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The FASEB Journal • Research Communication
Epistatic connections between microphthalmia-
associated transcription factor and endothelin
signaling in Waardenburg syndrome and other
pigmentary disorders
Kayo Sato-Jin,*
,1
Emi K. Nishimura
†,‡,1
, Eijiro Akasaka,* Wade Huber,
‡
Hajime Nakano,* Arlo Miller,
‡
Jinyan Du,
‡
Min Wu,
‡
Katsumi Hanada,*
Daisuke Sawamura,* David E. Fisher,
‡
and Genji Imokawa*
,§,2
*Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori,
Japan;
†
Department of Stem Cell Medicine, Cancer Research Institute, Kanazawa University,
Kanazawa, Ishikawa, Japan;
‡
Dana-Farber Cancer Institute Melanoma Program and Children’s
Hospital Boston, Department of Pediatric Hematology/Oncology, Harvard Medical School, Boston,
Massachusetts, USA; and
§
Tokyo University of Technology, School of Bionics, Hachioji, Tokyo, Japan
ABSTRACT Waardenburg syndrome (WS) is an in-
herited sensorineural deafness condition in humans
caused by melanocyte deficiencies in the inner ear and
forelock. Mutation of microphthalmia-associated tran-
scription factor (MITF) is known to produce WS type
IIA whereas mutations of either endothelin (EDN) or
its receptor endothelin receptor B (EDNRB) produce
WS type IV. However, a link between MITF haploinsuf-
ficiency and EDN signaling has not yet been estab-
lished. Here we demonstrate mechanistic connections
between EDN and MITF and their functional impor-
tance in melanocytes. Addition of EDN to cultured
human melanocytes stimulated the phosphorylation of
MITF in an EDNRB-dependent manner, which was
completely abolished by mitogen-activated protein ki-
nase kinase inhibition. The expression of melanocyte-
specific MITF mRNA transcripts was markedly aug-
mented after incubation with EDN1 and was followed
by increased expression of MITF protein. Up-regulated
expression of MITF was found to be mediated via
both the mitogen-activated protein kinase-p90 ribo-
somal S6 kinase-cAMP response element-binding pro-
tein (CREB) and cAMP-protein kinase A-CREB path-
ways. In addition, EDNRB expression itself was seen to
be dependent on MITF. The functional importance of
these connections is illustrated by the ability of EDN to
stimulate expression of melanocytic pigmentation and
proliferation markers in an MITF-dependent fashion.
Collectively these data provide mechanistic and epi-
static links between MITF and EDN/EDNRB, critical
melanocytic survival factors and WS genes.—Sato-Jin,
K., Nishimura, E. K., Akasaka, E., Huber, W., Nakano,
H., Miller, A., Du, J., Wu, M., Hanada, K., Sawamura,
D., Fisher, D. E., Imokawa, G. Epistatic connections
between microphthalmia-associated transcription fac-
tor and endothelin signaling in Waardenburg syndrome
and other pigmentary disorders. FASEB J. 22, 1155–1168
(2008)
Key Words: cAMP response element-binding protein 䡠 CREB 䡠 cy-
clin-dependent kinase 2 䡠 melanocyte 䡠 endothelin B receptor
Highly organized but complex interactions be-
tween environmental cues, signal transduction path-
ways, and transcription factors underlie the develop-
ment of a cell lineage. Mammalian pigmentation genes
provide an attractive system in which to analyze such
interactions in vivo because melanocyte distribution
and pigmentation are visually detectable. These prop-
erties have allowed for the identification of numerous
coat color mutations in mice, including a smaller
number of white spotting genes, which are essential for
melanocyte development or survival (as opposed to
pigmentation per se). In human skin, mutations or
hyperexpression of several pigmentation genes may be
associated with hypo- or hyperpigmentary disorders.
Melanocyte progenitors exit from the dorsal neural
tube and migrate toward the ventral midline through
the dorsolateral pathway to colonize the dermis, epi-
dermis, and hair follicles of the skin. They undergo
proliferation and differentiation dependent on envi-
ronmental cues during the colonization process. Once
in the skin, melanocytes may respond to exogenous
signals to stimulate their growth and melanogenesis,
thereby altering skin pigmentation levels. The molecu-
lar mechanisms whereby these cues are translated into
altered melanocyte cell fate, proliferation, or differen-
tiation are not well understood.
Mutations of the white spotting genes have been
similarly found in human hereditary deafness/depig-
1
These authors contributed equally to this work.
2
Correspondence: Tokyo University of Technology, School of
Bionics, Katayanagi Institute-W204, 1404-1 Katakura Hachioji,
Tokyo 192-0982 Japan. E-mail: imokawag@dream.ocn.ne.jp
doi: 10.1096/fj.07-9080com
11550892-6638/08/0022-1155 © FASEB
mentation syndromes. Recent studies have shown that
the transcription factor PAX3, which is responsible for
Waardenburg syndrome (WS) type I or III depending
on the precise mutation, modulates expression of the
central melanocytic transcriptional regulator microph-
thalmia-associated transcription factor (MITF) (1–3).
Similarly, the SOX10 transcription factor modulates
MITF expression and is mutated in WS type IV (1, 2, 4,
5). Additional studies have shown that KIT, which is
mutated in the piebaldism plus deafness condition
(Woolf’s syndrome), triggers a signaling pathway that
leads to MITF modulation via phosphorylation (6, 7).
Mutation of endothelin3 (EDN3) or its receptor
endothelin receptor B (EDNRB) causes loss of melano-
cytes as well as enteric ganglion cells (which are also
neural crest derivatives) both in humans with WS type
IV and in mutant mice (8 –10). EDNRB is required
between embryonic day 10 and 12.5 when melano-
blasts, which have just exited from the dorsal neural
tube, are proliferating and dispersing along the dorso-
lateral pathway (11). The significant increase in mela-
noblasts seen in wild-type embryos during this time
window is absent in Ednrb
s⫺l/s-l
and Edn3
ls/ls
mutant
mice (12, 13), indicating that EDN3/EDNRB signaling
is critical for melanoblast proliferation (and subse-
quent survival) during development.
EDN peptide derived from endothelial cells is a
potent vasoconstrictor of vascular smooth muscle (14).
