From the Division of Developmental and Newborn Biology, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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An imbalance between proteases and antiproteases is thought to play a role in the inflammatory injury that regulates wound healing. The activities of some proteases and antiproteases found in inflammatory fluids can be modified in vitro by heparin, a mast cell-derived glycosaminoglycan. Because syndecans, a family of cell surface heparan sulfate proteoglycans, are the major cellular source of heparin-like glycosaminoglycan, we asked whether syndecans modify protease activities in vivo.
Syndecan-1 and syndecan-4 ectodomains are shed into acute human dermal
wound fluids (Subramanian, S. V., Fitzgerald, M. L., and
Bernfield, M. (1997) J. Biol. Chem. 272, 14713-14720). Moreover, purified syndecan-1 ectodomain binds cathepsin
G (Kd = 56 nM) and elastase
(Kd = 35 nM) tightly and reduces the
affinity of these proteases for their physiological inhibitors. Purified syndecan-1 ectodomain protects cathepsin G from inhibition by
1-antichymotrypsin and squamous cell carcinoma antigen 2 and elastase from inhibition by
1-proteinase inhibitor
by decreasing second order rate constants for protease-antiprotease
associations (kass) by 3700-, 32-, and 60-fold,
respectively. Both enzymatic degradation of heparan sulfate and
immunodepletion of the syndecan-1 and -4 in wound fluid reduce these
proteolytic activities in the fluid, indicating that the proteases in
the wound environment are regulated by interactions with syndecan
ectodomains. Thus, syndecans are shed into acute wound fluids, where
they can modify the proteolytic balance of the fluid. This suggests a
novel physiological role for these soluble heparan sulfate
proteoglycans.
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INTRODUCTION |
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Multiple factors orchestrate the inflammatory response to tissue injury. These include proteases, antiproteases, cytokines, chemokines, and the growth factors derived from the plasma and cells associated with the injury, as well as from cells invading the injury site (1). Emigrating polymorphonuclear leukocytes release proteolytic enzymes into the injury site, including the most potent serine proteases, neutrophil elastase and cathepsin G (CatG).1 These enzymes aid wound repair by digesting extracellular proteins, releasing growth factors from extracellular matrix, and remodeling the tissue (2-4). However, these enzymes can also destroy tissues when proteolysis is prolonged, inappropriate, or excessive (5, 6).
Enormous local concentrations of proteases, estimated to be in the millimolar range for elastase and cathepsin G, are released into extracellular spaces during the leukocyte activation associated with tissue injury (7). Serine protease inhibitors (serpins), provide efficient control mechanisms to prevent undesirable extracellular protein degradation at the injury site (8). These antiproteases, mostly derived from plasma, share three principal properties: (i) they form 1:1 covalent complex with proteases, (ii) complex formation results in both inactivation of the protease and proteolytic cleavage of the serpin, and (iii) inhibition is essentially irreversible (9). The balance of proteases and antiproteases at the site of injury can regulate the extent of the inflammatory response during the repair process (10, 11).
Dermal wound repair requires harmonious protease-antiprotease
interactions or proteolytic balance. Excessive elastase action in the
wound bed can account for endothelial damage (12), degradation of the
epidermal/dermal junction (13), and the development of chronic skin
ulcers (14). Physiological neutrophil elastase inhibitors include
plasma-derived 2-macroglobulin and, most importantly,
1-proteinase inhibitor (also known as
1-PI,
1-antitrypsin, or
1-AT). The importance of
1-PI in
regulating the response to tissue injury is emphasized by the extensive
elastin and collagen fiber destruction leading to pulmonary emphysema
in the lungs of individuals with congenital
1-PI
deficiency (5). The major physiological cathepsin G inhibitor is
1-antichymotrypsin (
1-Achy), another
plasma-derived serpin (15). Inherited
1-Achy deficiency is pleiomorphic, but it is often associated with chronic active hepatitis and increased residual lung volumes (16). Another serpin that
inhibits cathepsin G is the squamous cell carcinoma antigen 2 (SCCA2),
a newly described product of skin and respiratory tract epithelia
(17).
