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In the inner ear, particularly in the stria vascularis, melanocytes are thought to play a role in the development of hearing. Aberrant migra­tion or survival of melanocytes within the inner ear, the iris, and midportions of the forehead and extremities explains the presence of congenital deafness, heterochromia irides, and patches of leukoderma, respectively, in patients with Waardenburg syndrome, the classic neuro­cristopathy. Also, aberrant migration or survival of enteric ganglion cells, another neural crest­derived cell population, provides an explana­tion for the association of aganglionic megacolon (Hirschsprung disease) with Waardenburg syndrome or rarely piebaldism.

The survival and migration of neural crest­derived cells during embryogenesis depends upon interactions between specific receptors on their cell surface and extracellular ligands. For example, KIT ligand (also known as steel factor or stem cell growth factor) binds to the transmembrane KIT receptor on melanocytes and melanocyte precur­sors (melanoblasts) (Figs 65.2 & 65.3); melanoblasts require expression of the KIT receptor in order to maintain their normal chemotactic 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 kit or steel can lead to white spotting. In the developing embryo, melanoblasts expressing endothelin receptor type B (EDNRB) are stimulated to migrate by endothelin­3 (ET3 [EDN3]), which is produced by the ecto­derm and dermamyotome. Mutations in one or both copies of EDN3 or EDNRB can result in Waardenburg syndrome plus aganglionic mega­colon (see Fig. 65.3).

Transcription factors represent another group of proteins that play an essential role during embryogenesis. Because transcription factors can bind DNA and influence the activity of other genes, they are able to regulate the complex interplay of various sets of genes that is required for embryonic development. Several of the genes that, when mutated, give rise to Waardenburg syndrome encode transcription factors (e.g. PAX3, MITF, SOX10; see Table 66.4). Fig. 65.4 demonstrates some of these interactions (e.g. PAX3 and SOX10 can control expression of MITF)8,9. MITF is sometimes referred to as the master regulator of melanocyte development and function given its modulation of mul­tiple differentiation genes and its early up­regulation in neural crest cells that will eventually become melanocytes and emigrate from the dorsal neural tube.

During embryogenesis, melanin­producing melanocytes are found diffusely throughout the dermis. They first appear in the head and neck region at ~10 weeks of gestation. However, by the end of gestation, active dermal melanocytes have “disappeared”, except in three primary anatomic locations – the head and neck, the dorsal aspects of the distal extremities, and the presacral area10. Some of the dermal melanocytes have clearly migrated into the epidermis, but, given the absolute numbers of cells in the two compartments, apoptosis of pigment cells has also occurred. The three sites where active dermal melanocytes are still present at the time of birth coincide with the most common sites for dermal melanocytoses and dermal melanocytomas (blue nevi). Hepatocyte growth factor may play a role in the survival and prolifera­tion of these dermal melanocytes as well as somatic activating muta­tions in GNA11 and GNAQ, which encode G proteins and are found in blue nevi (see Table 112.3).

As depicted in Fig. 65.1, melanocytes also migrate to the basal layer of the hair matrix and the outer root sheath of hair follicles. Cells that are actively producing melanin are easily recognized in the matrices of pigmented anagen hairs, whereas melanocytes within the outer root sheath are usually amelanotic and more difficult to identify11. It has been hypothesized that there are two populations of melanocytes in the skin, one in the interfollicular epidermis and the second in the hair

INTRODUCTIONIn order to understand the underlying pathophysiology of cutaneous disorders of hypopigmentation and hyperpigmentation, as well as the process of normal physiologic pigment production, an apprecia­tion of the structure and function of the melanocyte is required. A classic example of basic pathogenesis is type 1 oculocutaneous albi­nism (OCA1), in which pigmentary dilution of the skin, hair, and eyes is due to a reduction or absence of tyrosinase activity secondary to mutations in both copies of the tyrosinase gene (TYR). Within the realm of physiologic pigmentation, melanocytes in individuals with red hair often express variants of the melanocortin 1 receptor (MC1R)1. As a consequence of the altered amino acid sequences of the variant MC1Rs, their cell surface expression and interactions with melanocyte stimulating hormone (MSH) can be affected, leading to an increase in the production of pheomelanin as opposed to eumelanin. Based upon population genetics, genes that are mutated in OCA (e.g. TYR, OCA2, TYRP1, SLC45A2, SLC24A5) also influence normal pigment variation (Table 65.1)2–6.

The major sections in this chapter are:

• the origin and function of the melanocyte

• the formation and function of the melanosome

• regulation of melanin biosynthesis.

ORIGIN AND FUNCTION OF THE MELANOCYTEThe melanocyte is a neural crest­derived cell, and during embryogenesis precursor cells (melanoblasts) migrate along a dorsolateral then ventral pathway via the mesenchyme to reach the epidermis and hair follicles of the trunk (see Ch. 2). More recently, it was shown that cutaneous melanocytes can also arise from neural crest­derived Schwann cell precursors that migrate along nerves to the skin via a distinct ventral pathway7. Additional sites of melanocyte migration include the uveal tract of the eye (choroid, ciliary body, and iris), the leptomeninges, and the inner ear (cochlea) (Fig. 65.1). Presumably, the death of melanocytes within the leptomeninges, inner ear, and skin is responsible for the aseptic meningitis, auditory symptoms, and areas of vitiligo, respec­tively, seen in patients with the Vogt–Koyanagi–Harada syndrome (see Ch. 66).

Key features■ Themajordeterminantofnormalskincoloristheactivityof

melanocytes,i.e.thequantityandqualityofpigmentproduction,notthedensityofmelanocytes

■ Melanocytescontainauniqueintracytoplasmicorganelle,themelanosome,whichisthesiteofmelaninsynthesisanddeposition

■ Comparedwithlightlypigmentedskin,darklypigmentedskinhasmelanosomesthatcontainmoremelaninandarelarger;oncetransferredtokeratinocytes,themelanosomesaresinglydispersedanddegradedmoreslowly

■ Tyrosinaseisthekeyenzymeinthemelaninbiosyntheticpathway■ Twomajorformsofmelaninareproducedinmelanocytes:

brown–blackeumelaninandyellow–redpheomelanin■ Theproductionofeumelaninversuspheomelaninisinfluencedby

thebindingofmelanocytestimulatinghormonetothemelanocortin1receptor

65MelanocyteBiologyJean L. Bolognia and Seth J. Orlow

SEC TION 10PIGMENTARY DISORDERS

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ABSTRACTThe major determinant of normal skin color is the activity of melano­cytes, i.e. the quantity and quality of pigment production, not the density of melanocytes. Melanocytes contain a unique intracytoplasmic organelle, the melanosome, which is the site of melanin synthesis and deposition. Compared with lightly pigmented skin, darkly pigmented skin has melanosomes that contain more melanin and are larger; once transferred to keratinocytes, the melanosomes are singly dispersed and degraded more slowly. Tyrosinase is the key enzyme in the melanin biosynthetic pathway and the two major forms of melanin produced in melanocytes are brown–black eumelanin and yellow–red pheomelanin. The production of eumelanin versus pheomelanin is influenced by the binding of melanocyte stimulating hormone to the melanocortin 1 receptor.