We demonstrated previously that human epidermal
keratinocytes secrete EDN1 and EDN2 in response to
ultraviolet (UV) irradiation (15, 16). Those EDNs can
act on melanocytes as melanogens in conjunction with
their mitogenic properties, playing an essential role in
stimulating epidermal pigmentation in several hyper-
pigmentary disorders including UVB melanosis (17). A
number of in vitro experiments have also demonstrated
that EDN1 as well as the related factor EDN3 (which
binds and activates the same receptor) promote mela-
nocyte proliferation and differentiation in cell culture
experiments (18). Characterization of the signaling
pathways involved in those responses demonstrated
that the EDN-induced signal transduction pathway
dominates the activation of protein kinase C (PKC)
through EDNRB, resulting in activation of the mitogen-
activated protein kinase (MAPK) pathway via a conver-
gent point of Raf-1 (19 –21). EDN signaling is also
thought to target activation of p90 ribosomal S6 kinase
(RSK) family kinases downstream of MAPK (22). In
contrast to the action of EDN1, ligands such as basic
fibroblast growth factor (bFGF) and stem cell factor
(SCF), which are associated with tyrosine kinases, stim-
ulate melanization of human melanocytes only weakly
despite their distinct actions as mitogens (23–25).
Therefore, the signaling mechanisms involved in the
dual and potent biological effects of EDN1 on prolifer-
ation and differentiation of human melanocytes re-
mains unclear, and molecular targets of EDNs down-
stream of MAPK that underlie EDN-induced
melanoblast or melanocyte proliferation/differentia-
tion are not well understood.
MITF, one of the WS genes, encodes a tissue-re-
stricted transcription factor of the basic helix-loop-helix
leucine-zipper type (26), which recognizes E-box-con-
taining promoter/enhancer elements (27, 28). It reg-
ulates melanocytic pigmentation as a transcription fac-
tor regulating the key melanogenic enzymes tyrosinase,
TRP1 and TRP2 (29 –32), and is also associated with
melanocyte survival (26, 28). Mutation of MITF results
in WS type IIA in humans (33–35), causing hypopig-
mentation of the skin and hair and corresponding
white coat color and deafness in mice. Interestingly,
germline mutations at loci encoding MITF, EDNRB, or
its ligand EDN3, lead to strikingly similar defects in
melanocytes (10, 36, 37). The phenotypic overlap be-
tween MITF and EDN/EDNRB mutations in humans and
mouse suggests the existence of a common functionally
important pathway for melanocyte development and its
cellular responses to environmental stimuli.
Recently, new roles for MITF in melanocytes have
been proposed, such as regulating the apoptotic inhib-
itor BclII (38), the cyclin-dependent kinase inhibitor
gene p21 (Cip1) (39), p16/Ink4a (40), and/or cyclin-
dependent kinase 2 (CDK2) (41) in addition to regu-
lating transcription of melanogenic enzymes. Thus,
characterizing the dynamics of MITF and its modula-
tion within melanocytes during EDN stimulation, which
reflects in vivo abnormal pigmentary events, would
provide insights into the regulatory roles of melano-
cyte-specific MITF (MITF-M) in melanocytes. Here, we
used EDN stimulation of intracellular signaling to
examine the dynamics of MITF-M in normal human
melanocytes. We demonstrate that EDN signals modu-
late the MITF via three kinetically distinct mechanisms,
thereby connecting an essential growth factor/receptor
to a master transcription factor MITF, which is pivotal
to melanocyte fate.
MATERIALS AND METHODS
Cell culture and treatment
Primary human melanocytes from neonatal foreskins (pro-
vided by Dr. Ruth Halaban, Yale University, New Haven, CT,
USA) were maintained between passages 1 and 3 in F10
medium (GIBCO-Life Technologies, Inc., Rockville, MD,
USA) supplemented with 7% fetal bovine serum (FBS),
penicillin/streptomycin/glutamine (GIBCO-Life Technolo-
gies, Inc.), 1 ⫻ 10
⫺4
M 3-isobutyl-1-methylxanthine (Sigma-
Aldrich Corp., St. Louis, MO, USA), 50 ng/ml 12-O-tetra-
decanoyl phorbol-13-acetate (Sigma-Aldrich Corp.), 1 M
Na
3
VO
4
, and 1 ⫻ 10
⫺3
M N
6
,2⬘-O-dibutyryladenosine 3:5-
cyclic monophosphate (Sigma-Aldrich Corp.). Primary hu-
man melanocytes from another source (Cascade Biologics,
Portland, OR, USA) were maintained in medium 254 (Cas-
cade Biologics) supplemented with 3 ng/ml recombinant
bFGF, 5 g/ml insulin, 0.18 g/ml hydrocortisone, 5 g/ml
transferrin, 3 g/ml heparin, 10 ng/ml phorbol-12-myristate-
13-acetate (PMA), 0.2% (v/v) bovine pituitary extract (BPE),
and 0.5% (v/v) FBS (Cascade Biologics). The human mela-
noma cell line 501 MEL (gift of Dr. Ruth Halaban) was grown
in F10 medium with 10% FBS plus penicillin/streptomycin/
glutamine. In evaluation of signaling changes, human pri-
1156 Vol. 22 April 2008 SATO-JIN ET AL.The FASEB Journal
mary melanocytes were seeded in medium 254 or F10 me-
dium and at 24 h before the addition of ligands, the medium
was replaced with medium 254 depleted of FBS, PMA, bFGF,
and BPE or F10 medium supplemented only with penicillin/
streptomycin/glutamine. Human primary melanocytes were
stimulated with 10 nM EDN1 (Calbiochem, San Diego, CA,
USA or Sigma-Aldrich Corp.)/EDN3 (Calbiochem), 20
ng/ml recombinant human SCF (R&D Systems, Minneapolis,
MN, USA), and forskolin (FSK) (20 M) with or without 20
g/ml cycloheximide, 20 or 40 M mitogen-activated protein
kinase kinase (MEK) inhibitor PD98059 (New England Bio-
Labs, Ipswich, MA, USA or Sigma-Aldrich Corp.), 1 M PKC
inhibitor Go¨6983 (Calbiochem), 5 or 10 M protein kinase A
(PKA) inhibitor H89 (D. Western Therapeutics Institute, Inc,
Nagoya, Japan), 10 M p38 MAPK inhibitor SB203580 (Cal-
biochem), 100 nM phosphoinositol 3-kinase (PI3 kinase)
inhibitor wortmannin (Calbiochem), 1 M Akt inhibitor
SH-6 (Calbiochem), or 25 M proteasome inhibitor MG132
(Calbiochem) for the indicated times after 24 h of starvation
as described above. The 501 MEL human melanoma cells
were stimulated by EDN1/3 and FSK with or without the MEK
inhibitor PD98059 in F10 medium supplemented only with
penicillin/streptomycin/glutamine.
