From the UPRES-A CNRS 6021, IFR53
Biomolécules, Faculties of Sciences and Medicine and the
§ Laboratory of Biomolecular Spectroscopies and Structures,
IFR53 Biomolécules, Faculty of Sciences, University of Reims,
51687 Reims, France, ¶ Laboratory of Analytical Biochemistry,
University Claude Bernard, 69622 Lyon, France, and
Laboratory of
Organic Chemistry, Department of Chemistry, University of Potenza,
85100 Potenza, Italy
Received for publication, April 28, 2000, and in revised form, November 9, 2000
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ABSTRACT |
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We have established that treatment
of cultured human skin fibroblasts with tropoelastin or with
heterogenic peptides, obtained after organo-alkaline or leukocyte
elastase hydrolysis of insoluble elastin, induces a high expression of
pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The
identical effect was achieved after stimulation with a VGVAPG synthetic
peptide, reflecting the elastin-derived domain known to bind to the
67-kDa elastin-binding protein. This clearly indicated involvement of
this receptor in the described phenomenon. This notion was further
reinforced by the fact that elastin peptides-dependent
MMP-1 up-regulation has not been demonstrated in cultures preincubated
with 1 mM lactose, which causes shedding of the
elastin-binding protein and with pertussis toxin, which blocks the
elastin-binding protein-dependent signaling pathway
involving G protein, phospholipase C, and protein kinase C. Moreover,
we demonstrated that diverse peptides maintaining GXXPG
sequences can also induce similar cellular effects as a "principal"
VGVAPG ligand of the elastin receptor. Results of our biophysical
studies suggest that this peculiar consensus sequence stabilizes a type
VIII The extracellular matrix protein elastin is responsible for the
elastic properties of tissues such as lung, skin, and large arteries
(1-3). Due to its numerous cross-links and the extreme hydrophobicity
of its tropoelastin chains, elastin is highly resistant to proteolysis.
However, during inflammatory disorders, proteinases secreted from
polymorphonuclear neutrophils, such as elastase, cathepsin G, and
gelatinase B may cause significant elastolysis (4).
It has been established that
peptides derived from elastin or from the hydrophobic domains of
tropoelastin interact with cells via a cell surface-resided 67-kDa
elastin-binding protein identical to an enzymatically inactive,
alternatively spliced form of Matrix metalloproteinases (MMPs) are potent proteinases involved in a
broad range of normal and pathological processes (25). Their expression
is regulated through interaction between cells and extracellular matrix
via several classes of cell surface receptors (25). For example, a
single module within fibronectin can elicit a particular cell response,
resulting in either up- or down-regulation of collagenase (MMP-1)
production (26). Attracted by the wide range of cellular effects
induced by elastin-derived peptides, we investigated whether peptides
resulting from elastin degradation would also control MMPs (MMP-1,
i.e. collagenase-1; MMP-3, i.e. stromelysin-1)
expression and secretion and thereby contribute to further degradation
of other matrix components.
Results of the present study indicate that cultured human skin
fibroblasts up-regulate expression and secretion of pro-MMP-1 and
pro-MMP-3 after stimulation with products of organo-alkaline- and human
leukocyte elastase (HLE)-dependent elastin degradation. Our
data suggest that among many elastin-derived peptides, only those with
the GXXPG consensus sequence possess a conformation that
allows binding to the EBP and a consequent triggering of signals
responsible for the up-regulation pro-MMP-1 and pro-MMP-3. Finally, we
have established that both secreted pro-enzymes can be activated after
the addition of exogenous plasmin to the culture medium and then
degrade a collagen substrate. Our results thus raise the possibility
that elastin degradation could lead to collagenolysis during normal
and/or pathological conditions.
Samples Preparation and Reagents--
Bovine tropoelastin was
obtained from Elastin Products Co. (Universal Biological Ltd., London).
Alternatively, insoluble elastin was partially hydrolyzed by HLE
(Universal Biological Ltd., London). Briefly, 75 mg of elastin ground
to <100 mesh were dispersed in 7.5 ml of 100 mM Tris, 5 mM CaCl2, pH 8.0, and maintained under constant
mechanical stirring for 1 h at 37 °C. Human leukocyte elastase
(37.5 µg) was added, and the flasks were incubated for 1 h or
24 h at 37 °C. The reaction was stopped by the addition of 1 mM phenylmethanesulfonyl fluoride. Elastin dispersions were
then centrifuged at 12,000 × g for 15 min.
