J. Pineal Res.

2011; 51:94103

 2011 John Wiley & Sons A/S

Molecular, Biological, Physiological and Clinical Aspects of Melatonin

Doi:10.1111/j.1600-079X.2011.00866.x

Journal of Pineal Research

Melatonin protects against oxidative stress in granular corneal

dystrophy type 2 corneal fibroblasts by mechanisms that involve
membrane melatonin receptors
Abstract: Considering that oxidative stress plays a role in corneal broblast
degeneration during granular corneal dystrophy type 2 (GCD2) and
melatonin is an eective antioxidant, we examined the ability of melatonin to
protect against oxidative stress-induced cell death of primary cultured
normal and GCD2-homozygous corneal broblasts. Melatonin treatment
protected primary cultured normal and GCD2 corneal broblasts from
paraquat (PQ)-induced oxidative stress and caused increased expression
levels of Cu/Zn-superoxide dismutase (SOD1) and glutathione reductase
(GR) in both types of cells. Interestingly, catalase expression increased in
normal corneal broblasts, but decreased in GCD2 corneal broblasts after
melatonin treatment. Melatonin also reduced the levels of intracellular
reactive oxygen species and H2O2 in both cell types. In addition, the selective
melatonin receptor antagonist luzindole blocked melatonin-induced
expression of SOD1 and GR. The expression levels of melatonin receptors
1A (MT1) and 1B (MT2) were signicantly higher in GCD2 corneal
broblasts than in normal cells. These results suggest that increased
expression of melatonin receptors may be involved in the defense
mechanisms against oxidative stress in GCD2 corneal broblasts, and
melatonin may have potential therapeutic implications for GCD2 treatment.

Introduction
The cornea is composed of avascular tissue that maintains
transparency at the frontal surface of the eye [1] and contains
three major layers: the outer epithelium, a thick stroma with
corneal broblasts, and the inner endothelium. Corneal
tissue is chronically exposed to environmental oxidative
stimuli, such as solar ultra violet (UV) radiation and high
levels of oxygen [2], and is known to be particularly
susceptible to oxidative stress [3]. For example, the number
of corneal broblasts declines in normal corneas with age in
response to oxidative stress [4, 5]. Presumably as a result,
normal corneas have well-developed antioxidant defense
systems that contain direct free radical scavengers, including
vitamin E, vitamin C, b-carotene, and glutathione (GSH) [6,
7], and indirect antioxidant enzymes, such as superoxide
dismutase (SOD), glutathione peroxidase (GPx), glutathione
reductase (GR), and catalase [8]. The oxidative stress status
of a cell is the primary factor that aects the expression and
activities of these enzymes that protect cells from damage
induced by oxygen free radicals [9, 10]. However, factors that
undermine the activities of antioxidant enzymes may lead to
reactive oxygen species (ROS) accumulation and subsequent
oxidative damage to biological macromolecules [11, 12].
Granular corneal dystrophy type 2 (GCD2) is an
autosomal dominant disorder caused by point mutations
(R124H) in transforming growth factor-b-induced gene-h3
94

Seung-Il Choi1,2, Shorafidinkhuja

Dadakhujaev1,2, Hyunmi Ryu1,2,
Tae-im Kim1,2 and Eung Kweon
Kim1,2,3,4
1

Cornea Dystrophy Research Institute;

Department of Ophthalmology; 3Severance
Biomedical Science Institute, and 4Brain Korea
21 Project for Medical Science, Yonsei
University College of Medicine, Seoul, Korea
2

Key words: corneal fibroblasts, granular

corneal dystrophy type 2, melatonin,
melatonin receptors, oxidative stress
Address reprint requests to Eung Kweon Kim,
Department of Ophthalmology, Yonsei University College of Medicine, #134 Shinchondong, Seodaemun-ku 120-752, Seoul, Korea.
E-mail: eungkkim@yuhs.ac
Received November 23, 2010;
Accepted January 20, 2011.

(BIGH3). Age-dependent progressive accumulation of hyaline and amyloid are hallmarks of GCD2. This accumulation is characterized by the production of abnormal
transforming growth factor-b-induced protein (TGFBIp)
encoded by mutated BIGH3 in the corneal epithelia and
stroma that interferes with corneal transparency [1315].
More recently, it has been demonstrated that GCD2
primary cultured corneal broblasts are highly susceptible
to oxidative stress-induced cell death, when compared with
normal primary cultured corneal broblasts, and that
oxidative stress is involved in the corneal pathogenesis of
this disease [16]. Therefore, there is growing interest in the
therapeutic implications for antioxidant treatments for
GCD2.
Melatonin is produced in the pineal gland of all vertebrates
[17] and is involved in the control of various physiological
functions, such as coordination of seasonal and circadian
rhythms, anti-inammatory action and anti-cancer eects
[1822]. Melatonin and its metabolites have received much
attention due to their direct free radical scavenging [2326]
and antioxidant properties [27, 28]. Numerous studies have
documented that melatonin has anti-apoptotic eects in
normal cells [2931] attributable to its antioxidant properties
[32]. An anti-apoptotic mechanism of melatonin involved the
inhibition of Bad translocation from the cytosol to the
mitochondria by maintaining proteinprotein interactions
between 14-3-3b and p-Bad [33].