Adenovirus infection
Adenoviruses were used as described previously (42) and
were engineered to overexpress either wild-type human
MITF, R215del (dominant-negative MITF), or a green fluo-
rescence protein (GFP)/wee1-truncation hybrid (which tar-
gets GFP to the nucleus as vector control), all under the
control of the elongation factor ␣ promoter (42). Subconflu-
ent primary human melanocytes were incubated with concen-
trated adenoviruses in serum-free F10 medium supplemented
with 10 mM MgCl
2
for 30 min at a multiplicity of infection of
200 for each virus. After infection the medium was replaced
by fresh medium and cultured for indicated times until
stimulation.
Quantitative reverse transcriptase (RT) -polymerase chain
reaction (PCR)/TaqMan
RNA was isolated using the Ambion RNAqueous kit (Ambion,
Austin, TX, USA) and quantitated by spectrophotometry
(Beckman, Miami, FL, USA). TaqMan One-Step RT-PCR
Master Mix Reagents as well as GAPDH Control Reagents
(Applied Biosystems, Foster City, CA, USA) were used for
quantitative RT-PCR reactions, each containing 100 ng of
total sample RNA. Reactions were run for 40 cycles under the
following conditions: stage 1: 48°C, 30 min; stage 2: 95°C, 10
min; stage 3: 94°C, 20 s; and stage 4: 62°C, 1 min. The
message of human MITF, CDK2, and silver homolog (SILV)
was detected using the following primers (Integrated DNA
Technologies, Coralville, IA, USA) and TaqMan probe (Ap-
plied Biosystems): CDK2: forward 5⬘-ATG GAG AAC TTC
CAA AAG GTG GA-3⬘, reverse 5⬘-CAG GCG GAT TTT CTT
AAG CG-3⬘ primers and CDK2 probe 5⬘-6-FAM-ATC GGA
GAG GGC ACG TAC GGA GTT GT-TAMRA-3⬘; SILV: forward
5⬘-TCT GGG CTG AGC ATT GGG-3⬘, reverse 5⬘-AGA CAG
TCA CTT CCA TGG TGT GTG-3⬘ primers and SILV probe
5⬘-6-FAM-CAG GCA GGG CAA TGC TGG GC-TAMRA-3⬘; and
EDNRB: forward 5⬘-TGA GTC TAT GTG CTC TGA GTA TTG
ACA-3⬘, reverse 5⬘-ACC TAT GGC TTC AGG GAC AGC-3⬘
primers and EDNRB probe 5⬘-6-FAM-TGT TTT GAT TTG
GGT GGT CTC TGT GGT TCT-TAMRA-3⬘. All reactions were
run in triplicate on an ABI-PRISM 7700 instrument (Applied
Biosystems), and gene message levels were normalized to
GAPDH expression.
Quantitative RT-PCR/SYBR Green
RNA was isolated using an RNeasy mini-protocol kit (Qiagen,
Valencia, CA, USA) and quantitated by spectrophotometry
(Beckman). The first-strand cDNA was synthesized from total
RNA using Rever Tra Ace (TOYOBO, Nagoya, Japan). Reac-
tions were run under the following conditions: stage 1 30°C,
10 min; stage 2: 42°C, 20 min; and stage 3: 99°C, 5 min. iQ
SYBR Green Supermix Reagents (Bio-Rad Laboratories, Her-
cules, CA, USA) were used for quantitative RT-PCR reactions,
each containing 10 ng of total sample RNA. Reactions were
run for 40 cycles under the following conditions: stage 1:
95°C, 15 s; stage 2: 61°C for 30 s; and stage 3: 72°C, 30 s. The
message of human MITF-M and GAPDH was detected using
the following primers (FASMAC, Atsugi, Japan). MITF-M:
forward 5⬘-TCC GTC TCT CAC TGG ATT GGT G-3⬘, reverse
5⬘-CGT GAA TGT GTG TTC ATG CCT GG-3⬘; and GAPDH:
forward 5⬘-GCC ATC AAT GAC CCC TTC ATT-3⬘, reverse
5⬘-TTG ACG GTG CCA TGG AAT TT-3⬘. All reactions were
run in triplicate on a DNA Engine Opticon 2 Real-Time PCR
Detection System (Bio-Rad Laboratories), and gene message
levels were normalized to GAPDH expression.
Gel electrophoresis, immunoblotting,
and immunoprecipitation
For immunoblot analysis, cells were lysed in lysis buffer [50
mM Tris (pH 7.6), 150 mM NaCl, and 1% Triton X-100] plus
protease inhibitors (Complete mini-tablets; Boehringer
Mannheim, Mannheim, Germany) and phosphatase inhibi-
tors (20 mM NaPP, 10 mM NaF, and 1 mM Na
3
VO
4
, and 1
mM Na
3
VO
4
) and centrifuged at 13,000 rpm for 15 min. The
supernatant was harvested and lysed in 2⫻ loading buffer
[125 mM Tris (pH 6.8), 4.6% sodium dodecyl sulfate (SDS),
20% glycerol, and 0.04% pyronin Y]. The mixture was then
boiled for 5 min. Samples were solubilized in SDS sample
buffer plus 50 mM dithiothreitol and boiled for 5 min. Total
protein from cell cultures of human melanocytes was sub-
jected to Western blotting with anti-MITF (C5; NeoMarkers,
Fremont, CA, USA), anti-phospho-extracellular signal-regu-
lated kinase (ERK) 1/2 (Cell Signaling Technologies, Dan-
vers, MA, USA or Santa Cruz Biotechnology, Santa Cruz, CA,
USA), anti-phospho-cAMP response element-binding protein
(CREB) (Cell Signaling Technologies), anti-phospho-
MEK1/2 (Cell Signaling Technologies), anti-CREB (Cell Sig-
naling Technologies), anti-ERK (Santa Cruz Biotechnolo-
gies), anti-MEK (Santa Cruz Biotechnologies), anti--actin
(Sigma-Aldrich Corp.), and anti-␣-tubulin (Sigma-Aldrich
Corp.) antibody. Samples were run on SDS-PAGE gels, trans-
ferred onto nitrocellulose, blocked with 5% nonfat dry milk
in Tris-buffered saline (TBST) and probed with the respective
antibodies in TBST overnight at 4°C. Membranes were
washed 3 times for 15 min with TBST, probed with peroxi-
dase-conjugated secondary antibodies (Amersham Pharmacia
Biotech, Piscataway, NJ, USA or ICN Biomedicals Inc, Solon,
OH, USA) washed three times for 30 min in TBST and
developed by ECL (Amersham).
