DOI:10.1111/j.1600-0625.2009.01053.

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www.blackwellpublishing.com/EXD

Review Article

Transcription physiology of pigment formation in

melanocytes: central role of MITF
Jiri Vachtenheim1 and Jan Borovansky2
1
Laboratory of Molecular Biology, University Hospital, Prague, Czech Republic;2Department of Biochemistry and Experimental Oncology,
1st Faculty of Medicine, Charles University, Prague, Czech Republic
Correspondence: Jiri Vachtenheim, Laboratory of Molecular Biology, University Hospital, Prague 8 - Bulovka, 18000, Czech Republic,
Tel.: 420-266082272, Fax: 420-266082064, e-mail: jivach@upn.anet.cz

Accepted for publication 4 December 2009

Abstract: Melanin production is the primary mechanism

protecting human skin against the UV light-induced damage. The

polymeric compound melanin is synthesized within melanocytes
in the specialized subcellular organelles, termed melanosomes,
which are then transferred to surrounding keratinocytes. The
genes for melanin synthesis and deposition are coordinately
expressed in melanocytes. The transcription factor MITF, which
has been reported to activate more than 25 genes in pigment cells,
has emerged as an essential regulator not only for melanocyte
development, proliferation and survival, but also for the
expression of enzymes and structural proteins ensuring the
production of melanin. MITF is a transcriptional activator of

several genes which encode melanosome-localized proteins

involved both in melanin synthesis and in melanosome biogenesis
and transport, including genes whose mutations are associated
with human oculocutaneous and ocular forms of albinism. Here,
we outline the mechanisms of transcriptional regulation of genes
associated with the biosynthesis of melanin in melanocytes and
melanoma cells. MITF is crucial in this process, while several
other factors seem to have only an auxiliary role to play under
specific circumstances.
Key words: melanin melanoma microphthalmia

microphthalmia-associated transcription factor pigmentation

Please cite this paper as: Transcription physiology of pigment formation in melanocytes: central role of MITF. Experimental Dermatology 2010; 19:
617627.

MITF as an essential transactivator in

melanocytes
Pigmentation of the skin in humans and coat colour in
animals are prominent phenotypic features depending on
the type, mass and distribution of melanin, a polymeric
compound deposited in melanosomes. In the skin, only
melanocytes are specialized to produce melanin, but melanin-synthesizing cells are present also in the eye (in the
retinal pigmented epithelium, choroid and iris), inner ear,

leptomeninges, brain and heart. Several enzymes act

sequentially to synthesize this dark polymer, which is
deposited in a highly organized fashion together with the
melanosomal structural proteins to form a typical dense
structure of mature melanosomes.
Although the biochemical pathway from tyrosine to the
melanin polymer has been known for decades (1,2), the
findings of the last fifteen years have led to unveiling the
transcriptional regulation of genes which are necessary for
the synthesis and deposition of melanin. Remarkably, only

Abbreviations: AIM-1, absent in melanoma; a-MSH, melanocyte-stimulating hormone a; APE-1 Ref-1, apurinic apyrimidinic
endonuclease redox effector-1; ASP, agouti signalling protein; bHLH, basic-helix-loop-helix; Brm, Brahma; BRG-1, brahma-related
protein-1; BRN2, brain-2 POU class 3 homeobox transcription factor 2; CDK, cyclin-dependent kinase; CREB, cAMP response element
binding; CRF, corticotropin releasing factor; DCT, dopachrome tautomerase; DIA, diaphanous homolog 1 (DIAPH1); EDNRB, endothelin
receptor B; GPNMB, glycoprotein (transmembrane) nmb; GPR143, G protein-coupled receptor 143; HIF1a, hypoxia-inducible factor 1a;
HNF-1, hepatocyte nuclear factor 1; LZ, leucine zipper; MART-1, melanoma antigen recognized by T-cells 1; MET, hepatocyte growth
factor receptor; MITF, microphthalmia-associated transcription factor; ML-IAP, melanoma inhibitor of apoptosis; OA1, ocular albinism 1;
OCA, oculocutaneous albinism; PKA, protein kinase A; PMEL17, melanocyte protein 17; POMC, pro-opiomelanocortin; PPAR,
peroxisome proliferator activated receptor; ROCK, Rho-associated coiled-coil-containing protein kinase; RPE, retinal pigmented
epithelium; Skp2, S-phase kinase-associated protein 2; SLUG, Snail homolog 2 (SNAI2); SNF, sucrose non-fermenting; SOX10, SRY-box
10; SRY, sex determining region Y; SWI, switch; TAD, transactivation domain; TBX2, T-box 2 protein; TFEB, transcription factor EB;
TFEC, transcription factor EC; TFE3, transcription factor binding to enhancer E3; TRP-1, tyrosinase-related protein 1; TRP-2, tyrosinaserelated protein 2; TYR, tyrosinase.