Melatonin protects GCD2 corneal broblasts

In mammals, some of the specic functions of melatonin
are mediated by two dierent subtypes of G proteincoupled receptors, MT1 and MT2 [34]. Three dierent
membrane melatonin receptor subtypes are currently
known, as MT1, MT2, and MT3 [35]. Melatonin receptor
subtypes are present in the retina, cornea, ciliary body, lens,
choroid, and sclera of ocular tissues [36]. MT1 is present in
the endothelial cell layer, keratocytes, and the epithelial cell
layer of human corneas [37]. Additionally, melatonin
suppresses free radical-mediated ocular disease [38, 39].
On the basis of these ndings, several clinical trials have
investigated the usefulness of melatonin for treating agerelated macular degeneration and glaucoma [40, 41].
In the present study, we found a positive association
between antioxidant enzymes and melatonin receptors. Our
results clearly indicate that exogenously administered
melatonin may, at least in part, suppress cell death of
GCD2 corneal broblasts through the up-regulation of
several antioxidant enzymes. Therefore, melatonin application may be useful for the treatment of GCD2 and could be
further examined in future clinical trials alone or in
combination with other antioxidants.

Materials and methods

Isolation and culture of primary corneal fibroblasts
Primary corneal broblast cultures were prepared from
healthy corneas obtained from the eye bank of Yonsei
University Severance Hospital and from hetero- and
homozygotic GCD2 patients following penetrating or
lamellar keratoplasty. Donor condentiality was maintained according to the Declaration of Helsinki and was
approved by the Severance hospital IRB Committee
(CR04124), Yonsei University. GCD2 was diagnosed by
DNA sequencing analysis of BIGH3 gene mutations.
Corneas were washed three times with Dulbeccos modied
Eagles medium (DMEM; Gibco BRL, Grand Island, NY,
USA) containing 1000 units/mL penicillin and 1.0 mg/mL
streptomycin sulfate (Gibco BRL). Corneal epithelia were
removed by scraping the epithelial surface and detaching the
Descemets membrane with ne forceps and blades. Corneal
tissues were placed on 30-mm culture dishes and DMEM
containing 1000 units/mL penicillin, 1.0 mg/mL streptomycin sulfate, and 10% fetal bovine serum (Invitrogen-Life
Technologies, Carlsbad, CA, USA) was added. After
34 days, migratory broblasts from the corneal button were
identiable. These cultured primary corneal broblasts were
maintained in DMEM with 10% fetal bovine serum at 37C
with 95% humidity and 5% CO2. The medium was changed
every 3 days. When the cultured corneal broblasts were
approximately 8090% conuent, cells were subcultured with
0.25% trypsin and 5.0 mm EDTA at a 1:3 split. Cells from
passages 58 were used in the experiments.
Cell viability
Primary cultured corneal broblasts were plated in 96-well
plates at 10,000 cells per well overnight. Cell proliferation
was determined using a CellTiter 96 AQueous One
Solution Reagent Cell Proliferation Assay Kit using the

tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,

inner salt; MTS] (Cat. No. G3580; Promega, Madison, WI,
USA) as per the manufacturers protocol. Briey, the cells
were treated with dierent concentrations (100300 lm) of
PQ in the presence (50300 lm) or absence of melatonin
pretreatment. After 24 hr, the cells were washed and treated
with MTS, and the plate was incubated in the dark for 2 hr,
followed by an absorbance measurement at a wavelength of
490 nm using a microtiter plate reader and software
(Spcetramax PLUS and Softmax Pro; Molecular Devices
Corporation, Sunnyvale, CA, USA). For the determination of cell viability, the percent viability was calculated
as the absorbance of the PQ-treated sample/control
absorbance 100.
Induction and evaluation of cell death
Melatonin (100 lm) was added 12 hr prior to 100 lm
paraquat (PQ) (Sigma, St Louis, MO, USA) treatment, and
cell death was evaluated 12 hr after PQ treatment. Antagonism of melatonin receptors MT1 and MT2 was achieved
by adding 10 lm 2-benzyl-N-acetyltryptamine (luzindole)
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) 30 min
before the melatonin treatments. Cells were grown in an
incubator at 37C with 5% CO2 and 95% humidity. Cell
death was evaluated by ow cytometry in cells that were
stained with propidium iodide (FL2-H) and annexin-V
(FL1-H). The Annexin-V-FITC/PI assay was applied to
test whether cells were dying from apoptosis or necrosis.
The initiation of apoptosis is often accompanied by a
translocation of phosphatidylserine (PS) from the inner
leaet to the outer surface of the plasmalemma. PS
externalization typically precedes formation of membrane
blebs and DNA condensation.
Preparation of cell lysates and Western blot analysis
Cell lysates from primary cultured corneal broblasts were
prepared in radio-immunoprecipitation assay buer (RIPA
buer; 150 mm NaCl, 1% NP-40, 0.5% deoxycholate, 0.1%
SDS, and 50 mm Tris-HCl, pH 7.4) containing a protease
inhibitor tablet (Complete Mini Protease Inhibitor Tablet,
Roche, Indianapolis, IN # 1836170). Crude cell lysates were
centrifuged at 10,000 g for 10 min at 4C to remove nuclear
fragments and tissue debris. A portion of the supernatant was
used to determine the total protein concentration with a BCA
kit (Pierce, Rockford, IL, USA).
Total cellular proteins were electrophoresed in 10% Trisglycine sodium dodecyl sulfate (SDS) polyacrylamide gels.
Proteins were transferred onto PVDF membranes (Millipore
Corp., Bedford, MA, USA), blocked in 5% dry milk in TBST (0.02 m Tris and 0.15 m NaCl, pH 7.5 containing 0.1%
Tween 20) at room temperature for 1 hr, and washed three
times with TBS-T. Blots were incubated with primary
antibodies to Cu/Zn-SOD (1:1000 dilution; Cat. No.
SOD100; Stressgen, Victoria, BC, Canada), Mn-SOD
(1:1000 dilution; Cat. No. SOD110; Stressgen), catalase
(1:500 dilution; Cat. No. ab1877; Abcam, Cambridge, UK),
GPx (1:250 dilution; Cat. No. LF-PA0087; AbFrontier,
Seoul, Korea), GR (1:5000 dilution; Cat. No. LF-PA0087;
95

