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J. Biol. Chem., Vol. 278, Issue 36, 34483-34490, September 5, 2003
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3 LG4 Module Induces Matrix Metalloproteinase-1 through Mitogen-activated Protein Kinase Signaling*
¶
From the
Department of Dermatology and the
||Department of Biochemistry and Molecular
Pharmacology, Graduate School of Medicine, Chiba University, Chiba 260-8670,
Japan, the **Matrix Biology & Tissue Repair
Research Unit, Department of Dental Health and Biological Sciences, University
of Wales College of Medicine, Cardiff CF14 4XY, United Kingdom, and the
Graduate School of Environmental Earth
Science, Hokkaido University, Sapporo 060-0810, Japan
Received for publication, May 8, 2003 , and in revised form, June 23, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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3 chain (3 LG4), a component of
epithelial-specific laminin-5, has cell attachment activity and binds syndecan
(Utani, A., Nomizu, M., Matsuura, H., Kato, K., Kobayashi, T., Takeda, U.,
Aota, S., Nielsen, P. K., and Shinkai, H. (2001) J. Biol. Chem. 276,
28779–28788). Here, we show that recombinant 3 LG4 and a 19-mer
synthetic peptide (A3G756) within 3 LG4 active for syndecan binding
increased the expression of matrix metalloproteinase-1 (MMP-1) in
keratinocytes and fibroblasts. This induction was inhibited by heparin and
required de novo synthesis of proteins. In keratinocytes, A3G756
up-regulated interleukin (IL)-1
and MMP-1 expression and an IL-1
receptor antagonist thoroughly inhibited A3G756-mediated induction of MMP-1.
A3G756 also activated p38 mitogen-activated protein kinase (p38 MAPK) and
extracellular signal-related kinase (Erk). Studies with specific inhibitors of
MAPKs showed that p38 MAPK activation was necessary for both IL-1 and
MMP-1 induction, but Erk activation was required only for MMP-1 induction. In
fibroblasts, IL-1 receptor antagonist did not block A3G756-mediated induction
of MMP-1. These results indicated that induction of MMP-1 by 3 LG4 is
mediated through the IL-1 autocrine loop in keratinocytes but the
mechanism of the induction in fibroblasts is different. Our study suggests
that the laminin 3 LG4 module may play an important role in tissue
remodeling by inducing MMP-1 expression during wound healing. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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chains, 3 chains, and 3 
chains. Three chains
assemble into a cross-shaped heterotrimer () through
coiled-coil interaction at the long arm of the cross
(3,
4). Laminin-5 is specific to
epithelial cells and a component of the anchoring filament. Laminin-5 forms a
complex with the hemidesmosome apparatus by interacting with laminin-6 and -7,
collagen VII (5,
6), and fibulin-1 and -2
(7,
8). Laminin-5 consists of the
3, 3, and 2 chains. The 3 chain contains a large
globular module (G
module)1 at the C
terminus, which consists of a tandem repeat of five homologous LG modules
(LG1–LG5), each module containing about 200 amino acid residues
autonomous folding unit (9).
The LG subdomains of laminin chains have been shown to bind heparin,
31, 64
integrins, -dystroglycan, and syndecan (Ref.
10; for review, see Ref.
11) and are implicated as
active regions for various biological functions.
Syndecans, cell surface heparan sulfate proteoglycans, have been shown to
bind the G module of laminin chains and are involved in
laminin-mediated biological functions. We previously demonstrated that
keratinocytes and fibroblasts bound LG4 of the 3 G module via syndecans
(12). Neurite outgrowth of
PC12 cells was induced by 3 LG4 via syndecans
(13). We also showed that
HT1080 cells bound to the C-terminal G module of the laminin 4 chain
through syndecans (14). The
interaction of laminin 1 LG4 and syndecan family or heparan sulfate
proteoglycans was essential for embryonic basement membrane assembly
(15).
