Factors, and Receptors 1. IL-1, IL-6, and IFN-{alpha} and -{gamma} and their receptors A) IL-1, IL-6, AND IFN AND MELANOGENESIS. B) IL-1, IL-6, IFN, AND MELANOCYTE DISORDERS. 2. TNF-{alpha}, TNF-{beta} (lymphotoxin-{alpha}), and TGF-{beta}1 and their receptors A) TNF-{alpha}, TGF-{beta}1, AND MELANOGENESIS. B) TNF-{alpha}, LT-{alpha}, AND TGF-{beta} IN MELANOCYTE DISORDERS. D. Other Negative Regulators of Melanogenesis E. Summary VI. MISCELLANEOUS REGULATION OF MELANOGENESIS BY NUCLEAR RECEPTORS AND THEIR LIGANDS A. Glucocorticoids and Their Receptors 1. Glucocorticoid function and signaling in skin 2. Glucocorticoids and melanocyte biology B. Retinoids and Their Receptors 1. Retinoid function and signaling 2. Retinoids and melanocyte biology C. Summary VII. FUNCTIONAL REGULATION OF FOLLICULAR (HAIR) AND EPIDERMAL MELANIN UNITS A. The Epidermal Melanin Unit 1. Development of cutaneous pigmentary units 2. Melanocyte-keratinocyte interactions 3. Melanin transfer to keratinocytes B. Regulation of Hair Cycle-Coupled Follicular Melanogenesis 1. Development of hair follicle 2. Adult hair follicle 3. Follicular melanin unit VIII. UNIFIED CONCEPT FOR THE TRANSCRIPTIONAL REGULATION OF MELANOGENESIS: A KEY ROLE FOR MICROPHTALMIA-ASSOCIATED TRANSCRIPTION FACTOR IX. MELANOGENESIS AS MOLECULAR SENSOR AND TRANSDUCER OF ENVIRONMENTAL SIGNALS AND REGULATOR OF LOCAL HOMEOSTASIS X. COMMENTS AND FUTURE DIRECTIONS ACKNOWLEDGMENTS REFERENCES
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I. MELANIN PIGMENT |
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Melanins, the end-products of complex multistep transformations of L-tyrosine, are polymorphous and multifunctional biopolymers, represented by eumelanin, pheomelanin, neuromelanin, and mixed melanin pigment (323, 597, 598). Melanin biosynthesis can be initiated from either the hydroxylation of L-phenylalanine to L-tyrosine (nonobligatory step, operative in vivo) or directly from L-tyrosine, which is then hydroxylated to L-dihydroxyphenylalanine (L-DOPA) (obligatory step both in vitro and in vivo). L-DOPA serves as a precursor to both melanins and catecholamines, acting along separate pathways (Fig. 1). The next step, oxidation of L-DOPA to dopaquinone, is common to both eu- and pheomelanogenic pathways (597, 598). Eumelanogenesis involves the further transformation of dopaquinone to leukodopachrome, followed by a series of oxidoreduction reactions with production of the intermediates dihydroxyindole (DHI) and DHI carboxylic acid (DHICA), that undergo polymerization to form eumelanin (323, 573, 597). Pheomelanogenesis also starts with dopaquinone; this is conjugated to cysteine or glutathione to yield cysteinyldopa and glutathionyldopa, for further transformation into pheomelanin (323, 597, 598). Mixed melanin contains both eu- and pheomelanin. L-DOPA generation of catecholamines requires its enzymatic decarboxylation, hydroxylation, and methylation to produce dopamine, norepinephrine, and epinephrine, respectively (790). In vitro, all of these catecholamines can potentially convert into neuromelanin through several oxidation/reduction reactions (Fig. 1) (787); in vivo, only dopamine and cysteinyldopamine can serve as primary precursors to the pigment (101, 151, 930).
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Neuromelanins are macropolymers composed of aminochromes and noradrenalinochromes (101, 151, 522, 787, 930). Similar to other melanins, neuromelanins are brown/black pigment with stable paramagnetic properties, insoluble in organic solvents, bleached by hydrogen peroxide, and labeled by silver stain (930). Neuromelanins have mixed properties of both eu- and pheomelanins; they chelate metals and interact with several inorganic and organic compounds (9, 151, 428, 930). Other types of melanins may be generated by enzymatic oxidation of serotonin or tryptophan via tyrosinase; the resulting products have structures different from classical melanin (45). Melanin-like substances can also result from the tyrosinase-mediated transformation of opioid peptides that produce black to brown pigments with paramagnetic properties almost identical to DOPA-melanin (633).
