ARTICLE
Auteur(s) : Hee Jung Moon1, Sang Ho
Lee1, Mi Jeong Ku1, Byeng Chul
Yu1, Man Joong Jeon1, Seok Hoon
Jeong2, Valentin A Stonik3, Tatyana N
Zvyagintseva3, Svetlana P Ermakova3, Yong Hwan Lee1
1Institute of Natural Products for Health
Promotion and Department of Preventive Medicine, College
of Medicine, Kosin University, 34 Amnam-dong, Suh-gu, Busan,
602-702 Republic of Korea
2Department of Laboratory Medicine, College
of Medicine, Yonsei University; Seoul, 120-749, Korea
3Pacific Institute of Bioorganic Chemistry
of Far East Branch of the Russian Academy
of Sciences; Vladivostok -22, Russia
accepté le 29 Octobre 2008
Aging of the skin is primarily related to a reduction in type I
collagen levels, which is the principal component of skin dermis.
The skin dermis contains predominantly type I and type III
collagen, elastin, proteoglycans, and fibronectin. Because collagen
fibrils and elastin are responsible for the strength and resiliency
of skin, their disarrangement during photoaging causes the skin to
appear aged. Recently, it has been suggested that excessive matrix
degradation by UV induced matrix metalloproteinase-1 (MMP-1)
secreted by various cells, including keratinocytes, fibroblasts,
and inflammatory cells, contributes substantially to the connective
tissue damage that occurs during photoaging [1-4], through cleavage
of fibrillar collagen (type I and III in skin) at a single site
within its central triple helix [5]. This evidence suggests that
the expression of MMP-1 and the down-regulation of type I collagen
synthesis plays a major role in the process of photoaging. In the
absence of perfect repair, MMP-1 mediated collagen damage is
expected to accumulate with each successive UV exposure. Such
cumulative collagen damage is most likely a major contributor to
the phenotype of photoaged human skin [6].
The fucoidans are a family of sulphated polyfucose
polysaccharides. They have attracted considerable biotechnological
research interest since the discovery that they possess
anti-coagulant activities similar to those of heparin [7]. They
have been reported to produce antithrombotic, anti-inflammatory,
anti-tumour, anti-adhesive, and anti-viral effects [8]. Recently,
fucoidan was studied for potential biological activities in
anti-skin aging. Using in vitro models of dermal wound repair,
O’Leary et al. [9] reported that fucoidan modulates the effect of
TGF-β1 on fibroblast proliferation and wound repopulation during
wound repair. It has also been reported that L-fucose and
fucose-rich polysaccharide preparations are efficient modulators of
MMP-2 and MMP-9 activity [10], and that they increase elastic fibre
surface density in rat skin and tropoelastin biosynthesis in vitro
[11]. Percutaneous application of an L-fucose-containing
preparation produced an increase of skin thickness and a
densification of collagen bundles [12]. We have previously shown
that fucoidan inhibits UVB-induced MMP-1 expression in human skin
fibroblasts at the protein and mRNA levels [13].
In this study, we assessed the effects of fucoidan on the
inhibition of MMP-1 promoter activity and the increase of type I
procollagen synthesis in human skin fibroblasts.
Materials and methods
Cell culture
The normal human newborn foreskin fibroblast cell line, HS68 cell
(ATCC CRL 1635), was obtained from the American Type Culture
Collection (Rockville, MD). The cells were plated in 100 mm
tissue culture dishes and grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin (all from GIBCO; Grand Island, NY).
Fucoidan (Sigma; St. Louis, MO) was dissolved in distilled water.
For treatment, the cells were maintained in culture media without
FBS overnight, followed by treatment with fucoidan for 24 h.
The cells were rinsed twice with phosphate-buffered saline (PBS),
and all UVB irradiation exposures were performed under a thin layer
of PBS (GIBCO; Grand Island, NY). Immediately after irradiation,
the cells were incubated in serum-free fresh culture media
containing fucoidan.