Although laminin 3 LG4–5 is processed in the keratinocyte
culture medium (16,
17), the unprocessed laminin
3 chain was found in a cell layer of the provisional edge of the cell
sheet in cultured keratinocytes. In vivo, the unprocessed 3
chain has been identified especially in the newly synthesized epidermal
basement membrane in wounds but disappears from the mature basement membrane
(Refs. 18 and
19; for review, see Ref.
20).
Matrix metalloproteinase-1 (MMP-1) is expressed in the basal keratinocytes
at the leading edge of re-epithelization (Refs.
21 and
22; for review, see Ref.
23). At the wound edge, MMP-1
degrades collagen and promotes cell migration and wound closure. The similar
temporal and spatial localization of MMP-1 and unprocessed 3 chain led
us to study the relationship between MMP-1 induction and signaling pathways
induced by the laminin 3 LG4 module. In the present work, we
demonstrated that the laminin 3 LG4 module induced MMP-1 expression by
the activation of MAPK via syndecan receptor.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Reagents—Anti-phospho-p44/42 MAPK (Erk) polyclonal antibody,
anti-phospho-p38 MAPK polyclonal antibody, and anti-phospho-stress-activated
protein kinase/c-Jun N-terminal kinase polyclonal antibody were purchased from
Cell Signaling Technology (Beverly, MA). Polyclonal antibody to p38 MAPK was a
kind gift from Dr. Sudo (Antibiotics Laboratory, RIKEN, Saitama, Japan)
(25). Anti-Erk2 monoclonal
antibody was purchased from Upstate Biotechnology, Inc. (Waltham, MA).
Anti-human IL-1 monoclonal antibody (AF-201-NA) was obtained from
R&D Systems (Minneapolis, MN). Anti-human MMP-1 polyclonal antibody
(444209) was from Calbiochem (San Diego, CA). Peroxidase-conjugated
anti-rabbit and anti-mouse IgG antibodies and ECL (Amersham Biosciences,
Uppsala, Sweden) were used for Western blotting. Human syndecan-4 monoclonal
antibody (5G9) and human syndecan-2 antibody (L-18) were obtained from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA). Fluorescein isothiocyanate
(FITC)-conjugated anti-mouse IgG antibody and Cy3-conjugated anti-human IgG
antibody were purchased from Jackson ImmunoResearch Laboratories (West Grove,
PA). FITC-conjugated anti-heparan sulfate antibody (10E4) was obtained from
Seikagaku Kogyo (Tokyo, Japan). MAPK inhibitors SB202190 and PD98059 were from
Calbiochem and dissolved in dimethyl sulfoxide (Me2SO). IL-1
was obtained from Roche Diagnostics (Indianapolis, IN). Human IL-1 receptor
antagonist (IL-1RA, which traps IL-1) was purchased from R&D
Systems. Heparin was from Seikagaku Kogyo and cycloheximide from Sigma.
Recombinant Proteins—Recombinant 3 LG4 was expressed
as a chimera with a human IgG Fc portion at the C terminus and purified as
previously described with a minor modification
(12). Briefly, recombinant
proteins were expressed in 293 T cells by the Ca-P transfection kit
(Invitrogen). After 24 h, cells were incubated with Chinese hamster ovary
medium (Invitrogen) for another 2 days, followed by purification with protein
A-Sepharose (Amersham Biosciences).
Synthesis of A3G756 —Peptides were manually synthesized by the Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based solid-phase methods with a C-terminal amide as previously described (26). Peptides were purified by reverse phase high performance liquid chromatography (HPLC) using a Mightysil RP-18 column (Kanto Chemical Co., Inc., Tokyo, Japan) with a gradient of water/acetonitrile containing 0.1% trifluoroacetic acid. Purity and identity were confirmed by HPLC and by fast atom bombardment mass spectral analysis at the GC-MS & NMR Laboratory, Graduate School of Agriculture, Hokkaido University (Hokkaido, Japan). The A3G756 peptide (residues 1411–1429, KNSFMALYLSKGRLVFALG) showed syndecan-mediated cell attachment in a heparin dependent manner as strong as the previously identified 12-mer active peptide (residues 1412–1423, NSFMALYLSKGR) (12). Peptide S4 (LVAGAFFKRKLLLMNSSGY) was a scrambled peptide of A3G756, and cell adhesion and inhibition assays demonstrated that S4 had no adhesion activity.