B. Melanin Pigment in Skin Physiology and Pathology
Epidermal melanin has important evolutionary and physiological implications, particularly for unclothed humans. Thus high melanin content (racial pigmentation) protects the skin against ultraviolet (UV)-induced skin damage through its optical and chemical filtering properties (8). Indeed, skin pigment levels and anthropological origin are closely associated, with higher pigment amounts in regions of lower latitude and higher UV radiation levels. However, this connection may only be a recent human adaptation since early hominids may have possessed dark, dense, terminal body hair. A closely related primate, the chimpanzee, similar to most other nonhuman primates, exhibits white or lightly pigmented epidermis (591). Interestingly, chimpanzees have active melanocytes only in the epidermis of those areas directly exposed to UV radiation, e.g., face and friction surfaces (488).
The tendency toward relative hairlessness in modern humans has been explained by the need to maintain thermal balance under the progressive increase in demands for heat dissipation that results from the enhanced blood flow to the brain. Alternately or complementarily, hairlessness would also reduce parasitic infestations (549). Without concomitant increase in epidermal melanization, the end result of reduced hair coverage in humans residing in high UV radiation areas would be direct exposure to the adverse effects of that radiation. These include sunburn damage to the sweat glands with resultant suppression of sweating and abnormal thermoregulation (550), carcinogenesis, and nutrient inactivation by photolysis (e.g., folate) (74). Human populations living in areas with lower UV levels would adapt with lesser pigmentation, which also facilitates the cutaneous UV radiation-mediated conversion of 7-dehydrocholesterol to pre-vitamin D3 (876) In fact, if UV exposure of pigmented humans is limited in duration and/or intensity (e.g., northern latitudes), vitamin D3 deficiency and its associated pathologies may result, as seen in Southern Asians (Indians) living in northern European cities (267). Nevertheless, the value of the melanin pigmentation as a truly effective sunscreen for seasonal tanning is debatable since its sun protection factor (SPF) is only 1–2 (902). Additional properties of melanin may include a bactericidal potential via the production of orthoquinones (618), and contribution to the tensile strength of hair via cross-linking with proteins.
Hair color may have undergone a far more complex evolution than skin pigmentation. Although most humans are dark-haired and dark-eyed, melanization in skin, hair, and eyes do not closely correlate. Indeed, a large fraction of humans have dark eyes and hair but their skin would rate as "white," whereas in some western European populations, black hair commonly coexists with blue eyes. The occurrence of black scalp hair, a potent trap for radiant heat, may appear as a paradoxical development for primates and humans living in tropical climates; however, black scalp hair may provide some protection from sunstroke by helping with the salt balance through the highly efficient and fast ion exchange property by melanin (746, 902). In fact, the pigmented hair on the human scalp may have resulted from the littoral residence of Homo sapiens residing on sea coasts or riverbanks, with diet dominant in fish (many of which concentrate heavy metals). In this context, the capability to rapidly excrete toxic metals provided by the very high turnover of melanized cortical keratinocytes in the pigmented hair shaft would confer a selection advantage (46). Thus the long, melanized scalp hair with its capability to trap and/or bind chemicals, toxins, and heavy metals would prevent their access to living tissues. Pigmented hair may also provide antioxidant defense for the skin and hair follicles due to the high capacity of melanin for binding transition metals. This buffering capacity as applied to calcium would imply a role for melanin in cell function, since calcium is a critical second messenger in pigmentation signaling, acting in the transfer of melanosome to keratinocytes, and in epithelial cell differentiation (746).
A) SEXUAL DIFFERENCES IN SKIN MELANIZATION. The epidermis of adult human females is less melanized than in adult males, suggesting a gender-specific effect (626). One possible explanation for this discrepancy could be the higher need for vitamin D in women that is imposed by the increased intestinal calcium absorption of pregnancy and lactation (894).
B) MELANIN ROLE IN HUMAN PATHOLOGY. The main action of melanin in human skin appears to be attenuation of UV penetration to blood in dermal vessels. This may be inferred from the observation that peak UV absorption for oxyhemoglobin occurs at 545 nm, a value that in light-skinned individuals produces the strongest erythema reaction, and consequent pigmentary response. Also, when exposed to UV radiation, melanin can undergo photosensitization generating superoxide radicals and lethal injury in individual cells. Paradoxically, this action would however confer protection against the more deleterious outcome of cell neoplasia, consistent with the decreased proliferation rate of highly melanized normal skin cells (194), and with the close linkage between melanin production and photorepair of UV-induced DNA damage (212). Taken together, these data imply that melanin is important for skin homeostasis and that tanning itself represents a distress signal. The same pathophysiological explanation would apply to the localized pigmentation that follows the exposure of melanin to toxic compounds, and that may result in marked increases in melanin granules and melanin deposition (427).
