BJD

C L I N I C A L A N D LA B O R A T O R Y I N V E S T I G A T I O N S British Journal of Dermatology

Altered levels of Ets-1 transcription factor and matrix

metalloproteinases in melanocytes from patients with
vitiligo
R. Kumar, D. Parsad, A.J. Kanwar and D. Kaul*
Department of Dermatology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India
*Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India

Summary

Correspondence Background Vitiligo is characterized by the loss of functional melanocytes from the
Davinder Parsad. epidermis. Repigmentation in vitiligo is initiated by activation, proliferation and
E-mail: parsad@mac.com; dprs@sify.com migration of melanoblasts from the outer root sheath of hair follicles, or melano-
cytes from the border area of vitiligo lesions, into the depigmented epidermis.
Accepted for publication
15 March 2011 Cell migration plays a crucial role during repigmentation in vitiligo.
Objectives To investigate the role of matrix metalloproteinases (MMPs) and their
Funding sources transcription factor Ets-1 in vitiligo.
Financial support was received from the Indian Methods Skin biopsies were taken from 15 patients with vitiligo and six controls to
Council of Medical Research, New Delhi, India, culture melanocytes from clinically active perilesional and normal skin. Expres-
no. 3 ⁄ 1 ⁄ JRF ⁄ 70 ⁄ MPD-2006 (11586).
sion of MMP-1, MMP-2, MMP-9 and Ets-1 was examined by reverse transcrip-
Conflicts of interest tase-polymerase chain reaction analysis. Expression of Ets-1 was also confirmed
None declared. with Western blot analysis. Activity of MMP-2 and MMP-9 was assessed using
gelatin zymography.
DOI 10.1111/j.1365-2133.2011.10324.x Results The activity of MMP-2 and MMP-9 was significantly lower in patients with
vitiligo compared with the controls. The expression of MMP-2 and MMP-9 was
also significantly lower in patients with vitiligo. There was no expression of Ets-1
transcription factor at either the transcriptional or translational level in melano-
cytes cultured from patients with vitiligo.
Conclusion The absence of a basal level of expression of Ets-1 significantly decreases
the expression and activity of MMP-2 and MMP-9. Significant decreases in MMP-2
and MMP-9 activity could possibly reduce the migration of melanocyte precur-
sors (melanoblasts) from the outer root sheath of hair follicles or migration of
melanocytes from the border of vitiligo lesions into clinically depigmented epi-
dermis which is crucial to the repigmentation of vitiliginous skin.

Vitiligo vulgaris is a common depigmentation disorder result-
ing from the destruction of functional melanocytes in the
affected skin. The three prevailing pathomechanisms of vitiligo
are the immune hypothesis, the neural hypothesis and the
autocytotoxic hypothesis. Indeed, autoimmune, neural or
impaired redox status theories prevail, alone or in combin-
ation.1 In early attempts at culturing vitiligo melanocytes, it
was reported that they showed defective growth.2 Ultrastruc-
tural studies of vitiligo melanocytes demonstrated abnormal-
ities that consisted of dilation of rough endoplasmic
reticulum, circular rough endoplasmic reticulum and mem-
brane-bound compartmentalization of melanosomes.3 Vitiligo
melanocytes need to be cultured for a significantly longer per-
iod than normal melanocytes in order to obtain adequate
numbers for experimentation; once under experimental condi-
tions in which cultures were seeded at the same densities,
there was no significant difference in proliferation.4 An abnor-
mal morphology of cultured nonsegmental vitiligo melano-
cytes showing stubby dendrites was described by Jimbow
et al.5 We also reported in a previous study that melanocytes
cultured from patients with unstable vitiligo showed some sig-
nificant morphological changes.6 Melanoblasts in the outer
root sheath of the hair follicles are the reservoir cells for
repigmentation.7 Repigmentation in vitiligo is initiated by
activation, proliferation and migration of the melanoblasts from
the outer root sheath of hair follicles, or melanocytes from the
border area of vitiligo lesions, into depigmented epidermis.
Cell migration involves modifications of the architecture of

