From the ¶ Division of Pediatric Hematology/Oncology,
Children's Hospital and Dana Farber Cancer Institute, Harvard Medical
School, Boston, Massachusetts 02115, § St. George's
Hospital Medical School, London SW17 ORE, United Kingdom, and
INSERM U385, Biologie et Physiopathologie de la Peau,
Faculté de Médecine, 06107 Nice Cedex 2, France
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ABSTRACT |
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In mouse follicular melanocytes, production of
eumelanins (brown-black pigments) and pheomelanins (yellow-brownish
pigments) is under the control of two intercellular signaling molecules that exert opposite actions, -melanocyte-stimulating hormone (
MSH) which preferentially increases the synthesis of eumelanins, and agouti signal protein (ASP) whose expression favors the production of hair containing pheomelanins. In this study, we report that ASP does
not only affect mature melanocytes but can also inhibit the
differentiation of melanoblasts. We show that both
MSH and forskolin
promote the differentiation of murine melanoblasts into mature
melanocytes and that ASP inhibits this process. We present evidence
that the expression of a specific melanogenic transcription factor,
microphthalmia, and its binding to an M box regulatory element, is
inhibited by ASP. We also show that, in B16 murine melanoma cells, ASP
inhibits
MSH-stimulated expression of tyrosinase, tyrosine-related
proteins 1 and 2 through an inhibition of the transcription activity of
their respective promoters. Further, ASP inhibits
MSH-induced
expression of the microphthalmia gene and reduces the level of
microphthalmia in the cells. Our data demonstrate that ASP can regulate
both melanoblast differentiation and melanogenesis, pointing out the
key role of microphthalmia in the control of these processes.
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INTRODUCTION |
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Melanocytes are present in the skin and hair follicles as a dispersed population of differentiated cells, after they migrated along the dorsolateral pathway from the neural crest, as nonpigmented precursors, the melanoblasts (1). In mature melanocytes, melanin synthesis involves three specifically expressed enzymes: tyrosinase (2), tyrosinase-related protein 1 (TRP1)1 (3), and tyrosinase-related protein 2 (TRP2) (4) that control the quantity and the type of melanins. Indeed, two types of melanins are synthesized during melanogenesis; pheomelanins that are yellow to red pigments and eumelanins that are black to brown. Tyrosinase, which catalyzes the first and rate-limiting step of this process, is required for synthesis of both melanin types, while TRP1 and TRP2 appear to be involved mainly in eumelanin synthesis (5). Recently, a transcription factor, belonging to the basic helix-loop-helix family, named microphthalmia (Mi in mouse and MITF in human) and expressed in a limited number of tissues such as heart, mast cells, osteoclast precursors, and melanocytes, has been involved in melanocyte survival, development, and differentiation (6, 7). Indeed, mutations at the mouse mi locus lead to coat color dilution, white spotting or complete loss of pigmentation (6, 8). Similarly, mutation in the human homologue of the mouse microphthalmia gene has been linked to abnormal pigmentation observed in Waardenburg syndrome type II-A (9, 10). Further, studies have shown that Mi through binding to the M box (a highly conserved 10-base pair motif, GTCATGTGCT) strongly stimulates tyrosinase (11), TRP1 (12), and TRP2 (13) promoter activities, suggesting that Mi is involved in the tissue-specific expression of the melanogenic genes.
The relative amount of eumelanin and pheomelanin pigments in mammals is
controlled by the genetic loci agouti and
extension; extension encodes the receptor for
-melanocyte-stimulating hormone (
MSH), also called the
melanocortin 1 receptor (MC1R) and agouti encodes a
131-amino acid protein containing a signal sequence, the agouti-signal
protein (ASP) (14-18). ASP, which is produced in the dermal papilla
cells within the hair follicle, acts on follicular melanocytes to
switch them from eumelanin to pheomelanin production (19). In humans,
the mechanism that controls the class of melanin synthesized (eumelanin
or pheomelanin) has not been elucidated. It is well established that
MSH increases the synthesis of eumelanin in human melanocytes (18,
20, 21). A human homologue for the mouse agouti locus has
been cloned and its product functions similarly to the mouse protein
in vivo and in vitro (22, 23); however, its
physiological function in humans remains to be elucidated.
ASP seems to act as an antagonist of MSH signaling mediated by the
mouse melanocortin-1 receptor (MC1R). However, its mechanism of action
is still controversial. This effect appears to be mediated in part by
the ability of ASP to act as an inhibitor of
MSH binding to the MC1R
(24-26). On the other hand, some studies suggest that agouti protein
may act through a receptor distinct from the MC1R (27-30). Indeed ASP
also inhibits the melanogenic activity of agents such as forskolin and
dibutyryl cAMP, which mimic the effects of
MSH but act downstream of
the MC1R (31).