KEYWORDS:melanocyte, melanosome, tyrosinase, eumelanin, pheomelanin, melanocortin 1 receptor, MC1R, melanocyte stimulating hormone, MSH, pigmentation, agouti, melanin biosynthetic pathway

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Table 65.1 Disorders characterized by diffuse pigmentary dilution in which the genetic defect is known.SeeChapter66fordetailsofclinicalfindings.BLOC-1promotesendosomalmaturationbyrecruitingtheRab5GTPase-activatingproteinMsb3;BLOC-2targetsrecyclingendosomaltubulestomelanosomesforcargodelivery;BLOC-3functionsasaRab32/38guaninenucleotideexchangefactorthatiscapableofactivatingsmallGTPases(activatedRab32/38isneededfortransportoftyrosinaseandTYRP1tomelanosomes).AP-3,adaptorproteincomplex3;BLOC,biogenesisoflysosome-relatedorganellescomplex;DHICA,5,6-dihydroxyindole-2-carboxylicacid;ER,endoplasmicreticulum;MATP,membraneassociatedtransporterprotein;TYRP,tyrosinase-relatedprotein.

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

Disorder Gene Protein product Comments

Oculocutaneous albinism (OCA)[~40% of patients have OCA1 and ~50% have OCA2]

OCA1A TYR Tyrosinase •Completeabsenceoftyrosinaseactivityandmelaninproduction•RetentionofmisfoldedtyrosinaseproteinwithintheER

OCA1B TYR Tyrosinase •Decreasedtyrosinaseactivity;canproducepheomelanin•Variantwithtemperature-sensitivetyrosinase(normalactivityat35°C,butdiminishedat37°C)•Additionalvariants:minimalpigment,platinum,yellow

OCA2 OCA2 Pprotein(OCA2waspreviouslyknownasP)

•MelanosomaltransmembraneproteinthatisalsopresentintheER•PossiblefunctionsincluderegulatingorganellepH,facilitatingvacuolaraccumulationof

glutathione,andprocessing/traffickingoftyrosinase

OCA3 TYRP1 Tyrosinase-relatedprotein1* •TYRP1stabilizestyrosinaseinmiceandhumans,anditcanfunctionasaDHICAoxidase•Both“mutant”TYRP1andtyrosinaseareretainedintheERandthendegraded•Rufousphenotype≫brownphenotype;latterseeninOCA2

OCA4 SLC45A2 Solutecarrierfamily45member2(previouslyknownasMATP)

•Variablephenotype,withhairrangingfromwhitetoyellow–brown;mostcommoninAsians•Transmembranetransporterwitharoleintyrosinaseprocessingandintracellulartraffickingof

proteinstothemelanosome

OCA5 Notknown − •Locuslinkedto4q24

OCA6 SLC24A5 Solutecarrierfamily24member5

•Cationexchangerthatmaybeinvolvediniontransportinmelanosomes

OCA7 C10orf11 Chromosome10openreadingframe11

•Expressedinmelanoblastsandmelanocytes•Possibleroleinmelanocytedifferentiation

Hermansky–Pudlak syndrome (HPS)†

HPS1 HPS1 HPS1(BLOC-3subunit1) •Defectivetraffickingoforganelle-specificproteinstomelanosomes,lysosomesandcytoplasmicgranules(includingplateletdensegranulesandlyticgranulesincytotoxicTlymphocytes)

•Pulmonaryfibrosisandgranulomatouscolitis•ComponentofBLOC-3(seeFig.65.8)

HPS2 AP3B1 Adaptorrelatedproteincomplex3β1subunit

•AP-3recognizessortingsignalswithincytosolictailsofcargomoleculesandisinvolvedinthetraffickingofproteinsfromthetrans-Golginetworktoappropriateorganelles(seeFig.65.8)

•Pulmonaryfibrosis(somepatients)•AbnormaltargetingofCD1mayplayaroleinassociatedimmunodeficiency

HPS3 HPS3 HPS3(BLOC-2subunit1) •ComponentofBLOC-2

HPS4 HPS4 HPS4(BLOC-3subunit2) •Pulmonaryfibrosis•ComponentofBLOC-3

HPS5 HPS5 HPS5(BLOC-2subunit2) •ComponentofBLOC-2

HPS6 HPS6 HPS6(BLOC-2subunit3) •ComponentofBLOC-2

HPS7 DTNBP1 Dystrobrevinbindingprotein1 •ComponentofBLOC-1

HPS8 BLOC1S3 BLOC1S3(BLOC-1subunit3) •ComponentofBLOC-1

HPS9 BLOC1S6 BLOC1S6(BLOC-1subunit6) •ComponentofBLOC-1

HPS10 AP3D1 Adaptorrelatedproteincomplex3δ1subunit

•SeeAP-3above•Neurologicimpairment(e.g.seizures),immunodeficiency

Chédiak–Higashi syndrome

LYST Lysosomaltraffickingregulator

•Abnormalvesicletraffickingandfission/fusionoflysosome-relatedorganellesresultingiantorganelles(e.g.melanosomes,neutrophilgranules[lysosomes],plateletdensegranules)

Griscelli syndrome

GS1 MYO5A MyosinVa(attachesmelanosomestoactinfilaments)

• Inallthreeforms,melanosomesareretainedinthebodyofthemelanocyteratherthantraffickingtothetipsofdendritesfortransfertokeratinocytes(seeFig.65.10)

•Silveryhairseeninallthreeforms•MyosinVaexpressedinneurons(aswellasmelanocytes)anddysfunctionleadstoneurologic

abnormalities

GS2 RAB27A RAB27A(melanosomalmembraneGTPasethatbindsmelanophilin)

•GTPasealsoexpressedinhematopoieticcells;defectivereleaseofgranulecontentsfromcytotoxicTcellsleadstorecurrentinfectionsandhemophagocyticsyndrome

GS3 MLPH Melanophilin(linksmyosinVaandRAB27A)

•Melanophilinonlyexpressedinmelanocytes,soonlypigmentarydilutionintype3

MYO5A MyosinVaF-exondeletion •F-exononlyexpressedinmelanocytes

*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.

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pigmentation seen within the human race. In other words, a person who has minimal baseline pigmentation and an inability to tan has a similar density of melanocytes when compared with a person whose skin is dark brown to black in color. Indeed, even patients with the most severe form of OCA, OCA type 1A, have a normal complement of epidermal melanocytes.

The major determinant of normal skin color is the activity of the melanocytes, i.e. the quantity and quality of pigment production, not

follicle12. Based upon antigen expression and clinical observations, it is the former that is more sensitive to the destructive forces of vitiligo. As a result, repigmentation of patches of vitiligo in which the hairs are still pigmented relies upon activation and subsequent upward migra­tion 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 stem cell factor (SCF) which was shown to be essential for hair pigmentation, i.e., mouse hairs turn white when the SCF gene was deleted12a.

