PIGMENTARY DISORDERS SECTION 10 Melanocyte Biology 65 Jean L. Bolognia and Seth J. Orlow

In the inner ear, particularly in the stria vascularis, melanocytes are Key features thought to play a role in the development of hearing. Aberrant migra­ ■ The major determinant of normal color is the activity of tion or survival of melanocytes within the inner ear, the iris, and melanocytes, i.e. the quantity and quality of production, midportions of the forehead and extremities explains the presence of not the density of melanocytes congenital deafness, heterochromia irides, and patches of leukoderma, ■ Melanocytes contain a unique intracytoplasmic , the respectively, in patients with , the classic neuro­ , which is the site of synthesis and deposition cristopathy. Also, aberrant migration or survival of enteric ganglion cells, another -derived population, provides an explana­ ■ Compared with lightly pigmented skin, darkly pigmented skin has tion for the association of aganglionic megacolon (Hirschsprung disease) that contain more melanin and are larger; once with Waardenburg syndrome or rarely . transferred to , the melanosomes are singly dispersed The survival and migration of neural crest-derived cells during and degraded more slowly embryogenesis depends upon interactions between specific receptors on ■ is the key enzyme in the melanin biosynthetic pathway their cell surface and extracellular ligands. For example, KIT ligand ■ Two major forms of melanin are produced in melanocytes: (also known as steel factor or stem cell growth factor) binds to the –black eumelanin and yellow–red pheomelanin transmembrane KIT receptor on melanocytes and melanocyte precur­ ■ The production of eumelanin versus pheomelanin is influenced by sors (melanoblasts) (Figs 65.2 & 65.3); melanoblasts require expression the binding of melanocyte stimulating hormone to the of the KIT receptor in order to maintain their normal chemotactic 1 receptor migration directed by production of KIT ligand by the dermamyotome. Heterozygous germline mutations in KIT that decrease the ability of the KIT receptor to be activated by KIT ligand are responsible for human piebaldism, whereas in mice, mutations in either or steel can lead to white spotting. In the developing embryo, melanoblasts expressing receptor type B (EDNRB) are stimulated to INTRODUCTION migrate by endothelin-3 (ET3 [EDN3]), which is produced by the ecto­ derm and dermamyotome. Mutations in one or both copies of EDN3 In order to understand the underlying pathophysiology of cutaneous or EDNRB can result in Waardenburg syndrome plus aganglionic mega­ disorders of and , as well as colon (see Fig. 65.3). the process of normal physiologic pigment production, an apprecia­ Transcription factors represent another group of proteins that play an tion of the structure and function of the melanocyte is required. A essential role during embryogenesis. Because transcription factors can classic example of basic pathogenesis is type 1 oculocutaneous albi­ bind DNA and influence the activity of other genes, they are able to nism (OCA1), in which pigmentary dilution of the skin, , and regulate the complex interplay of various sets of genes that is required eyes is due to a reduction or absence of tyrosinase activity secondary for embryonic development. Several of the genes that, when mutated, to mutations in both copies of the tyrosinase gene (TYR). Within the give rise to Waardenburg syndrome encode transcription factors (e.g. realm of physiologic pigmentation, melanocytes in individuals with red PAX3, MITF, SOX10; see Table 66.4). Fig. 65.4 demonstrates some of hair often express variants of the (MC1R)1. these interactions (e.g. PAX3 and SOX10 can control expression of As a consequence of the altered sequences of the variant MITF)8,9. MITF is sometimes referred to as the master regulator MC1Rs, their cell surface expression and interactions with melanocyte of melanocyte development and function given its modulation of mul­ stimulating hormone (MSH) can be affected, leading to an increase in tiple differentiation genes and its early up-regulation in neural crest the production of pheomelanin as opposed to eumelanin. Based upon cells that will eventually become melanocytes and emigrate from the population genetics, genes that are mutated in OCA (e.g. TYR, OCA2, dorsal neural tube. TYRP1, SLC45A2, SLC24A5) also influence normal pigment variation During embryogenesis, melanin-producing melanocytes are found (Table 65.1)2–6. diffusely throughout the . They first appear in the head and neck The major sections in this chapter are: region at ~10 weeks of gestation. However, by the end of gestation, the origin and function of the melanocyte active dermal melanocytes have “disappeared”, except in three primary • the formation and function of the melanosome anatomic locations – the head and neck, the dorsal aspects of the distal • regulation of melanin biosynthesis. • extremities, and the presacral area10. Some of the dermal melanocytes have clearly migrated into the , but, given the absolute ORIGIN AND FUNCTION OF THE MELANOCYTE numbers of cells in the two compartments, apoptosis of pigment cells has also occurred. The three sites where active dermal melanocytes are The melanocyte is a neural crest-derived cell, and during embryogenesis still present at the time of birth coincide with the most common sites precursor cells (melanoblasts) migrate along a dorsolateral then ventral for dermal melanocytoses and dermal melanocytomas (blue nevi). pathway via the to reach the epidermis and hair follicles Hepatocyte growth factor may play a role in the survival and prolifera­ of the trunk (see Ch. 2). More recently, it was shown that cutaneous tion of these dermal melanocytes as well as somatic activating muta­ melanocytes can also arise from neural crest-derived Schwann cell tions in GNA11 and GNAQ, which encode G proteins and are found precursors that migrate along nerves to the skin via a distinct ventral in blue nevi (see Table 112.3). pathway7. Additional sites of melanocyte migration include the uveal As depicted in Fig. 65.1, melanocytes also migrate to the basal layer tract of the eye (choroid, ciliary body, and iris), the leptomeninges, and of the hair matrix and the of hair follicles. Cells that the inner ear (cochlea) (Fig. 65.1). Presumably, the death of melanocytes are actively producing melanin are easily recognized in the matrices of within the leptomeninges, inner ear, and skin is responsible for the pigmented anagen , whereas melanocytes within the outer root aseptic meningitis, auditory symptoms, and areas of , respec­ sheath are usually amelanotic and more difficult to identify11. It has tively, seen in patients with the Vogt–Koyanagi–Harada syndrome (see been hypothesized that there are two populations of melanocytes in the 1075 Ch. 66). skin, one in the interfollicular epidermis and the second in the hair non-print metadata ABSTRACT KEYWORDS: The major determinant of normal skin color is the activity of melano­ melanocyte, cytes, i.e. the quantity and quality of pigment production, not the melanosome, density of melanocytes. Melanocytes contain a unique intracytoplasmic tyrosinase, CHAPTER organelle, the melanosome, which is the site of melanin synthesis and eumelanin, deposition. Compared with lightly pigmented skin, darkly pigmented pheomelanin, 65 skin has melanosomes that contain more melanin and are larger; once melanocortin 1 receptor, transferred to keratinocytes, the melanosomes are singly dispersed and MC1R, degraded more slowly. Tyrosinase is the key enzyme in the melanin melanocyte stimulating hormone, biosynthetic pathway and the two major forms of melanin produced in MSH, melanocytes are brown–black eumelanin and yellow–red pheomelanin. pigmentation, The production of eumelanin versus pheomelanin is influenced by the agouti, binding of melanocyte stimulating hormone to the melanocortin 1 melanin biosynthetic pathway receptor. Melanocyte Biology