In vivo, it has been clearly shown that ASP decreases eumelanin synthesis due to a slight inhibition of the tyrosinase activity and to an almost complete loss of TRP1 and TRP2 expression (32). In human or murine cultured cells, ASP inhibits eumelanin synthesis, cell proliferation, tyrosinase activity and reduces the level of TRP1 without significantly altering the level of tyrosinase (30, 31, 33).
To further understand the molecular mechanisms involved in the
inhibition of melanogenesis by agouti protein, we studied its effect on
tyrosinase, TRP1, and TRP2 expression and on the transcription activities of the corresponding promoters in B16 melanoma cells. Further, we investigated the effect of ASP on microphthalmia, a
transcription factor that mediates, through its binding to an M box
motif upstream of the TATA box, the activation of tyrosinase, TRP1 and
TRP2 promoter by cAMP elevating agents (13, 34). In addition, we
address the question of whether ASP inhibits the cAMP- or
MSH-induced differentiation of melanoblasts into functional melanocytes. Using melb-a cells, a cloned immortal line of murine melanoblasts inducible to differentiate to melanocytes, we show that
MSH and forskolin promote the differentiation of melanoblasts into
melanocytes, a process that can be partially prevented by agouti signal
protein.
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EXPERIMENTAL PROCEDURES |
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Materials
Forskolin, melanocyte-stimulating hormone
([Nle4, D-Phe7]-
MSH), phorbol
12-myristate 13-acetate, p-nitrophenyl phosphate, sodium
fluoride, sodium orthovanadate,
-glycerophosphate,
4-(2-aminoethyl)benzenesulfonyl fluoride, aprotinin, and leupeptin
were from Sigma. Basic fibroblast growth factor (bFGF) was from
Promega. RPMI medium, Dulbecco's modified Eagle's medium, trypsin,
and LipofectAMINE reagent were from Life Technologies, Inc., and fetal
calf serum was from Hyclone. The C5 monoclonal antibody was raised
against a histidine fusion protein expressed from the amino-terminal
Taq-sac fragment of human MITF cDNA and produces a
specific gel mobility super shift with microphthalmia, but not with the
related proteins TFE3, TFEB, and TFEC (35). The
PEP7,
PEP1, and
PEP8 polyclonal antibodies were raised against the carboxyl termini
of, respectively, mouse tyrosinase, TRP1, and TRP2 proteins.
Peroxidase-conjugated anti-mouse and rabbit antibodies were from
Dakopatts. Mouse ASP was purified from the medium of a T. ni
baculovirus expression system (36).
Methods
Cell Culture--
The melb-a line was grown in RPMI 1640 medium
supplemented with 12-O-tetradecanoylphorbol-13-acetate (20 nM), bFGF (1 ng/ml), fetal calf serum (5%), and glutamine
(2 mM) without feeder cells. To induce differentiation,
12-O-tetradecanoylphorbol-13-acetate and bFGF were replaced
by forskolin (20 µM) or MSH (10 nM) and fetal calf serum was 10%. Melanoblasts were treated 24 h after plating and every 48 h for 6 days (a total of three treatments). In the conditions where ASP was added with forskolin or
MSH, a
15-min preincubation period was performed. B16-F10 melanoma cells were
grown in Dulbecco's modified Eagle's medium supplemented with 7%
fetal calf serum.
Western Blot Analysis--
Melb-a cells were treated with 10 nM MSH or 20 µM forskolin in the presence
or absence of 1 µM ASP for a total of 6 days as described
above. For B16 melanoma cells, the treatment period was 48 h. Cell
lysates were prepared in 0.1 M phosphate buffer containing
1% Triton X-100, 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 µg/ml leupeptin, and 10 µg/ml aprotinin. Equal amounts of protein (10 µg/lane) were separated on a 10% polyacrylamide gel
by electrophoresis. Following transblotting onto nitrocellulose membranes (1 h at 100 V) and blocking in 5% nonfat milk in saline buffer, the membranes were incubated with
PEP7,
PEP1, and
PEP8, each at 1:3000 dilution. Microphthalmia protein was detected
with the C5 monoclonal antibody at 1:10 dilution. To check equivalent loading and transfer of the gels, anti-ERK1 antibodies were used (1:3000). The membranes were then incubated with horseradish
peroxidase-conjugated anti-rabbit or mouse IgG (1:4000). The
immunoreactive bands were detected by chemiluminescence, using the ECL
Amersham kit.