By immunohistochemical staining, melanocytes are identified within the fetal epidermis as early as 50 days of gestation13. Melanin­containing melanosomes are recognizable by electron microscopy during the fourth month of gestation. Except in benign and malignant neoplasms, mela­nocytes reside within the basal layer of the epidermis, a location they maintain throughout life (Fig. 65.5). Although the cell body of the melanocyte sits on a specialized region of basal lamina, its dendrites come into contact with keratinocytes as far away as the mid stratum spinosum. This association of a melanocyte with ~30–40 surrounding keratinocytes to which it transfers melanosomes has been called the epidermal melanin unit14. However, melanocytes fail to form desmo­somal connections with neighboring keratinocytes; their interactions with keratinocytes are via cadherins.

Although there is variation in the density of epidermal melanocytes/mm2 when different regions of the body are analyzed via DOPA­stained epidermal sheets (Fig. 65.6), e.g. the density of melanocytes is greater in the genital region (~1500/mm2) compared with the back (~900/mm2), there are smaller differences between individuals when the same anatomic site is examined. This is despite the wide variation in

Fig. 65.1 Migration of melanocytes from the neural crest.Melanocytesmigratetotheuvealtractoftheeye(iris,ciliarybody,andchoroid),theleptomeninges,andthecochleaoftheinnerear,aswellastotheepidermisandhairfollicle.CutaneousmelanocytescanalsoarisefromSchwanncellprecursorslocatedalongnervesintheskin,whichalsooriginatefromtheneuralcrest.Theretinaactuallyrepresentsanoutpouchingoftheneuraltube.

MIGRATION OF MELANOCYTES FROM THE NEURAL CREST

Neural crest

Neural tube (optic cup)

Skin

Iris Choroid

Inner ear

Schwann cellprecursor along nerve

Eye

Medullaoblongata(leptomeninges)

Retina

Fig. 65.2 Activation of the KIT receptor on melanocytes.BecausetheKITreceptorisatyrosinekinasereceptor,ithastheabilitytophosphorylatethetyrosineresiduesofotherproteinsaswellasitself,i.e.autophosphorylation.HeterozygousgermlinemutationsinKITthatpreventtheactivationoftheKITreceptorbyKITligand,alsoreferredtoassteelfactor,leadtopiebaldism,whereassomaticactivatingmutationsinKITareseeninpatientswithmastocytosisaswellasmelanomasarisinginacralsites,mucosae,andchronicallyphotodamagedskin(seeFigs118.2&113.1).Aformoffamilialprogressivehyperpigmentationwithorwithouthypopigmentationcanresultfromheterozygousgain-of-functiongermlinemutationsinthegenethatencodesKITligand49.P,phosphorylation;Tyr,tyrosine.

KIT ligand(steel factor)

inactive active

Tyr-Tyr-

-Tyr

cell membrane

extracellular

intracellular

KITreceptor

ACTIVATION OF THE KIT RECEPTOR ON MELANOCYTES

PP

Fig. 65.3 Receptor–ligand interactions in precursors of melanocytes.Inthedevelopingembryo,melanoblastsexpressingendothelinreceptortypeB(EDNRB)arestimulatedtomigratebyendothelin-3(ET3)whichisproducedbytheectodermanddermamyotome.MelanoblastsalsorequireexpressionoftheKITreceptortomaintaintheirnormalchemotacticmigrationdirectedbyproductionofKITligand/steelfactorbythedermamyotome.

ET3

melanoblast or entericneural crest-derived cell

melanoblast

EDNRB KIT ligand(steel factor)

KITreceptor

RECEPTOR–LIGAND INTERACTIONS IN PRECURSORS OF MELANOCYTES

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FORMATION AND FUNCTION OF THE MELANOSOMEWithin the cytoplasm of melanocytes is a unique organelle known as the melanosome, in which melanin pigments are synthesized, depos­ited, and transported. The melanosome is most closely related to the lysosome16. Through compartmentalization, both organelles provide protection for the remainder of the cell – lysosomes protect against

the density of melanocytes15. Several factors play a role in deter­mining the level of melanocyte activity; they include specific character­istics of the individual melanosomes (e.g. their dimensions) as well as both baseline (constitutive) and stimulated (facultative) levels and activity of the enzymes involved in the melanin biosynthetic pathway. The latter are influenced by receptor­mediated interactions with extra­cellular ligands such as MSH which then influence the expression of transcription factors including MITF (see Fig. 65.4).

Fig. 65.4 Signal transduction pathways and transcription factors that contribute to melanocyte differentiation.MITFexpressionisactivatedearlyonduringthetransitionfrompluripotentneuralcrestcellstomelanoblastsandisrequiredformelanoblastsurvival8;mutationsinMITFleadtoWaardenburgsyndrome,aclassicneurocristopathy.MITFalsoregulatestheexpressionofmultiplepigmentgenesincludingthosethatencodetyrosinase,TYRP1,TYRP2,PMEL/PMEL17/gp100,andMART-1/Melan-A.AdditionaltranscriptionaltargetsareCDK2,CDKN2A,andBCL-2(whoseproteinproductisaninhibitorofapoptosis)9.SmallmoleculeinhibitorsofSIK(salt-induciblekinase)canupregulateMITF,andapplicationoftheseinhibitorstonormalhumanskinledtoanincreaseinpigmentation54.Inmice,Wntsignalinginmelanocytestemcellsiscriticalforhairpigmentation50.DetailsofhowactivationofG-protein-coupledreceptorsleadstoanincreaseinintracellularcAMPisshowninFig.65.15.Ofnote,EDNRBinteractswiththeGproteinsGNAQandGNA11,andactivatingmutationsinthegenesthatencodetheselattertwoproteinscanleadtoblueneviandphakomatosispigmentovascularis.cAMP,cyclicadenosinemonophosphate;CREB,cAMPresponse-elementbindingprotein;ET3,endothelin-3;EDNRB,endothelinreceptortypeB;LEF1,lymphoidenhancerbindingfactor1;MC1R,melanocortin1receptor;MITF,microphthalmia-associatedtranscriptionfactor;MSH,melanoctyestimulatinghormone;P,phosphorylation;PKA,proteinkinaseA;SOX10,SRY-boxcontaininggene10.

SIGNAL TRANSDUCTION PATHWAYS AND TRANSCRIPTION FACTORSTHAT CONTRIBUTE TO MELANOCYTE DIFFERENTIATION

α-MSH

β-Catenin

SOX10 LEF1 PAX3CREB

cAMP

PKA

Melanogenesis

Melanocyte differentiation

Melanocyte proliferation

Melanocyte survival

MITF promoter

RAS/RAF/MEK/ERK

MITF

MITF(activated)

Activation and Degradation

P

P

MC1R

KIT ligand

WNT

KIT receptor

ET3

EDNRB

Fig. 65.5 A melanocyte residing in the basal layer of the epidermis.Innormalskin,approximatelyeverytenthcellinthebasallayerisamelanocyte.Melanosomesaretransferredfromthedendritesofthemelanocyteintoneighboringkeratinocytesoftheepidermis,hairmatricesandmucousmembranes;notransferoccursinthepigmentepitheliumoftheretina.Theepidermalmelaninunitreferstotheassociationofamelanocytewith~30–40surroundingkeratinocytestowhichittransfersmelanosomes.