1075.e1 DISORDERS CHARACTERIZED BY DIFFUSE PIGMENTARY DILUTION IN WHICH THE GENETIC DEFECT IS KNOWN

Disorder Gene Protein product Comments Oculocutaneous (OCA) SECTION [~40% of patients have OCA1 and ~50% have OCA2] 10 OCA1A TYR Tyrosinase • Complete absence of tyrosinase activity and melanin production • Retention of misfolded tyrosinase protein within the ER

•

ers OCA1B TYR Tyrosinase Decreased tyrosinase activity; can produce pheomelanin • Variant with temperature-sensitive tyrosinase (normal activity at 35°C, but diminished at 37°C) • Additional variants: minimal pigment, platinum, yellow isord

D OCA2 OCA2 P protein (OCA2 was • Melanosomal transmembrane protein that is also present in the ER previously known as P) • Possible functions include regulating organelle pH, facilitating vacuolar accumulation of glutathione, and processing/trafficking of tyrosinase OCA3 TYRP1 Tyrosinase-related protein 1* • TYRP1 stabilizes tyrosinase in mice and humans, and it can function as a DHICA oxidase

igmentary • Both “mutant” TYRP1 and tyrosinase are retained in the ER and then degraded P • Rufous phenotype ≫ brown phenotype; latter seen in OCA2 OCA4 SLC45A2 Solute carrier family 45 • Variable phenotype, with hair ranging from white to yellow–brown; most common in Asians member 2 (previously known • Transmembrane transporter with a role in tyrosinase processing and intracellular trafficking of as MATP) proteins to the melanosome

OCA5 Not known − • Locus linked to 4q24

OCA6 SLC24A5 Solute carrier family 24 • Cation exchanger that may be involved in ion transport in melanosomes member 5

OCA7 C10orf11 Chromosome 10 open • Expressed in melanoblasts and melanocytes reading frame 11 • Possible role in melanocyte differentiation Hermansky–Pudlak syndrome (HPS)†

HPS1 HPS1 HPS1 (BLOC-3 subunit 1) • Defective trafficking of organelle-specific proteins to melanosomes, lysosomes and cytoplasmic granules (including platelet dense granules and lytic granules in cytotoxic T lymphocytes) • Pulmonary and granulomatous colitis • Component of BLOC-3 (see Fig. 65.8)

HPS2 AP3B1 Adaptor related protein • AP-3 recognizes sorting signals within cytosolic tails of cargo molecules and is involved in the complex 3 β1 subunit trafficking of proteins from the trans-Golgi network to appropriate (see Fig. 65.8) • Pulmonary fibrosis (some patients) • Abnormal targeting of CD1 may play a role in associated immunodeficiency

HPS3 HPS3 HPS3 (BLOC-2 subunit 1) • Component of BLOC-2

HPS4 HPS4 HPS4 (BLOC-3 subunit 2) • Pulmonary fibrosis • Component of BLOC-3

HPS5 HPS5 HPS5 (BLOC-2 subunit 2) • Component of BLOC-2

HPS6 HPS6 HPS6 (BLOC-2 subunit 3) • Component of BLOC-2

HPS7 DTNBP1 Dystrobrevin binding protein 1 • Component of BLOC-1

HPS8 BLOC1S3 BLOC1S3 (BLOC-1 subunit 3) • Component of BLOC-1

HPS9 BLOC1S6 BLOC1S6 (BLOC-1 subunit 6) • Component of BLOC-1

HPS10 AP3D1 Adaptor related protein • See AP-3 above complex 3 δ1 subunit • Neurologic impairment (e.g. seizures), immunodeficiency Chédiak–Higashi syndrome

LYST Lysosomal trafficking • Abnormal vesicle trafficking and fission/fusion of lysosome-related organelles result in giant regulator organelles (e.g. melanosomes, neutrophil granules [lysosomes], platelet dense granules)

GS1 MYO5A Myosin Va (attaches • In all three forms, melanosomes are retained in the body of the melanocyte rather than melanosomes to actin trafficking to the tips of dendrites for transfer to keratinocytes (see Fig. 65.10) filaments) • Silvery hair seen in all three forms • Myosin Va expressed in neurons (as well as melanocytes) and dysfunction leads to neurologic abnormalities

GS2 RAB27A RAB27A (melanosomal • GTPase also expressed in hematopoietic cells; defective release of granule contents from membrane GTPase that binds cytotoxic T cells leads to recurrent infections and hemophagocytic syndrome melanophilin)

GS3 MLPH Melanophilin (links myosin Va • Melanophilin only expressed in melanocytes, so only pigmentary dilution in type 3 and RAB27A)

MYO5A Myosin Va F-exon deletion • F-exon only expressed in melanocytes *Recognized by Mel-5 antibody. †Reflecting a founder effect, individuals of Puerto Rican origin have HPS1 and HPS3 (3 : 1 ratio); non-Puerto Ricans most commonly have HPS1, followed by HPS3 and HPS4 with ~70% of patients having one of these three types.

Table 65.1 Disorders characterized by diffuse pigmentary dilution in which the genetic defect is known. See Chapter 66 for details of clinical findings. BLOC-1 promotes endosomal maturation by recruiting the Rab5 GTPase-activating protein Msb3; BLOC-2 targets recycling endosomal tubules to melanosomes for cargo 1076 delivery; BLOC-3 functions as a Rab32/38 guanine nucleotide exchange factor that is capable of activating small GTPases (activated Rab 32/38 is needed for transport of tyrosinase and TYRP1 to melanosomes). AP-3, adaptor protein complex 3; BLOC, biogenesis of lysosome-related organelles complex; DHICA, 5,6-dihydroxyindole-2-carboxylic acid; ER, endoplasmic reticulum; MATP, membrane associated transporter protein; TYRP, tyrosinase-related protein. MIGRATION OF MELANOCYTES FROM THE NEURAL CREST ACTIVATION OF THE KIT RECEPTOR ON MELANOCYTES

CHAPTER KIT ligand (steel factor) 65 Eye KIT receptor extracellular Iris Choroid Retina intracellular Melanocyte Biology Skin

Neural tube (optic cup)

P Tyr- Neural crest Tyr- -Tyr P

inactive active

Schwann cell Fig. 65.2 Activation of the KIT receptor on melanocytes. Because the KIT precursor along nerve receptor is a kinase receptor, it has the ability to phosphorylate the tyrosine residues of other proteins as well as itself, i.e. autophosphorylation. Heterozygous germline mutations in KIT that prevent the activation of the KIT receptor by KIT ligand, also referred to as steel factor, lead to piebaldism, Inner ear whereas somatic activating mutations in KIT are seen in patients with Medulla mastocytosis as well as arising in acral sites, mucosae, and oblongata chronically photodamaged skin (see Figs 118.2 & 113.1). A form of familial (leptomeninges) progressive hyperpigmentation with or without hypopigmentation can result from heterozygous gain-of-function germline mutations in the gene that encodes KIT ligand49. P, phosphorylation; Tyr, tyrosine.

