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Mechanism of skin pigmentation

Article  in  Biotechnology and Bioprocess Engineering · August 2008

DOI: 10.1007/s12257-008-0143-z

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Biotechnology and Bioprocess Engineering 2008, 13: 383-395
DOI/10.1007/s12257-008-0143-z

Mechanism of Skin Pigmentation

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Department of Biological Engineering, Inha University, Incheon 402-751, Korea =
Abstract Melanin is a pigment that plays an important role in providing coloration and protecting human skin from the harm-
ful effects of UV light radiation. Human skin color is determined by the type and amount of melanins that are syn-
thesized and deposited within the melanosomes. In addition, the transfer of these specialized membrane-bound
organelles from melanocytes to surrounding keratinocytes also plays a role in dictating human skin color. In order
to investigate the principle features of skin pigmentation, the origin, function, and production ability of melanin
should be highly understood in terms of biological and pathophysiological aspects. Furthermore, a deep under-
standing of melanin synthesis will also contribute to cosmetics and drugs development. In this review, the proc-
esses of melanin biosynthesis, such as survival, proliferation, and differentiation of melanin cells, as well as the
biological regulation of human pigmentation were described. © KSBB

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INTRODUCTION
Melanin plays an important role in protecting human skin
from the harmful effects of UV sun radiation and in scaveng-
ing toxic drugs and chemicals. Visible pigmentation of the
skin, hair, and eyes depends primarily on the different func-
tions of melanocytes, a minor subset of cells that specialize
in the synthesis and distribution of the pigmented biopoly-
mer melanin. Melanocytes are derived from precursor cells
(called melanoblasts) during embryological development,
and melanoblasts destined for the skin originate from the
neural crest. The accurate migration, distribution, and func-
tion of melanoblasts/melanocytes determine the visible phe-
notype of organisms ranging from simple fungi to the most
complex animal species.
In this review, we will report the most recent findings in
the pigmentation system of the skin. Understanding the
process of producing melanin will provide the basic steps for
elucidating the mechanism of pigment-related diseases such
as melasma. Recently there has been a surge in the market
for pharmaceutical or functional cosmetics. This review em-
phasizes the mechanism of the pigmenting process and the
target genes that controlling melanin production and melano-
some transfer. Signal transduction inside melanocytes and
cytokine-mediated signaling between the mealnocyte and
keratonocyte is also reviewed in relation to melanin produc-
tion.
*Corresponding author
Tel: +82-32-860-7514 Fax: +82-32-872-4046
e-mail: ekkim@inha.ac.kr
MELANIN PIGMENT IN HUMAN SKIN
PHYSIOLOGY
Epidermal melanin has important evolutionary and
physiological implications, particularly for unclothed hu-
mans. Racial pigmentation has a high content of melanin
content that can protect the skin from ultraviolet radiation
(UV) − induced skin damage through its optical and chemi-
cal filtering properties [1]. Melanin can absorb UV light,
scavenge free radicals generated within the cytoplasm, and
shield the host from various types of ionizing radiation as
well as determine skin color when combine with other pig-
ments. In humans, the variation in skin color occurs at the
function level of the epidermal melanin unit. Basically, skin
color is defined by the density of melanocytes, the number,
size, and dispersion of melanosomes transferred to epidermal
keratinocytes, the nature of the pigment and its degradation
rate [2].
CELL ORIGIN AND DEVELOPMENT OF
MELANOCYTES
Melanocytes are highly dendritic cells that originate in the
neural crest, which is a transient population of cells localized
in the dorsal portion of the closing neural tube. It derives the
melanocyte lineage by expressing the Microphthalmia-
associated transcription factor (MITF). Melanoblasts origi-
nate in the neural crest and migrate to the epidermis where
hair follicle bulges promote the differentiation of melano-
blasts into melanocytes [3-5].
384
The early signals that induce formation of the neural crest
include members of the Wnt, fibroblast growth factor (FGF),
bone morphogenetic protein (BMP) families, etc. The Wnt/β
-catenin signaling pathway is the first activated, followed by
KIT, ET3/endothelin B, and then the hepatocyte growth fac-
tor (HGF)/MET. It is also possible that simultaneous activa-
tion of multiple receptors is required for the differentiation of
the melanocyte precursor [6,7].
Wnts
Wnts are secreted glycoproteins that play a critical role in
melanocyte determination by activating the Frizzled receptor
family. Wnt signaling also stimulates quiescent melanocyte
stem cells to enter the cell cycle for the generation of new
stem cells [3]. Wnt controls β-catenin via its influence on the
Ser/Thr kinase activity of the key enzyme glycogen synthase
kinase 3β (GSK3β). In the absence of Wnt, β-Catenin is
recruited to a multimolecular protein complex that includes
Axin, APC, and the kinase GSK-3β. β-Catenin is pho-
sphorylated by GSK-3β, and subsequently ubiquitinated and
targeted for proteasomal degradation. If Wnt binds to its
receptor Frizzled and potential co-receptor LRP-5/6, GSK-
3β phosphorylation of β-Catenin is suppressed, which results
in the accumulation of β-Catenin that is then translocated to
the nucleus. In the nucleus, it binds to LEF/TCF transcrip-
tion factors, which results in the activation of Wnt target
genes [8].
The survival of melanoblasts during development as well
as the proliferation and differentiation of the melanocyte
stem cells is most likely controlled by MITF. MITF is a Wnt
target gene that encodes a critical regulator of the melano-
cyte specific genes and has been shown to increase the in-
duction of melanoblast [9]. MITF binds the canonical E-box
sequence CACGTG as well as the nonpalindromic sequence
CACATG. Its expression is upregulated by MSH through
cAMP signaling followed by CREB phosphorylation and
activation of the melanocyte-specific MITF promoter and
may regulate multiple pigmentation genes by upregulating
MITF expression. The three major pigmentation enzymes
tyrosinase, Trp-1, and Trp-2, all contain consensus MITF
DNA binding elements in their promoter region [8]. In addi-
tion to activating the expression of p16INK4a and p21Cip1
to induce cell cycle arrest, MITF is also required for the pro-
liferation of melanoma cells. The effects of MITF on prolif-
eration most likely depends on the status of its post-
translational modifications that will in turn dictate its ability
to regulate the transcription of different target genes [10,11].
Mutation will affect many of the factors that are involved in
regulating the melanocyte-specific MITF-M promoter,
which can lead to reduced numbers of melanoblasts.
Steel Factor/C-kit Receptor
The melanoblasts have expression markers that are cha-
racteristic of the melanocyte lineage, most notably is the re-
ceptor tyrosine kinase Kit (C-KIT) [9]. C-KIT plays a pivotal
role in the normal growth and differentiation of embryonic
Fig. 1. Binding of the growth factor to the receptor on melano-
cytes.
melanoblasts [12]. Consequently, the survival and migration
of neural crest-derived cells during embryogenesis depends
upon interactions between specific receptors (C-KIT recep-
tor − a tyrosinase kinase receptor) on the cell surface and
their extracellular ligands (steel factor − mast/stem cell
growth factor) (Fig. 1).
The C-KIT receptor is a tyrosine kinase that belongs to the
platelet-derived growth factor (PDGF) receptor family. The
steel factors interact with KIT receptors on the cell mem-
brane surface, which leads to the replacement of the phos-
phate group on tyrosine residues, tyrosine transfer from the
inactive to the active form, and the control of the growth and
differentiation of embryonic melanoblasts [6].
HGF/MET
During development, melanocyte viability and prolifera-
tion depends on activation of MET by its ligand HGF (hepa-
tocyte growth factor). Targeted ablation of the MET gene in
mice caused a complete loss of melanoblasts, beginning at
the late stages of neural crest migration. In contrast, overex-
pression of HGF in various tissues or specifically in kertino-
cytes enhanced the ectopic localization of melanoblast and
increased the melanocyte population in the dermis [13,14].
Endothelins (ET)/Endothelin Receptor B (EDNRB)
Endothelins are growth factors that originate from endo-
thelial cells. Mutations in these growth factors produce sig-
nificant neural crest defects, including melanocyte defi-
ciency. There are three endothelin proteins, ET1, ET2, and
ET3, and two endothelin receptors, EDNRA and EDNRB.
ET3 binds to EDNRB on melanoblast ganglion cell precur-
sors, which is essential for the survival and migration of
melanoblast. Mutations in either ET3 or EDNRB produce a
substantial loss of melanocytes in human and mice [15,16].
Cadherins and Migration to the Skin
Cadherins are calcium-dependent surface receptors that
mediate cell adhesion, homing, and invasion into different
layers. The expression of cadherins dynamically changes as
 Biotechnol. Bioprocess Eng. 385=

