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S E C T I O N

11

DISORDERS OF MELANOCYTES

C H A P T E R 7 0

Biology of Melanocytes

Hee-Young ParkMarinya PongpudpunthJin LeeMina Yaar

EMBRYONIC DEVELOPMENT

Melanocytes are pigment-producingcells that originate from the dorsal por-tions of the closing neural tube in ver-tebrate embryos (Fig. 70-1).

1

They de-rive from pluripotent neural crest cellsthat differentiate into numerous celllineages, including neurons, glia, smoothmuscle, craniofacial bone, cartilage,and melanocytes (reviewed in refs. 2and 3). Progenitor melanoblasts mi-grate dorsolaterally between the meso-dermal and ectodermal layers to reachtheir final destinations in the hair folli-cles and the skin as well as inner earcochlea, choroids, ciliary body, and iris(reviewed in refs. 2 and 4). Pigment-producing cells can be found in fetalepidermis as early as the fiftieth day ofgestation.

Melanoblast migration and differenti-ation into melanocytes is influenced bya number of signaling molecules pro-duced by neighboring cells. These in-clude Wnt, endothelin-3 (ET3), bonemorphogenetic proteins (BMPs), steelfactor [SF; stem cell factor (SCF), c-Kit li-gand], and hepatocyte growth factor(HGF)/scatter factor.

5–10

By interactingwith their specific cell surface receptors,these molecules induce intracellular andintranuclear signaling to influence genetranscription and protein synthesis.

Wnt

Wnt is expressed in the dorsal neuraltube during neural crest cell migrationand directs the maturation of pluripo-tent neural crest cells into melanoblasts.

The Wnt family is composed of 16different secreted glycoproteins. Theybind and activate Frizzled, a transmem-brane heptahelical G protein–linked re-ceptor,

11

and induce the accumulationof

β

-catenin (Fig. 70-2A). Under baselineconditions, when Wnt does not bindFrizzled, cytosolic

β

-catenin is com-plexed with the enzyme glycogen syn-thase kinase 3

β

(GSK3

β

) that inducesrapid ubiquitin-mediated degradation of

β

-catenin by cellular proteosomes. Bind-ing of Wnt to its receptor Frizzled inhib-its GSK3

β

activity, leading to

β

-cateninaccumulation in the cytosol followed byits translocation to the nucleus (Fig. 70-2B).In the nucleus,

β

-catenin binds specifictranscription factors, and together thecomplex induces the transcription of mi-crophthalmia-associated transcription fac-tor (Mitf).

10

Mitf affects melanoblast dif-ferentiation by inducing the transcriptionof three enzymes that regulate melaninsynthesis: tyrosinase, tyrosinase-relatedprotein-1 (TRP-1), and 3,4 dihydroxyphe-nylalanine (DOPA)chrome tautomerase(TRP-2) (reviewed in ref. 12).

Bone Morphogenetic Proteins

BMPs are disulfide-linked dimeric pro-teins produced as large precursors.

13

They include more than 20 secretedproteins all sharing amino acid homol-ogy (reviewed in ref. 14). BMPs belongto the transforming growth factor-

β

family of secreted growth factors, andtheir signaling suppresses neural crestcell differentiation into melanoblastsand thus may be viewed as Wnt an-tagonists. Accordingly, there is a de-crease in BMP expression in the dorsal

neural tube at the time of melanoblastmigration.

8

Endothelins

ETs are a family of peptides, 21 aminoacids long, originally identified by their

BIOLOGY OF MELANOCYTES

AT A GLANCE

Melanoblasts

■

derive from the neural crest. Their migra-tion/survival in the epidermis is influ-enced by numerous factors.

Melanocytes

■

populate the epidermis, hair follicle, eye, cochlea and meninges.

■

synthesize melanin, an indole derivative of 3,4-dihydroxyphenylalanine that is stored in melanosomes.

■

are influenced by endocrine, paracrine and autocrine factors and by ultraviolet irradiation.

Melanosomes

■

display four maturation stages.

■

contain structural matrix proteins, mela-nogenic enzymes, pH maintaining pro-teins and free radical scavengers.

■

are transported to melanocyte dendrite tips and transferred to keratinocytes.

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592

vasoactive properties, that include threemembers, ET1, ET2, and ET3 (reviewedin ref. 15). They are produced via pro-teolysis of larger precursor molecules.Like Wnt, ETs bind and activate hepta-helical transmembrane G protein–linkedreceptors EdnrA and B. ET3, which issynthesized by ectodermal cells, and its

EdnrB receptor are particularly importantduring melanoblast migration along thedorso-lateral pathway (reviewed in ref.15), and their proper expression is re-quired for survival, proliferation, and/ormigration of melanoblasts.

Defects in either ET3 or its EdnrB re-ceptor result in prominent melanocyteloss.

16

It appears that the receptor playsa more critical role in melanocyte devel-opment as compared to its ligand, asET3 null mice do not have as severe de-pigmented phenotype as EdnrB nullmice. Because ETs and Ednrs also affectthe development of neural crest cellsother than melanoblasts, defects in ET3or EdnrB lead to disease symptomatol-ogy beyond pigmentary disorders, suchas that present in type IV Waardenburgsyndrome and in Hirschsprung syn-drome

16

(see also Chap. 71).

Steel Factor

Early melanoblast development requiresthe presence of the cytokine SF (mast/SCF, c-Kit ligand) and its tyrosine kinasetransmembrane receptor c-Kit.

17

SF isexpressed by epidermal keratinocytes,and, as soon as c-Kit is expressed onmelanoblasts, they begin their migra-tion to their final destination (reviewedin ref. 18).

Piebaldism is an autosomal dominantdisorder that results from mutations ofc-Kit or SF and leads to melanoblast fail-ure to migrate to the skin and/or survive

there

19,20

(see Chap. 71). Affected indi-viduals display broad depigmentedpatches, most prominent on the centralforehead and trunk. Interestingly, theventral aspect of the body is more fre-quently affected than the dorsal aspect,supposedly because it is the area far-thest from the dorsally located neuralcrest where melanoblast migration be-gins.

19,21

Complete absence of melano-cytes as well as abnormalities of the re-productive and hematopoietic systems,whose development also depends onSF/Kit receptor, have been observed inmice with homozygous loss of SF orc-Kit.

22,23

Hepatocyte Growth Factor

HGF (scatter factor) is the ligand for thetransmembrane tyrosine kinase receptorMet. In vitro studies performed on mu-rine neural crest cells show that HGF in-duces melanoblast proliferation and al-lows their differentiation into maturemelanocytes.

24

In addition, HGF regu-lates cadherin expression in melano-cytes, specifically downregulating E-cadherin, thus affecting melanoblasthoming (see Cadherins). Interestingly,in mice,

Met

null mutations do not ap-pear to affect melanoblast number ormigration, and no decrements in Metfunction have been identified to date inhuman pigmentary disorders. Thus, therole of HGF/Met signaling during mel-anocyte development is still unclear.

�

FIGURE 70-1

Migration of melanoblast pre-cursors. Migrating melanoblasts in an E11.5transgenic mouse embryo expressing the Lac Zreporter gene under the control of the melano-cyte-specific Dct (DOPAchrome tautomerase)promoter. The melanoblasts are visualized bystaining for

β

-galactosidase. (Used with permis-sion from Ian Jackson and Alison Wilkie, MedicalResearch Council, Human Genetics Unit, WesternGeneral Hospital, Edinburgh, Scotland.)

�

FIGURE 70-2

Regulation of microphthalmia-associated transcription factor (Mitf) transcription by Wnt signaling.

A.

When Wnt is not bound to Frizzled, amembrane-spanning domain receptor, Frizzled is inactive. Under these inactive conditions, glycogen synthase kinase 3

β

(GSK3

β

) is complexed with

β

-catenin,enhancing its ubiquitin-mediated degradation by cellular proteosomes.

B.

Wnt binding activates Frizzled, leading to GSK3

β

dissociation from

β

-catenin.

β

-Catenin then translocates to the nucleus, where it binds with specific transcription factors (Tr factor) to initiate Mitf transcription.

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593

Cadherins

Cadherins are a family of transmembraneglycoproteins (E-, P-, and N-) that pro-mote calcium-dependent cell-to-cell ad-hesion (reviewed in ref. 25). In melano-blasts, their cytoplasmic domains bind

β

-catenin. Murine studies show that E-cadherin is induced in melanoblasts be-fore their entry into the epidermis. Later,when melanoblasts migrate into the hairfollicle, E-cadherin expression is muted,and melanoblasts begin to express P-cadherin.

26,27

It is thought that coordinateexpression of E-cadherins by epidermalkeratinocytes and melanocytes plays arole in suppressing melanocyte prolifera-tion in the epidermis. Interestingly, attimes, some epidermal melanocytesswitch from E-cadherin to N-cadherin,and this switch allows them to escape thekeratinocyte-mediated growth suppres-sion and proliferate/aggregate in nests toform nevocellular nevi.

28

Generally, cad-herin expression by melanocytes matchescadherin expression of surrounding cells.

SITE-SPECIFIC MELANOCYTES

Melanocyte Stem Cells

Generally, stem cells are defined bytheir undifferentiated state and their ca-pacity to develop into several differenti-ated cell types. They are quiescent,slow-cycling cells that frequently arefound in niches where they are sur-rounded by differentiated cells that af-fect their behavior through the secretionof cytokines and growth factors (re-viewed in refs. 29 and 30).

Melanocyte stem cells reside in thehair follicle bulge (Fig. 70-3). They ex-press TRP-2 as well as the neural creststem cell intermediate filament nestin inaddition to other neural crest stem cellmarkers, including the transcription fac-tors Sox10 and Pax5 that participate inthe regulation of Mitf and TRP-2. Mel-anocyte stem cells can leave the bulgeregion and migrate/differentiate in theepidermis or the hair follicle.

Cutaneous Melanocytes

The largest number of melanocytes ispresent in the skin and hair follicles. Inmost furred mammals, melanocytes arefound only in the hair follicle, but in hu-mans melanocytes are also present in in-terfollicular epidermis, specifically in thebasal layer (reviewed in ref. 28). There isapproximately one melanocyte per fiveor six basal keratinocytes. Melanocytes

synthesize melanin, a pigmented poly-mer that is stored in cytosolic organellescalled

melanosomes

that are transferred tokeratinocytes through melanocyte den-dritic processes (Fig. 70-4). As keratino-cytes are continuously being desqua-mated, there is a constant need forsynthesis and transfer of melanosomes

from melanocytes to keratinocytes tomaintain cutaneous pigmentation.

