ARTICLE
Auteur(s) : Khine Khine Zaw, Yoko Yokoyama, Masatoshi Abe, Osamu
Ishikawa
Department of Dermatology, Gunma University Graduate School of
Medicine, 3-39-22, Showa-machi, Maebashi, Gunma, 371-8511,
Japan
accepté le 11 Avril 2006
Photoaged skin is characterized clinically by deep wrinkling,
laxity and dry leatheriness and histologically displays prominent
alterations in collagenous extracellular matrices in the dermis, a
diminished number of fibrils with reduced electron density and
cross-striations or filaments of degraded collagen [1-3]. Skin is
continuously in contact with oxygen (O2) and nowadays is
increasingly exposed to UV radiation. Therefore, interest in the
role of ROS that exert photo-oxidative damage on the skin has
recently increased [4, 5]. Oxygen radicals emerge from the
reduction of two unpaired electrons containing ground state
O2, which are super oxide radicals (O2-),
hydrogen peroxide (H2O2) and hydroxyl radical
(HO.). They are very potent oxidants that variably damage resident
cells and extracellular matrices [6].Due to the dangerously
destructive properties of superoxide radicals and other reactive
oxygen species, cells have developed several defensive mechanisms
to prevent or limit oxidative damage. Several enzyme systems have
been extensively studied including SOD, catalase, and glutathione
peroxidase (GSHPX) as well as antioxidants including α-tocopherol,
carotenoids, etc. Because of the limited number of experimental
models available, there are few data of in vitro studies to
evaluate the possible mechanism responsible for collagenous matrix
damage by ROS.We introduced a novel three-dimensional culture of
normal human dermal fibroblasts, in which multilayered fibroblasts
form a sheet-like structure and are embedded in self-generated
extra cellular matrices. This system can provide a more
physiological environment with fibroblasts in vitro [7, 8].In this
study, we investigated the scavenging effect of SOD and catalase on
ROS induced changes in the collagen and matrix metalloproteinases
mRNA expressions.
Materials and methods
Cell cultures
Human dermal fibroblasts were obtained from healthy young
individuals (7, 13 and 19 years old) after obtaining informed
consent. In our study, dermal fibroblast cultures were obtained
from healthy young individuals and from sun protected sites. We did
triplicate cultures in all cell lines to minimize possible
variation between different donors. Some major variation might be
present between dermal fibroblast cultures from different
patients/donors for example if the culture cells had taken from
different sites of the body (sun exposed and sun protected sites)
and different age groups (young and aged skin). Cells were
initiated from explant cultures and grown in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS)
in an atmosphere of 37 °C humidified air and 5%
CO2. At the second passage, fibroblasts were subcultured
into 3 flasks (75 cm2) and experiments were
conducted between 3-5 passages. Fibroblasts were seeded at 5 ×
105 cells/10 cm dish in DMEM supplemented with 1mM
magnesium salt of L-ascorbic acid 2-phosphate (Asc-2p, Wako Pure
Chemical Industries, Ltd, Osaka) for 14 days. The supplementation
of Asc-2p rendered cells to the organization of the self-produced
three-dimensional structure during this incubation period.
ROS and scavengers
To eliminate the effects of unknown factors in fetal calf serum, we
introduced a serum-free culture condition. The confluent
three-dimensional cultures were incubated in serum-free DMEM
without 1mM Asc-2p for 24 h, and the fibroblasts were then
exposed to ROS generated by hypoxanthine and xanthine oxidase
system and scavengers were added for corresponding incubation
periods, 6, 12, 24, 48, and 72 h. In brief, 100 mg/mL of
hypoxanthine and 10 mU/mL of XOD were both added to the cultures to
assess the effect of ROS on collagen metabolism, or both 100 mg/mL
of hypoxanthine and 10 mU/mL of XOD and scavengers, catalase
1 U/mL or SOD 10 U/mL, were added to cultures to assess the
effect of scavengers on the ROS-induced changes. The chemicals used
in the experiments were purchased from Sigma Chemical Co, USA.