Treatment of Cells—Keratinocytes were seeded at 1.0 x 105 in 12-well plastic dishes. After 24 h, early confluent cells were starved for 24 h in keratinocyte-SFM. Fibroblasts (2.0 x 105) were seeded in 12-well dishes, and were settled for several hours until attaching, followed by 24 h of starvation in DMEM containing 0.1% BSA. Then, recombinant protein, peptides, or cytokine were added in 0.4 ml of fresh medium. Cells were pretreated with 2 µg/ml cycloheximide for 2 h before stimulation. MAPK inhibitors, 30 µg/ml SB202190, 20 µg/ml PD98059, or Me2SO only were added 1 h before stimulation.
RT-PCR Analysis of MMP-1—Total RNA was prepared from cells
after 8-h incubation by RNAeasy kit (Qiagen GmbH, Hilden, Germany). cDNA was
synthesized from RNA (2.0 µg) with an oligo(dT) primer in a total volume of
21 µl by SPreamplification System (Invitrogen). cDNA (1 µl for
IL-1 or 0.1 µl for MMP-1 and GAPDH) was subjected to PCR for
amplification. In preliminary experiments, we determined the optimal number of
cycles within the linearity of reactions for each PCR product. The cycle
number was 35 cycles for IL-1 and was 25 cycles for MMP-1 and GAPDH. The
primers for PCR are as follows: human MMP-1 (830–1465 nt, GenBankTM
accession no. X54925
[GenBank]
), forward (5'-CATCCAAGCCATATATGGACG) and backward
(5'-GCAGTTGAACCAGCTATTAGC); human GAPDH (292–885 nt,
GenBankTM J04038
[GenBank]
), forward (5'-CCCATCACCATCTTCCAG) and backward
(5'-CCTGCTTCACCACCTTCT); human IL-1 (541–1331 nt,
GenBankTM M15330
[GenBank]
), forward (5'-AGCAACAAGTGGTGTTCTCCATG) and
backward (5'-CTAGGCTCTTTTACAGACACTGC). Using these primers, PCR was
performed by the Expand High Fidelity PCR System (Roche Diagnostics) at 94
°C for 4 min followed by individual cycles at 94 °C for 30 s, 58
°C for 30 s, and 72 °C for 1 min with an extension step of 7 min at 72
°C at the end of the last cycle. The products were separated on 1.5%
agarose gel. The bands were visualized with ethidium bromide staining. The
intensity of the products was calculated with NIH Image 1.60, and the values
were depicted as the ratio to those of GAPDH.
ELISA—Fibroblasts were treated with reagents in 0.4 ml of DMEM containing 0.1% BSA. The triplicate conditioned media were collected, and MMP-1 was measured by the Human Matrix Metalloproteinase 1 ELISA System (Amersham Bioscience) using precoated 96-well immunoplates. Absorbance was read at 450 nm with microplate reader. The MMP-1 level was calculated by standard curve with an internal control following the protocol from the manufacturer.
