Mechanism of skin pigmentation

Biotechnology and Bioprocess Engineering 2008, 13: 383-395 DOI/10.1007/s12257-008-0143-z Mechanism of Skin Pigmentation = i~ó=aỗ=mĩỡỗồệ=rúẫồI=aỡồệ=eỗ~ồệ=kệỡúẫồI=~ồầ=bỡồJhỏ=hỏóG= Department of Biological Engineering, Inha University, Incheon 402-751, Korea = Abstract 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 â KSBB hẫúùỗờầởW=óẫọ~ồỏồI=õẫờ~ớỏồỗúớẫI=ờẫệỡọ~ớỏỗồI=ớúờỗởỏồ~ởẫI=ỡọớờ~ợỏỗọẫớ=ờ~ầỏ~ớỏỗồ= = = = = INTRODUCTION Melanin plays an important role in protecting human skin from the harmful effects of UV sun radiation and in scavenging toxic drugs and chemicals Visible pigmentation of the skin, hair, and eyes depends primarily on the different functions of melanocytes, a minor subset of cells that specialize in the synthesis and distribution of the pigmented biopolymer melanin Melanocytes are derived from precursor cells (called melanoblasts) during embryological development, and melanoblasts destined for the skin originate from the neural crest The accurate migration, distribution, and function of melanoblasts/melanocytes determine the visible phenotype of organisms ranging from simple fungi to the most complex animal species In this review, we will report the most recent findings in the pigmentation system of the skin Understanding the process of producing melanin will provide the basic steps for elucidating the mechanism of pigment-related diseases such as melasma Recently there has been a surge in the market for pharmaceutical or functional cosmetics This review emphasizes the mechanism of the pigmenting process and the target genes that controlling melanin production and melanosome transfer Signal transduction inside melanocytes and cytokine-mediated signaling between the mealnocyte and keratonocyte is also reviewed in relation to melanin production *Corresponding author Tel: +82-32-860-7514 Fax: +82-32-872-4046 e-mail: ekkim@inha.ac.kr MELANIN PIGMENT IN HUMAN SKIN PHYSIOLOGY Epidermal melanin has important evolutionary and physiological implications, particularly for unclothed humans Racial pigmentation has a high content of melanin content that can protect the skin from ultraviolet radiation (UV) − induced skin damage through its optical and chemical filtering properties [1] Melanin can absorb UV light, scavenge free radicals generated within the cytoplasm, and shield the host from various types of ionizing radiation as well as determine skin color when combine with other pigments In humans, the variation in skin color occurs at the function level of the epidermal melanin unit Basically, skin color is defined by the density of melanocytes, the number, size, and dispersion of melanosomes transferred to epidermal keratinocytes, the nature of the pigment and its degradation rate [2] CELL ORIGIN AND DEVELOPMENT OF MELANOCYTES Melanocytes are highly dendritic cells that originate in the neural crest, which is a transient population of cells localized in the dorsal portion of the closing neural tube It derives the melanocyte lineage by expressing the Microphthalmiaassociated transcription factor (MITF) Melanoblasts originate in the neural crest and migrate to the epidermis where hair follicle bulges promote the differentiation of melanoblasts into melanocytes [3-5] 384 The early signals that induce formation of the neural crest include members of the Wnt, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) families, etc The Wnt/β -catenin signaling pathway is the first activated, followed by KIT, ET3/endothelin B, and then the hepatocyte growth factor (HGF)/MET It is also possible that simultaneous activation of multiple receptors is required for the differentiation of the melanocyte precursor [6,7] Wnts Wnts are secreted glycoproteins that play a critical role in melanocyte determination by activating the Frizzled receptor family Wnt signaling also stimulates quiescent melanocyte stem cells to enter the cell cycle for the generation of new stem cells [3] Wnt controls β-catenin via its influence on the Ser/Thr kinase activity of the key enzyme glycogen synthase kinase 3β (GSK3β) In the absence of Wnt, β-Catenin is recruited to a multimolecular protein complex that includes Axin, APC, and the kinase GSK-3β β-Catenin is phosphorylated by GSK-3β, and subsequently ubiquitinated and targeted for proteasomal degradation If Wnt binds to its receptor Frizzled and potential co-receptor LRP-5/6, GSK3β phosphorylation of β-Catenin is suppressed, which results in the accumulation of β-Catenin that is then translocated to the nucleus In the nucleus, it binds to LEF/TCF transcription factors, which results in the activation of Wnt target genes [8] The survival of melanoblasts during development as well as the proliferation and differentiation of the melanocyte stem cells is most likely controlled by MITF MITF is a Wnt target gene that encodes a critical regulator of the melanocyte specific genes and has been shown to increase the induction of melanoblast [9] MITF binds the canonical E-box sequence CACGTG as well as the nonpalindromic sequence CACATG Its expression is upregulated by MSH through cAMP signaling followed by CREB phosphorylation and activation of the melanocyte-specific MITF promoter and may regulate multiple pigmentation genes by upregulating MITF expression The three major pigmentation enzymes tyrosinase, Trp-1, and Trp-2, all contain consensus MITF DNA binding elements in their promoter region [8] In addition to activating the expression of p16INK4a and p21Cip1 to induce cell cycle arrest, MITF is also required for the proliferation of melanoma cells The effects of MITF on proliferation most likely depends on the status of its posttranslational modifications that will in turn dictate its ability to regulate the transcription of different target genes [10,11] Mutation will affect many of the factors that are involved in regulating the melanocyte-specific MITF-M promoter, which can lead to reduced numbers of melanoblasts Fig Binding of the growth factor to the receptor on melanocytes melanoblasts [12] Consequently, the survival and migration of neural crest-derived cells during embryogenesis depends upon interactions between specific receptors (C-KIT receptor − a tyrosinase kinase receptor) on the cell surface and their extracellular ligands (steel factor − mast/stem cell growth factor) (Fig 1) The C-KIT receptor is a tyrosine kinase that belongs to the platelet-derived growth factor (PDGF) receptor family