Fig. 65.1 Migration of melanocytes from the neural crest. Melanocytes migrate to the uveal tract of the eye (iris, ciliary body, and choroid), the leptomeninges, and the cochlea of the inner ear, as well as to the epidermis and . Cutaneous melanocytes can also arise from Schwann cell RECEPTOR–LIGAND INTERACTIONS IN PRECURSORS OF MELANOCYTES precursors located along nerves in the skin, which also originate from the neural crest. The retina actually represents an outpouching of the neural tube. melanoblast or enteric neural crest-derived cell follicle12. Based upon expression and clinical observations, it is the former that is more sensitive to the destructive forces of vitiligo. melanoblast As a result, repigmentation of patches of vitiligo in which the hairs are still pigmented relies upon activation and subsequent upward migra­ tion of the melanocytes present in the outer root sheath11. Of note, melanocyte stem cells have been identified within the lower portion of the hair follicle bulge, i.e. the lowermost permanent portion of the hair follicle (see Fig. 2.6). In addition, KROX20+ cells, which give rise to the hair shaft, have recently been identified within the hair bulb. These hair progenitor cells produce (SCF) which was shown to be essential for hair pigmentation, i.e., mouse hairs turn white when the SCF gene was deleted12a. KIT By immunohistochemical staining, melanocytes are identified within ET3 EDNRB KIT ligand 13 receptor the fetal epidermis as early as 50 days of gestation . Melanin-containing (steel factor) melanosomes are recognizable by electron microscopy during the fourth month of gestation. Except in benign and malignant neoplasms, mela­ Fig. 65.3 Receptor–ligand interactions in precursors of melanocytes. In the nocytes reside within the basal layer of the epidermis, a location they developing embryo, melanoblasts expressing endothelin receptor type B maintain throughout life (Fig. 65.5). Although the cell body of the (EDNRB) are stimulated to migrate by endothelin-3 (ET3) which is produced by melanocyte sits on a specialized region of basal lamina, its dendrites the and dermamyotome. Melanoblasts also require expression of the come into contact with keratinocytes as far away as the mid stratum KIT receptor to maintain their normal chemotactic migration directed by spinosum. This association of a melanocyte with ~30–40 surrounding production of KIT ligand/steel factor by the dermamyotome. keratinocytes to which it transfers melanosomes has been called the epidermal melanin unit14. However, melanocytes fail to form desmo­ somal connections with neighboring keratinocytes; their interactions pigmentation seen within the human race. In other words, a person with keratinocytes are via cadherins. who has minimal baseline pigmentation and an inability to tan has a Although there is variation in the density of epidermal melanocytes/ similar density of melanocytes when compared with a person whose mm2 when different regions of the body are analyzed via DOPA-stained skin is dark brown to black in color. Indeed, even patients with the epidermal sheets (Fig. 65.6), e.g. the density of melanocytes is greater most severe form of OCA, OCA type 1A, have a normal complement in the genital region (~1500/mm2) compared with the back (~900/ of epidermal melanocytes. 2 mm ), there are smaller differences between individuals when the same The major determinant of normal skin color is the activity of the 1077 anatomic site is examined. This is despite the wide variation in melanocytes, i.e. the quantity and quality of pigment production, not Fig. 65.4 Signal transduction pathways and SIGNAL TRANSDUCTION PATHWAYS AND TRANSCRIPTION FACTORS transcription factors that contribute to melanocyte THAT CONTRIBUTE TO MELANOCYTE DIFFERENTIATION differentiation. MITF expression is activated early on during the transition from pluripotent neural crest SECTION cells to melanoblasts and is required for melanoblast survival8; mutations in MITF lead to Waardenburg 10 WNT α-MSH syndrome, a classic neurocristopathy. MITF also regulates the expression of multiple pigment genes KIT ligand including those that encode tyrosinase, TYRP1, ers MC1R TYRP2, PMEL/PMEL17/gp100, and MART-1/Melan-A. ET3 Additional transcriptional targets are CDK2, CDKN2A,

isord cAMP and BCL-2 (whose protein product is an inhibitor of KIT receptor 9 D apoptosis) . Small molecule inhibitors of SIK β-Catenin (salt-inducible kinase) can upregulate MITF, and RAS/RAF/ EDNRB MEK/ERK PKA application of these inhibitors to normal led to an increase in pigmentation54. In mice, Wnt P signaling in melanocyte stem cells is critical for hair

igmentary MITF 50

P SOX10 LEF1 CREB PAX3 pigmentation . Details of how activation of (activated) G-protein-coupled receptors leads to an increase in intracellular cAMP is shown in Fig. 65.15. Of note, MITF promoter EDNRB interacts with the G proteins GNAQ and P GNA11, and activating mutations in the genes that encode these latter two proteins can lead to blue MITF nevi and phakomatosis pigmentovascularis. cAMP, Melanogenesis cyclic adenosine monophosphate; CREB, cAMP Melanocyte differentiation response-element binding protein; ET3, endothelin-3; EDNRB, endothelin receptor type B; LEF1, lymphoid Melanocyte proliferation enhancer binding factor 1; MC1R, melanocortin 1 Activation and Degradation Melanocyte survival receptor; MITF, microphthalmia-associated transcription factor; MSH, melanoctye stimulating hormone; P, phosphorylation; PKA, protein kinase A; SOX10, SRY-box containing gene 10.

Fig. 65.5 A melanocyte residing in the basal layer A MELANOCYTE RESIDING IN THE BASAL LAYER OF THE EPIDERMIS of the epidermis. In normal skin, approximately every tenth cell in the basal layer is a melanocyte. Melanosomes are transferred from the dendrites of the melanocyte into neighboring keratinocytes of the stratum epidermis, hair matrices and mucous membranes; no corneum transfer occurs in the pigment epithelium of the retina. The epidermal melanin unit refers to the stratum association of a melanocyte with ~30–40 granulosum surrounding keratinocytes to which it transfers melanosomes.