Although the activity of one class of serpins is accelerated by binding
to heparin or other glycosaminoglycans (GAGs) (9, 18),
1-PI and
1-Achy belong to the class of
serpins that function independently of heparin and other GAGs. However,
heparin can bind with high affinity to both neutrophil elastase and
cathepsin G (19, 20). This binding inhibits the enzymatic activities, but most importantly, it reduces the ability of the enzymes to interact
with serpins (19, 20). The heparin used clinically and in these studies
is a pharmaceutical product derived from processing of the heparin
proteoglycan within mast cells (21). The major physiological source of
the heparin-like GAG, heparan sulfate, is found in proteoglycans within
cells, at the cell surface and in the extracellular matrix (22).
Most cellular heparan sulfate derives from the syndecan family of cell surface proteoglycans. This family (currently known as syndecan 1-4 in mammals) consists of single transmembrane proteins containing conserved cytoplasmic and transmembrane domains and less well conserved extracellular domains (ectodomains), which bear variable numbers of GAG chains. All syndecans bear heparan sulfate, although syndecan-1 and -3 can also bear chondroitin sulfate. Syndecans bind many of the factors that orchestrate the inflammatory response to tissue injury as well as a variety of extracellular matrix components and adhesion molecules via their heparan sulfate chains and are individually expressed in distinct cell-, tissue-, and development-specific patterns (23).
Syndecan expression is highly regulated during wound repair. During cutaneous wound repair, keratinocytes migrating from the wound edge show loss of cell surface syndecan-1 (24). Concomitantly, syndecan-1 expression increases on the endothelial cells, and syndecan-4 expression increases on the dermal fibroblasts that form the granulation tissue (24, 25), apparently due to inductive action of neutrophil-derived antimicrobial peptides (26). Syndecans on cell surfaces can be cleaved near the plasma membrane, which releases the now soluble intact proteoglycan ectodomains into the surrounding milieu (27). This shedding is accelerated by activation of protease (e.g. thrombin) and growth factor receptors (epidermal growth factor family members) and by the direct action of proteases (e.g. plasmin) involved in wound repair (27). Moreover, soluble syndecan-1 and -4 ectodomains are detected in acute dermal wound fluids (27). Although syndecan expression and shedding are highly regulated during the response to tissue injury, the role these processes play in this response is not clear.
A key aspect of the response to tissue injury is the establishment and
maintenance of proteolytic balance at the wound site. The action of the
major proteases, neutrophil elastase and cathepsin G, must be countered
by their major inhibitors, 1-PI and
1-Achy, for normal wound repair to ensue. Loss of this
balance can prevent normal repair, potentially leading to chronic
wounds, which in the skin are difficult to treat satisfactorily (14).
We postulated that because activities of the major proteases in acute
wound fluids can be modified in vitro by heparin, soluble
syndecan ectodomains could be involved in establishing and maintaining
the proteolytic balance in wounds in vivo. We found
syndecan-1 and -4 ectodomains in acute human dermal wound fluids. We
also found that purified syndecan-1 ectodomain binds to both neutrophil
elastase and cathepsin G, markedly reducing their affinity for serpins
and thus protecting these enzymes from their physiological inhibitors.
Moreover, both degradation of endogenous heparan sulfate and removal of
syndecan-1 and -4 from wound fluids reduce proteolytic activities in
the fluid. Thus, syndecan ectodomains maintain the proteolytic balance in acute wound fluids, a novel physiological role for soluble heparan
sulfate proteoglycans.
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EXPERIMENTAL PROCEDURES |
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Materials--
Flat bottomed, low binding 96-well microtiter
plates were obtained from Costar (Cambridge, MA). Human trypsin and
mouse IgG were from Sigma, human neutrophil cathepsin G and human
plasma 1-PI were from Athens Research & Technology Inc.
(Athens, GA), human neutrophil elastase was from Calbiochem (La Jolla,
CA), and human plasma
1-Achy was from Biodesign
International (Kennebunk, ME). Purified glutathione
S-transferase-SCCA2 fusion protein was a kind gift from Dr.