The term “epidermal melanin unit”describes a single epidermal melanocytesurrounded by several epidermal ke-ratinocytes

31

(Fig. 70-5). Interestingly,signals from keratinocytes substantiallyregulate epidermal melanocyte survival,dendricity, melanogenesis, and the ex-pression of cell surface receptors

32

(seeSignaling Pathways Regulating Mel-anocyte Function). Most keratinocyte-derived signals are induced by ultravio-let (UV) irradiation.

Melanocyte density/mm

2

ranges fromapproximately 550 to > 1200, with thehighest concentrations found in the gen-italia and face.

33,34

Melanocyte den-sity is the same in individuals of differ-ent ethnic backgrounds,

34

and thuscutaneous pigmentation does not de-pend on melanocyte number, butrather on melanogenic activity withinthe melanocyte, the proportion of ma-ture melanosomes, and/or their trans-fer and distribution within the kerati-nocytes (reviewed in ref. 35). Indeed,in light-skinned individuals, melano-somes are smaller and are present inclusters within the keratinocytes,whereas in ethnic groups with darkercomplexion, melanosomes are larger,

�

FIGURE 70-4

Melanosomes are organizedinto supranuclear “caps” within keratinocytes.Note melanized dendritic melanocytes and adja-cent keratinocytes with the supranuclear “caps.”Melanin silver stained (Fontana-Masson) sectionof a heavily melanized human epidermis. (Bar =50

µ

M.) (From Byers HR et al: Role of cytoplasmicdynein in perinuclear aggregation of phagocy-tosed melanosomes and supranuclear melanincap formation in human keratinocytes.

J InvestDermatol

121

:813, 2003; with permission.)

�

FIGURE 70-3

Melanocyte stem cells in the hair follicle bulge. A stem cell melanocyte is shown in thehair follicle bulge, indicated by an

arrow

in the high-power insert. These cells stain positive for TRP-2 (

greenfluorescence

), an early marker of commitment to the melanocyte lineage, but are negative for the prolifera-tion marker Ki-67 (

red fluorescence

) that characterizes melanocytes migrating down the follicle to the der-mal papilla during the anagen phase of the hair cycle. (From Botchkareva NV et al: SCF/c-kit signaling is re-quired for cyclic regeneration of the hair pigmentation unit.

FASEB J

15

:645, 2001, with permission.)

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■

DISORDERS OF MELANOCYTES

594

darker, and are individually dispersedwithin the keratinocytes.

36

Hair Follicle Melanocytes

In contrast to interfollicular epidermalmelanocytes, the follicular melanin unitundergoes cyclic modifications in coor-dination with the hair cycle (Fig. 70-6).Melanocytes are located in the proximalhair bulb during anagen, and there is aratio of 1:5 between melanocytes andkeratinocytes and 1:1 between melano-cytes and basal layer keratinocytes.

37

Melanocytes proliferate, migrate, andundergo maturation during early to midanagen. Melanogenesis and melanintransfer to keratinocytes occur through-

out anagen. Melanocytes eventually ap-optose during late catagen. In mice, mel-anocyte proliferation and differentiationduring anagen depends on c-Kit expres-sion by melanocytes and SF synthesisby keratinocytes.

38

Similar to their role in the epidermis,in hair, melanocytes transfer melanin todifferentiated keratinocytes that ulti-mately form the hair shaft. They thusdetermine hair color by the amount ofmelanin transferred, as well as by theratio of eumelanin (black-brown) topheomelanin (red-yellow) (see MelaninBiosynthesis) (reviewed in ref. 39).

In hair, melanin does not appear tohave a protective effect, as UV irradia-tion does not reach the hair follicle. Still,

in furry animals hair color plays an im-portant role in camouflage, mimicry,and social communication.

40

It is alsospeculated that melanocytes restrain ke-ratinocyte proliferation, affect calciumhomeostasis, and protect against reac-tive oxygen species (ROS) during therapid proliferation and differentiation ofthe hair follicle.

37

Ocular Melanocytes

Ocular melanocytes are found in theuveal tract (reviewed in ref. 28). Unlikecutaneous melanocytes, ocular melano-cytes are in contact only with eachother, and they do not transfer theirmelanosomes (Fig. 70-7). It is proposed

�

FIGURE 70-5

The epidermal melanin unit.

A.

Representa-tion showing the relationship between basal melanocytes, ke-ratinocytes, and Langerhans cells, shown at the upper layer ofthe epidermis. (From Quevedo WC Jr. The control of color inmammals.

Am Zoology

9

:531, 1969, with permission.)

B.

Elec-tron micrograph of the epidermal-dermal junction of humanskin showing a dendritic melanocyte (M) among the basal ke-ratinocytes (K). k

′

represents a basal keratinocyte undergoingmitosis with condensed chromatin (

arrows

). (Bar = 10.0

µ

m.)(Illustration used with permission from Raymond E. Boissy, De-partment of Dermatology, University of Cincinnati, Cincinnati,Ohio.)

C.

Human epidermis immunostained for fibroblastgrowth factor-2 (FGF2). The figure shows basal keratinocyteswith peroxidase reaction indicating the presence of immunore-active FGF2.

Arrows

point to melanocytes. (Used with permis-sion from Glynis Scott, M.D., Department of Dermatology, Uni-versity of Rochester School of Medicine and Dentistry,Rochester, NY.)

D and E.

Distribution of melanosomes withinkeratinocytes in lightly pigmented Caucasian and darkly pig-mented African American skin. Melanosomes in lightly pig-mented Caucasian skin

(D)

are distributed in membrane-bound clusters. In contrast, in darkly pigmented African Amer-ican skin

(E)

the melanosomes are individually distributedthroughout the cytoplasm of epidermal keratinocytes. Melano-somes in both skin types are frequently concentrated over theapical pole of the nucleus (

arrows

). L = Langerhans cell. (Bar= 3.0

µ

m.) (From Minwalla L et al: Keratinocytes play a role inregulating distribution patterns of recipient melanosomes invitro.

J Invest Dermatol

117

:341, 2001.)

A B

C

D E

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that ocular melanocytes are exposed tohigh oxygen tension and melanin servesas protection against oxidative damage.Iris melanin may be required to protectcapillaries, muscles, and motor nervesthat control pupil contraction.

28

Melanocytes appear to be essential forthe development and function of the eyeand optic nerve (reviewed in ref. 41). It isthought that they may play a role in theproper routing of ipsilateral and contra-lateral neural fibers in the optic chiasmduring early development of the opticcup,

42,43

as in the absence of melanin, the

path of the highly intermixed optic nerveaxons is deranged, resulting in disruptionof the topographic relationship of thenerve fibers.

44,45

The importance of mel-anin for proper ocular function is demon-strated by the visual abnormalities ob-served in patients with albinism.

44

Otic Melanocytes

Melanocytes reside in the cochlea

28

andare important for hearing, as loss of oticmelanocytes leads to deafness. It isthought that otic melanocytes help in

the maintenance of the endolymphthrough the regulation of potassiumtransport.

46

The endolymphatic fluid inthe cochlea is one of the few extracellu-lar fluids with high concentrations ofpositively charged potassium, and mel-anocytes are believed to play a criticalrole in its maintenance, probably bytransporting potassium from areas ofhigh concentration to areas of low con-centration through plasma membraneionic channels.

47

In the absence of coch-lear melanocytes, the endolymphaticpotential is low, leading to deafness.

48

Loss of melanocytes at any age caneventually lead to deafness, and patientswith Waardenburg syndrome type 2 (seeChap. 71) are deaf because of lack ofmelanocytes in the inner ear.

49,50

Inter-estingly, the preservation of hearing in al-binos indicates that melanin productionwithin otherwise viable melanocytes isnot essential for hearing. However, it ap-pears that because albinos are more sus-ceptible than the normal population tohearing loss from noise and/or exposureto toxic agents,

51,52

melanin must pro-vide some protective effect.

Cephalic Melanocytes

Melanocytes are dispersed throughoutthe meninges and are particularly dense

�

FIGURE 70-7

Ocular tissue demonstratingthe choroid (C), the retinal pigment epithelium(R), and the photoreceptor cells of the retina (P).The connective tissue of the choroid contains nu-merous fibroblasts (F) and dendritic melanocytes(M) with cytoplasm filled with mature melano-somes. The retinal pigment epithelium is a singlelayer of polarized cuboidal melanocytes contain-ing relatively elongated mature melanosomesboth within the cell body of the retinal pigmentepithelium cells and in the numerous slender api-cal processes (

arrows

) that interdigitate betweenthe rod and cone photoreceptor cells. (Bar =10.0

µ

m.) (Illustration used with permission fromRaymond E. Boissy, Department of Dermatology,University of Cincinnati, Cincinnati, Ohio.)

�

FIGURE 70-6

Melanocytes in the hair.

A.

Pigmented human scalp hair follicle in full anagen withhigh levels of hair bulb melanogenesis. Mature melanin granules are transferred into cortical keratino-cyte. B. Scalp hair bulb. Representation of early catagen hair follicle showing loss of some bulbar melan-otic melanocytes via apoptosis. Arrows point at melanocytes located in epidermal, infundibular, and outerroot sheath regions. C. Transmission electron micrograph of section of an early catagen hair bulb show-ing apoptosis of melanotic melanocytes. Inset, high-power view of premelanosomes. D. Primary cultureof human scalp hair follicle melanocytes. Mature, fully differentiated (Diff; large arrow) and less differen-tiated (small arrows) are indicated. DP = dermal papilla. (From Tobin DJ, Paus R: Graying: Gerontobiologyof the hair follicle pigmentary unit. Exp Gerontol 36:29, 2001, with permission.)

B

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596

in the leptomeninges above the ponsand the medulla oblongata.53 They arethought to function as scavengers fortoxic cations and ROS.54,55

MELANIZATIONThe major differentiated function ofmelanocytes is to synthesize melanin inspecialized organelles within the mel-anocytes, the melanosomes, and totransfer melanosomes to neighboringkeratinocytes to provide protection fromUV irradiation (Fig. 70-8). Pigmentation,the synthesis and distribution of mela-nin in the epidermis, involves severalsteps: transcription of proteins requiredfor melanogenesis, melanosome biogen-esis, sorting of melanogenic proteinsinto the melanosomes, transport of mel-anosomes to the tips of melanocyte den-drites, and transfer of melanosomes tokeratinocytes. Disruption in any of theseevents results in hypopigmentation.