epidermis of tip stratum spinosum melanosomes

dendrite

basal melanocyte layer

fusion of dermis membranes

the density of melanocytes15. Several factors play a role in deter­ FORMATION AND FUNCTION OF mining the level of melanocyte activity; they include specific character­ THE MELANOSOME istics of the individual melanosomes (e.g. their dimensions) as well as both baseline (constitutive) and stimulated (facultative) levels and Within the cytoplasm of melanocytes is a unique organelle known as activity of the enzymes involved in the melanin biosynthetic pathway. the melanosome, in which melanin are synthesized, depos­ The latter are influenced by receptor-mediated interactions with extra­ ited, and transported. The melanosome is most closely related to the 16 1078 cellular ligands such as MSH which then influence the expression of lysosome . Through compartmentalization, both organelles provide transcription factors including MITF (see Fig. 65.4). protection for the remainder of the cell – lysosomes protect against pro-enzymes such as proteases and melanosomes protect against Fig. 65.7). In patients with OCA1 and OCA3, misfolded tyrosinase or melanin precursors (e.g. phenols, quinones) that can oxidize lipid aberrant tyrosinase-related protein 1 (TYRP1) plus tyrosinase polypep­ membranes. tides, respectively, accumulate within the endoplasmic reticulum (ER)17. The melanosome contains both matrix proteins (chiefly PMEL/ This leads to ER stress and activation of the unfolded protein response PMEL17/gp100), which form a scaffolding upon which the melanin (UPR)18. As a result of the UPR, which acts as a form of quality control, CHAPTER is deposited, and enzymes such as tyrosinase that regulate the biosyn­ these proteins become targets for destruction by proteasomes and there­ thesis of melanin. Following their formation via ribosomes, all these fore are not incorporated into melanosomes (see Fig. 65.7 inset). 65 proteins are found within the rough endoplasmic reticulum (RER; Several of the enzymes in the melanin biosynthetic pathway are glycoproteins that require the attachment of sugars in order to gain full function. For this reason, they undergo post-translational modification in the ER and the Golgi apparatus and then join up with the matrix proteins (e.g. PMEL/PMEL17/gp100) to initiate melanogenesis. The targeting of proteins to the plasma membrane versus intracytoplasmic organelles and the targeting of specific proteins to the correct type of organelle are complicated processes. This triaging requires the equiva­ Melanocyte Biology lent of traffic police within the cell, an example of which is adaptor protein complex 3 (AP-3). Mutations in the gene that encodes the β3A subunit of AP-3 are responsible for a subset of patients with Hermansky– Pudlak syndrome (HPS2)19. Other forms of HPS are due to dysfunction of the proteins that serve as components of biogenesis of lysosome- related organelles complexes (BLOCs; Fig. 65.8)20. It then follows that these patients have dysfunction of more than one intracytoplasmic organelle, i.e. not just melanosomes but other lysosome-related organ­ elles such as NK cell granules and platelet dense granules (see Table 65.1). The associated hypopigmentation can be explained by the failure to efficiently deliver melanogenic proteins, e.g. tyrosinase, TYRP1, to the melanosome. The progression of a melanosome from an organelle that lacks melanin to one that is fully melanized is arbitrarily divided, for conve­ Fig. 65.6 Dihydroxyphenylalanine (DOPA)-stained epidermal sheet. Following incubation with 5 mM L-DOPA for 4–5 hours, the epidermal nience, into four stages (Fig. 65.9). Proteins that play a key role melanocytes turn black because they contain the enzyme tyrosinase, which in the biogenesis of early-stage melanosomes are PMEL/PMEL17/ converts DOPA to black DOPA-melanin. Note the multiple dendrites and gp100, MART-1/Melan-A, and the type 1 (OA1) regular spacing of the melanocytes. G-protein-coupled receptor21. and processing of PMEL/

Fig. 65.7 Formation of FORMATION OF A MELANOSOME AND PATHOPHYSIOLOGY OF OCA1 a melanosome and pathophysiology of type 1 late myosin Va (OCA1). After glycosylation and processing within the endoplasmic reticulum (ER) and Golgi apparatus, several of the enzymes involved in the formation of melanin (including tyrosinase) are packaged T Golgi in vesicles and then combine with apparatus the matrix proteins (e.g. PMEL/ T M PMEL17/gp100). As more melanin is deposited within the RER melanosomes, they migrate into ribosome the dendrites in preparation for M their transfer into neighboring keratinocytes. In patients with proteasome OCA1 and OCA3 (triangular insert), tyrosinase polypeptides are SER retained too long within the lumen of the RER and then become targets for destruction within proteasomes. Thus, they are not incorporated into melanosomes. The primary matrix protein is dendrite T PMEL/PMEL17/gp100 (recognized by HMB45 antibody); formation of the matrix fibers requires cleavage actin of this protein by a proprotein 51 cytoskeleton nucleus convertase . Sorting of proteins to abnormal/ the correct organelles is a complex misfolded nascent process and it requires regulators protein such as adaptor protein complex 3 plasma (e.g. T in OCA1) membrane (AP-3; see Fig. 65.8); in the case of tyrosinase, specific dileucine residues in its cytoplasmic tail aid in the sorting. M, matrix proteins; RER, rough endoplasmic reticulum; SER, smooth endoplasmic 1079 reticulum; T, tyrosinase. Fig. 65.8 Regulation of protein trafficking by REGULATION OF PROTEIN TRAFFICKING BY ADAPTOR PROTEIN COMPLEX 3 (AP-3) adaptor protein complex 3 (AP-3) and biogenesis of AND BIOGENESIS OF LYSOSOME-RELATED ORGANELLES COMPLEXES (BLOCs) lysosome-related organelles complexes (BLOCs). Lysosome-related organelles include melanosomes, SECTION platelet dense granules, and lytic granules of cytotoxic lymphocytes and natural killer cells. 10 BLOC-2 Subtypes of Hermansky-Pudlak syndrome (HPS) that Lysosome are associated with a particular BLOC can have BLOC-1 HPS3 HPS5 similar clinical findings, e.g. patients with HPS1 and ers HPS7 HPS6 HPS4, both members of BLOC-3, develop pulmonary HPS8 fibrosis and granulomatous colitis. HPS9 isord BLOC-3 D HPS1 Lysosome- Golgi HPS4 related organelle AP-3 (e.g. igmentary

P melanosome) HPS2 HPS10

FOUR MAJOR STAGES OF EUMELANIN MELANOSOMES MOVEMENT OF MELANOSOMES WITHIN MELANOCYTE DENDRITES

Stage Description Electron micrographs

I Spherical; no melanin deposition

UVR II Oval; obvious matrix in the form of parallel longitudinal filaments; minimal deposition of melanin; high tyrosinase activity

III Oval; moderate deposition of melanin; high tyrosinase activity

IV Oval; heavy deposition of melanin; electron-opaque; minimum tyrosinase activity