For immunoprecipitation, cells were lysed with lysis buffer
as above plus protease inhibitors. The soluble fraction was
incubated overnight at 4°C with anti-MITF antibody (D5) or
anti-phospho-S73 MITF (43), and subsequently protein G
agarose beads (GIBCO-Life Technologies, Inc.) were added
and the solution was incubated for an additional1hat4°C.
Beads were washed three times with cold PBS, resuspended in
SDS sample buffer, and boiled for 5 min. The eluted proteins
were resolved on SDS-PAGE and immunoblotted with anti-
MITF antibody (C5).
1157MOLECULAR INTERACTIONS BETWEEN MITF AND ENDOTHELIN
Transient in vitro reporter assay
For reporter assays, 501 MEL cells were plated in a 96-well
black and white tissue culture plate (Wallac, Waltham, MA,
USA) to a density of 1 ⫻ 10
4
cells/well. The following day,
cells were transfected with 40 ng of promoter [pGL2.basic,
pGL2.MITF cAMP response element (CRE) wild-type (CRE
wt), or pGL2.MITF (⌬CRE) (CRE mut)] and 20 ng of
pRL/null (Promega, Southampton, UK) using FuGene6
transfection reagent (Roche Molecular Biochemicals,
Basel, Switzerland) as described previously (44). Cells were
allowed to incubate with the transfection mixture for 20 h.
The cells were then incubated with 20 M FSK, 10 nM
EDN1/3, or MEK inhibitor PD98059 for an additional 6 h.
Cells were washed once with PBS and lysed with 20 lof1⫻
passive lysis buffer (Promega). The assay samples were then
analyzed on a 96-well plate luminometer (EG&G Berthold,
Bad Wildbad, Germany) using a Dual-Luciferase kit (Pro-
mega). Luciferase signals were normalized to correspond-
ing Renilla signals. Results are expressed as fold activation
over unstimulated vector control transfection for each
individual promoter set and are plated as the mean ⫾ se
from at least three independent data points.
RESULTS
EDN signaling elicits phosphorylation of MITF
We first examined posttranslational modulation of
MITF via EDN signaling to assess whether treatment
with EDN1 stimulates the phosphorylation of MITF in
cultured human melanocytes. Western blotting of hu-
man melanocytes using antibody to MITF revealed that
there are two MITF species with relative mobilities
corresponding to molecular masses of 62 and 66 kDa
(Fig. 1A). The identities of the two MITF species with
different mobilities were suggested to be unphosphor-
ylated and phosphorylated MITFs because of their
changes with rapid kinetics and prior analyses by phos-
photryptic mapping (7). Activation of signaling by
EDN1 completely shifted the lower unphosphorylated
MITF band to the upper phosphorylated MITF band
with a peak at 10 min postincubation. This shift oc-
curred rapidly but transiently and the lower unphos-
phorylated band reappeared within 40 min. The mo-
bility shift was abrogated by BQ788, a selective EDNRB
Figure 1. A) EDN1 elicits a marked phosphorylation of MITF. Human primary melanocytes were stimulated with EDN1 (10 nM).
Lysates were harvested at the indicated times. EDN stimulation produced a mobility shift of MITF. Total protein blots were
probed for MITF and ERK1/2. Western blotting shows representative data. Densitometric data represent sem from three
independent experiments using melanocytes from the same donors. B) The EDNRB antagonist BQ788 abolishes the
EDN1/3-induced phosphorylation of ERK. Human primary melanocytes were treated with 10 nM EDN1/3 in the presence or
absence of BQ788 (10 M). Total protein blots were probed for MITF. C) MG132 prevents MITF degradation mediated through
proteasomal degradation. Human primary neonatal melanocytes were stimulated with EDN1 (10 nM) and cycloheximide
(CHX) (20 g/ml) in the presence or absence of MG132 protease inhibitor (25 ⌴). Lysates were harvested at the indicated
times and immunoblotted with MITF and ␣-tubulin antibodies. D) S73 of MITF is phosphorylated by EDN3 stimulation. Human
501 MEL melanoma cells were stimulated with EDN3 (10 nM). Lysates were immunoprecipitated (IP) with anti-phospho-S73
(pS73) MITF antibodies or anti-MITF antibody (D5) followed by Western blotting for MITF (C5).
1158 Vol. 22 April 2008 SATO-JIN ET AL.The FASEB Journal
receptor antagonist (Fig. 1B). Proteasome inhibition
using MG132 (Fig. 1C) stabilized MITF in the upper
phosphorylated form induced by EDN1 treatment,
suggesting prevention of MITF degradation mediated
through proteasomal degradation. Immunoprecipita-
tion with anti-phospho-S73 MITF antibody showed that
serine 73 of MITF is phosphorylated by EDN stimula-
tion (Fig. 1D).
EDN signaling elicits the phosphorylation of ERK1/2
and MEK
EDN1 stimulation also increased the phosphorylation
of ERK1/2 with a peak at 10 min postincubation (Fig.
2A), which correlated temporally with the shift in MITF
protein. The increased phosphorylation occurred rap-
idly but transiently from 2 to 20 min after incubation
with EDN1; the phosphorylation of ERK1/2 was mark-
edly diminished within 40 min postincubation. Further,
the increased phosphorylation of ERK1/2 was accom-
panied by increased phosphorylation of MEK during a
comparable time; the increased phosphorylation oc-
curred rapidly but transiently from 2 to 20 min postin-
cubation and almost disappeared within 40 min (Fig.
2B).
Phosphorylation of MITF is specifically abolished by
inhibitors of MEK and PKC
To elucidate the signaling mechanisms involved in
phosphorylation of MITF, we compared time courses of
phosphorylation of MITF and ERK1/2 and used several
specific signaling inhibitors at 10 min postincubation
when the phosphorylation of MITF reaches a maxi-
mum. Western blot analysis revealed that phosphoryla-
tion of ERK1/2 occurred at a time course similar to the
mobility shift of MITF with a peak at 10 –20 min
postincubation with EDN3 in human melanocytes and
human melanoma cells (501 MEL) (Fig. 3). Treatment
with the MEK inhibitor PD98059 (20 M),60min
before addition of EDN1, diminished the increased
phosphorylation of MITF in the presence or absence of
cycloheximide, a translation inhibitor (which blocks de
novo protein synthesis) (Fig. 4A, B). Three independent
experiments showed that MEK inhibition elicits a sig-
nificant decrease in EDN-induced MITF phosphoryla-
tion (Fig. 5A). The MEK inhibitor PD98059 also signif-
icantly decreased the EDN-induced phosphorylation of
Figure 2. A) EDN1 elicits a marked phosphorylation of ERK1/2 followed by the phosphorylation of MITF. Human primary
melanocytes were stimulated with EDN1 (10 nM). Lysates were harvested at indicated times. Total protein blots were probed for
phospho-ERK1/2 (pERK1/2) and ERK1/2. Western blotting shows representative data. Densitometric data represent sem from
three independent experiments using melanocytes from the same donors. B) EDN1 elicits a marked phosphorylation of MEK
followed by phosphorylation of MITF or ERK1/2. Human primary melanocytes were stimulated with EDN1 (10 nM). Lysates
were harvested at indicated times. Total protein blots were probed for phospho-MEK (pMEK) and MEK. Western blotting shows
representative data. Densitometric data represent sem from three independent experiments using melanocytes from the same
donors.