Choi et al.
AbFrontier), and b-actin (1:5000 dilution; Cat. No. A-5441;
Sigma Chemical Co., St Louis, MO, USA). Primary antibodies for MT1 (1:100 dilution; Cat. No. sc-13179) and MT2
(1:100 dilution; Cat. No. sc-13177) were purchased from
Santa Cruz Biotechnology. After washing three times with
TBS-T, the blots were incubated with secondary antibodies
conjugated to horseradish peroxidase (HRP) at room temperature for 1 hr. HRP-linked anti-mouse IgG (1:5000
dilution; Cat. No. NA931V; Amersham Pharmacia Biotechnology, Piscataway, NJ, USA) or anti-rabbit IgG (1:5000
dilution; Cat. No. NA934V; Amersham Pharmacia Biotechnology) were used as secondary antibodies. Immunoblots
were visualized using the ECL system (Pierce). The intensities
of immunoreactive protein bands were image-scanned and
optical densities of the bands were quantied using ImageJ
software, version 1.37 (Wayne Rasband, National Institute
of Health, Bethesda, MD, USA), corrected by background
subtraction, and normalized to the intensity of the corresponding b-actin protein bands.
RNA isolation and reverse transcription PCR
(RT-PCR)
For amplication of CAT and b-actin mRNA, total RNA
was isolated from normal and GCD2-homozygous corneal
broblasts by extraction in TRIZOL Reagent (Invitrogen
Life Technologies). cDNA synthesis and DNA amplication
was performed using a Superscript One-Step Reverse
Transcription (RT)-PCR System (Invitrogen Life Technologies) and primers specic for CAT (forward primer,
5-ATCTCGTTGGAAATAACACC-3, reverse primer,
5-AGAAACCTGATGCAGAGACT-3) and b-actin (forward primer, 5-GGACTTCGAGCAAGAGATGG-3, reverse primer, 5-AGCACTGTGTTGGCGTACAG-3).
ROS measurements
DCFH-DA (Sigma Chemical Co.) was dissolved in ethanol
to a nal concentration of 20 mm before use. Cells were
washed twice with PBS to remove the endogenous esterase
activity of the FBS. To measure ROS, cells were incubated
with 10 mm DCFH-DA at 37C for 30 min. Extracellular
DCFH-DA was removed with two PBS washes. The
uorescence intensity was recorded using a uorescent
microplate reader (HTS 7000; Perkin-Elmer Corp.,
Norwalk, CT, USA) equipped with an excitation lter of
485 nm and an emission lter of 535 nm.
To measure intracellular H2O2 levels, cells were washed
twice with PBS and incubated with 5 mm BES-H2O2 (Wako
Pure Chemical Co., Osaka, Japan) at 37C for 60 min.
Extracellular BES-H2O2 was removed with two PBS washes.
Fluorescence intensities were recorded using a luminescence
spectrometer (LS50B; Perkin-Elmer Corp.) equipped with an
excitation lter of 485 nm and an emission lter of 515 nm.
Data were calculated as the percentage of the uorescence
intensity of the normal control cultures.
Immunocytochemical staining
Normal and GCD2-homozygous corneal broblasts grown
on culture slides (Cat. No. REF 354108; BD Falcon,
96

