Disorders of Melanocytes

Chapter 72 :: Biology of Melanocytes

:: Hee-Young Park & Mina Yaar
derive from pluripotent neural crest cells that differenti-
BIOLOGY OF MELANOCYTES ate into numerous cell lineages including neurons, glia,
AT A GLANCE smooth muscle, craniofacial bone, cartiledge, and mela-
nocytes.2,3 Progenitor melanoblasts migrate dorsolater-
Melanoblasts ally between the mesodermal and ectodermal layers to
reach their final destinations in the hair follicles and the
derive from the neural crest. Their skin as well as inner ear cochlea, choroids, ciliary body,
migration/survival in the epidermis is and iris.2,4 Pigment-producing cells can be found in fetal
influenced by numerous factors. epidermis as early as the 50th day of gestation.
Melanoblast migration and differentiation into
Melanocytes melanocytes are influenced by a number of signal-
ing molecules produced by neighboring cells. These
populate the epidermis, hair follicle, eye, include Wnt, endothelin (ET)-3, bone morphogenetic
cochlea, and meninges. proteins (BMPs), steel factor (SF) (stem cell factor, c-Kit
ligand), and hepatocyte growth factor (HGF/scatter
synthesize melanin, an indole derivative factor).5–10 By interacting with their specific cell surface
of 3,4 di-hydroxy-phenylalanine (DOPA) receptors, these molecules induce intracellular and
that is stored in melanosomes. intranuclear signaling to influence gene transcription
and protein synthesis. Genetic defects in some of these
molecules are associated with human genetic dis-
are influenced by endocrine, paracrine,
eases: ETs (Waardenburg syndrome and Hirschsprung
and autocrine factors and by ultraviolet
disease) and c-Kit and stell factor with piebaldism.
(UV) irradiation.
Detailed discussion of each signaling molecule is avail-
able online.
Melanosomes

display four maturation stages.

SITE-SPECIFIC MELANOCYTES
contain structural matrix proteins,
melanogenic enzymes, pH-maintaining MELANOCYTE STEM CELLS
proteins, and free-radical scavengers.
Generally, stem cells are defined by their undifferen-
are transported to melanocyte dendrite tiated state and their capacity to develop into several
tips and transferred to epidermal differentiated cell types. They are quiescent, slow-
keratinocytes. cycling cells that frequently are found in niches where
they are surrounded by differentiated cells that affect
in skin, absorb UV radiation and protect their behavior through the secretion of cytokines and
against photodamage. growth factors.29,30
Melanocyte stem cells reside in the hair follicle bulge
(Fig. 72-1). They express TRP-2 as well as the neural
crest stem cell intermediate filament nestin in addition
to other neural crest stem cell markers including the
EMBRYONIC DEVELOPMENT transcription factors Sox10 and Pax5 that participate
in the regulation of microphthalmia-associated tran-
Melanocytes are pigment-producing cells that originate scription factor (MITF) and TRP-2. Melanocyte stem
from the dorsal portions of the closing neural tube in cells can leave the bulge region and migrate/differen-
vertebrate embryos1 (eFig. 72-0.1 in online edition). They tiate in the epidermis or the hair follicle.
11
Section 11

Figure 72-2 Melanosomes are organized into supranu-

Figure 72-1 Melanocyte stem cells in the hair follicle clear “caps” within keratinocytes. Note melanized dendritic
bulge. A stem cell melanocyte is shown in the hair follicle melanocytes and adjacent keratinocytes with the supra-
bulge, indicated by an arrow in the high-power insert. nuclear “caps.” Melanin silver-stained (Fontana-Masson)
These cells stain positive for TRP-2 (green fluorescence), an section of a heavily melanized human epidermis. (Bar =
::

early marker of commitment to the melanocyte lineage, 50 µm.) (From Byers HR et al: Role of cytoplasmic dynein in
Disorders of Melanocytes

but are negative for the proliferation marker Ki-67 (red perinuclear aggregation of phagocytosed melanosomes
fluorescence) that characterizes melanocytes migrating and supranuclear melanin cap formation in human kerati-
down the follicle to the dermal papilla during the anagen nocytes. J Invest Dermatol 121:813, 2003, with permission.)
phase of the hair cycle. (From Botchkareva NV et al: SCF/c-
kit signaling is required for cyclic regeneration of the hair
pigmentation unit. FASEB J 15:645, 2001, with permission.)
present in clusters within the keratinocytes, while in
ethnic groups with darker complexion, melanosomes
are larger, darker, and are individually dispersed
CUTANEOUS MELANOCYTES within the keratinocytes.36

The largest number of melanocytes are present in the

skin and hair follicles. While in most furred mam- HAIR FOLLICLE MELANOCYTES
mals melanocytes are found only in the hair follicle,
in humans, melanocytes are also present in interfol- In contrast with interfollicular epidermal melanocytes,
licular epidermis, specifically in the basal layer.28 There the follicular melanin unit undergoes cyclic modifica-
is approximately one melanocyte per five or six basal tions in coordination with the hair cycle (Fig. 72-4).
keratinocytes. Melanocytes synthesize melanin, a pig- Melanocytes are located in the proximal hair bulb dur-
mented polymer that is stored in cytosolic organelles ing anagen and there is a ratio of 1:5 between melano-
called melanosomes that are transferred to keratino- cytes and keratinocytes and 1:1 between melanocytes
cytes through melanocyte dendritic processes (Fig. and basal layer keratinocytes.37 Melanocytes prolifer-
72-2). As keratinocytes are continuously being desqua- ate, migrate, and undergo maturation during early
mated, there is a constant need for synthesis and trans- to mid anagen. Melanogenesis and melanin transfer
fer of melanosomes from melanocytes to keratinocytes to keratinocytes occurs throughout anagen. Melano-
in order to maintain cutaneous pigmentation. cytes eventually apoptose during late catagen. In mice,
The term “epidermal melanin unit” describes a sin- melanocyte proliferation and differentiation during
gle epidermal melanocyte surrounded by several epi- anagen depends on c-Kit expression by melanocytes
dermal keratinocytes31 (Fig. 72-3). Interestingly, signals and SF synthesis by keratinocytes.38
from keratinocytes substantially regulate epidermal Similar to their role in the epidermis, in hair, melano-
melanocyte survival, dendricity, melanogenesis and cytes transfer melanin to differentiated keratinocytes
the expression of cell surface receptors32 (see below). that ultimately form the hair shaft. They thus deter-
Most keratinocyte-derived signals are induced by mine hair color by the amount of melanin transferred,
ultraviolet (UV) irradiation. as well as by the ratio of eumelanin (black–brown) to
Melanocyte density/mm2 ranges from approxi- pheomelanin (red–yellow) (see below).39
mately 550 to >1,200 with the highest concentrations In hair, melanin does not appear to have a protective
found in the genitalia and face.33,34 Melanocyte den- effect, since UV irradiation does not reach the hair folli-
sity is the same in individuals of different racial back- cle. Still, in furry animals hair color plays an important
grounds,34 and thus cutaneous pigmentation does not role in camouflage, mimicry, and social communica-
depend on melanocyte number, but rather upon mela- tion.40 It is also speculated that melanocytes restrain
nogenic activity within the melanocyte, the proportion keratinocyte proliferation, affect calcium homeostasis,
of mature melanosomes, and/or their transfer and dis- and protect against reactive oxygen species (ROS) dur-
tribution within the keratinocytes.35 Indeed, in light- ing the rapid proliferation and differentiation of the
766 skinned individuals, melanosomes are smaller and are hair follicle.37
 11

A B

Chapter 72
::
Biology of Melanocytes
C

D E

Figure 72-3 The epidermal melanin unit. A. Representation showing the relationship between basal melanocytes, kera-
tinocytes, and Langerhans cells, shown at the upper layer of the epidermis. (From Quevedo WC Jr.: The control of color in
mammals. Am Zoology 9:531, 1969, with permission.) B. Electron micrograph of the dermal–epidermal junction of human
skin showing a dendritic melanocyte (M) among the basal keratinocytes (K). k′ represents a basal keratinocyte under-
going mitosis with condensed chromatin (arrows). (Bar = 10.0 µm.) (Illustration used with permission from Raymond E.
Boissy, Department of Dermatology, University of Cincinnati, Cincinnati, Ohio.) C. Human epidermis immunostained for
fibroblast growth factor-2 (FGF2). The figure shows basal keratinocytes with peroxidase reaction indicating the presence
of immunore-active FGF2. Arrows point to melanocytes. (Used with permission from Glynis Scott, MD, Department of Der-
matology, University of Rochester School of Medicine and Dentistry, Rochester, NY.) D and E. Distribution of melanosomes
within keratinocytes in lightly pigmented Caucasian and darkly pigmented African-American skin. Melanosomes in lightly
pigmented Caucasian skin (D) are distributed in membrane-bound clusters. In contrast, in darkly pigmented African-
American skin (E) the melanosomes are individually distributed throughout the cytoplasm of epidermal keratinocytes.
Melanosomes in both skin types are frequently concentrated over the apical pole of the nucleus (arrows). L = Langerhans cell.
(Bar = 3.0 µm.) (Used with permission from Minwalla L et al: Keratinocytes play a role in regulating distribution patterns of
recipient melanosomes in vitro. J Invest Dermatol 117:341, 2001.)
767
11 Melanocytes in the hair