2010 John Wiley & Sons A/S, Experimental Dermatology, 19, 617627

617

Vachtenheim and Borovansky

one transcription activator emerged as a universal regulator

of expression of several proteins accomplishing the assembly of the melanosome and its decoration with melanin.
This protein, MITF is a product of the mi locus in mice,
and numerous mutations in this locus are associated with
different phenotypes affecting coat colour, hearing and eye
development in mice (37). Mutations in this locus cause
the Waardenburg syndrome type II in humans (8). MITF
targets comprise not only pigmentation-associated genes.
The expression of an increasing number of genes with
divergent and even opposing functions in the cell relies on
MITF, including cyclin-dependent kinase inhibitors p21
and p16 (WAF1) (9,10), CDK2 (11), BCL2 (12), livin (13),
DIA1 (14), hypoxia-inducible factor 1a (HIF1a) (15), MET
(16), T-box 2 protein (TBX2) (17), SLUG (18) and apurinic apyrimidinic endonuclease redox effector-1 (APE1 Ref-1) (19) (Fig. 1a). Recently, beyond the already large
group of genes activated by MITF, other potential targets
were identified by using the microarrays in MITF-overexpressing human melanoma cells (20). Because severe MITF
mutations preclude the development of embryonic melanocytes (21) and MITF is required for the survival of adult
and even malignant melanocytes (22), the MITF protein is
believed to be an essential regulator of the life and differentiation of melanocytes.
Melanomas generally express high levels of MITF,
although these levels differ greatly among melanoma cell
lines and cells in the tumor tissue. MITF gene has been
found to be amplified in a fraction of melanomas and is

(a)

(b)

Figure 1. (a) microphthalmia-associated transcription factor (MITF)

regulates diverse biological processes in pigment cells by activating
expression of its downstream genes. Refer to text for further
description. (b) Schematic view of human MITF protein. Functional
domains of the molecule and the known phosphorylation sites (serines),
ubiquitination and sumoylation sites (lysines) are depicted. MITF has
two well-defined transcription activation domains (TAD) and a bHLH
structure followed by LZ with four leucines. So far, no specific functions
have been associated with the N-terminus or the C-terminal region of
MITF. The full-length protein contains 419 amino acids, but a splicing
variant lacking the six amino acids (ACIFPT) upstream of the basic
domain was also detected.

618

regarded as a lineage survival protein with a pro-oncogenic

function in melanomas (22,23). MITF controls not only
expression of pigmentation-related genes but also genes
involved in diverse biological processes in melanoma cells
(Fig. 1a) such as proliferation, invasiveness, resistance to
apoptosis and stress mediated by reactive oxygen species,
and possibly metastasis. It has been suggested that MITF
protein level inversely controls invasiveness and proliferation of melanoma cells through upregulation of DIA1, the
human homolog of Drosophila Diaphanous (14). High
expression of MITF activates DIA1 expression, accompanied by increased proliferation via accelerated degradation
of a CDK inhibitor p27 by S-phase kinase-associated protein 2 (Skp2), a DIA1-regulated gene. By contrast, low
MITF level favours decreased proliferation but increased
invasiveness via the Rho-ROCK-dependent pathway when
DIA1-mediated actin polymerization is attenuated (14).
The resistance of melanoma cells to apoptosis is at least
partly mediated by expression of BCL2 and livin (ML-IAP),
both of which are MITF transcriptional targets (12,13).
Another important MITF target is the protooncogene
MET, a protein tyrosine kinase and receptor for the hepatocyte growth factor (16), which is involved in antiapoptosis and invasion of melanoma cells (16,24). Furthermore,
TBX2, a member of the T-box family of developmentally
important transcription factors, plays a role in suppressing
senescence and maintaining proliferation of melanoma cells
(25).
The MITF-encoding locus on chromosome 3p (26)
encompasses multiple promoters (at least nine), each followed by the first exon which is then spliced into a shared
sequence (exons 29). Thus, reflecting tissue specificity, the
MITF isoforms arise from different promoters which are
selectively activated in melanocytes, macrophages, osteoclasts, heart muscle, or retinal pigmented epithelium (RPE)
(27,28). The melanocyte-specific promoter (M-MITF) is
closest to the common sequence, and the melanocyte-specific exon 1 is very short, coding only for 11 amino acid
residues. The melanocyte-specific MITF isoform, MITF-M,
is a 419-residue protein (Fig. 1b). MITF is a typical basichelix-loop-helix-leucine zipper (bHLH-LZ) transcription
factor with the bHLH region followed by a leucine zipper
located in the central part of the molecule while the strong
transcription activation domain is at the N-terminus (NTAD). The N-TAD was narrowed to several amino acid
residues (aa. 114132 with the core motif IISLE) which are
essential for the interaction with the CH3 region of
p300 CBP coactivators (29). MITF possesses also the second transactivation domain (TAD) placed at the C-terminus which is much weaker then N-TAD; nevertheless, the
removal of both TADs is necessary to completely disrupt
the transactivation potential of MITF. In fact, the MITF
molecule in which both TADs are deleted acts in a domi-