Gary Silverman, Children's Hospital, Boston, MA (17). For enzyme
substrates, succinyl-Ala-Ala-Pro-Phe-para-nitroanilide (Suc-AAPF-pNA)
for cathepsin G and N-benzoyl-Phe-Val-Arg-pNA for trypsin
were from Sigma, and (benzyloxycarbonyl
(CBZ)-Ala-Ala-Ala-Ala)2-R110 for elastase was from
Molecular Probes Inc. (Eugene, OR). Heparin (porcine intestinal mucosa)
was from Hepar Industries Inc. (Franklin, OH), and chondroitin-6
sulfate, chondroitin sulfate ABC lyase (chondroitinase ABC, EC
4.2.2.4), heparan sulfate lyase III (heparitinase, EC 4.2.2.8), and
heparan-sulfate lyase I (heparinase, EC 4.2.2.7) were from Seikagaku
America Inc. (Rockville, MD). The syndecan-1 ectodomain was purified to
homogeneity from the conditioned medium of NMuMG mouse mammary
epithelial cells (28). One mg of this syndecan-1 core protein contains
5 mg of HS.2 cDNA for
ectodomains of human syndecan-1 and -4 in glutathione S-transferase expression vector pGEX-2T (Amersham Pharmacia
Biotech) were expressed as fusion proteins in Escherichia
coli, induced by 0.2 mM
isopropyl-1-thio-
-D-galactopyranoside for 6 h at
37 °C, solubilized with 1% Triton X-100, and centrifuged
12,000 × g for 10 min. Supernatants were purified on
glutathione-agarose beads (Sigma); washed with PBS; eluted with 50 mM Tris, pH 8, and 5 mM reduced glutathione
(Janssen Chimica, New Brunswick, NJ); subjected to 10%
SDS-polyacrylamide gel electrophoresis; and detected with Coomassie
Blue. Antibodies used were polyclonal antisera HSE-1 against the
recombinant human syndecan-1 ectodomain (25); monoclonal antibodies
MCA-681 from Serotec (Oxford, United Kingdom) and DL-101 against human syndecan-1, 5G9 and 8C7 (25) against human syndecan-4, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and horseradish peroxidase-conjugated anti-mouse IgG were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA).
Affinity Co-electrophoresis (ACE) Analyses-- NMuMG cells were labeled with radiosulfate, and [35S]sulfate-labeled syndecan-1 ectodomain from conditioned medium was purified by DEAE and immunoaffinity chromatography (28). Binding of this ectodomain to elastase and cathepsin G was assessed by ACE as described elsewhere (31, 32). Briefly, 1% (w/v) low melt agarose gels were cast containing distinct lanes with various concentrations of protease (indicated in Fig. 1). [35S]Sulfate-labeled syndecan-1 ectodomain (12,500 cpm) was electrophoresed through these lanes. In competition assays, the same amount of syndecan-1 was mixed with 1 mg/ml chondroitin-6 sulfate or heparin prior to electrophoresis. The migration on syndecan-1 was detected on a PhosphoImager (Molecular Dynamics, Sunnyvale, CA). The pixel intensities were integrated and used to determine the migration distance of the major peak of 35S-labeled syndecan-1 in each protease-containing lane. These mobilities were plotted as a function of ligand concentration and used to estimate the apparent Kd values as described earlier (32).
Assays for Enzyme Inhibition--
The amounts of proteases and
serpins were calibrated by the method of Chase and Shaw (33). Trypsin
was calibrated by using p-nitrophenyl-p'-guanidinobenzoate (Sigma),
except that 100 mM Tris-HCl, pH 8.3, was used in place of
sodium barbiturate buffer. The concentration of 1-PI was
standardized against calibrated trypsin. Elastase and cathepsin G were
calibrated against the standardized
1-PI.
1-Achy was calibrated against the standardized cathepsin
G. Reaction buffers were 50 mM Hepes, 150 mM
NaCl, 5% N,N-dimethylformamide, pH 7.4, for
cathepsin G, and 50 mM Tris, 150 mM NaCl, 0.1 mg/ml bovine serum albumin, pH 7.4, for elastase.
Protease-Serpin Binding Stoichiometry-- Constant concentrations of syndecan-1 ectodomain or heparin were preincubated with protease for 15 min at 25 °C with increasing concentrations of serpin in the appropriate reaction buffer, and the residual enzyme activities were measured with an appropriate substrate. The concentrations for cathepsin G assays were 34 nM cathepsin G and 3 mM Suc-AAPF-pNA in cathepsin G reaction buffer. The concentrations for elastase assays were 34 nM elastase and 5 µM (CBZ-Ala-Ala-Ala-Ala)2-R110 in elastase reaction buffer.