 2011 The Authors

BJD  2011 British Association of Dermatologists 2011 165, pp285–291 285
286 Ets-1 transcription factor and MMPs in vitiligo, R. Kumar et al.
the cells, changes in cell adhesion and remodelling of the
extracellular matrix (ECM). These events require the coordi-
nated expression of several sets of genes.8 It is of major
importance to identify the molecular actors mediating the
transcriptional regulation of these genes.
The ECM is a network of macromolecules in which cells are
embedded. The primary components of ECM are collagens,
proteoglycans and a variety of multiadhesive matrix proteins
such as laminins and fibronectins. In many pathological condi-
tions, the balance between ECM synthesis and degradation is
disrupted, leading to abnormal ECM remodelling.9 ECM
remodelling and migration is mainly related to the transcrip-
tional activation of enzymes that are involved in ECM degrad-
ation such as matrix metalloproteinases (MMPs) and their
inhibitors (tissue inhibitors of metalloproteinases, TIMPs).
MMPs, also called matrixins, function in the extracellular
environment of cells and degrade both matrix and nonmatrix
proteins. They play central roles in morphogenesis, wound
healing, tissue repair, migration and remodelling in response
to an injury.10,11 MMP-1, also known as interstitial collagenase,
is a neutral zinc-dependent enzyme capable of hydrolysing
specific peptide sequences found in structural components of
the ECM.12,13 The primary function of MMP-2 and MMP-9 is
degradation of proteins in the ECM. MMP-2 proteolytically
digests gelatin (denatured collagen), and types IV, V, VII, IX
and X collagen whereas MMP-9 proteolytically digests decorin,
elastin, fibrillin, laminin, gelatin (denatured collagen), and
types IV, V, XI and XVI collagen. Induction of MMP-2 resulted
in significant increases of migration by melanoblasts on lami-
nin or on laminin-5 substrates, while concomitant treatment
with GM6001 blocked that induced migration.7 The expres-
sion of MMP-2 activity in supernatants derived from psoralen
plus ultraviolet A (PUVA)-treated melanocytes was signifi-
cantly increased compared with the control group. However,
neither MMP-2 nor MMP-9 activity in keratinocyte superna-
tants was stimulated by PUVA treatment.14
An important level of regulation of MMPs is inhibition by
several TIMPs15,16 and regulation of transcription by several
transcription factors, such as AP-1, SP-1 and members of the
Ets family alone or in combination.17–19 Among the large
Ets-1 gene family, ETS1, ETS2 and FLI1 seem to be consist-
ently involved in the induction and ⁄or repression of apopto-
sis, with consequent effects on cell proliferation and
differentiation.20,21 The Ets proteins comprise a superfamily
of winged helix-turn-helix DNA-binding proteins that share a
unique DNA-binding domain (Ets domain).22 These tran-
scription factors bind to a GGA (A ⁄T) core motif on the
enhancers and ⁄or promoters of target genes and are involved
in the control of cellular proliferation, differentiation and
apoptosis.23 There is evidence that Ets-1 also has a role in
embryonic development.24
The expression of MMPs and their transcription factor
Ets-1 was observed in situations involving extensive cell
migration and tissue remodelling. Although there has been
a considerable number of studies on the role of Ets-1 in
metastasis, no study on the role of Ets-1 in vitiligo has
BJD  2011 British Association of Dermatologists 2011 165, pp285–291
been reported. We undertook this investigation to demon-
strate the role of MMPs and their transcription factor Ets-1
in vitiligo.
Material and methods
Clinical samples
Patients with vitiligo with no ongoing treatment for their
disease for the previous 3 weeks were selected at the outpa-
tient clinic of the Department of Dermatology, Postgraduate
Institute of Medical Education and Research, Chandigarh,
India. The mean age of patients with unstable vitiligo was
26Æ2 years (range 18–40), and of the controls was 28Æ4 years
(range18–40). A biopsy was taken on clinically active perile-
sional and normal skin from 15 patients with vitiligo and six
controls with their written informed consent and this part of
the study was approved by the ethics committee at the Post-
graduate Institute of Medical Education.
Materials
Melanocyte growth medium and supplements were obtained
from PromoCell (Heidelberg, Germany). A Tri Reagent kit for
RNA isolation was obtained from Ambion (Austin, TX,
U.S.A.). A First-Strand cDNA Synthesis kit and all polymerase
chain reaction (PCR) reagents were obtained from Fermentas
(St Leon-Rot, Germany). The primary antibody for Ets-1 was
obtained from Santa Cruz Biotechnology (Santa Cruz, CA,
U.S.A.) and the secondary antibody was from Sigma (St Louis,
MO, U.S.A.). All primers, L-3,4-dihydroxyphenylalanine
(DOPA) and agarose were from Sigma.
Melanocyte culture
Primary culture of normal melanocytes was established by the
following procedure. Fresh biopsy specimens were obtained
under aseptic conditions in phosphate-buffered saline (PBS)
with antibiotics (penicillin and streptomycin), cut into small
pieces (5 · 5 mm) and incubated with trypsin–ethylenedia-
mine tetra-acetic acid (EDTA) (0Æ25% trypsin and 0Æ02%
EDTA) at 4 C overnight. The next day, trypsin activity was
neutralized by adding fetal calf serum in a 1 : 1 ratio. The
epidermis was separated from the dermis with sterile forceps.
Thorough pipetting was done to separate the cells and a
cell-rich suspension was formed. The solid waste of tissue
was removed and the suspension was centrifuged at 78 g for
5 min. The cell pellet was resuspended in serum-free medium
containing melanocyte basal medium from PromoCell with
supplements. The cell number was adjusted to
5 · 104 cells cm)2.
The culture was maintained at 37 C in a humidified, 95%
air and 5% CO2 atmosphere. The medium was changed at 3–
4-day intervals. The cultures were routinely examined for the
contamination as well as for the outgrowth of the cells. Mela-
nocytes from patients with vitiligo and controls were cultured
 2011 The Authors
 Ets-1 transcription factor and MMPs in vitiligo, R. Kumar et al. 287