Abnormalities in the transfer of melanosomes out of the melanocytes and into receiving keratinocytes represent the human counterpart of the dilute mutation in mice where the motor protein myosin V is defective. Disorders associated with aberrant melanosomes include the macromelanosomes and autophagic giant melanosome complexes of nevocellular nevi, lentigo simplex, malignant melanoma, and the neuroectodermal melanolysomal diseases that include the Elejalde, Chediak, Higashi, and Griscelli syndromes (515, 779, 780). These syndromes are more likely due to disordered melanosome biogenesis than alterations in melanosome degradation.
The most common pigment disorders are not disorders of melanin quality, but rather of the pigment-producing cell itself, which may be reduced in number, absent, or hyperactive and commonly, with regional localization. Hypomelanosis can either be acquired, e.g., vitiligo, or congenital via inheritance of mutations in pigment-related genes, e.g., albinisms and piebaldisms. Pigment excess (hypermelanosis) can be associated with inflammatory responses, as in keloid scars, or with local abnormal melanocyte function, as in dysplastic nevi or malignant melanoma.
C) MELANIN METABOLISM. Intact mature melanosomes pass from basal melanocytes into keratinocytes and their lysosomal compartment to become melanin dust in the upper nonviable layers of the epidermis. There is scant information on the actual mechanism of melanin breakdown or biodegradation. The melanin polymer appears to be resistant to enzymatic lysis, and it has been speculated that only phagosomal NADPH oxidase can degrade melanin itself via oxidative attack (68). Nevertheless, hair melanin granules, unlike those in overlying epidermis, tend to remain intact in the hair shaft. This is especially true for the eumelanogenic melanosomes of the black hair shaft such as seen in the hair of East-Asian (Oriental) individuals, the ethnic group with the highest density of pigment granules. In contrast, the pheomelanin granules characteristic of red and blonde hair, are partially digested. Both melanin types can be synthesized and released by the same melanocyte.
Most of the information on skin pigmentation has emerged from the intensive study of rodent coat color, as opposed to research focused specifically on epidermal pigmentation. As a uniquely mammalian trait, hair serves important functions most easily appreciated in furred mammals. These include thermal insulation, camouflage (for many species, melanin affords significant additional protective value, e.g., seasonal change of coat color in the arctic fox), social and sexual communication (involving visual stimuli, odorant dispersal, etc.) and sensory perception (e.g., whiskers). Many furred mammals, including the mouse, lack melanogenically active melanocytes in their adult truncal epidermis; instead, melanin is produced in the hair follicle bulb. There is, however, considerable variation in pigment patterns within and between furred mammals; for example, perifollicular melanocytes extend to the dermis in the hairy truncal skin of the adult Syrian golden hamster (602).
Mammalian hair color exhibits a wide range of shades. The highly variable color of murine pelage reflects variation in the copolymerization of eu- and pheomelanins, which results in the production of black, brown, yellow, gray, or white hair fibers. Natural eumelanins are produced by comixtures of DHI and DHICA, which provide variable contributions to pigmentation. For example, dilution mutant mice (e.g., slaty) exhibit a 30% reduction in total melanin compared with the black hair type, but the specific reduction in DHICA content can be >80% (548). DHICA melanins determine brown colors in the animal kingdom (538), although the ratio of DHI to DHICA in the brown mutation is similar to that seen in the black hair type. The light mutation at the brown locus results in the presentation of melanin only at the hair fiber tips due to premature death of follicular melanocytes (339). The silver mutation in mice is also associated with progressive graying caused by loss of melanocytes. Silver melanin is similar to brown and light melanins in those light-silver animals producing a diffuse "soluble" melanin within degenerating melanocytes.
Studies performed on the yellow (Ay/a) mouse and on the tortoise-shell guinea pig, with its potential to develop black, red, yellow, or white hair, have shown that the variation in melanin color is due to relative levels of glutathione reductase activity. Lowest levels are associated with black eumelanic hair, while highest levels are found in animals with lighter colors, and largely pheomelanosomes (40). Many of these color patterns map genetically to the extension locus (see elsewhere in this review). However, hair shaft color reflects not just the quantity and quality of the pigments produced by hair bulb melanocytes, but also the manner in which they are transferred to the hair shaft. Thus mouse coat color mutations, besides being associated with differences in melanin synthesis, can also be due to abnormalities in the formation of the melanosome and their transference to keratinocytes. These mutations are detected on qualitative and quantitative electron microscopy (224). For example, the albino locus is associated with a reduction in melanosome size, but data suggest that the albino locus, in addition to involve that structure, also has a functional (tyrosinase) role in the differentiation of mouse hair-bulb melanosomes. Data on melanosome length-to-width ratios indicate that the agouti locus determines melanosome shape, either spherical or elliptical. The agouti locus, even in the absence of melanization, directs melanosome shape via synthesis and deployment of agouti-locus-encoded matrix proteins, or via other structural actiors.