Determination of Rate Constant kass for Enzyme-Inhibitor Association-- The association rate constant for the interaction of serpins with free and syndecan-1-bound enzymes was determined under second order rate conditions (15). Equimolar amounts (34 nM) of enzyme with or without the syndecan-1 ectodomain and inhibitor were incubated at 25 °C for varying periods of time. The reaction was quenched by the addition of substrate, for which the enzyme has higher affinity, and the release of pNA or rhodamine was measured. The residual enzyme activities were used to calculate the concentration of free enzyme E. Protease standard curves used for calculation of free enzyme were done in the presence and absence of syndecan-1 ectodomain. The rate of change in the amount of free enzyme over time is described as
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(Eq. 1) |
Collection of Acute Wound Fluids--
Acute human dermal wound
fluids were collected from reduction mammoplasty patients (samples were
kindly provided by Dr. E. Eriksson, Brigham and Women's Hospital,
Boston, MA). Wound fluids were collected at 1-day intervals from
sterile closed-suction drains routinely placed in the subcutaneous
space after mammoplasty. After collection, fluids were centrifuged for
15 min at 200 × g and 4 °C to remove cells and
further for 15 min at 3300 × g and 4 °C to remove
debris. The supernatants were stored at 70 °C until use. Porcine
wound fluid was produced, collected, and processed as described
previously (26, 34).
Immunodetections-- Acute wound fluids collected from several patients were blotted on Immobilon-N membranes (Millipore, Bedford, MA) using a Dot Blot apparatus. Membranes were blocked for 60 min with 3% nonfat dry milk, 0.5% bovine serum albumin, 3% H2O2, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, and incubated for 60 min with polyclonal antisera HSE-1 for syndecan-1 and monoclonal antibody 5G9 for syndecan-4, followed by a 60-min incubation with horseradish peroxidase-conjugated anti-rabbit IgG or horseradish peroxidase-conjugated anti-mouse IgG, respectively. Syndecans were detected by ECL according to the manufacturer's instructions (Amersham Pharmacia Biotech).
Assays for Enzyme Activities in Wound Fluids-- Day 1 human wound fluids were treated with 150 milliunits/ml heparinase (Hase) and heparitinase (HSase) for 3 h at 37 °C in 50 mM Hepes, 150 mM NaCl, pH 7.4, to degrade endogenous heparan sulfate in the fluid. Five min after adding heparin (up to 5 µg/ml) to untreated or Hase/HSase-treated fluids, the chymotryptic and elastolytic activities in the samples were detected by adding the appropriate substrate (3 mM Suc-AAPF-pNA for chymotryptic and 5 µM (CBZ-Ala-Ala-Ala-Ala)2-R110 for elastolytic activities). Hydrolysis was measured over time at 405 nm with a UVmax plate reader (Molecular Devices) or at 488 nm with a FluorImager 575 (Molecular Dynamics).
Immunodepletion of Syndecan-1 and -4 Ectodomains from Wound Fluid-- Acute human dermal wound fluids precleared by centrifugation (0.5 ml) were sequentially incubated (at 4 °C for 30 min each) with a mixture of 20 µg/ml of each monoclonal antibody to syndecan-1 (DL101 and MCA-681) and syndecan-4 (5G9 and 8C7) or with 80 µg/ml of mouse IgG, 80 µg/ml rabbit anti-mouse IgG (Dako Corp., Carpinteria, CA), and protein A-Sepharose beads (Amersham Pharmacia Biotech). Beads were centrifuged, and supernatants were assayed for elastolytic activity in elastase reaction buffer by adding 5 µM (CBZ-Ala-Ala-Ala-Ala)2-R110 elastase substrate and measuring hydrolysis with time at 488 nm with a FluorImager 575 (Molecular Dynamics).
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RESULTS |
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Syndecan-1 Ectodomain Binds Elastase and Cathepsin G--
Because
heparin can protect elastase and cathepsin G against inhibition by
certain plasma-derived serpins (19, 20), we speculated that the soluble
syndecan ectodomains in wound fluid might act similarly.