Western Blotting—Keratinocytes and fibroblasts were treated
as described above. Conditioned media were collected. The media were
lyophilized and dissolved in 50 µl of sample buffer (20% glycerol, 2% SDS,
125 mM Tris-HCl, pH 6.8, 2% mercaptoethanol, and 0.15% bromphenol
blue), and 20 µl of sample was subjected to Western blotting. Cells were
washed with ice-cold PBS twice and were lysed on ice by the addition of 40
µl of lysis buffer (20 mM Tris-HCl, pH 7.5, 60 mM
glycerophosphate, 10 mM EGTA, 10 mM MgCl2, 10
mM NaF, 2 mM dithiothreitol, 1 mM
N3VO4, 1 mM phenylmethylsulfonyl
fluoride, 5 µg/ml leupeptin, and 1% Nonidet P-40) for 15 min. Cell lysates
were cleared by centrifugation at 20,000 x g at 4 °C for 15
min. Aliquot (15 µl) was boiled in sample buffer for 5 min and subjected to
12% SDS-PAGE, and transferred by electroblotting onto polyvinylidene
difluoride transfer membranes (ImmobilonTM, Millipore, Bedford, MA). The
membranes were blocked with 10% nonfat milk in Tris-buffered saline (150
mM NaCl, 40 mM Tris-HCl, pH 7.4) plus 0.1% Tween 20,
probed with the first antibodies (1:200), and visualized by the
peroxidase-conjugated second antibodies (1:2000–1:5000) and an ECL
system (Amersham Biosciences). Densitometric analysis was carried out using
NIH Image software version 1.60. To verify the amounts of the samples,
membrane was probed with anti-p38 MAPK or anti-Erk2 antibody after
stripping.
Syndecan-2 and -4 Overexpression in Mammalian Cells—cDNA of whole human syndecan-2 (577–1,245 nt, GenBankTM J04621 [GenBank] ) and cDNA of whole human syndecan-4 (9–623 nt, GenBankTM X67016 [GenBank] ) were amplified using mRNA derived from dermal fibroblasts as described previously (12). Briefly, PCR product was subcloned into the expression vector (27) containing a cytomegalovirus promoter and an internal ribosome entry site and puromycine-resistant gene (28). Human primary dermal fibroblasts were transfected by LipofectAMINE (Invitrogen), and clones producing syndecan-2 and -4 were selected with 0.4 µg/ml puromycin (Sigma). Both Western blotting and immunostaining with anti-syndecan-4 antibody confirmed selected syndecan-4-producing clones. The selected syndecan-2-expressing clones were analyzed by Western blotting with anti-syndecan-2 antibody and immunostaining with FITC-anti-heparan sulfate antibody.
Immunocytochemistry—Fibroblasts on cover glass that
overexpressed syndecan-2 and -4 were fixed by 4% paraformaldehyde/PBS for 15
min at room temperature and blocked with 5% normal donkey serum (Chemicon
International, Inc., Temecula, CA), 1% BSA (Sigma), 0.05% Nonidet P-40
(Sigma), PBS for 15 min. Cells were incubated with recombinant 3 LG4
(10 µg/ml) and anti-syndecan-4 monoclonal antibody (1:50) at 4 °C
overnight, followed by Cy3-conjugated anti-human IgG antibody (1:200) and
FITC-conjugated anti-mouse IgG antibody (1:200) for 30 min at 37 °C. For
syndecan-2 staining, FITC-conjugated anti-heparan sulfate antibody was used.
After washing, pictures were taken by Nikon fluorescent microscopy.
Molecular Modeling—A model of the 3 LG4–5
structure was generated using ProMod II version 3.5
(29) with the x-ray derived
coordinates of the mouse 2 LG4–5 module pair (Protein Data Bank
code 1dyk
[PDB]
) (30) as a template,
resulting in a final total energy of –3980 kJ/mol. As expected from
sequence alignments, the model shows disulfide bonds between Cys-1354 and
Cys-1617, Cys-1507 and Cys-1530, and Cys-1682 and Cys-1710.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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3 LG4 and A3G756 Induce MMP-1
Expression— We previously showed that laminin 3 LG4 and
peptide A3G756 within 3 LG4 bind keratinocytes and fibroblasts through
syndecans (12). We
hypothesized that this interaction regulates expression of MMP-1 during wound
healing. Therefore, we examined expression levels of MMP-1 in keratinocyte and
fibroblasts when these cells were incubated with 3 LG4 and A3G756.