MELANOSOMES

Melanosome Biogenesis

The melanosome is a unique mem-brane-bound organelle in which mela-nin biosynthesis takes place. Becausemelanosomes contain enzymes andother proteins also present in lyso-somes, they are thought to represent amodified version of the latter. Proteinscommon to both organelles include theliposomal-associated membrane pro-teins (LAMPs) that participate in au-tophagy and regulation of intra-vesicu-

lar pH (reviewed in ref. 56), as well asacid phosphatase, a marker enzyme forlysosomes.56 Also like lysosomes, mel-anosomes can endocytose receptorsthat are targeted for degradation.56

Depending on the type of melaninsynthesized, melanosomes can be di-vided into eumelanosomes and pheo-melanosomes (Fig. 70-9). Eumelano-somes are large (~0.9 × 0.3 µm), ellipticalin shape, and contain a highly structuredfibrillar glycoprotein matrix requiredfor eumelanin synthesis.40 Pheomelano-somes are smaller (~0.7 µm in diameter),spherical in shape, and their glycoproteinmatrix appears disorganized and loose.40

Although both eumelanosomes and pheo-melanosomes may be present within asingle melanocyte,57 once committed,they do not change.58

Melanosomes display four matura-tion stages (see Fig. 70-9). Stage I mel-anosomes or premelanosomes likely de-velop from the endoplasmic reticulum(ER).40 They have an amorphous matrixand display internal vesicles that formas a result of membrane invagination.Premelanosomes already contain theglycoprotein Pmel17 (gp100), but it re-quires further processing to become acomponent of the final fibrillar matrix.59

Stage II eumelanosomes have organizedstructured fibrillar matrix, but no active

melanin synthesis, whereas in stage IIpheomelanosomes, melanin synthesisalready takes place. Although no activemelanogenesis takes place in stage II eu-melanosomes, they already contain theenzyme tyrosinase. Deposition of mela-nin on the fibrillar matrix is found instage III eumelanosomes, whereas stageIV eumelanosomes are fully melanized,and their internal matrix is masked bymelanin deposits (reviewed in refs. 60and 61).

Melanogenic Proteins

The timely and organized sorting ofmelanogenic enzymes and structuralproteins to melanosomes is an integralpart of melanosomal maturation. Mel-anosome proteins express sorting sig-nals at their amino terminus, and thesedirect them into the ER and eventuallyinto the melanosomes (reviewed in refs.40, 60, and 61).

ENZYMES Tyrosinase. Tyrosinase is pres-ent in plants, insects, amphibians, andmammals. It was initially identified inthe early 1900s in mushroom extractsand was subsequently isolated and puri-fied in 1949 from murine melanomacells.62 Mouse and human tyrosinasegenes are 60 to 70 kb, and 50 kb long,

� FIGURE 70-8 Melanocytes cultured on ke-ratinocytes. Light micrograph showing dendriticmelanocytes from a Black donor loaded withmelanin and adjacent pigmented keratinocytesdue to transfer of melanosomes. (From HalabanR et al: bFGF as an autocrine growth factor forhuman melanomas. Oncogene Res 3:177, 1988,with permission.)

� FIGURE 70-9 Melanosome biogenesis. Electron microscopy of eumelanosome (a–f) and ofpheomelanosome (g–j) development. I, II, III, and IV in a–j represent the different maturation stages ofmelanosomes. [Scale bars are as follows (in µm): a = 0.20; b = 0.23; c = 0.24; d = 0.22; e = 0.20; f =0.35; h = 0.26; i = 0.26; j = 0.30; k = 0.5.] (From Slominski A et al: Melanin pigmentation in mamma-lian skin and its hormonal regulation. Physiol Rev 84:1155, 2004, with permission from the AmericanPhysiological Society.)

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597

respectively. The murine tyrosinase genemaps to chromosome 7, whereas hu-man tyrosinase gene maps to chromo-some 11. The human tyrosinase gene iscomposed of 5 exons and 4 introns,63

and tyrosinase messenger RNA is ap-proximately 2-kb long (gene bank accessnumber NM_000372).

Tyrosinase is synthesized in the ER asa precursor protein whose nascent chainis processed in the Golgi complexwhere sialic acid and neutral sugars areadded to the peptide via N- and O-glycosidic linkages through a processcalled glycosylation (reviewed in ref. 64)(Fig. 70-10). At least four forms of tyro-sinase, all differing with regards to theirdegree of glycosylation, have been iden-tified. The glycosylation steps havebeen shown to be important for properassociation of tyrosinase with melano-somes, as well as for its activity.64 Afterthe glycosylation steps, mature tyrosi-nase is folded in the ER, a step requiredfor appropriate trafficking/sorting of ty-rosinase into the Golgi apparatus andultimately into endosomes and finallyinto melanosomes. A strict controlmechanism guarantees the eliminationof defective tyrosinase.

Within the melanosome, tyrosinasespans the melanosomal outer mem-brane (Fig. 70-11). It has three domains:an inner melanosomal domain, a mel-anosomal transmembrane domain, anda cytoplasmic domain. The inner do-main that contains the catalytic region is

approximately 90 percent of the protein.It is followed by a short transmembranedomain and a cytoplasmic domain com-posed of approximately 30 amino acids(reviewed in ref. 65). Histidine residuespresent in the inner (catalytic) portion oftyrosinase bind copper ions, and the lat-ter are required for tyrosinase activity.

The biologic function of the tyrosinasecytoplasmic domain was not known fora long time. In a mouse model in which

the entire cytoplasmic domain is miss-ing, tyrosinase protein is inserted into thecellular plasma membrane instead of intothe melanosomal membrane, suggestingthat tyrosinase cytoplasmic domain is re-quired for proper trafficking of tyrosinaseinto melanosomes. Indeed, it was foundthat the motif EXXQPLL (glutamic acid-X-X-glutamine-proline-leucine-leucine,where “X” stands for any amino acid) inthe cytoplasmic domain is responsible

� FIGURE 70-10 Sorting of melanosomal proteins into melanosomes. Tyrosinase and tyrosinase-related protein-1 (TRP-1) are initially synthesized in the en-doplasmic reticulum (ER) and, after additional maturation steps (*) in the Golgi and Trans-Golgi network, are packaged in endosomes as a complex. TRP-2 fol-lows similar maturation steps. Melanosomes originate in the ER as stage I already containing the melanosomal proteins PMEL17 and MART-1. They then ma-ture to stage II melanosomes and fuse with tyrosinase/TRP-1 in a process directed by the adaptor protein 3 (AP-3). Melanosomes become progressively darkeras melanin biosynthesis takes place.

ER

Stage II

Stage IV

Stage III

Stage I

Golgi

Trans-Golgi network

TyrosinaseTRP-1

Tyrosinase/TRP-1

Endosome

AP-3

Tyrosinase/TRP-1

PMEL17/MART-1

TRP-2

Plasma membrane

TRP-2

******

****

**

� FIGURE 70-11 Schematic diagram of tyrosinase protein structure. Tyrosinase protein can be dividedinto three domains: the inner melanosomal domain, the transmembrane domain, and the cytoplasmicdomain. The inner domain contains the catalytic activity of the enzyme and the copper (Cu) binding sites.The cytoplasmic domain contains the sequence (EXXQPLL) that participates in cellular trafficking of tyro-sinase into melanosomes. The cytoplasmic domain also participates in regulating tyrosinase activitythrough phosphorylation of serine residues (Ser).

Cytoplasmicdomain

Melanosometransmembrane

domain

Innerdomain

Melanosome membrane

COOH

H2N

Cu

Cu

SerSer

EXXQ

PL L

SECTION 11■

DISORDERS OF MELANOCYTES

598

for tyrosinase trafficking into the mel-anosomes.66 In addition, protein kinaseC-β (PKC-β) (see Protein Kinase C-β)must phosphorylate two serine residueson this cytoplasmic domain to activatetyrosinase,65 and in the absence of thosephosphorylation events pigmentationdoes not occur.

Tyrosinase mutations, including mis-sense, nonsense frameshift, and deletionmutations that lead to inactivation ofthe enzyme, are found in oculocutane-ous albinism type I (see Chap. 71 andAlbinism database: http://albinismdb.med.umn.edu/). Such mutations may affecttyrosinase glycosylation, interfering withenzyme maturation, or may involvecopper binding sites, disrupting tyrosi-nase activity (reviewed in ref. 64).

Tyrosinase-Related Proteins. Two TRPs,TRP-1 and TRP-2, play important rolesin melanogenesis.67–69 They are struc-turally related to tyrosinase and share~40 percent amino acid homology. Also,similar to tyrosinase, TRP-1 and TRP-2are glycoproteins located within themelanosomes and span the melanoso-mal membrane.70 The conserved nu-cleotide and amino acid sequencesamong these three melanogenic en-zymes suggest that they originated froma common ancestral gene.71,72

TRP-1. In mice, TRP-1 maps to thebrown locus on chromosome 4 andspans ~18 kb on the genomic DNA.69,72

The human homolog of TRP-1 maps tochromosome 9 and spreads over 24 kbof the genomic DNA.72 Like tyrosinase,TRP-1 is synthesized in the ER and un-dergoes several glycosylation steps (seeFig. 70-10). The exact function of TRP-1in melanin biosynthesis, especially inhuman melanogenesis, is not wellunderstood. In mice, TRP-1 displaysa wide range of enzymatic activities,including DOPAchrome tautomerase,73

tyrosine hydroxylase,74 DOPA oxi-dase,69,75 catalase,76 and 5,6-dihydroxy-indole-2-carboxylic acid (DHICA) oxi-dase67,77 (see Melanin Biosynthesis).Conversely, in humans TRP-1 does notdisplay DHICA oxidase activity, and theadditional activities described abovehave not been reported to date for thisenzyme.