neural crest cells emerge from the neural tube. Cadherin ex-
pression is temporarily upregulated just prior to entering the
epidermis and is subsequently suppressed as migration con-
tinues out of the epidermis into hair follicles. Cadherins are
also regulators of invasion or metastatic behavior in melano-
cytic neoplasms [17,18].

LOCALIZATION, STRUCTURE, AND FUNCTION

OF MELANOCYTES

Melanocytes exist in various tissues in the body. They are

localized in the skin at the dermal-epidermal interface, in the
hair matrix, in the retinal pigment epithelium of the eye, the
uveal tract, the stria vascularis of the ear, the vestibular re-
gion of the inner ear, the leptomeninges, and mucous mem-
branes. In normal skin, melanocytes are located in the epi-
dermal layer and their dendrites are projected into the epi-
dermis where they transfer melanosomes to keratinocytes [4].
Approximately every tenth cell in the basal layer is a
melanocyte [6]. One melanocyte is associated with approxi-
mately 30~40 surrounding keratinocytes through its den-
drites. The entire unit is referred to as the “epidermal mela-
nin unit” (Fig. 2) [19].
The density of epidermal melanocytes varies at different
sites in the body. There are approximately 2,000 epidermal
melanocytes per square millimeter on the skin of the head
and forearm and approximately 1,000 on the rest of the body.
Individuals will experience an apparent change in the colora-
tion of their skin through the course of their life. Almost all
infants, including black infants, are lighter at birth and be-
come darker during the first week of postnatal life. The dor-
sal skin of the hand becomes mottled in color in old age as a
result of an age-induced decline in the number of epidermal
melanocytes. In addition, there is approximately an 8 to 10
percent reduction in the density of melanocytes for each dec-
ade of life in areas that are not exposed to sun light, except
for genital sites [20].
During embryogenesis, melanocytes diffuse throughout
the dermis. They first appear in the head and neck region at
approximately 10 weeks of gestation. However, by the end
of gestation, active dermal melanocytes “disappear”, except
in the head, neck, dorsal aspects of distal extremities, and
the presacral area. Therefore, at this stage a portion of the
dermal melanocytes have clearly migrated into the epider-
mis or died.
The major determinant of normal human skin color is the
melanogenic activity within the melanocytes and the quan-
tity and quality of pigment production, but not melanocyte
density [21]. Several factors play a role in determining the
level of melanocyte activity, including the specific charac-
teristics of the individual melanosomes (size, shape, type,
and color), baseline (constitutive) and stimulated (faculta-
tive) levels of activity of the enzymes involved in the
melanin biosynthetic pathway, the mode in which melano-
somes are transferred to the keratinocytes, and their distri-
bution in the keratinocytes. The latter are influenced by
receptor-mediated interactions with extracellular ligands
Fig. 2. “Epidermal melanin unit” of melanocytes located in the
stratum basal layer of the skin and associated with the
surrounding keratinocytes. Melanin is located inside
melanocyte and keratinocytes. The accumulation and
distribution of melanin to the basal layer will determine
the human skin color.
such as α-Melanocyte Stimulating Hormone (α-MSH) [4,6].
BIOSYNTHESIS, DISTRIBUTION, AND
TRANSPORT OF MELANOSOMES
Melanosomes are specialized members of the lysosomal
lineage, which originate in the smooth endoplasmic reticu-
lum. These membrane-bound granules, which are approxi-
mately 200 × 900 nm, are formed within melanocytes in the
skin and other places of the body. They are most closely
related to lysosomes, primarily because both of them contain
similar marker proteins and are positioned within the cyto-
plasm of melanocytes. The lysosome-associated membrane
proteins (Lamp) are also present in the outer membrane of
melanosomes [22]. Lysosomes protect against proenzymes
such as proteinases and melanosomes protect against mela-
nin precursors (phenols, quinones) that can oxidize lipid
membranes. The melanosome contains both matrix proteins,
which form scaffolds for melanin deposition, and proteins
(primarily enzymes) that regulate the biosynthesis of mela-
nin [4,6].
During development, melanosomes acquire some factors
and three genes-related melanogenic metalloenzyme, such as
tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosi-
nase-related protein 2 (TRP-2), which become fully func-
tional and properly folded through glycosylation.
The Glycosylation of Tyrosinase
Tyrosinase is the rate-limiting copper-containing enzyme
386
that contains virtually all of the catalytic activity necessary
for melanosome maturation and melanin synthesis. It con-
tains a signal peptide that is 529 amino acids, a C-terminal
transmembrane domain containing a 29 amino acid cytoso-
lic tail, 7 N-linked glycosylation sites (branched structure),
three cysteine (filled circles) clusters, and two copper-