Infundibular
Epidermal

C
Outer root
sheath
Section 11

Amelanotic

Bulbar melanogenic
::

Apoptotic
Disorders of Melanocytes

A B D

Figure 72-4 Melanocytes in the hair. A. Pigmented human scalp hair follicle in full anagen with high levels of hair bulb
melanogenesis. Mature melanin granules are transferred into cortical keratinocyte. B. Scalp hair bulb. Representation of
early catagen hair follicle showing loss of some bulbar melanotic melanocytes via apoptosis. Arrows point at melano-
cytes located in epidermal, infundibular, and outer root sheath regions. C. Transmission electron micrograph of section
of an early catagen hair bulb showing apoptosis of melanotic melanocytes. Inset, high-power view of premelanosomes.
D. Primary culture of 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: Gerontobiology of the hair follicle
pigmentary unit. Exp Gerontol 36:29, 2001, with permission.)

MELANIZATION
The major differentiated function of melanocytes is to
synthesize melanin in specialized organelles within
the melanocytes, the melanosomes, and to transfer
melanosomes to neighboring keratinocytes in order
to provide protection from UV irradiation (Fig. 72-5).
Melanogenesis, the synthesis and distribution of
melanin in the epidermis, involves several steps: tran-
scription of proteins required for melanogenesis; mela-
nosome biogenesis; sorting of melanogenic proteins
into the melanosomes; transport of melanosomes to
the tips of melanocyte dendrites and transfer of mela-
nosomes to keratinocytes. Disruption in any of these
events results in hypopigmentation.

MELANOSOMES
MELANOSOME BIOGENESIS
Figure 72-5 Melanocytes cultured on keratinocytes.
The melanosome is a unique membrane-bound organ- Light micrograph showing dendritic melanocytes from a
elle in which melanin biosynthesis takes place. Because black donor loaded with melanin and adjacent pigmented
768 melanosomes contain enzymes and other proteins also keratinocytes due to transfer of melanosomes.
 11

Chapter 72
B C

D F

::
Biology of Melanocytes
G H I J

Figure 72-6 Melanosome biogenesis. Electron microscopy of eumelanosome (A–F) and of pheomelanosome (G–J) de-
velopment. I, II, III, and IV in A–J represent the different maturation stages of melanosomes. [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 mammalian skin and its hormonal regulation. Physiol Rev 84:1155, 2004, with permission from the Ameri-
can Physiological Society.)

present in lysosomes, they are thought to represent become a component of the final fibrillar matrix.59 Stage
a modified version of the latter. Proteins common to II eumelanosomes have organized structured fibrillar
both organelles include the lysosome-associated mem- matrix, but no active melanin synthesis, whereas in
brane proteins (LAMPs) that participate in autophagy stage II pheomelanosomes melanin synthesis already
and regulation of intravesicular pH,56 as well as acid takes place. Although no active melanogenesis takes
phophatase, a marker enzyme for lysosomes.56 Also place in stage II eumelanosomes, they already contain
like lysosomes, melanosomes can endocytose recep- the enzyme tyrosinase. Deposition of melanin on the
tors that are targeted for degradation.56 fibrillar matrix is found in stage III eumelanosomes,
Depending on the type of melanin synthesized, while stage IV eumelanosomes are fully melanized and
melanosomes can be divided into eumelanosomes their internal matrix is masked by melanin deposits.60,61
and pheomelanosomes (Fig. 72-6). Eumelanosomes
are large (∼0.9 × 0.3 µm), elliptical in shape and con- MELANOGENIC PROTEINS
tain a highly structured fibrillar glycoprotein matrix
required for eumelanin synthesis.40 Pheomelanosomes
The timely and organized sorting of melanogenic
are smaller (∼0.7 µm in diameter), spherical in shape
enzymes and structural proteins to melanosomes is an
and their glycoprotein matrix appears disorganized
integral part of melanosomal maturation. Melanosome
and loose40 Although both eumelanosomes and phe-
proteins express sorting signals at their amino-terminus
omelanosomes may be present within a single melano-
and these direct them into the ER and eventually into
cyte,57 once committed, they do not change.58
the melanosomes.40,60,61
Melanosomes display four maturation stages (Fig.
72-6). Stage I melanosomes or premelanosomes likely
ENZYMES
develop from the endoplasmic reticulum (ER).40 They
have an amorphous matrix and display internal ves- Tyrosinase. Tyrosinase is present in plants, insects,
icles that form as a result of membrane invagination. amphibians, and mammals. It was initially identified
Premelanosomes already contain the glycoprotein in the early 1900s in mushroom extracts and was sub-
Pmel17 (gp100) but it requires further processing to sequently isolated and purified in 1949 from murine 769
11 Sorting of melanosomal proteins into melanosomes
cytoplasmic domain. The inner domain that contains
the catalytic region is approximately 90% of the pro-
Plasma membrane tein. It is followed by a short transmembrane domain,
and a cytoplasmic domain composed of approximately
30 amino acids.65 Histidine residues present in the
inner (catalytic) portion of tyrosinase bind copper (Cu)
Stage II
Endosome ions and the latter are required for tyrosinase activity.
Stage III The biological function of the tyrosinase cytoplasmic
AP-3 domain was not known for a long time. In a mouse
Tyrosinase/
Tyrosine/
Stage II Stage I model where the entire cytoplasmic domain is missing,
TRP-1
TRP-1 tyrosinase protein is inserted into the cellular plasma
TRP-2 PMEL17/
MART-1 membrane instead of into the melanosomal membrane,
suggesting that tyrosinase cytoplasmic domain is
Trans-Golgi network
required for proper trafficking of tyrosinase into mela-
nosomes. Indeed, it was found that the motif EXXQPLL
Section 11

Golgi (glutamic acid-X-X-glutamine-proline-leucine-leucine,

where “X” stands for any amino acid) in the cytoplas-
ER mic domain is responsible for tyrosinase trafficking
into the melanosomes.66 In addition, protein kinase C-β
(PKC-β) (see below) must phosphorylate two serine
::