Abnormal transfer of melanin granules into the hair shaft can also lead to variation in pigmentation of mouse coat. An example of this is the dilute mouse, where reduction in coat color is due to mutation of the gene encoding for myosin Va (885). Thus, whereas mature melanosomes of normal melanocytes are located at the dendrite tips, in dilute mice they are retained in the perinuclear region of the cell due to a defect in protein motor capacity to transport melanosome along the cytoskeletal tracks to the dendrite tips. The discovery of this mouse gene, later identified as the first candidate gene for the Griscelli locus (Griscelli syndrome patients display severe immunodeficiency with diluted hair pigmentation), has stimulated a new field of study concerned with the function of molecular motors in vesicle/organelle transportation (849).
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II. BIOCHEMISTRY AND CELLULAR AND MOLECULAR BIOLOGY OF MELANOGENESIS |
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Under physiological conditions, melanin synthesis in melanocytes is restricted to melanosomes, and its enzymatic and structural elements are organized and assembled separately in a process resembling lysosome formation (333, 335, 487, 515, 597), although membrane traffic pathways differ between melanosomes and lysosomes (187). In general, melanosome structure correlates with the type of melanin produced, e.g., eumelanosomes are elliptical and contain fibrillar matrix while pheomelanosomes shape is variable with predominantly rounded contour and contains vesiculoglobular matrix (515). Melanosome development involves four steps. Stage I corresponds to the early matrix organization. In stage II, the matrix is already organized but without melanin formation (eumelanosomes); in pheomelanosomes melanin is already formed at this stage. In stage III there is deposition of melanin. In stage IV, melanosomes are fully melanized (completely filled with melanin) (Fig. 3). Under pathological conditions (e.g., melanoma), this orderly process is deregulated; for example, tyrosinase may already be activated at stage I of melanosome formation, whereas melanin can be deposited within organelles as a "granular type" without fibrillar or vesiculoglobular matrix (65, 334). These granular melanosomes produce eumelanin (Fig. 3C). There is no evidence for differences in melanosome biogenesis between follicular and epidermal melanocytes. Thus, in black hair follicles, melanocytes contain the largest number and most electron-dense melanosomes (eumelanosomes), each with a fibrillar matrix; in brown hair, bulb melanocytes are somewhat smaller, and in blonde hair melanosomes are poorly melanized, often with only the melanosomal matrix visible. Red hair pheomelanosomes contain a vesicular matrix, but melanin is deposited irregularly, in blotches (Fig. 3). Of interest, both eumelanogenic and pheomelanogenic melanosomes can coexist in the same human cell (316), but not within the same pathway, e.g., there is a switch committing melanosomes to either eu- to pheomelanin synthesis (547). These structural principles apply to follicular melanocytes and also probably to human epidermal melanocytes and rodent cutaneous melanocytes (181, 515).
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Another theory postulates earlier involvement of tyrosine hydroxylase to produce L-DOPA that acts as a necessary cofactor for tyrosinase enzyme action (457). The initial hydroxylation reactions, as well as L-DOPA stability, require an acidic environment that is provided by premelanosomes, where pH is regulated by a proton pump system (145, 487, 601). However, once L-DOPA is present, efficient formation of melanin pigment requires an increased pH (preferably neutral or basic) (19, 191), since acidification inhibits melanin synthesis (191, 487). The mechanism(s) for delivery of MRPs is under intensive investigation, and the timing of their actual incorporation into melanosomes is still unclear. Proper folding and assembly of MRPs in the ER requires their interaction with calnexin, glycosylation in the TGN, and the interaction of tyrosinase with TyrP1 or P protein (262, 385). MRP transport requires the formation of special vesicles, with assembly of coat proteins on the cytoplasmic side of the TGN to select MRPs for melanosomal delivery (333, 692). The adapter protein-3 (AP-3) that binds a dileucine motif in the cytoplasmic tail of tyrosinase-related proteins (TRPs) is important for the transport of TRPs from the TGN to melanosomes (335, 692). Small GTP binding proteins such as Rab 5 and 7, and phosphatidylinositol 3-kinase are involved in the intracellular trafficking of MRPs (333). An updated model of melanosome formation and trafficking of MRPs (Fig. 4) (132) includes tyrosinase, TyrP1, TyrP2, MART-1, P and gp100, and also the pathological misrouting of tyrosinase and TyrP1 (385). Proteomic analysis of early melanosomes has provided new information on the recruitment of organelle specific proteins and on membrane remodeling crucial for melanosome formation, movement, and transfer (Fig. 5) (33).