Ultraviolet irradiation
The UV light source originated from a Philips TL 20W/12RS
fluorescent sun lamp (Amsterdam, Holland) with an emission spectrum
of 285-350 nm (peak at 310-315 nm). The cells were then exposed to
a 100 mJ/cm2 dose of UVB light.
Western blotting
The cells were lysed with a buffer (50 mM Tris-HCl (pH 7.4),
150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 μg/μL
aprotinin, 10 μg/μL leupeptin, 5 mM
phenylmethanesulfonyluoride (PMSF), and 1 mM DTT containing 1%
Triton X-100). The supernatant extracts were centrifuged at 12,000
× g for 10 min at 4 °C and used for western blot
analysis. Equal amounts of protein were resolved in gradient (10%)
SDS PAGE gels (Invitrogen; Carlsbad, CA) and electrophoretically
transferred to nitrocellulose membranes. The membranes were
subsequently blocked with 5% skim milk in TBST (20 mM Tris-HCl
(pH 7.6), 137 mM NaCl, and 0.05% Tween 20) and incubated with the
indicated antibodies. Western blotting was performed using the
anti-human MMP-1 (Calbiochem; San Diego, CA) and the type 1
procollagen antibodies (Santa Cruz Biotechnology; Santa Cruz, CA).
The proteins resulting from the western blot were visualized by
enhanced chemiluminescence.
RNA extraction and reverse transcriptase (RT)-polymerase
chain reaction (PCR)
To assay for the MMP-1 mRNA, total RNA was isolated using the
procedure of Chomczynski and Sacchi [14]. RNA concentration was
quantified by UV spectrophotometer at 260 nm and the purity
was determined using the A260/A280
ratio. All samples were reverse-transcribed using
moloneymurine leukemia virus reverse transcriptase (Bioneer;
Daejeon, Korea) and 30 pM oligo dT19 in a total reaction volume of
20 μL containing 5 × RT buffer (250 mM Tris-HCl, pH 8.3;
375 mM KCl; 15 mM MgCl2; and 50 mM DTT),
and 1 mM dNTPs. The RT-PCR assay was performed to specifically
quantify the mRNA level. In all of the assays, the cDNA was
amplified using a standardized program (5 min denaturing
steps, 30 cycles of 30 seconds at 94 °C, 30 seconds at
55 °C, and 30 seconds at 72 °C, melting point analysis in
1 °C steps, and a final cooling step) using a Gene Amp PCR
2400 (Applied Biosystem; Foster City, CA). The primers used for
β-actin were forward 5′-GGA CCT GAC AGA CTA CCT CA-3′, reverse
5′-GTT GCC AAT AGT GAT GAC CT -3′, and for MMP-1 they were forward
5′-GGT GAT GAA GCA GCC CAG-3′ and reverse 5′-CAG TAG AAT GGG AGA
GTC-3′. For the type I procollagen they were forward 5′-CTC GAG GTG
GAC ACC ACC CT-3′ and reverse 5′-CAG CTG GAT GGC CAC ATC GG-3′.
Plasmid constructs
We used a genomic DNA as a PCR template and primers at
–2,270 bp and + 30 bp to generate a fragment containing a
Sac I site at 5’ end and a Hind III site at 3’ end. PCR for human
MMP-1 promoter was carried out using a GeneAmp® PCR
System 2700 (Applied Biosystem; Foster City, CA). PCR was performed
in 95 °C for 1 min followed by 30 cycles of 94 °C
for 1 min, 62 °C for 1 min, and 72 °C for
1 min. The PCR products were purified by QIAquick PCR
purifiction kit (Qiagen, Hilden, Germany) and then enzyme digestion
by Sac I and Hind III was carried out. After the gel
electrophoresis, each DNA of 2.3 kb size was extracted from
gel by gel extraction kit (Qiagen, Hilden, Germany). –2,300 MMP-1
promoter was subcloned into pGEM® T easy vector
according to the manufacturer’s instructions. Some of the white
colonies were checked to see whether they were ligated or not by
Mini preparation and ligated DNA was sequenced. pGL3-basic vector
was digested by Sac I restriction enzyme at 37 °C overnight.