A MELANOCYTE RESIDING IN THE BASAL LAYER OF THE EPIDERMIS

phagocytosisof tip

stratumcorneum

stratumgranulosum

epidermis

dermis

stratumspinosum

basallayer

melanocyte

fusion ofmembranes

melanosomes

keratinocyte

dendrite

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Fig. 65.7). In patients with OCA1 and OCA3, misfolded tyrosinase or aberrant tyrosinase­related protein 1 (TYRP1) plus tyrosinase polypep­tides, respectively, accumulate within the endoplasmic reticulum (ER)17. This leads to ER stress and activation of the unfolded protein response (UPR)18. As a result of the UPR, which acts as a form of quality control, these proteins become targets for destruction by proteasomes and there­fore are not incorporated into melanosomes (see Fig. 65.7 inset).

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 post­translational 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 equiva­lent of traffic police within the cell, an example of which is adaptor protein complex 3 (AP­3). Mutations in the gene that encodes the β3A subunit of AP­3 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 lysosome­related 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 lysosome­related organ­elles 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 conve­nience, into four stages (Fig. 65.9). Proteins that play a key role in the biogenesis of early­stage melanosomes are PMEL/PMEL17/gp100, MART­1/Melan­A, and the ocular albinism type 1 (OA1) G­protein­coupled receptor21. Cleavage and processing of PMEL/

Fig. 65.6 Dihydroxyphenylalanine (DOPA)-stained epidermal sheet.Followingincubationwith5mML-DOPAfor4–5hours,theepidermalmelanocytesturnblackbecausetheycontaintheenzymetyrosinase,whichconvertsDOPAtoblackDOPA-melanin.Notethemultipledendritesandregularspacingofthemelanocytes.

Fig. 65.7 Formation of a melanosome and pathophysiology of oculocutaneous albinism type 1 (OCA1).Afterglycosylationandprocessingwithintheendoplasmicreticulum(ER)andGolgiapparatus,severaloftheenzymesinvolvedintheformationofmelanin(includingtyrosinase)arepackagedinvesiclesandthencombinewiththematrixproteins(e.g.PMEL/PMEL17/gp100).Asmoremelaninisdepositedwithinthemelanosomes,theymigrateintothedendritesinpreparationfortheirtransferintoneighboringkeratinocytes.InpatientswithOCA1andOCA3(triangularinsert),tyrosinasepolypeptidesareretainedtoolongwithinthelumenoftheRERandthenbecometargetsfordestructionwithinproteasomes.Thus,theyarenotincorporatedintomelanosomes.TheprimarymatrixproteinisPMEL/PMEL17/gp100(recognizedbyHMB45antibody);formationofthematrixfibersrequirescleavageofthisproteinbyaproproteinconvertase51.Sortingofproteinstothecorrectorganellesisacomplexprocessanditrequiresregulatorssuchasadaptorproteincomplex3(AP-3;seeFig.65.8);inthecaseoftyrosinase,specificdileucineresiduesinitscytoplasmictailaidinthesorting.M,matrixproteins;RER,roughendoplasmicreticulum;SER,smoothendoplasmicreticulum;T,tyrosinase.

RER

SER

nucleus

Golgiapparatus

proteasome

abnormal/misfolded nascentprotein(e.g. T in OCA1)

ribosome

dendrite

late endosome myosin Va

T

T

M

M

T

plasmamembrane

actincytoskeleton

FORMATION OF A MELANOSOME AND PATHOPHYSIOLOGY OF OCA1

pro­enzymes such as proteases and melanosomes protect against melanin precursors (e.g. phenols, quinones) that can oxidize lipid membranes.

The melanosome contains both matrix proteins (chiefly PMEL/PMEL17/gp100), which form a scaffolding upon which the melanin is deposited, and enzymes such as tyrosinase that regulate the biosyn­thesis of melanin. Following their formation via ribosomes, all these proteins are found within the rough endoplasmic reticulum (RER;

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dilution, whereas in the latter the hypopigmentation is circumscribed. Phagocytosis of melanosomes by keratinocytes can be triggered by acti­vation of the keratinocyte growth factor (KGF) receptor (KGFR/FGFR2b) by KGF, also referred to as fibroblast growth factor 726, as well as by acti­vation of protease­activated receptor 2 (PAR­2)27. Inhibitors of PAR­2 receptor–ligand interactions, e.g. inhibitors of serine proteases, have led to cutaneous hypopigmentation in animal models28.

In summary, it is the activity of the melanocytes and their interac­tions with neighboring keratinocytes, not their density, that is the major determinant of normal skin color. The activity of a melanocyte is reflected in the number and size of melanized melanosomes it pro­duces as well as in its efficiency at transferring those melanosomes to keratinocytes. For example, primarily stage II and stage III melano­somes are seen in lightly pigmented skin, whereas primarily stage IV melanosomes are seen in darkly pigmented skin (Table 65.2). An

PMEL17/gp100, an amyloidogenic protein, leads to a fibrillar matrix that acts as a scaffold upon which melanin is deposited22. As melanin is deposited within the melanosome, the organelle migrates via micro­tubules into the dendrites in preparation for transfer into the neighbor­ing keratinocytes, either within the epidermis or within the anagen hair matrix. In addition to microtubules, proteins such as kinesin and dynein are involved in the movement of melanosomes (Fig. 65.10)23.

Within the dendrites, there is a specialized myosin protein, myosin Va, that aids in the process of melanosome transfer by assisting attach­ment between the actin cytoskeleton beneath the plasma membrane 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 illustrated by the three different forms of Griscelli syndrome in which there are mutations in the respective genes (see Table 65.1)24,25. The various phenotypes are explained by the tissue­specific expression of these three genes, e.g. neurons versus cytotoxic T cells. All three genes are expressed in melanocytes and as expected, the associated diffuse pigmentary dilution is a reflection of a lack of transfer of melano­somes from the melanocytes to nearby keratinocytes. Histologically, numerous melanosomes are seen to congregate within the center of the melanocytes.

To reiterate, normal pigmentation of the skin is dependent on an orderly transfer of melanosomes from melanocytes to keratinocytes. When this transfer is disrupted, either in inherited diseases such as Griscelli syndrome or in acquired diseases such as atopic dermatitis, the result is hypopigmentation. In the former there is diffuse pigmentary

Fig. 65.8 Regulation of protein trafficking by adaptor protein complex 3 (AP-3) and biogenesis of lysosome-related organelles complexes (BLOCs).Lysosome-relatedorganellesincludemelanosomes,plateletdensegranules,andlyticgranulesofcytotoxiclymphocytesandnaturalkillercells.SubtypesofHermansky-Pudlaksyndrome(HPS)thatareassociatedwithaparticularBLOCcanhavesimilarclinicalfindings,e.g.patientswithHPS1andHPS4,bothmembersofBLOC-3,developpulmonaryfibrosisandgranulomatouscolitis.