Tyrosinase/ residues on this cytoplasmic domain to activate tyrosi-

Disorders of Melanocytes

TRP-1 TRP-2
nase,65 and in the absence of those phosphorylation
events pigmentation does not occur.
Figure 72-7 Sorting of melanosomal proteins into me-
Tyrosinase mutations including missense, nonsense
lanosomes. Tyrosinase and tyrosinase-related protein-1
(TRP-1) are initially synthesized in the endoplasmic re- frameshift, and deletion mutations that lead to inac-
ticulum (ER) and, after additional maturation steps (*) tivation of the enzyme are found in oculocutaneous
in the Golgi and trans-Golgi network, are packaged in albinism type I (see Chapter 73 and Albinism data-
endosomes as a complex. TRP-2 follows similar matura- base: http://albinism-db.med.unm.edu/). Such muta-
tion steps. Melanosomes originate in the ER as stage I tions may affect tyrosinase glycosylation interfering
already containing the melanosomal proteins PMEL17 and with enzyme maturation, or may involve Cu-binding
MART-1. They then mature to stage II melanosomes and sites disrupting tyrosinase activity or premature termi-
fuse with tyrosinase/TRP-1 in a process directed by the nation of tyrosinase protein that causes truncation of
adaptor protein 3 (AP-3). Melanosomes become progres- cytoplasmic domain.64
sively darker as melanin biosynthesis takes place.
Tyrosinase-Related Proteins. Two tyrosinase-
related proteins, (1) TRP-1 and (2) TRP-2, play impor-
tant roles in melanogenesis.67–69 They are structurally
melanoma cells.62 Mouse and human tyrosinase genes
related to tyrosinase and share ∼40% amino acid homol-
are 60–70 kb, and 50 kb long, respectively. The murine
ogy. Also, similar to tyrosinase, TRP-1 and TRP-2 are
tyrosinase gene maps to chromosome 7, whereas
glycoproteins located within the melanosomes and
human tyrosinase gene maps to chromosome 11. The
span the melanosomal membrane.70 The conserved
human tyrosinase gene is composed of five exons and
nucleotide and amino acid sequences among these
four introns,63 and tyrosinase mRNA is approximately
three melanogenic enzymes suggest that they origi-
2 kb long (genebank access number NM_000372).
nated from a common ancestral gene.71,72 Mutations
Tyrosinase is synthesized in the ER as a precursor
of TRP-1 and TRP-2 in mice after coat color (“brown”
protein whose nascent chain is processed in the Golgi
and “slaty” mice, respectively) and polymorphisms of
complex where sialic acid and neutral sugars are
these gene products are implicated in lighter hair and
added to the peptide via N- and O-glycosidic linkages
skin color in European population studies. Their func-
through a process called glycosylation64 (Fig. 72-7).
tions are incompletely understood, but the proteins
At least four forms of tyrosinase, all differing with
regards to their degree of glycosylation, have been complex with tyrosinase and may stablilize it.
identified. The glycolsylation steps have been shown Protein Kinase C-β. PKC constitutes a family
to be important for proper association of tyrosinase of at least 12 isoforms86 among that PKC-β has been
with melanosomes, as well as for its activity.64 Follow- shown to be involved in regulating tyrosinase activ-
ing the glycosylation steps, mature tyrosinase is folded ity.87 The mechanisms through which PKC mediates a
in the ER, a step required for appropriate trafficking/ wide range of membrane generated signals and their
sorting of tyrosinase into the Golgi apparatus and ulti- relevance to melanocyte biology are further discussed
mately into endosomes and finally into melanosomes. below (see Section “Signaling Pathways Regulating
A strict control mechanism guarantees the elimination Melanocyte Functions”). PKC-β phosphorylates serine
of defective tyrosinase. residues on the cytoplasmic domain of tyrosinase, thus
Within the melanosome, tyrosinase spans melano- activating tyrosinase.88 Still, the means by which PKC-
somal outer membrane (eFig. 72-7.1 in online edition). β-mediated phosphorylation of tyrosinase leads to the
It has three domains: (1) an inner melanosomal domain, enzyme activation is not well elucidated. It has been
770 (2) a melanosomal transmembrane domain, (3) and a suggested that phosphorylation of tyrosinase causes a
 Activation of tyrosinase by protein kinase
However, there are no known hypopigmentary disor-
ders in humans linked to mutations of Pmel17.
11
Plasma membrane MART-1/Melan A. MART-1, also known as Melan
A, is a membrane-associated protein98 that is present in
stage I and stage II melanosomes and forms a complex
PKC-β with Pmel17 (Fig. 72-7). MART-1 affects the expres-
sion, stability, trafficking, and processing of Pmel17
Rack-1 PKC-β P
within the melanosomes.98 To date, no hypopigmented
Rack-1 P
phenotypes associated with nonfunctional MART-1
TYR TYR have been identified.
Melanin
TRANSPORT PROTEINS
Inactive Active
Heterotetrameric Adaptor Protein Com-
plexes. Sorting of membrane-associated proteins

Chapter 72
Figure 72-8 Activation of tyrosinase by protein kinase including tyrosinase, TRP-1, TRP-2, and Pmel17 and
C-β (PKC-β). Under baseline conditions, there is no activa- directing them to the appropriate cytosolic organelles is
tion of PKC-β, and tyrosinase (TYR) is not phosphorylated. facilitated by heterodimeric adapter protein complexes
Activated PKC-β binds receptors for activated C-kinase-I (APs).99,100 AP-3 and possibly also AP-1 facilitate tyrosi-
(RACK-I), the complex translocates to the melanosome, nase transport from endosomes to melanosomes101 (Fig.

::
and phosphorylates serine residues on the cytoplasmic 72-7). Patients with Hermansky–Pudlak syndrome—an

Biology of Melanocytes
tail of tyrosinase. Tyrosinase phosphorylation activates the autosomal recessive disorder of oculocutaneous albi-
enzyme to catalyze melanin biosynthesis. nism, platelet dysfunction, and pulmonary disease (see
Chapter 73)—have defects in specific subunits of the
complex to form between tyrosinase and TRP-1,89 an AP-3 adaptor protein complex and as a result display
event known to stabilize tyrosinase and increase its several anomalies associated with cellular transport
enzymatic activity.79 of molecules.102 Studies suggest that a molecule of the
In melanocytes, activated PKC-β is associated with kinesin family, microtubule-associated motor proteins,
melanosomes and the enzyme is found in close prox- is involved in endosomal sorting and positioning of
imity to the melanosomal membrane.88 While struc- melanosomal proteins via interaction with AP-1.103,104
tural differences among PKC isoforms may contribute
to their associations with particular subcellular frac-
P-Protein. The P-protein (pink-eyed dilution) is a
transmembrane protein with 12 membrane-spanning
tions, receptors for activated C-kinase (RACK), unique
domains whose sequence is homologous to that of
for each PKC isoform, primarily determine the translo-
other transmembrane transport proteins, including
cation of specific PKC isoforms to specific cellular com-
anion transporters97,105,106 thought to function as a
partments to activate its target on the membrane90–92
transport protein.107 Studies have identified the pro-
(Fig. 72-8). RACK-I is the PKC-β partner,90 and in
tein as an ATP-associated proton pump responsible
human melanocytes, the activated PKC-β/RACK-I
for maintaining acidic environment within the mela-
complex translocates to the melanosome membrane to
nosomes.108 Other proposed functions of P-protein
allow tyrosinase phosphorylation (Fig. 72-8)93
include stabilizing the tyrosinase/TRP-1/TRP-2
complex and/or transporting tyrosine into the mela-
STRUCTURAL PROTEINS. Fibrillar matrix pro- nosomes.105 Individuals lacking functional P-protein
teins within the melanosomes are required for proper
display occulocutaneous albinism type 2, largely due
deposition of melanin. Pmel17 and MART-1 are two
to improper melanosomal pH.108–110 Also, Angelman
such melanosomal structural matrix proteins.
and Prader–Willi syndromes display deletion muta-
Pmel17. Pmel17, also known as gp100 and the sil- tions that include the P-locus on chromosome 15.
ver locus product, is a glycoprotein recognized by the
SLC24A5. SLC24A5 is a melanosomal protein whose
antibodies HMB45, HMB50, and NKI-β.94 It plays a
structure and homology to cation exchange proteins
critical role in fibril matrix formation within eumelano-
suggests that it is a melanosome-associated cation
somes.59,95 Pmel17 transcription is induced by α-MSH
exchanger.111 Mutations in slc24a5 in zebrafish lead to
through MITF and it is synthesized as a precursor pro-
hypopigmentation of the organism.111 The ancestral
tein in the ER and the protein undergoes glycosylation
human homolog is expressed by darker complexioned
and eventual cleavage (Fig. 72-7). After its synthesis,
individuals including Africans and Asians, while
Pmel17 is transported to stage I melanosomes to form
lighter complexioned Europeans tend to express a
a fibrillar structure that is the backbone of eumelano-
variant allele.111
some matrix,94 contributes to melanosome ellipsoid
shape and promotes melanin polymerization.94 Mela- SCAVENGER PROTEIN
nosomes lacking Pmel17 cannot transit to stage II and
have no active melanogenesis.96 It has been suggested Lysosome-Associated Membrane Proteins.
that loss of functional Pmel17 results in melanin cyto- LAMPs are linked to melanosome membranes and/
toxicity, perhaps through leakage of melanin interme- or matrix. They are thought to protect melanosomal
diates from abnormal melanosomes into the cytosol.94,97 integrity by acting as scavengers of free radicals that 771
11 are produced during melanin biosynthesis.112 Because
LAMPs are also present in lysosomes, it is thought that
MITF Role in Melanocyte Proliferation and Sur-
vival. MITF promotes melanoycte survival by
melanosomes and lysosomes share a common ances- upregulating the expression of a major antiapop-
tral origin.112 totic protein BCl2.127 It is frequently overexpressed
or amplified in melanomas, contributing to their
REGULATORY PROTEINS increased survival.128–131 Mutations in MITF are found
in the pigmentary disorder Waardenburg syndrome
Microphthalmia-Associated type 2132(see Chapter 73).
A role for MITF in melanocyte proliferation has
Transcription Factor also been proposed, as under certain conditions,
Gene and Protein. MITF, a basic-helix-loop-helix MITF induces the expression of the cell cycle-asso-
(bHLH) and leucine zipper transcription factor, has ciated kinase Cdk2 that is involved in the progres-
been termed the master gene for melanocyte sur- sion of cells from G1 into S phase of the cell cycle.133
vival and is a key factor regulating the transcription MITF also suppresses the expression of p21, a protein
of the major melanogenic proteins, tyrosinase, TRP-1, that inhibits Cdk2 activation.133,134 Conversely, under
Section 11