binding sites that are essential for catalytic activity and for
the cellular trafficking of tyrosinase, which facilitates
transport from the rough endoplasmic reticulum (RER)
through the Golgi complex to the melanosome. This en-
zyme is highly important enzyme because of its ability to
induce melanin biosynthesis when expressed in heterolo-
gous cellular systems, even in the absence of any other
melanocyte specific proteins [23,24].
In addition to tyrosinase, Trp-1 and Trp-2 are tyrosinase
gene family proteins that are highly glycosylated and pri-
marily N-linked. They must be correctly and fully glycosy-
lated in the ER and Golgi complex before being transported
from the trans-Golgi network (TGN) to melanosomes [22].
Tyrosinase glycosylation is a post-translational process that
occurs within the endoplasmic reticulum (ER) and the
Golgi apparatus and is catalyzed by oligosaccharyltrans-
ferase (OT), a protein complex localized in the lumen of
the ER. The normal structure and function of tyrosinase
requires the attachment of sugars that are essential for the
correct folding [25]. This process is initiated on the cytoso-
lic surface of the ER membrane. The ER contains many
soluble molecular chaperone and folding enzymes that in-
teract with tyrosinase to achieve correct folding. N-linked
glycans are attached to the protein in the ER and are bound
to the polypeptide chain through an N-glycosidic bond. 2
N-acetylglucosamines and 5 mannoses are then added onto
the oligosaccharide and the oligosaccharide is transferred
to the Asn side chain in the Asn−Xaa−Thr/Ser sequence
motif by the oligosaccharyltransferase enzyme complex.
This process begins when the ER α-glucosidase removes 3
glucose residues in the core glycan and 2 terminal α-1, 2
linked mannose residues. The trimming of the glycan chain
by ER α-mannosidase I and II to create Man9 oligoman-
nose and Man7 glycoprotein is then folded and transported
to the Golgi [25].
In the Golgi apparatus, 2 mannoses from the Man7GlcNAc2
glycans are remove by the Golgi α-mannosidase I to become
Man5GlcNAc2 glycans. Through N-acetyl glucosamine I
(NAG I) activity, these glycans transfer 1 N-acetyl glucosa-
mine to form a hybrid structure of Man5GlcNAc3 glycans.
Two mannose residues are then removed from this hybrid
structure resulting in the formation of Man3GlcNAc3 glycans.
Finally the addition of N-acetyl glucosamine, galactoses,
fucose, and sialic acids forms the complex structure which
escapes terminal glycosylation [23,24].
The newly synthesized tyrosinase polypeptide is then sub-
jected to the quality control system in the ER, which moni-
tors protein maturation to ensure that defective proteins are
not transported throughout the cell. The system includes
calnexin, calreticulin, which are lectin chaperones that bind
specifically to glycoproteins containing monoglucosylated
glycans. Finally, incorrectly folded and misassembled
Fig. 3. The role of calnexin in melanosome biogenesis.
glycoproteins are recognized by glucosyl transferase, which
then targets them for ER retention [4,24].
The immature misfolded tyrosinases that remain bound to
calnexin and calreticulin are repaired and eventually attain
their correct three-dimensional structure. Calnexin has a spe-
cific affinity for monoglucosylated asparagine-like oligosac-
charides (Glu1Man9-8GlucNAc2), which are formed from the
removal of the first and second glucoses (G) in the original
triglucosylated core oligosaccharide (Glu3Man9GlucNAc2)
by the sequential action of glucosidase I and II. Only prop-
erly folded glycoproteins are transported to TGN, from
which newly synthesized glycoproteins are selectively trans-
ported to secretory granules, such as lysosome, endosome,
and melanosome through a M6P recognition system. By
virtue of a specific cargo-recognition system Lamp-1, -2, and
-3 are associated with the outer membrane of late endosomes,
lysosomes and melanosomes, and tyrosinase and Trp-1 are
transported to melanosomes and late endosomes (Fig. 3)
[22,26].
The quality control test in the ER may also involve the
production of the tyrosinase cofactor DOPA, which is pro-
duced by the oxidation of tyrosine. DOPA can bind to and
stabilize nascent tyrosinase, making it competent for trans-
port. Other proteins, such as Trp1 and Trp-2, can stabilize
tyrosinase in the ER and increase its activity. Therefore, the
presence of melanogenic complexes is important not only for
enzymatic optimization but also for the trafficking of pro-
teins out of the ER [4,26].
It has been well established that the cytoplasmic domain is
critical for melanogenic function and for the cellular traffick-
ing of tyrosinase. The cytoplasmic domain facilitates trans-
port from the RER through the Golgi complex to the
melanosome. Protein kinase C-β (PKC-β) is a signal trans-
duction enzyme that is required for the activation of tyrosi-
nase. PKC-β co-localizes with tyrosinase at the melanosomal
membrane and activates tyrosinase by phosphorylating the
serine residues on the C-terminal of the cytoplasmic domain
[27,28].