Nevertheless, the presence of TRP-1appears to be required for melanin syn-thesis in human melanocytes, as TRP-1absence results in the pigmentary disor-der oculocutaneous albinism type 378 (seeChap. 71). Because recently TRP-1 wasshown to influence tyrosinase activity by

forming a complex with tyrosinase, it ispossible that TRP-1 plays a role in tyrosi-nase activation and/or stabilization.79,80 Aknown TRP-1 function in both murineand human melanocytes at least in vitrois to increase the ratio of eumelanin topheomelanin.81,82 TRP-1 may also play arole in melanosomal biogenesis, as sup-pression of TRP-1 is associated withstructurally abnormal melanosomes.61

TRP-2. In mice, TRP-2, also known asDOPAchrome tautomerase, maps to theSlaty locus on chromosome 14, whereashuman TRP-2 maps to chromosome13.68 Mouse and human TRP-2 share 84percent nucleotide homology.83 Like ty-rosinase and TRP-1, TRP-2 is aglycoprotein84 that is synthesized in theER and undergoes several maturationsteps in the Golgi and trans-Golgi net-work (see Fig. 70-10). Like tyrosinase andTRP-1, it eventually localizes to melano-somes, and it spans the melanosomalmembrane. Within the melanosome,TRP-2 is complexed with tyrosinase andTRP-1. During melanin synthesis, TRP-2converts DOPAchrome to the carboxy-lated derivative DHICA. As is the casefor tyrosinase, TRP-2 also requires metalions for its enzymatic activity, but it iszinc rather than copper that is requiredfor TRP-2 function.85

Protein Kinase C-β. PKC constitutes a fam-ily of at least 12 isoforms (reviewed in ref.86) among which PKC-β has been shownto be involved in regulating tyrosinase ac-tivity.87 The mechanisms through whichPKC mediates a wide range of mem-brane-generated signals and their rele-vance to melanocyte biology are further

discussed below (see Signaling PathwaysRegulating Melanocyte Functions). PKC-βphosphorylates serine residues on the cy-toplasmic domain of tyrosinase, thus acti-vating tyrosinase.88 Still, the means bywhich PKC-β–mediated phosphorylationof tyrosinase leads to the enzyme activa-tion is not well elucidated. It has beensuggested that phosphorylation of tyrosi-nase causes a complex to form betweentyrosinase and TRP-1,89 an event knownto stabilize tyrosinase and increase its en-zymatic activity.79

In melanocytes, activated PKC-β is as-sociated with melanosomes, and the en-zyme is found in close proximity to themelanosomal membrane.88 Althoughstructural differences among PKC iso-forms may contribute to their associa-tions with particular subcellular frac-tions, receptors for activated C-kinase(RACK), unique for each PKC isoform,primarily determine the translocation ofspecific PKC isoforms to specific cellularcompartments to activate its target onthe membrane90–92 (Fig. 70-12). RACK-Iis the PKC-β partner,90 and in humanmelanocytes, the activated PKC-β/RACK-I complex translocates to the mel-anosome membrane to allow tyrosinasephosphorylation (see Fig. 70-12).93

STRUCTURAL PROTEINS Fibrillar matrixproteins within the melanosomes arerequired for proper deposition of mel-anin. Pmel17 and MART-1 are twosuch melanosomal structural matrixproteins.

Pmel17. Pmel17, also known as gp100and the silver locus product, is a glyco-protein recognized by the antibodies

� FIGURE 70-12 Activation of tyrosinase by protein kinase C-β (PKC-β). Under baseline conditions,there is no activation of PKC-β, and tyrosinase (TYR) is not phosphorylated. Activated PKC-β binds re-ceptors for activated C-kinase-I (RACK-I), the complex translocates to the melanosome, and phosphory-lates serine residues on the cytoplasmic tail of tyrosinase. Tyrosinase phosphorylation activates the en-zyme to catalyze melanin biosynthesis.

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HMB45, HMB50, and NKI-beteb (re-viewed in ref. 94). It plays a critical rolein fibril matrix formation within eumel-anosomes.59,95 Pmel17 transcription is in-duced by α-melanocyte-stimulating hor-mone (MSH) through Mitf, and it issynthesized as a precursor protein in theER, and the protein undergoes glycosyla-tion and eventual cleavage (see Fig. 70-10). After its synthesis, Pmel17 is trans-ported to stage I melanosomes to form afibrillar structure that is the backbone ofeumelanosome matrix,94 contributes tomelanosome ellipsoid shape, and pro-motes melanin polymerization.94 Mel-anosomes lacking Pmel17 cannot transitto stage II and have no active melanogen-esis (reviewed in ref. 96). It has been sug-gested that loss of functional Pmel17 re-sults in melanin cytotoxicity, perhapsthrough leakage of melanin intermedi-ates from abnormal melanosomes intothe cytosol.94,97 However, there are noknown hypopigmentary disorders in hu-mans linked to mutations of Pmel17.

MART-1/Melan A. MART-1, also knownas Melan A, is a membrane-associatedprotein98 that is present in stage I and IImelanosomes and forms a complex withPmel17 (see Fig. 70-10). MART-1 affectsthe expression, stability, trafficking, andprocessing of Pmel17 within the melano-somes.98 To date, no hypopigmentedphenotypes associated with nonfunc-tional MART-1 have been identified.

ADDITIONAL MELANOGENIC PROTEINS PProtein. The p protein (pink-eyed dilu-tion) is a transmembrane protein with 12membranes spanning domains whose se-quence is homologous to that of othertransmembrane transport proteins, includ-ing anion transporters97,99,100 thought tofunction as a transport protein.101 Studieshave identified the protein as an adenosinetriphosphate–associated proton pump re-sponsible for maintaining acidic environ-ment within the melanosomes (reviewedin ref. 102). Other proposed functions of pprotein include stabilizing the tyrosinase/TRP-1/TRP-2 complex and/or transportingtyrosine into the melanosomes.99 Individ-uals lacking functional p protein displayoculocutaneous albinism type 2, largelydue to improper melanosomal pH.102–104

Also, Angelman and Prader-Willi syn-dromes display deletion mutations that in-clude the p locus on chromosome 15.

Heterotetrameric Adaptor Protein Com-plexes. Sorting of membrane-associatedproteins, including tyrosinase, TRP-1,TRP-2, and Pmel17, and directing them

to the appropriate cytosolic organellesare facilitated by heterodimeric adaptorprotein complexes (APs).105,106 AP-3 andpossibly also AP-1 facilitate tyrosinasetransport from endosomes tomelanosomes107 (see Fig. 70-10). Pa-tients with Hermansky-Pudlak syn-drome, an autosomal recessive disorderof oculocutaneous albinism, platelet dys-function, and pulmonary disease (seeChap. 71), have defects in specific sub-units of the AP-3 adaptor protein com-plex and as a result display severalanomalies associated with cellular trans-port of molecules.108

SLC24A5. SLC24A5 is a melanosomalprotein whose structure and homologyto cation exchange proteins suggests thatit is a melanosome-associated cationexchanger.109 Mutations in slc24α5 inzebrafish lead to hypopigmentation ofthe organism.109 The ancestral humanhomolog is expressed by darker com-plexioned individuals, including Africansand Asians, whereas lighter-complex-ioned Europeans tend to express a vari-ant allele.109

Lysosomal-Associated Membrane Proteins.LAMPs are linked to melanosome mem-branes and/or matrix. They are thoughtto protect melanosomal integrity by act-ing as scavengers of free radicals that areproduced during melanin biosynthe-sis.110 Because LAMPs are also present inlysosomes, it is thought that melano-somes and lysosomes share a commonancestral origin.110

REGULATORY PROTEINS Microphthalmia-Associated Transcription Factor. GE N EAND PROTEIN. Mitf, a basic-helix-loop-helix and leucine zipper transcriptionfactor, has been termed the master genefor melanocyte survival and is a key fac-tor regulating the transcription of themajor melanogenic proteins, tyrosi-nase, TRP-1, TRP-2,12 PKC-β,111 andMART-1.112 Mitf binds to conservedconsensus elements in gene promoters,specifically the M- (AGTCATGTGCT)and E- (CATGTG) boxes.113 It can bindas a homodimer or a heterodimer withanother related family member (re-viewed in ref. 114). Mitf appears to be akey regulator determining cell fate, astransfection of human Mitf complemen-tary DNA into mouse fibroblasts con-verts these cells into dendritic cells ex-pressing melanocyte-specific genes.115

Mitf comprises a family of nine iso-forms, Mitf-M, -A, -B, -H, -C, -D, -E, -J,and -Mc.116,117 Mitf-M expression is

highly specific for melanocytic cells.118

Melanocytes express in addition otherMitf isoforms, specifically, Mitf-A, -B,and -E.112 In melanocytes, it is the Mitf-M isoform that stimulates transcriptionof tyrosinase and PKC-β.112 The biologicrole of other Mitf isoforms in normalmelanocytes is not known.

REGULATION OF MICROPHTHALMIA-ASSOCIATED TRANSCRIPTION FACTORACTIVITY AND EXPRESSION. The ac-tivity and stability of Mitf are modulatedby phosphorylation of the protein. Mitfactivity is increased on its phosphoryla-tion by the mitogen-activated protein ki-nase-2 (MAP kinase-2), whose activity isin turn induced by binding of SF/kit/SCFto c-Kit receptor119 (see Fig. 70-12). Phos-phorylated Mitf binds to another protein,p300/CBP, that belongs to a coactivatorfamily of proteins and acts to enhanceMitf transcriptional activity.119,120 An-other kinase that is activated by SF/c-Kitinteraction is p90RSK, which alsophosphorylates Mitf, but at a differentsite from that phosphorylated by MAPkinase-2.121 These phosphorylationevents both activate Mitf and at thesame time decrease the stability of theprotein, as phosphorylated Mitf is tar-geted for degradation by proteosomes(Fig. 70-13A).121,122

The expression of Mitf is under thecontrol of several transcription factors,including Sox10 (mutated in Waarden-burg syndrome type 4, see Chap. 71)and Pax3. Mitf expression is also con-trolled by the cyclic adenosine mono-phosphate (cAMP)-response elementbinding protein (CREB) and Lef1 trans-cription factor, which participates inWnt signaling. These transcription fac-tors bind to specific sites within MITFpromoter regions to induce Mitf trans-cription (reviewed in ref. 114). The pro-moter region of the MITF gene containsa cAMP-response element (CRE) that in-teracts with CREB when the cAMP-de-pendent pathway is activated.123,124

Therefore, cAMP-elevating agents suchas α-MSH induce the expression of Mitf(see Fig. 70-13B).

MICROPHTHALMIA-ASSOCIATED TRANS-CRIPTION FACTOR ROLE IN MELANO-CYTE PROLIFERATION AND SURVIVAL.Mitf promotes melanocyte survival byupregulating the expression of a majoranti-apoptotic protein, BCl2.125 It is fre-quently overexpressed or amplified inmelanomas, contributing to their in-creased survival.126–129 A role for Mitf inmelanocyte proliferation has also been

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proposed, as under certain conditions,Mitf induces the expression of the cell cy-cle–associated kinase Cdk2 that is in-volved in the progression of cells from G1into S phase of the cell cycle.130 Mitf alsosuppresses the expression of p21, a pro-tein that inhibits Cdk2 activation.130,131

Conversely, under different conditions,Mitf can induce p21 expression, and it canalso stimulate the expression of p16INK4a,a protein that inhibits the activation of ki-nases required for progression throughthe cell cycle, thus promoting cell cycle ar-rest.132,133 Because Mitf cooperates withother transcription factors to induce its ef-fects, it is to be expected that these trans-cription factors would influence Mitf ac-tivity, resulting in either stimulation orinhibition of melanocyte proliferation.