TRP-2,12 PKC-β113 and MART-1.114 In melanocytes, it is

the MITF-M isoform that stimulates transcription of
tyrosinase and PKC-b.114 MITF binds to conserved con-
The melanocortin receptor and ligands
sensus elements in gene promoters, specifically the M-
(AGTCATGTGCT) and E- (CATGTG) boxes.115 It can bind ACTH β-endorphin
::

as a homodimer or a heterodimer with another related A H2N-SYSMEHFRWGKPVGKKRRPV YGGFMTSEKSQTPLV

KVYPNGAEDESAEAFPLEF-OH TLFKNAIIKNAYKKGE
Disorders of Melanocytes

family member.116 MITF appears to be a key regulator 138-176 237-267

determining cell fate, as transfection of human MITF
cDNA into mouse fibroblasts converts these cells into
dendritic cells expressing melanocyte-specific genes.117
77-88 217-234
MITF comprises a family of nine isoforms: (1) MITF-
H2N-YVMGHFRWDRFG-OH H2N-DEGPYRMEHFRWGSPPKD-OH
M, (2) -A, (3) -B, (4) -H, (5) -C, (6) -D, (7) -E, (8) -J, and γ-MSH 138-152 β-MSH
(9) -Mc.118,119 MITF-M expression is highly specific for Ac-SYSMEHFRWGKPVGK-NH2
melanocytic cells.120 Melanocytes express in addition α-MSH
other MITF isoforms specifically, MITF-A, -B, and,
-E114The biologic role of other MITF isoforms in normal B
Extracellular
melanocytes is not known.
D84E
Regulation of MITF Activity and Expression. The V6OL D294H
activity and stability of MITF are modulated by phos-
phorylation of the protein. MITF activity is increased
TM
upon its phosphorylation by the mitogen-activated
protein kinase-2 (MAP kinase-2), whose activity is in
R160W
turn induced by binding of SF/kit/stem cell factor
to c-Kit receptor121 (Fig. 72-8). Phosphorylated MITF R151C R160W Intracellular
binds to another protein, p300/CBP, which belongs
to a coactivator family of proteins and acts to enhance Loss of function mutants associated with red hair
MITF transcriptional activity.121,122 Another kinase that and melanoma
is activated by SF/c-Kit interaction is p90RSK that also Common in red head or blond light skin
phosphorylates MITF but at a different site from that
phosphorylated by MAP kinase-2.123 These phosphor- Figure 72-9 The melanocortin receptor (MCR) and its
ylation events both activate MITF and at the same time ligands. A. Structure of the proopiomelanocortin precur-
decrease the stability of the protein, as phosphory- sor. Standard abbreviations for amino acids are used. The
lated MITF is targeted for degradation by proteosomes synthetic superactive α-melanocyte-stimulating hormone
(eFig. 72-8.1A in online edition).123,124 (α-MSH) analogue [Nle4 D-Phe7]-α-MSH is modified by
The expression of MITF is under the control of sev- the exchange of methionine (M) with norleucine and L-
eral transcription factors, including Sox10 (mutated in phenylalanine (F) with D-phenylalanine. In red are critical
Waardenburg’s syndrome type 4, see Chapter 73) and amino acids required for binding to the MCR. B. Schematic
Pax3. MITF expression is also controlled by the cAMP- representation of the human MC1R receptor. Each of the
response element binding protein (CREB) and Lef1 318 amino acid residues in the polypeptide chain of the
transcription factor that participates in Wnt-signaling. receptor is represented by an empty circle. Branched
These transcription factors bind to specific sites within structures represent N-linked glycosylation sites. Reduced
function mutants (red circles), variants common in red- or
MITF promoter regions to induce MITF transcription.116
blond-haired and fair skinned individuals (orange circles),
The promoter region of the MITF gene contains a cAMP- and the conserved C-terminal cysteine (green circle), the
response element (CRE) that interacts with CREB when possible site for fatty acid acylation and anchoring to
the cAMP-dependent pathway is activated.125,126 There- the plasma membrane, are indicated. Ac = acetylated;
fore, cAMP-elevating agents like α-MSH induce the ACTH = adrenocorticotropic hormone; NH2 = amidated;
772 expression of MITF (eFig. 72-8.1B in online edition). TM = transmembrane domain.
 Eumelanin Pheomelanin
11

Chapter 72
A B

::
Figure 72-10 Eumelanin and pheomelanin presentation in mice. A. Two mice with different coat colors are shown. The

Biology of Melanocytes
one on the left displays brown/black coat color due to eumelanin, and the one on the right displays red/yellow coat color
due to pheomelanin. B. Representative hair shafts of these mice. (From Sharov et al: Bone morphogenic protein (BMP)
signaling controls hair pigmentation by means of cross-talk with the melanocortin receptor 1 pathway. PNAS 102:93, 2004,
with permission.)

different conditions, MITF can induce p21 expression protein expressed in both humans and mice, whose
and it can also stimulate the expression of p16INK4a, expression in mice leads to yellow coat color, antago-
a protein that inhibits the activation of kinases nizes α-MSH by competitive binding to MC1R. It thus
required for progression through the cell cycle, thus blocks adenylate cyclase activation144–146 and favors
promoting cell cycle arrest.135,136 Because MITF coop- pheomelanin over eumelanin synthesis (Fig. 72-10).
erates with other transcription factors to induce its However, the role of agouti in human pigmentation is
effects, it is to be expected that these transcription poorly documented.
factors would influence MITF activity, resulting Polymorphisms within the MC1R gene are largely
in either stimulation or inhibition of melanocyte responsible for the different skin/hair color among dif-
proliferation. ferent racial groups.147 At least 30 MC1R variants have
Mice bearing null mutations of MITF display loss of been identified and nine of them display loss of func-
melanocytes, deafness and failure of differentiation of tion148,149 (Fig. 72-9B), not being able to induce intracel-
retinal pigment epithelium.12 lular cAMP production in response to α-MSH despite
adequate receptor/ligand binding. Other MC1R vari-
Melanocortin 1 Receptor. Melanocortin recep- ants have reduced affinity for α-MSH.148,149 Three
tors (MCRs) comprise a family of five related receptors MC1R variants, each with only a single amino acid
(MC1R, MC2R, MC3R, MC4R, and MC5R). Each has substitution, have been associated with red/yellow
seven transmembrane domains and they belong to the hair and fair skin150 of Northern Europeans and Aus-
G-protein-coupled receptor superfamily.137 MC3R and tralians.151–156 Mice expressing a loss-of-function MC1R
MC4R are mainly found in the central nervous system, variant receptor also fail to respond to UV irradiation
are absent in melanocytes,138 and are thought to control with increased pigmentation despite an increased
energy intake. MC2R is expressed in the adrenal cor- level of epidermal α-MSH,157 but do tan if provided
tex and MC5R is expressed in peripheral adipocytes.139 forskolin, a chemical enhancer of pigmentation that
MC1R is expressed in a number of cells such as endo- bypasses the receptor to directly increase cAMP, dem-
thelial cells, fibroblasts,140 and keratinocytes,140 but the onstrating that the intracellular melanogenic pathway
highest expression is found in melanocytes.140 is functional in such individuals.155
α-MSH and adrenocorticotropic hormone (ACTH),
a 39 amino acid proopiomelanocortin-derived
peptide that contains the α-MSH sequence (Fig. MELANIN BIOSYNTHESIS
72-9A),141–143 activate MC1R (Fig. 72-9B) Receptor–
ligand interaction leads to G-protein-dependent acti- Two types of melanins are synthesized within melano-
vation of the enzyme adenylate cyclase followed by somes: (1) eumelanin and (2) pheomelanin.158 Eumela-
increased intracellular cAMP level141 inducing MITF nin is dark, brown–black, and insoluble, whereas
transcription and upregulating the level of melano- pheomelanin is light, red–yellow sulfur-containing,
genic proteins including tyrosinase40 promoting the and soluble.158 Melanins are indole derivatives of
synthesis of brown/black eumelanin.141 Agouti, a 3,4 di-hydroxy-phenylalanine (DOPA) and they are 773
11 Melanin biosynthesis
DOPA is oxidized into DOPAquinone,162 DOPAqui-
none is further converted to DOPAchrome and DOPA-
chrome can be converted to 5,6-dihydroxyindole (DHI)
Tyrosine
or to 5,6-dihydroxyindole-2-carboxylic acid (DHICA).
Tyrosinase The latter reaction is catalyzed by the enzyme DOPA-
chrome tautomeras or TRP-2. The level of brown ver-
sus black eumelanin appears to correlate with DHI/
DOPA
DHICA ratio, with a higher ratio leading to the forma-
Tyrosinase tion of black eumelanin and a lower ratio to brown
eumelanin.163 DOPAquinone can also combine with
DOPAquinone
glutathione or cyteine to form cysteinylDOPA, which
then becomes the yellow/red, soluble, low-molecular-
weight pheomelanin.163 Interestingly, tyrosinase also
DHI DOPAchrome CysteinylDOPA
catalyzes a more distant step in melanin biosynthe-
sis, namely DHI conversion to indole-5,6-quinone. In
Section 11