2010 John Wiley & Sons A/S, Experimental Dermatology, 19, 617627

Transcription of pigmentation-related genes

nant negative fashion in that it inhibits the activity of the

wild-type protein (30).
Similarly, as other bHLH transcription regulators, MITF
recognizes the nucleotide sequence CANNTG (E-box) but
the association is maximal only if this motif is 5-flanked
with T (on either strand) (31). These T-flanked boxes were
identified as essential cis-acting elements in virtually all
MITF-responsive promoters. MITF is believed to bind the
cis-element as a homodimer to stimulate the target genes
in pigment cells. However, it is also able to form heterodimers with the related factors transcription factor binding
to enhancer E3 (TFE3), transcription factor EC (TFEC)
and transcription factor EB (TFEB) in vitro, and these
dimers are capable of binding the E-box in gel shift assays
(32). As there are no additional data to substantiate the
physiological significance of these heterodimers in melanocytic transcriptional regulation, it seems unlikely that the
TFE proteins play any major role in regulating the pigment
formation.
At least four different transcription factors participate in
the transactivation of the MITF gene in melanocytes: the
paired box-containing transcription factor PAX3, a sex
determining region Y (SRY) family member SOX10, the
Wnt b-catenin pathway effector LEF-1 and the cAMP
pathway effector cAMP response element binding (CREB)
(Fig. 2), with the last two proteins being probably more
important for the maintenance of MITF levels in melanoma cells (regulation of MITF expression has been
reviewed previously, e.g. in 7,27,3335). The cut-homeodomain transcription factor Onecut-2 (36) and activated
PPARc (37) were also shown to stimulate MITF promoter.
In addition, the POU domain homeobox transcription
factor hepatocyte nuclear factor 1(HNF1a) may also be
involved in MITF regulation via binding to its enhancer
(38), and the dimerization partner of HNF1a, DcoH
(pterin-4a-carbinolamine dehydratase) was shown to be
overexpressed in melanoma when compared with nevi
(39). Intriguingly, DcoH is an enzyme catalysing regeneration of tetrahydrobiopterin, a cofactor of phenylalanine
hydroxylase and tyrosine hydroxylase, and an allosteric
inhibitor of tyrosinase (reviewed in 38). A recent report
describes a direct transcriptional repression of the MITF
promoter by a POU domain-containing factor brain2 POU class 3 homeobox transcription factor 2 (BRN2)
(40); however, this protein is nearly absent in normal melanocytes suggesting that its possible importance as a regulator of MITF expression is restricted to melanomas.
In addition to the transcriptional control, the MITF level
is regulated by protein degradation. Clearly, phosphorylation of two serine residues (S73 by ERK2 and S409 by
RSK) after the stimulation of cells with the c-kit ligand
Steel leads to protein destabilization (41), presumably via
the enhanced ubiquitination on lysine 201 followed by deg-

2010 John Wiley & Sons A/S, Experimental Dermatology, 19, 617627

Figure 2. Regulation of microphthalmia-associated transcription factor

(MITF) expression and activation of its target genes related to pigment
formation in melanocytes. Arrows show transactivation, horizontal bars
show transcriptional repression. Transcription is designated by stair-step
horizontal arrows. Dashed arrows denote non-transcriptional regulation.
Besides pigment-related genes shown here, additional MITF targets
regulating cell proliferation, apoptosis, or invasiveness of melanoma
cells were identified (see Fig. 1). In addition to melanocortin one
receptor (MC1R), at least two other receptors, b2-AR (b2 adrenergic
receptor) and MC4R (melanocortin 4 receptor), have been reported to
be involved in cAMP signalling (132,133), but it is unknown whether
their expression is regulated by MITF.

radation by a proteasome (42). The phosphorylation at S73

was shown to be necessary for the interaction with the
p300 coactivator (43). However, the truncated MITF construct lacking the N-terminus including S73 or the S73A
mutant lost none of the transactivation potential (44), indicating that interaction of MITF with p300 is not a critical
event for constitutive activation. More likely, it might play
a physiological role in transient signal-induced changes of
MITF activity. Other posttranslational modifications
include phosphorylation on S298 (45), an activating event,
and sumoylation on K182 and K316 (46), which may affect
the activity differently depending on the target promoter
(47). Nonetheless, the current data do not point to a specific covalent modification on the MITF molecule such as
would critically activate or disable its function. What they
do suggest is that adjustment of the intracellular level of
the protein, the degradation of which is controlled by
phosphorylation and ubiquitination, may be a decisive
mechanism regulating the pigment production and other
MITF-modulated processes such as proliferation or cell survival (as mentioned above) in normal and malignant melanocytes.

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Vachtenheim and Borovansky

Regulation of transcription of the three

melanogenesis-controlling enzymes
Tyrosinase (TYR), the first rate-limiting and tri-functional
enzyme in the melanogenic biochemical route, has been
studied as a prototypic target among the MITF-regulated
genes. TYR catalyses the conversion of tyrosine to dopaquinone and the oxidation of DOPA (formed at the redox
exchange between dopaquinone and cyclodopa, a cyclization product of dopaquinone) back to dopaquinone
(48,49). Thus, DOPA acts as a cofactor which greatly
accelerates the first tyrosinase-catalysed step. Dopaquinone
is also the branching point between eumelanogenesis and
pheomelanogenesis; it reacts with cysteine to form cysteinyldopa, a precursor of red pheomelanins. A second
enzyme in the pathway, tyrosinase-related protein 2 (TRP2) (DCT, dopachrome tautomerase), enables a rapid conversion of dopachrome (50), a cyclodopa-derived quinone,
to 5,6-dihydroxyindol-2-carboxylic acid (DHICA). The last
enzyme, tyrosinase-related protein 1 (TRP-1), is a DHICA
oxidase (51) which facilitates the formation of carboxy
group-containing eumelanins. However, in the absence of
TRP-2, dopachrome is decarboxylated to dihydroxyindol
(DHI) which is also polymerized to melanin (the
DHI-melanin is darker than DHICA-containing melanin).
DHI can serve as a third substrate for tyrosinase (52),
which thus promotes also this last oxidative reaction in the
pathway. Melanogenesis is spatially restricted only to melanosomes because it is a potentially toxic pathway producing several highly reactive intermediates (quinones). These
can react with and damage the critical cellular macromolecules if synthesized in an inappropriate place in the cell
(5355).