The DNA was precipitated to exchange reaction buffer. The DNA was
digested by Hind III restriction enzyme at 37 °C overnight and
gel electrophoresis was performed. The DNA of 4.8 kb size was
extracted from gel by gel extraction kit (Qiagen, Hilden, Germany).
–2,300 MMP-1 promoter subcloned into pGEM® T easy vector
was digested using SacI and Hind III restriction enzyme. They were
gel electrophoresed and extracted from gel. The prepared pGL3-basic
vector and –2,300 MMP-1 promoter were ligated in the same way as a
pGEM® T easy vector system.
Transient transfection and luciferase assay
The cells were seeded in 6 well plates at 3 × 105
cells/well with 2 mL of media and grown for 24 h.
Transfection experiments were carried out with the Lipofectamin
2000 (Invitrogen; Carlsbad, CA) according to the manufacturer’s
instructions. Transfection efficiency was measured by the X-gal
staining method to optimize the condition. The plasmids used were
2.5 μg of test plasmid and 0.5 μg of pCMV-β galactosidase
as an internal standard to adjust transfection efficiency. Four
hours after the transfection, the cells were washed twice with PBS
and treated with 1, 10, or 100 μg/mL of fucoidan in serum-free
media overnight. The cells were then washed twice with PBS and
irradiated with UVB at a dose of 100 mJ/cm2.
Luciferase activity was determined with a luminometer (TD 20/20,
Promega, Sunnyvale, CA) and luciferase activity was normalized for
variation in transfection efficiency by dividing relative light
units (RLU) by β-galactosidase activity.
Statistical analysis
Data were expressed as the mean ± SD and were analyzed by analysis
of variance (ANOVA) followed by Duncan’s test. The significance
level was p < 0.05.
Results
Effect of fucoidan on UVB induced MMP-1
expression
The cells were treated for 24 h with various treatment
concentrations of fucoidan (1, 10 or 100 μg/mL) followed by UVB
irradiation (100 mJ/cm2). The cells were further
incubated for an additional 24 h. Fucoidan treatment
significantly inhibited the expression of MMP-1 in a dose-dependent
manner (figure
1).
Effect of fucoidan on UVB induced MMP-1 mRNA
expression
To study the inhibitory effects of fucoidan on UVB-induced MMP-1
mRNA expression at the transcription level, RT-PCR analysis was
performed using total RNA isolated from the cells. As shown in
figure 2, the
result was consistent with the finding in figure 1. UVB-induced MMP-1
mRNA expression was significantly inhibited by the action of
fucoidan; treatment of fucoidan with 1, 10 or 100 μg/mL inhibited
MMP-1 expression by 70.7%, 77.2% and 81.6%, respectively, compared
to UVB irradiation alone (p < 0.05) (figure 2). Furthermore,
when the cells were treated with fucoidan without UVB irradiation,
the expression of MMP-1 mRNA was not changed compared to a control
(data not shown).
Effect of fucoidan on MMP-1 promoter activity
Time-dependent regulation (0-24 h) of the MMP-1 promoter by
UVB irradiation was examined by luciferase assay. MMP-1 promoter
activity gradually increased in a time-dependent manner with
maximal induction at 24 h in UVB-induced cells (figure 3). Because
previous studies have shown that fucoidan can inhibit the
expression of MMP-1 mRNA in UVB-induced cell, the effect of
fucoidan on MMP-1 promoter activity was assessed. Fucoidan
inhibited UVB-induced MMP-1 promoter activity by 8.5% at
1 μg/mL, by 45.7% at 10 μg/mL, by 57.8% at 100 μg/mL,
compared to UVB irradiation alone (p < 0.05) (figure 4).
Effect of fucoidan on type I procollagen protein
and mRNA expressions
We assessed the effect of fucoidan on type I procollagen synthesis.