RT-PCR analysis demonstrated that recombinant 3 LG4 and peptide A3G756
stimulated MMP-1 mRNA expression in keratinocytes, whereas control peptide S4
(scrambled A3G756 peptide sequence) did not change MMP-1 mRNA
(Fig. 1, A and
B). This increase was strongly blocked by the addition of
heparin into the medium (Fig. 1, A
and B). The heparin-dependent MMP-1 induction and
syndecan binding to the A3G756 peptide suggest that the induction process is
mediated through syndecans.
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Because IL-1 is known to induce MMP-1 expression
(31,
32), we next examined whether
A3G756 increases IL-1 mRNA levels. In both keratinocytes and
fibroblasts, A3G756 significantly increased IL-1 mRNA expression
(Fig. 1C). We also
confirmed that IL-1 induced MMP-1 expression in these cell types,
agreeing with previous reports (Fig.
1D). Because a 4-h incubation with A3G756 was not
sufficient to induce MMP-1 gene expression (data not shown), de novo
synthesis of some factors might be necessary in this induction process.
Pretreatment of keratinocytes with cycloheximide, an inhibitor of translation,
totally diminished the up-regulation of MMP-1 gene expression by A3G756
(Fig. 1E). These
results indicate that de novo protein synthesis is required for
A3G756-mediated MMP-1 induction. On the contrary, cycloheximide did not
inhibit up-regulation of IL-1 gene expression (data not shown),
suggesting that the up-regulation of IL-1 by A3G756 did not require new
synthesis of a factor(s). Thus, IL-1 is a likely candidate responsible
for the synthesis of a new factor involved in the up-regulation of MMP-1 by
A3G756.
IL-1 Receptor Antagonist Blocks MMP-1 Induction by A3G756 in
Keratinocytes but Not in Fibroblasts—To further investigate the
mechanism of IL-1 action, we next examined the effect of IL-1 receptor
antagonist on A3G756-mediated MMP-1 induction. In keratinocytes, IL-1RA (IL-1
receptor antagonist) at 0.3 µg/ml inhibited the increase of MMP-1 gene
expression by A3G756 (Fig.
2A). On the contrary, in fibroblasts A3G756 could
up-regulate MMP-1 expression in the presence of an even higher concentration
of IL-1RA (1.0 µg/ml) (Fig.
2B), which is the concentration used by other groups for
blocking the IL-1 autocrine loop in dermal fibroblasts
(33,
34). Taken together, the
results show that the IL-1 autocrine loop is an essential pathway for
A3G756-mediated induction of MMP-1 in keratinocytes, but it is not necessary
in fibroblasts, even though A3G756 was able to induce IL-1 gene
expression (Fig. 1D)
and exogenously added IL-1 increased MMP-1 gene expression
(Fig. 1C).
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Increase of MMP-1 Protein Levels by A3G756 —Increase of the
MMP-1 protein by A3G756 in the keratinocytes was demonstrated by Western
blotting of the conditioned medium. MMP-1 protein levels increased in the
12–33-h incubations, but not in the 6- and 8-h incubation, whereas
protein levels of IL-1 increased within the 6–12-h incubation
(Fig. 3A). Although
IL-1 showed rather rapid disappearance from the conditioned medium, the
observation that IL-1 expression preceded the MMP-1 expression were
consistent with the facts that newly synthesized IL-1 induced MMP-1
expression in the keratinocytes. The increase of basal expression of MMP-1 at
33-h incubation without peptide stimulation likely represents the accumulation
of MMP-1 protein as also seen in the fibroblasts
(Fig. 3C). These data
confirm that up-regulation of MMP-1 mRNA levels by A3G756 results in increased
protein levels. MMP-1 protein levels in fibroblasts were also examined by
ELISA using the conditioned medium. A dose-dependent increase in MMP-1 protein
levels was observed by incubation of fibroblasts with various amounts of
A3G756 in a 24-h incubation (Fig.