Tyrosinase TRIP-2 mice, the enzyme TRP-1 (also called DHICA oxidase)
converts DHICA to indole-5,6-quinone carboxylic acid.
Indole 5,6-quinone
Indole 5,6-quinone Alanyl-hydroxy- However, the TRP-1 role in human melanin biosynthe-
carboxylic acid benzothiazine sis is not well established.
Tyrosinase The main function of melanin is to provide protec-
::

or TRIP-2 tion against UV-induced DNA damage by absorbing

Disorders of Melanocytes

and scattering UV radiation (280–400 nm). Accord-

Pheomelanin
ingly, energy absorption by melanin is maximal in
DHI melanin DHICA melanin
black brown red/yellow this portion of the electromagnetic spectrum, and
insoluble poorly soluble soluble decreases gradually across the visible light spectrum.
high MW intermediate MW low MW UV absorbed by melanin is converted into heat, a less
toxic form of energy.164 Still, in vitro studies conducted
by several investigators suggest that melanin’s capac-
ity to act as a sunscreen is limited and that melanin,
Figure 72-11 Melanin biosynthesis. Melanin biosynthesis when incorporated into a cream and spread over the
begins with the amino acid tyrosine that is converted to
skin, absorbs only 50%–75% of incident sunlight. Nat-
DOPA (3,4-dihydroxyphenylalanine) in the rate-limiting
urally, it is possible that in vivo, by virtue of localizing
step of melanin biosynthesis catalyzed by tyrosinase.
DOPA is subsequently converted to DOPAquinone by above the nucleus, melanin in melanosomes achieves a
the same enzyme. DHI (5,6-dihydroxyindole) and DHICA higher level of protection.
(5,6-dihydroxyindole-2-carboxylic acid) are then formed Melanin intermediates as well as melanin itself
to produce either black or brown eumelanin. Alternatively, can also be harmful to the cell because, depend-
through incorporation of glutathione or cysteine, DOPA- ing on their molecular weight and polymerization
quinone can form pheomelanin. MW = molecular weight; state, they can promote UVA (320–400 nm)-induced
TRP = tyrosinase-related protein. DNA damage, most likely through the generation
of ROS.165 It has been suggested that the increased
incidence of UV-induced melanomas in light-
skinned, red-hair individuals is not only due to
formed in melanosomes through a series of oxidative decreased ability of pheomelanin to protect against
steps159 (Fig. 72-11). Melanosomal pH affects the activ- UV-induced DNA damage, but may also be due to
ity of the melanogenic enzymes and influences mela- mutagenic capacity of pheomelanin and possibly
nin polymerization. other melanin intermediates as a result of their pro-
The synthesis of both types of melanin involves the oxidant capacity.166
rate-limiting catalytic step in which the amino acid
tyrosine is oxidized by the enzyme tyrosinase (also
called tyrosine oxidase, DOPA oxidase, monophenol, MELANOCYTE DENDRITES
DOPA: oxygen oxidoreductase) to DOPA, a first step
in a reaction known as the Raper–Mason pathway160 Melanocyte dendrites are branching protoplasmic
(Fig. 72-11). Conversion of tyrosine to DOPA is thought processes that interact with keratinocytes. Actin is a
to be the critical rate-limiting step in melanogenesis, as major structural component of melanocyte dendrites,
inhibition of this reaction blocks melanin synthesis.161 and actin filament disruption leads to dendrite loss.167
In both reactions, DOPA acts as a cofactor and also as a Cocultures of keratinocytes and melanocytes demon-
substrate for tyrosinase. Although the exact interaction strate that keratinocyte-derived factors play a role in
between tyrosinase and its substrates is not completely melanocyte dendricity.168 These factors include ET-1,
understood, in vitro kinetic studies suggest that dis- nerve growth factor (NGF), α-MSH, ACTH, prosta-
tinct sites mediate tyrosinase binding to tyrosine and glandins (PGs) E2 and F2α168 and β-endorphin.169 Inte-
to DOPA, and that binding to DOPA causes a confor- grins, receptors that mediate actin-extracellular matrix
mational change in tyrosinase, resulting in increased contact are likely to play a role in dendrite formation
774 affinity for both tyrosine and DOPA. as well.170
 Another group of proteins, the Rho family, also
plays a role in melanocyte dendrite formation. Rho
tional,181 consistent with a cooperative forward and
backward pull of kinesin173 and dynein,176 respectively.
11
proteins become active when they bind GTP and inac- For melanosomes with net centrifugal movement, the
tive when binding GDP.171,172 It appears that when Rho bidirectional movement appears to terminate with
is activated, dendrites retract; while when its family myosin-Va (encoded by dilute locus)-dependent mela-
member Rac is activated, dendrites form.172 Indeed, nosomal capture in the actin-rich periphery of the den-
it is currently assumed that by increasing cAMP lev- drite (Fig. 72-12).181
els, α-MSH inhibits Rho, enhancing melanocyte den- Additional proteins that participate in melanosome
dricity. Thus, the equilibrium between Rho and Rac transport include Rab27a (encoded by ashen locus) that
appears to be an important factor influencing melano- mediates myosin-Va binding to melanosomes through
cyte dendricity. another linker protein-melanophilin (encoded by
leaden locus) (Fig. 72-12).182 In the absence of myosin-
Va, melanosomes do not collect in dendrite tips.
MELANOSOME TRANSPORT Mutations in any of the above gene products results
in decreased cutaneous pigmentation. Griscelli syn-

Chapter 72
WITHIN MELANOCYTES drome, a rare autosomal recessive disorder in which
individuals display dilute skin and hair color, is the
Melanosomes are transferred from their site of origin result of mutations of myosin-Va, Rab27a, or mela-
in melanocyte perikaryon to the tips of melanocyte nophilin182 (see Chapter 73). Myosin-Va and Rab27a
dendrites. Melanosome transport takes place on micro- are closely located on chromosome 15.183–186 Because

::
tubules that are arranged parallel to the long axis of the myosin-Va is also expressed in the brain, mutations