[35S]Sulfate-labeled syndecan-1 ectodomain purified from
the conditioned media of NMuMG cells was incubated with nitrocellulose
filters containing dots of purified human neutrophil cathepsin G and
elastase, and serum-derived 1-PI and
1-Achy. The syndecan-1 ectodomain bound to cathepsin G
and elastase at picomolar levels of protease, whereas no binding to the
antiproteases was detected at 10-fold higher concentrations (data not
shown). ACE (32) of [35S]sulfate-labeled syndecan-1
ectodomain with cathepsin G and elastase confirmed this binding and
yielded apparent Kd values of 56 nM for
cathepsin G and 35 nM for elastase (Fig.
1, A and C).
Heparin (1 mg/ml) completely abolished binding to the proteases, whereas chondroitin sulfate (1 mg/ml) had little or no effect, indicating that the binding is mainly due to the heparan sulfate chains
on syndecan-1 (Fig. 1, B and D). The ACE profiles
with both enzymes showed heterogeneity in syndecan-1 ectodomain binding at concentrations near the Kd values (80 nM), suggesting that there are subfractions of the
ectodomain that differ in their avidity for the proteases (data not
shown).
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Binding of Syndecan-1 Ectodomain to the Protease Reduces the Effect
of Antiprotease--
To determine whether the binding of the
syndecan-1 ectodomain to the proteases affects their rate of
interaction with a serpin, rate constants
(kass) for these interactions were measured
in the presence and absence of soluble syndecan-1 ectodomain (Table I). The protease and serpin form a 1:1
complex. Because neither heparin or the syndecan-1 ectodomain alters
this stoichiometry (Fig. 2), the
kass were determined under second order
conditions (15). Equimolar amounts (34 nM) of protease and
serpin were incubated in the presence or absence of the syndecan-1
ectodomain at concentrations indicated in Table I. After various times, complex formation was quenched by adding substrate, and the remaining free enzyme activity was measured as described under "Experimental Procedures." The kass for the interaction was
calculated from linear regressions (Equation 1). The
kass for cathepsin G with 1-antichymotrypsin decreased over 3700-fold and with
SCCA2 over 32-fold in the presence of the syndecan-1 ectodomain (Table
I). The kass for elastase with
1-proteinase inhibitor was decreased 60-fold by the
syndecan-1 ectodomain (Table I). For comparison, second order rate
constants were also measured for protease-serpin complex formation in
the presence of a heparin concentration equivalent to that of the
syndecan-1 ectodomain HS (Table I). At the concentrations tested, the
soluble syndecan-1 ectodomain decreased the rates of protease-serpin
association at least as effectively as authentic heparin. The
association rate for cathepsin G and
1-antichymotrypsin was reduced to a significantly greater extent in the presence of
soluble syndecan-1 ectodomain than in the presence of equivalent heparin concentration (Table I).
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Soluble Syndecan-1 Ectodomain Modifies Protease Activities via
Interactions of its Heparan Sulfate Chains--
The effect of the
purified syndecan-1 ectodomain on protease activity was assessed in the
presence and absence of serpin. The ectodomain was preincubated with
cathepsin G or elastase for 15 min and assayed for protease activity
with or without equimolar concentrations of serpins (Fig.
3). Syndecan-1 ectodomain alone reduced
cathepsin G activity in a concentration-dependent manner, reaching maximal inhibition (35%) at 2 µg/ml as core protein (Fig. 3A). However, the syndecan-1 ectodomain markedly decreased
the ability of both 1-Achy and SCCA2 to inhibit
cathepsin G activity (Fig. 3A). In the absence of
ectodomain, these serpins completely inhibit the protease, but with
increasing concentrations of ectodomain, their inhibitory activity is
reduced and ultimately abolished (Fig. 3A). The syndecan-1
ectodomain was more effective in reducing cathepsin G inhibition by
SCCA2 (ED50 = 0.2 µg/ml; Fig. 3A) than by
1-Achy (ED50 = 0.5 µg/ml; Fig.
3A). Analogous findings were obtained with elastase (Fig.
3B). The ectodomain alone reduced elastase activity,
reaching maximal inhibition of approximately 40%. But the ectodomain
also decreased the ability of
1-PI to inhibit the
protease (ED50 = 1.0 µg/ml; Fig. 3B). These
results indicate that syndecan-1 ectodomain alone reduces the
activities of cathepsin G and elastase to an extent nearly identical to
that observed with heparin (19, 20). Moreover, binding of the
syndecan-1 ectodomain to the proteases protects them from inhibition by
serpins (Fig. 3).