REGULATION OF PROTEIN TRAFFICKING BY ADAPTOR PROTEIN COMPLEX 3 (AP-3)AND BIOGENESIS OF LYSOSOME-RELATED ORGANELLES COMPLEXES (BLOCs)

Lysosome

Lysosome-relatedorganelle (e.g. melanosome)

BLOC-1

HPS7HPS8HPS9

AP-3

HPS2HPS10

Golgi

BLOC-2

HPS3HPS5HPS6

BLOC-3

HPS1HPS4

Fig. 65.9 Four major stages of eumelanin melanosomes.Courtesy, Raymond Boissy, PhD.

Stage Description Electron micrographs

I Spherical; no melanin deposition

Oval; obvious matrix in the form of parallellongitudinal filaments; minimal deposition ofmelanin; high tyrosinase activity

Oval; moderate deposition of melanin;high tyrosinase activity

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

II

III

IV

FOUR MAJOR STAGES OF EUMELANIN MELANOSOMES

Fig. 65.10 Movement of melanosomes within melanocyte dendrites.Asmelaninisdepositedwithinmelanosomes,theymigratealongmicrotubulesfromthecellbodyintodendritesinpreparationfortransfertokeratinocytes.Kinesinanddyneinserveasmolecularmotorsformicrotubule-associatedanterogradeandretrogrademelanosomaltransport,respectively,andUVRresultsinaugmentedanterogradetransportviaincreasedkinesinanddecreaseddyneinactivity.MyosinVa,whichislinkedtothemelanosomalRAB27AGTPasebymelanophilin,capturesmaturemelanosomeswhentheyreachthecellperipheryandattachesthemtotheactincytoskeleton.

MOVEMENT OF MELANOSOMES WITHIN MELANOCYTE DENDRITES

Microtubule

Actin filament

Melanosome

Kinesin

Dynein

UVR

Myosin Va

Melanophilin

RAB27A

Dynactin

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additional factor is the rate of degradation of the melanosomes once they are transferred to the surrounding keratinocytes and this is related in part to the size of the individual melanosomes. The smaller mela­nosomes of lightly pigmented skin are clustered in groups of two to ten within secondary lysosomes in the keratinocytes and are degraded by the mid stratum spinosum (Table 65.3)15. In darkly pigmented skin, the melanosomes are larger and singly dispersed within lysosomes of the keratinocytes; they are degraded more slowly, such that melanin granules can still be found in the stratum corneum.

REGULATION OF MELANIN BIOSYNTHESISThis section will begin with a review of the melanin biosynthetic pathway and then examine the factors, both external and internal, that 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 regu­latory enzyme in the pathway is tyrosinase, which controls the initial biochemical reactions in this pathway (Fig. 65.11). It should then come as no surprise that the initial investigations into the molecular basis of OCA focused on the gene that encodes tyrosinase.

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 found in the hair, skin, or eyes (see Table 65.1). However, in OCA1B, where there is decreased enzyme activity, pheomelanin is produced, especially in the hair as the patient ages. The formation of pheomelanin requires less tyrosinase activity than does the formation of eumelanin (see Fig. 65.11) and therefore the formation of pheomelanin can be thought of as a default pathway.

The activity of tyrosinase is enhanced by DOPA and is stabilized by tyrosinase­related protein 1 (TYRP1) (see below). Competitive inhibi­tors of tyrosinase activity include hydroquinone, which is used to treat disorders of hyperpigmentation such as melasma, and L­phenylalanine. In patients with phenylketonuria (PKU), there is a diffuse pigmentary

Fig. 65.11 The melanin biosynthetic pathway.ThepathwayincludesthesitesofdysfunctioninOCA1(tyrosinase)andOCA3(TYRP1).Thetwomajorformsofmelaninintheskinandhairarebrown–blackeumelaninandyellow–redpheomelanin.Theenzymesaretransmembraneproteinslocatedwithinthemelanosome.DHI,5,6-dihydroxyindole;DHICA,5,6-dihydroxyindole-2-carboxylicacid;DOPA,dihydroxyphenylalanine;MW,molecularweight;TYRP,tyrosinase-relatedprotein.Adapted from Hearing VJ. Determination of melanin synthetic pathways. J Invest Dermatol. 2011;131:E8–E11.

HO HO

HO

NH2 NH2

CO2H CO2HHO

Dopachrome tautomerase(DCT)/TYRP2

Tyrosinase or TYRP1

Tyrosinase

Cysteine

Tyrosine DOPAquinone CycloDOPA then DOPA DOPAquinone

DQ

DOPA

DQ

DOPA

DHICA

DHICA-melaninbrown, slightly soluble,

intermediate MW

Pheomelaninyellow/red, alkali-soluble, low MW

DHI-melaninblack, insoluble,

high MW

Eumelanin

*Or 2-S-cysteinylDOPA

DOPAchrome DHI

5-S-cysteinylDOPA*

THE MELANIN BIOSYNTHETIC PATHWAY

HO NH CO2H

HO

NH2

CO2H

+ CO2

O

ONH2

CO2HO

O

O

HONH2

NH2

CO2H

CO2H

CD-quinones

o-Quinoneimine

1, o-Benzothiazine intermediates

HO

HO NH

HO

N CO2H

S

O2O2

Tyrosinase

Table 65.2 Variation of predominant melanosomal stages with level of cutaneous pigmentation.

VARIATION OF PREDOMINANT MELANOSOMAL STAGES WITH LEVEL OF CUTANEOUS PIGMENTATION

Pigmentation of skin

Predominant melanosomal stages

Melanocytes Keratinocytes

Fair II,III OccasionalIII

Medium II,III,IV III,IV

Dark IV>III IV

Table 65.3 Melanosomes in lightly pigmented versus darkly pigmented skin.

MELANOSOMES IN LIGHTLY PIGMENTED VERSUS DARKLY PIGMENTED SKIN

Lightly pigmented skin

Darkly pigmented skin

Melanization StagesII,III StageIV

Size(diameter) 0.3–0.5microns 0.5–0.8microns

Numberpercell <20 >200

Distributionofmelanosomeswithinthelysosomesofkeratinocytes

Groupsof2–10 Single

Degradation Fast Slow

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Among the multiple protein products of the proopiomelanocortin (POMC) gene are adrenocorticotropic hormone (ACTH), β­endorphin, and the three forms of MSH (α, β, and γ) (Fig. 65.13); in humans, α­MSH represents the major biologically active form of MSH. The primary site of expression of POMC is the pituitary gland; however, other sites of expression include the testis, the endothelium, and, of particular importance, epidermal keratinocytes. Although MSH is clas­sically associated with the pigmentary system, this peptide has a wide range of biologic properties, including suppression of inflammation and regulation of body weight. For example, mutations in POMC can result in severe early­onset obesity, adrenal insufficiency, and red hair33. It then follows that mutations in the genes that encode the melanocortin receptors to which the protein products of POMC bind can lead to similar clinical findings; for example, mutations in MC4R are associ­ated with morbid obesity and variant alleles in MC1R are associated with red hair (see below). Of note, subcutaneous afamelanotide (4­norleucyl­7­phenylalanine­α­MSH), which has enhanced binding to MC1R compared to α­MSH, can lead to cutaneous hyperpigmentation. When subcutaneous implants of afamelanotide were administered monthly, patients with erythropoietic protoporphyria experienced fewer phototoxic reactions and could tolerate more direct sunlight without pain while patients with vitiligo had more rapid and extensive repig­mentation in response to treatment with UVB.

