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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- numbers for experimentation; once under experimental condi-
ing from the destruction of functional melanocytes in the tions in which cultures were seeded at the same densities,
affected skin. The three prevailing pathomechanisms of vitiligo there was no significant difference in proliferation.4 An abnor-
are the immune hypothesis, the neural hypothesis and the mal morphology of cultured nonsegmental vitiligo melano-
autocytotoxic hypothesis. Indeed, autoimmune, neural or cytes showing stubby dendrites was described by Jimbow
impaired redox status theories prevail, alone or in combin- et al.5 We also reported in a previous study that melanocytes
ation.1 In early attempts at culturing vitiligo melanocytes, it cultured from patients with unstable vitiligo showed some sig-
was reported that they showed defective growth.2 Ultrastruc- nificant morphological changes.6 Melanoblasts in the outer
tural studies of vitiligo melanocytes demonstrated abnormal- root sheath of the hair follicles are the reservoir cells for
ities that consisted of dilation of rough endoplasmic repigmentation.7 Repigmentation in vitiligo is initiated by
reticulum, circular rough endoplasmic reticulum and mem- activation, proliferation and migration of the melanoblasts from
brane-bound compartmentalization of melanosomes.3 Vitiligo the outer root sheath of hair follicles, or melanocytes from the
melanocytes need to be cultured for a significantly longer per- border area of vitiligo lesions, into depigmented epidermis.
iod than normal melanocytes in order to obtain adequate Cell migration involves modifications of the architecture of
the cells, changes in cell adhesion and remodelling of the been reported. We undertook this investigation to demon-
extracellular matrix (ECM). These events require the coordi- strate the role of MMPs and their transcription factor Ets-1
nated expression of several sets of genes.8 It is of major in vitiligo.
importance to identify the molecular actors mediating the
transcriptional regulation of these genes.
Material and methods
The ECM is a network of macromolecules in which cells are
embedded. The primary components of ECM are collagens,
Clinical samples
proteoglycans and a variety of multiadhesive matrix proteins
such as laminins and fibronectins. In many pathological condi- Patients with vitiligo with no ongoing treatment for their
tions, the balance between ECM synthesis and degradation is disease for the previous 3 weeks were selected at the outpa-
disrupted, leading to abnormal ECM remodelling.9 ECM tient clinic of the Department of Dermatology, Postgraduate
remodelling and migration is mainly related to the transcrip- Institute of Medical Education and Research, Chandigarh,
tional activation of enzymes that are involved in ECM degrad- India. The mean age of patients with unstable vitiligo was
ation such as matrix metalloproteinases (MMPs) and their 26Æ2 years (range 18–40), and of the controls was 28Æ4 years
inhibitors (tissue inhibitors of metalloproteinases, TIMPs). (range18–40). A biopsy was taken on clinically active perile-
MMPs, also called matrixins, function in the extracellular sional and normal skin from 15 patients with vitiligo and six
environment of cells and degrade both matrix and nonmatrix controls with their written informed consent and this part of
proteins. They play central roles in morphogenesis, wound the study was approved by the ethics committee at the Post-
healing, tissue repair, migration and remodelling in response graduate Institute of Medical Education.
to an injury.10,11 MMP-1, also known as interstitial collagenase,
is a neutral zinc-dependent enzyme capable of hydrolysing
Materials
specific peptide sequences found in structural components of
the ECM.12,13 The primary function of MMP-2 and MMP-9 is Melanocyte growth medium and supplements were obtained
degradation of proteins in the ECM. MMP-2 proteolytically from PromoCell (Heidelberg, Germany). A Tri Reagent kit for
digests gelatin (denatured collagen), and types IV, V, VII, IX RNA isolation was obtained from Ambion (Austin, TX,
and X collagen whereas MMP-9 proteolytically digests decorin, U.S.A.). A First-Strand cDNA Synthesis kit and all polymerase
elastin, fibrillin, laminin, gelatin (denatured collagen), and chain reaction (PCR) reagents were obtained from Fermentas
types IV, V, XI and XVI collagen. Induction of MMP-2 resulted (St Leon-Rot, Germany). The primary antibody for Ets-1 was
in significant increases of migration by melanoblasts on lami- obtained from Santa Cruz Biotechnology (Santa Cruz, CA,
nin or on laminin-5 substrates, while concomitant treatment U.S.A.) and the secondary antibody was from Sigma (St Louis,
with GM6001 blocked that induced migration.7 The expres- MO, U.S.A.). All primers, L-3,4-dihydroxyphenylalanine
sion of MMP-2 activity in supernatants derived from psoralen (DOPA) and agarose were from Sigma.