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Soluble Syndecan-1 and -4 Ectodomains Can Alter the Proteolytic Balance of Wound Fluids-- Wound fluids accumulating during dermal wound repair contain syndecan-1 and -4. Aliquots of cell-free human acute dermal wound fluids, collected days 1-3 after mammoplasty, were applied to cationic membranes. Antiserum to human syndecan-1 (HSE-1) and monoclonal antibody to human syndecan-4 (5G9) detected syndecan-1 and -4, respectively (Fig. 5). However, antibodies against the cytoplasmic domains of syndecan-1 and -4 failed to detect these proteoglycans (data not shown) (27), and neither proteoglycan was detected in human plasma (data not shown) (27).
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DISCUSSION |
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In this study, we provide new insights into the regulation of
protease-antiprotease balance during tissue injury. We show that
syndecan-1 and -4, cell surface heparan sulfate proteoglycans, are shed
into wound fluids as soluble ectodomains. These acute dermal wound
fluids contain the neutrophil-derived proteases cathepsin G and
elastase, as well as the plasma-derived antiproteases
1-PI and
1-Achy. Both degradation of
endogenous HS in the wound fluid and immunodepletion of syndecan-1 and
-4 from the wound fluids alter the protease-antiprotease balance of the
fluid. In vitro studies show that this balance results from
binding of the HS chains on the ectodomains to cathepsin G and
elastase. This interaction reduces the ability of the antiprotease to
inhibit protease activity. These results indicate that syndecan-1 and
-4 are shed into inflammatory fluids, where they modify the proteolytic
balance of the fluids. The findings also suggest a novel physiological
role for soluble heparan sulfate proteoglycans and new approaches to
modulate the protease balance of inflammatory fluids.
Soluble Syndecan Ectodomains as Heparin-like Mediators at the Wound Site-- Wound repair requires precise temporal and spatial regulation of a panoply of effectors, including chemokines, growth factors, extracellular components, cell adhesion proteins, proteases, and antiproteases. Many of these proteins bind heparin and heparan sulfate under physiological conditions and with high affinities (35). During repair of skin injury, cellular expression of syndecan-1 and -4 is altered (24, 25), and cell surface syndecan-1 and -4 are converted to soluble molecules by juxtamembrane cleavage of their extracellular domains (ectodomains), a process known as shedding (27). Recent studies have shown that syndecan shedding is a highly regulated process that is stimulated by certain agents released at the site of tissue injury (27). Shedding instantly converts a cell surface proteoglycan into a soluble effector.
The functions of these soluble ectodomains are not clear. Syndecans on cell surfaces can act as co-receptors for heparin-binding growth factors; notably, the action of FGF-2 requires a growth factor-heparan sulfate proteoglycan-FGFR1 complex (36). However, because the soluble ectodomains retain all their HS, they can bind the same ligands as the cell surface syndecans, enabling them to be potential inhibitors of these ligand interactions. On the other hand, the soluble ectodomains place HS chains containing heparin-like domains into the wound environment. These chains can interact with heparin-binding proteins and peptides involved in the repair. The inflammatory phase of tissue repair is characterized by plasma exudation and the involvement of neutrophils that produce and secrete the matrix remodeling enzymes elastase and cathepsin G. Although heparin binds and accelerates activity of some serpins (9), heparin does not interact with the serpins that regulate these enzymes. Rather, the enzymes themselves bind heparin, which reduces their affinity for the serpin and protects them from inhibition (19, 20). Our results indicate that the HS chains on the soluble syndecan ectodomains mimic this action of heparin and thus regulate the activity of the neutrophil-derived proteases in the wound environment (Fig. 4). Indeed, the syndecan-1 ectodomain HS chains are at least as effective in decreasing the protease-antiprotease interaction as an equal concentration of heparin (Table I). The binding affinities of the syndecan-1 ectodomain for the protease approximate that for heparin (Fig. 1). As previously observed with heparin (19, 20), the interaction between the syndecan-1 ectodomain and free protease inhibits the protease activity (Fig. 3) but does not alter the stoichiometry of protease binding to the serpin (Fig. 2).Proteolytic Balance in Wound Repair-- Proteolysis is important for fibrinolysis, growth factor mobilization and activation, cell migration into the wound site, reepithelialization, angiogenesis, and extracellular matrix degradation (37). An imbalance of proteolytic activity disrupts normal wound repair and capillary morphogenesis (38, 39). If the soluble ectodomains also act like heparin to accelerate the activity of heparin-activatable serpins (viz. antithrombin III, protease nexin I, plasminogen activator inhibitor-1, and others), the ectodomain could regulate several aspects of proteolysis during wound repair.