The steel factors interact with KIT receptors on the cell membrane surface, which leads to the replacement of the phosphate group on tyrosine residues, tyrosine transfer from the inactive to the active form, and the control of the growth and differentiation of embryonic melanoblasts [6] HGF/MET During development, melanocyte viability and proliferation depends on activation of MET by its ligand HGF (hepatocyte growth factor) Targeted ablation of the MET gene in mice caused a complete loss of melanoblasts, beginning at the late stages of neural crest migration In contrast, overexpression of HGF in various tissues or specifically in kertinocytes enhanced the ectopic localization of melanoblast and increased the melanocyte population in the dermis [13,14] Endothelins (ET)/Endothelin Receptor B (EDNRB) Endothelins are growth factors that originate from endothelial cells Mutations in these growth factors produce significant neural crest defects, including melanocyte deficiency There are three endothelin proteins, ET1, ET2, and ET3, and two endothelin receptors, EDNRA and EDNRB ET3 binds to EDNRB on melanoblast ganglion cell precursors, which is essential for the survival and migration of melanoblast Mutations in either ET3 or EDNRB produce a substantial loss of melanocytes in human and mice [15,16] Steel Factor/C-kit Receptor Cadherins and Migration to the Skin The melanoblasts have expression markers that are characteristic of the melanocyte lineage, most notably is the receptor tyrosine kinase Kit (C-KIT) [9] C-KIT plays a pivotal role in the normal growth and differentiation of embryonic Cadherins are calcium-dependent surface receptors that mediate cell adhesion, homing, and invasion into different layers The expression of cadherins dynamically changes as Biotechnol Bioprocess Eng 385= neural crest cells emerge from the neural tube Cadherin expression is temporarily upregulated just prior to entering the epidermis and is subsequently suppressed as migration continues out of the epidermis into hair follicles Cadherins are also regulators of invasion or metastatic behavior in melanocytic neoplasms [17,18] LOCALIZATION, STRUCTURE, AND FUNCTION OF MELANOCYTES Melanocytes exist in various tissues in the body They are localized in the skin at the dermal-epidermal interface, in the hair matrix, in the retinal pigment epithelium of the eye, the uveal tract, the stria vascularis of the ear, the vestibular region of the inner ear, the leptomeninges, and mucous membranes In normal skin, melanocytes are located in the epidermal layer and their dendrites are projected into the epidermis where they transfer melanosomes to keratinocytes [4] Approximately every tenth cell in the basal layer is a melanocyte [6] One melanocyte is associated with approximately 30~40 surrounding keratinocytes through its dendrites The entire unit is referred to as the “epidermal melanin unit” (Fig 2) [19] The density of epidermal melanocytes varies at different sites in the body There are approximately 2,000 epidermal melanocytes per square millimeter on the skin of the head and forearm and approximately 1,000 on the rest of the body Individuals will experience an apparent change in the coloration of their skin through the course of their life Almost all infants, including black infants, are lighter at birth and become darker during the first week of postnatal life The dorsal skin of the hand becomes mottled in color in old age as a result of an age-induced decline in the number of epidermal melanocytes In addition, there is approximately an to 10 percent reduction in the density of melanocytes for each decade of life in areas that are not exposed to sun light, except for genital sites [20] During embryogenesis, melanocytes diffuse throughout the dermis They first appear in the head and neck region at approximately 10 weeks of gestation However, by the end of gestation, active dermal melanocytes “disappear”, except in the head, neck, dorsal aspects of distal extremities, and the presacral area Therefore, at this stage a portion of the dermal melanocytes have clearly migrated into the epidermis or died The major determinant of normal human skin color is the melanogenic activity within the melanocytes and the quantity and quality of pigment production, but not melanocyte density [21] Several factors play a role in determining the level of melanocyte activity, including the specific characteristics of the individual melanosomes (size, shape, type, and color), baseline (constitutive) and stimulated (facultative) levels of activity of the enzymes involved in the melanin biosynthetic pathway, the mode in which melanosomes are transferred to the keratinocytes, and their distribution in the keratinocytes The latter are influenced by receptor-mediated interactions with extracellular ligands Fig “Epidermal melanin unit” of melanocytes located in the stratum basal layer of the skin and associated with the surrounding keratinocytes Melanin is located inside melanocyte and keratinocytes The accumulation and distribution of melanin to the basal layer will determine the human skin color such as α-Melanocyte Stimulating Hormone (α-MSH) [4,6] BIOSYNTHESIS, DISTRIBUTION, AND TRANSPORT OF MELANOSOMES Melanosomes are specialized members of the lysosomal lineage, which originate in the smooth endoplasmic reticulum These membrane-bound granules, which are approximately 200 × 900 nm, are formed within melanocytes in the skin and other places of the body They are most closely related to lysosomes, primarily because both of them contain similar marker proteins and are positioned within the cytoplasm of melanocytes The lysosome-associated membrane proteins (Lamp) are also present in the outer membrane of melanosomes [22] Lysosomes protect against proenzymes such as proteinases and melanosomes protect against melanin precursors (phenols, quinones) that can oxidize lipid membranes The melanosome contains both matrix proteins, which form scaffolds for melanin deposition, and proteins (primarily enzymes) that regulate the biosynthesis of melanin [4,6] During development, melanosomes acquire some factors and three genes-related melanogenic metalloenzyme, such as tyrosinase, tyrosinase-related protein (TRP-1), and tyrosinase-related protein (TRP-2), which become fully functional and properly folded through glycosylation The Glycosylation of Tyrosinase Tyrosinase is the rate-limiting copper-containing enzyme 386 that