Microtubule Myosin Va Fig. 65.9 Four major stages of eumelanin melanosomes. Courtesy, Raymond Boissy, Actin filament Melanophilin PhD. Melanosome RAB27A Kinesin Dynactin PMEL17/gp100, an amyloidogenic protein, leads to a fibrillar matrix Dynein that acts as a scaffold upon which melanin is deposited22. As melanin Fig. 65.10 Movement of melanosomes within melanocyte dendrites. As is deposited within the melanosome, the organelle migrates via micro­ melanin is deposited within melanosomes, they migrate along tubules into the dendrites in preparation for transfer into the neighbor­ from the cell body into dendrites in preparation for transfer to keratinocytes. ing keratinocytes, either within the epidermis or within the anagen hair Kinesin and dynein serve as molecular motors for -associated matrix. In addition to microtubules, proteins such as kinesin and anterograde and retrograde melanosomal transport, respectively, and UVR dynein are involved in the movement of melanosomes (Fig. 65.10)23. results in augmented anterograde transport via increased kinesin and Within the dendrites, there is a specialized myosin protein, myosin decreased dynein activity. Myosin Va, which is linked to the melanosomal Va, that aids in the process of melanosome transfer by assisting attach­ RAB27A GTPase by melanophilin, captures mature melanosomes when they ment between the actin cytoskeleton beneath the plasma membrane reach the cell periphery and attaches them to the actin cytoskeleton. and the organelle itself. RAB27A and melanophilin also play a role in ensuring this attachment. The importance of the interactions between these three proteins – myosin Va, RAB27A, and melanophilin – is dilution, whereas in the latter the hypopigmentation is circumscribed. illustrated by the three different forms of Griscelli syndrome in which Phagocytosis of melanosomes by keratinocytes can be triggered by acti­ there are mutations in the respective genes (see Table 65.1)24,25. The vation of the keratinocyte growth factor (KGF) receptor (KGFR/FGFR2b) various phenotypes are explained by the tissue-specific expression of by KGF, also referred to as growth factor 726, as well as by acti­ these three genes, e.g. neurons versus cytotoxic T cells. All three genes vation of protease-activated receptor 2 (PAR-2)27. Inhibitors of PAR-2 are expressed in melanocytes and as expected, the associated diffuse receptor–ligand interactions, e.g. inhibitors of serine proteases, have led pigmentary dilution is a reflection of a lack of transfer of melano­ to cutaneous hypopigmentation in animal models28. somes from the melanocytes to nearby keratinocytes. Histologically, In summary, it is the activity of the melanocytes and their interac­ numerous melanosomes are seen to congregate within the center of tions with neighboring keratinocytes, not their density, that is the the melanocytes. major determinant of normal skin color. The activity of a melanocyte To reiterate, normal pigmentation of the skin is dependent on an is reflected in the number and size of melanized melanosomes it pro­ orderly transfer of melanosomes from melanocytes to keratinocytes. duces as well as in its efficiency at transferring those melanosomes to When this transfer is disrupted, either in inherited diseases such as keratinocytes. For example, primarily stage II and stage III melano­ 1080 Griscelli syndrome or in acquired diseases such as atopic dermatitis, the somes are seen in lightly pigmented skin, whereas primarily stage IV result is hypopigmentation. In the former there is diffuse pigmentary melanosomes are seen in darkly pigmented skin (Table 65.2). An additional factor is the rate of degradation of the melanosomes once they are transferred to the surrounding keratinocytes and this is related VARIATION OF PREDOMINANT MELANOSOMAL STAGES WITH LEVEL in part to the size of the individual melanosomes. The smaller mela­ OF CUTANEOUS PIGMENTATION nosomes of lightly pigmented skin are clustered in groups of two to ten Predominant melanosomal stages within secondary lysosomes in the keratinocytes and are degraded by CHAPTER the mid stratum spinosum (Table 65.3)15. In darkly pigmented skin, Pigmentation of skin Melanocytes Keratinocytes 65 the melanosomes are larger and singly dispersed within lysosomes of Fair II, III Occasional III the keratinocytes; they are degraded more slowly, such that melanin granules can still be found in the stratum corneum. Medium II, III, IV III, IV Dark IV > III IV

REGULATION OF MELANIN BIOSYNTHESIS Table 65.2 Variation of predominant melanosomal stages with level of cutaneous pigmentation. This section will begin with a review of the melanin biosynthetic pathway and then examine the factors, both external and internal, that Melanocyte Biology can influence the level of melanin production. The “starting material” for the production of melanin, both the brown–black eumelanin and the yellow–red pheomelanin, is the amino acid tyrosine. The key regu­ latory enzyme in the pathway is tyrosinase, which controls the initial MELANOSOMES IN LIGHTLY PIGMENTED VERSUS DARKLY biochemical reactions in this pathway (Fig. 65.11). It should then come PIGMENTED SKIN as no surprise that the initial investigations into the molecular basis Lightly pigmented Darkly pigmented of OCA focused on the gene that encodes tyrosinase. skin skin In OCA1A, the form of OCA where mutations in both copies of the tyrosinase gene lead to complete loss of enzyme activity, no melanin is Melanization Stages II, III Stage IV found in the hair, skin, or eyes (see Table 65.1). However, in OCA1B, Size (diameter) 0.3–0.5 microns 0.5–0.8 microns where there is decreased enzyme activity, pheomelanin is produced, Number per cell 20 200 especially in the hair as the patient ages. The formation of pheomelanin < > requires less tyrosinase activity than does the formation of eumelanin Distribution of Groups of 2–10 Single (see Fig. 65.11) and therefore the formation of pheomelanin can be melanosomes within the thought of as a default pathway. lysosomes of keratinocytes The activity of tyrosinase is enhanced by DOPA and is stabilized by tyrosinase-related protein 1 (TYRP1) (see below). Competitive inhibi­ tors of tyrosinase activity include hydroquinone, which is used to treat Degradation Fast Slow disorders of hyperpigmentation such as , and L-phenylalanine.

In patients with phenylketonuria (PKU), there is a diffuse pigmentary Table 65.3 Melanosomes in lightly pigmented versus darkly pigmented skin.

THE MELANIN BIOSYNTHETIC PATHWAY

Tyrosinase Tyrosinase

CO2H O CO2H HO CO2H O CO2H HO CO2H

NH NH NH NH NH 2 2 2 2 Cysteine 2 HO O HO O HO Tyrosine DOPAquinone CycloDOPA then DOPA DOPAquinone S 5-S-cysteinylDOPA* NH2 CO2H Dopachrome tautomerase (DCT)/TYRP2 DQ HO O HO DOPA

+ CO2 CD-quinones HO NH CO2H HO N CO2H HO NH DHICA DOPAchrome DHI

Tyrosinase or TYRP1 DQ o-Quinoneimine O2 O2 DOPA 1, o-Benzothiazine intermediates

DHICA-melanin Eumelanin DHI-melanin Pheomelanin brown, slightly soluble, black, insoluble, yellow/red, alkali- intermediate MW high MW soluble, low MW

*Or 2-S-cysteinylDOPA

Fig. 65.11 The melanin biosynthetic pathway. The pathway includes the sites of dysfunction in OCA1 (tyrosinase) and OCA3 (TYRP1). The two major forms of melanin in the skin and hair are brown–black eumelanin and yellow–red pheomelanin. The enzymes are transmembrane proteins located within the melanosome. DHI, 5,6-dihydroxyindole; DHICA, 5,6-dihydroxyindole-2-carboxylic acid; DOPA, dihydroxyphenylalanine; MW, molecular weight; TYRP, tyrosinase-related protein. 1081 Adapted from Hearing VJ. Determination of melanin synthetic pathways. J Invest Dermatol. 2011;131:E8–E11. dilution due to elevated levels of L-phenylalanine resulting from a Among the multiple protein products of the deficiency in the enzyme L-phenylalanine hydroxylase that converts (POMC) gene are adrenocorticotropic hormone (ACTH), β-endorphin, L-phenylalanine to L-tyrosine15. The characteristic blonde hair of PKU and the three forms of MSH (α, β, and γ) (Fig. 65.13); in humans, can undergo darkening when the patient is on a low-phenylalanine diet. α-MSH represents the major biologically active form of MSH. The SECTION Of note, tyrosinase is a copper-requiring enzyme and it has two copper- primary site of expression of POMC is the pituitary gland; however, binding sites. Rare cases of copper deficiency can lead to diffuse cutane­ other sites of expression include the testis, the endothelium, and, of 10 ous pigmentary dilution, and in patients with Menkes disease, where particular importance, epidermal keratinocytes. Although MSH is clas­ a transmembrane Cu2+-transporting ATPase that delivers copper to the sically associated with the pigmentary system, this peptide has a wide ers trans-Golgi network and melanosomes is dysfunctional29, the kinky range of biologic properties, including suppression of inflammation and hair is hypopigmented. regulation of body weight. For example, mutations in POMC can result 33 isord In a test tube, L-DOPA can spontaneously oxidize to form melanin, in severe early-onset obesity, adrenal insufficiency, and red hair . It D an insoluble biopolymer. For this reason, it was originally thought that then follows that mutations in the genes that encode the melanocortin tyrosinase was the sole enzyme involved in melanin biosynthesis. receptors to which the protein products of POMC bind can lead to However, by the late 1970s, it was becoming clear that there were similar clinical findings; for example, mutations in MC4R are associ­ additional control points in the pathway (see Fig. 65.11). For example, ated with morbid obesity and variant alleles in MC1R are associated