RNA extraction and Northern blot analysis
At the time of harvest, medium and cell layer were collected
separately. The cell layer was rinsed three times with cold
phosphate buffer saline, and total RNA was isolated by a
single-step method using a commercially available acid guanidinium
thiocyanate -phenol extraction reagent (Isogen, Nippongene, Toyama,
Japan). Ten mg of total RNA were denatured in formaldehyde and
electrophoresed in 1.2% agarose-1.1 mol/L formaldehyde gels. The
electrophoresed RNA was then transferred to a nylon membrane, and
crossed-linked by exposure to 120 mJ/cm2 of 312 nm
UV radiation in a spectro UV cross-linker (Spectronics Corporation,
Westberg, NY, USA). The same filter was hybridized repeatedly with
each specific probe labelled with α-[32p] dCTP by the
random priming method (Gibco BRL, Gaitherburg, MD, USA). After
hybridization, the filter was washed and exposed to Kodak X-Omat
films.
The following human sequence-specific cDNAs were used for
hybridization: a 1.4 kb cDNA, Hf 677-6, for proα1(I) collagen mRNA,
a 0.9 kb cDNA, pH, for proα1(III) collagen mRNA, a 0.7 kb cDNA, K4,
for MMP-1 (generously provided by Dr Hatamochi, Chiba University,
Japan), and 1.5 kb cDNA, pCU19, for MMP-2, a 0.6 kb-long cDNA,
pBlue script, for TIMP-1 mRNA (kindly provided by Dr H. Sato,
Cancer Research Institute, Kanazawa University, Japan).
Assay of carboxyterminal propeptide of type I procollagen
(PICP)
PICP content in the culture media was measured with an enzyme
immunoassay kit (Takara, Tokyo, Japan) according to the
manufacturer’s protocol. The experiment was done in triplicate
culture, and measurement of PICP was performed in duplicate for
each well.
Gelatin zymography
Gelatin zymography was used to determine the proteolytic activity
of MMP-2. The total protein content in the culture media was
quantified by BCA protein assay (PIERCE Co, USA) and an equal
amount of protein solution (15 μL/lane) was subjected to
zymography. Unboiled samples were loaded and electrophoresed in a
zymogram-PAGE containing 0.1% gelatin gel (TEFCO Co, Japan) at
4 °C. After electrophoresis, the gelatin gel was washed twice
in 2.53% Triton-X 100 (v/v) for 30 min. Subsequently, the gels
were rinsed three times in incubation buffer (0.1 M Tris-HCL, pH
7.4, 10 mM CaCl2 and 1 mM ZnCl2) for
15 min each, and incubated in the same buffers at 37 °C
for 24 h. After immersion in fixation solution (45% methanol
and 10% acetic acid) at room temperature for 10 min, the gel
was stained with Coomassie brilliant blue and destained in
distilled water or 45% methanol and 10% acetic acid. The wet gel
was photographed against a light box.
Western immunoblotting
MMP-1 was semiquantified by western blot. Culture media of equal
protein content (10 μg/lane) were separated using 10% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Proteins were electroblotted onto a nitrocellulose membrane and
detected with anti-human MMP-1 mouse monoclonal IgG antibody (Fuji
chemical Co, Japan) and peroxidase-conjugated anti-mouse
immunoglobulins (Dako, Denmark) using ECL chemiluminescence
(Amersham Life Science, England).
Collagenase activity assay for MMP-1
Collagenase activity was assayed in the two fibroblast strains
obtained from 13 and 19 year-old males. Culture supernatant were
collected at indicated times and used for assay. Collagenase
activity was determined using a rapid collagenase assay kit
(Chondrex, LLC, USA) according to manufacturer’s protocol. The
measurement was performed in triplicate for each well. One unit of
collagenolytic activity was defined as the cleavage of 1 mg of
collagen per min.
Densitometric evaluation and statistical analysis
X films were scanned with Bioimage Gel print 2000 i/VGA (Genomic
Solutions Inc, USA) and the relative intensities of the bands were
quantified using Basic Quantifier (Genomic Solutions Inc, USA)
computer software package.
Statistical analyses were performed by ANOVA test.
Results
ROS-induced changes in mRNA expression of normal human dermal
fibroblasts in the three-dimensional culture.
As shown in ( figure
1 ), the mRNA expression of GAPDH was uneffected in all
tested groups. The levels of mRNA for proα1(I) and proα1(III)
collagen were significantly decreased in a time-dependent manner
with maximum at 48 and 72 h. In contrast, ROS increased the
mRNA expressions of MMP-1 and MMP-2 with a maximum at 48 and
72 h after exposure. TIMP-1 mRNA levels were also increased
but not as prominently as others.
Effects of scavengers on ROS-induced changes in mRNA expression.
