Conformational dependence of collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in cultured fibroblasts.

Abstract

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 beta-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.

Full text

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Conformational Dependence of Collagenase (Matrix
Metalloproteinase-1) Up-regulation by Elastin Peptides
in Cultured Fibroblasts*
Received for publication, April 28, 2000, and in revised form, November 9, 2000
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M003642200
Bertrand Brassart‡, Patrick Fuchs§, Eric Huet‡, Alain J. P. Alix§, Jean Wallach¶, Antonio M.
Tamburro?, Fre ´de ´ric Delacoux‡, Bernard Haye‡, Herve ´ Emonard‡, William Hornebeck‡, and
Laurent Debelle‡**
From the ‡UPRES-A CNRS 6021, IFR53 Biomole ´cules, Faculties of Sciences and Medicine and the §Laboratory of
Biomolecular Spectroscopies and Structures, IFR53 Biomole ´cules, 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
We have established that treatment of cultured hu-
man skin fibroblasts with tropoelastin or with hetero-
genic peptides, obtained after organo-alkaline or leuko-
cyte elastase hydrolysis of insoluble elastin, induces a
high expression of pro-collagenase-1 (pro-matrix metal-
loproteinase-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-regula-
tion has not been demonstrated in cultures preincu-
bated 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 pro-
tein 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 stud-
ies suggest that this peculiar consensus sequence stabi-
lizes a type VIII ?-turn in several similar, but not iden-
tical, peptides that maintain a sufficient conformation
to be recognized by the elastin receptor. We have also
established that GXXPG elastin-derived peptides, in ad-
dition 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 re-
sults may be, therefore, relevant to pathobiology of in-
flammation, in which elastin-derived peptides bearing
the GXXPG conformation (created after leukocyte-de-
pendent 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 exacer-
bate local connective tissue damage.
The extracellular matrix protein elastin is responsible for the
elastic properties of tissues such as lung, skin, and large arter-
ies (1–3). Due to its numerous cross-links and the extreme
hydrophobicity of its tropoelastin chains, elastin is highly re-
sistant to proteolysis. However, during inflammatory disor-
ders, proteinases secreted from polymorphonuclear neutro-
phils, 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 ?-galactosidase (5). The binding of elastin peptides to
the elastin-binding protein (EBP)1has been shown to be re-
sponsible 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).
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). At-
tracted 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 secre-
tion 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 or-
gano-alkaline- and human leukocyte elastase (HLE)-dependent
elastin degradation. Our data suggest that among many elas-
* This work was supported by grants from the Association Re ´gionale
pour l’Enseignement et la Recherche Scientifique et Technologique
(ARERS) and the Re ´gion Champagne-Ardenne (to W. H.), by an Asso-
ciation 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. This 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 Biomole ´cules, 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.
1The abbreviations used are: EBP, elastin-binding protein; CD, cir-
cular dichroism; HLE, human leukocyte elastase; kE, ?-elastin; MMP,
matrix metalloproteinase; MMP-1, collagenase-1 or matrix metallopro-
teinase-1; MMP-3, stomelysin-1 or matrix metalloproteinase-3.
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 276, No. 7, Issue of February 16, pp. 5222–5227, 2001
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
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tin-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 collag-
enolysis during normal and/or pathological conditions.
EXPERIMENTAL PROCEDURES
Samples Preparation and Reagents—Bovine tropoelastin was ob-
tained from Elastin Products Co. (Universal Biological Ltd., London).
?-Elastin was prepared from insoluble bovine elastin purified from calf
ligamentum nuchae using the hot alkali procedure (27).
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 ?g) 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 ?-elastin as standard rather than bovine serum albumin.
Lactose, interleukin-1?, D-609 (tricyclodecan-9-yl-xanthogenate, po-
tassium) 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 an-
tibody were obtained from Calbiochem. Rabbit polyclonal anti-human
MMP-3 antibody came from Valbiotech (Paris, France). Other reagents
were from Life Technologies, Inc.
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 chromatog-
raphy 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 ab-
sence of lactose and different cell-signaling effectors. The culture su-
pernatant was then harvested, and cellular debris were pelleted (500 ?
g, 10 min, 22 °C).
Collagen Degradation—Petri dishes were coated with 150 ?g of3H-
radiolabeled type I collagen from rat tail tendon in 18 mM acetic acid
(24000 cpm/100 ?g 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 ?g/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, electrophore-
sed in a 0.1% SDS-10% polyacrylamide gel under reducing conditions,
and transferred onto Immobilon-P membranes (Millipore, Saint-Quen-