Figure 3. EDN3 stimulation produced a mobility shift of MITF
accompanied by ERK phosphorylation. Human primary me-
lanocytes and human 501 MEL melanoma cells were stimu-
lated with EDN3 (10 nM) for the indicated times, and total
protein blots were probed for MITF (top), phospho-ERK1/2
(pERK1/2) and ERK1/2 (center), and ␣-tubulin (bottom).
1159MOLECULAR INTERACTIONS BETWEEN MITF AND ENDOTHELIN
ERK1/2 (Fig. 5B). Although MEK inhibition was in-
complete, it also suppressed a corresponding degree of
MITF phosphorylation.
Similarly, treatment with the PKC inhibitor Go¨6983
(1 M), 60 min before addition of EDN1, significantly
abolished the increased phosphorylation of MITF (Fig.
6A), which was accompanied by marked inhibition in the
phosphorylation of ERK1/2 (Fig. 6B), consistent with the
possibility that suppression of MITF phosphorylation by
the PKC inhibitor was mediated via the inhibition of
ERK1/2 phosphorylation. In contrast, inhibitors of other
kinases such as PKA (H89, 5 M), p38 MAPK (SB203580,
10 M), PI3 kinase (wortmannin, 100 nM), and Akt
(SH-6, 1 M) did not significantly inhibit the phosphor-
ylation of MITF (Fig. 7A–D).
EDN signaling stimulates the expression of MITF
Because evidence suggests that MITF expression is
regulated by a signal transduction pathway using cAMP
as a second messenger (45, 46) and because we have
already shown that EDN signaling exerts a rapid and
marked increase in the level of cAMP in human mela-
nocytes (19), we determined whether EDN1 alters
MITF expression using quantitative RT-PCR analysis
and Western blotting. Within 40 min after EDN1 stim-
ulation there was an increase in the intensity of MITF-M
mRNA transcripts, relative to control GAPDH levels
(Fig. 8A). This increase occurred transiently with a
peak at 40 to 80 min postincubation and returned to
the control level within 3 h. Western blotting analysis
during 24 h after incubation with EDN1 revealed that
within2hoftreatment there was an increase in the
intensity of MITF protein (upper ⫹ lower bands),
relative to control -actin levels (Fig. 8B). This increase
occurred transiently with a peak at 2 to 3 h postincu-
bation and returned to the control level within 24 h.
The increased production of MITF protein was signifi-
cantly diminished by the PKA inhibitor H89 (Fig. 8C),
suggesting that the stimulation was dependent on the
cAMP-PKA pathway.
Figure 4. PD98059 abolishes EDN1-induced phosphorylation
of MITF. Human primary melanocytes were stimulated with
EDN1 (10 nM) and without (A) or with (B) cycloheximide
(CHX) (20 g/ml) in the presence or absence of MEK
inhibitor PD98059 (20 M). Lysates were harvested at indi-
cated times and immunoblotted with MITF and ␣-tubulin or
-actin antibodies.
Figure 5. PD98059 abolishes the EDN1-induced phosphorylation of MITF and ERK1/2. Human primary melanocytes were
stimulated with or without EDN1 (10 nM) in the presence or absence of PD98059 (20 M). Lysates were harvested at 10 min
and immunoblotted with MITF and ERK1/2 (A) or with phospho-ERK1/2 (pERK1/2) and ERK1/2 (B). Western blotting shows
representative data. Densitometric data represent mean ⫾ sd from three independent experiments using melanocytes from the
same donors. *P ⬍ 0.05.
1160 Vol. 22 April 2008 SATO-JIN ET AL.The FASEB Journal
EDN signaling activates CREB factors
To examine whether the transcription of the MITF
gene is dependent on binding of CREB to the MITF
promoter, luciferase reporter assays were performed
using plasmids containing the melanocyte-restricted
MITF promoter (46) driving luciferase in 501 MEL
melanoma cells (47). These assays showed that EDN (as
well as SCF) significantly induces MITF promoter activ-
ity (Fig. 9 and data not shown). The melanocytic MITF
promoter has a CRE consensus sequence where CREB/
ATF family members bind and transactivate (48). It has
been shown that whereas cAMP signaling stimulates the
MITF promoter via this conserved CRE (45, 46), ERK/
MAPK activation in melanocytes stimulates RSK, which
is known to phosphorylate and activate CREB factors in
other lineages (49, 50). We therefore examined the
possibility that activation of the MITF promoter is
mediated by the CRE site. As shown in Fig. 9 (black
bars), EDN-induced MITF promoter activation was
abrogated when the CRE site was mutated. Further-
more, the EDN responsiveness was also lost with MEK
inhibitor PD98059 treatment before EDN stimulation.
These data are consistent with the possibility that EDN
signaling induces MITF gene transcription using CREB
phosphorylation/activation significantly via the MAPK-
p90RSK pathway. Indeed, as seen in Fig. 10A, Western
blot analysis of EDN1/3 or SCF-treated melanocytes
using anti-phospho-ERK and anti-phospho-CREB
showed that both EDN and SCF stimulations induce
Figure 6. Go¨6983 diminishes the EDN1-induced phosphorylation of MITF and ERK1/2. Human primary neonatal melanocytes
were stimulated with or without EDN1 (10 nM) in the presence or absence of PKC inhibitor Go¨6983 (1 M). Lysates were
harvested at 10 min and immunoblotted with MITF and ERK1/2 (A) or with phospho-ERK1/2 (pERK1/2) and ERK1/2 (B).
Western blotting shows representative data. Densitometric data represent mean ⫾ sd from three independent experiments
using melanocytes from the same donors. *P ⬍ 0.05.