Supernatants were removed, and pellets were washed three times with 10 ml of distilled water, lyophilized, and weighed. Due to the low Tyr
content of elastin, the protein content of supernatants (hydrolysates)
was determined (28) using
Lactose, interleukin-1 Peptides Synthesis--
The synthetic peptides VGVAPG, GVAPGV,
VAPGVG, APGVGV, PGVGVA, GVGVAP, PGAIPG, and LGTIPG were purchased from
Ansynth Service B.V. (Roosendaal, The Netherlands) or synthesized
according to classical solid phase synthesis (10, 22). Purity of the
peptides was confirmed by high performance liquid chromatography and by fast atom bombardment mass spectrometry.
Cell Culture--
Human skin fibroblast strains were established
from explants of human adult skin biopsies obtained from informed
healthy volunteers (age 21-41 years). Cells were grown as monolayer
cultures in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum and 2 mM glutamine in the presence of 5%
CO2. Cells at subcultures 5 to 10 were used. Fibroblasts
were grown to subconfluency in 10% serum containing medium. Cell
cultures were washed twice with phosphate-buffered saline and incubated
for 6 or 24 h in serum-free medium with or without elastin-like
material in the presence or absence of lactose and different
cell-signaling effectors. The culture supernatant was then harvested,
and cellular debris were pelleted (500 × g, 10 min,
22 °C).
Collagen Degradation--
Petri dishes were coated with 150 µg
of 3H-radiolabeled type I collagen from rat tail tendon in
18 mM acetic acid (24000 cpm/100 µg collagen). Human skin
fibroblasts were grown on this collagen matrix in a medium supplemented
with 1% fetal calf serum in the presence or absence of kE (50 µg/ml)
and/or plasmin (0.48 units/ml). After 24 h, the culture
supernatant was harvested, and its radioactivity was measured.
Western Blot Analysis--
After medium concentration, the
individual fractions were adjusted to the same protein concentration,
electrophoresed in a 0.1% SDS-10% polyacrylamide gel under reducing
conditions, and transferred onto Immobilon-P membranes (Millipore,
Saint-Quentin-en-Yvelines, France). The membranes were saturated with
5% calf serum, 0.1% Tween 20 in Tris-buffered saline for
2 h, incubated for 1 h with sheep polyclonal anti-human MMP-1
or rabbit polyclonal anti-human MMP-3 antibodies, and then incubated
with the alkaline phosphatase-conjugated anti-sheep or anti-rabbit
antibodies for 1 h at room temperature. Immuno complexes were
visualized with nitro blue tetrazolium 5-bromo-4-chloro-3-indolyl phosphate reagent. The molecular masses corresponding to the stained bands were determined, and they were further quantified by densitometry with the Bio1D software (Vilber-Lourmat, Marne-la-Vallée,
France). Linear range of intensity of the bands was assessed using
purified pro-MMP-1 or pro-MMP-3 as standard. Linearity was between 10 and 200 ng of enzyme.
Northern Blot Analysis--
Confluent cultures were washed twice
with serum-free medium and then incubated under serum-free conditions
for 2 h. Subsequently, elastin-like material was added to the
medium, and incubation was continued for 6 or 24 h. The cultures
were then washed with phosphate-buffered saline, and total RNA was
extracted from fibroblasts as described (29).
After washing, the blots were exposed to Kodak X-Omat film at
CD Spectroscopy--
The CD spectra were recorded in 0.1-cm path
length cylindrical cells on a JASCO J-810 dichrograph by averaging
three consecutive scans. The samples were dissolved in water at
concentrations ranging from 1 to 3 × 10 Structural Predictions--
The Statistical Analysis--
Experiments were performed in
triplicate. Results are expressed as means ± S.E. Differences
between control means and treated groups were assessed using the
unpaired Student's t test.
Elastin and Elastin Peptides Up-regulate Pro-MMP-1 Production and
Expression by Fibroblasts--
As previously reported (33), human skin
fibroblasts in culture produced a low level of pro-MMP-1 (Fig.