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Melanosomes also contain the lysosome-associated membrane proteins (LAMP) that protect the lysosomal membrane (333, 335, 654) and, as already mentioned, the presence of LAMP-1, -2, and -3 proteins in melanosomes supports a common ancestral origin for melanosomes and lysosomes. It has been suggested that LAMP-1 protects melanosomal integrity by acting as a scavenger of free radicals produced during melanogenesis, while the membrane-bound calcium binding protein calnexin (p90) of 90 kDa would participate in the assembly of melanosomal proteins and regulation of tyrosinase (335, 654). Melanosomes contain a proton pump that allows regulation of intramelanosomal pH; melanosomes also internalize cell surface melanocyte-stimulating hormone (MSH) receptors via the endocytic pathway (487). These properties together with the incorporation of lysosomal enzymes such as acid phosphatase and the lysosomal protective protein LAMP strengthen the view that lysosomes and melanosomes share a common pathway of organellogenesis (333, 335, 487, 607, 654). The MSH-induced delivery of MSH receptor to melanosomes points to a possible intracellular mechanism for specific and precise regulatory function of MSH (415, 487, 561).
The mechanism of pheomelanosome formation is less precisely defined than for eumelanosomes. Briefly, vesiculoglobular bodies are incorporated into stage I melanosomes. At stage II melanosomes, pheomelanin is deposited on a vesiculoglobular matrix (333, 335, 515, 597, 708), indicating the presence of tyrosinase activity at an earlier phase than in the eumelanogenic pathway. The process of pheomelanogenesis depends on the presence of tyrosinase activity, which is comparatively low, and on the availability of cysteine to conjugate dopaquinone formed by tyrosinase action (515, 597, 598).
Because melanosomes are metabolically active organelles, their activity is bound to affect the function of the host melanocyte or keratinocyte (746). Thus melanosomes modify the cellular energy-yielding metabolism by switching oxidative catabolism to anaerobic glycolysis (684), altering the intracellular NAD/NADH and NADP/NADPH ratios (682) and/or stimulating the pentose phosphate pathway (683). The presence of pigment granules that can regulate intracytosolic calcium concentration, or reversibly bind cations or bioregulatory compounds such as catecholamines, serotonin, and prostaglandins, may also affect the function of the host cell (736, 746). Based on this, it has been further proposed that melanosomes could serve as an accurate indicator for cellular responses to the environment (262). Results of proteomics analyses appear to support the notion that melanosomes are complex organelles (Fig. 5) (33) that regulate the function of melanocytes and perhaps other surrounding cell types (262, 385, 736–738, 746).
B. Biochemistry of Melanogenesis
1. Introduction to melanogenesis
Cutaneous pigmentation is under complex genetic control regulated by more than 150 alleles spread over 90 loci (262, 263, 328, 385, 515, 673, 708). Protein products of these loci acting as enzymes, structural proteins, transcriptional regulators, transporters, receptors, and growth factors have a wide array of functions and cellular targets (263, 515). Among them are the important structural, enzymatic, and regulatory melanosomal proteins coded by albino(c)/TYR, brown(b)/TYRP1, slaty(slt)/TYRP2/DCT, silver(slt)/SILV, pink-eyed dilute(p)/P/OCA2, underwhite(uw)/LOC51151, MART1, and OA1 loci (263, 515).
An early obligatory and rate-limiting step in melanogenesis is the hydroxylation of L-tyrosine to L-DOPA catalyzed by either the tyrosine hydroxylase activity of tyrosinase (EC 1.14.18.1) (264, 413, 464, 575, 597) or possibly by tyrosine hydroxylase itself (457). Once L-DOPA is formed, further steps of melanogenesis (series of oxidoreduction reactions and intramolecular transformations) can occur spontaneously, at varying rates depending on hydrogen ion concentration, presence, and concentration of metal cations, reducing agents, thiols, and oxygen (597). Most importantly, the velocity and specificity of the pathway are regulated by the melanogenesis-related enzymes (MREs) of which the most important is tyrosinase (264, 575, 597).