Proteases in wounds co-exist with their physiological inhibitors, and thus their activity is finely regulated to provide optimal activity for repair. This activity results from a balance, involving enzyme production and activation counterpoised by enzyme degradation and inhibition. The involvement of syndecan ectodomains in regulating proteolytic balance could explain several observations, including the variability of elastase activity and the inconsistency of fibronectin degradation in wound fluids (11). Our finding that both HS degradation and immunodepletion of syndecan ectodomains reduce the proteolytic activity of acute wound fluids (Figs. 6 and 7) indicates that these soluble proteoglycans contribute to balanced proteolytic activity in the wound environment. Alterations in proteolytic balance are thought to be one reason why acute wounds do not heal properly and become chronic (11, 40). The high levels of proteolytic activity in chronic wound fluid have led to the proposal that misregulated proteases contribute to the inability of chronic wounds to heal even when treated with exogenous matrix and growth factors (41, 42). Whether alterations in the levels of syndecan ectodomains could lead to loss of proteolytic balance and thus to development of chronic wounds needs investigation.Abnormalities in Proteolytic Balance-- Optimal proteolytic activity is needed for normal wound repair. Formation of the fibrin-rich provisional matrix produced after tissue injury is an initial step in the repair process. Once the fibrin clot has formed, migrating keratinocytes at the wound edge and emigrating neutrophils produce a variety of serine proteases and matrix metalloproteases to degrade this matrix and close the wound. An imbalance of proteases and serpins contributes to chronic inflammatory conditions, such as rheumatoid arthritis, pulmonary fibrosis, emphysema, and the development of vascular plaques of atherosclerosis and amyloid plaques in the central nervous system in Alzheimer's disease (9). Whether or not syndecans have a role in regulating proteolytic activities in these events is not known, but in light of our data, this possibility seems worth investigating.
We have found that syndecan-1 and -4 ectodomains act within human acute wound fluids to maintain proteolytic balance. Although no evidence so far exists, the ectodomains of other heparan sulfate proteoglycans that can be shed, such as glypican-1 and CD-44 (Refs. 43 and 29, respectively), might act similarly. Altered proteolytic balance in the wound environment has the potential to interfere with therapeutic procedures ranging from growth factor application to skin grafting. Thus, syndecan expression and shedding should be considered in evaluating and attempting to modify the response to tissue injury. ![]() |
ACKNOWLEDGEMENTS |
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We thank Drs. Bohdan Pomahaz and Elof Eriksson for providing wound fluid samples, Drs. Gary Silverman and Marilyn Fitzgerald for discussions, and Dimitry Leyfer and Olga Goldberger for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by the Emil Aaltonen Foundation, the Finnish Cultural Foundation, and the Maud Kuistila Foundation (to V. K.) and by National Institutes of Health Grants CA-28735 and HL-56398 (to M. B.).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: Harvard Medical
School, Children's Hospital, Enders 9, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-355-6366; Fax: 617-355-7677; E-mail:
bernfield{at}a1.tch.harvard.edu.
1
The abbreviations used are: CatG, neutrophil
cathepsin G; 1-Achy,
1-antichymotrypsin;
1-PI,
1-proteinase inhibitor; SCCA2, squamous cell carcinoma antigen 2; Suc-AAPF-pNA,
succinyl-Ala-Ala-Pro-Phe-para-nitroanilide; ACE, affinity
co-electrophoresis; HS, heparan sulfate; Hase, heparinase; HSase,
heparitinase; GAG, glycosaminoglycan; serpin, serine protease inhibitor; CBZ, benzyloxycarbonyl.
2 M. Kato, H. Wang, V. Kainulainen, M. L. Fitzgerald, D. Ornitz, S. Ledbetter, and M. Bernfield, submitted for publication.
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REFERENCES |
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