1; Control). The two
immuno-reactive species had an apparent molecular mass of 57 and 53 kDa
and corresponded to the glycosylated and nonglycosylated pro-MMP-1
isoforms, respectively. We found that the level of pro-MMP-1 was
considerably enhanced when cells were incubated in the presence of 50 µg/ml elastin for 24 h (Fig. 1; Elastin). Incubation
of fibroblasts, with the supernatant withdrawn after a 24-h hydrolysis of elastin by elastase (50 µg of elastin-derived peptides/ml), resulted in a 2-fold stronger stimulation (Fig. 1; HLE
lysate). The corresponding insoluble elastin pellet had lost this
potentiality (Fig. 1; HLE pellet), suggesting that the
elastin peptides promoting pro-MMP-1 production were totally released
from insoluble elastin by HLE.
Interestingly, tropoelastin, kE, and the VGVAPG synthetic peptide could
also stimulate pro-MMP-1 production (Fig. 1). The fact that
tropoelastin also exhibited an effect excluded the possibility that
elastin-cross-linked regions could be involved. Consequently, the
strong stimulation observed after incubation with kE (Fig. 1), a
mixture of elastin-derived peptides of a diverse length, particularly
rich in fragments originating from the hydrophobic domains of
tropoelastin, was explained. As VGVAPG proved an efficient stimulator
of pro-MMP-1 production (Fig. 1), we hypothesized that this sequence
and similar ones could be responsible for pro-MMP-1 up-regulation.
The kE-induced pro-MMP-1 production stimulation was correlated with
enhanced expression of MMP-1 mRNA levels (Fig.
2), suggesting that elastin peptides
up-regulated MMP-1 at the expression level. Standardization of data
using a 36B4 cDNA probe (Fig. 2) demonstrated that, after 24 h
of culture, MMP-1 mRNA levels were increased 8-fold.
We point out here that although elastin, elastolysate, tropoelastin,
and kE concentrations as low as 50 µg/ml proved sufficient to
stimulate pro-MMP-1 production, comparable stimulation levels could
only be reached with 200 µg/ml VGVAPG, a major ligand domain of the
elastin receptor. Although this concentration was 3-4 orders of
magnitude higher than the one required for some other elastin peptide-induced activities such as chemotaxis, the used concentration range was similar with those needed for enzyme excretion (34) and
proliferation (15).
The 67-kDa EBP Mediates the Effect of Elastin Peptides on Pro-MMP-1
Production by Fibroblasts--
Elastin peptides bearing the VGVAPG
sequence have been shown as a principal ligand of the 67-kDa EBP (35).
It has also been established that the EBP interaction with this
elastin-derived domain was only possible in the absence of
galactosugars, which otherwise may bind to a separate galactolectin
binding domain of the EBP and make this molecule unreceptive for
elastin. Thus, the addition of such galactosugar-bearing moieties as
lactose blocks the specific interaction between elastin peptides and
the EBP (5).
Indeed, the addition of 1 mM lactose to the fibroblast
culture medium resulted in a substantial (35%) inhibition of
kE-stimulated pro-MMP-1 production (Fig.
3). In the same conditions,
VGVAPG-stimulating effect was inhibited by 80% (Fig. 3). These data
strongly suggested that binding of VGVAPG on the 67-kDa EBP could
explain pro-MMP-1 up-regulation. It needs to be emphasized, however,
that stimulation of pro-MMP-1 by interleukin-1
An additional experiment aiming at the elucidation of the signaling
pathways triggered upon interaction between elastin peptides and EBP
has also been carried out. Several inhibitors of
EBP-dependent intracellular signaling have been tested. The
results listed in Table I clearly
indicate the involvement of a pertussis toxin-sensitive G protein,
phospholipase C, and protein kinase C (but not phospholipase D and
protein-tyrosine kinase) in the intracellular signaling pathways
leading to pro-MMP-1 up-regulation after exposure to elastin-derived
ligands. These results further implicate involvement of the EBP in the
signaling pathways leading to up-regulation of MMP-1 and are consistent
with the previously described EBP-dependent signaling
during elastin peptide-stimulated chemotaxis of leukocytes (17). It
must also be stressed that elastin peptide-dependent induction of pro-MMP-1 could not be blocked by an interleukin-1 receptor antagonist (Table I).
Peptides Containing the GXXPG Consensus Sequence Up-regulate
Pro-MMP-1--
The multiple hydrophobic VGVAPG sequences occur
exclusively in tropoelastin region encoded by exon 24 (36). In bovine
tropoelastin, it repeats twice, and in human tropoelastin, it repeats
six times (37). Since the synthetic peptide reflecting VGVAPG sequence proved so efficient in stimulation of pro-MMP-1 production (Fig. 1), we
also tested whether other domains bearing a similar conformation could
evoke similar cellular effects.
