Journal of Cell Science 104, 1001-1011 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
1001
Stromal fibroblasts synthesize collagenase and stromelysin during longterm tissue remodeling
Marie T. Girard1, Masao Matsubara1, Claire Kublin2, Marilyn J. Tessier1, Charles Cintron2
and M. Elizabeth Fini1,*
1MGH/Harvard
Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA and
Department of Dermatology, Harvard Medical School
2Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
02114, USA
*Author for correspondence
SUMMARY
The process of connective tissue remodeling is an important mechanism contributing to tissue morphogenesis in
development and homeostasis. Although it has long been
known that remodeling tissues actively mediate collagenolysis, little is understood about the molecular
mechanisms controlling this cell-regulated process. In
this study, we examined the biosynthesis of collagenase
and the related metalloproteinase, stromelysin, during
remodeling of repair tissue deposited after mechanical
injury to the rabbit cornea. Neither enzyme was synthesized by uninjured corneas; however, synthesis and
secretion was detectable within one day after injury.
Collagenase accumulated in its latent form while
stromelysin appeared to be partially activated. Enzymes
were synthesized by cells having a fibroblast phenotype.
These cells were found within the stroma. New synthesis was correlated with accumulation of enzyme-specific
INTRODUCTION
The morphology of a connective tissue can undergo
dynamic alterations through progressive synthesis, degradation and resynthesis of its extracellular matrix (ECM)
components. This process, called tissue remodeling, is an
important mechanism contributing to the morphogenesis of
repair tissue deposited after injury (Clark, 1988). Early
repair tissue is composed of a haphazard arrangement of
collagen fibrils and an abnormal complement of proteoglycan and collagen types. Remodeling transforms the structure and molecular composition of repair tissue. In skin,
these changes contribute to an increase in tensile strength,
a property which is important to the survival of the organism. However, despite such changes, the structure of repairing skin never returns to normal and its original function
is never restored. In contrast, studies using a rabbit model
have demonstrated that remodeling of corneal repair tissue
can result in functional regeneration (Cintron and Kublin,
1977). The opaque repair tissue deposited in the cornea
mRNA. Highest levels of enzyme synthesis were
observed in the repair tissue. However, stromal cells
outside of the repairing area also synthesized both
enzymes. The level of synthesis decreased in a gradient
radiating from the repair tissue. Total synthetic levels
in a given area of cornea were dependent on both the
number of cells expressing enzyme and the rate of
enzyme synthesis. Synthesis of collagenase was detected
in repair tissue as long as nine months after injury. Our
findings provide direct support for the hypothesis that
new collagenase synthesis by cells in repair tissue is the
first step in collagen degradation during long-term
tissue remodeling.
Key words: collagenase, extracellular matrix, metalloproteinase,
wound healing, tissue remodeling, cornea
shortly after injury has a disorganized structure which is
similar to the structure of repairing skin. Remodeling, over
a period of months to years, gradually reorganizes corneal
repair tissue so that its structure comes to approximate the
uninjured cornea (Cintron et al., 1973; Cintron and Kublin,
1977; Cintron et al., 1978, 1981, 1988). Changes resulting
from remodeling eventually restore the cornea to normal
transparency at the site of injury. It is this unique regenerative aspect that makes the cornea particularly interesting
for the study of mechanisms controlling remodeling.
The cornea is composed of an epithelium, a stroma and
an endothelium. These tissues are arranged in simple layers
that can be separated easily from one another. In addition,
each of these tissues is remarkably homogeneous in cell
type and extracellular matrix composition. These structural
features have made possible a precise documentation of the
cellular events that occur during corneal repair (Weimar,
1960). Within hours after injury, the corneal epithelium
begins to resurface the damaged area by migrating as a
sheet from the edge of the wound. Also at this time, the
1002 M. T. Girard and others
quiescent cells of the underlying stroma transform to a
fibroblast phenotype (Weimar, 1957). By 30-36 hours after
injury, these fibroblasts begin mitosis and migrate into the
damaged area. It is thought that these fibroblasts deposit
the ECM comprising early repair tissue (Ross et al., 1970).
A few inflammatory cells, including macrophages and polymorphonuclear neutrophils (PMNs), also accumulate in the
damaged area, but these cells disappear during the first few
weeks after injury. Over time, the cells in repair tissue continue to synthesize matrix molecules and thus mediate the
synthetic phase of the remodeling process (Cionni et al.,
1986).