All of the three enzymes operating in melanogenesis have

been shown to be transcriptional targets of MITF (Fig. 2)
with an identical TCATGTG sequence in their proximal
promoters quite close to the start of transcription. Promoter-reporter studies revealed that promoters of tyrosinase (56,57), TRP-1 (58,59) and TRP-2 (58) were activated
by cotransfected MITF. The human tyrosinase promoter
contains the M-box (extended E-box, AGTCATGTGCT)
located about 100 bp upstream of the transcription start
and the E-box sequence ACATGTGA at the initiator. Paradoxically, not the M-box but this initiator E-box is more
important for the promoter function (56). The identified
well-defined MITF-binding motifs in the tyrosinase promoter and in other promoters of MITF-responsive genes
are summarized in Table 1.
While MITF ensures a continuous transcription of the
tyrosinase gene under normal circumstances and after
hormonal stimulation through the cAMP signalling, the
increase of tyrosinase production as the UV response is
mediated, at least in part, by another bHLH factor, the
ubiquitous USF-1 (60). Upon UV irradiation, USF-1 is
phosphorylated by the p38 stress kinase and this modification is required for the USF-1-mediated transactivation
of the tyrosinase promoter (60). In addition, the p53
tumor suppressor protein was also shown to participate
in increased melanogenesis after UV irradiation because
p53 upregulates the tyrosinase and TRP-1 promoters in
reporter assays, and potential binding sites for p53 were
identified in the TRP-1 promoter (61). Further, tyrosinase
mRNA level was increased via a p53-dependent mechanism upon UV irradiation of melanoma cells in culture,
and p53 was required for the thymidine dinucleotideinduced increase of tyrosinase in mouse epidermis (62).

Table 1. Functionally relevant MITF-binding motifs in proximal promoters of MITF-regulated human genes which participate in the production
and deposition of melanin

Target gene

TYR
TRP-1
TRP-2
GPNMB
Melastatin 1
MART-1
PMEL17
OA1
RAB27A

M-box or
E-box

Position relative
to the transcription
start

AGTCATGTGCT
ACATGTGA
AATCATGTGCT
GGTCATGTGCT
GCACATGAGT
GCTCACATGCT
TCACGTGTG
TCACATGAA
GGCACATGATG
ACAGCTGA
CCATATGA

)107 to )97
)13 to )6
)211 to )201
)137 to )127
)46 to )37
)63 to )53
)715 to )707
+ 583 to + 591
)33
)46 to )39
)58 to )51

Reference

56
66
66,75
101, (http://www.ncbi.nlm.nih.gov/Genomes)1
112 (http://www.ncbi.nlm.nih.gov/Genomes)
99, 130 (http://www.ncbi.nlm.nih.gov/Genomes)
99 (http://www.ncbi.nlm.nih.gov/Genomes)
105
117,131

MART-1, melanoma antigen recognized by T-cells 1; OA1, ocular albinism 1.

1
Human genome searches: http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9606).

620

2010 John Wiley & Sons A/S, Experimental Dermatology, 19, 617627

Transcription of pigmentation-related genes

Another transcription factor, a dimer of DcoH HNF1alpha, was also found to be involved in tyrosinase transcription in skin melanocytes (63). According to Hou and
coworkers, the mouse embryonic melanocytes require a
coordinated action of Mitf and Sox10 for tyrosinase
induction, because both pigmentation and tyrosinase
expression in Sox10-deficient neural tube explant cultures
were rescued only by exogenous Sox10, which acts
upstream of MITF, but not by exogenous MITF alone
(64). The MITF-related bHLH protein TFE3, the closest
to MITF in evolution, was reported to bind E-boxes of
both TYR and TRP-1 promoters in vitro. Also, exogenous
TFE3 up-regulated the promoter-reporters (65). However,
because endogenous TFE3 failed to form dimers with
MITF or bind to the M-boxes, its physiological role in
melanogenesis is disputable.
The promoter of the human TRP-1 gene possesses the
M-box (AATCATGTGCT) which is localized about 210 bp
upstream of the start of transcription and, unlike the
mouse promoter, the human one harbours the TATA
sequence (66). While the M-box is necessary for promoter
upregulation by MITF (58), the TRP-1 promoter is contacted and its activity is elevated also by Pax3 (67), the
MITF promoter-activating transcription factor in melanocytes. Furthermore, TBX2, a T-box protein family member
expressed in pigmented cells, is capable of transrepressing
the TRP-1 promoter (68).
In what is an intriguing feature of the TRP-1 gene, its
transcription is frequently and selectively attenuated or
completely extinguished in melanoma cell lines and tumor
tissues, whereas tyrosinase, TRP-2 and MITF itself are normally expressed. Weaker expression of TRP-1, as determined by in situ hybridization, was also revealed in nevi:
the lower dermal layer of intradermal nevi showed a
reduced signal when compared with the expression in the
upper dermal layer, while a high expression was detected in
the basal epidermal layer in junctional nevi (69). Similarly,
in dermis-invading lesions of primary melanoma, the staining with the anti-TRP-1 antibody was also fading (70).
Transcriptional blockage was observed upon the addition
of a known inducer of differentiation hexamethylene bisacetamide (HMBA) to the medium of melanoma cells in culture (71). A complete extinction of TRP-1 was seen in
several human melanoma cell lines (7274). Although
lower MITF binding to the TRP-1 E-box was observed in
TRP-1-negative cell lines (73), this cannot account for the
complete loss of TRP-1 mRNA. Thus, the mechanism
which may involve a putative TRP-1-specific repressor
remains to be elucidated. This might be of importance
because TRP-1 is a melanocytic antigen recognized by activated T-lymphocytes; consequently, TRP-1-negative melanomas can evade eradication by DNA vaccines directed to
TRP-1 epitopes.