As outlined in Table 65.4, there are five major melanocortin receptors, all of which have seven transmembrane domains (Fig. 65.14). Although the MC1R is present on a variety of cells within the skin, from endothe­lial cells to fibroblasts, the highest density of this receptor is found on melanocytes34. As in the case of the β­adrenergic receptor, the MC1R is a G­protein­coupled receptor, i.e. it uses proteins that bind guanosine triphosphate (GTP) and guanosine diphosphate (GDP) as intermediary messengers (Fig. 65.15). Following the binding of MSH to the MC1R, the protein pair interacts with a complex of G proteins. The GTP­Gsα subunit then activates adenylate cyclase, leading to increased produc­tion of cyclic adenosine monophosphate (cAMP) within the melanocyte. An increase in the intracellular concentration of cAMP leads to an increase in tyrosinase activity and eumelanin production via MITF (see Fig. 65.4). If the MC1R is dysfunctional and fails to initiate a significant rise in the intracellular level of cAMP, then pheomelanogenesis is favored (Fig. 65.16). Of note, the majority of individuals with red hair are compound heterozygotes or homozygotes for a variant R allele in the 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

dilution due to elevated levels of L­phenylalanine resulting from a deficiency in the enzyme L­phenylalanine hydroxylase that converts L­phenylalanine to L­tyrosine15. The characteristic blonde hair of PKU can undergo darkening when the patient is on a low­phenylalanine diet. Of note, tyrosinase is a copper­requiring enzyme and it has two copper­binding sites. Rare cases of copper deficiency can lead to diffuse cutane­ous pigmentary dilution, and in patients with Menkes disease, where a transmembrane Cu2+­transporting ATPase that delivers copper to the trans­Golgi network and melanosomes is dysfunctional29, the kinky hair is hypopigmented.

In a test tube, L­DOPA can spontaneously oxidize to form melanin, an insoluble biopolymer. For this reason, it was originally thought that tyrosinase was the sole enzyme involved in melanin biosynthesis. However, by the late 1970s, it was becoming clear that there were additional control points in the pathway (see Fig. 65.11). For example, dopachrome tautomerase, also known as tyrosinase­related protein 2 (TYRP2), which like TYRP1 shares similarities in its amino acid sequence with tyrosinase, converts DOPAchrome to 5,6­dihydroxyindole­2­carboxylic acid (DHICA). In mice and humans, TYRP1 stabilizes tyrosinase30 and mutations in both copies of TYRP1 lead to OCA3 (see Table 65.1).

Decreased function of yet another transmembrane protein, the P protein, leads to OCA231 (Fig. 65.12). Based upon its amino acid sequence, a prediction was made that the P protein was involved in the transport of small molecules across the membrane of the melanosome. Tyrosine, the initial precursor in the melanin biosynthetic pathway, was considered the most likely candidate for transmembrane transport. However, the nature of what might be transported by the P protein remains unclear. Data suggest that the P protein regulates processing and trafficking of tyrosinase, possibly via control of pH or glutathione content within intracellular compartments32.

Melanins represent a group of complex polymers whose functions vary from camouflage to the quenching of oxidative free radicals gener­ated via exposure to UV radiation (UVR). The level and type of melanin production is a complex interplay of the activity of the various enzymes involved in the biosynthetic pathway as well as the activity of proteins such as the P protein and those that stabilize the activity of tyrosinase (e.g. TYRP1). Several factors are known to influence the activity of these key proteins of melanogenesis, and they include α­MSH, basic fibroblast growth factor (bFGF), endothelin­1, KIT ligand, and UVR (see below).

Fig. 65.12 The P protein within the lipid bilayer.Thepolypeptidehas12putativetransmembranedomains.Thesolutecarrierfamily45member2(previouslyknownasmembrane-associatedtransporterprotein),whichisdysfunctionalinOCA4,hasasimilarconfigurationwithintheplasmamembrane.Adapted from Rinchik EM, Bultman SJ, Horsthemke B, et al. A gene for the mouse pink-eyed dilution locus and for human type II oculocutaneous albinism. Nature. 1993;361:72–6.

THE P PROTEIN WITHIN THE LIPID BILAYER

NH3+

− −

−

+

1 471 08

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

3 253 74

3 253 45

4 914 42

6 716 12

7 247 73

6 076 46

5 335 03

6 676 08

7 957 16

8 73

Cytoplasmic

Luminal

COO–

8 43

4 704 10

1 691 99

3 133 92

3 073 27

4 044 94

6 736 84

7 127 81

6 846 54

5 415 30

6 697 10

7 777 08

8 818 21

3 583 28

Fig. 65.13 Post-translational processing of the POMC polypeptide.PituitaryhypersecretionofACTHand/orα-MSHcanleadtogeneralizedhyperpigmentationinpatientswithAddisondisease.ac,acetylated;ACTH,adrenocorticotropichormone;des,desacetyl;END,endorphin;JP,joiningpeptide;LPH,lipotropichormone;MSH,melanocytestimulatinghormone;PC,prohormone-convertingenzyme;POMC,proopiomelanocortin.

POST-TRANSLATIONAL PROCESSING OF THE POMC POLYPEPTIDE

POMC1 241

ACTH β-LPHJPPC1

PC2

Pro γ-MSH

β-ENDACTH1-17

des α-MSH

ac α-MSH

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Fig. 65.14 Melanocortin 1 receptor (MC1R) within the plasma membrane of a melanocyte.MC1RhasseventransmembranedomainsandisaG-coupledreceptor(seeFig.65.15).TherearenumerousgeneticvariantsoftheMC1R.ThenullorhypomorphicRallelesD84E,R151C,R160W,andD294Hhaveredhairoddsratiosof62,118,50,and94,respectively;thelowpenetrancerallelesofV60L,V92M,andR163Qhaveredhairoddsratiosof6,5,and2,respectively52.Ingeneral,R/Rindividualshaveredhairandlightskin.

Cytoplasm

MELANOCORTIN-1 RECEPTOR (MC1R) WITHIN THE PLASMA MEMBRANE OF A MELANOCYTE

NH2

Major R alleles: D84ER151CR160WD294H

COOH

Plasma membrane

Table 65.4 Major forms of the melanocortin receptor (MCR).ACTH,adrenocorticotropichormone;MSH,melanocytestimulatinghormone.