Tyrosinase Activity Stain--
L-Dopa colorimetric
stain for tyrosinase was performed as described previously (37).
Briefly, 5 µg of total protein extracts were mixed with Laemmli
sample buffer without -mercaptoethanol, boiling was avoided, and the
samples were separated on a 12% polyacrylamide gel. Gels were
equilibrated in 50 mM phosphate buffer (pH 6). Colorimetric
staining was carried out by incubating the gels for about 15 min at
37 °C in a solution of 1.5 mM L-dopa and 4 mM 3-methyl-2-benzothiazolinone hydrazone in 10 mM phosphate buffer (pH 6.8).
Transfections and Luciferase Assays--
B16 melanoma cells were
seeded in 24-well dishes, and transient transfections were performed
the following day using 2 µl of LipofectAMINE and 0.35 µg of total
plasmid DNA in a 200-µl final volume as indicated in the figure
legends. After transfection, cells were incubated with 3 nM
MSH, in the presence or in the absence of 0.6 µM ASP.
pCMV-
-galactosidase was transfected with the test plasmids to
control the variability in transfection efficiency. Twenty-four hours
after transfection, soluble extracts were harvested in 50 µl of lysis
buffer and assayed for luciferase and
-galactosidase activities. The
reporter plasmids, containing the 2.2-kilobase pair fragment of the
mouse tyrosinase promoter, the 1.1-kilobase pair fragment of the mouse
TRP1 promoter, and the 0.6-kilobase pair fragment of the human TRP2
promoter upstream from the luciferase reporter gene, were previously
described (13, 34). The reporter plasmid containing the 2.1-kilobase
pair fragment 5' of the transcription start site of the
microphthalmia-associated transcription factor (MITF) gene was isolated
from SpMITF 1, kindly provided by Dr. Shibahara (38).
Nuclear Extracts and Gel Mobility Shift Assays--
Melb-a cells
were stimulated for six days with 10 nM MSH or 20 µM forskolin in the presence or absence of 1 µM ASP, and nuclear extracts were prepared essentially as
described previously (39), except that phosphatase inhibitors (1 mM NaVO4, 5 mM NaF, 20 mM
-glycerophosphate, and 10 mM
p-nitrophenyl phosphate) were added to the nuclear
extraction buffer. A double-stranded synthetic tyrosinase M box probe
(5'-GAAAAAGTCATGTGCTTTGCAGAAGA-3') was
-32P-end-labeled
with T4 polynucleotide kinase. One µg of nuclear protein was
preincubated in binding buffer containing 10 mM Tris, pH
7.5, 100 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 4% glycerol, 80 µg/ml salmon sperm DNA, 0.1 µg poly(dI·dC), 0.2% bovine serum albumin, 2 mM
MgCl2, and 2 mM spermidine for 15 min on ice.
Then 200,000 cpm of 32P-labeled probe was added to the
binding reaction for 10 min at room temperature. For antibody
supershift assay, nuclear extracts were preincubated with 0.3 µl of
the C5 monoclonal antibody directed against microphthalmia. DNA-protein
complexes were resolved by electrophoresis on a 4% polyacrylamide gel
(37.5:1 acrylamide/bisacrylamide) in 0.5% TBE buffer (22.5 mM Tris borate, 0.5 mM EDTA, pH 8) for 2 h
at 100 V. After fixation in 7% acetic acid, gels were dried and
autoradiographed. Quantitation of the bands were performed using
the NIH Image 1.54 software.
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RESULTS |
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Molecular Mechanisms Involved in the Inhibition of Melanogenesis in B16 Cells by ASP
ASP Inhibits the Expression of Tyrosinase, TRP1, and TRP2--
In
B16 melanoma cells, it was shown that ASP inhibits tyrosinase activity
and production of melanin (33). However, regulation concerning the
three melanogenic enzymes has not been investigated. Therefore, we
performed Western blot experiments on B16 cells exposed for 48 h
to 10 nM MSH in the presence or absence of 1 µM ASP (Fig. 1). ASP almost
completely abolished the
MSH-induced expression of tyrosinase.
Potential inhibition of tyrosinase expression by ASP was not
detectable under basal conditions owing to its low level of
expression in B16 melanoma cells. In addition, ASP reduced both
basal and
MSH-stimulated expressions of TRP1 and TRP2.
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ASP Inhibits Transcription Activity of the Tyrosinase, TRP1, and
TRP2 Promoters--
To gain more insight into the mechanisms by which
ASP modulates the expression of the three melanogenic enzymes, we
studied the transcription regulation of their promoters. For this
purpose, B16 cells were transiently transfected with the corresponding promoters and exposed to MSH in the presence or absence of ASP. Tyrosinase, TRP1, and TRP2 promoter activities were stimulated upon
MSH treatment (between 4- and 7-fold over basal levels). ASP
partially inhibited basal activities (about 50% for all three promoters) and completely prevented the effect of
MSH on these promoter activities (Fig. 2).