plus ultraviolet A (PUVA)-treated melanocytes was signifi-
cantly increased compared with the control group. However,
Melanocyte culture
neither MMP-2 nor MMP-9 activity in keratinocyte superna-
tants was stimulated by PUVA treatment.14 Primary culture of normal melanocytes was established by the
An important level of regulation of MMPs is inhibition by following procedure. Fresh biopsy specimens were obtained
several TIMPs15,16 and regulation of transcription by several under aseptic conditions in phosphate-buffered saline (PBS)
transcription factors, such as AP-1, SP-1 and members of the with antibiotics (penicillin and streptomycin), cut into small
Ets family alone or in combination.17–19 Among the large pieces (5 · 5 mm) and incubated with trypsin–ethylenedia-
Ets-1 gene family, ETS1, ETS2 and FLI1 seem to be consist- mine tetra-acetic acid (EDTA) (0Æ25% trypsin and 0Æ02%
ently involved in the induction and ⁄or repression of apopto- EDTA) at 4 C overnight. The next day, trypsin activity was
sis, with consequent effects on cell proliferation and neutralized by adding fetal calf serum in a 1 : 1 ratio. The
differentiation.20,21 The Ets proteins comprise a superfamily epidermis was separated from the dermis with sterile forceps.
of winged helix-turn-helix DNA-binding proteins that share a Thorough pipetting was done to separate the cells and a
unique DNA-binding domain (Ets domain).22 These tran- cell-rich suspension was formed. The solid waste of tissue
scription factors bind to a GGA (A ⁄T) core motif on the was removed and the suspension was centrifuged at 78 g for
enhancers and ⁄or promoters of target genes and are involved 5 min. The cell pellet was resuspended in serum-free medium
in the control of cellular proliferation, differentiation and containing melanocyte basal medium from PromoCell with
apoptosis.23 There is evidence that Ets-1 also has a role in supplements. The cell number was adjusted to
embryonic development.24 5 · 104 cells cm)2.
The expression of MMPs and their transcription factor The culture was maintained at 37 C in a humidified, 95%
Ets-1 was observed in situations involving extensive cell air and 5% CO2 atmosphere. The medium was changed at 3–
migration and tissue remodelling. Although there has been 4-day intervals. The cultures were routinely examined for the
a considerable number of studies on the role of Ets-1 in contamination as well as for the outgrowth of the cells. Mela-
metastasis, no study on the role of Ets-1 in vitiligo has nocytes from patients with vitiligo and controls were cultured
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- PCR reaction was finished by a final elongation step at 72 C
son et al.25 Primary melanocytes and human skin fibroblasts for 10 min. All PCR products were analysed by separation on
(negative control) were plated into 24-well tissue culture a 2% agarose gel stained with ethidium bromide.
plates. When confluent, medium was removed, and cells were
washed in PBS and fixed in 5% formalin at 4 C for 30 min.
Gelatin zymography
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 Activity of MMPs was determined by gelatin zymography.
were fixed in 10% formalin for 60 min. Formalin was Conditioned medium was normalized to equal cell numbers
removed and cells were air-dried. 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-
RNA isolation and cDNA synthesis
ples were electrophoresed on 10% polyacrylamide gels copoly-
Total RNA was isolated using the Tri Reagent kit (Ambion) merized with 1 mg mL)1 gelatin. After electrophoresis, the
and cDNA was synthesized using the First-Strand cDNA Syn- gel was washed twice in 2% Triton-X-100 for 10 min each
thesis kit (Fermentas) following the manufacturers’ protocols. 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.