Biology of Melanocytes
dendrite and is controlled by two classes of microtu- of this gene may also cause neurological abnormali-
bule-associated motor proteins: (1) kinesins173–175 and ties. Rab27a also plays a role in immuno-regulation
(2) cytoplasmic dyneins176–180 (Fig. 72-12). Both motor and individuals with mutations of this gene display
proteins act as short cross-bridge structures connecting abnormalities of the immune system. Mutations of
the organelle to the microtubules. melanophilin result only in the distinctive hypopigmen-
Centrifugal, anterograde organelle movement is tation that characterizes the syndrome.186
mediated primarily by kinesin, whereas centripetal
movement is controlled by cytoplasmic dynein. Stud-
ies examining melanosomal transport suggest that TO KERATINOCYTES
their microtubule-dependent movement is bidirec-
Transfer of melanosomes from melanocytes to neigh-
boring keratinocytes is a critical step in normal
pigmentation. Studies suggest several ways for mela-
Melanosome transport across melanocyte dendrites nosomal transfer, including exocytosis, cytophagocy-
tosis, fusion of plasma membranes, and transfer by
Melanocyte
dendrite tip
membrane vesicles.187
The exocytosis pathway of melanosomal trans-
fer involves fusion of the melanosomal membrane
with the melanocyte plasma membrane, melanosome
release into the intercellular space and phagocytosis by
Melanosomes Rab27 surrounding keratinocytes. Cytophagocytosis is a term
MIph
indicating the phagocytosis of a live cell or a portion of
it. With regard to keratinocytes, they cytophagocytose
the tip of a melanocyte dendrite, which then fuses with
MyoVa lysosomes inside the keratinocyte, is transported to a
Dynein Kinesin supranuclear location where the phagolysosome mem-
Actin branes break up releasing the melanosomes. Fusion of
keratinocyte and melanocyte plasma membranes cre-
ates a space through which melanosomes are trans-
ferred from the melanocyte to the keratinocyte. Indeed,
high-resolution photography shows the presence of
filopodia—slender, filliform, pointed cytoplasmic pro-
jections at the tip of melanocyte dendrites.188 These
filopodia adhere and fuse with keratinocyte plasma
Figure 72-12 Schematic diagram of melanosome trans- membrane prior to melanosome transfer. Another
port across melanocyte dendrites. Melanosomes move
way of melanosomal transfer involves shedding of
bidirectionally along melanocyte dendrites. They are
attached to microtubules through the motor proteins melanosome-filled vesicles followed by phagocytosis
kinesin (anterograde) and dynein (retrograde). At the tip of these vesicles by keratinocytes, or their fusion with
of the dendrite, melanosomes are captured in the actin- keratinocyte plasma membrane.
rich periphery. Myosin-Va (MyoVa) mediates melanosome The molecular and cellular mechanisms involved
binding to actin through the linker proteins Rab27a and in melanosome phagocytosis have been partially
melanophilin (Mlph). elucidated. It appears that keratinocytes express a 775
11 seven-transmembrane G-protein coupled receptor
called protease activated receptor-2 (PAR-2). PAR-2
ENDOTHELIN-1. ET-1 appears to play a role in
mature melanocytes, inducing melanogenesis by acti-
is activated when serine proteases cleave the extra- vating tyrosinase and increasing TRP-1 levels.203,204
cellular portion of the receptor, exposing a new seg- ET-1 also leads to melanocyte proliferation203,204 and
ment that acts as a tethered (attached) ligand.189,190 promotes dendrite formation.205 Cultured keratino-
Activation of PAR-2 increases keratinocyte phago- cytes synthesize and secrete ET-1,204–206 and UV irradia-
cytic activity.189,190 tion stimulates ET-1 production by keratinocytes.204,205
Interestingly, and consistent with its role in mela- ET-1 can also cooperate synergistically with other
nosome phagocytosis, UV induces the activity and growth factors/cytokines to further influence melano-
expression of PAR-2.191 UV effect on PAR-2 activity cyte function.
and expression is more pronounced in individuals ET-1 upregulates MC1R level and increases MC1R
with skin phototypes II and III than in those with skin affinity for α-MSH.207,208 Similar to α-MSH, ET-1 dis-
phototype I.191 Keratinocyte growth factor receptor plays photoprotective effects on melanocytes, enhanc-
has also been implicated in enhancing phagocytosis of ing thymine dimer repair, decreasing the level of
melanosmes by keratinocytes.192
Section 11

UV-induced hydrogen peroxide, and inducing the

level of antiapoptotic proteins.201,209
REGULATION OF MELANOCYTE STEEL FACTOR (SF). Like other keratinocyte-
FUNCTION derived factors, SF is induced by UV-irradiation, and
in guinea pigs, anti-Kit antibodies block UV-induced
::

Melanocyte behavior in skin is largely influenced by tanning. SF can also act synergistically with other
Disorders of Melanocytes

signals from neighboring keratinocytes as well as auto- cytokines such as IL-3, IL-6, IL-7, IL-9, and granulo-
crine signals and environmental factors such as UV cyte-macrophage-colony stimulating factor to regu-
irradiation (also see Section “UV Irradiation and Mela- late UV-induced melanogenesis and melanocyte
nocytes”). The synthesis and secretion of most kerati- survival.210,211
nocyte-derived factors is increased by UV irradiation,
but it is also evident that UV can directly stimulate
INFLAMMATORY MEDIATORS. Several inflam-
matory mediators can affect skin pigmentation.
melanocyte dendricity and melanin production.171,193
PGs—arachidonic acid-derived metabolites, and
Melanocytes receive both positive and negative para-
leukotrienes—lipid compounds related to PGs, both
crine signals that modulate their proliferation and dif-
mediators of inflammatory responses, affect mela-
ferentiated function.
nocyte function. Their level is elevated in sunburned
skin212 and in a variety of inflammatory dermatoses,
MELANOGENIC STIMULATORS including atopic dermatitis213 (see Chapter 14) and
psoriasis214 (see Chapter 18).
PROOPIOMELANOCORTIN AND DERIVED Human melanocytes express several PG recep-
PEPTIDES. It is well documented that MSH and tors including the receptors for PGE2 and PGF2α.215,216
ACTH are potent stimulators of melanogenesis. Indeed, PGF2α stimulates melanocyte dendrite forma-
They belong to a family of peptides derived from tion and activates tyrosinase,215,216 and UV irradiation
the precursor proopiomelanocortin (POMC) that is upregulates the level of PG receptors on melano-
synthesized, in addition to the pituitary gland, also cytes.215,216 Similarly, leukotrienes B4 and C4 increase
by epidermal keratinocytes. Interestingly, POMC melanin synthesis and stimulate melanocyte prolif-
expression in keratinocytes is induced by UV, phor- eration and motility.217 Interestingly, melanocytes also
bol esters, and interleukins.194,195 In rodents, α-MSH contribute to cutaneous inflammatory responses, as
stimulates melanogenesis and favors eumelanin they synthesize and release IL-8 when stimulated by
over pheomelanin production, but systemic admin- the proinflammatory cytokines IL-1 and TNF-α.218
istration of α-MSH, β-MSH, and ACTH to people Melanocytes also respond to histamine released
increases skin pigmentation predominantly in sun- by mast cells during cutaneous inflammation. Hista-
exposed areas.196,197 However, in certain disease con- mine binds H1 and H2 receptors to induce melanocyte
ditions characterized by abnormally high levels of dendricity and upregulate tyrosinase level.219,220 These
ACTH, such as Addison disease198 or Nelson’s syn- effects are decreased when melanocytes are pretreated
drome199 (ACTH secreting pituitary adenoma), more with the H2 receptor antagonist famotidine.220
generalized hyperpigmentation of the skin has been
observed.200 NEUROTROPHINS. Neurotrophins (NTs) are
Aside from its effect on melanogenic proteins and a family of molecules that enhance neuronal sur-
eumelanin synthesis, α-MSH was also reported to vival in the central and peripheral nervous sys-
enhance the repair of UV-induced DNA damage in tems. They include NGF,221 NT-3,222–224 NT-4,225 and
melanocytes, specifically the repair of pyrimidine brain-derived neurotrophic factor.226,227 Melanocytes
dimers, and also to reduce the level of UV-induced express the low-affinity receptor common to all
hydrogen peroxide in the cell.201 In addition, α-MSH NTs, p75NTR,228 as well as the high-affinity receptors
was shown to regulate melanosomal pH.202 These data for NGF (TrkA) and NT3 (TrkC).229 Keratinocyte-
suggest a role for POMC-derived peptides beyond derived NGF, whose expression is upregulated by
776 merely stimulating melanogenesis. UV irradiation, is chemotactic for melanocytes and
induces their dendricity.230 Both NGF and NT-3,
the latter expressed by dermal fibroblasts, increase
SIGNALING PATHWAYS
11
melanocyte survival. Specifically, after UV irradia- REGULATING MELANOCYTE
tion, NGF supplementation increases the level of the
antiapoptotic Bcl-2 protein, reducing melanocyte
FUNCTION
apoptotic cell death.231,232 Thus, in addition to other
keratinocyte-derived cytokines, NGF may help pre- Growth factors, cytokines, hormones, and other
serve the population of cutaneous melanocytes that ligands for receptors expressed on melanocytes exert
would otherwise be depleted by UV damage. their biologic effect by interacting with their specific
cell surface receptors, generating a signaling cascade
BASIC FIBROBLAST GROWTH FACTOR. Basic involving activation or inhibition of protein kinases
fibroblast growth factor (bFGF), named for its ability to and leading to distinct patterns of protein phosphory-
stimulate the growth of fibroblasts, was one of the first lation. Two types of kinases participate in cellular sig-
identified melanocyte mitogens.233,234 It is produced by naling: (1) serine/threonine kinases and (2) tyrosine
keratinocytes, but lacks a secretory signal and hence is kinases that by definition phosphorylate serine and/or