Type I procollagen protein expression was increased by 1.51-fold at
1 μg/mL, 2.08-fold at 10 μg/mL, and 2.26-fold at 100 μg/mL of
fucoidan compared to UVB irradiation alone (p < 0.05) (figure 5).
Also, to study the up-regulation effects of fucoidan on the
synthesis of type I procollagen at mRNA level, RT-PCR analysis was
performed. As expected, UVB-induced type I procollagen mRNA
expression was increased by fucoidan. Fucoidan significantly
increased type I procollagen mRNA expression in a dose-dependent
manner (p < 0.05) (figure 6).
Discussion
Collagen is the main component of the extracellular matrix of
dermal connective tissue, and its concentration decreases with
chrono- and photoaging. Once collagen is initially cleaved by
MMP-1, MMP-3, and other MMPs, collagen breakdown is further
promoted. The enzyme mainly responsible for collagen breakdown in
skin is, MMP-1 (fibroblast collagenase), which cleaves types I,
III, VII, VIII and X collagen.
Varani et al. [15] reported that with increasing age, MMP-1
levels rise and collagen synthesis declines for sun-protected human
skin in vivo. Hence, the development of MMP-1 inhibitors and
methods to increase synthesis of type I procollagen is considered
to be a promising strategy for skin cancer therapy and photoaging.
For example, some flavonoid compounds, such as naringenin,
apigenin, wogonin, kaempferol, and quercetin have already been
reported to regulate MMP-1 and type I procollagen expression levels
[16].
Using various in vitro experiments, we previously reported an
inhibitory effect of fucoidan on MMP-1 expression and elucidated
its inhibitory pathways [13], yet the effects of fucoidan on MMP-1
transcription have not been investigated. Recently Benbow and
Brinckerhoff [17] suggested that MMP gene expression may be
regulated in a cell-type specific manner that includes
transcriptional and post-transcriptional mechanisms. Also, Sun et
al. [18] reported that the activity of MMP-1 was stringently
regulated at three levels: the promoter, the activation of
pro-enzyme, and the inhibition of active enzyme. Murphy et al. [19]
reported that the regulation of MMPs occurred primarily at the
level of transcription activity.
We found that UVB stimulated MMP-1 promoter activity in a
time-dependent manner, confirmed that MMP-1 activation is regulated
by UVB, and observed that UVB-induced MMP-1 promoter activity was
maximally and significantly inhibited by fucoidan at 100 μg/mL in
vitro compared to UVB irradiation alone. In an earlier study, the
binding sites for activator protein-1 (AP-1) were found to be
important to MMP-1 promoter regulation [20], and further studies
have also indicated that a suppressor of AP-1 inhibits MMP-1
promoter regulation [21-24]. Therefore, additional studies are
needed to identify the critical regulatory-transcriptional factors
and the MMP-1 promoter regulatory regions controlled by
fucoidan.
We also found that fucoidan significantly inhibited UVB-induced
MMP-1 mRNA expression in a dose-dependent manner, and, as expected,
the UVB-induced MMP-1 protein expression was also inhibited by
fucoidan treatment compared to UVB irradiation alone.
To confirm the effect of fucoidan on type I procollagen
synthesis, we performed RT-PCR and western blot analysis. Two
mechanisms contribute to reduced type I procollagen synthesis: i)
UV irradiation induces the transcription factor AP-1. By binding
and sequestering factors that are part of a transcriptional complex
required for type I procollagen transcription, AP-1 interferes with
collagen production [25, 26]. Transcription factor AP-1 has also
been shown to decrease collagen synthesis by blocking the effects
of transforming growth factor-β (TGF-β), a major profibrotic
cytokine, and sequestering one of the signaling proteins it
activates both directly and indirectly [26-30]. ii) Ultraviolet
irradiation also interferes with TGF-β-dependent type I procollagen
gene expression by down-regulating type II TGF-β receptors, within
8 hours of irradiation, rendering the cells unresponsive to TGF-β
effects [6]. Senni et al. [31] reported that polysaccharides were
able to stimulate dermal fibroblast proliferation and extracellular
matrix deposition in vitro, to control important parameters
involved in connective tissue breakdown. Similar to their results,
we observed that fucoidan treatment increased type I procollagen
mRNA and protein synthesis, in a dose-dependent manner.