Transport of the Tyrosinase Gene Family from the
Golgi to Melanosomes
After glycosylation, proper folding, stabilizing, and possi-
bly oligomerizing within the Golgi apparatus, tyrosinases are
packaged into COPII-coated transport vesicles that bud from
the smooth ER. The vesicles traffic through the ER-Golgi
intermediate compartment (ERGIC) and combine with ma-
trix proteins to transport tyrosinase and Tyrp1 through late
endosomes to early melanosomes.
The sorting of tyrosinase and other melanocyte-specific
proteins from the Golgi to melanosomes is determined by
recognition signals in the cytoplasmic segment of the protein,
which are typically short stretches of six amino acid residues
including a di-leucine motif [29].
There are two distinct types of sorting signals for intracel-
lular localization of tyrosines to melanosome, a tyrosinase-
based signal and a di-leucine motif signal in the tyrosine tail,
which acquires the presence of Trp-1, silver (Pmel17 and gp
100) and P-protein through the binding of an adapter-like
protein complex AP3.
The sorting system has adapter proteins, such as clathrin,
which is a coat protein that assembles under the presence of
two distinct but closely related adapter protein (AP) com-
plexes. Clathins bind to different sorting signals on the cyto-
plasmic tails of protein and functions at various subcellular
locations for the transport of proteins between different or-
ganelle components [30,31]. AP-1, which specifically binds
to tyrosinase-based signals, is a distinct sorting pathwayand
that involves transport from the TGN to early endosomes.
Proteins that use the AP1 pathway need to subsequently use
AP3 or another sorting mechanism to transport from early
endosomes to melanosomes. AP2 is involved in sorting and
transporting proteins from the plasma membrane back into
the cell and to early endosomes. In order to use this system,
proteins need to first be sorted out of the melanocyte and
then brought back in. AP-1 and AP-2 are heterotetramers
that provide clathrins with membrane specificity at their as-
sembly by enabling them to recognize the sorting motifs in
the cytoplasmic domains of trafficking membrane proteins.
Cargo proteins, which are sorted by AP-1 and AP-2 into
clathrin-coated vesicles, are subsequently delivered to the
endocytic lysosomal system. The third AP complex (AP-3)
is more distantly related to AP-1 and AP-2. AP3 is involved
in tyrosinase sorting, which also transports cargo from the
TGN to the plasma membrane. From an immune perspective,
a number of melanosome proteins must have at least a tran-
sient expression on the cell surface. In additional, AP-3 does
not bind to the cytoplasmic tails of other non-melanosomal
membrane proteins that contain di-leucine and tyrosine-
based sorting signals. This finding indicates that AP-3 must
only transport a subset of cargo proteins, specifically the
tyrosinase gene family proteins, through the di-leucine-based
motifs [32,33]. Finally, AP4 may also play a role in the traf-
ficking of melanosome-specific proteins. Sorting signals are
typically found at the carboxyl termini of proteins and sev-
eral types are dileucine and tyrosine-based sorting signals.
Tyrosinases only weakly interact with AP-1 and AP-2, but
Biotechnol. Bioprocess Eng. 387=
Fig. 4. Trafficking and sorting of melanosomal proteins to
melanosomes correlate with the maturation stages of
melanosomes from I to IV. This process involves not only
gp100 but also the AP family. Cleavage of the amino
terminus plays a critical role in the maturation of the
amorphous vesicular stage I to the fibrillar stage II of
melanosome.
these proteins have a high affinity for the QPLL signal at the
cytoplasmic tail of the AP-3. Cytoplasmic tail with di-
leucine-based signals, will direct these glycoproteins to
melanosomes. This tail is important for the intracellular
transport of the tyrosinase gene family proteins from the
TGN to melanosomes [34].
The processing of gp100, which results in the cleavage of
the amino terminus, plays a critical role in the maturation of
the amorphous vesicular stage I melanosome to the fibrillar
stage II. The gp100 has been shown to localize in the ER,
AP3 vesicles, cis-Golgi, and trans-Golgi network. In addi-
tion, gp100 does not transport to early and late endosomes,
but rather traffics directly to Stage I melanosomes. Except
for gp100, the other melanosomal proteins, such as tyrosi-
nase, are further glycosylated and processed through the
trans-Golgi network (TGN). From that point they can poten-
tially use the AP3 sorting system, which traffics proteins to
early or late endosomes and then to lysosomes and melano-
somes (Fig. 4) [1].
Melanosome Maturation
Melanosomes develop through a series of morphologically
defined stages (Fig. 6) from an unpigmented (stage I) to a
striated organelle enriched in melanin (stage IV). In the sub-
sequent stages of maturation, melanosomes are relatively
nondescript membrane-bound vesicles (stage I) found in the
peri-nuclear area. Melanosomes under eumelanogenesis
(eumelanosomes) are always ellipsoidal in shape and are
located in well-organized lamellae filaments, elongated fi-
brillar and membrane bound organelles termed a premelano-
some (stage II). Once the fibrillar matrix had been generated,
melanin synthesis begins and the electron dense pigment is
deposited on the fibrils, which become electron dense upon
388
Fig. 5. Electron microscopy of melanosome development during
eumelanogenesis (a~f) and pheomelanogensis (g~j) in
normal melanocytes.
melanin deposition (stage III). Finally in the case of darkly
pigmented tissues, melanin synthesis and deposition contin-
ues until fully pigmented (stage IV). In contrast, melano-
somes under pheomelanogenesis (pheome-lanosomes) are
always spherical in shape and contain only internal granular
materials at all four stages of their maturation (Fig. 5)
[35,36].
Stage I melanosomes is the initial site for transport of the
tyrosinase gene family proteins. In this stage, melanosomes
are aspherical organelles that have an irregular structure and
contain matrix filaments and internal membranous vesicles,
which are formed by the invagination of the outer limiting
membrane. They likely correspond to the late-coated en-
dosomal multivesicular bodies found in nonmelanogenic
cells.
At stage II, the melanosomes become elongated and form
ordered striations which act as templates for melanin polym-
erization, which commences in stage III melanosomes.
Stage III melanosomes is characterized by the deposition
of electron dense material on the matrix. This stage pos-
sesses functional oxidoreductases, which triggers the melan-
somes to darken. Silver is a type I membrane glycoprotein
that plays a structural role in the formation of these striations
because silver expression in nonmelanogenic cells support
the formation of striations within multivescular bodies.
Melanin biosynthesis is dependent on proper pH homeosta-
sis, osmotic pressure, transport of the substrate into the
melanosome lumen and other uncharacterized activities such
as that controlled by the OA1 protein. Proper pH and os-
motic pressure are likely to require the activity of multiple
spanning membrane transport proteins (pink-eyed dilution
protein, P-protein, and antigen in melanoma-1/membrane-
associated transporter protein (AIM-1/MATP) that have a
role in ionic regulation in the melanosomes and possibly
even in the ER and the Golgi [8,15,52]. If the normal P-
protein is absent, the melanosomal structure is disrupted and
Fig. 6. The melanin biosynthetic pathway according to Raper-
Mason [44].
tyrosinase and Trp1 are misrouted to other sites. P-proteins,
in trafficking or sorting of melanocyte-specific proteins, exit
the Golgi apparatus and are transported to premelanosomes
[37,38].
Stage IV melanosomes, after complete opacification of the
melanosomal content by melanin deposition, will be trans-
ported to dendritic tips [39].
Melanin Biosynthesis
Melanin is the major product of melanocytes and is the
main determinant of differences in skin color. Melanin is
synthesized in two main forms: eumelanin and the pheome-
lanin. The amino acid tyrosine is the starting material for the
production of melanin in the synthesis of both the brown-
black eumelanin and the yellow-red pheomelanin. The key
regulatory enzyme in this pathway is tyrosinase, which has
tyrosine hydroxylase, DOPA oxidase, and dihydroxyindole
oxidase activity, therefore, it can catalyze multiple steps in
the biosynthesis of melanin (Fig. 6) [6,40,41].
Tyrosinase controls the initial chemical reaction, an im-
portant step in melanin biosynthesis, which is the hydrolysis
of tyrosine to L-DOPA (3,4-dihydroxy-phenylalanin). L-
DOPA enhances tyrosinase activity by allowing oxygen to
bind to the active site of tyrosinase. The oxygen-bound form
of tyrosine uses tyrosine and DOPA as substrates. Subse-
quently, L-DOPA is catalytically oxidized to L-DOPAquinone
(3,4-dihydroxy-phenylalanin quinone) by the met-form of the
enzyme. L-DOPA is a reactive intermediate, which is either
processed into eumelanin or pheomelanin. In the absence of
thiol compounds, DOPAquinone undergoes cyclization and is
converted DOPAchrome. TRP-2 (DOPAchrome tautomerase)
catalyses the tautomerization of DOPAchrome into 5, 6-
dihydroxyindole-2-carboxylic acid (DHICA). This step leads
to a much slower oxidation and polymerization, which results
in a more soluble, lower molecular weight and lighter colored
melanin known as DHICA-melanin. At the same time, the
decarboxylation of DOPAchrome leads to the formation of
indole-5,6-quinine from 5, 6-dihydroxyindole (DHI) and oxi-
date DHI. Trp1 and tyrosinase catalyze the conversion of
DHICA to carboxylated indole-quinone for the final produc-