The availability of L-tyrosine for enzymatic oxidation is a central component of melanogenesis, regulated at the level of transport both through the plasma membrane, and from the cytosol into the melanosome (592, 593). Another source of L-tyrosine, L-phenylalanine (taken up actively by melanocytes via neutral amino acid Na+-Ca2+-ATPase antiporter system) (673, 790) and hydroxylated by phenylalanine hydroxylase (PAH) (EC 1.14.16.1) can generate relatively high intracellular concentrations of L-tyrosine, sufficient to initiate melanogenesis (673, 676). Thus initiation of melanogenesis is dependent on either transport of L-tyrosine from the extracellular space or intracellular hydroxylation of L-phenylalanine by PAH regulated by availability of reduced tetrahydropteridine cofactor (661, 670, 673).
The type of melanin produced is determined by the enzymatic library available and its prevalent metabolism (Fig. 1). For example, in the central nervous system, enzymatic decarboxylation of DOPA by aromatic amino acid decarboxylase (AAD) yields catecholamines that may be oxidated to neuromelanins. It is nevertheless unclear whether oxidation of catecholamines in substantia nigra with transformation into neuromelanins is catalyzed enzymatically or whether it is dependent on physicochemical factors (e.g., pH, presence of metal cations, and thiols concentration) (930). The involvement of macrophage inhibitory factor in the formation of neuromelanin has been already demonstrated (262, 467, 638, 664), but not the involvement of peroxidase (930). In melanocytes, the presence of tyrosinase allows rapid oxidation of tyrosine or L-DOPA to dopaquinone, initiating eu- or pheomelanogenesis pathways. Similarly, high concentrations of metal ions such as Mn2+ or Cu2+ also rapidly oxidized DOPA to melanin. Metal cations such as copper, zinc, and iron are also involved in the rearrangement of dopachrome to DHICA, thus affecting the composition of the melanin polymer (597, 598).
The formation of eu- or pheomelanin is directly determined by the presence/absence of cysteine (actively transported through the melanosomal membrane) (262, 594, 597, 598), and of GSH in fully reduced thiolate state and redox potential (high GSH for eumelanin and low for pheomelanin). Therefore, the presence and actual activity in the melanocyte of antioxidant enzymes such as catalase, superoxide dismutases, glutathione peroxidase, glutathione reductase, and thioredoxin reductase/thioredoxin would modify quantitatively or qualitatively the melanogenic pathway (664). When concentration of sulfhydryl compounds is low, dopaquinone is converted to dopachrome, initiating the eumelanogenic pathway. High concentrations of cysteine and glutathione lead to their conjugation with dopaquinone and corresponding formation of cysteinyldopa (5-S-cysteinyldopa is the major isomer while 2-S-cysteinyldopa, 6-S-cysteinyldopa, and 2,5-dicysteinyldopa are minor isomers) and glutathionyldopa (597, 598). The transformation of oxidized glutathione (GSSG) to reduced glutathione (GSH) by glutathione reductase, which requires NADPH, is crucial for the formation of glutathionyldopa (GSDOPA). Hence, the NADPH/NADP recycling system and consequently the pentose phosphate shunt are indirectly involved in the regulation of melanogenesis (682, 683). GSDOPA is further transformed by glutamyltranspeptidase to cysteinyldopa (323), which serves as the starting point of pheomelanogenesis. The velocity of the postcysteinyldopa steps of pheomelanogenesis is increased by peroxidase and tyrosinase through oxidative transformation of benzothiazinylalanines (597, 598).
2. Tyrosinase, the main enzyme regulating melanin synthesis
The key regulatory enzyme of melanogenesis, tyrosinase (EC 1.14.18.1), is encoded by the TYR or c-locus that maps to chromosome 11q14–21 in humans (32) and chromosome 7 in mice, respectively, and is composed of five exons and four introns (387, 515, 820). The posttranscriptional processing of pro-tyrosinase mRNA generates several alternatively spliced products (351, 407, 590, 702) of which some are translated to protein products with only one expressing tyrosinase activity (500, 642). It has been proposed that products of translation of alternatively spliced tyrosinase mRNA could serve as regulatory protein (736, 739), acting for example as "receptors" for L-tyrosine and L-DOPA (739). It must be noted that enzymatically nonfunctional tyrosinase proteins can be expressed in nonmelanocytic cells of neural crest origin (254, 826).