contains virtually all of the catalytic activity necessary for melanosome maturation and melanin synthesis It contains a signal peptide that is 529 amino acids, a C-terminal transmembrane domain containing a 29 amino acid cytosolic tail, N-linked glycosylation sites (branched structure), three cysteine (filled circles) clusters, and two copperbinding sites that are essential for catalytic activity and for the cellular trafficking of tyrosinase, which facilitates transport from the rough endoplasmic reticulum (RER) through the Golgi complex to the melanosome This enzyme is highly important enzyme because of its ability to induce melanin biosynthesis when expressed in heterologous cellular systems, even in the absence of any other melanocyte specific proteins [23,24] In addition to tyrosinase, Trp-1 and Trp-2 are tyrosinase gene family proteins that are highly glycosylated and primarily N-linked They must be correctly and fully glycosylated in the ER and Golgi complex before being transported from the trans-Golgi network (TGN) to melanosomes [22] Tyrosinase glycosylation is a post-translational process that occurs within the endoplasmic reticulum (ER) and the Golgi apparatus and is catalyzed by oligosaccharyltransferase (OT), a protein complex localized in the lumen of the ER The normal structure and function of tyrosinase requires the attachment of sugars that are essential for the correct folding [25] This process is initiated on the cytosolic surface of the ER membrane The ER contains many soluble molecular chaperone and folding enzymes that interact with tyrosinase to achieve correct folding N-linked glycans are attached to the protein in the ER and are bound to the polypeptide chain through an N-glycosidic bond N-acetylglucosamines and mannoses are then added onto the oligosaccharide and the oligosaccharide is transferred to the Asn side chain in the Asn−Xaa−Thr/Ser sequence motif by the oligosaccharyltransferase enzyme complex This process begins when the ER α-glucosidase removes glucose residues in the core glycan and terminal α-1, linked mannose residues The trimming of the glycan chain by ER α-mannosidase I and II to create Man9 oligomannose and Man7 glycoprotein is then folded and transported to the Golgi [25] In the Golgi apparatus, mannoses from the Man7GlcNAc2 glycans are remove by the Golgi α-mannosidase I to become Man5GlcNAc2 glycans Through N-acetyl glucosamine I (NAG I) activity, these glycans transfer N-acetyl glucosamine to form a hybrid structure of Man5GlcNAc3 glycans Two mannose residues are then removed from this hybrid structure resulting in the formation of Man3GlcNAc3 glycans Finally the addition of N-acetyl glucosamine, galactoses, fucose, and sialic acids forms the complex structure which escapes terminal glycosylation [23,24] The newly synthesized tyrosinase polypeptide is then subjected to the quality control system in the ER, which monitors protein maturation to ensure that defective proteins are not transported throughout the cell The system includes calnexin, calreticulin, which are lectin chaperones that bind specifically to glycoproteins containing monoglucosylated glycans Finally, incorrectly folded and misassembled Fig The role of calnexin in melanosome biogenesis glycoproteins are recognized by glucosyl transferase, which then targets them for ER retention [4,24] The immature misfolded tyrosinases that remain bound to calnexin and calreticulin are repaired and eventually attain their correct three-dimensional structure Calnexin has a specific affinity for monoglucosylated asparagine-like oligosaccharides (Glu1Man9-8GlucNAc2), which are formed from the removal of the first and second glucoses (G) in the original triglucosylated core oligosaccharide (Glu3Man9GlucNAc2) by the sequential action of glucosidase I and II Only properly folded glycoproteins are transported to TGN, from which newly synthesized glycoproteins are selectively transported to secretory granules, such as lysosome, endosome, and melanosome through a M6P recognition system By virtue of a specific cargo-recognition system Lamp-1, -2, and -3 are associated with the outer membrane of late endosomes, lysosomes and melanosomes, and tyrosinase and Trp-1 are transported to melanosomes and late endosomes (Fig 3) [22,26] The quality control test in the ER may also involve the production of the tyrosinase cofactor DOPA, which is produced by the oxidation of tyrosine DOPA can bind to and stabilize nascent tyrosinase, making it competent for transport Other proteins, such as Trp1 and Trp-2, can stabilize tyrosinase in the ER and increase its activity Therefore, the presence of melanogenic complexes is important not only for enzymatic optimization but also for the trafficking of proteins out of the ER [4,26] It has been well established that the cytoplasmic domain is critical for melanogenic function and for the cellular trafficking of tyrosinase The cytoplasmic domain facilitates transport from the RER through the Golgi complex to the melanosome Protein kinase C-β (PKC-β) is a signal transduction enzyme that is required for the activation of tyrosinase PKC-β co-localizes with tyrosinase at the melanosomal membrane and activates tyrosinase by phosphorylating the serine residues on the C-terminal of the cytoplasmic domain [27,28] Biotechnol Bioprocess Eng 387= Transport of the Tyrosinase Gene Family from the Golgi to Melanosomes After glycosylation, proper folding, stabilizing, and possibly oligomerizing within the Golgi apparatus, tyrosinases are packaged into COPII-coated transport vesicles that bud from the smooth ER The vesicles traffic through the ER-Golgi intermediate compartment (ERGIC) and combine with matrix proteins to transport tyrosinase and Tyrp1 through late endosomes to early melanosomes The sorting of tyrosinase and other melanocyte-specific proteins from the Golgi to melanosomes is determined by recognition signals in the cytoplasmic segment of the protein, which are typically short stretches of six amino acid residues including a di-leucine motif [29] There are two distinct types of sorting signals for intracellular localization of tyrosines to melanosome, a tyrosinasebased signal and a di-leucine motif signal in the tyrosine tail, which acquires the presence of Trp-1, silver (Pmel17 and gp 100) and P-protein through the binding of an adapter-like protein complex AP3 The sorting system has adapter proteins, such as clathrin, which is a coat protein