Fig. 7. The molecular machinery that is involved in melanosome
movement in melanocytes. Anterograde movement, to-
wards the plus end of microtubules at the cell periphery is
achieved by kinesin. According to the formation of com-
plex Rab27a, melanophilin and myosin Va. In the periph-
ery region of the cell, melanosomes display short-range
movements on actin filaments. The microtubule- and ac-
tin-dependent motors are coordinated to achieve the in-
tracellular dispersion of melanosomes. Retrograde move-
ment, towards the minus end of microtubules in the cen-
ter of the cell is dependent on dynein. Single black ar-
rows indicate direction of melanosome movement.
tion of brown-black eumelanins. DHICA-melanins have re-
duced photoabsorption, no phototoxicity and less cytotoxicity
[31,41-43].
The formation of pheomelanin requires less tyrosinase ac-
tivity than does the formation of eumelanin. They are pro-
duced from cysteinyldopa and benzothiazine metabolites
when DOPAquinone combines with cysteine or glutathione
to form the intermediate products cysteinyl-DOPA and
alanyl-hydroxyl-benzothiazine. Pheomelanins have a yellow-
ish-red color, are soluble in alkali and have a low molecular
weight. In contrast, pheomelanin has very little photoabsorp-
tion, a high phototoxic potential, and low cytotoxicity. The
ratios of pheomelanic and eumelanic monomers determine
the final color of the skin and hair [44].
During the induction of new melanogenesis, tyrosinase
and TRP-1 coordinate together to upregulate Lamp-1, which
is continuously coated on the inner surface of the melano-
somal membrane. Their high content of N- and O-linked
oligosaccharides serve as a scavenger to toxic melanin in-
termediates that are produced by tyrosinase [45].
Melanosome Transport to Dendritic Tips
Melanosomes become enriched with pigment in the cell
body at stage IV and travel to the dendritic tip in a centrifu-
gal manner. At the dendritic tip, melanosomes are subse-
quently transferred to neighboring keratinocytes in the epi-
dermis. Melanosomes move in a bi-directional manner on
Biotechnol. Bioprocess Eng. 389=
microtubules in the dendrites until they are captured at the
dendrited tips (Fig. 7) [4].
This process requires protein motors, cytoskeletal tracks,
and adapters or effectors that attach the organelles to the
motors. There are two mechanisms involved in the centrifu-
gal movement of melanosomes to the microtubules and cell
periphery. Long-range movement by kinesin and dynein or
short-range movement at the cell periphery by the Rab 27a,
melanophilin, myosin Va complex [43].
The long-range movement of melanosomes proceeds to-
wards the plus ends of microtubules; therefore it may be
powered by motors of the kinesin superfamily. Kinesins are
molecular motors that are involved in microtubule and ATP
dependent transport of organelles, protein complexes and
mRNA [43,46,47]. Kinesins consist of two globular heads,
which is formed by two kinesin heavy chains (KHCs), and a
fan-like end, which is formed by two kinesin light chains.
The KHC possesses a motor domain, which binds to the
microtubules, a-helical coiled-coil stalk domain, which is
involved in dimer formation, and a C-terminal tail domain.
Conventional kinesin (kinesin I) is highly expressed in
mammalian melanocytes, and has been implicated in
melanosome transport. Anti-sense oligonucleotides that in-
terfered with the synthesis of kinesin I inhibited the bi-
directional movement of melanosomes along microtubules
and promoted perinuclear aggregation [48]. Retrograde
movement towards the microtubules’ minusends is achieved
by cytoplasmic dynein, which associates to its cargo via the
multisubunit complex dynactin. Cytoplasmic dynein was
implicated in both human and frog melanosome movement.
This microtubule-activated ATPase is a member of the
dynein superfamily of proteins and is composed of two
heavy chains, multiple intermediate chains, light intermedi-
ate chains, and light chains. Another associated subunit is the
dynactin complex, which may serve in general regulatory
roles [46,47].
In short-range movements, melanosomes still undergo
rapid bi-directional and microtubule-dependent movements
between the center and the periphery of the cell. This mode
of movement provides a means of transporting the melano-
somes to the cell periphery. In the cell periphery, the acto-
myosin system functions to ensure that the mature melano-
somes are captured and stay in position for subsequent trans-
fer. The Rab family is involved in the transport of the vesicle
carriers along microtubules and actin filaments, tethering or
docking of vesicles to the acceptor membrane, and fusion of
the vesicles with the membrane of the acceptor compartment.
In melanocytes, Rab27a associates with the cytosolic leaflet
of the melanosome membrane and appears to be the key
melanosome-associated protein of the tethering complex.
The association of Rab27a with melanosomes does not de-
pend on melanophilin and myosin Va. In fact, the mecha-
nism of Rab27a − melanosome association is currently un-
known, however, unraveling these targeting mechanisms
will help in fully understanding this process. Melanophilin
contains at least three functional domains. The N-terminal
domain binds to Rab27a and is conserved throughout the Slp
family. Over-expression of this domain in melanocytes in-
390
duces melanosome clustering, which serves as a Rab27a-