Figure 7. Other inhibitors do not elicit the EDN1 induced phosphorylation of
MITF or ERK1/2. Human primary melanocytes were stimulated with or without
EDN1 (10 nM) in the presence or absence of PKA inhibitor H89 (5 M) (A),
p38 MAPK inhibitor SB203580 (10 M) (B), PI3 kinase inhibitor wortmannin
(100 nM) (C) or Akt inhibitor SH-6 (1 M) (D). Lysates were harvested at 10
min and immunoblotted with MITF, phospho-ERK1/2 (pERK1/2), and
ERK1/2. Similar results were seen in three independent experiments using
melanocytes from the same donors.
1161MOLECULAR INTERACTIONS BETWEEN MITF AND ENDOTHELIN
significant MAPK and CREB phosphorylation, whereas
FSK treatment did not induce comparable ERK/MAPK
phosphorylation, although it did trigger strong CREB
phosphorylation that was not abrogated by MEK inhib-
itor (Fig. 10B).
The time course study of the phosphorylation of
CREB revealed that CREB was markedly phosphory-
lated by EDN stimulation with a broad peak from 3
through 30 min postincubation (Fig. 11A). The CREB
phosphorylation induced by EDN was abrogated to a
different extent depending on postincubation times
by the PKA inhibitor (H89) or the MEK inhibitor
(PD98059) (Fig. 11B–E). Although CREB phosphor-
ylation was significantly abolished at 5 min postincu-
bation by the MEK inhibitor but not by the PKA
inhibitor (Fig. 11B, C), the induced phosphoryla-
tions at 15 and 30 min postincubation were signifi-
cantly abrogated by the PKA inhibitor but not by the
MEK inhibitor (Fig. 11B, D, E), suggesting a biphasic
activation of CREB due to the differential time
course of signaling in the MAPK-RSK and cAMP-PKA
pathways. These findings, taken together, suggest
that EDN signaling up-regulates MITF expression
significantly through both MAPK-RSK and cAMP-
Figure 8. A) EDN1 increases transcription of MITF-M. Human
primary melanocytes were treated with 10 nM EDN1 and were
harvested and solubilized after the incubation times. Total
RNA extracts from treated or control human melanocytes
were reverse-transcribed and the cDNAs were PCR amplified
with specific primer sets. **P ⬍ 0.01; *P ⬍ 0.05. B) EDN1
stimulates production of MITF protein. Human primary
melanocytes were stimulated with EDN1 (10 nM). Lysates
were harvested at the indicated times. Total protein blots
were probed for MITF and -actin. Western blotting shows
representative data. Densitometric data represent sem from
three independent experiments using melanocytes from the
same donors. C) Human primary melanocytes were stimu-
lated with or without EDN1 (10 nM) in the presence or
absence of H89 (5 M). Lysates were harvested at3hand
immunoblotted with MITF and -actin. Western blotting
shows representative data. Densitometric data represent
mean ⫾ sd from three independent experiments using
melanocytes from the same donors. *P ⬍ 0.05.
Figure 9. EDN1/3 Significantly induces MITF promoter ac-
tivity. 501 MEL melanoma cells were transfected with the
human MITF promoter (⫺387 to ⫹97) or CREB binding site
(CRE) mutant promoter driving luciferase. Transfected cells
were treated with FSK or EDN1/3. Further, in one case,
EDN3 was added after PD98059 (20 M) treatment. Lucif-
erase activity was corrected for transfection efficiency using
constitutive Renilla (sea pansy) luciferase activity. Luciferase
activities were normalized with no promoter control
(pGL2.basic). The value of no stimulation control was nor-
malized to 1. Data represent sem from three independent
experiments using melanocytes from the same donors.
1162 Vol. 22 April 2008 SATO-JIN ET AL.The FASEB Journal
PKA pathways, which converge on CREB and exhibit
differential (complementary) kinetics.
EDN induces MITF-dependent transactivation of the
cell cycle regulator CDK2 and the melanosomal
protein SILV
The above results place MITF downstream of EDN
signals and suggest that significant aspects of EDN-
induced melanocyte proliferation or differentiation
may be mediated by MITF. We have recently found that
SILV and CDK2 are both MITF target genes (41, 51).
Quantitative RT-PCR revealed that EDN stimulation
significantly increased the expression levels of SILV and
CDK2 in primary melanocytes (Fig. 12). We therefore
asked whether up-regulation of these targets by EDN
was mediated by MITF. To test this hypothesis, EDN3
stimulation was repeated in the presence of control or
dominant-negative MITF adenoviruses (52) in primary
melanocytes (Fig. 12). The increased expression levels
of SILV and CDK2 induced by EDN administration were
both selectively blocked by dominant-negative MITF.
The dominant-negative MITF virus itself did not affect
MAPK activation (data not shown). These results dem-
onstrate that EDN stimulates expression of SILV and
CDK2, two genes that are involved in melanocyte dif-
ferentiation and proliferation, respectively, and this
stimulated expression is dependent on endogenous
MITF.
Expression of EDN receptor EDNRB is dependent
on MITF
Analysis of MITF expression in human melanocytes
stimulated with EDN1/3 suggested that the EDN-MITF
signaling pathways are regulated by multiple feedback
pathways. Our previous microarray analysis suggested
that expression of EDNRB is regulated via MITF in
human melanocytes (38). Thus, we examined EDNRB
expression by human melanocytes infected with adeno-
virus expressing the wild-type and dominant-negative
form of MITF. As shown in Fig. 13, EDNRB mRNA
expression was significantly induced with wild-type
MITF and inhibited by dominant-negative MITF, indi-
cating that EDNRB expression is dependent on MITF.