As shown in ( figure
2 ), catalase (1U/mL) completely abrogated the ROS-induced
reduction of proα1(I) and proα1(III) collagen mRNA expressions,
with levels higher than the control. Also, the ROS-induced
elevation of MMP-1, MMP-2 and TIMP-1 mRNA was significantly
suppressed by catalase (1U/mL). In contrast, SOD (10 U/mL) did not
block the ROS-induced changes ( (figure 3) ).
Levels of PICP.
PICP levels were reduced in ROS treated cultures prominently at
48 h and 72 h of exposure. The addition of catalase
increased the PICP to certain levels but SOD showed no effect on
ROS-induced PICP reduction (table 1)( Table
1 ).
Protein levels of MMP-1 and collagenolytic activity in culture
supernatants after ROS exposure.
The protein level of MMP-1 was analyzed by western blot using
human specific antibodies against MMP-1. Control and experimental
samples showed immunoreactive bands for MMP-1 of 58 and 48 kDa (
(figure 4) ).
The 48 kDa band corresponds to the active form displaying the
highest intensities. MMP-1 proteins were increased at 48 and
72 h after ROS treatment. Catalase abolished the ROS-induced
increase of MMP-1 proteins at the corresponding exposure time. SOD
showed no effect on ROS-induced MMP-1 proteins.
The collagenolytic activity of MMP-1 was assessed using a
collagenase activity assay. ROS stimulated collagenolytic activity
of MMP-1 with a maximum at 48 and 72 h after exposure. Again,
catalase completely abrogated the action of ROS while SOD showed no
prevention activity on ROS-induced changes.
Table 1 Carboxy termal propeptide of type I procollagen
levels in culture media
|
Times
|
6 h
|
12 h
|
24 h
|
48 h
|
72 h
|
|
none
|
|
|
|
|
9.62 ± 4.85*
|
|
CAT alone
|
|
|
|
|
6.45 ± 1.36
|
|
SOD alone
|
|
|
|
|
5.98 ± 0.29
|
|
ROS
|
1.61 ± 0.04
|
2.64 ± 0.03
|
0.38 ± 0.11
|
1.38 ± 0.14
|
0.43 ± 0.18
|
|
ROS+CAT
|
4.44 ± 1.93
|
5.21 ± 0.64
|
7.50 ± 0.3
|
2.63 ± 0.76
|
2.24 ± 0.2
|
|
ROS+SOD
|
2.03 ± 0.1
|
6.83 ± 2.41
|
2.92 ± 0.5
|
0.60 ± 0.26
|
0.14 ± 0.03
|
Discussion
Morphological appearances of photoaged skin are invariably
reflected by the biochemical alterations in dermal collagen
metabolism including reduced levels of type I and type III collagen
precursors [9]. In human skin, the induction of matrix-degrading
metalloproteinase mRNA, protein and activity has been described as
accelerating both collagen breakdown and other substrates of the
dermal extracellular components in vivo a few hours after
ultraviolet B irradiation [10]. Matrix damage and imperfect
restoration which lead to the accumulation of altered matrices such
as disorganized collagen fibril and abnormal elastin-containing
materials may be induced by recurrent exposure to sun. These
changes are considered to form solar scar and eventually,
observable wrinkles of photoaged skin [11, 12].
In the present study, we first demonstrated that MMP-1, MMP-2,
proα1(I) and α1(III) collagen are regulated by ROS at the
transcriptional or posttranscriptional stage.
It is well documented that UVA [13-15] and singlet oxygen
[16-18] stimulate the production of MMP-1 mRNA in monolayer
cultured human dermal fibroblasts. Berstein et al. [19]
demonstrated an equal amount of mRNA expression of α1(I) and
α1(III) collagen between fibroblast cultures was established in
photodamaged skin compared to sun protected skin. In contrast,
Tanaka et al [20] reported that ROS both decreased collagen
production and increased GAGs synthesis in cultured human dermal
fibroblasts. Catalase, not SOD, could cancel the ROS-changes as
seen our study.
We first demonstrated that ROS induced significant reduction in
mRNA expressions of proα1(I) and proα1(III) collagen, but,
increased the mRNA expressions of MMP-1, MMP-2 and TIMP-1 in
three-dimensional cultured of dermal fibroblasts. Data from our
experiments provide an overall picture of mRNA changes involved in
collagen metabolism. To determine the effect of ROS at the cellular
level, the effect on m-RNA impression is very basic but the amount
and function of the protein gene products are also important. We
performed Collagenase activity assay for MMP-1 and assay of
carboxyterminal propeptide of type I procollagen (PICP). MMP-1
proteins were increased at 48 and 72 h after ROS treatment in
Collagenase activity assay for MMP-1. Catalase abolished the
ROS-induced increase of MMP-1 proteins at the corresponding
exposure time. SOD showed no effect on ROS- induced MMP-1 proteins.