Less information is available concerning the mechanisms
controlling the degradative phase of repair tissue remodeling. Culturing of tissue fragments on collagen gels has
demonstrated that tissue isolated from skin (Grillo and
Gross, 1967) or cornea in the early stages of repair (Brown
and Weller, 1970) actively mediates collagenolysis. However, collagenolysis during the long-term remodeling of
repair tissue has not been examined.
Documentation of the origin of those cells producing collagenase in early repair tissue is still not complete. The
fibroblast at the edge of the granulation tissue was recently
identified as the predominant collagenase-synthesizing cell
in healing skin (Porras-Reyes et al., 1991). Fibroblasts have
also been identified as collagenase-producing cells in a
pathological skin model (Hembrey and Erlich, 1986). However, the origin of these fibroblasts, dermis or subcutaneous
connective tissue, has not been established (Grillo and
Gross, 1967). Since the origin of the fibroblasts found in
repair tissue of the cornea is known, the corneal model
could help to define the origin of collagenase-synthesizing
cells found in early repair tissue.
Other important questions also remain unanswered. In
vitro experiments have revealed that net collagenolytic
activity is determined by many interacting factors, including the level of enzyme synthesis and the degree to which
enzyme is processed to its active form. The contribution of
these factors to collagenolytic activity during tissue repair
has not been examined. To begin defining the mechanisms
which regulate the process of remodeling in repair tissue,
we have investigated collagenase biosynthesis in a corneal
repair model. We have also analyzed biosynthesis of the
related metalloproteinase, stromelysin, which is part of the
proteolytic cascade that generates activated collagenase
from its inactive form.
MATERIALS AND METHODS
Corneal surgery and collection of tissue
To make a full-thickness corneal injury, a penetrating keratectomy
was performed with a 2.0 mm trephine on one eye of New Zealand
White Rabbits (2.5 kg) as previously described (Matsubara et al.,
1991). This procedure surgically removes all three layers of the
central cornea. It is a simple procedure resulting in reproducible
healing with minimal complications and allows easy recovery of
repair tissue (Cintron et al., 1973). The contralateral eye was left
unwounded to serve as a control. After injury, at times specified
in the text, animals were killed by lethal injection of sodium pentobarbital, and 2.0 mm-diameter fragments of corneal epithelium
or stroma were isolated with a trephine as described previously
(Matsubara et al., 1991). Fragments were isolated from the repair
(i.e. site of injury), repair-adjacent (i.e. 1.0 mm from the repair
area) and repair-peripheral (i.e. 2.0 mm from the limbus) areas of
the cornea. In some cases, epithelium and stroma were isolated as
a single unit. Following collection, each tissue fragment was either
(1) cultured immediately, (2) disrupted for preparation of primary
cell cultures (described below) or (3) placed in an individual tube
and frozen immediately in liquid nitrogen.
Biosynthetic labelling of secreted proteins from
tissue explants
Tissue fragments were cultured in equal volumes (200 µl) of
serum-free medium consisting of Minimal Essential Medium
(MEM; Gibco, Grand Island, NY) supplemented with antibiotics/antimycotics (Gibco) in 96-well cluster dishes. Explant cultures were incubated at 37°C in a humidified 5% CO2-air atmosphere. [35S]methionine (Amersham, Arlington Heights, IL) was
included in the medium at a final concentration of 80 µCi/ml.
Explant-conditioned medium was collected for analysis after 48
h, centrifuged to remove debris, and frozen at −20°C for later use.
Gel electrophoresis and autoradiography
Equal-volume samples (10 µl) of explant-conditioned medium
were diluted 2:5 in sample buffer, and proteins were reduced by
the addition of β-mercaptoethanol. Electrophoresis of samples was
performed on 8% SDS-polyacrylamide gels (Laemmli, 1970) as
previously described (Fini and Girard, 1990). Gels were dried and
autoradiographed without intensifying screens and over a range of
times to ensure the linear response of the film. The relative
amounts of individual proteins in each gel lane were quantified
by volume densitometry (Molecular Dynamics, Sunnyvale, CA)
and compared.