binding region. The Rab27a binding domain consists of two
stretches of amino acids referred to as the Sl phomology
domain-1 (SHD1) and the Sl phomology domain-2 SHD2.
The SHD1 is both necessary and sufficient for Rab27a spe-
cific binding, whereas the SHD2 is involved in enhancing
the Rab27a binding affinity to melanophilin. Melanophilin
specifically interacts with the globular tail of myosin Va via
a coiled coil domain within a central domain. The C-terminal
domain immunoprecipitates with actin and may be required
for peripheral melanosome distribution. Myosin Va is an
actin-based motor that belongs to the large myosin super-
family and shares a common N-terminal motor domain that
binds to actin and generates force through the hydrolysis of
ATP. The tail domain of myosin is postulated to direct the
interaction with the cargo, which ultimately determines the
functional specificity of the motor. Myosin Va is also found
to colocalize with F-actin in the dendrites and respective tips
where the melanosomes normally accumulate. This suggests
that the interaction of myosin Va with cortical actin is re-
sponsible for the peripheral capture of melanosomes. Myosin
Va, containing exons D and F, is expressed in skin and other
tissues and are essential for the interaction of myosin Va
with melanophilin and consequently for the association of
myosin Va with melanosomes [44]. An increase in micro-
tubule based transport is observed in the absence of compo-
nents needed for actomyosin based transport. Myosin V con-
tributes to the dispersion of melanosomes by counteracting
the action of dynein, particularly by shortening the length of
dynein-driven runs and by ensuring that the regular motion
of melanosomes is towards the microtubule plus-end. My-
osin Va operates in concert with actin filaments that line the
cell periphery to move granules to dendritic tips. In the ab-
sence of Myosin Va, the bi-directional movement of
melanosomes on microtubules is normal. However, under
these conditions the melanosomes fail to accumulate at the
dendritic tips. Rab proteins are adapter proteins that are lo-
calized on the melanosome membrane and are involved in
the regulation of intracellular vesicular transport in concert
with effector molecules. They link to melanophilin and in
turn link to myosin Va and F-actin, thus, providing specific-
ity in the trafficking steps of intracellular vesicles by promot-
ing the binding and fusion of vesicles destined for specific
locations within the cell [4,32,49].
Melanosome Transfer to Keratinocytes
Melanosomes that are enriched in pigment at the dendritic
tips are translocated to adjacent keratinocytes where melanin
forms a photoprotective cap over the keratinocyte nuclei [50].
Four mechanisms of the transfer have been proposed (Fig.
8) [51-54]. The first mechanism suggests that the dendrite
tips of the melanocytes are phagocytosed by the keratinocyte
in the presence of keratinocyte receptors (Fig. 8A).
In this mechanism it has been postulated that melanocytes
extend their dendrite to contact with a keratinocyte. Then the
dendrite tip is squeezed and pinched off and the cytoplasmic
reticulum is filled with melanosomes. These melansomes
A
M
K
B C D
M
K
Fig. 8. Different modes of melanin transfer [50].
will form a phagolysosome by fusion with lysosomes result-
ing in the degradation of the melanocyte membranes. The
phagolysosome will then be transported to the supranuclear
region and disintegrates into smaller vesicles, followed by
dispersion into the cytoplasm.
The second mechanism is based on regulated exocytosis.
In this process, the membranes of cytoplasmic organelles
fuse with the melanocyte plasma membrane and release
melanin into the intercellular space. Then keratinocytes take
up the melanin by phagocytosis (Fig. 8B) [53].
The third mechanism proposes that the presence of cog-
nate SNARE protein on melanocyte and keratinocyte plasma
membrane is responsible for the transportation to keratino-
cytes. In this mechanism, SNARE proteins initiate the direct
fusion of the two outer membranes. The resultant filopodia
then extends from the dendrite tips and cell body of melano-
cytes, adheres to the surface of neighbouring keratinocytes
and creates a channel that will support the transfer of
melanosomes from one cell to the other cell (Fig. 8C).
The final mechanism postulates that the transfer of mela-
nin from melanocytes to keratinocytes occurs through mem-
brane vesicles (Fig. 8D). Proteins and lipids destined for
transfer are concentrated in the plasma membrane and con-
tribute to the formation of extracellular vesicles, which sheds
the melanosomes and travels to distant cells. After this proc-
ess is complete, the vesicles will be ingested by the keratino-
cytes through phagocytosis or fuse with the keratinocyte
plasma membrane. Once inside the keratinocyte, melanins
are free and ultimately determine skin color.
In these mechanisms, PAR-2, which has an important role
in phagocytosis, is a major regulator of melanosome transfer.
PAR-2 is expressed in keratinocytes and enhances the
phagocytosis rate of keratinocytes, which leads to increased
melanosome transfer.
PAR-2 can potentially affects pigment modulation
through keratinocyte phagocytosis (Fig. 9). UVB-induced
PAR-2 activation could provide immediate photo-protection
by the rapid transfer of available melanosomes. This would