Mice bearing null mutations of MITFdisplay loss of melanocytes, deafness,and failure of differentiation of retinalpigment epithelium.12 Mutations in MITF

are found in the pigmentary disorderWaardenburg syndrome type 2134 (seeChap. 71).

Melanocortin 1 Receptor. Melanocortin re-ceptors (MCRs) comprise a family offive related receptors (MC1R, MC2R,MC3R, MC4R, and MC5R). Each hasseven transmembrane domains, andthey belong to the G-protein–coupledreceptor superfamily.135 MC3R andMC4R are mainly found in the centralnervous system, are absent in melano-cytes,136 and are thought to control en-ergy intake. MC2R is expressed in theadrenal cortex, and MC5R is expressedin peripheral adipocytes.137 MC1R is ex-pressed in a number of cells, such as en-dothelial cells, fibroblasts,138 and kerati-nocytes,138 but the highest expression isfound in melanocytes.138

α-MSH and adrenocorticotropic hor-mone (ACTH), a 39 amino acid proopio-

melanocortin–derived peptide that con-tains the α-MSH sequence (Fig. 70-14A)(reviewed in refs. 139–141), activateMC1R (see Fig. 70-14B). Receptor ligandinteraction leads to G-protein–dependentactivation of the enzyme adenylate cy-clase followed by increased intracellularcAMP level (reviewed in ref. 139), induc-ing Mitf transcription and upregulating thelevel of melanogenic proteins, includingtyrosinase,40 promoting the synthesis ofbrown/black eumelanin (reviewed in 139).Agouti, a protein expressed in both hu-mans and mice, whose expression in miceleads to yellow coat color, antagonizesα-MSH by competitive binding to MC1R.It thus blocks adenylate cyclase activa-tion142–144 and favors pheomelanin overeumelanin synthesis (Fig. 70-15). How-ever, the role of agouti in human pigmen-tation is poorly documented.

Polymorphisms within the MC1Rgene are largely responsible for the dif-

� FIGURE 70-13 Microphthalmia-associated transcription factor (Mitf) regulation. A. Mitf is activated when steel factor (SF) binds to its cell surface receptorc-Kit. SF/c-Kit interaction activates mitogen-activated protein (MAP) kinases, which then phosphorylate Mitf. Phosphorylated Mitf then recruits the co-transcrip-tion activator CBP/p300, and the complex binds to the M- or E-boxes (M/E box) within the promoter of target genes to regulate their transcription. Mitf can alsobe phosphorylated at a different site by p90/RSK kinase. Both phosphorylation events lead to Mitf ubiquitination and subsequent proteosome-mediated deg-radation. B. On binding to melanocortin receptor-1 (MC1R), α-melanocyte-stimulating hormone (α-MSH)/adrenocorticotropic hormone (ACTH) activate the en-zyme adenylate cyclase that upregulates the cyclic adenosine monophosphate (cAMP) level, leading to cAMP-response element binding protein (CREB) activa-tion and binding its DNA consensus sequence CRE in the Mitf promoter to induce Mitf transcription.

α-MSH/ACTH

A B

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ferent skin/hair color among differentethnic groups.145 At least 30 MC1R vari-ants have been identified, and nine ofthem display loss of function146,147 (seeFig. 70-14B), not being able to induce in-tracellular cAMP production in responseto α-MSH despite adequate receptor/li-gand binding. Other MC1R variantshave reduced affinity for α-MSH.146,147

Three MC1R variants, each with only asingle amino acid substitution, have beenassociated with red/yellow hair and fairskin148 of Northern Europeans and Aus-tralians.149–154 Mice expressing a loss-of-function MC1R variant receptor also failto respond to UV irradiation with in-creased pigmentation despite an in-

creased level of epidermal α-MSH,155 butdo tan if provided forskolin, a chemicalenhancer of pigmentation that bypassesthe receptor to directly increase cAMP,demonstrating that the intracellular mel-anogenic pathway is functional in suchindividuals.155

MELANIN BIOSYNTHESISTwo types of melanins are synthesizedwithin melanosomes: eumelanin andpheomelanin.156 Eumelanin is dark,brown-black, and insoluble, whereaspheomelanin is light, red-yellow, sulfur-containing, and soluble.156 Melanins areindole derivatives of DOPA, and they are

formed in melanosomes through a seriesof oxidative steps (reviewed in ref. 157)(Fig. 70-16). Melanosomal pH affects theactivity of the melanogenic enzymes andinfluences melanin polymerization.

The synthesis of both types of mela-nin involves the rate-limiting catalyticstep in which the amino acid tyrosine isoxidized by the enzyme tyrosinase (alsocalled tyrosine oxidase, DOPA oxidase,monophenol, L-DOPA:oxygen oxidoreduc-tase) to L-DOPA, a reaction known as theRaper-Mason pathway158 (see Fig. 70-16).Conversion of tyrosine to L-DOPA isthought to be the critical rate-limitingstep in melanogenesis, as inhibition ofthis reaction blocks melanin synthe-sis.159 In both reactions, L-DOPA acts asa co-factor and also as a substrate for ty-rosinase. Although the exact interactionbetween tyrosinase and its substrates isnot completely understood, in vitro ki-netic studies suggest that distinct sitesmediate tyrosinase binding to tyrosineand to L-DOPA, and that binding to L-DOPA causes a conformational change

� FIGURE 70-15 Eumelanin and pheomelaninpresentation in mice. A. Two mice with differentcoat colors are shown. The one on the left dis-plays brown/black coat color due to eumelanin,and the one on the right displays red/yellow coatcolor due to pheomelanin. B. Representative hairshafts of these mice. (From Sharov et al: Bonemorphogenic protein (BMP) signaling controlshair pigmentation by means of cross-talk with themelanocortin receptor 1 pathway. PNAS 102:93,2004, with permission.)

A

B

� FIGURE 70-14 The melanocortin receptor (MCR) and its ligands. A. Structure of the proopiomelanocor-tin precursor. Standard abbreviations for amino acids are used. The synthetic superactive α-melanocyte-stimulating hormone (α-MSH) analogue [Nle4 D-Phe7]-α-MSH is modified by the exchange of methionine(M) with norleucine and L-phenylalanine (F) with D-phenylalanine. In red are critical amino acids required forbinding to the MCR. B. Schematic representation of the human MC1R receptor. Each of the 318 amino acidresidues in the polypeptide chain of the receptor is represented by an empty circle. Branched structuresrepresent N-linked glycosylation sites. Reduced function mutants (red circles), variants common in red- orblond-haired and fair skinned individuals (orange circles), and the conserved C-terminal cysteine (green cir-cle), the possible site for fatty acid acylation and anchoring to the plasma membrane, are indicated. Ac =acetylated; ACTH = adrenocorticotropic hormone; NH2 = amidated; TM = transmembrane domain.

ACTHH2N-SYSMEHFRWGKPVGKKRRPV

KVYPNGAEDESAEAFPLEF-OH 138-176

β-endorphin YGGFMTSEKSQTPLV TLFKNAIIKNAYKKGE237-267

β-MSH138-152Ac-SYSMEHFRWGKPVGK-NH2

α-MSH

217-234 H2N-DEGPYRMEHFRWGSPPKD-OH

77-88H2N-YVMGHFRWDRFG-OH

γ-MSH

A

B

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in tyrosinase, resulting in increased af-finity for both tyrosine and L-DOPA.

L-DOPA is oxidized into DOPA-quinone,160 DOPAquinone is furtherconverted to DOPAchrome, and DOPA-chrome can be converted to 5,6-dihy-droxyindole (DHI) or to 5,6-dihydroxy-indole-2-carboxylic acid (DHICA). Thelatter reaction is catalyzed by the en-zyme DOPAchrome tautomeras or TRP-2. The level of brown versus black eu-melanin appears to correlate with theDHI/DHICA ratio, with a higher ratioleading to the formation of black eumela-nin and a lower ratio to brown eumela-nin.161 DOPAquinone can also combinewith glutathione or cysteine to formcysteinylDOPA, which then becomesthe yellow/red, soluble, low-molecular-weight pheomelanin.161 Interestingly, ty-rosinase also catalyzes a more distantstep in melanin biosynthesis, namelyDHI conversion to indole-5,6-quinone.

In mice, the enzyme TRP-1 (also calledDHICA oxidase) converts DHICA to in-dole-5,6-quinone carboxylic acid. How-ever, the TRP-1 role in human melaninbiosynthesis is not well established.

The main function of melanin is to pro-vide protection against UV-induced DNAdamage by absorbing and scattering UVradiation (280 to 400 nm). Accordingly,energy absorption by melanin is maximalin this portion of the electromagneticspectrum and decreases gradually acrossthe visible light spectrum. UV absorbedby melanin is converted into heat, a lesstoxic form of energy (reviewed in ref.162). Still, in vitro studies conducted byseveral investigators suggest that mela-nin’s capacity to act as a sunscreen is lim-ited, and that melanin, when incorporatedinto a cream and spread over the skin, ab-sorbs only 50 percent to 75 percent of in-cident sunlight. Naturally, it is possiblethat in vivo, by virtue of localizing above

the nucleus, melanin in melanosomesachieves a higher level of protection.

Melanin intermediates as well as mela-nin itself can also be harmful to the cellbecause, depending on their molecularweight and polymerization state, theycan promote UVA (320 to 400 nm)-induced DNA damage, most likelythrough the generation of ROS.163 It hasbeen suggested that the increased inci-dence of UV-induced melanomas in light-skinned, red-haired individuals is notonly due to decreased ability of pheomel-anin to protect against UV-induced DNAdamage, but may also be due to mu-tagenic capacity of pheomelanin and pos-sibly other melanin intermediates as a re-sult of their pro-oxidant capacity.164

MELANOCYTE DENDRITESMelanocyte dendrites are branching pro-toplasmic processes that interact with ke-

� FIGURE 70-16 Melanin biosynthesis. Melanin biosynthesis begins with the amino acid tyrosine that is converted to L-DOPA (3,4 dihydroxyphenylalanine) inthe rate-limiting step of melanin biosynthesis catalyzed by tyrosinase. L-DOPA is subsequently converted to DOPAquinone by the same enzyme. DHI (5,6-dihy-droxyindole) and DHICA (5,6-dihydroxyindole-2-carboxylic acid) are then formed to produce either black or brown eumelanin. Alternatively, through incorpora-tion of glutathione or cysteine, DOPAquinone can form pheomelanin. MW = molecular weight; TRP = tyrosinase-related protein.