for 20–30 days. Cells were split at confluence by trypsin treat- Table 2 Polymerase chain reaction conditions used for amplification
ment for 5 min at room temperature. with cycles of denaturation, annealing and extension
To collect the conditioned medium, 2 · 104 cells cm)2
were placed in 10 cm2 culture dishes and allowed to adhere Gene Denaturation Annealing Extension
for 24 h. Conditioned medium was collected after 48 h incu- b-Actin 94 C for 45 s 58 C for 30 s 72 C for 45 s
bation at 37 C. It was centrifuged at 500 g to remove cell ETS1 94 C for 45 s 60 C for 30 s 72 C for 45 s
debris and stored at )20 C until used. MMP1 94 C for 15 s 66 C for 20 s 72 C for 10 s
MMP2 94 C for 15 s 66 C for 20 s 72 C for 10 s
MMP9 94 C for 15 s 66 C for 20 s 72 C for 10 s
3,4-Dihydroxyphenylalanine staining TIMP1 94 C for 15 s 66 C for 20 s 72 C for 10 s

To verify that the cells cultured from the skin biopsies exhibit-
ed the characteristic signatures of melanocytes, DOPA staining
was done following a modified method described by Edmond-
son et al.25 Primary melanocytes and human skin fibroblasts
(negative control) were plated into 24-well tissue culture
plates. When confluent, medium was removed, and cells were
washed in PBS and fixed in 5% formalin at 4 C for 30 min.
After fixation, cells were incubated with 0Æ1% L-DOPA in PBS
at 37 C for 3Æ5 h. L-DOPA was then removed, and the cells
were fixed in 10% formalin for 60 min. Formalin was
removed and cells were air-dried.
RNA isolation and cDNA synthesis
Total RNA was isolated using the Tri Reagent kit (Ambion)
and cDNA was synthesized using the First-Strand cDNA Syn-
thesis kit (Fermentas) following the manufacturers’ protocols.
Polymerase chain reaction amplification
For analysis of gene expression, PCR amplification was per-
formed using the GeneAmp PCR System 9700 (Applied Bio-
systems, Foster City, CA, U.S.A.). All primers were synthesized
by Sigma. The primer sequences used are given in Table 1.
PCR amplification of cDNA was performed in a reaction con-
taining 10 · polymerase buffer, MgCl2, dNTPs, primers and
Taq DNA polymerase, 2 lL cDNA template and sterile RNAse-
free water added to a total volume of 25 lL. All PCR reagents
were from Fermentas. We first amplified a housekeeping gene
encoding b-actin, in order to monitor RNA quality and cDNA
synthesis and to ensure that equivalent amounts of cDNA were
used in all PCR amplifications. All PCR reactions were pre-
ceded by a denaturation step at 94 C for 4 min followed by
35 cycles of denaturation, annealing and extension; PCR con-
ditions for the different cDNAs are shown in Table 2. Every
PCR reaction was finished by a final elongation step at 72 C
for 10 min. All PCR products were analysed by separation on
a 2% agarose gel stained with ethidium bromide.
Gelatin zymography
Activity of MMPs was determined by gelatin zymography.
Conditioned medium was normalized to equal cell numbers
and concentrated using Centricon (10-kDa cut-off; Millipore,
Bedford, MA, U.S.A.). Aliquots of 25 lL of each sample were
mixed with an equal amount of nonreducing buffer. The sam-
ples were electrophoresed on 10% polyacrylamide gels copoly-
merized with 1 mg mL)1 gelatin. After electrophoresis, the
gel was washed twice in 2% Triton-X-100 for 10 min each
and then incubated in incubation buffer at 37 C overnight.
After incubation the gel was stained with 0Æ5% Coomassie
Brilliant Blue and destained with gel-clear destaining solution.
Gelatinolytic activities were detected as transparent bands
against a background of Coomassie Brilliant Blue-stained
gelatin.
Western blot analysis
Total protein was isolated using the Tri Reagent kit following
the manufacturer’s protocols Proteins were then separated on
10% polyacrylamide gel and transferred electrophoretically to
polyvinylidene difluoride membranes. The blots were incu-
bated using anti-Ets-1 primary antibodies (Santa Cruz) (at
1 : 500 dilution) for 3 h at 37 C, and then washed several
times. Horseradish peroxidase-conjugated antimouse IgG was
used as a secondary antibody (Sigma) at 1 : 1000 dilution for
1 h. Blots were developed using the diaminobenzidine tetra-
chloride–H2O2 system.