Chapter 72
presumed to affect melanocytes through cell–cell con- threonine residues and tyrosine residues, respectively,
tact. It binds tyrosine kinase transmembrane receptors on their specific target proteins. This section reviews
to induce its mitogenic effect in the presence of cAMP the major signaling pathways that affect melanocyte
elevating factors. Like other keratinocyte-derived behavior in skin.
cytokines, it is upregulated in response to UV irradia-

::
tion.234,235 Keratinocyte growth factor, another member cAMP/PKA-DEPENDENT PATHWAY

Biology of Melanocytes
of the FGF family of proteins, has been shown to pro-
mote melanosome transfer from melanocytes to kera- cAMP, one of the first identified intracellular second
tinocytes.192 messengers, plays a key role in diverse biological func-
tions such as cellular metabolism, growth, and dif-
NITRIC OXIDE. Nitric oxide (NO) is a diffusible free ferentiation.253,254 It also mediates α-MSH effect255 and
radical displaying pleiotropic bioregulatory effects in was one of the first recognized regulators of mamma-
diverse cells and tissues.236,237 Melanocytes and kera- lian pigmentation256 The intracellular level of cAMP
tinocytes produce NO in response to inflammatory is upregulated by a membrane-associate enzyme
cytokines,238–241 and NO production in keratinocytes is called adenylate cyclase that is activated upon recep-
induced by UV irradiation.242 NO increases tyrosinase tor–ligand interaction in receptors that are coupled
activity and melanogenesis242 and is thus an autocrine to GTP-binding proteins like MC1R257 (eFigs. 72-8.1
as well as paracrine molecule that affects melanocyte and 72-12.1 in online edition). cAMP is also elevated
behavior in skin. by reagents such as choleragen or isobutylmethylxan-
thine. Providing melanocytes with dibutyryl cAMP, a
CATECHOLAMINES. Catecholamines are a group cAMP analog, increases the intracellular level of cAMP
of signaling molecules, primarily functioning as neu- and induces signaling that leads to melanogenesis.258
rotransmitters and as endocrine hormones.243 Cate- cAMP-dependent protein kinase (PKA) mediates
cholamines bind either α1-adrenergic receptors (AR) or most of the biologic actions of cAMP.257 PKA is a serine/
β2-AR and can induce melanogenesis through PKC-β threonine kinase consisting of two regulatory subunits
or cAMP-dependent pathways.169,244 and two catalytic subunits.257 It exists in the cytosol in
an inactive form and binding of cAMP to its regulatory
subunits releases the catalytic subunits, activating the
MELANOGENIC INHIBITORS enzyme.257 PKA phosphorylates the cAMP responsive
element-binding protein (CREB) that binds its DNA con-
Numerous reports have suggested the existence of sensus sequence CRE in the MITF promoter to induce
endogenous melanogenic inhibitors,245,246 but only MITF transcription (eFig. 72-8.1 in online edition). cAMP
few specific molecules have been identified. One elevation also affects other target genes by increasing or
group of inhibitors includes sphingolipids, a class of decreasing their transcription259 (eFig. 72-12.1 in online
membrane-associated lipids247 that act as signal trans- edition). In vitro, PKA effect can be antagonized by the
ducers. Sphingolipids were shown to decrease melano- inhibitor PKI that acts as a pseudosubstrate for the cat-
genesis, at least in part by enhancing MITF degradation alytic subunit of PKA and thus prevents it from phos-
via ubiquitin-meditated pathways.248,249 Another mela- phorylating its endogenous substrates.260
nogenic inhibitor, BMP-4, downregulates tyrosinase
expression in melanocytes,250 also in part via its effects
on MITF.251 Interestingly, physiologic doses of UV irra- PKC-DEPENDENT PATHWAY
diation, a potent melanogenic stimulator, decrease the
expression of BMP receptors on melanocytes,250 pre- PKC is a serine/threonine kinase involved in diverse
sumably eliminating its inhibition during UV-induced cellular functions, including growth, transforma-
tanning. Mice that transgenically overexpress the tion, and differentiation.261 PKC resides as an inactive
physiologic BMP antagonist noggin have a darker coat enzyme in the cytoplasm, and it is activated by diacyl-
color than wild-type mice and their hairs have a higher glycerol (DAG), a component cleaved from the plasma
eumelanin to pheomelanin ratio.252 membrane when cell surface receptors interact with 777
11 their ligands. DAG can also be released from the mem-
brane by UV irradiation (eFig. 72-12.1 in online edi-
to melanin synthesis.244 Therefore, numerous path-
ways may act in tandem to regulate melanogenesis.
tion). DAG induces PKC translocation to membranes
where the latter is activated261 to induce phosphory-
lation of serine/theonine residues on target proteins UV IRRADIATION AND
like tyrosinase. Phorbol esters mimic DAG action and MELANOCYTES
initially activate PKC.261 However, within 24 hours the
entire cellular reserves of PKC are depleted, and when
melanocytes are treated with phorbol esters they can TANNING RESPONSE
no longer signal through PKC.87
The critical role of PKC in melanogenesis was first Melanocyte survival, proliferation, and differentiated
suggested by the observation that addition of DAG, function are influenced by environmental factors, the
the endogenous activator of PKC, to cultured human most important of which is UV irradiation. UV irra-
melanocytes rapidly increased total melanin content,262 diation induces tanning, the so-called facultative skin
and this increase was blocked by a PKC inhibitor.262 color, an increase above baseline or constitutive skin
Section 11

Moreover, topical application of DAG to guinea pig pigmentation that provides protection against future
skin increased epidermal melanin content.263 UV irradiation.271 Tanning is divided into immediate
The expression of the 12 PKC isoforms varies among tanning and delayed tanning.
different tissues.86 Each isoform is thought to carry
out a distinct biological function. Human melanocytes IMMEDIATE TANNING. Immediate tanning or
::

express PKC-α, -β, -ε, -δ, and -ς264,265 and the PKC-β iso- immediate pigment darkening occurs within 5–10
Disorders of Melanocytes

form is specifically involved in regulating tyrosinase minutes of exposure and fades within minutes to days
activity (see above). ET-1 and histamine also utilize depending on the UV dose and the complexion of the
the PKC-dependent pathway (in addition to cAMP- individual (Fig. 72-13B). As summarized in Table 72-1,
dependent pathway) to exert their regulatory effects immediate tanning does not provide photoprotection
on melanocyte function.266,267 and does not increase epidermal melanin level.272 It is
primarily produced by UVA irradiation, although vis-
ible light can also induce immediate tanning.273 Imme-
RECEPTOR TYROSINE KINASES diate tanning is only visible in darker individuals, and
it is thought to represent melanosomal relocation from
Melanocytes express several distinct tyrosine kinase the perikaryon to melanocyte dendrites.274
receptors that bind BMP, bFGF, HGF, and c-Kit ligand.
Receptor–ligand interaction activates an intracellular DELAYED TANNING. Delayed tanning, summa-
tyrosine kinase domain on the receptor, phosphorylat- rized in Table 72-1 and shown in Figure 72-13A, occurs
ing the receptor and subsequently activating a series of within 3–4 days after UV exposure.271,272 UV is arbi-
kinases called mitogen-activated protein (MAP) kinases, trarily divided into UVC (100–280 nm), UVB (280–320
or other intracellular signaling molecules (eFig. 72-12.1 nm), and UVA (320–400 nm). The UVC portion of the
in online edition). Through a chain reaction involving spectrum is generally not present in terrestrial sunlight
phosphorylation of proteins like MITF, the signals are because it is absorbed by the atmospheric ozone layer.
transferred to the nucleus to activate or suppress the Delayed tanning is affected by both UVB and UVA.
transcription of genes that participate in melanocyte The action spectrum that produces delayed tanning
proliferation, melanogenesis, and/or survival. is the same as for UV-induced erythema (sunburn),
with UVB wavelengths far more effective than UVA.264
Especially in darker skinned individuals, suberythe-
β2- AND α1-ADRENERGIC RECEPTORS mogemic UV doses may be effective as well. Delayed
tanning peaks between 10 days and 3–4 weeks depend-
Studies suggest that pathways that increase intracel- ing on the absorbed UV dose and the individual’s skin
lular cAMP are also involved in regulation of mela- type, then fades gradually over a few weeks. Histo-
nogenesis. POMC-deficient mice (POMC −/−) that lack logically, there are increased epidermal melanocytes,
melanocortin ligands still display normal black coat melanocyte dendriticity, and melanosome transfer to
color.269 Histological and electron paramagnetic reso- keratinocytes with greater melanization of individual
nance spectrometry of the hair follicles showed nor- melanosomes.272,274 Overall, total epidermal melanin is
mal structure and eumelanin pigmentation.269 This increased, providing additional photoprotection from
study suggests that either MC1R has adequate basal UV irradiation.
activity to induce pigmentation or that pathways that
do not involve melanocortin can also induce melano-
genesis. Indeed, melanocytes express both β2-AR and DIRECT AND INDIRECT EFFECTS OF
α1-AR, respectively.270,244 α1-AR interacts with mela- UV IRRADIATION
nocyte derived-norepinephrine and increases the
level of DAG169,244 inducing melanogenesis in a PKC- UV irradiation affects melanization, melanocyte pro-
β-dependent pathway. Also, keratinocytes produce liferation and survival both directly and indirectly
epinephrine, which then binds to β2-AR expressed on through its effect on keratinocytes, inducing the syn-
778 melanocytes and increases the level of cAMP, leading thesis and secretion of paracrine keratinocyte factors.
 11