DISCUSSION
Despite increasing knowledge of the functions of MITF
in melanoblasts/melanocytes during their develop-
ment and of the roles of EDNs in several pigmentary
disorders (10, 33–37), the effects of EDN-activated
intracellular signaling on MITF dynamics, including its
phosphorylation, have received little attention. Thus, it
remains to be clarified as to how the activation of the
PKC, MAPK, and cAMP pathways initiated by EDN
receptor binding (19 –21) are linked to survival as well
as to the stimulation of mitogenesis and melanogenesis
of melanocytes in association with the dynamics of
MITF. Although MAPK is known to link MITF to KIT
signaling in melanocytes via MITF phosphorylation (7,
53), it had not been established whether stimulation of
human melanocytes with EDN affects the phosphoryla-
tion of MITF due to the activation of the MAPK
pathway. Here we demonstrate that EDN1 signaling
elicits a marked phosphorylation of MITF with a peak at
10 min postincubation with EDN1. This phosphoryla-
tion of MITF occurs at Ser
73
in an EDNRB-sensitive
manner and is accompanied by or preceded by the
phosphorylation of ERK1/2 and MEK, which indicates
activation of the MAPK pathway. Treatment with a MEK
inhibitor markedly suppresses the phosphorylation of
MITF. The inhibition of MITF phosphorylation is ac-
companied by a significant suppression of the phos-
phorylation of ERK1/2 at a comparable time. Because
ERK2 is the kinase responsible for this MITF phosphor-
ylation (7) and because the increased phosphorylation
of ERK1 and 2 occurs with response kinetics similar to
those of EDN1, these findings strongly suggest that
activation of the MAPK pathway by EDN1 directly
Figure 10. A) EDN1/3 and SCF elicit a marked phosphoryla-
tion of CREB and ERK1/2. Human primary melanocytes were
stimulated with EDN1 (10 nM), EDN3 (10 nM), and SCF (20
ng/ml). Total protein blots were probed for MITF, phospho-
CREB (pCREB), phospho-ERK1/2 (pERK1/2), and ␣-tubu-
lin. B) FSK induces a marked CREB phosphorylation that is
not abrogated by PD98059. Human primary melanocytes
were stimulated with FSK (20 M) in the presence or absence
of 20 M PD98059 for 15 min. Total protein blots were
probed for MITF, phospho-CREB, phospho-ERK1/2, and
␣-tubulin.
1163MOLECULAR INTERACTIONS BETWEEN MITF AND ENDOTHELIN
triggers MITF phosphorylation in human melanocytes
similar to the stimulation by SCF.
In the signaling pathway initiated by EDN1 activation
of the EDNRB in human melanocytes, we found that
the activation of PKC is linked probably at a convergent
point of Raf-1 to the phosphorylation of ERK1/2, which
indicates activation of the MAPK pathway (21). In
addition, we demonstrated that EDN1 signaling elicits a
marked increase in intracellular cAMP levels in a
PKC-dependent manner, indicating cross-talk between
PKC and the cAMP pathway (19). Based on the above
observations regarding EDN signaling, we used several
specific signaling inhibitors in this study to show that
MITF phosphorylation is significantly abolished by a
Figure 11. A) EDN1 elicits a
marked phosphorylation of CREB
with a peak at 3–15 min. Human
primary melanocytes were stimu-
lated with EDN1 (10 nM). Lysates
were harvested at indicated times.
EDN stimulation produced phos-
phorylation of CREB. Total pro-
tein blots were probed for phos-
pho-CREB (p-CREB), phospho-
ERK1/2 (pERK1/2), ERK1/2, and
-actin. Western blotting shows
representative data. Densitometric
data represent sem from three in-
dependent experiments using melanocytes from the same donors. B) PD98059 or H89 diminishes the EDN1-induced
phosphorylation of CREB. Human primary neonatal melanocytes were stimulated with EDN1 (10 nM) in the presence or
absence of PD98059 (40 M) and H89 (10 M). Lysates were harvested at indicated times and immunoblotted with
phospho-CREB, CREB, phospho-ERK1/2, and -actin. C–E) PD98059 and H89 diminishes the EDN1-induced phosphorylation
of CREB with a different time course. Human primary neonatal melanocytes were stimulated with or without EDN1 (10 nM)
in the presence or absence of PD98059 (40 M) and H89 (10 M). Lysates were harvested at 5 min (C), 15 min (D), or 30 min
(E) and immunoblotted with phospho-CREB, CREB, and -actin. Western blotting shows representative data. Densitometric
data represent mean ⫾ sd from three independent experiments using melanocytes from the same donors. **P ⬍ 0.01; *P ⬍
0.05; n.s., not significant.
Figure 12. EDN induces MITF-dependent trans-
activation of cell cycle regulator CDK2 and
melanosomal protein SILV. CDK2 (A) and
SILV (B) mRNA expressions were examined in
human primary melanocytes infected with ade-
novirus (ad.) expressing the dominant-negative
(dn) form of MITF and stimulated with EDN3.
Data represent sem from three experiments
using melanocytes from the same donors.
1164 Vol. 22 April 2008 SATO-JIN ET AL.The FASEB Journal
specific PKC inhibitor, but not by p38 MAPK, PI3
kinase, Akt, or PKA inhibitors. The fact that the inhi-
bition of MITF phosphorylation by the PKC inhibitor is
accompanied by a significant suppression of ERK1/2
phosphorylation suggests that the MITF phosphoryla-
tion induced by EDN signaling is not directly triggered
by the activation of PKC but is evoked by the activation
of ERK1/2, which occurs subsequently as a result of the
activation of PKC.
In contrast to MITF Ser
73
phosphorylation, which is
associated with the signal transduction pathway involv-
ing MAPK, MITF expression is probably regulated by a
signal transduction pathway using cAMP as a second
messenger as the MITF promoter contains a CRE (48).
In fact, elevated levels of intracellular cAMP, triggered
either by ␣-melanocyte-stimulating hormone (␣-MSH)
or by FSK, lead to rapid and potent induction of the
MITF promoter (45, 46). Because EDN1 signaling
results in a marked increase in cAMP levels in human
melanocytes in a PKC-dependent manner (19), we
asked whether EDN1 stimulation alters MITF expres-
sion via CREB phosphorylation. In this connection, our
study demonstrates that EDN1 treatment elicits a rapid
increase in MITF-M mRNA transcripts with a peak at
40–80 min postincubation. This is followed by a rapid
and transient increase in MITF protein with a peak at
2–3 h after EDN1 stimulation. As for signaling mecha-
nisms underlying stimulated MITF expression, we
found that 1) the increased production of MITF pro-
tein was significantly diminished by the PKA inhibitor
H89, 2) EDN-induced MITF promoter activation was
abrogated when the CRE site was mutated, and 3) EDN
stimulation induces a significant CREB phosphoryla-
tion in concert with MAPK phosphorylation. These
findings strongly suggest that the increased expression
of MITF after EDN stimulation is mediated via CREB
activation. As for the signaling pathway leading to
CREB activation after EDN stimulation, we demon-
strated that both the potent induction of the MITF
promoter and CREB phosphorylation by EDN stimula-
tion are abrogated by the MEK inhibitor (PD98059).
Because CREB phosphorylation after FSK stimulation
was not significantly affected by the MEK inhibitor, our
results suggest the possibility that the MAPK-RSK path-
way is also implicated in EDN-induced MITF gene
expression through CREB phosphorylation/activation.