Our data indicate that fucoidan may prevent UVB-induced MMP-1
expression and inhibit the down-regulation of type I procollagen
synthesis. We suggest that fucoidan is a potential therapeutic
agent for the prevention and treatment for photoaging of the skin.
Further in vivo studies are necessary to elucidate the
anti-photoaging effects of fucoidan.
Acknowledgements
This work was supported by the Korea Foundation for International
Cooperation of Science & Technology (KICOS) through a grant
provided by the Korean Ministry of Science & Technology (MOST)
in 2006 (M60606000001-06A0600-00140). The authors have declared no
conflict of interest.
References
1 Fisher GJ, Datta SC, Talwar HS, et al.
Molecular basis of sun induced premature skin ageing and retinoid
antagonism. Nature 1996; 379: 335-9.
2 Chung JH, Seo JY, Choi HR, et al.
Modulation of skin collagen metabolism in aged and photoaged human
skin in vivo. J Invest Dermatol 2001; 117: 1218-24.
3 Chung JH, Seo JY, Lee MK, et al.
Ultraviolet modulation of human macrophage metalloelastase in human
skin in vivo. J Invest Dermatol 2002; 119: 507-12.
4 Moon HI, Kim MR, Woo ER, Chung JH.
Triterpenoid from Styrax japonica SIEB. et ZUCC, and its effects on
the expression of matrix metalloproteinases-1 and type 1
procollagen caused by ultraviolet irradiated cultured primary human
skin fibroblasts. Biol Pharm Bull 2005; 28: 2003-6.
5 Fisher GJ, Talwar HS, Lin J, et al.
Retinoic acid inhibits induction of c-Jun protein by ultraviolet
radiation that occurs subsequent to activation of mitogen-activated
protein kinase pathways in human skin in vivo. J Clin Invest 1998;
101: 1432-40.
6 Quan T, He T, Kang S, Voorhees JJ,
Fisher GJ. Solar ultraviolet irradiation reduces collagen in
photoaged human skin by blocking transforming growth factor-beta
type II receptor/Smad signaling. Am J Pathol 2004; 165: 741-51.
7 Doctor VM, Hill C, Jackson GJ. Effect of
fucoidan during activation of human plasminogen. Thromb Res 1995;
79: 237-47.
8 Berteau O, Mulloy B. Sulfated fucans, fresh
perspectives: structures, functions, and biolo gical properties of
sulfated fucans and an overview of enzymes active toward this class
of polysaccharide. Glycobiology 2003; 13: 29-40.
9 O’Leary R, Rerek M, Wood EJ. Fucoidan modulates
the effect of transforming growth factor (TGF)-beta1 on fibroblast
proliferation and wound repopulation in in vitro models of dermal
wound repair. Biol Pharm Bull 2004; 27: 266-70.
10 Isnard N, Péterszegi G, Robert AM,
Robert L. Regulation of elastase-type endopeptidase activity,
MMP-2 and MMP-9 expression and activation in human dermal
fibroblasts by fucose and a fucose-rich polysaccharide. Biomed
Pharmacother 2002; 56: 258-64.
11 Robert L, Fodil-Bourahla I, Bizbiz L,
Robert AM. Effect of L-fucose and fucose-rich polysaccharides
on elastin biosynthesis, in vivo and in vitro. Biomed Pharmacother
2004; 58: 123-8.
12 Fodil-Bourahla I, Bizbiz L, Schoevaert D,
Robert AM, Robert L. Effect of L-fucose and fucose-rich
oligo- and polysaccharides (FROP-s) on skin aging: penetration,
skin tissue production and fibrillogenesis. Biomed Pharmacother
2003; 57: 209-15.
13 Moon HJ, Lee SR, Shim SN, et al. Fucoidan
inhibits UVB-induced MMP-1 expression in human skin fibroblasts.