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ASP Inhibits Microphthalmia Protein Expression and Promoter
Activity--
We have recently proposed that microphthalmia, a
transcription factor of the basic helix-loop-helix family, is involved
in the regulation of tyrosinase, TRP1, and TRP2 expression by
cAMP-elevating agents (13). Hence, we studied the effects of ASP
treatment on the expression of microphthalmia and on its promoter
activity. As seen by Western blot analysis, a 4-h incubation with
MSH-stimulated microphthalmia expression but this was completely
inhibited upon ASP addition (Fig. 3,
left). Further, microphthalmia
promoter activity was up-regulated by
MSH (about 3-fold over basal),
and this effect was dramatically blocked in the presence of ASP (Fig. 3, right).
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Effects of ASP on Differentiation of Murine Melanoblasts into Melanocytes
Inhibition of Tyrosinase Activity and Expression of the Melanogenic
Enzymes by ASP--
Melb-a cells underwent differentiation in the
presence of 20 µM forskolin or 10 nM MSH
for 1 week. We then evaluated tyrosinase activity by carrying out
tyrosinase activity stains in polyacrylamide electrophoresis gel (Fig.
4). We observed an increased activity following
MSH and forskolin treatments that was significantly reduced by ASP. ASP also inhibited basal activity of tyrosinase.
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Microphthalmia Expression Is Up-regulated during cAMP-induced
Melanoblast Differentiation; Its Binding to the M Box Is Inhibited by
ASP--
Next we studied the effects of MSH and ASP treatment on
the expression of microphthalmia and its binding to the M box. Western blot experiments showed that microphthalmia is constitutively expressed
in melanoblasts and that its expression is transiently enhanced by
MSH treatment with a maximum at 3 h and noticeably less
stimulation by 18 h (Fig. 6).
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DISCUSSION |
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Skin pigmentation involves a set of complex developmental
mechanisms occurring outside as well as inside the embryonic skin, such
as (i) emergence of the melanocyte lineage among neural crest cells,
(ii) the migration of melanocyte precursors from the neural primordium
to the epidermis and hair follicles, and (iii) cell proliferation and
final maturation of melanoblasts into melanin-producing cells, as
reviewed in Bennett (40). MSH is thought to play an important role
in the differentiation of mouse melanocytes in the epidermis and hair
bulb by inducing tyrosinase activity, melanin formation, increased
dendricity and transfer of melanosomes. It is now well established that
structure and function of melanocytes are controlled by both genetic
factors and the local tissue environment. In vivo,
MSH
promotes the production of eumelanin, while expression of the agouti
gene promotes the production of pheomelanin. In vitro
studies have shown that ASP could induce pheomelanogenesis in eumelanic
melanocytes (30, 33) demonstrating that ASP alone is sufficient to
elicit such changes. It has also been shown that ASP could
down-regulate the stimulation of melanogenesis induced by forskolin and
MSH (30, 31). In this report, we show that ASP can by itself inhibit
spontaneous and induced-differentiation of melanoblasts into
melanocytes. Syntheses of tyrosinase, TRP1, and TRP2 are down-regulated
and total tyrosinase activity is decreased in basal-, forskolin-, and
MSH-stimulated conditions by ASP. We have also shown that in B16
melanoma cells, used as a model of partially differentiated
melanocytes, ASP almost completely inhibited the expression of
tyrosinase stimulated by
MSH. TRP1 and TRP2 expressions were
down-regulated under these conditions. It is interesting that
inhibition by ASP of melanoblast differentiation induced by
MSH
involves a weak effect on tyrosinase expression as compared with the
inhibition exerted on TRP1 and TRP2 expression in the same conditions.
This observation is consistent with the fact that tyrosinase expression
is indispensable for pheomelanogenesis while decrease in TRP1 and TRP2
is a requisite. Thus in a long term process such as differentiation,
ASP preserves the acquisition of tyrosinase enzyme and prevents or
strongly reduces TRP1 expression which may lead to a population of
melanocytes producing pheomelanins instead of eumelanins. This
observation is to be compared with the marked inhibition exerted by ASP
on
MSH-induced expression of tyrosinase in B16 melanoma cells. This
suggests the existence of different mechanisms between inhibition of
melanoblast differentiation and inhibition of melanogenesis in
differentiated melanocytes. Moreover, the fact that ASP does inhibit
basal and forskolin-induced expression of tyrosinase in melanoblasts
suggests a specific and different mechanism for ASP inhibition of the
cAMP-induced events.