that assembles under the presence of two distinct but closely related adapter protein (AP) complexes Clathins bind to different sorting signals on the cytoplasmic tails of protein and functions at various subcellular locations for the transport of proteins between different organelle components [30,31] AP-1, which specifically binds to tyrosinase-based signals, is a distinct sorting pathwayand that involves transport from the TGN to early endosomes Proteins that use the AP1 pathway need to subsequently use AP3 or another sorting mechanism to transport from early endosomes to melanosomes AP2 is involved in sorting and transporting proteins from the plasma membrane back into the cell and to early endosomes In order to use this system, proteins need to first be sorted out of the melanocyte and then brought back in AP-1 and AP-2 are heterotetramers that provide clathrins with membrane specificity at their assembly by enabling them to recognize the sorting motifs in the cytoplasmic domains of trafficking membrane proteins Cargo proteins, which are sorted by AP-1 and AP-2 into clathrin-coated vesicles, are subsequently delivered to the endocytic lysosomal system The third AP complex (AP-3) is more distantly related to AP-1 and AP-2 AP3 is involved in tyrosinase sorting, which also transports cargo from the TGN to the plasma membrane From an immune perspective, a number of melanosome proteins must have at least a transient expression on the cell surface In additional, AP-3 does not bind to the cytoplasmic tails of other non-melanosomal membrane proteins that contain di-leucine and tyrosinebased sorting signals This finding indicates that AP-3 must only transport a subset of cargo proteins, specifically the tyrosinase gene family proteins, through the di-leucine-based motifs [32,33] Finally, AP4 may also play a role in the trafficking of melanosome-specific proteins Sorting signals are typically found at the carboxyl termini of proteins and several types are dileucine and tyrosine-based sorting signals Tyrosinases only weakly interact with AP-1 and AP-2, but Fig Trafficking and sorting of melanosomal proteins to melanosomes correlate with the maturation stages of melanosomes from I to IV This process involves not only gp100 but also the AP family Cleavage of the amino terminus plays a critical role in the maturation of the amorphous vesicular stage I to the fibrillar stage II of melanosome these proteins have a high affinity for the QPLL signal at the cytoplasmic tail of the AP-3 Cytoplasmic tail with dileucine-based signals, will direct these glycoproteins to melanosomes This tail is important for the intracellular transport of the tyrosinase gene family proteins from the TGN to melanosomes [34] The processing of gp100, which results in the cleavage of the amino terminus, plays a critical role in the maturation of the amorphous vesicular stage I melanosome to the fibrillar stage II The gp100 has been shown to localize in the ER, AP3 vesicles, cis-Golgi, and trans-Golgi network In addition, gp100 does not transport to early and late endosomes, but rather traffics directly to Stage I melanosomes Except for gp100, the other melanosomal proteins, such as tyrosinase, are further glycosylated and processed through the trans-Golgi network (TGN) From that point they can potentially use the AP3 sorting system, which traffics proteins to early or late endosomes and then to lysosomes and melanosomes (Fig 4) [1] Melanosome Maturation Melanosomes develop through a series of morphologically defined stages (Fig 6) from an unpigmented (stage I) to a striated organelle enriched in melanin (stage IV) In the subsequent stages of maturation, melanosomes are relatively nondescript membrane-bound vesicles (stage I) found in the peri-nuclear area Melanosomes under eumelanogenesis (eumelanosomes) are always ellipsoidal in shape and are located in well-organized lamellae filaments, elongated fibrillar and membrane bound organelles termed a premelanosome (stage II) Once the fibrillar matrix had been generated, melanin synthesis begins and the electron dense pigment is deposited on the fibrils, which become electron dense upon 388 Fig The melanin biosynthetic pathway according to RaperMason [44] Fig Electron microscopy of melanosome development during eumelanogenesis (a~f) and pheomelanogensis (g~j) in normal melanocytes melanin deposition (stage III) Finally in the case of darkly pigmented tissues, melanin synthesis and deposition continues until fully pigmented (stage IV) In contrast, melanosomes under pheomelanogenesis (pheome-lanosomes) are always spherical in shape and contain only internal granular materials at all four stages of their maturation (Fig 5) [35,36] Stage I melanosomes is the initial site for transport of the tyrosinase gene family proteins In this stage, melanosomes are aspherical organelles that have an irregular structure and contain matrix filaments and internal membranous vesicles, which are formed by the invagination of the outer limiting membrane They likely correspond to the late-coated endosomal multivesicular bodies found in nonmelanogenic cells At stage II, the melanosomes become elongated and form ordered striations which act as templates for melanin polymerization, which commences in stage III melanosomes Stage III melanosomes is characterized by the deposition of electron dense material on the matrix This stage possesses functional oxidoreductases, which triggers the melansomes to darken Silver is a type I membrane glycoprotein that plays a structural role in the formation of these striations because silver expression in nonmelanogenic cells support the formation of striations within multivescular bodies Melanin biosynthesis is dependent on proper pH homeostasis, osmotic pressure, transport of the substrate into the melanosome lumen and other uncharacterized activities such as that controlled by the OA1 protein Proper pH and osmotic pressure are likely to require the activity of multiple spanning membrane transport proteins (pink-eyed dilution protein, P-protein, and antigen in melanoma-1/membraneassociated transporter protein (AIM-1/MATP) that have a role in ionic regulation in the melanosomes and possibly even in the ER and the Golgi [8,15,52] If the normal Pprotein is absent, the melanosomal structure is disrupted and tyrosinase and Trp1 are misrouted to other sites P-proteins, in trafficking or sorting of melanocyte-specific proteins, exit the