Peptides corresponding to circular permutation of the VGVAPG sequence
(VGVAPG, GVAPGV, VAPGVG, APGVGV, PGVGVA, GVGVAP) were used to identify
the precise sequences and/or motifs involved in pro-MMP-1
up-regulation. Two other 67-kDa EBP binding peptides were also used for
comparison: PGAIPG and LGTIPG, from elastin (9) and laminin B1 chain
(35), respectively. As presented in Fig.
4, only those peptides bearing the
GXXPG sequence could induce pro-MMP-1, suggesting that this
consensus motif was important for correct binding to the EBP. VGVAPG
induced pro-MMP-1 to a substantial level (1 ng/h/105
fibroblasts), and GVAPGV was 20% more efficient. The MMP-1 stimulation efficiency was GVAPGV > VGVAPG > LGTIPG > PGAIPG,
suggesting that the nature of the residues found at X
positions in GXXPG could determine the affinity of the
peptide pattern for binding to EBP. Apolar side chains seemed to be
preferred but not required, as demonstrated by the presence of a Thr
residue in LGTIPG.
Pro-MMP-1 Up-regulation by EDPs Depends on the Peptide
Conformation--
To understand the importance of the GXXPG
sequence for EBP binding, the conformation of the peptides was
investigated using CD spectroscopy. Our CD spectra were in excellent
agreement with those obtained by others for similar peptides (12, 38).
They were characterized by a dominant negative band centered around 200 nm, commonly associated with the
Indeed, two groups could be distinguished within the spectra of active
peptides: LGTIPG and PGAIPG exhibited a negative maximum around 196 nm
and a shoulder at 220 nm, whereas those of VGVAPG and GVAPGV yielded a
highly symmetric minimum around 200 nm. Interestingly the most active
peptides were from the second group. These findings suggested that
activity of the peptides originated from the presence of several folded
peptides within a population of unordered conformers.
Our prediction data (Table II) suggested
that all the active peptides contained a type VIII
Although a type VIII
The CD spectra of peptides could not be interpreted in terms of the
possible presence of type VIII Elastin Peptides Up-regulate Pro-MMP-3, a Potential Activator of
Pro-MMP-1--
To further explore the physiopathological significance
of pro-MMP-1 stimulation by elastin peptides, we investigated whether the EBP-dependent cellular response would also involve
activation of up-regulated pro-MMP-1 to MMP-1. Since pathways of
pro-MMP-1 activation involve MMP-3 and/or the plasmin system (40), we also tested these possibilities.
Western blotting indicated that fibroblasts stimulated either with kE
or with VGVAPG significantly up-regulated expression of proteins
reacting with anti-MMP-3 antibody. Two immuno-reactive bands
corresponding to pro-MMP-3 glycosylated (60 kDa) and nonglycosylated (57 kDa) isoforms were observed and the accumulation of pro-MMP-3 in
the medium was decreased when cells were treated with 1 mM lactose (Fig. 7). Furthermore the
appearance of these pro-MMP-3 bands was consistent with an increased
MMP-3 gene expression in kE-stimulated fibroblasts (Fig.
8). The demonstrated up-regulation of
MMP-3 expression was strikingly parallel to that observed for MMP-1,
suggesting that both MMP-1 and MMP-3 genes were coregulated by elastin
peptides.
The Up-regulation of Pro-MMPs by Elastin Peptides Can Lead to
Collagenolysis--
To further justify the pathophysiological
relevance of the observed overexpression of both pro-MMPs in cells
stimulated by the elastin-derived peptides, the involvement of plasmin
was additionally tested. Fibroblasts were cultured on a layer of
radiolabeled type I collagen in the presence or absence of kE and/or
plasmin to detect collagenolysis. Activation of pro-MMP-1 to MMP-1 was
simultaneously monitored by detection of the active form of the enzyme
by Western blots and by its ability to degrade radiolabeled collagen
(Fig. 9).
Our data indicate that the addition of plasmin to the culture media
lead to activation of all secreted pro-MMP-1 to MMP-1 (48 and 42 kDa)
in both untreated and kE-stimulated fibroblasts. A similar activation
pattern was observed for MMP-3 (data not shown). Apparently, in
cultures of unstimulated fibroblasts, the basic level of detected MMPs
was not sufficient to up-regulate a basic level of collagenolysis. In
kE-stimulated fibroblasts, the addition of plasmin triggered a massive
activation of up-regulated pro-MMP-1 to a collagenolytic enzyme (Fig.