Tyrosine

Tyrosinase

L-DOPA

Tyrosinase

DOPAquinone

DHI DOPAchrome CysteinyIDOPA

Tyrosinase

TRP-2

Indole 5,6-quinoneIndole 5,6-quinone

carboxylic acidAlanyl-hydroxy-benzothiazine

Tyrosinase or TRP-2

Glutathione or cysteine

DHICA

Pheomelaninred/yellow

solublelow MW

DHICA melaninbrown

poorly solubleintermediate MW

DHI melaninblack

insolublehigh MW

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ratinocytes. Actin is a major structuralcomponent of melanocyte dendrites, andactin filament disruption leads to dendriteloss.165 Co-cultures of keratinocytes andmelanocytes demonstrate that keratino-cyte-derived factors play a role in mel-anocyte dendricity (reviewed in ref. 166).These factors include ET1, nerve growthfactor (NGF), α-MSH, ACTH, prostaglan-dins E2 (PGE2) and F2α (PGF2α),166 and β-endorphin.167 Integrins, receptors that me-diate actin-extracellular matrix contact,are likely to play a role in dendrite forma-tion as well.168

Another group of proteins, the Rhofamily, also plays a role in melanocytedendrite formation. Rho proteins becomeactive when they bind guanosine triphos-phate and inactive when binding gua-nosine diphosphate.169,170 It appears thatwhen Rho is activated, dendrites retract;whereas when its family member Rac isactivated, dendrites form.170 Indeed, it iscurrently assumed that by increasingcAMP levels, α-MSH inhibits Rho, en-hancing melanocyte dendricity. Thus, theequilibrium between Rho and Rac ap-pears to be an important factor influenc-ing melanocyte dendricity.

MELANOSOME TRANSPORT

Within Melanocytes

Melanosomes are transferred from theirsite of origin in melanocyte perikaryonto the tips of melanocyte dendrites. Mel-anosome transport takes place on micro-tubules that are arranged parallel to thelong axis of the dendrite and is controlledby two classes of microtubule-associatedmotor proteins: kinesins171–173 and cyto-plasmic dyneins174–178 (Fig. 70-17). Bothmotor proteins act as short cross-bridgestructures connecting the organelle to themicrotubules.

Centrifugal, anterograde organellemovement is mediated primarily by ki-nesin, whereas centripetal movement iscontrolled by cytoplasmic dynein. Stud-ies examining melanosomal transportsuggest that their microtubule-depen-dent movement is bidirectional,179 con-sistent with a cooperative forward andbackward pull of kinesin171 and dy-nein,174 respectively. For melanosomeswith net centrifugal movement, the bi-directional movement appears to termi-nate with myosin-Va (encoded by dilutelocus)–dependent melanosomal capturein the actin-rich periphery of the den-drite (see Fig. 70-17).179

Additional proteins that participate inmelanosome transport include Rab27a

(encoded by ashen locus), that mediatesmyosin-Va binding to melanosomesthrough another linker protein-melanophi-lin (encoded by leaden locus) (see Fig. 70-17).180 In the absence of myosin-Va, mel-anosomes do not collect in dendrite tips.

Mutations in any of the above geneproducts results in decreased cutaneouspigmentation. Griscelli syndrome, a rareautosomal recessive disorder in whichindividuals display dilute skin and haircolor, is the result of mutations of myo-sin-Vα, Rab27α or melanophilin180 (seeChap. 71). Myosin-Vα and Rab27α areclosely located on chromosome 15.181–184

Because myosin-Va is also expressed inthe brain, mutations of this gene mayalso cause neurologic abnormalities.Rab27a also plays a role in immunoregu-lation, and individuals with mutationsof this gene display abnormalities ofthe immune system. Mutations of mel-anophilin result only in the distinctivehypopigmentation that characterizesthe syndrome.184

To Keratinocytes

Transfer of melanosomes from melano-cytes to neighboring keratinocytes is a

critical step in normal pigmentation.Studies suggest several ways for mel-anosomal transfer, including exocyto-sis, cytophagocytosis, fusion of plasmamembranes, and transfer by membranevesicles (reviewed in ref. 185).

The exocytosis pathway of melano-somal transfer involves fusion of themelanosomal membrane with the mel-anocyte plasma membrane, melano-some release into the intercellular space,and phagocytosis by surrounding kerati-nocytes. Cytophagocytosis is a term indi-cating the phagocytosis of a live cell or aportion of it. With regard to keratino-cytes, they cytophagocytose the tip of amelanocyte dendrite, which then fuseswith lysosomes inside the keratinocyte,which is transported to a supranuclearlocation where the phagolysosomemembranes break up, releasing the mel-anosomes. Fusion of keratinocyte andmelanocyte plasma membranes createsa space through which melanosomesare transferred from the melanocyte tothe keratinocyte. Indeed, high-resolu-tion photography shows the presence offilopodia, slender, filiform, pointed, cy-toplasmic projections at the tip of mel-anocyte dendrites.186 These filopodia

� FIGURE 70-17 Schematic diagram of melanosome transport across melanocyte dendrites. Melano-somes move bidirectionally along melanocyte dendrites. They are attached to microtubules through themotor proteins kinesin (anterograde) and dynein (retrograde). At the tip of the dendrite, melanosomes arecaptured in the actin-rich periphery. Myosin-Va (MyoVa) mediates melanosome binding to actin throughthe linker proteins Rab27a and melanophilin (Mlph).

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adhere and fuse with keratinocyteplasma membrane before melanosometransfer. The fourth way of melanoso-mal transfer involves shedding of mel-anosome-filled vesicles followed by ph-agocytosis of these vesicles bykeratinocytes or their fusion with kerat-inocyte plasma membrane.

The molecular and cellular mecha-nisms involved in melanosome phago-cytosis have been partially elucidated. Itappears that keratinocytes express aseven transmembrane G-protein–coupledreceptor called protease-activated receptor-2 (PAR-2). PAR-2 is activated whenserine proteases cleave the extracellu-lar portion of the receptor, exposing anew segment that acts as a tethered(attached) ligand.187,188 Activation ofPAR-2 increases keratinocyte phago-cytic activity.187,188

Interestingly, and consistent with itsrole in melanosome phagocytosis, UVinduces the activity and expression ofPAR-2.189 UV effect on PAR-2 activityand expression is more pronounced inindividuals with skin phototypes II andIII than in those with skin phototypeI.189 Keratinocyte growth factor recep-tor has also been implicated in enhanc-ing phagocytosis of melanosomes bykeratinocytes.190

REGULATION OF MELANOCYTE FUNCTION

Melanocyte behavior in skin is largelyinfluenced by signals from neighboringkeratinocytes as well as autocrine sig-nals and environmental factors such asUV irradiation (also see Ultraviolet Irra-diation and Melanocytes). The synthe-sis and secretion of most keratinocyte-derived factors is increased by UVirradiation, but it is also evident that UVcan directly stimulate melanocyte den-dricity and melanin production.169,191

Melanocytes receive both positive andnegative paracrine signals that modulatetheir proliferation and differentiatedfunction.

Melanogenic Stimulators

PROOPIOMELANOCORTIN AND DERIVED PEP-TIDES It is well documented that MSHand ACTH are potent stimulators ofmelanogenesis. They belong to a familyof peptides derived from the precursorproopiomelanocortin (POMC) that issynthesized, in addition to the pituitarygland, also by epidermal keratinocytes.Interestingly, POMC expression in ke-ratinocytes is induced by UV, phorbol

esters, and interleukins (ILs).192,193 In ro-dents, α-MSH stimulates melanogenesisand favors eumelanin over pheomelaninproduction, but systemic administrationof α-MSH, β-MSH, and ACTH to peo-ple increases skin pigmentation pre-dominantly in sun-exposed areas.194,195

However, in certain disease conditionscharacterized by abnormally high levelsof ACTH, such as Addison disease196 orNelson syndrome197 (ACTH-secretingpituitary adenoma), more generalizedhyperpigmentation of the skin hasbeen observed.198

Aside from its effect on melanogenicproteins and eumelanin synthesis, α-MSH was also reported to enhance therepair of UV-induced DNA damage inmelanocytes, specifically the repair ofpyrimidine dimers, and also to reducethe level of UV-induced hydrogen perox-ide in the cell.199 These data suggest arole for POMC-derived peptides beyondmerely stimulating melanogenesis.

ENDOTHELIN-1 ET1 appears to play a rolein mature melanocytes, inducing mela-nogenesis by activating tyrosinase andincreasing TRP-1 levels.200,201 ET1 alsoleads to melanocyte proliferation200,201

and promotes dendrite formation.202

Cultured keratinocytes synthesize andsecrete ET1,201–203 and UV irradiationstimulates ET1 production by keratino-cytes.201,202 ET1 can also cooperate syn-ergistically with other growth factors/cy-tokines to further influence melanocytefunction.

ET1 upregulates the MC1R level andincreases MC1R affinity for α-MSH.204,205

Similar to α-MSH, ET1 displays photo-protective effects on melanocytes, en-hancing thymine dimer repair, decreas-ing the level of UV-induced hydrogenperoxide, and inducing the level of anti-apoptotic proteins.199,206

STEEL FACTOR Like other keratinocyte-derived factors, SF is induced by UV ir-radiation, and, in guinea pigs, anti-Kitantibodies block UV-induced tanning.SCF can also act synergistically withother cytokines such as IL-3, IL-6, IL-7,IL-9, and granulocyte-macrophage col-ony-stimulating factor to regulate UV-induced melanogenesis and melanocytesurvival.207,208

INFLAMMATORY MEDIATORS Several in-flammatory mediators can affect skinpigmentation. PGs (arachidonic acid–derived metabolites) and leukotrienes(lipid compounds related to PGs), bothmediators of inflammatory responses,

affect melanocyte function. Their levelis elevated in sunburned skin209 and in avariety of inflammatory dermatoses, in-cluding atopic dermatitis210 (see Chap.14) and psoriasis211 (see Chap. 18).