tin-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 phos-
phatase-conjugated anti-sheep or anti-rabbit antibodies for 1 h at room
temperature. Immuno complexes were visualized with nitro blue tetra-
zolium 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 (Vil-
ber-Lourmat, Marne-la-Valle ´e, France). Linear range of intensity of the
bands was assessed using purified pro-MMP-1 or pro-MMP-3 as stand-
ard. 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
?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, Va-
lencia, 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).
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 con-
centrations ranging from 1 to 3 ? 10?3M. The data are presented in
terms of mean residue molar ellipticity expressed in deg cm2dmol?1in
the 185–250-nm spectral range.
Structural Predictions—The ?-turns propensities (Pt) have been cal-
culated using our software COUDES (TURNS).2They 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 Ptis
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-dimen-
sional structures of proteins (32).
Statistical Analysis—Experiments were performed in triplicate. Re-
sults are expressed as means ? S.E. Differences between control means
and treated groups were assessed using the unpaired Student’s t test.
RESULTS
Elastin and Elastin Peptides Up-regulate Pro-MMP-1 Pro-
duction 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 corre-
sponded 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 ?g/ml elastin for 24 h (Fig. 1; Elastin). Incuba-
tion of fibroblasts, with the supernatant withdrawn after a
24-h hydrolysis of elastin by elastase (50 ?g of elastin-derived
2P. Fuchs and A. J. P. Alix, article in preparation.
FIG. 1. Influence of elastin material on the production of pro-
MMP-1 by human skin fibroblasts in culture. Western blots anal-
yses of pro-MMP-1 production after a 24-h incubation. Elastin, 50 ?g of
insoluble elastin/ml; HLE lysate, supernatant of elastin digestion by
HLE after 24 h (50 ?g of elastin peptides/ml); HLE pellet, pellet of
elastin digestion by HLE after 24 h (50 ?g of remaining insoluble
elastin/ml); TE, tropoelastin (50 ?g/ml); kE, ?-elastin (50 ?g/ml);
VGVAPG, 200 ?g of VGVAPG/ml. Statistically significant differences
between stimulated and control are indicated (*, p ? 0.01).
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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 elas-
tin peptides promoting pro-MMP-1 production were totally re-
leased 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 produc-
tion (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 cor-
related 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, tro-
poelastin, and kE concentrations as low as 50 ?g/ml proved
sufficient to stimulate pro-MMP-1 production, comparable
stimulation levels could only be reached with 200 ?g/ml
VGVAPG, a major ligand domain of the elastin receptor. Al-
though 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 bear-
ing 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-stim-
ulated 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? could not be blocked by lactose, and lactose
alone had no effect on pro-MMP-1 accumulation (data not
shown).
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 sig-
naling during elastin peptide-stimulated chemotaxis of leuko-
cytes (17). It must also be stressed that elastin peptide-depend-
ent 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 se-
quences 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 syn-
thetic 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
FIG. 2. Influence of kE on MMP-1 mRNA levels in human skin
fibroblasts. Northern blot analysis of 20 ?g of total RNA from un-
stimulated fibroblasts at 6 h (control 6h) or 24 h (control 24h) and
treated with 50 ?g 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 stimu-
lated and control are indicated (*, p ? 0.01).
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 fibro-
blast production after a 24-h incubation. ?-Elastin concentration
was 50 ?g/ml.
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VGVAPG sequence (VGVAPG, GVAPGV, VAPGVG, APGVGV,
PGVGVA, GVGVAP) were used to identify the precise se-
quences 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 lami-
nin 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/105fibroblasts), 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 sim-
ilar peptides (12, 38). They were characterized by a dominant
negative band centered around 200 nm, commonly associated
with the ?-?* 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 en-
hance 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 min-
imal intensities, about ?9000 deg cm2dmol?1(Fig. 5) as com-
pared with those of the second group. These results suggested
that the intensity of pro-MMP-1 production to the tested pep-
tides could be conformational-dependent, although those pep-
tides were substantially unordered.
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 pep-
tides 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 ?-turn (39) in the GXXP se-
quence (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.
Although a type VIII ?-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.
The CD spectra of peptides could not be interpreted in terms
of the possible presence of type VIII ?-turns because, to our
knowledge, CD spectra concerning this particular class of
?-turns were not available. However, considering that the es-
sential 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 pres-