Polymerase chain reaction amplification
Gelatinolytic activities were detected as transparent bands
For analysis of gene expression, PCR amplification was per- against a background of Coomassie Brilliant Blue-stained
formed using the GeneAmp PCR System 9700 (Applied Bio- gelatin.
systems, Foster City, CA, U.S.A.). All primers were synthesized
by Sigma. The primer sequences used are given in Table 1.
Western blot analysis
PCR amplification of cDNA was performed in a reaction con-
taining 10 · polymerase buffer, MgCl2, dNTPs, primers and Total protein was isolated using the Tri Reagent kit following
Taq DNA polymerase, 2 lL cDNA template and sterile RNAse- the manufacturer’s protocols Proteins were then separated on
free water added to a total volume of 25 lL. All PCR reagents 10% polyacrylamide gel and transferred electrophoretically to
were from Fermentas. We first amplified a housekeeping gene polyvinylidene difluoride membranes. The blots were incu-
encoding b-actin, in order to monitor RNA quality and cDNA bated using anti-Ets-1 primary antibodies (Santa Cruz) (at
synthesis and to ensure that equivalent amounts of cDNA were 1 : 500 dilution) for 3 h at 37 C, and then washed several
used in all PCR amplifications. All PCR reactions were pre- times. Horseradish peroxidase-conjugated antimouse IgG was
ceded by a denaturation step at 94 C for 4 min followed by used as a secondary antibody (Sigma) at 1 : 1000 dilution for
35 cycles of denaturation, annealing and extension; PCR con- 1 h. Blots were developed using the diaminobenzidine tetra-
ditions for the different cDNAs are shown in Table 2. Every chloride–H2O2 system.
(a) (b)
(a) C C N L N L (a) C C N L N L
Pro-MMP-2 MMP-2
Active-MMP-2 MMP-9
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 Discussion
(n = 6) (n = 15) skin (n = 15)
Melanocytes cultured from patients with unstable vitiligo
Fig 4. Levels of matrix metalloproteinase (MMP)-2 and MMP-9 were exhibited abnormal morphological characteristics in the form
determined in the culture supernatants. Data are presented as of a large perinuclear area with small dendrites. It seems that
mean ± SEM (statistical significance: a, vs. control; b, vs. vitiligo these dendrites were retracted whereas in melanocytes cul-
normal; *P < 0Æ05; **P < 0Æ01). tured from the controls, dendrites were long and normally
spread as we have shown in our previous study.
controls. We also compared the expression of MMP-1, MMP- In the present study, we found that expression of the MMP
2, MMP-9 and TIMP-1 between normal skin and perilesional transcription factor Ets-1 was absent at both the transcriptional
skin of patients with vitiligo. Our results demonstrated insig- and translational levels in vitiligo melanocytes whereas expres-
nificant differences in the expression of MMP-1, MMP-2, sion was present in the control melanocytes. A survey of the lit-
MMP-9 and TIMP-1 between normal skin and perilesional skin erature showed that ours is the first report to assess the Ets-1
of patients with vitiligo. 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
The expression of Ets-1 at mRNA and protein level
mice are characterized by a previously unreported white-spotted
The study of MMP transcription factor Ets-1 at both transcrip- coat colour, suggesting a defect in melanocyte proliferation,
tional level by RT-PCR and translational level by Western blot survival or migration.26 Inactivation of the Ets-1 proto-oncogene
C C N L
tion of melanocyte precursors (melanoblasts) from the outer
(a)
ETS-1
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,
b-Actin
but such migratory cells must penetrate ECM tissue barriers
in vivo. Therefore for repigmentation, timely degradation of
ETS-1 ECM is an important feature of migration.