3B). The MMP-1 protein level reached plateau at 24
µg/ml A3G756. Within the 8-h incubation, protein levels of MMP-1 did not
change significantly, but after 16 h of incubation, it increased and the
reached its maximum level by 24 h (Fig.
3C). AG756-mediated induction of MMP-1 protein was
inhibited by heparin but not by IL-1 receptor antagonist in fibroblasts
(Fig. 3D). Exogenous
IL-1 induced MMP-1 expression in fibroblasts
(Fig. 3D), and IL-1
receptor antagonist blocked the IL-1-mediated induction (data not
shown). The failure of the IL-1 receptor antagonist to inhibit A3G756-mediated
MMP-1 induction in fibroblasts suggests that IL-1 is not necessary for
MMP-1 induction, although exogenous IL-1 induced MMP-1 expression. These
results were consistent with those of mRNA changes in fibroblasts.
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A3G756 Activates MAPK Signaling—Because IL-1 is known
to activate MAPKs, activation of MAPKs was analyzed by Western blotting with
anti-phosphorylated MAPK antibodies (Fig.
4). Treatment of keratinocytes with recombinant 3 LG4 or
A3G756 activated p38 MAPK (Fig.
4A). Activation of p38 MAPK by A3G756 was observed after
15 min of incubation and reached the maximum level at 2 h. A3G756 also
activated Erk within 6 h of incubation
(Fig. 4B). During
these incubation periods, c-Jun N-terminal kinase was not activated (data not
shown). As expected, exogenous IL-1 activated both p38 MAPK and Erk in
keratinocytes at 15-min incubation (Fig.
4A, 4B). Activations of p38 MAPK and Erk by A3G756 were
completely blocked by the addition of heparin
(Fig. 4, A and
B). These results indicate that A3G756 induces rapid
activation of p38 MAPK and slow activation of Erk in keratinocytes. The
activation of Erk was drastically inhibited by the addition of IL-1 receptor
antagonist, but p38 MAPK activation was partially blocked
(Fig. 4C). These
findings suggest that the Erk activation observed at 6-h incubation was
predominantly mediated by the IL-1 autocrine loop. Although at least a
part of p38 MAPK activation at 6-h incubation with A3G756 was mediated by the
IL-1 autocrine loop, there should be another pathway, e.g. a
direct activation of p38 MAPK.
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In fibroblasts, p38 MAPK was phosphorylated at 15-min incubation and returned to a basal level after 1-h incubation with A3G756 (Fig. 4D). Erk activation was not detectable from 15 min to 6 h of incubation, and p38 MAPK was not activated from 2 to 6 h of incubation (data not shown). These different profiles of MAPK activation indicate that A3G756-syndecan binding transduces signals in a cell type-specific manner.
The Effects of MAPK Inhibitors on A3G756 Induction of MMP-1 or
IL-1—Because A3G756 activated p38 MAPK and/or Erk,
we examined whether MAPK inhibitors block the up-regulation of MMP-1 by
A3G756. RT-PCR analysis showed that SB202190, a specific inhibitor of p38
MAPK, blocked the up-regulation of MMP-1 and IL-1 in keratinocytes
(Fig. 5A). PD98059, an
inhibitor of Erk, blocked the up-regulation of MMP-1, but did not change the
IL-1 induction (Fig.
5A). Together with the observation that Erk activation
was inhibited by IL-1 receptor antagonist, these results indicate that Erk is
involved in the up-regulation of MMP-1 through the IL-1 autocrine loop
in keratinocytes.