Immunoprecipitation of collagenase and
stromelysin
Conditioned media from like tissue explants were pooled, and
equal volumes were analyzed by immunoprecipitation using standard techniques (Fini and Girard, 1990). Normal sheep serum or
sheep antisera directed against either rabbit collagenase or rabbit
stromelysin (gifts from Dr Constance Brinckerhoff, Dartmouth
Medical School) were utilized as primary antibodies. Immunoprecipitated proteins were visualized by gel electrophoresis. The
relative amounts of specific immunoprecipitated proteins synthesized by tissues derived from different areas of cornea were compared after quantitation by volume densitometry (Molecular
Dynamics, Sunnyvale, CA).
Collagenolytic activity
Collagenolytic activity in conditioned medium from stromal
explants was assayed using the radioactive collagen fibril film
method (Johnson-Wint, 1980a). In some cases, medium was
treated with 0.01% L-(tosylamino 2-phenyl) ethyl chloromethylketone-trypsin (Worthington Biochemical Corp., Freehold, NJ) for
7 min at 37°C to convert proenzyme to active enzyme. Trypsin
was then inactivated by the addition of a five-fold excess of soybean trypsin inhibitor (Worthington, Biochemical Corp.). Activated and unactivated samples were divided into 200 µl samples
and placed into wells of a 96-well cluster plates for collagenolytic
assay.
Primary cell culture and analysis by
immunofluorescence and in situ hybridization
Primary cultures of epithelial and stromal cells were prepared from
equal-diameter fragments of combined epithelium/stroma, as pre-
Collagenase synthesis during tissue remodeling 1003
kDa
kDa
Fig. 1. Biochemical forms and tissue and spatial localization of collagenase and stromelysin in repairing corneas. Equal-sized tissue
fragments from repairing (4) and control (4) corneas were isolated as described in Materials and Methods. Each fragment was cultured
separately in an equal volume of serum-free medium containing [35S]methionine for 48 h. Equal volumes of conditioned media from each
type of sample were then pooled and immunoprecipitated with non-immune serum (N), collagenase antiserum (C) or stromelysin
antiserum (S). PERIPH, peripheral cornea; CEN, central cornea. Size standards are indicated in kilodaltons (kDa). Protein bands visible
in immunoprecipitations performed with non-immune serum represent non-specific background of the procedure.
viously described (Fini and Girard, 1990). To allow cells to attach
and spread on the glass slide, released cells were placed in 8chamber, Tissue-Tek slides (VWR Scientific, Boston, MA) and
incubated for 18 h in MEM containing antibiotics/antimycotics
and 10% calf serum (Hyclone, Logan, UT).
Monensin (1 µM final concentration) was added to cultures for
4 h prior to processing for immunofluorescence. Cells were fixed
in 10% sodium phosphate-buffered formalin (pH 7.0) and double,
indirect immunofluorescence was performed using standard methods (Harlow and Lane, 1988). The primary antibodies used were
a sheep antiserum directed against rabbit collagenase, described
above (used at 1:50 final dilution) and a murine monoclonal antibody directed against human vimentin (used at 1:25 final dilution;
Sigma, St. Louis, MO). The secondary antibodies were fluorescein isothiocyanate (FITC)-conjugated, donkey anti-sheep IgG
(used at 1:50 final dilution) and rhodamine isothiocyanate (RITC)conjugated, rabbit anti-mouse IgG (used at 1:50 final dilution).
Both secondary antibodies were purchased from Jackson
ImmunoResearch Laboratories (West Grove, PA). Cells were
viewed and photographed using a Zeiss Axiophot (Atlantex and
Zeiler Instrument Corp., Avon, MA) equipped for epi-illumination.
Cells to be analyzed for in situ hybridization were fixed in 4%
paraformaldehyde and processed further as described by Pardue
(1985). A 35S-labeled antisense RNA probe was synthesized by
in vitro transcription (Melton et al., 1984) from a rabbit
stromelysin cDNA template (Fini et al., 1987), which had been
cloned into the vector, pBluescript (Stratagene, La Jolla, CA). The
hybridization mixture contained 35S-labeled probe (107 cts per
min/ml), 4×SSC (0.6 M NaCl, 0.06 M sodium citrate), 50% deionized formamide, 1 ×Denhardt’s solution and 20 mM dithiothreitol.
Hybridization was carried out at 50°C overnight.
Percentages of fluorescent or radiolabeled cells were calculated
by dividing the number of positive cells in at least 20, non-overlapping fields by the total number of cells in these fields.