Fig. 9. PAR-2 activation and inhibition and the effects on kerati-
nocyte phagocytosis and melanosome transfer. The acti-
vation of PAR-2 by UV radiation and the presence of
trypsin result in an enhanced melanosome transfer,
which leads to pigmentation. The presence of trypsin in-
hibitors follow by inhibiting PAR-2 activation and reduced
melanosome transfer, which leads to depigmentation.
increase the ability of keratinocytes to ingest melanosomes
through a process of keratinocyte-melanocyte interaction.
PAR-2 activation leads to skin darkening, but inhibition of
PAR-2 activation by serine protease inhibitors reduces pig-
ment transfer and leads to depigmentation. The soybean
trypsin inhibitor (STI) have been shown to inhibit PAR-2
cleavage, and reduce phagocytosis and melanosome transfer;
thereby completely inhibiting UVB-induced pigmentation
[55].
Other factors, such as keratinocyte growth factor receptor
(KGFR), α-MSH, Rab3, Rab27a, cadherins, lectin, ect. also
contribute to events that enhance the phagocytosis rate of
keratinocyte, such as regulating melanosome exocytosis,
effect the docking of melanosomes at the plasma membrane,
and mediate melanocytes-keratinocytes adhesion and expres-
sion of genes associated with exocytosis. All of these proc-
esses are essential for the transfer of melanocytes to kerati-
nocytes [51].
FACTORS THAT INFLUENCE SKIN
PIGMENTATION
Skin pigmentation is influenced by many factors. These
factores belong to three groups: ultraviolet light (UVR),
drugs and genetic componensts (α-melanocyte stimulating
hormone (α-MSH), agouti signal protein (ASP), basic fibro-
blast growth factor (β-FGF), and endothelin-1 (ET-1)) (Fig.
10) [32,56,57].
Role of Ultraviolet Radiation in Melanogenesis
Solar ultraviolet radiation (UV) is a major environmental
factor that dramatically alters the homeostasis of the skin by
affecting the survival, proliferation, and differentiation of
various cutaneous cell types. The effects of UV on the skin
Biotechnol. Bioprocess Eng. 391=
Fig. 10. Factor-induced skin pigmentation. UV irradiation, growth
factors or endothelin-1, drugs and the activation of pro-
tein kinase A by α-MSH and its receptor MC1-R as well
as protein kinase C by TPA and Ca2+ can induce skin
pigmentation.
include direct damage to DNA, apoptosis, growth arrest, and
stimulation of melanogenesis. Long-term effects of UV in-
clude photoaging and photocarcinogenesis. Melanin, particu-
larly eumelanin, represents the major photoprotective
mechanism of the skin. Melanin limits the extent of UV
penetration through the epidermal layers and scavenges reac-
tive oxygen radicals that may lead to oxidative DNA damage.
The extent of UV-induced DNA damage and the incidence
of skin cancer are inversely correlated with the total melanin
content of the skin [52].
When UV radiation reaches the skin, reflection, scattering,
and absorption takes place. UV rays penetrate into the deep
layers of the subcutaneous tissue, particularly by the corneo-
cytes. UV ray absorption is increased by the content of aro-
matic acids, such as tyrosine, tryptophan, and phenylalanine
and urocanic acid, which forms in the keratinocyte by the
process of keratinisation. Urocanic acid is presence mainly
in the stratum corneum and is important for skin moisture
mainternance, and stimulation of stratum corneum thicken-
ing and melanin synthesis by melanocytes [52].
Facultative pigmentation is often divided into immediate
pigment darkening (IPD) and delayed pigment darkening
(DPD). IPD is a transitory darkening of the skin, which is
observed within seconds of UVA (320~400 nm) exposure
and is typically resolved within 1~3 days. IPC involves
structural changes in melanocytes and keratinocytes and a
chemical modification of pre-existing melanin. The mecha-
nism for IPD include photo-oxidation of melanin, changes in
cytoskeletal distribution, translocation of melanosomes from
the perinuclear region to the dendrites, increased melano-
somes transfer, and changes in the melanosome distribution
pattern within keratinocytes [58]. DPD results from an in-
crease in the number of melanocytes in addition to an in-
crease in the number of melanosomes in melanocytes and
keratinocytes. DPD typically occurs within 2~3 days after
392
Fig. 11. Interactions that control pigment production. α-MSH and
ASP interact with MC1-R. Activity of the MC1-R is en-
hanced by binding with α-MSH, which results in eume-
lanogenesis. The dysfunction of the MC1-R or the com-
bination of ASP blocking MC1-R can lead to pheome-
lanogenesis.
UVB (290~320 nm) exposure. A major stimulant for faculta-
tive pigmentation is UVR, which is the most potent stimu-
lant for growth and differentiation of melanocytes. Mito-
genic signals, in addition to UVR, have been shown to regu-
late melanocytic proliferation. Melanin not only functions as
a sunscreen to absorb UV and prevent DNA damage, but
also an antioxidant and radical scavenger, which play impor-
tant roles in protecting cells from such damage [32,59,60].
The activation and differentiation of melanocytes can be
induced directly by UVR or indirectly through their interac-
tion with surrounding UV-irradiated keratinocytes. UVR can
increase the synthesis of β-fibroblast growth factor (β-FGF)
in keratinocytes, which in turn stimulates proliferation and
melanogenesis of epidermal melanocytes [61,62].
In addition to increased synthesis of β-FGF, exposure of
keratinocytes to UVR results in the upregulation of other
keratinocyte-derived cytokines such as endothelin-1, which
is a small peptide originally isolated from endothelial cells.
Endothelin-1 plays an important role in stimulating melano-
cyte proliferation and melanization through the G protein-
coupled endothelin B receptor-mediated signal transduction
pathway and can also lead to an increase in tyrosinase activ-
ity and increase in melanin production.
THE ACTIVATION OF PROTEIN KINASE A OR C
Exposure of melanocytes to tetradecanol phorbol acetate
(TPA), can lead to increased melanin formation via the acti-
vation of protein kinase C (PKC). In addition, an enhance-
ment in pigment production will result from melanocytes
exposure to agents that increase intracytoplasmic levels of
cAMP such as cholera toxin, dibutyryl cAMP, and α-MSH.
The interactions between α-MSH and ASP are critical for
the switch to produce eumelanin or pheomelanin (Fig. 11)
[63,64]. α-MSH produced from UVR stimulated keratino-
cytes promotes eumelanin synthesis, whereas ASP promotes
pheomelanin synthesis. The effects of α-MSH are mediated
by the MSH receptor, which is known as the melanocortin 1
receptor (MCR-1) and is expressed at high levels in melano-
cytes. α-MSH binding to MC1-R enhances the expression of
the MSH receptor in melanocytes, activates the PKA path-
way, stimulates melanocyte proliferation and differentiation,
and results in an increased melanogenesis by the melanocyte
[11,65]. α-MSH binds to the MC1-R and this protein pair
interacts with a complex of G proteins, which uses
guanosine triphosphate (GTP) and guanosine diphosphate
(GDP) as intermediary messengers. The GTP-Gsα subunit
then activates adenylate cyclase, which leads to an increase
in production of cyclic adenosine monophosphate (cAMP)
within the melanocyte. An increase in the intracellular con-
centration of cAMP then causes an increase in tyrosinase
activity and eumelanin production. If the MC1-R receptor is
dysfunctional and fails to initiate a significant rise in the in-
tracellular level of cAMP, pheomelanins are produced [9,66].
Agouti Signal Protein (ASP)
Agouti signal protein (ASP) acts as a competitive antago-
nist of α-MSH for MSH-R binding. MSH-R in melanocytes
is considered to be a control point for pigmentation. MSH-R
is also present on other cells such as monocytes, endothelial
cells, and keratinocytes. When ASP is present, as a result of
its inhibition of MC1-R function and down-regulation of the
PKA pathway, melanocytes switch into their pheomelano-
genic mode and express melanosomal proteins at basal levels
except for tyrosinase, which continues to be expressed at
very low levels [67,68].
Activation of Tyrosinase
The expression of only tyrosinase still results in a low
level of melanin production. The sulfhydryl content of
melanosomes, in the form of cysteine, is not exhausted faster
than it can be transported into the melanosome and the
dopaquinone is converted to cysteinyldopa only to produce
pheomelanin. Furthermore, since the other melanosomal
proteins are not expressed, the melanosome never matures
beyond stage I. Therefore, the melanin produced does not
have a physical substrate to polymerize; hence the pheome-
lanosomes have splotchy irregular deposits of melanin. In
contrast, upon stimulation of differentiation by UV or α-
MSH, expression of all known melanosomal proteins is up-
regulated and melanosomes then undergo normal maturation
to the fibrillar stage II and beyond [68]. The high levels of
tyrosinase result in an increased synthesis of melanin. In
addition, the exhaustion of intra-melanosomal sulfhydryl