Chapter 72
::
Biology of Melanocytes
A B

Figure 72-13 A. Delayed tanning. Four template test sites in a phototype III individual were exposed to repeated ery-
themogenic doses of ultraviolet B (UVB) (+UVA) delivered in 24-hour intervals, and the photograph was taken 10 days
after the last exposure. The tan in the more heavily pigmented test sites persisted for 2 months. B. Immediate tanning in a
phototype III individual. Four template test sites were exposed to various doses of UVA, and the photograph was taken 2
hours after the end of exposure. After 48 hours, the tan had almost completely faded.

DIRECT EFFECTS. UV irradiation triggers several assumed to be the result of oxidative damage mediated
biological reactions through its interaction with cellu- through UVA absorption by cellular chromophores like
lar chromophores that absorb photons. Photochemical melanin precursors that act as photosensitizers leading
reactions affect proliferation, survival, and the differ- to the generation of ROS and free radicals.275 UVB irra-
entiated function of melanocytes. Most UVA effects are diation is directly absorbed by cellular DNA, leading

TABLE 72-1
Immediate Tanning versus Delayed Tanning

Immediate Delayed
Onset Minutes 3–4 days
Peak intensity Minutes to few hours 10–28 days
Fading Within 24 hours Weeks
Mechanism Redistribution of melanosomes ↑Keratinocyte-derived melanogenic cytokines
↑Tyrosinase level and activity
↑Melanin synthesis
↑Melanocyte dendriticity
↑Melanosome number
↑Melanosome transfer
↑Melanocyte proliferation
Photoprotection Unchanged Increased
Change in skin color Undetectable in fair skin Obvious in most light-skinned and all dark-skinned
Subtle in darker skin individuals

From Dillman AM: Photobiology of skin pigmentation. In: Pigmentation and Pigmentary Disorders, edited by N Levine. Boca Raton, CRC Press,
1993, pp. 61-94; Gilchrest BA et al: Mechanisms of ultraviolet light-induced pigmentation. Photochem Photobiol 63(1):1-10, 1996; Ortonne JP: The
effects of ultraviolet exposure on skin melanin pigmentation. J Int Med Res 18(Suppl. 3):8C-17C, 1990; and Sturm RA: Human pigmentation genes
and their response to solar UV radiation. Mutat Res 422(1):69-76, 1998. 779
11 to the formation of DNA lesions, mainly cyclobutane
dimers and pyrimidine (6–4) pyrimidone photoprod-
on melanocytes, resulting in increased production of
eumelanin.286
ucts.276 DNA damage repair systems are activated, at It has also been shown that p53 transcriptionally
least in part through the tumor suppressor p53 pro- upregulates the hepatocyte nuclear factor-1α (HNF-
tein. It has been shown that plasma membrane lipids 1α) that is a transcription factor for tyrosinase.287 Thus,
are also affected by UV irradiation to release DAG,277 even in keratinocyte absence, UV directly activates
which then activates PKC-β to stimulate melanogen- p53 and HNF-1α in melanocytes to increase tyrosinase
esis by activating tyrosinase (see above). transcription. Furthermore, UV is known to increase
H2O2 that activates p53.288 Thus, tanning may be
INDIRECT EFFECTS. Key keratinocyte paracrine fac- viewed as part of a p53-mediated DNA damage adap-
tors induced by UV irradiation and their effects on mela- tive response aimed at protecting the skin from subse-
nocytes are summarized in eTable 72-1.1 in online edition. quent UV irradiation.282,283,289
These factors can act alone and/or synergistically to mod-
ulate melanocyte function. Interestingly, UV irradiation
also induces the level of TNF-α and IL-1, cytokines that MELANOCYTE AGING AND
Section 11

inhibit melanogenesis, suggesting a fine-tuned epider- PHOTOAGING

mal equilibrium between melanogenic stimulators and
inhibitors after UV irradiation, with the final outcome of
Epidermal melanocyte aging is affected by both
increased melanogenesis and melanocyte proliferation.
genetic and environmental factors. With aging, there
::

is a decrease in the density of epidermal melanocytes

THE ROLE OF DNA DAMAGE IN (number per unit area of skin surface), approximately
Disorders of Melanocytes

MELANOGENESIS 10% per decade.290 However, the number of DOPA-

positive melanocytes is greater in chronically sun-
Interestingly, the action spectrum for tanning is vir- exposed skin than in sun-protected skin,290 possibly
tually the same as that for the formation of thymine due to melanocyte proliferation after sun exposure
dimers,278,279 and UV-induced melanogenesis can be and/or UV-induced keratinocyte-derived paracrine
augmented in pigment cells by treatment with T4 endo- factors. Melanocyte loss is especially notable in hair
nuclease V,276 an enzyme that acts exclusively to enhance follicles with age, with total loss of melanocytes in
the repair of UV-induced DNA damage. Moreover, approximately half of all scalp follicles by middle
treatment of melanocytes with agents that act exclu- age.37 Hair graying (depigmentation) occurs over the
sively by damaging DNA, unlike UV that has multiple entire body but is usually first noted on the scalp, per-
cellular targets, also stimulates melanogenesis.280 haps because of the long anagen (growth) cycle and
A central role for DNA damage and/or its repair resulting requirement for melanocyte proliferation and
in stimulating melanogenesis is further suggested sustained high level of melanogenesis.
by the fact that p53, a tumor-suppressor protein In vitro melanocytes derived from older indi-
and transcription factor termed the Guardian of the viduals show decreased proliferative capacity com-
Genome, when activated, upregulates the level of pared to those derived from younger individuals.
tyrosinase mRNA and protein, enhancing melano- Also, with aging in vitro, there is a general increase
genesis.281–284 Thus, tanning may be viewed as part of in the levels of total melanin as well as in the level
a p53-mediated DNA damage adaptive response that differentiation-associated proteins such as MITF,
protects the skin during subsequent exposure to UV TRP-1, and TRP-2291,292 and decrease in the level of
irradiation. proliferation-associated proteins such as cyclin D1
and E.291,292
p53 IN UV-INDUCED MELANOGENESIS. A
central role for DNA damage and/or its repair in stim- KEY REFERENCES
ulating melanogenesis is further supported by the fact
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upregulates the level of tyrosinase mRNA and protein,
enhancing melanogenesis.281–285 In p53 knockout mice, DVD contains references and additional content
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lates melanogenesis, compared to the far greater “tan- 23. Mizoguchi M: Melanocyte development: With a message
ning” response in p53 wild-type mice.282 These findings of encouragement to young women scientists. Pigment
were expanded by Cui et al,286 who found a p53 con- Cell Res 17(5):533-544, 2004
39. Ito S: Biochemistry and physiology of melanin. In: Pig-
sensus sequence in the POMC gene promoter, thus mentation and Pigmentary Disorders, edited by N Levine.
establishing a continuous signaling pathway from UV- Boca Raton, FL, CRC Press, 1993, pp. 34-59
induced DNA damage to the final tanning response. 40. Slominski A et al: Melanin pigmentation in mammalian
It was shown in mice keratinocytes that following UV skin and its hormonal regulation. Physiol Rev 84(4):1155-
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65. Park HY, Gilchrest BA: Signaling pathways mediating
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α-MSH, a key physiologic inducer of melanogenesis. 171. Scott G. Rac and rho: The story behind melanocyte den-
780 Keratinocyte-derived α-MSH then stimulates MC1R drite formation. Pigment Cell Res 15(5):322-330, 2002