MAJOR FORMS OF THE MELANOCORTIN RECEPTOR

Receptor Distribution, major (minor) Ligands

MC1R* Melanocytes(keratinocytes,fibroblasts,endothelialcells,antigen-presentingcells)

α-MSH,ACTH>β-MSH

MC2R Adrenalcortex(adipocytes) ACTH

MC3R† Brain(gut,placenta) α-,β-,γ–MSH,ACTH

MC4R† Brain α-,β-MSH,ACTH

MC5R Peripheraltissues,e.g.sebaceousglands,fibroblasts,adipocytes

α-,β-MSH,ACTH

*Antagonistic ligands – agouti protein (mouse) and agouti signaling protein (ASIP; human).†Antagonistic ligand – agouti-related protein (AGRP).

used to describe the banding of hairs seen in some mammals, including dogs, foxes and mice, that is due to alternating production of eumelanin and pheomelanin (Fig. 65.17). The production of agouti protein by the cells in the hair follicle papillae is cyclic, and, when the agouti protein is present, it effectively competes with MSH and the formation of pheomelanin within pheomelanosomes is favored36,37 (see Fig. 65.16). Compared to eumelanosomes, pheomelanosomes are characterized by a more spherical shape and the presence of an unstructured matrix with vesicular bodies.

An enhancement of pigment production can be seen following expo­sure of melanocytes to agents that increase intracytoplasmic levels of cAMP such as cholera toxin, forskolin38, dibutyryl cAMP, and MSH (see Fig. 65.15). The activation of protein kinase A (PKA) by cAMP leads to the phosphorylation of various proteins, which can result in their acti­vation. One of the proteins that is phosphorylated by PKA is the cAMP response­element binding protein (CREB), which functions as a tran­scription factor, regulating the expression of other genes, including MITF (see Fig. 65.4). Patients with the McCune–Albright syndrome (polyostotic fibrous dysplasia) are mosaics for an activating mutation in the gene that encodes the G protein Gsα (see Fig. 65.15). As a result, the cAMP cascade is permanently “turned on” and, with continued transcription of the CREB­controlled genes, there is hyperplasia of the bones and endocrine organs, albeit in a mosaic pattern. Presumably within the melanocytes of the segmental café­au­lait macules typical

of this disorder, the increase in intracellular cAMP leads to increased tyrosinase activity and eumelanin production.

Exposure of melanocytes to phorbol esters, e.g. tetradecanoyl phorbol acetate (TPA), can lead to increased melanin formation via the activa­tion of protein kinase C (PKC) (Fig. 65.18). Growth factors, including bFGF and KIT ligand, can also lead to an increase in the pigment content within melanocytes whereas KIT receptor inhibitors (e.g. ima­tinib) can lead to hypopigmentation. Endothelin­1, a small peptide originally isolated from endothelial cells, is produced by keratinocytes and can lead to an increase in tyrosinase activity followed by an increase in melanin production.

Multiple genes have been implicated as playing a role in normal pigment variation in humans, including: (1) MC1R and ASIP; (2) several genes that play a role in OCA, e.g. TYR, OCA2, TYRP1, SLC45A2, SLC24A5 (see Table 65.1); (3) KITLG which encodes KIT ligand; (4) TPCN2 which encodes an ion channel transporter; and (5) IRF4 which encodes interferon regulatory factor 42–4. The presence of a variant (rather than the African ancestral) allele of SLC24A5, whose protein product is a putative cation exchanger in the melanosomal membrane, correlates with lighter skin color5. In addition, variants in OCA2 are thought, at least in part, to determine the normal phenotypic variation in human eye color2. A haplotype with several polymorphisms in ASIP (and presumably a gain of ASIP function) has been associated with red 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 WNT­activated transcription factor LEF1 is associated with blonde hair41, and in mice, decreased lef1 leads to light­colored hair. Of note, some loss­of­function muta­tions in MC1R confer a risk for developing cutaneous melanoma, inde­pendent of pigmentary phenotype35.

In humans, graying or whitening of hair is a normal aging phenom­enon. There is evidence that reactive oxygen species accumulate within affected hair follicles and lead to oxidative damage of hair follicle mela­nocytes. Both hair bulb melanocytes and precursor cells in the bulge region can be affected42. In addition, melanocytes that disappear fail to express TYRP2 and SOX10. The role of SCF production by KROX20+ cells within the hair bulb was discussed previously.

Ultraviolet Radiation (UVR)Following a single exposure to UVR, an increase in the size of mela­nocytes can be observed, along with an increase in tyrosinase activ­ity15. Repeated exposures to UVR lead to an increase in the number

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increase in tyrosinase activity. In addition to an increase in melanocyte size and number, tyrosinase activity, and transfer of melanosomes to keratinocytes, the response to PUVA includes an alteration in size and aggregation pattern of melanosomes, i.e. from smaller and grouped to larger and singly dispersed (see Table 65.3).

UVR may work by increasing one or more of the following:

• transcription of the tyrosinase gene (via MITF)

• the number or activity of MC1R on melanocytes

• the expression of POMC and its derivative peptides by keratinocytes and several cell types within the dermis (e.g. endothelial cells, sebocytes, lymphocytes)

• the release of diacylglycerol from the plasma membrane, which activates protein kinase C

• an activation of the nitrous oxide/cGMP pathway

• the production of cytokines and growth factors by keratinocytes (e.g. endothelin­1)

of stage IV melanosomes transferred to keratinocytes, as well as an increase in the number of active melanocytes. When chronically sun­exposed sites (e.g. the upper outer arm) are compared with non­ sun­exposed sites (e.g. the upper inner arm), the density of mela­nocytes is up to two times greater in sun­exposed sites43. Melano­cytes, like other neural­derived tissues, have a low mitotic rate, and whether this increase in number represents an increase in mitotic rate or an activation of “inactive” melanocytes or melanocyte precursors is not known.

Following exposure to UVA irradiation, an immediate pigmentary darkening can be observed, which occurs within minutes and fades over 20–30 minutes. It is clinically most obvious in darkly pigmented skin and is thought to represent oxidation of pre­existing melanin or melanin precursors. Given its transient nature, it does not provide photoprotec­tion. Delayed tanning is visible within 24–72 hours of exposure to UVB and UVA radiation and represents new pigment production via an

Fig. 65.15 Activation of a G-protein-coupled receptor such as the melanocortin 1 receptor (MC1R).InthecaseoftheMC1R,theincreaseintheintracellularconcentrationofcAMPleadstoanincreaseintyrosinaseactivityandeumelaninproduction.GDP,guanosinediphosphate;GTP,guanosinetriphosphate;P,phosphategroup;S,stimulatory.Adapted from Alberts B. Molecular Biology of the Cell. Garland Publishing; 1989.