Labware, Franklin Lakes, NJ, USA) were permeabilized

and xed in methanol at )20C for 3 min. Cells were
washed in PBS, blocked with 10% bovine serum albumin
(Sigma) in PBS for 10 min, and incubated with primary
antibodies in blocking buer for 1 hr at room temperature.
Cells were incubated with secondary antibodies for 1 hr at
RT. Coverslips were mounted on the glass slides with
Vectashield mounting medium (Vector Labs Inc., Burlingame, CA, USA). Cells were viewed under a Leica TCS SP5
confocal microscope (Leica Microsystems, Wetzlar,
Germany). The primary antibodies were polyclonal antiMT1 (1:50 dilution; Cat. No. sc-13179; Santa Cruz
Biotechnology) and the secondary antibody was uorescein
isothiocyanate (FITC; green)-labeled anti-rabbit IgG (1:200
dilution; Jackson ImmunoResearch Laboratories, West
Grove, PA, USA).
Flow cytometric analysis
Flow cytometric analysis was performed according to the
manufacturers protocol (Annexin V Fluorescent in situ
Apoptosis Detection Kit, BioBud Company, Seoul, Korea).
Approximately 1 104 cells were plated into 100-mm2
tissue culture dishes (Corning, Corning, NY). On day 0,
medium was replaced with fresh medium containing
100 lm melatonin. After 12 hr, medium was replaced with
medium containing various melatonin with or without of
100 lm PQ, and cells were harvested after 12 hr. Cells were
treated with RNase and washed with PBS, pH 7.4 (Cat. No.
10010; Invitrogen Life Technologies), and 1 106 cells were
resuspended in 5 lL PI and 5 lL annexin V incubation
buer for 15 min at room temperature in the dark. Binding
buer (500 lL) was added to each sample, and the samples
were analyzed on a FACS Calibur ow cytometer (BectonDickinson Immunocytometry Systems, San Jose, CA,
USA).
Statistical analysis
Data were statistically evaluated for signicance
(P < 0.05) with one-way analysis of variance (ANOVA)
followed by NewmanKeuls multiple comparison tests.
Data are expressed as means S.D. All data were
processed using the Graph Pad Prism version 4.0 statistical package (Graph Pad Software Inc., San Diego, CA,
USA).

Results
To test the ability of melatonin to protect corneal
broblasts against oxidative stress-induced cell death, both
normal and GCD2 cultured primary corneal broblasts
were treated with 100 lm melatonin for 12 hr prior to
exposure to 100 lm PQ. An MTS assay showed that cell
viability signicantly decreased to 83.5 5.9% in normal
and 62.1 7.3% in GCD2-homozygous corneal broblasts following treatment with 100 lm PQ alone
(P < 0.05, Fig. 1A,B). Treatment with 100 lm melatonin
signicantly prevented loss of the viability of normal
(94.3 1.9%) and GCD2-homozygous (80.5 2.9%)
corneal broblasts, as compared to normal (83.5 5.9%)

Melatonin protects GCD2 corneal broblasts

Cell viability (%)

100

120

(B)
Cell viavilty (%)

120

(A)

80
60
40
20

80
60
40
20
0

Mel (M)

PQ (M)

100

Cells

100

100

Mel (M)

100

PQ (M)

100

Cells

NOR
None

(C)

100

100 M PQ

100 M PQ + 50 M Mel

100

100

100

HO
100 M PQ + 100 M Mel

100 M PQ + 300 M Mel

1.3
0.2

0.7
0.1

5.6
0.5

1.9
0.2

4.6
0.4

* 1.1
0.2

* 2.6
0.6

* 0.8
0.1

* 1.7
0.2

* 0.6
0.2

97.1
1.4

0.9
0.1

91.4
0.8

1.2
0.1

93.0
1.0

1.3
0.1

* 94.9
0.8

* 1.6
0.2

* 96.7
1.1

1.1
0.2

2.9
0.1

6.4
0.5

7.5
0.6

5.2
0.6

* 5.8
0.3

4.7
0.4

* 5.1
0.3

* 4.0
0.2

* 4.4
0.2

* 3.6
0.3

89.6
1.3

1.0
0.2

84.7
1.1

2.6
0.2

86.4
1.0

* 3.2
0.1

* 87.7

* 3.2
0.1

* 89.3

2.8
0.2

NOR

HO
1.4

1.7

Fig. 1. Melatonin prevents PQ-induced apoptotic cell death. Corneal broblasts were pre-incubated for 12 hr with 50500 lm melatonin
(Mel), and then treated with 100 lm paraquat (PQ) for 24 hr. Cell viability of (A) normal corneal broblasts (NOR) and (B) GCD2homozygous corneal broblasts (HO) was determined by MTS assays. Data represent means S.D. of at least three independent
experiments. *P < 0.05. (C) Flow cytometric analysis of apoptotic cell death. Cells were labeled with annexin V-uorescein isothiocyanate
(FITC) and PI 24 hr after PQ treatment. Viable cells are double-negative for annexin and PI staining (lower left quadrants), early-stage
apoptotic cells are represented by high-annexin and low-PI staining (lower right quadrants), late-stage apoptotic cells are double-positive for
annexin and PI staining (upper right quadrants), and necrotic cells are represented by low-annexin and high-PI staining (upper left
quadrants). *P < 0.05, homozygous versus normal cultures.