Golgi apparatus and are transported to premelanosomes [37,38] Stage IV melanosomes, after complete opacification of the melanosomal content by melanin deposition, will be transported to dendritic tips [39] Melanin Biosynthesis Melanin is the major product of melanocytes and is the main determinant of differences in skin color Melanin is synthesized in two main forms: eumelanin and the pheomelanin The amino acid tyrosine is the starting material for the production of melanin in the synthesis of both the brownblack eumelanin and the yellow-red pheomelanin The key regulatory enzyme in this pathway is tyrosinase, which has tyrosine hydroxylase, DOPA oxidase, and dihydroxyindole oxidase activity, therefore, it can catalyze multiple steps in the biosynthesis of melanin (Fig 6) [6,40,41] Tyrosinase controls the initial chemical reaction, an important step in melanin biosynthesis, which is the hydrolysis of tyrosine to L-DOPA (3,4-dihydroxy-phenylalanin) LDOPA enhances tyrosinase activity by allowing oxygen to bind to the active site of tyrosinase The oxygen-bound form of tyrosine uses tyrosine and DOPA as substrates Subsequently, L-DOPA is catalytically oxidized to L-DOPAquinone (3,4-dihydroxy-phenylalanin quinone) by the met-form of the enzyme L-DOPA is a reactive intermediate, which is either processed into eumelanin or pheomelanin In the absence of thiol compounds, DOPAquinone undergoes cyclization and is converted DOPAchrome TRP-2 (DOPAchrome tautomerase) catalyses the tautomerization of DOPAchrome into 5, 6dihydroxyindole-2-carboxylic acid (DHICA) This step leads to a much slower oxidation and polymerization, which results in a more soluble, lower molecular weight and lighter colored melanin known as DHICA-melanin At the same time, the decarboxylation of DOPAchrome leads to the formation of indole-5,6-quinine from 5, 6-dihydroxyindole (DHI) and oxidate DHI Trp1 and tyrosinase catalyze the conversion of DHICA to carboxylated indole-quinone for the final produc- Biotechnol Bioprocess Eng 389= Fig The molecular machinery that is involved in melanosome movement in melanocytes Anterograde movement, towards the plus end of microtubules at the cell periphery is achieved by kinesin According to the formation of complex Rab27a, melanophilin and myosin Va In the periphery region of the cell, melanosomes display short-range movements on actin filaments The microtubule- and actin-dependent motors are coordinated to achieve the intracellular dispersion of melanosomes Retrograde movement, towards the minus end of microtubules in the center of the cell is dependent on dynein Single black arrows indicate direction of melanosome movement tion of brown-black eumelanins DHICA-melanins have reduced photoabsorption, no phototoxicity and less cytotoxicity [31,41-43] The formation of pheomelanin requires less tyrosinase activity than does the formation of eumelanin They are produced from cysteinyldopa and benzothiazine metabolites when DOPAquinone combines with cysteine or glutathione to form the intermediate products cysteinyl-DOPA and alanyl-hydroxyl-benzothiazine Pheomelanins have a yellowish-red color, are soluble in alkali and have a low molecular weight In contrast, pheomelanin has very little photoabsorption, a high phototoxic potential, and low cytotoxicity The ratios of pheomelanic and eumelanic monomers determine the final color of the skin and hair [44] During the induction of new melanogenesis, tyrosinase and TRP-1 coordinate together to upregulate Lamp-1, which is continuously coated on the inner surface of the melanosomal membrane Their high content of N- and O-linked oligosaccharides serve as a scavenger to toxic melanin intermediates that are produced by tyrosinase [45] Melanosome Transport to Dendritic Tips Melanosomes become enriched with pigment in the cell body at stage IV and travel to the dendritic tip in a centrifugal manner At the dendritic tip, melanosomes are subsequently transferred to neighboring keratinocytes in the epidermis Melanosomes move in a bi-directional manner on microtubules in the dendrites until they are captured at the dendrited tips (Fig 7) [4] This process requires protein motors, cytoskeletal tracks, and adapters or effectors that attach the organelles to the motors There are two mechanisms involved in the centrifugal movement of melanosomes to the microtubules and cell periphery Long-range movement by kinesin and dynein or short-range movement at the cell periphery by the Rab 27a, melanophilin, myosin Va complex [43] The long-range movement of melanosomes proceeds towards the plus ends of microtubules; therefore it may be powered by motors of the kinesin superfamily Kinesins are molecular motors that are involved in microtubule and ATP dependent transport of organelles, protein complexes and mRNA [43,46,47] Kinesins consist of two globular heads, which is formed by two kinesin heavy chains (KHCs), and a fan-like end, which is formed by two kinesin light chains The KHC possesses a motor domain, which binds to the microtubules, a-helical coiled-coil stalk domain, which is involved in dimer formation, and a C-terminal tail domain Conventional kinesin (kinesin I) is highly expressed in mammalian melanocytes, and has been implicated in melanosome transport Anti-sense oligonucleotides that interfered with the synthesis of kinesin I inhibited the bidirectional movement of melanosomes along microtubules and promoted perinuclear aggregation [48] Retrograde movement towards the microtubules’ minusends is achieved by cytoplasmic dynein, which associates to its cargo via the multisubunit complex dynactin Cytoplasmic dynein was implicated in both human and frog melanosome movement This microtubule-activated ATPase is a member of the dynein superfamily of proteins and is composed of two heavy chains, multiple intermediate chains, light intermediate chains, and light chains Another associated subunit is the dynactin complex, which may serve in general regulatory roles [46,47] In short-range movements, melanosomes still undergo rapid bi-directional and microtubule-dependent movements between the center and the periphery of the cell This mode of movement provides a means of transporting the melanosomes to the cell periphery In the cell periphery, the actomyosin system functions to ensure that the mature melanosomes are captured and stay in position for subsequent transfer The Rab family is involved in the transport of the vesicle carriers along