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In fibroblasts, PD98059 inhibited both MMP-1 and IL-1 mRNA induction,
whereas SB202190 inhibited only IL-1 mRNA induction
(Fig. 5B). There are
two differences in the responses to MAPK inhibitors. One is that both p38 MAPK
and Erk activation are required for IL-1 mRNA induction. The other is
that blockade of IL-1 mRNA induction by SB202190 did not abolish the
MMP-1 induction. These results suggest that IL-1 is not involved in
A3G756-mediated MMP-1 induction in fibroblasts. Similar patterns were obtained
in MMP-1 protein levels estimated by ELISA in fibroblasts
(Fig 5C). PD98059, but
not SB202190, completely blocked the increase of MMP-1 protein levels by
A3G756. Only Erk activation is necessary for A3G756-mediated MMP-1, although
Erk activation could not be demonstrated in fibroblasts by Western blotting as
mentioned (Fig.
4D).
Colocalization of 3 LG4 and
Syndecans—Previously we showed that 3 LG4 and A3G756 bind
keratinocytes and fibroblasts through syndecans in solid phase binding assays
(12). Here we demonstrate that
soluble 3 LG4 is colocalized with syndecans on the cell surface of
dermal fibroblasts (Fig. 6).
For this experiment, we obtained dermal fibroblast cell lines that were stably
transfected with the expression vector for syndecan-2 or syndecan-4.
Incubation of syndecan-expressing fibroblasts with 3 LG4 showed
colocalization of 3 LG4 with syndecan-2 or syndecan-4 in double
immunostaining (Fig. 6). The
3 LG4 binding was completely diminished by the addition of heparin (30
µg/ml) in both clones during the incubation (data not shown). These results
confirm that 3 LG4 interacts with syndecans on the cell surface.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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3 LG4 module of laminin-5 induces
MMP-1 expression by activating the MAPK signaling pathway. We also found that
this process involves the IL-1 autocrine loop in keratinocytes. The
3 LG4-mediated induction of MMP-1 occurs through the interaction of
syndecans with the A3G756 sequence within the module. Therefore, we are
interested in the structure of the 3 LG4 module and the location of the
active A3G756 site within the module structure. Although the structure of the
LG4–5 module of laminin 3 is not known, the crystal structure of
the LG4–5 domain pair of laminin 2 was reported
(30).
Fig. 7 shows the
structure-sequence-based alignment of human 3 LG4–5 generated
using the murine 2 LG4–5 structure as a template. The basic
residues, Lys (Lys-1421) and Arg (Arg-1423), critical for syndecan binding
(12) are exposed and clustered
within the sequence of A3G756 (residues 1411–1429; KNSFMALYLSKGRLVFALG)
within the LG4 module, which consists of 14 strands forming a
-sheet sandwich. These two residues are located at one end of a cleft
formed by the loop connecting strands. This cleft contains several
further surface-exposed basic residues that might provide a specific
orientation to the extended glycosaminoglycan chains by interaction with their
sulfate groups. This would be consistent with the active bases of 2
LG4–5 for -dystroglycan and heparin binding. These results
suggest that the A3G756 site of laminin 3 is exposed on the surface of
the 3 LG4 structure and is active for syndecan binding.
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It has been reported that exogenous IL-1 activates p38 MAPK and leads
to up-regulation of MMP-1 expression in fibroblasts
(31,
32). Inhibitor of p38 MAPK
blocked A3G756-mediated IL-1 expression in keratinocytes
(Fig. 5). However, IL-1
receptor antagonist could not completely inhibit p38 MAPK activation in
keratinocytes (Fig.
4C). Therefore, p38 MAPK is likely involved in both
pathways, one from syndecan to IL-1 up-regulation and the other from
IL-1 receptor to MMP-1 up-regulation. Erk activation was detected as
early as 6 h after incubation with keratinocytes. The activation of Erk was
drastically inhibited by the addition of IL-1 receptor antagonist. Further,
PD98059, an inhibitor of Erk, did not block A3G756-mediated IL-1
induction in keratinocytes. Therefore, it is likely that Erk mediates
signaling from IL-1 receptor to MMP-1 gene expression. Because Erk
activation by exogenous IL-1 in keratinocytes is mediated by epidermal
growth factor receptor transactivation
(35), Erk activation via the
syndecan-IL-1 pathway may be induced by a similar mechanism.