Human melanocytes express severalPG receptors, including the receptorsfor PGE2 and PGF2α.212,213 Indeed, PGF2αstimulates melanocyte dendrite forma-tion and activates tyrosinase,212,213 andUV irradiation upregulates the level ofPG receptors on melanocytes.212,213 Sim-ilarly, leukotrienes B4 and C4 increasemelanin synthesis and stimulate mel-anocyte proliferation and motility.214

Interestingly, melanocytes also con-tribute to cutaneous inflammatory re-sponses, as they synthesize and releaseIL-8 when stimulated by the pro-inflam-matory cytokines IL-1 and tumor necro-sis factor-α.215

Melanocytes also respond to hista-mine released by mast cells during cu-taneous inflammation. Histamine bindsH1 and H2 receptors to induce mel-anocyte dendricity and upregulate ty-rosinase level.216,217 These effects aredecreased when melanocytes are pre-treated with the H2 receptor antagonistfamotidine.217

NEUROTROPHINS Neurotrophins (NTs) area family of molecules that enhance neu-ronal survival in the central and periph-eral nervous systems. They includeNGF,218 NT3,219–221 NT4,222 and brain-derived neurotrophic factor.223,224 Mel-anocytes express the low affinity recep-tor common to all NTs, p75NTR,225 aswell as the high affinity receptors forNGF (TrkA) and NT3 (TrkC) (reviewedin ref. 226). Keratinocyte-derived NGF,whose expression is upregulated by UVirradiation, is chemotactic for melano-cytes and induces their dendricity.227

Both NGF and NT3, the latter expressedby dermal fibroblasts, increase melano-cyte survival. Specifically, after UVirradiation, NGF supplementation in-creases the level of the anti-apoptoticBcl2 protein, reducing melanocyte ap-optotic cell death.228,229 Thus, in addi-tion to other keratinocyte-derived cyto-kines, NGF may help preserve thepopulation of cutaneous melanocytesthat would otherwise be depleted byUV damage.

BASIC FIBROBLAST GROWTH FACTOR Basicfibroblast growth factor (bFGF), namedfor its ability to stimulate the growth offibroblasts, was one of the first identi-fied melanocyte mitogens.230,231 It isproduced by keratinocytes, but lacks a

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secretory signal and hence is presumed toaffect melanocytes through cell-cell con-tact. It binds tyrosine kinase transmem-brane receptors to induce its mitogeniceffect in the presence of cAMP-elevatingfactors. Like other keratinocyte-derivedcytokines, it is upregulated in responseto UV irradiation.231,232 Keratinocytegrowth factor, another member of theFGF family of proteins, has been shownto promote melanosome transfer frommelanocytes to keratinocytes.190

NITRIC OXIDE Nitric oxide (NO) is a dif-fusible free radical displaying pleiotropicbioregulatory effects in diverse cells andtissues.233,234 Melanocytes and keratino-cytes produce NO in response to inflam-matory cytokines,235–238 and NO produc-tion in keratinocytes is induced by UVirradiation.239 NO increases tyrosinase ac-tivity and melanogenesis239 and is thus anautocrine as well as paracrine moleculethat affects melanocyte behavior in skin.

Melanogenic Inhibitors

Numerous reports have suggested theexistence of endogenous melanogenic in-hibitors,240,241 but only few specific mol-ecules have been identified. One groupof inhibitors include sphingolipids, aclass of membrane-associated lipids (re-viewed in ref. 242) that act as signaltransducers. Sphingolipids were shownto decrease melanogenesis, at least inpart by enhancing Mitf degradation viaubiquitin-meditated pathways.243,244 An-other melanogenic inhibitor, BMP-4,downregulates tyrosinase expression inmelanocytes,245 also in part via its effectson Mitf.246 Interestingly, physiologicdoses of UV irradiation, a potent melano-genic stimulator, decrease the expressionof BMP receptors on melanocytes,245 pre-sumably eliminating its inhibition duringUV-induced tanning. Mice that transgen-ically overexpress the physiologic BMPantagonist noggin have a darker coatcolor than wild-type mice, and theirhairs have a higher eumelanin-pheomel-anin ratio.247

SIGNALING PATHWAYS REGULATING MELANOCYTE FUNCTION

Growth factors, cytokines, hormones,and other ligands for receptors ex-pressed on melanocytes exert their bio-logic effect by interacting with theirspecific cell surface receptors, generatinga signaling cascade involving activationor inhibition of protein kinases, andleading to distinct patterns of protein

phosphorylation. Two types of kinasesparticipate in cellular signaling: serine/threonine and tyrosine kinases that bydefinition phosphorylate serine and/orthreonine residues and tyrosine resi-dues, respectively, on their specific tar-get proteins. This section reviews themajor signaling pathways that affectmelanocyte behavior in skin.

Cyclic Adenosine Monophosphate/PKA–Dependent Pathway

cAMP, one of the first identified intracel-lular second messengers, plays a key rolein diverse biologic functions such as cel-lular metabolism, growth, and differenti-ation.248,249 It also mediates α-MSH ef-fect (reviewed in ref. 250) and was one ofthe first recognized regulators of mam-malian pigmentation.251 The intracellu-lar level of cAMP is upregulated by amembrane-associated enzyme calledadenylate cyclase that is activated on re-ceptor/ligand interaction in receptorsthat are coupled to guanosine triphos-phate–binding proteins like MC1R252

(see Fig. 70-13; Fig. 70-18). cAMP is alsoelevated by reagents such as choleragenor isobutylmethyl xanthine. Providingmelanocytes with dibutyryl cAMP, acAMP analog, increases the intracellularlevel of cAMP and induces signaling thatleads to melanogenesis.253

cAMP-dependent protein kinase (PKA)mediates most of the biologic actions ofcAMP.252 PKA is a serine/threonine ki-nase consisting of two regulatory sub-units and two catalytic subunits.252 It ex-ists in the cytosol in an inactive form,and binding of cAMP to its regulatorysubunits releases the catalytic subunits,activating the enzyme.252 PKA phospho-rylates the CREB that binds its DNA con-sensus sequence CRE in the Mitf pro-moter to induce Mitf transcription (seeFig. 70-13). cAMP elevation also affectsother target genes, increasing or decreas-ing their transcription254 (see Fig. 70-18).In vitro, PKA effect can be antagonizedby the protein kinase inhibitor that actsas a pseudo substrate for the catalyticsubunit of PKA and thus prevents itfrom phosphorylating its endogenoussubstrates.255

Protein Kinase C–Dependent Pathway

PKC is a serine/threonine kinase in-volved in diverse cellular functions, in-cluding growth, transformation, and dif-ferention.256 PKC resides as an inactiveenzyme in the cytoplasm, and it is acti-vated by diacylglycerol (DAG), a compo-

nent cleaved from the plasma membranewhen cell surface receptors interact withtheir ligands. DAG can also be releasedfrom the membrane by UV irradiation(see Fig. 70-18). DAG induces PKC trans-location to membranes where the latteris activated256 to induce phosphoryla-tion of serine/theonine residues on targetproteins like tyrosinase. Phorbol estersmimic DAG action and initially activatePKC.256 However, within 24 hours theentire cellular reserves of PKC are de-pleted, and when melanocytes aretreated with phorbol esters they can nolonger signal through PKC.

The critical role of PKC in melano-genesis was first suggested by the obser-vation that addition of DAG, the endog-enous activator of PKC, to culturedhuman melanocytes rapidly increasedtotal melanin content,257 and this in-crease was blocked by a PKC inhibi-tor.257 Moreover, topical application ofDAG to guinea pig skin increased epi-dermal melanin content.258

The expression of the 12 PKC iso-forms varies among different tissues.86

Each isoform is thought to carry out adistinct biologic function. Human mel-anocytes express PKC-α, -β, -ε, -δ, and-ζ,259,260 and the PKC-β isoform is specif-ically involved in regulating tyrosinaseactivity (see Protein Kinase C-β). ET1and histamine also utilize the PKC-dependent pathway (in addition to thecAMP-dependent pathway) to exerttheir regulatory effects on melanocytefunction.261,262

Receptor Tyrosine Kinases

Melanocytes express several distinct ty-rosine kinase receptors that bind BMP,bFGF, HGF, and c-Kit ligand. Receptor/ligand interaction activates an intracel-lular tyrosine kinase domain on the re-ceptor, phosphorylating the receptorand subsequently activating a series ofkinases called MAP kinases, or other in-tracellular signaling molecules (see Fig.70-18). Through a chain reaction in-volving phosphorylation of proteinslike Mitf, the signals are transferred tothe nucleus to activate or suppress thetranscription of genes that participate inmelanocyte proliferation, melanogene-sis, and/or survival.

Cyclic Guanosine Monophosphate Pathway

NO and histamine activate the enzymeguanylate cyclase that increases the intra-cellular level of cyclic guanosine mono-

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phosphate in melanocytes235–237,239,263

to induce melanogenesis. Inhibition ofNO and cyclic guanosine monophos-phate signaling impedes UV-inducedtanning.239

ULTRAVIOLET IRRADIATION AND MELANOCYTES

Tanning Response

Melanocyte survival, proliferation, anddifferentiated function are influenced byenvironmental factors, the most impor-tant of which is UV irradiation. UV irra-diation induces tanning, so-called fac-ultative skin color, an increase above

baseline or constitutive skin pigmenta-tion that provides protection against fu-ture UV irradiation (reviewed in ref.264). Tanning is divided into immediatetanning and delayed tanning.

IMMEDIATE TANNING Immediate tanning,or immediate pigment darkening, occurswithin 5 to 10 minutes of exposure andfades within minutes to days dependingon the UV dose and the complexion ofthe individual (Fig. 70-19B). As summa-rized in Table 70-1, immediate tanningdoes not provide photoprotection anddoes not increase epidermal melaninlevel.265 It is primarily produced by UVAirradiation, although visible light can also

induce immediate tanning.266 Immedi-ate tanning is only visible in darker indi-viduals and is of greyish-brown color,and it is thought to represent melanoso-mal relocation from the perikaryon tomelanocyte dendrites.267

DELAYED TANNING Delayed tanning, sum-marized in Table 70-1 and shown in Fig.70-19A, occurs within 3 to 4 days afterUV exposure.264,265 UV is arbitrarily di-vided into UVC (100 to 280 nm), UVB(280 to 320 nm), and UVA (320 to 400nm). The UVC portion of the spectrumis generally not present in terrestrialsunlight because it is absorbed by theatmospheric ozone layer. Delayed tan-

� FIGURE 70-18 Signaling pathways regulating melanogenesis. Melanogenesis is stimulated through cyclic adenosine monophosphate (cAMP) or protein ki-nase C (PKC)–dependent pathways. α-Melanocyte-stimulating hormone (MSH)/adrenocorticotropic hormone (ACTH) binding to melanocortin receptor-1 (MC1R)activates the enzyme adenylate cyclase, increasing cAMP levels, activating the enzyme PKA, and inducing gene transcription. Ultraviolet (UV) irradiation upreg-ulates the levels of α-MSH, ACTH, and their cognate receptor MC1R to stimulate cAMP production. Steel factor, by binding to c-Kit, leads to receptor autophos-phorylation and activation of mitogen-activated protein (MAP) kinases. MAP kinases phosphorylate (activate) microphthalmia-associated transcription factor(Mitf), leading to transcription of the melanogenic enzymes tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2. UV irradiation (as well as cell surfacereceptors not shown in this figure) releases diacylglycerol (DAG) from the cell membrane. DAG activates PKC-β, which then phosphorylates serine residues ontyrosinase, activating the enzyme. Nitric oxide (NO) activates the enzyme guanylate cyclase that increases the intracellular level of cyclic guanosine monophos-phate (cGMP). UV irradiation, by decreasing the level of bone morphogenetic protein (BMP) receptors, prevents BMP-4–mediated inhibition on melanogenesis.P = phosphate group.