THE ACTIVATION OF A G-PROTEIN-COUPLED RECEPTORSUCH AS THE MELANOCORTIN1 RECEPTOR (MC1R)

Effector hormone

Hormone receptor

Adenylate cyclaseGDP

GDP

GDP

GTP

GDP

GTP ATP cAMP**In melanocytes, ↑ tyrosinase activity

and eumelanin production

GTP

Gs�Gs�

Gs�

P

Fig. 65.16 Interaction of melanocyte stimulating hormone (MSH) and agouti protein with the melanocortin 1 receptor (MC1R).AThereisbaselineactivityoftheMC1R,enhancedbybindingwithMSH.AgoutiproteinrepresentsanantagonistligandfortheMC1Rwhosebindingcanleadtopheomelanogenesis.DysfunctionoftheMC1Rcanalsoleadtopheomelanogenesis,asinthecaseofhumanswithredhair.BInteractionsareactuallymorecomplex,inthattobefullyeffective,agoutirequiresattractinandmahogunin;theformeraidsinthebindingofagoutitoMC1Rwhilethelatteractsonthecytosolicside.Thereisalsoaneutralagonist,β-defensin,whichinterfereswithbothagonistandantagonistbinding.Adapted from Schiaffino MV. Signaling pathways in melanosome biogenesis and pathology. Int J Biochem Cell Biol. 2010;42:1094–104.

INTERACTION OF MSH AND AGOUTI PROTEIN WITH THEMELANOCORTIN 1 RECEPTOR (MC1R)

α-MSH

MC1R

DysfunctionalMC1R

Agouti protein

A

B

Extracellular

Intracellular

α-MSH Agouti Attractinβ-Defensin

Gsα

MC1R

Adenylate cyclase

Mahogunin

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with the production of oxygen radicals following the UV irradiation of pheomelanins, probably contributes to the increased incidence of both cutaneous melanoma and keratinocyte carcinomas in persons with red hair. In addition, chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure48.

For table on genes associated with physiologic variation in human pigmentation and references visit www.expertconsult.com

• the induction of an SOS response to UVR­induced DNA damage34,44,45

• transactivation of the POMC promoter by p5346,47

• the ratio of kinesin to dynein, affecting melanosome transport (see Fig. 65.10).

The inability of the majority of red­haired individuals to develop a tan following exposure to UVR can be explained, at least in part, by dysfunction of their melanocyte MC1R. This phenomenon, along

Fig. 65.17 Formation of agouti hairs – underlying physiology.AFoxhairswithalternatingeumelaninandpheomelaninproductionwithinindividualhairs,apatternreferredtoasagouti.BExplanationfortheagoutipatternbasedondifferentligandsinteractingwiththemelanocortin1receptor(MC1R).Bothgain-of-functionmutationsattheagoutilocusandloss-of-functionofMC1Rcanleadtoyellow-hairedmice.IntheCNS,anabundanceofagoutiproteinleadstoobesity,andyellowmicewithgain-of-functionmutationsattheagoutilocusarealsoobese53.

MSHAgouti protein

MC1R

FORMATION OF AGOUTI HAIRS – UNDERLYING PHYSIOLOGY

A B

Fig. 65.18 Mechanisms of UVR-induced pigmentation.Theseincludeanincreaseinoneormoreofthefollowing:(1)expressionofproopiomelanocortin(POMC)anditsderivativepeptidesbycellswithintheskin,inparticularkeratinocytes;(2)thenumberofmelanocortin1receptors(MC1R)onmelanocytes;(3)thereleaseofdiacylglycerol(DAG)fromtheplasmamembrane,whichactivatesproteinkinaseC;(4)theinductionofanSOSresponsetoUVR-inducedDNAdamage;(5)nitricoxide(NO)production,whichactivatesthecGMPpathway;and(6)productionofcytokinesandgrowthfactorsbykeratinocytes.Asaresult,thereisenhancedtranscriptionofthegenesthatencodemicrophthalmia-associatedtranscriptionfactor(MITF)andmelanogenicproteinsincludingtyrosinase,tyrosinase-relatedprotein1(TYRP1),TYRP2,andPMEL/PMEL17/gp100.Inaddition,melanocytedendricityandtransferofmelanosomestokeratinocytesisstimulatedviaincreasedactivityofRac1(involvedindendriteformation),theratioofkinesintodynein,andexpressionofprotease-activatedreceptor-2(PAR-2;involvedinmelanosometransfer).TPA,tetradecanoylphorbolacetate.

+

+MITF

Keratinocyte Melanocyte

TPA

MC1R

Cytokines andgrowth factors: IL-1Endothelin-1bFGF KIT ligand/SCFProtein

kinase A

POMC

Ca2+

DAG

Protein kinase C

cAMP

α-MSH

Genes formelanogenic

proteins

MECHANISMS OF UVR-INDUCED PIGMENTATION

cGMP

NO

Protein kinase G

Keratinocyte

p53

+

+

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GENES ASSOCIATED WITH PHYSIOLOGIC VARIATION IN HUMAN PIGMENTATION

Gene Protein

Selected variant(s): amino acid alteration (if applicable) or SNP

Skin color, sun sensitivity, and freckles Hair color Eye color

Skin cancer risk*

Population frequencies of variant haplotype

European East Asian African

TYR Tyrosinase Ser192Tyr Freckles – – SCC 0.4 0 0

Arg402Gln Fair, sun-sensitive

Light (weak) Blue (vs green) Melanoma, BCC

0.4 0 0.07

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

Arg419Gln – – Green/hazel Melanoma, BCC

0.07 – 0

His615Arg – – Brown – 0 0.5 0

rs12913832 T>C in HERC2†

Fair Light Blue Melanoma (weak)

0.75 – –

SLC45A2 MATP Glu272Lys Dark Dark Non-blue – 0 0.4 0.05

Leu374Phe Fair Light Blue Melanoma, NMSC

0.95 0.01 0

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-sensitive, freckles

Red/light – Melanoma, NMSC

0.3 0 0

r alleles§ Variable Variable – Melanoma, NMSC

0.3 0.4 0.01

ASIP ASIP g.8818 A>G Dark Dark Brown – 0.15 – 0.6

ASIP haplotype¶ Fair, sun-sensitive, freckles

Red/light Light Melanoma, NMSC

0.1–0.3 0–0.2 0–0.1

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 regulatory factor 4

rs12203592 C>T Fair, sun-sensitive

Dark Blue – 0.8 0 0

rs1540771 G>A Sun-sensitive, freckles

Dark (weak)

– – 0.5 0.2 0.05

*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.

eTable 65.1 Genes associated with physiologic variation in human pigmentatione1–e6.Grayhighlight=polymorphismassociatedwithdarkerpigmentation.ThegenesinthefirstfourrowsarethoseresponsibleforOCAtypes1to4.ASIP,agoutisignalingprotein;BCC,basalcellcarcinoma;MATP,membrane-associatedtransporterprotein;MC1R,melanocortin1receptor;NCKX5,Na-Ca-Kexchanger5;NMSC,non-melanomaskincancer;SCC,squamouscellcarcinoma;SLC24A4/5,solutecarrierfamily24,member4/5;SLC45A2,solutecarrierfamily45,member2;TPCN2,two-poresegmentchannel2;TYRP1,tyrosinase-relatedprotein1.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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