2010 John Wiley & Sons A/S, Experimental Dermatology, 19, 617627

TRP-2 enzyme (DCT) is expressed very early during

melanoblast differentiation in the developing embryo,
approximately when MITF begins to be expressed. The 5regulatory region of the TRP-2 gene contains the M-box
(GGTCATGTGCT) positioned about 135 nucleotides
upstream of the transcription start (75) and the promoter
responds to MITF coexpression (58). Another important
factor specifically involved in the transcription of TRP-2 is
SOX10, which also promotes expression of MITF (see
above). The human TRP-2 promoter-reporter construct
was shown to be activated by SOX10 (76,77) and SOX10
and MITF (Fig. 2) act in a synergistic manner to activate
the promoter (78,79). These data are in accord with the
observation that mouse heterozygous embryos carrying the
Sox10dom mutation transiently lack Dct (around days 11
12) in the melanoblast lineage, and MITF alone is incapable of triggering the Dct transcription in these early MITFpositive cells (77). In a recent report, another member of
the Sox family, Sox5, inhibited the Sox10-stimulated activity of the Dct promoter in melanocytes (80). In the TRP-2
expression, yet another transcription factor, LEF-1, is
thought to play a role in conjunction with MITF. LEF-1
protein, one of the effectors of the Wnt b-catenin pathway
at target promoters, was shown to physically interact and
cooperate with MITF in the transactivation of TRP-2 promoter. However, not only the MITF-LEF-1 interaction, but
also the cis-acting motif in the promoter (surprisingly, not
recognized by LEF-1) are required for the TRP-2 promoter
stimulation (81). A CRE-like element in the TRP-2 promoter might also contribute to gene expression through
direct regulation by CREB protein (58).
The distal enhancers were described for mouse tyrosinase
and Trp1 genes (82). An upstream regulatory region for
human tyrosinase, too, is reportedly located about )9 kb
upstream from the start of transcription (83,84). This
sequence, which showed a homology with a similar distal
locus found in the mouse and functioned as an enhancer
in transfection assays, may prove to be important for the
pigment cell-specific expression of human tyrosinase.
It is also noteworthy that the three human promoters of
melanogenic enzymes contain either a typical TATA box
(tyrosinase and TRP-1 promoters) (66,85) or a TATA-like
element (TRP-2 promoter) (66,75), but their function in
tissue-specific transcription is presumably omissible. At
least in the case of tyrosinase, the TATA sequence seems to
be superfluous for the promoter activity in reporter assays
(our unpublished results). Although other recognition elements are also present in the tyrosinase, TRP-1 and TRP-2
promoters, none have been shown to be crucial for expression and the promoter deletion analyses pointed to the
MITF-binding elements as regulators of the promoters
activity with MITF being the essential physiological transactivator.

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Vachtenheim and Borovansky

MITF conveys hormonal and stress

responses to pigmentation genes
The pigmentation is stimulated hormonally by a-MSH and
its receptor melanocortin one receptor (MC1R) via the
cAMP signalling cascade. The POMC (pro-opiomelanocortin) gene encodes a precursor that is processed to form the
a-MSH, ACTH and b-endorphin. The POMC gene is active
mainly in the pituitary, but POMC-derived peptides are
produced also in peripheral tissues including skin melanocytes (35,86). Although a-MSH is a potent inductor of pigmentation in mammalian skin, b-MSH and ACTH possess
melanogenic activities as well (hormonal regulation of skin
pigmentation has been reviewed, e.g. in 35,87,88).
The hormonal stimulation by a-MSH is mediated
through the MC1R resulting in increased intracellular
cAMP level and activation of protein kinase A (PKA). This
notorious hormonal signalling pathway has specific consequences in melanocytes as the MITF promoter possesses a
CRE element which is bound by CREB proteins that are
phosphorylated by PKA and activate transcription of MITF.
It is not surprising, therefore, that pharmacological agents
that increase the cAMP level in the melanocyte are extremely potent in upregulating the endogenous level of MITF,
which consequently triggers transcription of downstream
melanogenic factors. Although increase of MITF mRNA
and protein is only a transient response, the resulting
induction of melanogenesis is a prominent outcome of this
hormonally triggered signalling cascade. It is noteworthy
that MSH can control melanogenesis also independently of
MC1R, possibly by acting directly in melanosomes (38).
Remarkably, the MC1R gene was identified as an MITF target (Fig. 2), as its promoter-reporter activity responded to
MITF cotransfection in human melanoma cells (89). Thus,
a positive feedback loop is created after hormonal stimulation resulting in an increased expression of receptor molecules in the pigment cell. Intriguingly, yet another receptor
is upregulated by MITF; the expression of endothelin
receptor B (EDNRB), which serves as a receptor for peptides endothelins, is also regulated by MITF (90). Moreover, signalling by endothelins 1 and 3 activates MAP
kinases with subsequent phosphorylation of MITF and
stimulation of MITF expression as well (90). As with the aMSH signalling, endothelins induce CREB protein phosphorylation with consequent activation of MITF expression
in this pathway.
Hormonal signalling is also involved in skin responses to
UV irradiation, although the effect of UV light on pigmentation is complex. UVB can induce expression of POMC
and MC1R genes in melanocytes and keratinocytes (87,91).
In addition, expression of corticotropin-releasing hormone
(CRH) is also stimulated by UVB in melanocytes, which is
mediated by the CREB-PKA signalling with consequent