Cell migration has been associated with the activation of
b-Actin ECM degradation. This degradation involves activation of
MMPs. Therefore, we hypothesize that in vitiligo the absence
(b) of the Ets-1 transcription factor could possibly decrease the
120 expression of MMPs (MMP-2 and MMP-9). However, the
100
expression of TIMP-1 was not significantly decreased. There-
fore, activity of MMP-2 and MMP-9 was absent in the vitiligo
% expression
80
melanocytes compared with the control melanocytes.
60 In conclusion, our results suggest that there was no expres-
mRNA expression
sion of the Ets-1 transcription factor in the melanocytes of
40 Protein expression
patients with vitiligo. The absence of a basal level of expres-
20 sion of the Ets-1 transcription factor can affect the migration
of vitiligo melanocytes. Further studies are required with a
0
Control Vitiligo normal skin Vitiligo perilesional greater number of patients with vitiligo to confirm the expres-
(n = 6) (n = 15) skin (n = 15) 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
Fig 6. Expression of Ets-1. (a) Representative images showing reverse could significantly decrease the expression and activity of
transcriptase-polymerase chain reaction products and Western blot MMP-2 and MMP-9. A significant decrease in MMP-2 and
products in melanocytes from controls (C), and normal (N) and
MMP-9 activity could reduce the migration of melanocyte pre-
perilesional (L) skin of patients with vitiligo. (b) The signal intensities
cursors (melanoblasts) from the outer root sheath of hair folli-
of the Western blot products were measured using SCION IMAGE
cles or the migration of melanocytes from the border of
analysis software (http://www.scioncorp.com/). The relative levels of
target expression were determined by normalizing their individual vitiligo lesions into the depigmented epidermis which is
band intensity to b-actin band intensity. crucial to the repigmentation of vitiliginous skin.
increased T-cell apoptosis and terminal B-cell differentiation.27 What’s already known about this topic?
It has been reported that T cells lacking the Ets-1 transcription
factor showed defective activation and survival.21 Therefore, as • Repigmentation in vitiligo is initiated by activation, pro-
our results revealed, the absence of Ets-1 expression in vitiligo liferation and migration of melanoblasts from the outer
melanocytes might be the reason for defective melanocyte root sheath of hair follicles, or melanocytes from the bor-
survival, proliferation and migration in vitiligo. der area of vitiligo lesions, into depigmented epidermis.
Importantly, Ets-1 regulates the expression of various mem- • The expression of matrix metalloproteinases (MMPs) and
bers of the MMP family (MMP-1, MMP-2, MMP-3, MMP-9 and their transcription factor Ets-1 is observed in situations
MMP-13).28,29 In this study, we investigated the expression of involving extensive cell migration and tissue remodelling.
MMP-1, MMP-2 and MMP-9 and their inhibitor TIMP-1. Our • Although there has been considerable study on the role
results showed that the expression of MMP-2 and MMP-9 was of Ets1 in metastasis, no studies on the role of Ets-1 in
significantly lower in patients with vitiligo compared with the vitiligo have been reported.
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 What does this study add?
patients with vitiligo and this difference might be because of • We report that the absence of a basal level of expression
the different conditions in vitiligo normal and perilesional skin. of Ets-1 significantly decreases the expression and activity
We also studied the activity of MMP-2 and MMP-9 by zymo- of MMP-2 and MMP-9. Significant decreases in MMP-2
graphy. Surprisingly, no activity of MMP-2 and MMP-9 was and MMP-9 activity affect the migration of melanocyte
detected in vitiligo melanocytes compared with the control. precursors (melanoblasts) from the outer root sheath of
Melanocytes are a prototype of a migratory cell, migrating hair follicles, or migration of melanocytes from the border
from the neural crest to the epidermis during fetal life,30 and of vitiligo lesions, into clinically depigmented epidermis
from the hair follicle infundibula to the depigmented epider- which is crucial to the repigmentation of vitiliginous skin.
mis during adult life.31 It is known that in vitiligo, the migra-