The collagenolytic activity of MMP-1 was assessed using a
collagenase activity assay. ROS stimulated collagenolytic activity
of MMP-1 with a maximum at 48 and 72 h after exposure. Again,
catalase completely abrogated the action of ROS while SOD showed no
prevention activity on ROS-induced changes ( (figure 4) ). PICP levels
were reduced in ROS treated cultures prominently at 48 h and
72 h of exposure in assay of carboxyterminal propeptide of
type I procollagen (PICP). The addition of catalase increased the
PICP to certain levels but SOD showed no effect on ROS-induced PICP
reduction (table 1). The present study suggests that the dermal
collagen changes in photoaged skin may result from both decreased
collagen synthesis and increased expression of enzymes that degrade
the collagen matrix.
Several enzymatic steps are involved in collagen degradation.
The induction of interstitial collagenase cleaves collagen into
three-quarter and quarter-length fragments [21] which are
susceptible to further proteolytic digestion by type IV
collagenase. Type IV collagenase has the ability to degrade
collagen type IV, V, VII and gelatin [22, 23]. Their activities are
regulated by a tissue inhibitor of matrix metalloproteinases
(TIMP-1) which prevents excessive matrix degradation [24]. In the
present study, we demonstrated the time-dependent induction of
MMP-1 activity ( (figure
4) ). It might be plausible that the action of TIMP-1 is
not adequate to counterbalance the action of interstitial
collagenase, resulting in accelerated collagen degradation.
We also investigated the scavenging effect of SOD and catalase
on ROS-induced changes in both the collagen and matrix
metalloproteinase mRNA expressions. The enzyme, xanthine oxidase,
catalyzes hypoxanthine as a substrate in aerobic conditions and
produces superoxide radicals (O2–), and derived oxidants
such as hydrogen peroxide (H2O2) and hydroxyl
radicals (HO.). O2– undergoes dismutation reaction in
the presence of SOD to form H2O2 and
O2. In this study, ROS was experimentally generated
outside the cells in culture human skin fibroblasts. Among them,
H2O2, can diffuse and across biological
membranes because of its small size, solubility and lack of
charges. It can be reduced to HO which is a highly reactive and
largely indiscriminate oxidant. O2– can collaborate with
H2O2 in the production of HO. The reduction
of H2O2 can occur in the presence of reduced
metal cations such as Fe(II) or Cu(I)6. If these metal
cations are bound to DNA or to cell membranes, then the HO will be
generated adjacent to, and will react preferentially with, these
critical targets. Catalase dismutates H2O2
into O2 and H2O. Although ROS-induced MMP-1
production was completely inhibited by catalase, SOD showed no
scavenging effects. These data indicate that
H2O2 may play a pivotal role in mediating the
induction of MMP-1. The suppression of type α1 (I) and α1 (III)
collagen mRNA expressions in ROS-treated cells was restored by
catalase scavenging. It is of interest that the levels of type α1
(I) and α1 (III) collagen mRNA increased in cultures with catalase
alone or slightly higher in ROS-treated cultures together with
catalase with a maximum level at 48 h and 72 h ( (figure 2) ). However,
SOD showed no recovery from ROS-induced changes at a corresponding
exposure time ( (figure
3) ). Gelatinolytic activities of MMP-2 were increased with
a maximum level at 48 h and 72 h in either ROS, ROS plus
catalase or ROS plus SOD treated culture cells. The reason for the
disparity between the results of mRNA and activity of MMP-2 is
difficult to explain. There is a possibility that although
O2– or HO. can be eliminated by the addition of SOD, the
H2O2 formed in this reaction may activate the
preexisting form proMMP-2.
In summary, we demonstrated that H2O2 may
participate in the mRNA expression of MMP-1, MMP-2, TIMP-1, type α1
(I) and α1 (III) collagen in normal dermal fibroblasts in vitro and
catalase could abrogate these changes. Understanding the role of
various ROS in the regulation of collagen metabolism could improve
the regulation of photoaging by novel antioxidant agents.
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