and GCD2-homozygous (62.1 7.3%) corneal broblasts treated with PQ alone (P < 0.05, Fig. 1A,B).
Treatment with melatonin or DMSO alone, regardless
of concentrations, showed no signicant eects on cell
viability.
We performed annexin-V/PI ow cytometric analyses to
determine whether the observed cell death was apoptotic or
necrotic (Fig. 1C). Annexin-V/PI staining indicated that
cell death occurred via apoptosis following the 12-hr
incubation with 100 lm PQ in the presence of 0, 100, or
300 lm melatonin (Fig. 1C), after preincubation with or
without 50300 lm melatonin for 12 hr. Cells treated with
PQ displayed signicant decreases in cell viability (normal:
91.4 0.8%; GCD2-homozygous: 84.7 1.1%) as compared to untreated cells (normal: 97.1 1.4%; GCD2homozygous: 89.6 1.3%). After exposure to 50, 100, or
300 lm melatonin for 12 hr previously, PQ treatment
signicantly prevented loss of viability of normal cells
treated with this drug to 93.0 1.0%, 94.9 0.8%, and
96.7 1.1%, respectively. Similarly, the decrease of viability of PQ-treated GCD2-homozygous cells was signicantly prevented to 86.4 1.0%, 87.7 1.4%, and
89.3 1.7% following pretreatment with 50, 100 or
300 lm melatonin, respectively (P < 0.05, Fig. 1C).

Melatonin directly protects cells from oxidative stress by

acting as an oxygen free radical scavenger, and indirectly as
an antioxidant enzyme regulator. Based on these properties
of melatonin, we studied the possible regulation of antioxidant enzymes by melatonin in normal and GCD2 corneal
broblasts. Cells were incubated with 0.10.3 mm melatonin for 14 hr, and Cu/Zn-SOD, Mn-SOD, catalase, GPx,
and GR proteins were quantied by Western blot analysis.
Levels of both Cu/Zn-SOD and GR proteins increased in a
dose-dependent manner in normal and GCD2 corneal
broblasts (Fig. 2A, B and D). However, no changes in
Mn-SOD and GPx expression were observed in either type
of cells treated with melatonin (Fig. 2A). Decreased levels
of catalase in GCD2 corneal broblasts before melatonin
treatment (Fig. 2C) were similar to previously reported
data [16], while unchanged levels of Cu/Zn-SOD, Mn-SOD,
GPx, and GR before treatment in this report may be due to
individual variations of GCD2 corneal broblasts as
compared to normal corneal broblasts (Fig. 2A, B and
D). Interestingly, catalase expression increased in normal
corneal broblasts, while it decreased in GCD2 corneal
broblasts (Fig. 2A,C).
To determine whether the down regulation of catalase in
GCD2 corneal broblasts is regulated by transcriptional or
97

Choi et al.
(B)

Relative levels (%) of

Cu/Zn-SOD expression

(A)

Cu/Zn-SOD
Mn-SOD
Catalase

250

*
*

150

50
0

100 200 300

Cells

GR

100

Mel (M)

GPx

200

100 200 300

NOR

HO

(C)

100 200 300

Cells

100 200 300

NOR

HO

Relative levels (%) of

catalase expression

Mel (M)

125

150

-Actin

100

75
50

25
0

Mel (M)

100 200 300

Cells

(E)
(D)

120

40
20
0

100 200 300

Cells

25

100 200 300

NOR

800
600
400
200

Cells
Mel (M)

Relative levels of H2O2

fluorescence (%)

Relative levels of ROS

fluorescence (%)

(H)

HO

*
*

10
5
0

HO

100 200

15

100 200

Cells
1000

20

Mel (M)

(G)

100 200
NOR

(F)

60

Mel (M)

Cells

80

HO

CAT
-Actin
Mel (M)

100

100 200 300

NOR

Relative levels of
CAT expression

Relative levels (%) of

GR expression

140

HO

HO

HO

Cells

DMSO

200

Mel (M)

100 200

NOR

HO

700
600
500
400
300
200
100
0

NOR

NOR

HO

HO

HO

DMSO

200

Fig. 2. Eects of melatonin on the expression of antioxidant enzymes in normal and GCD2 corneal broblasts. (A) Western blot analysis of
Cu/Zn-SOD, Mn-SOD, glutathione peroxidase (GPx), glutathione reductase (GR), catalase and b-actin in 50 lg of total proteins from
normal and GCD2 corneal broblasts treated without or with 100, 200 or 300 lm melatonin for 12 hr. Relative levels of (B) Cu/Zn-SOD,
(C) catalase, and (D) GR after normalization to their respective b-actin protein signals in normal (black) and GCD2 (gray) corneal
broblasts. (E) Eect of melatonin on CAT mRNA levels in normal (NOR) and GCD2 (HO) corneal broblasts. (F) Relative levels of CAT
mRNA in normal and GCD2 corneal broblasts after normalization to b-actin signals. Data represent the means S.D. of three individual
experiments. Eect of melatonin on reactive oxygen species (G) and H2O2 (H) generation in normal and GCD2 corneal broblasts. Data
represent the means S.D. of four individual experiments.