α-MSH/ACTH

PKC-β

PKC-β

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ning is affected by both UVB and UVA.The action spectrum that produces de-layed tanning is the same as for UV-induced erythema (sunburn), with UVBwavelengths far more effective thanUVA.264 Especially in darker-skinned in-dividuals, sub-erythemogenic UV dosesmay be effective as well. Delayed tan-ning peaks between 10 days and 3 to 4weeks, depending on the absorbed UVdose and the individual’s skin type,then fades gradually over a few weeks.Histologically, there are increased epi-dermal melanocytes, melanocyte den-dricity, and melanosome transfer to ke-ratinocytes, with greater melanizationof individual melanosomes.265,267 Over-all, total epidermal melanin is increased,

providing additional photoprotectionfrom UV irradiation.

Direct and Indirect Effects of Ultraviolet Irradiation

UV irradiation affects melanization andmelanocyte proliferation and survival,both directly and indirectly, through itseffect on keratinocytes, inducing thesynthesis and secretion of paracrine ke-ratinocyte factors.

DIRECT EFFECTS UV irradiation triggersseveral biologic reactions through its in-teraction with cellular chromophoresthat absorb photons. Photochemical re-actions affect proliferation, survival, andthe differentiated function of melano-cytes. Most UVA effects are assumed tobe the result of oxidative damage medi-ated through UVA absorption by cellularchromophores like melanin precursors

that act as photosensitizers, leading tothe generation of ROS and free radi-cals.268 UVB irradiation is directly ab-sorbed by cellular DNA, leading to theformation of DNA lesions, mainly cy-clobutane dimers and pyrimidine (6-4)pyrimidone photoproducts.269 DNAdamage repair systems are activated, atleast in part through the tumor suppres-sor p53 protein. It has been shown thatplasma membrane lipids are also affectedby UV irradiation to release DAG,270

which then activates PKC-β to stimulatemelanogenesis by activating tyrosinase(see Protein Kinase C-β).

INDIRECT EFFECTS Key keratinocyte para-crine factors induced by UV irradiationand their effects on melanocytes aresummarized in Table 70-2. These fac-tors can act alone and/or synergisticallyto modulate melanocyte function. Inter-estingly, UV irradiation also induces the

� FIGURE 70-19 A. Delayed tanning. Fourtemplate test sites in a phototype III individualwere exposed to repeated erythemogenic dosesof ultraviolet B (UVB) (+UVA) delivered in 24-hourintervals, and the photograph was taken 10 daysafter the last exposure. The tan in the moreheavily pigmented test sites persisted for 2months. B. Immediate tanning in a phototype IIIindividual. Four template test sites were exposedto various doses of UVA, and the photograph wastaken 2 hours after the end of exposure. After 48hours, the tan had almost completely faded.

A

B

TABLE 70-1Immediate Tanning vs. Delayed Tanning

IMMEDIATE DELAYED

Onset Minutes278,282 3–4 days278,282

Peak intensity Minutes to a few hours278,282 10–28 days278,282

Fading Within 24 h278,282 Weeks265,278,282

Mechanism Redistribution of melanosomes278,283

↑Keratinocyte-derived melanogenic cyto-kines↑Tyrosinase level and activity ↑Melanin synthesis ↑Melanocyte dendricity↑Melanosome number ↑Melanosome transfer↑Melanocyte proliferation265,278,282–284

Photoprotection Unchanged278 Increased278,282

Change in skin color

Undetectable in fair skin278,283 Obvious in most light-skinned and all dark-skinned individuals283Subtle in darker skin

TABLE 70-2Ultraviolet Irradiation-Induced Keratinocyte-Derived Paracrine Cytokines

KERATINOCYTE-DERIVED FACTORPROLIF-ERATION DENDRICITY

MELANO-GENESIS

MELANOSOMAL TRANSFER SURVIVAL

Basic fibroblast growth factor ↑↑Endothelin-1 ↑ ↑ ↑Interleukin-1 α/1 β ↓ ↑ ↓Adrenocorticotropic hormone ↑ ↑ ↑α-Melanocyte-stimulating hormone

↑ ↑ ↑ ↑

Prostaglandin E2/prostaglandin F2α

↑ ↑ ↑

Granulocyte-macrophage colony-stimulating factor

↑ ↑

Nitric oxide ↑Tumor necrosis factor-α ↓Nerve growth factor ↑ ↑

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level of tumor necrosis factor-α andIL-1, cytokines that inhibit melano-genesis, suggesting a fine-tuned epi-dermal equilibrium between melano-genic stimulators and inhibitors afterUV irradiation, with the final outcomeof increased melanogenesis and melano-cyte proliferation.

Role of DNA Damage in Melanogenesis

Interestingly, the action spectrum fortanning is virtually the same as that forthe formation of thymine dimers,271,272

and UV-induced melanogenesis can beaugmented in pigment cells by treat-ment with T4 endonuclease V,273 an en-zyme that acts exclusively to enhancethe repair of UV-induced DNA damage.Moreover, treatment of melanocyteswith agents that act exclusively bydamaging DNA, unlike UV that hasmultiple cellular targets, also stimulatesmelanogenesis.274

A central role for DNA damage and/or its repair in stimulating melanogene-sis is further suggested by the fact thatp53, a tumor suppressor protein andtranscription factor termed the Guardianof the Genome, when activated, upregu-lates the level of tyrosinase messengerRNA and protein, enhancing melano-genesis.275–278 Thus, tanning may beviewed as part of a p53-mediated DNAdamage adaptive response that protects

the skin during subsequent exposure toUV irradiation.

MELANOCYTE AGING AND PHOTOAGING

Epidermal melanocyte aging is affected byboth genetic and environmental factors.With aging, there is a decrease in the den-sity of epidermal melanocytes (numberper unit area of skin surface), approxi-mately 10 percent per decade.279 How-ever, the number of DOPA-positive mel-anocytes is greater in chronically sun-exposed skin than in sun-protected skin,279

possibly due to melanocyte proliferationafter sun exposure and/or UV-induced ke-ratinocyte-derived paracrine factors. Mel-anocyte loss is especially notable in hairfollicles with age, with total loss of mel-anocytes in approximately one-half of allscalp follicles by middle age.37 Hair gray-ing (depigmentation) occurs over the en-tire body but is usually first noted on thescalp, perhaps because of the long anagen(growth) cycle and resulting requirementfor melanocyte proliferation and sus-tained high level of melanogenesis.

In vitro melanocytes derived fromolder individuals show decreased prolif-erative capacity compared to those de-rived from younger individuals. Also,with aging in vitro, there is a general in-crease in the levels of total melanin aswell as in the level of differentiation-associated proteins such as Mitf, TRP-1,and TRP-2280,281 and decrease in the

level of proliferation-associated proteinssuch as cyclin D1 and cyclin E.280,281

KEY REFERENCES

The full reference list for all chapters is available at www.digm7.com.

3. Westerhof W: The discovery of thehuman melanocyte. Pigment Cell Res19:183, 2006

23. Mizoguchi M: Melanocyte develop-ment: With a message of encourage-ment to young women scientists.Pigment Cell Res 17:533, 2004

39. Ito S: Biochemistry and physiology ofmelanin, in Pigmentation and PigmentaryDisorders, edited by Levine N. BocaRaton, FL, CRC Press, 1993, p 34

40. Slominski A et al: Melanin pigmentationin mammalian skin and its hormonal reg-ulation. Physiol Rev 84:1155, 2004

65. Park HY, Gilchrest BA: Signaling path-ways mediating melanogenesis. CellMol Biol 45:919, 1999

169. Scott G: Rac and rho: The story behindmelanocyte dendrite formation. Pig-ment Cell Res 15:322, 2002

180. Matesic LA, Copeland NG, Jenkins NA: Agenetic approach to the study of vesicletransport in the mouse, in Mechanisms ofSuntanning, edited by Ortonne JP, BallottiR. Nice, Martin Dunitz, 2002, p 199

185. Van Den Bossche K, Naeyaert JM, Lam-bert J: The quest for the mechanism ofmelanin transfer. Traffic 7:769, 2006

251. Lerner AB: My 60 years in pigmenta-tion. Pigment Cell Res 12:131, 1999

264. Gilchrest BA et al: The photobiology ofthe tanning response, in The PigmentarySystem: Physiology and Pathophysiology,edited by Nordlund JJ et al. New York,Oxford Press, 1998, p 359

C H A P T E R 7 1

Albinism and Other Genetic Disorders of PigmentationThomas J. Hornyak

EPIDEMIOLOGY

Epidemiology of Albinism

Oculocutaneous albinism (OCA) is themost common inherited disorder ofgeneralized hypopigmentation, with anestimated frequency of 1 in 20,000 inmost populations. Four different types

of OCA have been described. OCAtypes 1 and 2 (OCA1, OCA2) are mostfrequent and account for approximately40 percent and 50 percent, respectively,of OCA cases worldwide. OCA2 occursin approximately 1 in 30,000 to 1 in36,000 Caucasians and 1 in 10,000 to 1in 17,000 blacks in the United States,1–3

but is reported at higher frequenciesranging from 1 in 1400 to 1 in 7000 insub-Saharan Africa.4,5 OCA3 and OCA4are far less frequent, although the rufousOCA phenotype, described later, associ-ated with OCA3 in southern Africanblacks has been reported at an incidenceof approximately 1 in 8500.6

Hermansky-Pudlak syndrome (HPS)is rare except in the Caribbean island ofPuerto Rico, particularly in the north-western region where the majority ofpatients are found, and has an incidence

there of 1 in 1800.7 Chédiak-Higashisyndrome (CHS) is also quite rare.

Epidemiology of Congenital Disorders of Pigmentation

Waardenburg syndrome (WS) is proba-bly less frequent than OCA. The high-est reported incidence is 1 in 20,000 inKenya, and most estimates of its inci-dence in the Netherlands, where it wasoriginally reported, are in the range of1 in 40,000. Incidences of WS withdeafness are lower, ranging between 1in 50,000 and 1 in 212,000. WS hasbeen described occurring at varyingfrequencies in the congenitally deaf,ranging from 0.9 percent to 2.8 percentin some studies to 2 percent to 5 per-cent in others. The incidence of pie-