9).
It has been shown previously that interaction of
elastin-derived peptides with the cell surface EBP leads to
modulation of diverse gene expression and multiple cellular effects
(17, 20). Our data demonstrate that tropoelastin and elastin
degradation products are potent inducers of collagenolytic enzyme
expression in human skin fibroblasts. Interestingly, such an
effect was attained even at elastin-derived peptide concentrations
close to those determined in physiological fluids (41) and potentiated
at higher concentrations, often detected during inflammatory processes
(33). The fact that this effect could be largely inhibited in the
presence of lactose and reproduced by stimulation with VGVAPG and other peptides bearing the GXXPG consensus sequence suggests
involvement of the EBP in signaling triggering pro-MMP-1 and pro-MMP-3
up-regulation.
Among elastin-derived peptides, only those containing the
GXXPG conformation have been identified as the ligands of
the cell surface EBP and stimulators of numerous cellular effects (9). Our present results broaden the range of the biological activity of
those peculiar peptides and provides a molecular explanation of their
binding to the cell surface receptor.
The assembly of tropoelastin into mature elastic fibers is also
directed by the EBP (42). Therefore, it could be assumed that neither
tropoelastin bound to this chaperone protein during its secretion from
cells nor insoluble elastin surrounded in tissues by a mantle of
microfibrils (43) could interact directly with cells under
physiological conditions.
The VGVAPG cell recognition domains are accessible on the surface of
growing elastic fibers as shown using specific monoclonal antibodies
(44). In mature fibers, however, these hydrophobic sequences remain
probably masked and simply contribute to the global elasticity of the
polymer. However, after tissue injury and release of potent elastolytic
enzymes by leukocytes, these domains could be unmasked and/or cleaved
of the elastin polymer so that they could bind to the EBP of the
adjacent cells.
In fact, we demonstrated that peptides capable of interaction
with the EBP can be released from the elastin network by HLE. We
therefore suggest that these peptides bound to the EBP and triggered a
pathway of intracellular signals leading to the described up-regulation
of pro-MMP-1 and pro-MMP-3 production.
Matrix proteins like elastin, laminins, collagens, fibrillins, or
fibronectin contain several GXXPG consensus sequences. As seen here, the stimulation of MMP-1 expression could also be achieved using the laminin-derived LGTIPG peptide. Therefore, it is reasonable to assume that small peptides bearing GXXPG sequences,
released from other matrix proteins could also interact with the cell
surface EBP. We therefore propose that peptides bearing
GXXPG conformation, regardless their origin, could serve as
stimulators of pro-MMP-1 production once released. This seems to be
particularly relevant to inflammatory processes in which HLE and other
proteinases released by infiltrating leukocytes could degrade elastin
and other matrix proteins, leading to accumulation of peptides, which
in turn could interact with EBP and lead to the local accumulation of
pro-MMP-1 and its activator pro-MMP-3. Since these pro-enzymes could be activated to active proteinases by plasmin (40, 45), degradation of
local collagen could follow. In summary, we suggest that initial elastolysis could lead to a consequent degradation of collagen and
other matrix components. This phenomenon could play an important part
in the mechanisms controlling connective tissue remodeling during
normal and/or pathological processes.
-turn in several similar, but not identical, peptides that
maintain a sufficient conformation to be recognized by the elastin
receptor. We have also established that GXXPG
elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation
of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we
found that the presence of plasmin in the culture medium activated
these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of
inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after
leukocyte-dependent proteolysis) bind to the elastin
receptor of local fibroblasts and trigger signals leading to expression
and activation of MMP-1 and MMP-3, which in turn exacerbate local
connective tissue damage.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (5). The binding of
elastin peptides to the elastin-binding protein (EBP)1 has
been shown to be responsible for chemotaxis to the peptides (6-12),
stimulation of cell proliferation (13-16), ions flux modifications (17, 18), vasorelaxation (19-22), and enzymes secretion (23, 24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Elastin was prepared from insoluble bovine elastin purified from
calf ligamentum nuchae using the hot alkali procedure (27).
-elastin as standard rather than bovine
serum albumin.