98

Melatonin protects GCD2 corneal broblasts

(A)
Cu/Zn-SOD
GR
-Actin
0

0 300 300 300 100 300 500 0

10

10 15

(C)

200

Relative levels (%) of

GR expression

(B)
Relative levels (%) of
Cu/Zn-SOD expression

Fig. 3. Melatonin regulates the levels of

antioxidant enzymes via a melatonin
receptor-mediated mechanism. Normal
corneal broblasts were pre-incubated for
30 min with or without 10 lm luzindole
(Luzin), and exposed to 100 lm melatonin
for 14 hr. (A) Western blot analysis of Cu/
Zn-SOD, glutathione reductase (GR), and
b-actin with 50 lg of total proteins from
normal corneal broblasts. Relative levels
of (B) Cu/Zn-SOD and (C) GR after
normalization to b-actin protein signals.

Mel (M)
Luzin (M)

150
100
50

Mel (M)

Luzin (M)

125
100
75
50
25
0

0 300 300 300 100 300 500 0

Mel (M)

10

Luzin (M)

post-transcriptional mechanisms, we analyzed the expression of the catalase-encoding CAT mRNA through
RT-PCR analysis of normal and GCD2 corneal broblasts
treated with or without melatonin. Interestingly, melatonin
treatment increased the level of CAT mRNA expression in
normal and GCD2 corneal broblasts (Fig. 2E,F). Therefore, the decreased catalase level in GCD2 corneal broblasts is controlled by a post-transcriptional mechanism as
we previously described [broblasts (Fig. 2E,F)]. Therefore,
the decreased catalase level in GCD2 corneal broblasts is
controlled by a post-transcriptional mechanism as we
previously described [16].
We examined whether melatonin inhibits intracellular
ROS and H2O2 generation in normal and GCD2 corneal
broblasts. Intracellular levels of ROS and H2O2 were
measured using DCFH-DA and BES-H2O2, respectively.
BES-H2O2 used to detect cell-derived H2O2 based on a
nonoxidative uorescence mechanism that is highly selective for H2O2. The intracellular ROS and H2O2 levels were
elevated signicantly in homozygous (8.3 1.2-fold)
(Fig. 2G) and homozygous (5.5 1.2-fold) (Fig. 2H) corneal broblasts, respectively, as compared with normal
corneal broblasts. These results are similar to previously
published estimates [16]. Corneal broblasts were incubated
for 14 hr with 200 lm melatonin. As shown in Fig. 2G,
signicantly lower ROS (5.7 0.5-fold) and H2O2
(3.3 0.5-fold) levels were detected in homozygous corneal broblasts treated with melatonin as compared to
nontreatment homozygous corneal broblasts. These
results suggest that melatonin may decrease levels of ROS
and H2O2 via direct oxygen free radical scavenging mechanisms, because expression levels of two major H2O2scavenging enzymes, catalase and GPx, were not altered
following melatonin treatment in GCD2 corneal broblasts
(Fig. 2A).
The direct free radical scavenging activity of melatonin is
independent of its receptor-mediated actions. For example,
the melatonin receptor antagonist luzindole does not block
the antioxidant eects of melatonin [42, 43]. Melatonin
reduces oxidative damage indirectly by regulating antiox-

10 15

150

10

300 300 300 100 300 500

5

10

15

0
0

idant enzyme expression after it binds to specic receptors

[44]. We investigated the indirect antioxidant mechanisms
of melatonin on corneal broblasts and assessed whether
melatonin receptors are involved in the regulatory mechanisms of antioxidant enzymes. Cells were treated with
10 lm luzindole, a melatonin receptor antagonist 30 min
after melatonin treatment. As illustrated in Fig. 3, luzindole
abolished the eect of melatonin on up-regulating the
expression of Cu/Zn-SOD (Fig. 3A,B) and GR (Fig. 3A,C).
Thus, the antioxidant activity of melatonin could be
mediated via its receptor-mediated actions in corneal
broblasts.
To conrm the involvement of melatonin receptors in
melatonin protection against oxidative stress-induced cell
death, luzindole was used in combination with melatonin.
Luzindole signicantly attenuated the eects of melatonininduced protection (Fig. 4A,B), suggesting that melatonin
worked via its receptors to prevent both normal and
GCD2-homozygous corneal broblast cell death. Therefore, the protection rendered by melatonin to corneal
broblasts is receptor-mediated and melatonin may be an
eective protective agent for attenuating corneal broblast
degeneration in GCD2 pathogenesis.
However, it remained unclear as to whether these eects
of melatonin treatment functioned through the melatonin
receptors. Therefore, we analyzed the expression of melatonin receptors in normal and GCD2 corneal broblasts. In
a previous study, MT1 was found in corneal broblasts of
the endothelial and epithelial cell layer of human eye [37].
Here, we also identied MT1 and MT2 in corneal
broblasts (Fig. 5). Approximately 37- and 39-kDa bands
representing MT1 and MT2, respectively, were detected in
normal and GCD2 corneal broblasts (Fig. 5A). Interestingly, expression of MT1 was signicantly higher in GCD2
heterozygotic (26.7 4.9) and homozygotic (26.3 3.2)
corneal broblasts than in normal corneal broblasts
(2.2 0.9) (Fig. 5A,B). Similarly, levels of MT2 were also
signicantly higher in GCD2 heterozygotic (11.4 3.6)
and homozygotic (11.0 2.5) corneal broblasts than in
normal corneal broblasts (3.8 1.4) (Fig. 5A,C). In
99