Table 1 Primers used for reverse

transcriptase-polymerase chain reaction Gene Forward primer Reverse primer
products
b-Actin 5¢-CATGTACGTTGCTATCCAGGC-3¢ 5¢-CTCCTTAATGTCACGCACGAT-3¢
ETS1 5¢-AGCCGACTCTCACCATCA-3¢ 5¢-TCTGCAAGGTGTCTGTCTGG-3¢
MMP1 5¢-GAGCAAACACATCTGAGGTACAGGA-3¢ 5¢-TTGTCCCGATGATCTCCCCTGACA-3¢
MMP2 5¢-AGATCTTCTTCTTCAAGGACCGGTT-3¢ 5¢-GGCTGGTCAGTGGCTTGGGGTA-3¢
MMP9 5¢-GCGGAGATTGGGAACCAGCTGTA-3¢ 5¢-GACGCGCCTGTGTACACCCACA-3¢
TIMP1 5¢-CATCCTGTTGTTGCTGTGGCTGAT-3¢ 5¢-GTCATCTTGATCTCATAACGCTGG-3¢

 2011 The Authors

BJD  2011 British Association of Dermatologists 2011 165, pp285–291
288 Ets-1 transcription factor and MMPs in vitiligo, R. Kumar et al.
Determination of matrix metalloproteinase (MMP)-2 and
MMP-9 levels
Levels of MMP-2 and MMP-9 were determined in the culture
supernatant with use of commercially available enzyme-linked
immunosorbent assay (ELISA) kits (Calbiochem, La Jolla, CA,
U.S.A.) according to the manufacturer’s instructions.
Statistical analysis
A value of P < 0Æ01 was considered as highly significant and
all values of P < 0Æ05 were considered significant. All the anal-
yses were performed using SPSS 14.0 (SPSS Inc., Chicago, IL,
U.S.A.)
Results
Morphological characteristics of melanocyte cultures
Human skin melanocytes from patients with vitiligo and from
controls were cultured for 20–30 days and then passaged after
80% confluence. Melanocytes from patients with unstable viti-
ligo and controls were confirmed by DOPA staining (Fig. 1),
whereas fibroblasts remained unstained. Melanocytes cultured
from patients with unstable vitiligo showed a large perinuclear
area and the dendrites were small with clubbed ends
(Fig. 2b). It seems that these dendrites were retracted whereas
Fig 1. Representative image showing cultured melanocytes stained
with 3,4-dihydroxyphenylalanine.
(a) (b)
BJD  2011 British Association of Dermatologists 2011 165, pp285–291
in the control melanocytes (Fig. 2a) the perinuclear region
was normal in size and the dendrites were long and normally
spread. Melanocytes were then used for RNA and protein
extraction after a second passage.
Activity of matrix metalloproteinase-2 and -9 in
supernatant medium of melanocytes cultures
Supernatant medium with melanocyte cultures was used to
assess the activity of MMP-2 and MMP-9 by zymography
(Fig. 3). Our results demonstrate that levels of the inactive
forms (pro)-MMP-2 and pro-MMP-9 were significantly lower
in patients with vitiligo compared with the controls. The level
of pro-MMP-2 was almost the same in normal skin and peri-
lesional skin of patients with vitiligo whereas pro-MMP-9 was
significantly lower in the perilesional skin compared with the
normal skin of patients with vitiligo. Our important finding
from zymography was that the active forms of MMP-2 and
MMP-9 were not detected in patients with vitiligo compared
with the controls.
Levels of matrix metalloproteinase-2 and -9 in
supernatant medium of melanocyte cultures
Levels of MMP-2 and MMP-9 were determined in the culture
supernatants using a commercially available ELISA kit (Fig. 4).
Our results demonstrated that MMP-2 and MMP-9 levels were
significantly lower in the patients with vitiligo. The MMP-9
level was significantly lower in the perilesional skin compared
with vitiligo normal skin whereas there was no significant dif-
ference in MMP-2 level between vitiligo normal and peri-
lesional skin.
The expression of matrix metalloproteinase-1, -2, -9 and
tissue inhibitor of metalloproteinase-1 at mRNA level
Next we examined the mRNA expression of MMP-1, MMP-2,
MMP-9 and TIMP-1, using semiquantitative reverse transcrip-
tase (RT)-PCR; the data showed (Fig. 5) that MMP-1, MMP-2,
MMP-9 and TIMP-1 transcripts were expressed by melano-
cytes. Expression of MMP-2 and MMP-9 was significantly
lower in melanocytes from patients with vitiligo compared
with the controls. The mRNA expression of TIMP-1 was also
investigated; there was no significance decrease in mRNA
expression of TIMP-1 between patients with vitiligo and
Fig 2. Representative images showing
cultured melanocytes from (a) control and
(b) patients with unstable vitiligo.
 2011 The Authors
 Ets-1 transcription factor and MMPs in vitiligo, R. Kumar et al. 289