igmentary dopachrome tautomerase, also known as tyrosinase-related protein 2 with red hair (see below). Of note, subcutaneous P (TYRP2), which like TYRP1 shares similarities in its amino acid (4-norleucyl-7-phenylalanine-α-MSH), which has enhanced binding to sequence with tyrosinase, converts DOPAchrome to 5,6-dihydroxyindole- MC1R compared to α-MSH, can lead to cutaneous hyperpigmentation. 2-carboxylic acid (DHICA). In mice and humans, TYRP1 stabilizes When subcutaneous implants of afamelanotide were administered tyrosinase30 and mutations in both copies of TYRP1 lead to OCA3 (see monthly, patients with erythropoietic protoporphyria experienced fewer Table 65.1). phototoxic reactions and could tolerate more direct without Decreased function of yet another transmembrane protein, the P pain while patients with vitiligo had more rapid and extensive repig­ protein, leads to OCA231 (Fig. 65.12). Based upon its amino acid mentation in response to treatment with UVB. sequence, a prediction was made that the P protein was involved in the As outlined in Table 65.4, there are five major melanocortin receptors, transport of small molecules across the membrane of the melanosome. all of which have seven transmembrane domains (Fig. 65.14). Although Tyrosine, the initial precursor in the melanin biosynthetic pathway, was the MC1R is present on a variety of cells within the skin, from endothe­ considered the most likely candidate for transmembrane transport. lial cells to , the highest density of this receptor is found on However, the nature of what might be transported by the P protein melanocytes34. As in the case of the β-adrenergic receptor, the MC1R is remains unclear. Data suggest that the P protein regulates processing a G-protein-coupled receptor, i.e. it uses proteins that bind guanosine and trafficking of tyrosinase, possibly via control of pH or glutathione triphosphate (GTP) and guanosine diphosphate (GDP) as intermediary content within intracellular compartments32. messengers (Fig. 65.15). Following the binding of MSH to the MC1R, represent a group of complex polymers whose functions the protein pair interacts with a complex of G proteins. The GTP-Gsα vary from camouflage to the quenching of oxidative free radicals gener­ subunit then activates adenylate cyclase, leading to increased produc­ ated via exposure to UV radiation (UVR). The level and type of melanin tion of cyclic adenosine monophosphate (cAMP) within the melanocyte. production is a complex interplay of the activity of the various enzymes An increase in the intracellular concentration of cAMP leads to an involved in the biosynthetic pathway as well as the activity of proteins increase in tyrosinase activity and eumelanin production via MITF (see such as the P protein and those that stabilize the activity of tyrosinase Fig. 65.4). If the MC1R is dysfunctional and fails to initiate a significant (e.g. TYRP1). Several factors are known to influence the activity of rise in the intracellular level of cAMP, then pheomelanogenesis is these key proteins of melanogenesis, and they include α-MSH, basic favored (Fig. 65.16). Of note, the majority of individuals with red hair fibroblast growth factor (bFGF), endothelin-1, KIT ligand, and UVR (see are compound heterozygotes or homozygotes for a variant R allele in the below). gene that encodes the MC1R1,35 (see Fig. 65.14). MC1R also interacts with a protein known as the agouti protein (mouse) or agouti signaling protein (ASIP; human). Agouti is a term

THE P PROTEIN WITHIN THE LIPID BILAYER

POST-TRANSLATIONAL PROCESSING OF THE POMC POLYPEPTIDE

+ Cytoplasmic NH3 1 POMC 241 COO–

174 352 352 407 419 533 617 670 676 742 759 837 180 347 354 401 424 530 621 664 680 737 761 834 PC1 Pro γ-MSH JP ACTH β-LPH

1 2 3 4 5 6 7 8 9 10 11 12

− PC2 ACTH1-17 β-END − − + 196 331 370 385 440 514 637 648 696 721 777 818 199 329 372 382 449 503 648 645 701 718 780 812

des α-MSH

Luminal

ac α-MSH

Fig. 65.12 The P protein within the lipid bilayer. The polypeptide has 12 Fig. 65.13 Post-translational processing of the POMC polypeptide. Pituitary putative transmembrane domains. The solute carrier family 45 member 2 hypersecretion of ACTH and/or α-MSH can lead to generalized (previously known as membrane-associated transporter protein), which is hyperpigmentation in patients with Addison disease. ac, acetylated; ACTH, dysfunctional in OCA4, has a similar configuration within the plasma adrenocorticotropic hormone; des, desacetyl; END, endorphin; JP, joining 1082 membrane. Adapted from Rinchik EM, Bultman SJ, Horsthemke B, et al. A gene for the mouse peptide; LPH, lipotropic hormone; MSH, melanocyte stimulating hormone; PC, pink-eyed dilution locus and for human type II oculocutaneous albinism. Nature. 1993;361:72–6. prohormone-converting enzyme; POMC, proopiomelanocortin. used to describe the banding of hairs seen in some mammals, including of this disorder, the increase in intracellular cAMP leads to increased dogs, foxes and mice, that is due to alternating production of eumelanin tyrosinase activity and eumelanin production. and pheomelanin (Fig. 65.17). The production of agouti protein by the Exposure of melanocytes to phorbol esters, e.g. tetradecanoyl phorbol cells in the hair follicle papillae is cyclic, and, when the agouti protein acetate (TPA), can lead to increased melanin formation via the activa­ is present, it effectively competes with MSH and the formation of tion of protein kinase C (PKC) (Fig. 65.18). Growth factors, including CHAPTER pheomelanin within pheomelanosomes is favored36,37 (see Fig. 65.16). bFGF and KIT ligand, can also lead to an increase in the pigment Compared to eumelanosomes, pheomelanosomes are characterized by content within melanocytes whereas KIT receptor inhibitors (e.g. ima­ 65 a more spherical shape and the presence of an unstructured matrix with tinib) can lead to hypopigmentation. Endothelin-1, a small peptide vesicular bodies. originally isolated from endothelial cells, is produced by keratinocytes An enhancement of pigment production can be seen following expo­ and can lead to an increase in tyrosinase activity followed by an increase sure of melanocytes to agents that increase intracytoplasmic levels of in melanin production. cAMP such as cholera toxin, forskolin38, dibutyryl cAMP, and MSH (see Multiple genes have been implicated as playing a role in normal Fig. 65.15). The activation of protein kinase A (PKA) by cAMP leads to pigment variation in humans, including: (1) MC1R and ASIP; (2) several the phosphorylation of various proteins, which can result in their acti­ genes that play a role in OCA, e.g. TYR, OCA2, TYRP1, SLC45A2, vation. One of the proteins that is phosphorylated by PKA is the cAMP SLC24A5 (see Table 65.1); (3) KITLG which encodes KIT ligand; (4) Melanocyte Biology response-element binding protein (CREB), which functions as a tran­ TPCN2 which encodes an ion channel transporter; and (5) IRF4 which scription factor, regulating the expression of other genes, including encodes interferon regulatory factor 42–4. The presence of a variant MITF (see Fig. 65.4). Patients with the McCune–Albright syndrome (rather than the African ancestral) allele of SLC24A5, whose protein (polyostotic fibrous dysplasia) are mosaics for an activating mutation product is a putative cation exchanger in the melanosomal membrane, 5 in the gene that encodes the G protein Gsα (see Fig. 65.15). As a result, correlates with lighter skin color . In addition, variants in OCA2 are the cAMP cascade is permanently “turned on” and, with continued thought, at least in part, to determine the normal phenotypic variation transcription of the CREB-controlled genes, there is hyperplasia of the in color2. A haplotype with several polymorphisms in ASIP and endocrine organs, albeit in a mosaic pattern. Presumably (and presumably a gain of ASIP function) has been associated with red within the melanocytes of the segmental café-au-lait macules typical or blonde hair, freckling and a tendency to burn, while variants that lead to destabilized ASIP mRNA have been associated with darker skin phototypes3,39,40. A polymorphism in the regulatory region of KITLG that reduces its responsiveness to the WNT-activated transcription MAJOR FORMS OF THE MELANOCORTIN RECEPTOR factor LEF1 is associated with blonde hair41, and in mice, decreased lef1 leads to light-colored hair. Of note, some loss-of-function muta­ Receptor Distribution, major (minor) Ligands tions in MC1R confer a risk for developing cutaneous , inde­ MC1R* Melanocytes (keratinocytes, α-MSH, ACTH > pendent of pigmentary phenotype35. fibroblasts, endothelial cells, β-MSH In humans, graying or whitening of hair is a normal aging phenom­ antigen-presenting cells) enon. There is evidence that reactive oxygen species accumulate within MC2R Adrenal cortex () ACTH affected hair follicles and lead to oxidative damage of hair follicle mela­ nocytes. Both hair bulb melanocytes and precursor cells in the bulge MC3R† Brain (gut, placenta) -, -, –MSH, ACTH α β γ region can be affected42. In addition, melanocytes that disappear fail to MC4R† Brain α-, β-MSH, ACTH express TYRP2 and SOX10. The role of SCF production by KROX20+ MC5R Peripheral tissues, e.g. sebaceous α-, β-MSH, ACTH cells within the hair bulb was discussed previously. glands, fibroblasts, adipocytes *Antagonistic ligands – agouti protein (mouse) and agouti signaling protein (ASIP; human). Radiation (UVR) †Antagonistic ligand – agouti-related protein (AGRP). Following a single exposure to UVR, an increase in the size of mela­