The mechanisms of ASP action are only partially elucidated. Competitive
inhibition of melanocortin binding has been proposed as a first
hypothesis since ASP antagonizes the effects of MSH and inhibits
125I-labeled nucleoside diphosphate-
MSH binding (24). A
classic antagonist used at concentrations 100 times above the ligand
concentrations as we did, should have completely blocked the effects of
MSH. However, in some of our experiments, this was not the case.
These results suggest that in certain conditions, ASP does not
antagonize completely the effect of
MSH through the MC1R. Moreover,
ASP can inhibit basal melanogenesis and spontaneous differentiation as
well as forskolin-induced melanogenic events; this observation weakens
the antagonist-only hypothesis. The effects of ASP on basal and
forskolin-induced differentiation fit better with the hypothesis that
ASP would be an inverse agonist of the MC1 receptor. Inverse agonists,
ligands that suppress spontaneous receptor signaling activity, have
been described for a growing number of G-protein coupled receptors: the
2-adrenoreceptor of myocardial cells (41), the
5-hydroxytryptamine receptors in NIH-3T3 fibroblasts (42), and the
calcitonin receptors (43). Hence, ASP appears likely to act both as an
antagonist of
MSH binding on MC1R and an inverse agonist to block
both the effects induced by cAMP elevating agents and basal effects.
We show that down-regulation of differentiation in melb-a cells and of
melanogenesis in B16 cells involve inhibition of tyrosinase, TRP1 and
TRP2 expression to different extents. In B16 cells, we also demonstrate
that this down-regulation results from inhibition of the tyrosinase,
TRP1, and TRP2 promoters. Besides the fact that we described for the
first time an inhibitory effect on the transcription activity of genes
for the enzymes involved in melanogenesis, it is interesting that, in
melanoma cells, ASP exhibited a strong inhibition on both basal and
MSH-stimulated activities of the corresponding promoters.
Recently, we suggested that microphthalmia, through the binding to M
box, mediates the effect of cAMP on tyrosinase, TRP1, and TRP2 promoter
activities (13). In B16 cells, ASP inhibits the activity of the
microphthalmia promoter and markedly reduces the expression of the
protein. Since microphthalmia has been shown to transactivate
tyrosinase (11), TRP1 (12), and TRP2 (13) promoters, we propose that
the inhibition of microphthalmia expression by ASP is responsible for
the decrease in melanogenic enzyme expression. Beside its putative role
in cAMP-induced melanogenesis, the microphthalmia gene plays a crucial
role in development and survival of melanocytes and controls the
tissue-specific expression of the melanogenic genes. Therefore, we
focused our attention on the expression of microphthalmia in
melanoblasts during differentiation and the effect of ASP on this
process. We observed that microphthalmia is constitutively expressed in
melanoblasts, and that MSH and forskolin increase its expression as
well as its binding to the M box. ASP inhibited the
MSH and
forskolin-stimulated expression and binding of microphthalmia to the M
box. Our results suggest that
MSH and forskolin promote
differentiation of melanoblasts into melanocytes and that ASP partially
blocks this cAMP-stimulated differentiation and also spontaneous
(basal) differentiation, probably through an inhibition of
microphthalmia gene expression.
Taken together, our studies indicate a key regulatory role for agouti signal protein in melanocyte differentiation and in melanogenesis, and point to the involvement of microphthalmia in these processes.
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ACKNOWLEDGEMENTS |
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We thank Dr. D. Willard for the mouse agouti protein (Glaxo Research Institute, Research Triangle Park, NC) and Dr. V. Hearing (Bethesda, MD) for providing anti-tyrosinase, TRP1, and TRP2 antibodies. We are grateful to K. Bille for excellent technical assistance and to Dr. R. Busca for critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported by La Ligue Nationale Contre le Cancer and Association pour la Recherche sur le Cancer (Grant 8402).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: INSERM U385,
Biologie et Physiopathologie de la Peau, Faculté de
Médecine, Avenue de Valombrose 06107, Nice Cedex 2, France. Tel.:
(33) 4 93 37 77 90; Fax: (33) 4 93 81 14 04; E-mail:
ballotti{at}unice.fr.
1
The abbreviations used are: TRP,
tyrosinase-related protein; ASP, agouti signal protein; MSH,
-melanocyte-stimulating hormone; mi, microphthalmia; bFGF, basic
fibroblast growth factor; MC1R, melanocortin 1 receptor; PAGE,
polyacrylamide gel electrophoresis.
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REFERENCES |
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