(a) C C N L N L (a) C C N L N L

Pro-MMP-9 MMP-9 MMP-1

Active-MMP-9
MMP-2

Pro-MMP-2 MMP-2
Active-MMP-2 MMP-9

(b) 120 TIMP-1

100 Pro-MMP-2 b-Actin

80 Pro-MMP-9
(b)
% activity

Control (n = 6)
Active form
60 Vitiligo normal skin (n = 15)
a* a* 120 Vitiligo perilesional skin (n = 15)
40 a*
100
a*

% mRNA expression
20 b*
80
0
60
Control Vitiligo normal skin Vitiligo perilesional
a*
(n = 6) (n = 15) skin (n = 15) 40 a*
a*
20 a**
Fig 3. Zymography of gelatinolytic enzymes [matrix metalloproteinase
0
(MMP)-2 and MMP-9]. (a) Representative gel images showing
MMP-1 MMP-2 MMP-9 TIMP-1
zymography products in the supernatant of cultured melanocytes from
control (C), vitiligo normal skin (N) and vitiligo perilesional skin (L).
Fig 5. Expression of matrix metalloproteinase (MMP)-1, MMP-2,
(b) The signal intensities of the products were measured using SCION
MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1. (a)
IMAGE analysis software (http://www.scioncorp.com/). The bar
Representative agarose gel images showing ethidium bromide-stained
diagram shows the inactive (pro-) and active forms of MMP-2 and
reverse transcriptase-polymerase chain reaction (RT-PCR) products in
MMP-9 in control, vitiligo normal and vitiligo perilesional skin. Data
the melanocytes from controls (C), and normal (N) and perilesional
are presented as mean ± SEM (statistical significance: a, vs. control; b,
(L) skin of patients with vitiligo. (b) The signal intensities of the
vs. vitiligo normal; *P < 0Æ05; **P < 0Æ01).
RT-PCR products were measured using SCION IMAGE analysis
software (http://www.scioncorp.com/). The relative levels of target
mRNA expression were determined by normalizing their individual
MMP-2 and MMP-9 levels (ng mL–1)

25
MMP-2 band intensity to b-actin band intensity. Data are presented as
20 MMP-9 mean ± SEM (statistical significance: a, vs. control; b, vs. vitiligo
normal; *P < 0Æ05; **P < 0Æ01).
15
a*
10 a* analysis revealed the absence of Ets-1 expression in patients
with vitiligo, whereas Ets-1 expression was detected in the
a**
5 b* control melanocytes (Fig. 6).