622

stimulation of POMC expression through the CRH-R1

receptor (91). The POMC gene has been reported to be
p53-responsive following UV irradiation, and the POMC
promoter contains a p53 binding site which is necessary for
its highest activity (92). The in vivo tanning response and
POMC mRNA induction were dependent on p53, further
suggesting that the p53 protein is an important mediator
of UV-induced melanogenesis increasing the transcription
of POMC (92). However, the concept that activated p53 is
the only essential stimulator of UV-induced pigmentation
via induction of POMC transcription was challenged from
several reasons, mainly because the POMC-knockout mice
have been known to display normal melanin pigmentation,
and a more complex regulation was suggested (93). A
direct regulation of tyrosinase transcription after UV irradiation is mentioned above.
Hormonal regulation also underlies the switch of pigment type formation in skin. The a-MSH signalling on the
MC1R receptor is inhibited by the agouti signalling protein
(ASP, ASIP in humans) that can compete with a-MSH in
binding to MC1R. High expression of ASP is associated
with yellow bands in mouse hair. Thus, MC1R and its
ligands, a-MSH and ASIP, regulate switching between eumelanin and pheomelanin synthesis in melanocytes (35,94).
ASP was demonstrated to downregulate MITF gene expression, and hence its targets, by antagonizing the effect of aMSH, thus favouring pheomelanogenesis by reduced production of eumelanin (95). Interestingly, a similar profile
of genes was inversely regulated by ASP and a-MSH in a
microarray analysis (96). Recently, it has been shown that
the receptor-binding domain of ASIP efficiently antagonizes
the MSH-MC1R signalling by reducing the cAMP level,
while it induces no changes in pigmentation, demonstrating that the negative regulation of differentiation by agouti
signalling is independent of the cAMP-CREB pathway (94).
Other hormones such as steroids can also influence pigmentation (reviewed in 35,87), and a recent report
describes that even cholesterol is capable of increasing
expression of MITF and its target genes in melanocytes,
possibly through the upregulation of the CREB protein
(97).

MITF regulates expression of

non-enzymatic melanosomal proteins
Biogenesis of melanosomes
Early stage melanosomes (stage I and II) display a striatal
structure formed by amyloid-like fibrils upon which the
polymer melanin is deposited in later stages (stage III and
IV). PMEL17 (also known as SILV or GP100), a critical
structural protein required for the formation of the characteristic fibrilar structure, can by itself drive the formation
of fibrilar structures even if expressed exogenously in non-

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Transcription of pigmentation-related genes

pigmented cells (98). Pmel17 is a product of the mouse silver locus, and the human SILV protein is recognized by
the widely used melanoma diagnostic antibody HMB-45.
In melanoma cells, PMEL17 was found to be a MITF target
(Fig. 2) with protein and mRNA levels correlating well with
those for MITF, and PMEL17 mRNA was upregulated
upon transfer of wt MITF and downregulated by a dominant-negative MITF mutant (99). The MITF-binding site
critical for the promoter activity is located, quite exceptionally, in the first intron and conforms the TYR family
consensus TCATGTG. The Pmel17 transcript was not
detectable in RPE of MITF-mutant mouse embryos, further
supporting the necessity of MITF expression for the
Pmel17 gene transcription (100).
Recently, a transmembrane glycoprotein osteoactivin
(GPNMB), expressed in osteoclasts, macrophages and melanoma cells, has been identified as a MITF-regulated gene
both in pigmented cells (101) and in osteoclasts (102).
Although Gpnmb manifests partial similarity to the silver
protein and localizes to melanosomes, its precise role in
melanocyte physiology is not known. Mouse Mitf mutant
embryos do not express Gpnmb while in normal embryos,
the protein appears early in the development of RPE and
melanoblasts. The E-box CACATGA in the Gpnmb promoter is located next to the AP1 site and is highly conserved during evolution. This motif is important for the
reporter expression in transfected cells; however, even the
E-box-deleted construct was capable of directing the transgene expression in zebrafish melanoblasts, indicating that
the remaining sequences in the short (89 bp) promoter are
sufficient for in vivo expression (101).
MLANA (also termed melanoma antigen recognized by
T-cells 1(MART-1) or Melan-A) is yet another transmembrane protein associated with melanosome biogenesis.
MLANA is in a complex with PMEL17 and influences its
stability and processing within the melanosome (103).
MLANA has also been recognized as a MITF transcriptional target (Fig. 2) with two E-boxes in the human promoter, the more proximal one being the TCACGTG motif
(99). MLANA promoter activity depends on cotransfected
MITF and is bound by MITF in chromatin immunoprecipitation assays. The MLANA protein and mRNA levels correlate with MITF levels in melanoma cell lines (99). Thus,
the expression of this melanosomal matrix protein is also
believed to require MITF.
The ocular, X-linked albinism type 1 is caused by mutations in the ocular albinism 1(OA1) gene (also named
GPR143) (104). Its protein product is a melanosome membrane protein expressed exclusively in melanocytes and
RPE cells. OA1 shares a similarity with members of a
superfamily of the G protein-coupled receptors. Melanocytes carrying the mutated OA1 gene display giant abnormal melanosomes, implicating its product in melanosome