Table 65.4 Major forms of the melanocortin receptor (MCR). ACTH, nocytes can be observed, along with an increase in tyrosinase activ­ adrenocorticotropic hormone; MSH, melanocyte stimulating hormone. ity15. Repeated exposures to UVR lead to an increase in the number

Fig. 65.14 Melanocortin 1 receptor (MC1R) within MELANOCORTIN-1 RECEPTOR (MC1R) WITHIN THE PLASMA MEMBRANE OF A MELANOCYTE the plasma membrane of a melanocyte. MC1R has seven transmembrane domains and is a G-coupled receptor (see Fig. 65.15). There are numerous genetic Major R alleles: variants of the MC1R. The null or hypomorphic R NH2 D84E alleles D84E, R151C, R160W, and D294H have red hair R151C odds ratios of 62, 118, 50, and 94, respectively; the R160W low penetrance r alleles of V60L, V92M, and R163Q D294H have red hair odds ratios of 6, 5, and 2, respectively52. In general, R/R individuals have red hair and .

Plasma membrane

COOH

Cytoplasm 1083 THE ACTIVATION OF A G-PROTEIN-COUPLED RECEPTOR INTERACTION OF MSH AND AGOUTI PROTEIN WITH THE SUCH AS THE MELANOCORTIN1 RECEPTOR (MC1R) MELANOCORTIN 1 RECEPTOR (MC1R)

SECTION A Effector hormone MC1R 10 Hormone receptor

ers α-MSH

Gs␤ Gs␣ isord Gs␥ D GDP Adenylate cyclase

Dysfunctional MC1R igmentary P

GDP

Agouti protein

GTP GDP B α-MSH β-Defensin Agouti Attractin

MC1R Extracellular

GTP

Adenylate cyclase Gsα GTP ATP cAMP * Intracellular *In melanocytes, ↑ tyrosinase activity Mahogunin and eumelanin production

Fig. 65.16 Interaction of melanocyte stimulating hormone (MSH) and agouti protein with the melanocortin 1 receptor (MC1R). A There is baseline activity of the MC1R, enhanced by binding with MSH. Agouti protein represents an antagonist ligand for the MC1R whose binding can lead to GDP pheomelanogenesis. Dysfunction of the MC1R can also lead to P pheomelanogenesis, as in the case of humans with red hair. B Interactions are actually more complex, in that to be fully effective, agouti requires attractin and mahogunin; the former aids in the binding of agouti to MC1R while the Fig. 65.15 Activation of a G-protein-coupled receptor such as the melanocortin 1 receptor (MC1R). In the case of the MC1R, the increase in the latter acts on the cytosolic side. There is also a neutral agonist, β-defensin, intracellular concentration of cAMP leads to an increase in tyrosinase activity which interferes with both agonist and antagonist binding. Adapted from Schiaffino and eumelanin production. GDP, guanosine diphosphate; GTP, guanosine MV. Signaling pathways in melanosome biogenesis and pathology. Int J Biochem Cell Biol. triphosphate; P, phosphate group; S, stimulatory. Adapted from Alberts B. Molecular 2010;42:1094–104. Biology of the Cell. Garland Publishing; 1989.

of stage IV melanosomes transferred to keratinocytes, as well as an increase in tyrosinase activity. In addition to an increase in melanocyte increase in the number of active melanocytes. When chronically size and number, tyrosinase activity, and transfer of melanosomes to sun-exposed sites (e.g. the upper outer arm) are compared with non- keratinocytes, the response to PUVA includes an alteration in size and sun-exposed sites (e.g. the upper inner arm), the density of mela­ aggregation pattern of melanosomes, i.e. from smaller and grouped to nocytes is up to two times greater in sun-exposed sites43. Melano­ larger and singly dispersed (see Table 65.3). cytes, like other neural-derived tissues, have a low mitotic rate, and UVR may work by increasing one or more of the following: whether this increase in number represents an increase in mitotic rate transcription of the tyrosinase gene (via MITF) or an activation of “inactive” melanocytes or melanocyte precursors is • the number or activity of MC1R on melanocytes not known. • the expression of POMC and its derivative peptides by Following exposure to UVA irradiation, an immediate pigmentary • keratinocytes and several cell types within the dermis (e.g. darkening can be observed, which occurs within minutes and fades over endothelial cells, sebocytes, lymphocytes) 20–30 minutes. It is clinically most obvious in darkly pigmented skin the release of diacylglycerol from the plasma membrane, which and is thought to represent oxidation of pre-existing melanin or melanin • activates protein kinase C precursors. Given its transient nature, it does not provide photoprotec­ • an activation of the nitrous oxide/cGMP pathway 1084 tion. Delayed tanning is visible within 24–72 hours of exposure to UVB the production of and growth factors by keratinocytes and UVA radiation and represents new pigment production via an • (e.g. endothelin-1) Fig. 65.17 Formation of agouti hairs – underlying FORMATION OF AGOUTI HAIRS – UNDERLYING PHYSIOLOGY physiology. A Fox hairs with alternating eumelanin and pheomelanin production within individual hairs, a pattern referred to as agouti. B Explanation for the A B agouti pattern based on different ligands interacting CHAPTER with the melanocortin 1 receptor (MC1R). Both gain-of-function mutations at the agouti locus and 65 loss-of-function of MC1R can lead to yellow-haired mice. In the CNS, an abundance of agouti protein leads to obesity, and yellow mice with gain-of- function mutations at the agouti locus are also obese53. Melanocyte Biology