The structure of tyrosinase protein is highly conserved among different species and shows high homology with other tyrosinase-related proteins including tyrosinase-related protein 1 (TRP1/TYRP1) and tyrosinase-related protein 2 (TRP2/TYRP2/DCT) (Fig. 6). The NH2-terminal domain of tyrosinase comprises the NH2-terminal signal peptide (important for intracellular trafficking and processing), the EGF-like/cysteine-rich domain, two histidine-rich regions binding copper with a cysteine-rich region between them (the important catalytic domain), as well as the COOH-terminal hydrophobic transmembrane segment and cytoplasmic tail (387, 389, 515, 702). The transmembrane and cytoplasmic domains are necessary for targeting the enzyme to the melanosome (333, 335, 692), while the NH2 terminus cysteine-rich region may serve as a protein binding/regulatory domain unrelated to enzymatic function. Newly synthesized tyrosinase has a molecular mass of 55–58 kDa and an isoelectric point of 4.2. Proper folding of tyrosinase protein in the endoplasmic reticulum (ER) appears to be crucial for its further transport to Golgi apparatus. Proteolytic cleavage of the transmembrane portion of the newly synthesized enzyme generates two soluble molecular forms: a 53-kDa unmodified protein, or a 65-kDa glycosylated tyrosinase, which may be active in the melanosome or secreted into the extracellular environment. After glycosylation in the trans-Golgi complex, tyrosinase increases in size to 65–75 kDa or even 80 kDa (140, 141, 264, 335, 655). The higher molecular mass forms of tyrosinase (140, 655, 720, 722, 741) may represent dimmers, tight complexes with other melanogenic proteins (542), or high-molecular-weight tyrosinase proteins. It is still unclear how or when copper ions (necessary for enzymatic activity) are integrated into apotyrosinase. However, recent data suggest that the Menkes copper transporter (MNK) is required for copper loading of tyrosinase enzyme and consequently its activation (581). The catalytic site of tyrosinase is represented by two copper atoms ligated to six histidine residues.
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Tyrosinase catalyzes three distinct reactions in the melanogenic pathway: hydroxylation of monophenol (L-tyrosine), dehydrogenation of catechol (L-DOPA), and dehydrogenation of DHI; L-DOPA serves as cofactor in the first and third reactions (264, 374, 413, 575). Both ortho hydroxylation of tyrosine and dehydrogenation of DOPA may proceed in a single step in which the substrate binding site for L-tyrosine and L-DOPA is the same, and the reaction involving electrons exchange with copper atoms generates orthoquinone and water as final products (reviewed in Refs. 400, 401, 515, 619). It has been proposed that production of DOPA during tyrosine oxidation by tyrosinase is the result of spontaneous reductive cyclization of dopaquinone to cycloDOPA that undergoes redox exchange with dopaquinone to yield dopachrome and DOPA. The latter is necessary for copper reduction and formation of deoxytyrosinase. Thus the stability, folding, and activation of tyrosinase are critically controlled by redox conditions. Activation of inactive enzyme (met-tyrosinase) involves the reduction of its two Cu2+ sites to two Cu1+ centers (fully active form; deoxytyrosinase) requiring a two-electron reduction step. L-DOPA is the most efficient electron donor necessary to start tyrosine hydroxylation, although ascorbic acid, dopamine, and superoxide anion radicals can potentially activate the enzyme (903). The effect of ascorbic acid on the monophenolase activity of tyrosinase has been explained by its reducing action on enzymatically generated quinines, thus inducing accumulation of L-DOPA, the main electron donor to the Cu2+-Cu2+ enzyme active site (629). A similar mechanism may be responsible for the reduction in lag period for tyrosine hydroxylation by catecholamines, by reduced tri- and diphosphopyridine nucleotides, and by high concentrations of tetrahydropteridine (587). However, these explanations may be valid only at relatively high pH (6.8 or higher), since at low pH (5.0), tyrosine hydroxylation proceeds independently of L-DOPA (in the presence of ascorbic acid as the only reductant). At acid pH tyrosine loses its ability to bind at its allosteric site and cresolase activity is not inhibited by excess of tyrosine (145) and since DOPA oxidase activity is inhibited by acidic pH the excess of L-DOPA could escape catalytic site and diffuse to other cellular compartments. Tyrosinase activity can also be inhibited by interactions with cysteine, by chelation of copper ions, by competitive occupancy of the catalytic site (for example, by L-phenylalanine), by feedback inhibition by intermediates of melanogenesis, and by direct inactivation by melanin pigment. Tyrosinase activity in vitro is thus regulated by the local chemical environment and, by the process of melanogenesis itself including its final product melanin.