2010 John Wiley & Sons A/S, Experimental Dermatology, 19, 617627

biogenesis. One evolutionarily conserved binding site for

MITF (CACATGA), only 28 nucleotides upstream from the
transcription start, was found in the mouse Oa1 gene promoter (nt -33 in the human promoter) (105). Thus, as the
target sequence on the opposite strand is in keeping with
the classical consensus motif TCATGTG, the mouse Oa1
promoter (and very likely also the human OA1 promoter)
was assumed to be a direct target regulated by MITF in
pigment cells (Fig. 2). The integrity of this site was indeed
essential for the promoter activity and binding to MITF in
gel shift assays (105). MITF protein also occupied the
human promoter in melanoma cells. As with several other
MITF target genes, a relatively short fragment of the proximal Oa1 promoter (containing the E-box) was sufficient to
direct the tissue-specific expression: when introduced into
mouse retina, the transgene driven by this promoter
sequence was expressed in RPE (105).
Another transporter protein localized in melanosomes is
AIM-1 (absent in melanoma, also called MATP for membrane-associated transporter protein, and also designated as
SLC45A2). Mutations of this gene were shown to cause
OCA4 (106). MATP protein like another melanosomal protein, the P protein regulating the intramelanosomal pH
(107), contains 12 transmembrane regions and is required
for a correct tyrosinase processing in melanosomes, because
homozygously mutated mouse melanocytes (uw uw) display a defect during maturation of melanosomes, which are
only slightly melanized, irregularly shaped and have a disorganized fibrilar structure (108). Remarkably, tyrosinase
(and also TRP-1 and TRP-2) are secreted from uw uw
mutated melanocytes in vesicular bodies. Human AIM-1
gene contains a repetitive sequence in the 5-regulatory
region with numerous E-boxes, including the consensus
TCATGTG sequences, and ectopic MITF upregulates AIM1 mRNA in human melanoma cells (109). Nevertheless, the
positive regulation of Aim-1 might be indirect as the
endogenous MITF does not seem to associate with the
Aim-1 promoter (109).
Two members of a large family of transient receptor
potential (TRP) proteins, melastatin 1 (TRPM1) and melastatin 7 (TRPM7), were found expressed in melanomas
and nevi. While melastatin 1 (also known as MLSN1) was
expressed in normal melanocytes, nevi and primary melanomas, it was not detected in metastatic lesions (110).
Contrary to this, TRPM7 expression was found in several
metastatic melanoma cell lines (111). Whilst it is unknown
whether TRP7 expression is MITF-dependent, melastatin 1
was identified as a MITF target gene, the promoter of
which responded exceptionally sharply to MITF cotransfection (112,113). The human promoter region contains
several E-boxes. The most proximal of these (at around nt
-58) is presumably the main element important for
promoter activity (112). Chromatin immunoprecipitation

623

Vachtenheim and Borovansky

revealed that MITF resides on the TRPM1 promoter in

melanoma cells. Furthermore, mRNA levels of TRPM1 and
MITF correlated very closely both in melanoma cells and
in normal melanocytes. Also, melastatin 1 mRNA was lacking in RPE derived from MITF mutant embryos, thus indicating a dependency on MITF in developing pigmented
cells (112).

Melanosome transport
Movement towards the cell periphery and transfer to the
surrounding keratinocytes mark the physiological route of
melanosomes in skin melanocytes. Molecular interaction
essential for the melanosome trafficking in the dendrite tips
results in the formation of trimolecular complex consisting
of RAB27A, a small GTPase protein, its effector melanophilin (SLAC2A), and an actin-based protein myosin Va (114
116). MITF was shown to upregulate the transcription of
the RAB27A gene and its promoter-reporter and, conversely, siRNA-mediated knockdown of MITF was found to
impair the normal distribution of melanosomes in mouse
B16 melanoma cells via downregulation of Rab27a (117).
The MITF-dependent transcriptional regulation of RAB27A
(Fig. 2) is phylogenetically conserved because MITF was
also required for melanosome transport and dendricity in
Xenopus melanophores (118). Notably, the expression of
melanophilin, myosin Va, as well as another GTPase Rab7,
which is involved in the microtubule-based transport of
early-stage melanosomes (114) and in the maturation of
TRP-1 and Pmel17 Silv proteins within the melanosome
(119,120), was found not to be influenced by MITF (117).
OA1 gene transcription gene is directly activated by
MITF (see above). Besides its function in the biogenesis of
melanosomes, studies on mouse melanocytes demonstrated
the Oa1 proteins importance for melanosome transport to
the cell periphery (121). MITF therefore regulates melanosome traffic by sustained expression of at least two proteins, Rab27a and Oa1.
Additional specialized proteins have been identified in
the Golgi-lysosome-melanosome network. These include
RAB38 (chocolate in mice), the P protein (its defects were
found in OCA2, known as pink-eyed in mice), melanophilin, SLC24A5 (potassium-dependent sodium-calcium
exchanger), and several proteins of the BLOC (biogenesis
of lysosome-related organelles complexes), which are
altered in HermanskyPudlak syndrome subtypes (122). In
spite of that, the mechanism of their transcription and possible MITF involvement still await elucidation.