We showed that exogenous IL-1 stimulated MMP-1 gene expression and
that A3G756 up-regulated IL-1 gene expression in fibroblasts
(Fig. 1). Therefore, the
involvement of the IL-1 autocrine loop in A3G756-mediated induction of
MMP-1 in fibroblasts is possible. However, in fibroblasts, IL-1
autocrine loop and MMP-1 up-regulation likely occur by an independent
mechanism. Unlike keratinocytes, p38 MAPK activation was not needed for
A3G756-mediated induction of MMP-1 in fibroblasts
(Fig. 5B). It was
reported that, in fibroblasts, basic fibroblast growth factor-mediated
induction of MMP-1 required activation of Erk, but not p38 MAPK
(32). Signaling from syndecan
may up-regulate and/or activate another factor(s) for MMP-1 up-regulation
through Erk, independent of the IL-1 autocrine pathway in
fibroblasts.
There are four mammalian syndecans, all of which are transmembrane heparan
sulfate proteoglycans. Syndecans bind to extracellular matrices as a cell
surface receptor and store fibroblast growth factors and vascular endothelial
growth factors (for review, see Refs.
36 and
37). Syndecan-1 is the major
syndecan of epithelial cells, syndecan-2 is that of mesenchymal cells, and
syndecan-3 is that of neural tissues. Syndecan-4 is widely distributed.
Usually, more than one syndecan are expressed in a single cell type. The
carboxyl terminus of a short cytoplasmic module is conserved in the syndecan
family and can interact with syndecan type-specific PDZ-domain-containing
proteins. With these signal-transducing molecules, syndecans may have unique
functions other than cell adhesion. It is conceivable that a different set of
syndecan isoforms expressed at the cell surface of keratinocytes (syndecan-1
and -4) and fibroblasts (syndecan-2 and -4) may play a key role in regulating
different responses to laminin 3 LG4. Activating antibody to
51 integrin increases MMP-1 gene expression
via the IL-1 autocrine loop in fibroblasts
(38). Recently, it was
reported that the signal from syndecan-4, but not from syndecan-2,
transactivates integrin 1
(39). Taken all together,
these results suggest that signals from syndecan binding to laminin 3
LG4 may activate integrins, resulting in MMP-1 induction.
Keratinocytes are known to produce MMPs when they contact collagen fibers
at a wound site. MMP-1 degrades collagen I, allowing keratinocytes to migrate
on the wound granulation tissue under desiccated eschar. It is also possible
that syndecan binding to laminin 3 LG4 contributes to re-epithelization
with two different activities such as cell adhesion and MMP-1 induction. The
identification of the precise mechanisms of how syndecan binding to laminin
3 LG4 can induce MMP-1 gene expression may lead to the development of
clinical applications for wound healing.
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FOOTNOTES |
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Both authors contributed equally to this work.
¶ To whom correspondence should be addressed: Dept. of Dermatology, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8670, Japan. Tel.: 81-43-222-7171 (ext. 5332); Fax: 81-43-226-2128; E-mail: utani{at}derma01.m.chiba-u.ac.jp.
1 The abbreviations used are: G module, globular module; MMP-1, matrix
metalloproteinase-1; MAPK, mitogen-activated protein kinase; IL-1,
interleukin-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse
transcriptase; Erk, extracellular signal-related kinase; PBS,
phosphate-buffered saline; FITC, fluorescein isothiocyanate; nt,
nucleotide(s); BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent
assay; DMEM, Dulbecco's modified Eagle's medium; HPLC, high performance liquid
chromatography.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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