, D-609 (tricyclodecan-9-yl-xanthogenate,
potassium) and alkaline phosphatase-conjugated anti-sheep antibody were
from Sigma. Pertussis toxin, cholera toxin, RO 31-8220, plasmin,
pro-MMP-1, pro-MMP-3, and sheep polyclonal anti-human MMP-1 antibody
were obtained from Calbiochem. Rabbit polyclonal anti-human MMP-3
antibody came from Valbiotech (Paris, France). Other reagents were from
Life Technologies, Inc.
80 °C using intensifying screens. The human 2.1-kilobase MMP-1 cDNA and 1.9-kilobase MMP-3 cDNA probes were kind gifts from
Dr. Angel (Deutsches Krebsforschungs-zentrum, Heidelberg,
Germany) and Dr. Saus (Valencia Foundation of Biomedical
Investigations, Valencia, Spain), respectively. The radioactive bands
were quantified by densitometry and normalized using the human
1.06-kilobase 36B4 cDNA probe generously provided by Prof. Chambon
(University of Strasbourg, Strasbourg, France). 36B4 is a reporter gene
encoding the human acidic ribosomal phosphoprotein PO (30).
3 M. The data are presented in
terms of mean residue molar ellipticity expressed in deg
cm2 dmol
1 in the 185-250-nm
spectral range.
-turns propensities
(Pt) have been calculated using our software COUDES
(TURNS).2 They represented
the propensity (31) for a tetrapeptide to belong to a particular type
of
-turn. If Pt > 1, the tetrapeptide is considered as a
probable turn; if Pt < 1, the tetrapeptide is not taken as a
turn. The calculation of Pt is based on the simple product of
four individual residue propensities. These residue propensities,
defined for each type of turn and taking into account the location of
the residue in the turn (i.e. the position 1, 2, 3, or 4),
were determined from a reference set of 205 known three-dimensional
structures of proteins (32).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Influence of elastin material on the
production of pro-MMP-1 by human skin fibroblasts in culture.
Western blots analyses of pro-MMP-1 production after a 24-h incubation.
Elastin, 50 µg of insoluble elastin/ml; HLE
lysate, supernatant of elastin digestion by HLE after 24 h
(50 µg of elastin peptides/ml); HLE pellet, pellet of
elastin digestion by HLE after 24 h (50 µg of remaining
insoluble elastin/ml); TE, tropoelastin (50 µg/ml); kE,
-elastin (50 µg/ml); VGVAPG, 200 µg of VGVAPG/ml. Statistically
significant differences between stimulated and control are indicated
(*, p < 0.01).
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Fig. 2.
Influence of kE on MMP-1 mRNA levels in
human skin fibroblasts. Northern blot analysis of 20 µg of total
RNA from unstimulated fibroblasts at 6 h (control
6h) or 24 h (control 24h) and treated with
50 µg of kE/ml for 6 h (kE 6h) or 24 h (kE
24h). Histograms of the ratio MMP-1 mRNA versus
mRNA from the constitutive 36B4 gene is provided. Statistically
significant differences between stimulated and control are indicated
(*, p < 0.01).
could not be
blocked by lactose, and lactose alone had no effect on pro-MMP-1
accumulation (data not shown).
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Fig. 3.
Western blot analysis with anti-pro-MMP-1
antibody shows that 1 mM lactose inhibits the kE- and
VGVAPG-induced production of pro-MMP-1 in a 24-h culture of human skin
fibroblast production after a 24-h incubation. -Elastin
concentration was 50 µg/ml.
Exploration of signal transduction pathways leading to the
elastin-mediated MMP-1 induction by cultured human skin fibroblasts
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Fig. 4.
Influence of elastin- and laminin-derived
peptides on the production of pro-MMP-1 by human skin fibroblasts.
Western blots analyses of pro-MMP-1 production after a 24-h incubation.
All synthetic peptides were used at 200 µg/ml. Statistically
significant differences between stimulated and control are indicated
(*, p < 0.01; **, p < 0.001).
-
* electronic transition of
disordered peptides (Fig. 5).
Nevertheless, two spectral groups could be defined: one in which the
negative band was centered around 195 nm (filled symbols)
and another exhibiting a band around 200 nm (open symbols).
Strikingly, the spectra of peptides that did not enhance pro-MMP-1
production all belonged to the first group, whereas those of active
peptides were in the second one. This separation was underlined by the
consistency of group 1 minimal intensities, about
9000 deg
cm2 dmol
1 (Fig. 5) as compared with those of the
second group. These results suggested that the intensity of pro-MMP-1
production to the tested peptides could be
conformational-dependent, although those peptides were
substantially unordered.