Agouti MSH MC1R protein

Fig. 65.18 Mechanisms of UVR-induced MECHANISMS OF UVR-INDUCED PIGMENTATION pigmentation. These include an increase in one or more of the following: (1) expression of proopiomelanocortin (POMC) and its derivative peptides by cells within the skin, in particular keratinocytes; (2) the number of melanocortin 1 receptors (MC1R) on melanocytes; (3) the release of diacylglycerol (DAG) from the plasma membrane, which activates protein kinase C; (4) the induction of an SOS response to UVR-induced DNA damage; (5) MC1R α-MSH POMC nitric oxide (NO) production, which activates the cGMP pathway; and (6) production of cytokines and growth factors by keratinocytes. As a result, there is enhanced transcription of the genes that encode Cytokines and microphthalmia-associated transcription factor (MITF) growth factors: and melanogenic proteins including tyrosinase, IL-1 tyrosinase-related protein 1 (TYRP1), TYRP2, and Endothelin-1 PMEL/PMEL17/gp100. In addition, melanocyte cAMP bFGF dendricity and transfer of melanosomes to Protein KIT ligand/SCF keratinocytes is stimulated via increased activity of kinase A Rac1 (involved in dendrite formation), the ratio of DAG kinesin to dynein, and expression of protease- + Keratinocyte activated receptor-2 (PAR-2; involved in melanosome transfer). TPA, tetradecanoyl phorbol acetate. p53 Ca2+ + MITF Protein kinase C + NO Genes for melanogenic + TPA proteins cGMP

Protein kinase G Keratinocyte Melanocyte

the induction of an SOS response to UVR-induced DNA with the production of oxygen radicals following the UV irradiation of • damage34,44,45 pheomelanins, probably contributes to the increased incidence of both transactivation of the POMC promoter by p5346,47 cutaneous melanoma and keratinocyte carcinomas in persons with red • the ratio of kinesin to dynein, affecting melanosome transport (see hair. In addition, chemiexcitation of melanin derivatives induces DNA • Fig. 65.10). photoproducts long after UV exposure48. The inability of the majority of red-haired individuals to develop a tan following exposure to UVR can be explained, at least in part, For table on genes associated with physiologic variation in human 1085 by dysfunction of their melanocyte MC1R. This phenomenon, along pigmentation and references visit www.expertconsult.com Online only content

GENES ASSOCIATED WITH PHYSIOLOGIC VARIATION IN HUMAN PIGMENTATION

Selected variant(s): Skin color, sun Population frequencies of amino acid alteration sensitivity, and Skin cancer variant haplotype CHAPTER Gene Protein (if applicable) or SNP Hair color risk* European East Asian African 65 TYR Tyrosinase Ser192Tyr Freckles – – SCC 0.4 0 0 Arg402Gln Fair, sun- Light (weak) Blue (vs green) Melanoma, 0.4 0 0.07 sensitive BCC TYRP1 TYRP1 rs1408799 C>T Sun-sensitive Light Blue Melanoma 0.7 0.02 0.2 rs2733832 C>T– Light Blue – 0.6 0.02 0.07 OCA2 (P) P protein Arg305Trp – – Brown – 0.07 0.04 0.02 Melanocyte Biology Arg419Gln – – Green/hazel Melanoma, 0.07 – 0 BCC His615Arg – – Brown – 0 0.5 0 rs12913832 T>C in Fair Light Blue Melanoma 0.75 – – HERC2† (weak) SLC45A2 MATP Glu272Lys Dark Dark Non-blue – 0 0.4 0.05 Leu374Phe Fair Light Blue Melanoma, 0.95 0.01 0 NMSC SLC24A5 NCKX5 Ala111Thr Light – – – 0.99 0.01 0.02 SLC24A4 SLC24A4 rs12896399 G>T Sun-sensitive Light Blue (vs green) – 0.6 0.5 0.01 MC1R MC1R R alleles‡ Fair, sun- Red/light – Melanoma, 0.3 0 0 sensitive, freckles NMSC r alleles§ Variable Variable – Melanoma, 0.3 0.4 0.01 NMSC ASIP ASIP g.8818 A>G Dark Dark Brown – 0.15 – 0.6 ASIP haplotype¶ Fair, sun- Red/light Light Melanoma, 0.1–0.3 0–0.2 0–0.1 sensitive, freckles NMSC KITLG KIT ligand rs642742 A>G Fair – – – 0.85 0.8 0.07 rs12821256 T>C– Light – – 0.15 0 0 TPCN2 TPCN2 Met484Leu – Light – – 0.2 0 0 Gly734Gln – Light – – 0.4 0.2 0.02 IRF4 Interferon rs12203592 C>T Fair, sun- Dark Blue – 0.8 0 0 regulatory sensitive factor 4 rs1540771 G>A Sun-sensitive, Dark – – 0.5 0.2 0.05 freckles (weak) *Independent of pigmentary phenotype. †Intronic regulatory region that determines expression of the OCA2 gene; other intronic polymorphisms in the HERC2 gene have also been linked to decreased pigmentation, with the same proposed mechanism. ‡High-penetrance “red hair color” alleles, e.g. Asp84Glu, Arg151Cys, Arg160Trp, and Asp294His. §Low-penetrance alleles, e.g. Val60Leu, Val92Met, and (especially in East Asians) Arg163Gln. ¶Single long (~1.8 Mb) haplotype that includes multiple single nucleotide polymorphisms (SNPs; e.g. rs4911414 G>T and rs4911442 A>G) and encompasses several smaller haplotype blocks.

e1–e6 eTable 65.1 Genes associated with physiologic variation in human pigmentation . Gray highlight = polymorphism associated with darker pigmentation. The genes in the first four rows are those responsible for OCA types 1 to 4. ASIP, agouti signaling protein; BCC, basal cell carcinoma; MATP, membrane-associated transporter protein; MC1R, melanocortin 1 receptor; NCKX5, Na-Ca-K exchanger 5; NMSC, non-melanoma skin cancer; SCC, squamous cell carcinoma; SLC24A4/5, solute carrier family 24, member 4/5; SLC45A2, solute carrier family 45, member 2; TPCN2, two-pore segment channel 2; TYRP1, tyrosinase-related protein 1. From Schaffer JV, Bolognia JL. The biology of the melanocyte. In: Rigel DS, et al (eds). Cancer of the Skin, 2nd edn. St Louis, MI: Elsevier, 2011:23–39.

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