Choi et al.
120

100

(B)

Cell viavility (%)

Cell viavility (%)

(A)

80
60
40
20

120

100

80
60
40
20

Mel (M)

100

100

100

Mel (M)

100

100

PQ (M)

100

100

100

PQ (M)

100

100

100

Luzin (M)

10

10

Luzin (M)

10

10

Cells

NOR

Cells

100

HO

Fig. 4. Melatonin prevents paraquat (PQ)-induced cell death via a melatonin receptor-mediated mechanism. (A) Normal (NOR) and (B)
granular corneal dystrophy homozygous (HO) corneal broblasts were pre-incubated for 30 min with 10 lm luzindole, exposed to 100 lm
melatonin for 12 hr, and further incubated with or without 100 lm PQ for 24 hr. Cell viability was determined by MTS assay. Values
represent means S.D. of at least three independent experiments. *P < 0.05.
NOR

(A)

HE

MT1
MT2

39

-Actin

45

(C)

*
Relative levels of
MT2 expression

40
30
20
10

(D)

kDa
37

(B)
Relative levels of
MT1 expression

HO

NOR
MT1

*
15

HO
MT1

10
5
0

NOR

HE

HO

NOR

HE

HO

Fig. 5. Melatonin receptor expression is higher in granular corneal dystrophy type 2 (GCD2) corneal broblasts than in normal corneal
broblasts. (A) Western blot analysis of MT1 and MT2 receptors in normal (NOR), GCD2-heterozygous (HE), and GCD2-homozygous
(HO) corneal broblasts. Relative levels of (B) MT1 and (C) MT2 after normalization to b-actin protein signals. (D) Immunostaining of
melatonin receptor in normal and GCD2 corneal broblasts. Cell preparations were done as described in Materials and methods. These
confocal micrographs show immunoreactivity (green) for MT1 in normal and GCD2 corneal broblasts. Data represent the means S.D.
of three individual experiments. *P < 0.05 compare to normal corneal broblasts.

addition, confocal immunostaining using the anti-MT1

antibody identied MT1 expression in normal and GCD2
corneal broblasts (Fig. 5C).

Discussion
The present study shows for the rst time that melatonin
treatment protects normal, as well as GCD2 corneal
broblasts from oxidative stress via MT1 and MT2, which
are highly expressed in GCD2 corneal broblasts. These
results are important because corneal tissue is chronically
exposed to environmental oxidative stimuli, and is particularly susceptible to oxidative stress.
The number of corneal broblasts declines in normal
corneas with age in response to oxidative stress. Agedependent progressive accumulation of mutant TGFBIp is
100

a hallmark of GCD2 pathogenesis. Cultured primary

GCD2 corneal broblasts are highly susceptible to oxidative stress-induced cell death, as compared with normal
corneal broblasts [16]. These ndings suggest that antioxidant therapy has the potential to prevent, delay, or
ameliorate GCD2. Although the optimal therapeutic
antioxidant treatment must be individually tailored and
modied due to the complexity of oxidative pathobiology,
we have demonstrated the potential therapeutic eect of
melatonin in degeneration of GCD2 corneal broblasts.
Melatonin is a well-tolerated and safe drug used for
several purposes with daily doses ranging from 0.1300 mg
[45]. Additionally, it was previously demonstrated that
melatonin is distinct from classical antioxidants. Melatonin
acts on a broad-range of oxygen free radicals by direct
scavenging [46, 47], and its antioxidative prole extends to