0
Control Vitiligo normal skin Vitiligo perilesional
(n = 6) (n = 15)
Fig 4. Levels of matrix metalloproteinase (MMP)-2 and MMP-9 were
determined in the culture supernatants. Data are presented as
mean ± SEM (statistical significance: a, vs. control; b, vs. vitiligo
normal; *P < 0Æ05; **P < 0Æ01).
controls. We also compared the expression of MMP-1, MMP-
2, MMP-9 and TIMP-1 between normal skin and perilesional
skin of patients with vitiligo. Our results demonstrated insig-
nificant differences in the expression of MMP-1, MMP-2,
MMP-9 and TIMP-1 between normal skin and perilesional skin
of patients with vitiligo.
The expression of Ets-1 at mRNA and protein level
The study of MMP transcription factor Ets-1 at both transcrip-
tional level by RT-PCR and translational level by Western blot
Discussion
skin (n = 15)
Melanocytes cultured from patients with unstable vitiligo
exhibited abnormal morphological characteristics in the form
of a large perinuclear area with small dendrites. It seems that
these dendrites were retracted whereas in melanocytes cul-
tured from the controls, dendrites were long and normally
spread as we have shown in our previous study.
In the present study, we found that expression of the MMP
transcription factor Ets-1 was absent at both the transcriptional
and translational levels in vitiligo melanocytes whereas expres-
sion was present in the control melanocytes. A survey of the lit-
erature showed that ours is the first report to assess the Ets-1
expression in vitiligo melanocytes. Ets-1 is a transcription factor
which regulates the expression of many key genes that are
involved in cell growth, survival and migration. Ets-1 knockout
mice are characterized by a previously unreported white-spotted
coat colour, suggesting a defect in melanocyte proliferation,
survival or migration.26 Inactivation of the Ets-1 proto-oncogene

 2011 The Authors

BJD  2011 British Association of Dermatologists 2011 165, pp285–291
290 Ets-1 transcription factor and MMPs in vitiligo, R. Kumar et al.
C C N L
(a)
ETS-1
b-Actin
ETS-1
b-Actin
(b)
120
100
% expression
80
60
mRNA expression
40 Protein expression
20
0
Control Vitiligo normal skin Vitiligo perilesional
(n = 6) (n = 15) skin (n = 15)
Fig 6. Expression of Ets-1. (a) Representative images showing reverse
transcriptase-polymerase chain reaction products and Western blot
products in melanocytes from controls (C), and normal (N) and
perilesional (L) skin of patients with vitiligo. (b) The signal intensities
of the Western blot products were measured using SCION IMAGE
analysis software (http://www.scioncorp.com/). The relative levels of
target expression were determined by normalizing their individual
band intensity to b-actin band intensity.
increased T-cell apoptosis and terminal B-cell differentiation.27
It has been reported that T cells lacking the Ets-1 transcription
factor showed defective activation and survival.21 Therefore, as
our results revealed, the absence of Ets-1 expression in vitiligo
melanocytes might be the reason for defective melanocyte
survival, proliferation and migration in vitiligo.
Importantly, Ets-1 regulates the expression of various mem-
bers of the MMP family (MMP-1, MMP-2, MMP-3, MMP-9 and
MMP-13).28,29 In this study, we investigated the expression of
MMP-1, MMP-2 and MMP-9 and their inhibitor TIMP-1. Our
results showed that the expression of MMP-2 and MMP-9 was
significantly lower in patients with vitiligo compared with the
controls whereas there was no significant change in MMP-1
expression. Our results demonstrated a difference in the expres-