MITF cofactors and regulation of

endogenous genes
The melanosomal enzymes and non-enzymatic proteins,
the transcription of which is controlled by MITF, constitute

624

only a subset of MITF-responsive genes. It is less clear

whether MITF utilizes the same cofactors, coregulators, or
cooperates with additional transcription factors to specifically regulate so many target genes involved in so diverse
cellular processes in pigment cells. The p300 and CBP proteins, which are global transcription coactivators serving as
cofactors for many different transcription factors (123),
were found to bind MITF and stimulate transcription from
the responsive promoter-reporters (29,43). Therefore, they
are presently thought to be MITF coactivators. P300 CBP
proteins are histone acetyltransferases capable of acetylating
histones and non-histone proteins, many of which are transcription factors. Acetylation of N-terminal lysine residues
on histone tails by p300 CBP and other histone acetyltransferases neutralizes the negative charge and reduces the
interaction between histones and DNA. Although it is likely
that MITF recruits p300 CBP to target promoters causing
the hyperacetylation of surrounding histones, MITF itself
might be acetylated by these, and possibly by other, histone
acetyltransferases. MITF has several potential acetylation
sites and is acetylated in vitro (our unpublished results),
but whether the acetylation influences its activity or helps
to discriminate among the many targets remains to be
established.
Endogenous genes often require local chromatin decondensation in the promoter regions. This is accomplished,
besides the histone acetylation, by the action of chromatin
remodelling complexes. The SWI SNF complex which utilizes Brm or Brg1 ATPases is necessary for the efficient
transcription of endogenous Tyr, Trp-1, dct and Pmel17
genes in mouse fibroblasts transfected with MITF, because
expression of these genes was found attenuated by dominant negative mutants of Brm and Brg1 (124). On the contrary, the MC1R gene expression was not decreased by
these mutants. Thus, the expression of differentiation-specific genes might require the SWI SNF enzymes in melanocytes. Transcription of TYR, but not other markers, can be
triggered by MITF also in the human non-melanocytic
(osteosarcoma) cell line U2OS (74), but not in H1299 lung
carcinoma cells, in which Brg1 is not expressed. However,
co-delivery of exogenous Brg1 together with MITF into
H1299 cells elicits the expression of endogenous tyrosinase
(125), indicating that a similar requirement of SWI SNF
for the tyrosinase expression exists in human cells.
As MITF is a crucial activator of melanogenesis-related
genes, an increase or decrease of its level should be
reflected by changes in transcriptional activity of the downstream genes. While it is believed that the inhibition of
MITF expression or activity leads to the decreased levels of
its targets and eventually to the proliferation block or
induction of apoptosis in melanoma cells (12,22), exogenously increased MITF level does not have any effect on
tyrosinase or Trp-1 expression (126). So, perhaps, addi-

2010 John Wiley & Sons A/S, Experimental Dermatology, 19, 617627

Transcription of pigmentation-related genes

tional factors or coactivators may be required as well as

MITF, or some unknown mechanism may restrict the production of pigment cell markers at constant lower levels
and only stress conditions such as UV light might trigger
the specific coordinated response to facilitate melanogenesis.
Interaction with other proteins, as demonstrated for bcatenin, could be another level of regulation of MITF activity on the downstream promoters (127). Transcription of
MITF itself is up-regulated by the Wnt b-catenin pathway,
which is frequently deregulated in many cancers including
melanomas. The identified direct interaction between MITF
and b-catenin can result in the redistribution of b-catenin
from LEF-1 target promoters to MITF targets. MITF can
thus exploit b-catenin as its own cofactor in melanomas
(127). Last, the Rb tumor suppressor protein was reported
to interact with MITF (128) and activate transcription of
the MITF target p21(WAF1), thus causing cell cycle arrest
(9). Although the Rb) ) mouse melanocytes displayed pigmentation defects in vitro (129), it remains unclear whether
Rb directly participates in the transactivation of melanogenic factors.

Conclusions
The published data lead to a notion that MITF represents a
melanocyte-specific communication hub integrating intracellular signals and transcription responses in cell differentiation, proliferation and survival. The synthesis of
melanosomal enzymes and structural proteins is coordinated
already at the transcriptional level because MITF regulates
their gene promoters. Hormonal and UV light-induced
changes in pigmentation seem to be more complex and are
further regulated by transcription factors p53 and USF-1. In
addition to pigmentation, MITF regulates also proliferation,
survival and invasiveness of malignant melanocytes by regulating transcription of genes involved in these processes.
Further experimentation is needed to elucidate how this
central position of MITF in the melanocyte physiology influences the sets of the transcriptional targets to be regulated
according to the requirements of the pigment cell.
Acknowledgements
This work was supported by grant NR 9319-03 (Ministry of Health) and
from institutional projects MZO00064211 (MH) and MSM21620808
(MEYS), Czech Rep.

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