Melatonin protects GCD2 corneal broblasts

the activation of several antioxidant enzymes [46, 47]. The
activity of c-glutamylcysteine synthetase is induced by
melatonin, thereby stimulating the production of another
intracellular antioxidant, glutathione [48]. The antioxidative action of melatonin is more eective than vitamin E
[49, 50], b-carotene [51], vitamin C [51, 52], and garlic oil
[53]. Furthermore, melatonin is amphiphilic and thus
able to enter both lipophilic and hydrophilic cellular
environments [47]. Therefore, melatonin is a potent
endogenous antioxidant that is lipid- [54] and water-soluble
[55], is able to act in all compartments of the cell, and can
protect DNA [56], and possibly proteins [57] and lipids [58].
Melatonin signicantly reduces ROS production and cell
death caused by PQ treatments [58]. Therefore, melatonin is
a potential therapeutic agent for oxidative stress-induced
corneal diseases, including GCD2. The antioxidative eect
of melatonin likely functions, in part, through an indirect
antioxidant system that up-regulates the expression of
Cu/Zn-SOD, Mn-SOD, and GR in normal and GCD2
corneal broblasts.
Interestingly, although several studies have shown
increased levels and activity of catalase in response to
melatonin treatments [5961], catalase expression decreased
in GCD2 corneal broblasts following melatonin treatment. These results indicate that currently unknown mechanisms are involved in the regulation of catalase expression
by melatonin in GCD2 corneal broblasts. Autophagy may
be involved in the regulation of catalase expression, since
catalase is selectively degraded in autophagy-activated cells
and catalase mRNA expression increased after melatonin
treatment [62]. Moreover, we have recently found that
autophagy is activated in GCD2 corneal broblasts (S.I.
Choi, B.Y. Kim, S. Dadakhujaev, H. Ryu, T.M. Kim, J.Y.
Kim and E.K. Kim, manuscript was submitted for publication). Thus, these results indicate that melatonin may
have no eect on catalase expression in cells in which
autophagy is activated because catalase could be degraded
in response to autophagy activation [16, 62]. Since melatonin treatments regulate the expression of other antioxidant enzymes via melatonin receptors in corneal broblasts,
we cannot rule out another possibility that dierential
regulation of catalase in response to melatonin may be
correlated with increased expression of the melatonin
receptors in GCD2 corneal broblasts. Our data indicated
that two melatonin receptors, MT1 and MT2, are highly
expressed in primary cultured GCD2 corneal broblasts,
suggesting that increased expression of these receptors may
be involved in the defense mechanisms against oxidative
stress in GCD2 corneal broblasts. However, some questions about dierential expression and regulation of the
melatonin receptors in normal and GCD 2 corneal broblasts following melatonin treatments still remain.
Cu/Zn-SOD expression was more dramatically increased
by the melatonin treatment than that of the other enzymes in
both normal and GCD2 corneal broblasts. Cu/Zn-SOD is
constitutively expressed [63], and presumably represents a
ready defense against oxidative stress. Cu/Zn-SOD is also
the major contributor to the total dismutating activity of
cells [64]. Therefore, it is biologically plausible that this
enzyme may be more easily induced by melatonin than the
other enzymes analyzed in this study. On the other hand, the

detoxifying eciency of SOD is closely dependent on its

cooperation with catalase and GPx. Catalase and GPx are
the predominant enzymes in the regulation of H2O2 intracellular levels. GPx plays a major role in detoxifying H2O2.
However, in this study, expression of GPx was unchanged by
melatonin treatment in two types of cells. Therefore, our
data do not support a previous study that demonstrated a
twofold increase in GPx activity after melatonin treatment
[65]. The regulation of GPx expression by melatonin may
depend on the specic cell type. Cellular protection by
antioxidant enzymes requires an appropriate relationship
between the enzymes. Enhancement of SOD increases H2O2
generation, and if H2O2 is not eciently removed by GPx
and/or catalase, the eect of increased SOD may be the cause
of oxidative stress by production of OH radicals.
Superoxide dismutase plays a protective role, even when
other antioxidant enzymes remain unchanged. For example, the exogenously administered human Cu/Zn-SOD gene
ameliorates neuronal loss in several neurodegenerative
disease models [66]. Similarly, brain damage is markedly
decreased in transgenic mice that overexpress the Cu/ZnSOD gene [67]. Therefore, the reduced cell death of GCD2
corneal broblasts in response to melatonin could occur by
increased SOD alone. However, melatonin treatment could
not reverse the decrease in expression of catalase in GCD2
corneal broblasts. Nevertheless, we cannot exclude the
possibility of direct free radical scavenging activities of
melatonin. To nd a more eective therapeutic agent,
future studies will examine the benets of co-administering
melatonin and other antioxidants that are able to upregulate catalase and GPx expression.
The results from the present study demonstrated for the
rst time increased expression of the melatonin receptors
MT1 and MT2 in GCD2 corneal broblasts. The
up-regulation of melatonin receptors may be involved in
the defense mechanisms against oxidative stress in GCD2
corneal broblasts, although further studies will be necessary to evaluate the mechanisms involved in this process.
Furthermore, melatonin treatment protects normal, as well
as GCD2 corneal broblasts from oxidative stress through
direct free radical scavenging by melatonin or, at least in
part, the up regulation of several antioxidant enzymes via
its receptors.
In conclusion, melatonin may be a potent drug for the
prevention and treatment of GCD2 and age-related ocular
disorders that are associated with oxidative stress.

Acknowledgements
This work was supported by the Converging Research
Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education,
Science, and Technology (2010K001134) and by a Midcareer Researcher Program grant through the NRF funded
by the MEST (No. 2010-0000324).

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