3. TRP1 and TRP2 as modifiers of pathway velocity
Two additional TRPs stimulate eumelanin synthetic rate: TRP1, product of TYRP1 (human) or b (mouse) locus, and TRP2 product of TYRP2/DCT (human) and slaty locus (mice). The gene for TYRP1 is 37 kb long and contains eight exons separated by seven introns (515, 656). The TYRP1 proteins is encoded by exons 2–8, with exon 1 containing a noncoding sequence (329, 700). However, functional analysis of the human TYRP1 promoter showed that the downstream region, containing 5-untranslated region (exon 1) and intron 1 had enhancer activity for the gene (703, 704). Alternative splicing of TYRP1 pre-mRNA generates at least two isoforms, one coding for the correct protein and another containing a deletion of 103 bp at the 5'-end of exon 8 to generate soluble TYRP1 protein without transmembrane domain because of frame shift (700). The potential for production of multiple alternatively spliced isoforms had been predicted based on analysis of the TYRP1 gene sequence (329). The gene for TYRP2 is 60 kb long and contains at least eight exons and seven introns; all eight exons encode the final protein (84). Similar to TYR and TYRP1, TYRP2 transcription and processing also generates several alternatively spliced forms (356, 445, 585). These include the correct TYRP2 mRNA, and the isoforms TYRP2–6b (contains an in frame insertion of two novel exons from intron 6), TYRP2-INT2 (retains an intron 2 with stop codon), TYRP2-LT (has an extended 3'-untranslated end), and TYRP2–8b (contains a novel exon 8b replacing exon 8) (356, 445, 585). TYRP2-LT codes for a protein identical to TYRP2. TYRP2–6b codes a protein with sequence almost identical to TYRP2 with the in-frame insertion of 33 amino acids; most likely this represents a fully functional enzyme. The remaining TYRP2-INT2 and TYRP2–8b isoforms correspond to truncated enzymatically inactive soluble proteins without transmembrane domains (445, 585).
TYRP1 and TYRP2/DCT proteins share 
40% amino acid homology with tyrosinase, with which they also have structural similarity (84, 329, 387, 515, 700). The proteins contain an NH2-terminal signal sequence, EGF-like domains, and other cysteine-rich region, two histidine-rich metal binding domains, and a COOH-terminal transmembrane segment with short cytoplasmic tail. However, different exons code for different homologous segments in tyrosinase and tyrosinase-related proteins (Fig. 6). In TYRP2, the metal binding domain binds zinc, while in TYRP1 it weakly binds iron. The COOH terminus and transmembrane domains are crucial for targeting the enzyme to the melanosome. Newly synthesized TYRP1 and TYRP2 have 55 kDa molecular mass and after posttranslational folding are targeted to the Golgi apparatus for further processing (262, 333, 335, 385). Mature and glycosylated proteins of 70–75 kDa are sorted from the Golgi apparatus to melanosomes. Because of the presence of sequences homologous to EGF in TYRPs, they can form multimeric complexes of 200–700 kDa, which may be important in the regulation of melanogenesis (330, 452, 542), or in the synthesis and assembly of the melanogenic apparatus (452, 736, 739).
In the mouse, TYRP1 acts as a DHICA oxidase to generate indole-5,6-quinone-carboxylic acid (329, 336, 368). However, some authors have proposed that TYRP1 does not catalyze the reaction in humans exhibiting instead tyrosine hydroxylase activity at low concentration of substrate (62). TYRP1 activity appears to be important for eumelanogenesis, as suggested by its lack or defective expression in cells displaying an active pheomelanogenic pathway (142, 722). An additional function of TYRP1 may be the securing of appropriate processing of tyrosinase (see above) and stabilization of its enzymatic activity and, possibly, maintenance of melanosomal structure integrity (262, 410, 656, 657). TYRP-2 acts as dopachrome tautomerase (EC 5.3.2.3) catalyzing transformation of dopachrome to DHICA (330, 843, 920). TYRP2, similar to TYRP1, is considered to be a eumelanogenic enzyme and also stabilizes tyrosinase activity. Most recently, a role for TYRP2 in melanocyte survival has been demonstrated (reviewed in Ref. 262). Thus both TYRP1 and TYRP2 can act as enzymes modifying eumelanogenesis velocity, as regulators/stabilizers of the eumelanogenic apparatus in vivo and, perhaps, as regulators of other functions of the melanocyte.
4. Tetrahydropteridines, phenylalanine, and tyrosine hydroxylase as regulators of melanogenesis
A recent theory proposes that tetrahydropteridines, phenylalanine, and tyrosine hydroxylase participate in the regulation of the
-glutamyltranspeptidase); b: oxidation of cysteinyldopa (by peroxidase); c: intramolecular cyclization of cysteinyldopaquinone (may be facilitated by peroxidase); I: DOPA decarboxylation (by AAD); II: hydroxylation of dopamine (by DBH); III: methylation of norepinephrine (by PNMT).