microtubules and actin filaments, tethering or docking of vesicles to the acceptor membrane, and fusion of the vesicles with the membrane of the acceptor compartment In melanocytes, Rab27a associates with the cytosolic leaflet of the melanosome membrane and appears to be the key melanosome-associated protein of the tethering complex The association of Rab27a with melanosomes does not depend on melanophilin and myosin Va In fact, the mechanism of Rab27a − melanosome association is currently unknown, however, unraveling these targeting mechanisms will help in fully understanding this process Melanophilin contains at least three functional domains The N-terminal domain binds to Rab27a and is conserved throughout the Slp family Over-expression of this domain in melanocytes in- 390 duces melanosome clustering, which serves as a Rab27abinding region The Rab27a binding domain consists of two stretches of amino acids referred to as the Sl phomology domain-1 (SHD1) and the Sl phomology domain-2 SHD2 The SHD1 is both necessary and sufficient for Rab27a specific binding, whereas the SHD2 is involved in enhancing the Rab27a binding affinity to melanophilin Melanophilin specifically interacts with the globular tail of myosin Va via a coiled coil domain within a central domain The C-terminal domain immunoprecipitates with actin and may be required for peripheral melanosome distribution Myosin Va is an actin-based motor that belongs to the large myosin superfamily and shares a common N-terminal motor domain that binds to actin and generates force through the hydrolysis of ATP The tail domain of myosin is postulated to direct the interaction with the cargo, which ultimately determines the functional specificity of the motor Myosin Va is also found to colocalize with F-actin in the dendrites and respective tips where the melanosomes normally accumulate This suggests that the interaction of myosin Va with cortical actin is responsible for the peripheral capture of melanosomes Myosin Va, containing exons D and F, is expressed in skin and other tissues and are essential for the interaction of myosin Va with melanophilin and consequently for the association of myosin Va with melanosomes [44] An increase in microtubule based transport is observed in the absence of components needed for actomyosin based transport Myosin V contributes to the dispersion of melanosomes by counteracting the action of dynein, particularly by shortening the length of dynein-driven runs and by ensuring that the regular motion of melanosomes is towards the microtubule plus-end Myosin Va operates in concert with actin filaments that line the cell periphery to move granules to dendritic tips In the absence of Myosin Va, the bi-directional movement of melanosomes on microtubules is normal However, under these conditions the melanosomes fail to accumulate at the dendritic tips Rab proteins are adapter proteins that are localized on the melanosome membrane and are involved in the regulation of intracellular vesicular transport in concert with effector molecules They link to melanophilin and in turn link to myosin Va and F-actin, thus, providing specificity in the trafficking steps of intracellular vesicles by promoting the binding and fusion of vesicles destined for specific locations within the cell [4,32,49] Melanosome Transfer to Keratinocytes Melanosomes that are enriched in pigment at the dendritic tips are translocated to adjacent keratinocytes where melanin forms a photoprotective cap over the keratinocyte nuclei [50] Four mechanisms of the transfer have been proposed (Fig 8) [51-54] The first mechanism suggests that the dendrite tips of the melanocytes are phagocytosed by the keratinocyte in the presence of keratinocyte receptors (Fig 8A) In this mechanism it has been postulated that melanocytes extend their dendrite to contact with a keratinocyte Then the dendrite tip is squeezed and pinched off and the cytoplasmic reticulum is filled with melanosomes These melansomes A M K B C D M K Fig Different modes of melanin transfer [50] will form a phagolysosome by fusion with lysosomes resulting in the degradation of the melanocyte membranes The phagolysosome will then be transported to the supranuclear region and disintegrates into smaller vesicles, followed by dispersion into the cytoplasm The second mechanism is based on regulated exocytosis In this process, the membranes of cytoplasmic organelles fuse with the melanocyte plasma membrane and release melanin into the intercellular space Then keratinocytes take up the melanin by phagocytosis (Fig 8B) [53] The third mechanism proposes that the presence of cognate SNARE protein on melanocyte and keratinocyte plasma membrane is responsible for the transportation to keratinocytes In this mechanism, SNARE proteins initiate the direct fusion of the two outer membranes The resultant filopodia then extends from the dendrite tips and cell body of melanocytes, adheres to the surface of neighbouring keratinocytes and creates a channel that will support the transfer of melanosomes from one cell to the other cell (Fig 8C) The final mechanism postulates that the transfer of melanin from melanocytes to keratinocytes occurs through membrane vesicles (Fig 8D) Proteins and lipids destined for transfer are concentrated in the plasma membrane and contribute to the formation of extracellular vesicles, which sheds the melanosomes and travels to distant cells After this process is complete, the vesicles will be ingested by the keratinocytes through phagocytosis or fuse with the keratinocyte plasma membrane Once inside the keratinocyte, melanins are free and ultimately determine skin color In these mechanisms, PAR-2, which has an important role in phagocytosis, is a major regulator of melanosome transfer PAR-2 is expressed in keratinocytes and enhances the phagocytosis rate of keratinocytes, which leads to increased melanosome transfer PAR-2 can potentially affects pigment modulation through keratinocyte phagocytosis (Fig 9) UVB-induced PAR-2 activation could provide immediate photo-protection by the rapid transfer of available melanosomes This would Biotechnol Bioprocess Eng 391= Fig PAR-2 activation and inhibition and the effects on keratinocyte phagocytosis and melanosome transfer The activation of PAR-2 by UV radiation and the presence of trypsin result in an enhanced melanosome transfer, which leads to pigmentation The presence of trypsin inhibitors follow by inhibiting