Biol Pharm Bull 2008; 31: 284-9.
14 Chomczynski P, Sacchi N. Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction. Anal Biochem 1987; 162: 156-9.
15 Varani J, Warner RL, Gharaee-Kermani M,
et al. Vitamin A antagonizes decreased cell growth and
elevated collagen-degrading matrix metalloproteinases and
stimulates collagen accumulation in naturally aged human skin. J
Invest Dermatol 2000; 114: 480-6.
16 Lim H, Kim HP. Inhibition of mammalian
collagenase,matrix metalloproteinases-1, by naturally-occurring
flavonoids. Planta Med 2007; 73: 1267-74.
17 Benbow U, Brinckerhoff CE. The AP-1 site and MMP
gene regulation: what is all the fuss about? Matrix Biol 1997; 15:
519-26.
18 Sun Y, Sun Y, Wenger L, Rutter JL,
Brinckerhoff CE, Cheung HS. p53 down-regulates human
matrix metalloproteinase-1 (Collagenase-1) gene expression. J Biol
Chem 1999; 274: 11535-40.
19 Murphy KA, Villano CM, Dorn R, White LA.
Interaction between the aryl hydrocarbon receptor and retinoic acid
pathways increases matrix metalloproteinase-1 expression in
keratinocytes. J Biol Chem 2004; 279: 25284-93.
20 Doyle GA, Pierce RA, Parks WC. Transcriptional
induction of collagenase-1 in differentiated monocyte like (U937)
cells is regulated by AP-1 and an upstream C/EBP-beta site. J Biol
Chem 1997; 272: 11840-9.
21 Südel KM, Venzke K, Knussmann-Hartig E,
et al. Tight control of matrix metalloproteinase-1 activity in
human skin. Photocem Photobiol 2003; 78: 355-60.
22 Wang XY, Bi ZG. UVB-irradiated human keratinocytes
and interleukin-1alpha indirectly increase MAP kinase/AP-1
activation and MMP-1 production in UVA-irradiated dermal
fibroblasts. Chin Med J 2006; 119: 827-31.
23 Oh JH, Kim A, Park JM, Kim SH,
Chung AS. Ultraviolet B-induced matrix metalloproteinase-1 and
-3 secretions are mediated via PTEN/Akt pathway in human dermal
fibroblasts. J Cell Physiol 2006; 209: 775-85.
24 Ramos MC, Steinbrenner H, Stuhlmann D,
Sies H, Brenneisen P. Induction of MMP-10 and MMP-1 in a
squamous cell carcinoma cell line by ultraviolet radiation. Biol
Chem 2004; 385: 75-86.
25 Karin M, Liu ZG, Zandi E. AP-1 function and
regulation. Curr Opin Cell Biol 1997; 9: 240-6.
26 Chung KY, Agarwal A, Uitto J, Mauviel A.
An AP-1 binding sequence is essential for regulation of the human
alpha2(I) collagen (COL1A2) promoter activity by transforming
growth factor-beta. J Biol Chem 1996; 271: 3272-8.
27 Verrecchia F, Tacheau C, Schorpp-Kistner M,
Angel P, Mauviel A. Induction of the AP-1 members c-Jun
and JunB by TGF-beta/Smad suppresses early Smad-driven gene
activation. Oncogene 2001; 20: 2205-11.
28 Pessah M, Marais J, Prunier C, et al.
c-Jun associates with the oncoprotein Ski and suppresses Smad2
transcriptional activity. J Biol Chem 2002; 277: 29094-100.
29 Massagué J. TGF-β signal transduction. Annu Rev Biochem
1998; 67: 753-91.
30 Massagué J. How cells read TGF-β signals. Nat Rev Mol
Cell Biol 2000; 1: 169-78.
31 Senni K, Gueniche F, Foucault-Bertaud A,
et al. Fucoidan a sulfated polysaccharide from brown algae is
a potent modulator of connective tissue proteolysis. Arch Biochem
Biophys 2006; 445: 56-64.
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