This finding is consistent with our results (see Fig. 10A)
that SCF stimulation induces CREB phosphorylation
concomitant with ERK phosphorylation despite the fact
that SCF signaling does not up-regulate intracellular
cAMP levels (19). It is known that CREB is activated by
cAMP-dependent protein kinase as well as by all three
members of the RSK family (RSK1–3) in cells stimu-
lated by activators of the Ras-MEK-ERK1/2 cascade
(49). Therefore, as depicted in Fig. 14, it is likely that
EDN signaling triggers MITF expression via CREB
phosphorylation/activation due to both cAMP-PKA
and MAPK-p90RSK cascades, which occur with differ-
ent (biphasic) kinetics, PKA being much later than
tMAPK.
␣-MSH, KIT, and EDN signaling pathways are impli-
cated in the modulation and the expression of MITF,
but they do so in very different ways. ␣-MSH stimulation
of melanocytes up-regulates cAMP, which increases the
transcription of MITF through a CRE in the MITF
promoter (45, 46). In contrast, KIT stimulation elicits a
very rapid MAPK-mediated phosphorylation of MITF,
which induces the enhanced recruitment of p300/
CREB-binding protein (54), the coactivator family that
interacts with and modulates the transcriptional activity
of MITF. All of that occurs over the course of minutes.
On the other hand, EDN stimulation not only elicits
very rapid MAPK-mediated phosphorylation of MITF
within minutes but also profoundly increases MITF
protein expression within hours. These dual actions
Figure 13. EDNRB expression is dependent on MITF func-
tion. EDNRB mRNA expression were examined in human
primary melanocytes infected with adenovirus expressing the
wild-type (WT) or dominant-negative (DN) form of MITF.
Data represent sem from three experiments using melano-
cytes from the same donors.
Figure 14. Schematic diagram of the EDN-MITF signaling
pathway. EDN signals profoundly regulate the central mela-
nocyte transcription factor MITF in two ways: 1) up-regula-
tion of the MITF gene and 2) direct MITF phosphorylation
through MAPK. This pathway forms a feedback loop with
MITF-dependent up-regulation of EDNRB gene expression.
1165MOLECULAR INTERACTIONS BETWEEN MITF AND ENDOTHELIN
triggered by EDN stimulation are likely to be associated
with the dual potent effects of EDN on both the
proliferation and the differentiation of human melano-
cytes (15).
It is well known that KIT-mediated MAPK phosphor-
ylation triggers a short-lived MITF activation as well as a
net degradation of MITF mediated by proteasomes (7,
53). Our data also showed that a rapid disappearance of
phosphorylated MITF during EDN stimulation occurs
via its rapid degradation through proteasomes. EDN1
signaling elicits the increased production of MITF
protein and sustains MITF protein at more than control
levels over 24 h after the addition of EDN1. The
increased production of MITF protein is significantly
diminished by the PKA inhibitor H89, suggesting that
the stimulation occurs at least via the cAMP pathway.
Thus, it is likely that the cAMP-mediated new produc-
tion of MITF protein may compensate to some extent
for MITF degradation, which is stimulated by MAPK-
mediated MITF phosphorylation and subsequent ubiq-
uitination.
In this study, we demonstrate a molecular link be-
tween MITF and EDN/EDNRB, mutations, which pro-
duce WS type II and IV, respectively. Our findings show
that EDN signaling modulates the MITF by up-regula-
tion of MITF gene expression and MITF phosphoryla-
tion, which is associated with altered half-life. These
effects are both mediated by the ERK/MAPK pathway
but follow distinct kinetics. Furthermore, our transfec-
tion studies using dominant-negative MITF demon-
strate that expression of EDNRB is dependent on MITF
function, suggesting that EDNRB is a downstream
target of MITF. These findings connect several central
regulators of melanocyte development/proliferation/
differentiation into a signaling pathway that is dis-
rupted in WS types II and IV. The deafness and white
forelock (spotting) seen in patients with WS types I–IV
and Woolf syndrome thus probably results from MITF
dosage insufficiency due to disruption of 1) signaling
pathways that modulate MITF expression (EDN3,
EDNRB, and KIT), 2) transcription factors that regu-
late MITF expression (PAX3 and SOX10), or 3) muta-
tions within MITF itself. The finding of WS type II MITF
null mutant alleles (34) is consistent with the conclu-
sion that MITF dosage (haploinsufficiency) is the crit-
ical determinant of this autosomal dominant (heterozy-
gous) clinical syndrome. In addition, the discovery of
signaling pathways that modulate MITF promoter activ-
ity might theoretically permit therapeutic up-regulation
of the remaining wild-type MITF allele in these patients.
This possibility may also exist through the MSH path-
way, which signals via cAMP to CREB and similarly
stimulates MITF expression in melanocytes (45, 46).
As for signaling mechanisms underlying EDN-in-
duced proliferation and differentiation (melanin syn-
thesis) in association with MITF function, the present
study demonstrated that EDN induces MITF-depen-
dent transactivation of the CDK2 and SILV genes. CDK2
is a major cell cycle regulator important for S phase
progression (54). Therefore, a direct link of MITF with
CDK2 gene transactivation during EDN signaling may
contribute to the EDN-induced stimulation of melano-
cyte proliferation (21). SILV (PMEL17) is a melanoso-
mal protein (55) whose mutation in mice (si/si) results
in reduced melanosome number and the silver pig-
ment color defect (56). Details of the transcriptional
regulation of these genes by MITF have been described
previously (41, 51). The induction of SILV gene trans-
activation during EDN signaling is also consistent with
EDN-induced stimulation of melanin synthesis (includ-
ing melanosome formation) in human melanocytes
(21) as MITF can act as a transcription factor for
multiple components in the pigmentation pathway
(29–32).
In conclusion, as depicted in Fig. 14, EDN signals
profoundly regulate the central melanocyte transcrip-
tion factor MITF in two ways: 1) direct MITF phosphor-
ylation through MAPK and 2) up-regulation of MITF
gene expression. This pathway is also definitively asso-
ciated with up-regulation of EDNRB gene expression.
Thus, our findings associate EDN/EDNR signaling with
MITF modulation as a central regulator of melanocyte
fate, leading to the formation of a feedback loop with
strong epistatic connections. The dynamics of MITF
phosphorylation and production initiated by the acti-
vation of EDN/EDNR provides a deep insight into the
pathogenesis of WS types II and IV as well as the
biological mechanisms underlying other EDN-related
hyper- or hypopigmentary disorders.
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Received for publication August 7, 2007.
Accepted for publication October 18, 2007.
1168 Vol. 22 April 2008 SATO-JIN ET AL.The FASEB Journal