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Fig. 5.
CD spectra of the elastin synthetic peptides
in aqueous solution. The filled symbols correspond to
spectra of peptides unable to induce pro-MMP-1 production.
-turn (39) in the
GXXP sequence (Fig. 6). The
glycyl residue after the GXXP turn was also necessary for
the biological activity, since GVGVAP was inactive even if it comprised
the GXXP sequence.
Propensities for -turns within elastin- and laminin-derived
hexapeptides
-turns occurring in active peptides are shown in bold
face. In these cases, the GXXP pattern is always followed by
a glycyl residue.
, propensity is lower than
one.
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Fig. 6.
The type VIII -turn
conformation proposed for the active peptides, as applied to the
sequence GVAP. C
carbons are shown as black
spheres, and the backbone bending is depicted as a gray
ribbon.
-turn might be adopted by the active peptides,
it must be stressed that the peptides consist of only six
residues and were thus very flexible. Therefore, they could form a type
VIII
-turn but probably not as their dominant conformation. The fact
that a type VIII
-turn together with the PG sequence was a common
feature of the biologically active hexapeptides indicates that this
conformation could well constitute the dominant structure needed for
the binding to their target, i.e. cell surface EBP.
-turns because, to our knowledge, CD
spectra concerning this particular class of
-turns were not
available. However, considering that the essential dihedrals (i.e. those pertaining to residues i+1 and i+2,
respectively) were in the right-handed helical and
regions of
a Ramachadran plot, one might suggest that the type VIII
-turn
should give dominant negative contribution to the CD spectrum at
wavelengths longer than 190 nm. In the case of the active peptides,
their CD spectra were fully compatible with the presence of type VIII
-turn and open (unordered) conformations.
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Fig. 7.
Western blot analysis with anti-pro-MMP-3
antibody indicates that 50 µg/ml kE and
200 µg/ml VGVAPG peptides induce pro-MMP-3
production in 24-h cultures of human skin fibroblasts. The effect
was partially blocked in cultures treated with 1 mM
lactose.
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Fig. 8.
kE induces MMP-3 gene expression.
Northern blots analyses of 20 µg of total RNA from unstimulated
fibroblasts at 6 h (control 6h) or 24 h
(control 24h) and treated with 50 µg of kE/ml for 6 h
(kE 6h) or 24 h (kE 24h). The corresponding
levels of the constitutive 36B4 gene mRNA are provided.
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Fig. 9.
The induction of pro-MMP-1 production by kE
(50 µg/ml) can lead to collagenolysis.
Upper panel, Western blot analysis of MMP-1 production after
incubation with kE in the presence or absence of plasmin (0.48 units/ml). Lower panel, levels of radioactive collagen
released to the conditioned media of human skin fibroblasts cultured
for 24 h on the top of radiolabeled collagen substrate. The
control value represents cultures performed in the absence of both
plasmin and kE. Statistically significant differences between
stimulated and control are indicated (*, p < 0.01).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank F. Charton, L. Rittié, and M. Decarme for their skillful technical assistance and Dr. A. Hinek (Hospital for Sick Children, Toronto, Canada) for helpful advice and assistance with English.
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FOOTNOTES |
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* This work was supported by grants from the Association Régionale pour l'Enseignement et la Recherche Scientifique et Technologique (ARERS) and the Région Champagne-Ardenne (to W. H.), by an Association pour la Recherche sur le Cancer (ARC) fellowship (to B. B.), by a grant from the Ligue contre le Cancer (to H. E.), and by CNRS (UPRES-A 6021).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: FRE CNRS 2260, IFR53 Biomolécules, Laboratory of Biochemistry, Faculty of Sciences, University of Reims Champagne-Ardenne, 51687 Reims Cedex 2, France. Tel.: 33 3 26 91 34 35; Fax: 33 3 26 91 31 68; E-mail: laurent.debelle@univ-reims.fr.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M003642200
2 P. Fuchs and A. J. P. Alix, article in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
EBP, elastin-binding
protein;
CD, circular dichroism;
HLE, human leukocyte elastase;
kE, -elastin;
MMP, matrix metalloproteinase;
MMP-1, collagenase-1 or
matrix metalloproteinase-1;
MMP-3, stomelysin-1 or matrix
metalloproteinase-3.
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REFERENCES |
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