PAR-2 activation and reduced melanosome transfer, which leads to depigmentation increase the ability of keratinocytes to ingest melanosomes through a process of keratinocyte-melanocyte interaction PAR-2 activation leads to skin darkening, but inhibition of PAR-2 activation by serine protease inhibitors reduces pigment transfer and leads to depigmentation The soybean trypsin inhibitor (STI) have been shown to inhibit PAR-2 cleavage, and reduce phagocytosis and melanosome transfer; thereby completely inhibiting UVB-induced pigmentation [55] Other factors, such as keratinocyte growth factor receptor (KGFR), α-MSH, Rab3, Rab27a, cadherins, lectin, ect also contribute to events that enhance the phagocytosis rate of keratinocyte, such as regulating melanosome exocytosis, effect the docking of melanosomes at the plasma membrane, and mediate melanocytes-keratinocytes adhesion and expression of genes associated with exocytosis All of these processes are essential for the transfer of melanocytes to keratinocytes [51] FACTORS THAT INFLUENCE SKIN PIGMENTATION Skin pigmentation is influenced by many factors These factores belong to three groups: ultraviolet light (UVR), drugs and genetic componensts (α-melanocyte stimulating hormone (α-MSH), agouti signal protein (ASP), basic fibroblast growth factor (β-FGF), and endothelin-1 (ET-1)) (Fig 10) [32,56,57] Role of Ultraviolet Radiation in Melanogenesis Solar ultraviolet radiation (UV) is a major environmental factor that dramatically alters the homeostasis of the skin by affecting the survival, proliferation, and differentiation of various cutaneous cell types The effects of UV on the skin Fig 10 Factor-induced skin pigmentation UV irradiation, growth factors or endothelin-1, drugs and the activation of protein kinase A by α-MSH and its receptor MC1-R as well as protein kinase C by TPA and Ca2+ can induce skin pigmentation include direct damage to DNA, apoptosis, growth arrest, and stimulation of melanogenesis Long-term effects of UV include photoaging and photocarcinogenesis Melanin, particularly eumelanin, represents the major photoprotective mechanism of the skin Melanin limits the extent of UV penetration through the epidermal layers and scavenges reactive oxygen radicals that may lead to oxidative DNA damage The extent of UV-induced DNA damage and the incidence of skin cancer are inversely correlated with the total melanin content of the skin [52] When UV radiation reaches the skin, reflection, scattering, and absorption takes place UV rays penetrate into the deep layers of the subcutaneous tissue, particularly by the corneocytes UV ray absorption is increased by the content of aromatic acids, such as tyrosine, tryptophan, and phenylalanine and urocanic acid, which forms in the keratinocyte by the process of keratinisation Urocanic acid is presence mainly in the stratum corneum and is important for skin moisture mainternance, and stimulation of stratum corneum thickening and melanin synthesis by melanocytes [52] Facultative pigmentation is often divided into immediate pigment darkening (IPD) and delayed pigment darkening (DPD) IPD is a transitory darkening of the skin, which is observed within seconds of UVA (320~400 nm) exposure and is typically resolved within 1~3 days IPC involves structural changes in melanocytes and keratinocytes and a chemical modification of pre-existing melanin The mechanism for IPD include photo-oxidation of melanin, changes in cytoskeletal distribution, translocation of melanosomes from the perinuclear region to the dendrites, increased melanosomes transfer, and changes in the melanosome distribution pattern within keratinocytes [58] DPD results from an increase in the number of melanocytes in addition to an increase in the number of melanosomes in melanocytes and keratinocytes DPD typically occurs within 2~3 days after 392 Fig 11 Interactions that control pigment production α-MSH and ASP interact with MC1-R Activity of the MC1-R is enhanced by binding with α-MSH, which results in eumelanogenesis The dysfunction of the MC1-R or the combination of ASP blocking MC1-R can lead to pheomelanogenesis vation of protein kinase C (PKC) In addition, an enhancement in pigment production will result from melanocytes exposure to agents that increase intracytoplasmic levels of cAMP such as cholera toxin, dibutyryl cAMP, and α-MSH The interactions between α-MSH and ASP are critical for the switch to produce eumelanin or pheomelanin (Fig 11) [63,64] α-MSH produced from UVR stimulated keratinocytes promotes eumelanin synthesis, whereas ASP promotes pheomelanin synthesis The effects of α-MSH are mediated by the MSH receptor, which is known as the melanocortin receptor (MCR-1) and is expressed at high levels in melanocytes α-MSH binding to MC1-R enhances the expression of the MSH receptor in melanocytes, activates the PKA pathway, stimulates melanocyte proliferation and differentiation, and results in an increased melanogenesis by the melanocyte [11,65] α-MSH binds to the MC1-R and this protein pair interacts with a complex of G proteins, which uses guanosine triphosphate (GTP) and guanosine diphosphate (GDP) as intermediary messengers The GTP-Gsα subunit then activates adenylate cyclase, which leads to an increase in production of cyclic adenosine monophosphate (cAMP) within the melanocyte An increase in the intracellular concentration of cAMP then causes an increase in tyrosinase activity and eumelanin production If the MC1-R receptor is dysfunctional and fails to initiate a significant rise in the intracellular level of cAMP, pheomelanins are produced [9,66] Agouti Signal Protein (ASP) UVB (290~320 nm) exposure A major stimulant for facultative pigmentation is UVR, which is the most potent stimulant for growth and differentiation of melanocytes Mitogenic signals, in addition to UVR, have been shown to regulate melanocytic proliferation Melanin not only functions as a sunscreen to absorb UV and prevent DNA damage, but also an antioxidant and radical scavenger, which play important roles in protecting cells from such damage [32,59,60] The activation and differentiation of melanocytes can be induced directly by UVR or indirectly through their interaction with surrounding UV-irradiated keratinocytes UVR can increase the synthesis of β-fibroblast growth factor (β-FGF) in keratinocytes, which in turn stimulates proliferation and melanogenesis of epidermal melanocytes [61,62] In addition to increased