sion of MMP-9 between normal skin and perilesional skin of
patients with vitiligo and this difference might be because of
the different conditions in vitiligo normal and perilesional skin.
We also studied the activity of MMP-2 and MMP-9 by zymo-
graphy. Surprisingly, no activity of MMP-2 and MMP-9 was
detected in vitiligo melanocytes compared with the control.
Melanocytes are a prototype of a migratory cell, migrating
from the neural crest to the epidermis during fetal life,30 and
from the hair follicle infundibula to the depigmented epider-
mis during adult life.31 It is known that in vitiligo, the migra-
BJD  2011 British Association of Dermatologists 2011 165, pp285–291
tion of melanocyte precursors (melanoblasts) from the outer
root sheath of hair follicles or migration of melanocytes from
the margins of vitiligo lesions into clinically depigmented epi-
dermis is crucial to the repigmentation of vitiliginous skin,
but such migratory cells must penetrate ECM tissue barriers
in vivo. Therefore for repigmentation, timely degradation of
ECM is an important feature of migration.
Cell migration has been associated with the activation of
ECM degradation. This degradation involves activation of
MMPs. Therefore, we hypothesize that in vitiligo the absence
of the Ets-1 transcription factor could possibly decrease the
expression of MMPs (MMP-2 and MMP-9). However, the
expression of TIMP-1 was not significantly decreased. There-
fore, activity of MMP-2 and MMP-9 was absent in the vitiligo
melanocytes compared with the control melanocytes.
In conclusion, our results suggest that there was no expres-
sion of the Ets-1 transcription factor in the melanocytes of
patients with vitiligo. The absence of a basal level of expres-
sion of the Ets-1 transcription factor can affect the migration
of vitiligo melanocytes. Further studies are required with a
greater number of patients with vitiligo to confirm the expres-
sion of Ets-1 and its role in the migration of vitiligo melano-
cytes. Possibly, the absence of a basal level expression of Ets-1
could significantly decrease the expression and activity of
MMP-2 and MMP-9. A significant decrease in MMP-2 and
MMP-9 activity could reduce the migration of melanocyte pre-
cursors (melanoblasts) from the outer root sheath of hair folli-
cles or the migration of melanocytes from the border of
vitiligo lesions into the depigmented epidermis which is
crucial to the repigmentation of vitiliginous skin.
What’s already known about this topic?
• Repigmentation in vitiligo is initiated by activation, pro-
liferation and migration of melanoblasts from the outer
root sheath of hair follicles, or melanocytes from the bor-
der area of vitiligo lesions, into depigmented epidermis.
• The expression of matrix metalloproteinases (MMPs) and
their transcription factor Ets-1 is observed in situations
involving extensive cell migration and tissue remodelling.
• Although there has been considerable study on the role
of Ets1 in metastasis, no studies on the role of Ets-1 in
vitiligo have been reported.
What does this study add?
• We report that the absence of a basal level of expression
of Ets-1 significantly decreases the expression and activity
of MMP-2 and MMP-9. Significant decreases in MMP-2
and MMP-9 activity affect the migration of melanocyte
precursors (melanoblasts) from the outer root sheath of
hair follicles, or migration of melanocytes from the border
of vitiligo lesions, into clinically depigmented epidermis
which is crucial to the repigmentation of vitiliginous skin.
 2011 The Authors