ence of type VIII ?-turn and open (unordered) conformations.
Elastin Peptides Up-regulate Pro-MMP-3, a Potential Activa-
tor of Pro-MMP-1—To further explore the physiopathological
significance of pro-MMP-1 stimulation by elastin peptides, we
TABLE I
Exploration of signal transduction pathways leading to the elastin-
mediated MMP-1 induction by cultured human skin fibroblasts
Cells were preincubated with several inhibitors of intracellular sig-
naling for 3 h and then stimulated with kE (50 ?g/ml) for 24 h.
Effector pro-MMP-1Activity
ng/h/105cells
2.10 ? 0.31
%
None
G protein inhibitor
Cholera toxin (2.5 ?g/ml)
Pertussis toxin (100 ng/ml)
Phospholipase C inhibitor
D-609 (20 ?g/ml)
Protein kinase C inhibitor
RO 31–8220 (5 ?M)
Phospholipase D inhibitor
Propanol-1 (130 mM)
Interleukin-1 receptor antagonist
Interleukin-1ra (40 nM)
Protein-tyrosine kinase inhibitor
Herbimycin A (10 ?M)
100
2.51 ? 0.17
1.10 ? 0.12
119
52
0.10 ? 0.01 5
0.34 ? 0.0616
2.24 ? 0.16 106
2.03 ? 0.2496
2.37 ? 0.17113
FIG. 4. Influence of elastin- and laminin-derived peptides on
the production of pro-MMP-1 by human skin fibroblasts. West-
ern blots analyses of pro-MMP-1 production after a 24-h incubation. All
synthetic peptides were used at 200 ?g/ml. Statistically significant
differences between stimulated and control are indicated (*, p ? 0.01;
**, p ? 0.001).
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.
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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 expres-
sion of proteins reacting with anti-MMP-3 antibody. Two im-
muno-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 de-
creased 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-regula-
tion of MMP-3 expression was strikingly parallel to that ob-
served 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 pathophysiolog-
ical relevance of the observed overexpression of both pro-MMPs
in cells stimulated by the elastin-derived peptides, the involve-
ment of plasmin was additionally tested. Fibroblasts were cul-
tured on a layer of radiolabeled type I collagen in the presence
or absence of kE and/or plasmin to detect collagenolysis. Acti-
vation 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 fibro-
blasts. A similar activation pattern was observed for MMP-3
(data not shown). Apparently, in cultures of unstimulated fi-
FIG. 7. Western blot analysis with anti-pro-MMP-3 antibody
indicates that 50 ?g/ml kE and 200 ?g/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.
FIG. 8. kE induces MMP-3 gene expression. Northern blots anal-
yses of 20 ?g of total RNA from unstimulated fibroblasts at 6 h (control
6h) or 24 h (control 24h) and treated with 50 ?g of kE/ml for 6 h (kE 6h)
or 24 h (kE 24h). The corresponding levels of the constitutive 36B4 gene
mRNA are provided.
FIG. 9. The induction of pro-MMP-1 production by kE (50 ?g/
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).
TABLE II
Propensities for ?-turns within elastin- and laminin-derived
hexapeptides
The type VIII ?-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.
Tetrapeptidic
sequence
Type I Type II Type VIII Type I? Type II?
Inactive VAPGVG 1 VAPG
2 APGV
3 PGVG
1 APGV
2 PGVG
3 GVGV
1 PGVG
2 GVGV
3 VGVA
1 GVGV
2 VGVA
3 GVAP
—
1.03
—
1.03
—
—
—
—
—
—
—
—
——
—
—
—
—
—
—
—
—
—
—
3.17
—
—
1.74
—
2.65
—
2.51
—
2.56
—
5.75
—
5.75
—
52.95
—
52.95
—
4.24
—
4.24
—
4.24
—
—
APGVGV
—
—
—
—
—
—
—
—
—
PGVGVA
GVGVAP
Active
VGVAPG 1 VGVA
2 GVAP
3 VAPG
1 GVAP
2 VAPG
3 APGV
1 LGTI
2 GTIP
3 TIPG
1 PGAI
2 GAIP
3 AIPG
—
—
—
—
—
1.03
—
—
—
—
—
—
—
—
—
—
—
——
—
—
—
—
—
—
—
—
—
—
—
5.75
—
1.74
—
1.74
—
—
—
—
—
—
—
3.17
—
3.17
—
—
—
7.55
—
—
8.08
—
GVAPGV
52.95
—
—
—
—
—
—
LGTIPG
PGAIPG
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.
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Page 6

broblasts, 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 activa-
tion of up-regulated pro-MMP-1 to a collagenolytic enzyme
(Fig. 9).
DISCUSSION
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 prod-
ucts are potent inducers of collagenolytic enzyme expression in
human skin fibroblasts. Interestingly, such an effect was at-
tained even at elastin-derived peptide concentrations close to
those determined in physiological fluids (41) and potentiated at
higher concentrations, often detected during inflammatory pro-
cesses (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 trigger-
ing 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 mono-
clonal antibodies (44). In mature fibers, however, these hydro-
phobic sequences remain probably masked and simply contrib-
ute 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 de-
scribed 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. There-
fore, 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 inflam-
matory processes in which HLE and other proteinases released
by infiltrating leukocytes could degrade elastin and other ma-
trix proteins, leading to accumulation of peptides, which in
turn could interact with EBP and lead to the local accumula-
tion of pro-MMP-1 and its activator pro-MMP-3. Since these
pro-enzymes could be activated to active proteinases by plas-
min (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 compo-
nents. This phenomenon could play an important part in the
mechanisms controlling connective tissue remodeling during
normal and/or pathological processes.
Acknowledgments—We thank F. Charton, L. Rittie ´, 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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