synthesis of β-FGF, exposure of keratinocytes to UVR results in the upregulation of other keratinocyte-derived cytokines such as endothelin-1, which is a small peptide originally isolated from endothelial cells Endothelin-1 plays an important role in stimulating melanocyte proliferation and melanization through the G proteincoupled endothelin B receptor-mediated signal transduction pathway and can also lead to an increase in tyrosinase activity and increase in melanin production THE ACTIVATION OF PROTEIN KINASE A OR C Exposure of melanocytes to tetradecanol phorbol acetate (TPA), can lead to increased melanin formation via the acti- Agouti signal protein (ASP) acts as a competitive antagonist of α-MSH for MSH-R binding MSH-R in melanocytes is considered to be a control point for pigmentation MSH-R is also present on other cells such as monocytes, endothelial cells, and keratinocytes When ASP is present, as a result of its inhibition of MC1-R function and down-regulation of the PKA pathway, melanocytes switch into their pheomelanogenic mode and express melanosomal proteins at basal levels except for tyrosinase, which continues to be expressed at very low levels [67,68] Activation of Tyrosinase The expression of only tyrosinase still results in a low level of melanin production The sulfhydryl content of melanosomes, in the form of cysteine, is not exhausted faster than it can be transported into the melanosome and the dopaquinone is converted to cysteinyldopa only to produce pheomelanin Furthermore, since the other melanosomal proteins are not expressed, the melanosome never matures beyond stage I Therefore, the melanin produced does not have a physical substrate to polymerize; hence the pheomelanosomes have splotchy irregular deposits of melanin In contrast, upon stimulation of differentiation by UV or αMSH, expression of all known melanosomal proteins is upregulated and melanosomes then undergo normal maturation to the fibrillar stage II and beyond [68] The high levels of tyrosinase result in an increased synthesis of melanin In addition, the exhaustion of intra-melanosomal sulfhydryl Biotechnol Bioprocess Eng 393= A B C Fig 12 Drug-induced skin pigmentation (A) Minocyline pigmentation, (B) flagellate pigmentation from bleomycin, (C) amiodarone photosensitivity cause the dopaquinone generated to cyclize and form dopachrome This process results in the predominant synthesis of eumelanin The eumelanin is then deposited on the melanosomal matrix followed by increased levels of visible pigmentation in the tissue Drug-induced Skin Pigmentation Drug-induced skin pigmentation is quite common and accounts for 10~20% of all cases of acquired hyperpigmentation Pigmentation may be induced by a wide variety of drugs; the most common ones include non-steroidal anti-inflammatory drugs (NSAIDs), phenytoin, antimalarials, amiodarone, antipsychotic drugs, cytotoxic drugs, tetracyclines, and heavy metals (Fig 12) [8] Drug-induced skin pigmentation may result from increased melanin synthesis, increased lipofuscin synthesis or cutaneous deposition of drug-related material Certain heavy metals, such as iron, may be deposited in the dermis following damage to dermal vessels If deposited in sufficient quantities a distinctive change in skin color may be observed without any significant increase in melanin Some drugs react with melanin in different ways, including to form a drug-pigment complex, induce accumulation of melanin as a non-specific post-inflammatory change in predisposed individuals or induce pigmentation directly by accumulating and reacting with other substances in the skin [8,69] CONCLUSION AND FINAL REMARKS Over the past few years, significant advances have been made in the understanding of the molecular mechanisms of skin pigmentation A better understanding of these mechanisms could result in better control of human skin color In addition, this insight could be used to discover new depigmenting agents and validate their efficacy and safety This review summarizes the current understanding of pigmentation in mammalian skin This is a process that is carryied out within melanosomes via a series of oxidative reactions involving the substrate tyrosine and melanogenic tyrosinase enzymes The first event in this process is the development and migration of dendritic precursor cells, known as melanoblast, which originate in the neural crest After migration, melanoblasts differentiate into melanocytes in the presence of MITF, Sox, Bcl-2, CREB, etc Then, melanogenic genes, such as tyrosinase, TRP-1, TRP-2, and melanosomal matrix components are induced and synthesized Tyrosinase undergoes posttranslational processing and glycosylation, which controls its activity and directs its transport to melanocytes In melanocyte, tyrosinase is localized to the melansomes, where its functions in concert with other melanogenic proteins to generate melanin Melanosomes act as carriers of tyrosinase, where melanin synthesis is initiated and eventually transported to keratinocytes Melanin is distributed within the epidermis and determines skin color However, many more important questions remain unanswered, such as how the dendrite interacts with the keratinocyte membrane at the site of attachment and what is the mechanism of regulation of the microtubule and actin based motors Moreover, further studies should aim at a better understanding of keratinocyte phagocytosis, the regulation of PAR-2 expression and signaling in keratinocytes, the natural activators of PAR-2 in skin, the termination of PAR-2 signaling as well as the identification of key molecules that are involved in the dendrite-keratinocyte 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carbohydrates in tyrosinase are required for its recognition by human MHC class II-restricted CD4+ T cells Eur J Immunol 31: 2690-2701  ... been made in the understanding of the molecular mechanisms of skin pigmentation A better understanding of these mechanisms could result in better control of human skin color In addition, this insight... synthesis of eumelanin The eumelanin is then deposited on the melanosomal matrix followed by increased levels of visible pigmentation in the tissue Drug-induced Skin Pigmentation Drug-induced skin pigmentation. .. associated with exocytosis All of these processes are essential for the transfer of melanocytes to keratinocytes [51] FACTORS THAT INFLUENCE SKIN PIGMENTATION Skin pigmentation is influenced by