A B C genes, such as tyrosinase, TRP-1, TRP-2, and melanosomal
Fig. 12. Drug-induced skin pigmentation. (A) Minocyline pigmen-
tation, (B) flagellate pigmentation from bleomycin, (C)
amiodarone photosensitivity.
cause the dopaquinone generated to cyclize and form
dopachrome. This process results in the predominant synthe-
sis of eumelanin. The eumelanin is then deposited on the
melanosomal matrix followed by increased levels of visible
pigmentation in the tissue.
Drug-induced Skin Pigmentation
Drug-induced skin pigmentation is quite common and ac-
counts for 10~20% of all cases of acquired hyperpigmentation.
Pigmentation may be induced by a wide variety of drugs; the
most common ones include non-steroidal anti-inflammatory
drugs (NSAIDs), phenytoin, antimalarials, amiodarone, antip-
sychotic drugs, cytotoxic drugs, tetracyclines, and heavy met-
als (Fig. 12) [8].
Drug-induced skin pigmentation may result from in-
creased melanin synthesis, increased lipofuscin synthesis or
cutaneous deposition of drug-related material.
Certain heavy metals, such as iron, may be deposited in
the dermis following damage to dermal vessels. If deposited
in sufficient quantities a distinctive change in skin color may
be observed without any significant increase in melanin.
Some drugs react with melanin in different ways, includ-
ing to form a drug-pigment complex, induce accumulation of
melanin as a non-specific post-inflammatory change in pre-
disposed individuals or induce pigmentation directly by ac-
cumulating and reacting with other substances in the skin
[8,69].
CONCLUSION AND FINAL REMARKS
Over the past few years, significant advances have been
made in the understanding of the molecular mechanisms of
skin pigmentation. A better understanding of these mecha-
nisms could result in better control of human skin color. In
addition, this insight could be used to discover new depig-
menting agents and validate their efficacy and safety. This
review summarizes the current understanding of pigmenta-
tion in mammalian skin. This is a process that is carryied out
within melanosomes via a series of oxidative reactions in-
volving the substrate tyrosine and melanogenic tyrosinase
enzymes. The first event in this process is the development
and migration of dendritic precursor cells, known as
melanoblast, which originate in the neural crest. After migra-
tion, melanoblasts differentiate into melanocytes in the pres-
ence of MITF, Sox, Bcl-2, CREB, etc. Then, melanogenic
Biotechnol. Bioprocess Eng. 393=
matrix components are induced and synthesized. Tyrosinase
undergoes posttranslational processing and glycosylation,
which controls its activity and directs its transport to melano-
cytes. In melanocyte, tyrosinase is localized to the melan-
somes, where its functions in concert with other melanogenic
proteins to generate melanin. Melanosomes act as carriers of
tyrosinase, where melanin synthesis is initiated and eventually
transported to keratinocytes. Melanin is distributed within the
epidermis and determines skin color. However, many more
important questions remain unanswered, such as how the den-
drite interacts with the keratinocyte membrane at the site of
attachment and what is the mechanism of regulation of the
microtubule and actin based motors. Moreover, further studies
should aim at a better understanding of keratinocyte phagocy-
tosis, the regulation of PAR-2 expression and signaling in
keratinocytes, the natural activators of PAR-2 in skin, the ter-
mination of PAR-2 signaling as well as the identification of
key molecules that are involved in the dendrite-keratinocyte
interaction. Therefore, a further and better understanding of
these issues will continue to provide important insights about
the skin pigmentation process.
Acknowledgements This work was supported by the
Korea Science and Engineering Foundation (KOSEF) grant
funded by the Korea Government (MEST) R0A-2007-000-
10015-0.
Received May 19, 2008; accepted June 30, 2008
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