References
1 Le Poole IC, Das PK, van den Wijngaard RM et al. Review of the
etiopathomechanism of vitiligo: a convergence theory. Exp Dermatol
1993; 2:145–53.
2 Puri N, Mojamdar M, Ramaiah A. In vitro growth characteristics of
melanocytes obtained from adult normal and vitiligo subjects.
J Invest Dermatol 1987; 88:434–8.
3 Boissy RE, Liu Y-Y, Medrano EE, Nordlund JJ. Structural aberration
of the rough endoplasmic reticulum and melanosome compart-
mentalization in long-term cultures of melanocytes from vitiligo
patients. J Invest Dermatol 1991; 97:395–404.
4 Headley SJ, Metcalfe R, Gawkrodger DJ et al. Vitiligo melanocytes
in long-term culture show normal constitutive and cytokine-
induced expression of intercellular adhesion molecule-1 and major
histocompatibility complex class I and class II molecules. Br J Der-
matol 1998; 139:965–73.
5 Jimbow K, Chen H, Park JS, Thomas PD. Increased sensitivity of
melanocytes to oxidative stress and abnormal expression of tyrosi-
nase-related protein in vitiligo. Br J Dermatol 2001; 144:55–65.
6 Kumar R, Parsad D, Kanwar AJ. Role of apoptosis and melanocytor-
rhagy: a comparative study of melanocyte adhesion in stable and
unstable vitiligo. Br J Dermatol 2011; 164:187–91.
7 Lei TC, Vieira WD, Hearing VJ. In vitro migration of melanoblasts
requires matrix metalloproteinase-2: implications to vitiligo ther-
apy by photochemotherapy. Pigment Cell Res 2002; 15:426–32.
8 Jiang WG, Puntis MC, Hallett MB. Molecular and cellular basis of
cancer invasion and metastasis: implications for treatment. Br J Surg
1994; 81:1576–90.
9 Trojanowska M. Ets factors and regulation of the extracellular
matrix. Oncogene 2000; 19:6464–71.
10 Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors
of metalloproteinases: structure, function, and biochemistry. Circ
Res 2003; 92:827–39.
11 Nagase H, Woessner JF Jr. Matrix metalloproteinases. J Biol Chem
1999; 274:21491–4.
12 Woessner JF Jr. Matrix metalloproteinases and their inhibitors in
connective tissue remodeling. FASEB J 1991; 5:2l45–54.
13 Birkedal-Hansen H, Moore WG, Bodden MK et al. Matrix metallo-
proteinases: a review. Crit Rev Oral Biol Med 1993; 4:197–250.
14 Wu CS, Lan CCE, Wang LF et al. Effects of psoralen plus ultraviolet
A irradiation on cultured epidermal cells in vitro and patients with
vitiligo in vivo. Br J Dermatol 2007; 156:122–9.
15 Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metallo-
proteinases: evolution, structure and function. Biochim Biophys Acta
2000; 1477:267–83.
16 Borden P, Heller RA. Transcriptional control of matrix metallopro-
teinases and the tissue inhibitors of matrix metalloproteinases. Crit
Rev Eukaryot Gene Expr 1997; 7:159–78.
 2011 The Authors
BJD  2011 British Association of Dermatologists 2011 165, pp285–291
Ets-1 transcription factor and MMPs in vitiligo, R. Kumar et al. 291
17 Jinnin M, Ihn H, Mimura Y et al. Matrix metalloproteinase-1 up-
regulation by hepatocyte growth factor in human dermal fibro-
blasts via ERK signaling pathway involves Ets-1 and Fli1. Nucleic
Acids Res 2005; 33:3540–9.
18 Huang HC, Liu SY, Liang Y et al. Transforming growth factor-beta1
stimulates matrix metalloproteinase-9 production through ERK
activation pathway and upregulation of Ets-1 protein. Zhonghua Yi
Xue Za Zhi 2005; 85:328–31.
19 Chakraborti S, Mandal M, Das S et al. Regulation of matrix metallo-
proteinases: an overview. Mol Cell Biochem 2003; 253:269–85.
20 Huang CC, Papas TS, Bhat NK. A variant form of Ets-1 induces
apoptosis in human colon cancer cells. Oncogene 1997; 15:851–6.
21 Muthusamy N, Barton K, Leiden JM. Defective activation and sur-
vival of T cells lacking the Ets-1 transcription factor. Nature 1995;
377:639–42.
22 Sharrocks AD, Brown AL, Ling Y, Yates PR. The ETS-domain tran-
scription factor family. Int J Biochem Cell Biol 1997; 29:1371–87.
23 Pei H, Yordy JS, Leng Q et al. EAPII interacts with ETS1 and modu-
lates its transcriptional function. Oncogene 2003; 22:2699–709.
24 Kola I, Brookes S, Green AR et al. The Ets1 transcription factor is
widely expressed during murine embryo development and is asso-
ciated with mesodermal cells involved in morphogenetic processes
such as organ formation. Proc Natl Acad Sci USA 1993; 90:
7588–92.
25 Edmondson SR, Russo VC, McFarlane AC et al. Interactions between
growth hormone, insulin-like growth factor I, and basic fibroblast
growth factor in melanocyte growth. J Clin Endocrinol Metab 1999;
84:1638–44.
26 Nagarajan P, Parikh N, Garrett-Sinha LA, Sinha S. Ets-1 induces
dysplastic changes when expressed in terminally differentiating
squamous epidermal cells. PLoS ONE 2009; 4:e4179.
27 Bories JC, Willerford DM, Grévin D et al. Increased T-cell apoptosis
and terminal B-cell differentiation induced by inactivation of the
Ets-1 proto-oncogene. Nature 1995; 377:635–8.
28 Westermarck J, Seth A, Kahari VM. Differential regulation of inter-
stitial collagenase (MMP-1) gene expression by ETS transcription
factors. Oncogene 1997; 14:2651–60.
29 Reisdorff J, En-Nia A, Stefanidis I et al. Transcription factor Ets-1
regulates gelatinase a gene expression in mesangial cells. J Am Soc
Nephrol 2002; 13:1568–78.
30 Holbrook KA. Melanocytes in human embryonic and fetal skin: a
review and new findings. Pigment Cell Res 1988; 1 (Suppl. 1):
6–17.
31 Staricco RG. Activation of amelanotic melanocytes in the outer root
sheath of the hair follicle following ultraviolet exposure. J Invest
Dermatol 1962; 39:163–4.