Extraction of caseinases and zymography
Frozen corneal tissue fragments, prepared as described above,
were thawed and immediately extracted with a solution of 2%
sodium dodecyl sulfate (SDS) and analyzed by zymography
(Heussen and Dowdle, 1980) as previously described (Matsubara
et al., 1991). The entire extract prepared from each tissue sample
was electrophoresed without reduction on 11% SDS-polyacrylamide gels containing beta-casein (Sigma, St. Louis, MO) at a
concentration of 0.05%. After staining, the location of caseinases
on the gel could be identified as clear bands, where the casein
substrate had been digested, on a blue-stained casein background.
Since proenzymes are activated by a conformational change produced by the SDS in the gel without an alteration in molecular
size, both procaseinases and activated caseinases can be visualized using this technique. Molecular sizes of caseinases were
determined by comparison with reduced size standards run on the
same gel.
RESULTS
Biochemical forms and tissue and spatial
localization of collagenase and stromelysin
synthesized after corneal injury
Isolated epithelium and stroma from control and surgicallyinjured corneas were cultured separately in the presence of
[35S]methionine to label newly synthesized proteins.
Immunoprecipitation analyses were then performed (Fig. 1)
on equal volumes of explant-conditioned medium samples.
Collagenase antiserum specifically precipitated a 57 kDa
and a 53 kDa protein, of the appropriate sizes to be the glycosylated and unmodified proenzyme forms of collagenase,
respectively. Only explants from the stromal layer of repair
tissue produced immunoprecipitable collagenase and
stromelysin. These enzymes were not synthesized by the
epithelial layer of repair tissue or by the stroma or epithelium of control corneas.
Stromelysin antiserum specifically precipitated a 51 kDa
protein of the appropriate size to be prostromelysin, as well
as a protein doublet migrating at a molecular mass of 40
1004 M. T. Girard and others
kDa. This doublet co-electrophoresed with the autoproteolytically-activated form of stromelysin produced in vitro
by treating enzyme with the organomercurial, 4aminophenylmercuric acetate (data not shown). Like collagenase, stromelysin was only synthesized by the stromal
repair tissue.
To confirm that collagenase synthesized by repairing
stromas is in its latent form, we evaluated the collagenolytic
activity of conditioned medium from stromal explants harvested one week after injury. We were unable to detect collagenolytic activity in these samples. However after trypsin
treatment, which removes the N-terminal peptide from procollagenase, we measured collagenolytic activity in two out
of four samples. The level of activity was at the lower limit
of the assay. Considering the quantity of procollagenase
protein that we had observed by immunoprecipitation in 7day stromal wound samples, this level was much less than
we had expected. This result probably indicates that enzyme
inhibitors are also present in the medium and contribute to
collagenase latency.
Temporal expression of collagenase in
remodeling repair tissue
The temporal expression of collagenase in repair tissue
deposited over nine months after injury was documented in
a time course study. A more extensive spatial analysis of
enzyme expression was performed in this set of experiments
than in the one described above. For analysis of collagenase expression within one week after injury, equal-diameter fragments of tissue were isolated from corneas which
had been undergoing repair for one, three, five or seven
days. Fragments were harvested from repair, repair-adjacent and peripheral regions of injured corneas and from
similar locations in control corneas. Newly-synthesized and
secreted proteins from epithelial and stromal explants were
analyzed by immunoprecipitation. Relative autoradiographic band densities of immunoprecipitated procollagenase in samples from injured corneas are summarized in
Table 1.
As in the previous experiment, collagenase was produced
by explants from the stromal layer of repairing corneas.
Enzyme expression by explants was easily detectable as
soon as one day after injury. In general, collagenase expression increased over the time course, reaching its peak at the
seventh (last) day of analysis. Collagenase synthesis was
not specifically localized to the repair tissue. A gradient of
collagenase expression radiated from the area of repairing
stroma. Enzyme expression decreased precipitously with
increasing distance from the repair tissue. In fact, collagenase expression in the area most peripheral to the repairing
area would have been missed if a long autoradiographic
exposure had not been used. This explains why peripheral
expression was not noticed in the previous experiment.
However, even with the longest autoradiographic exposures, immunoprecipitable collagenase was not detectable
in conditioned medium samples from epithelium at any
location in repairing corneas, nor was enzyme synthesis
detectable in control corneal explants.
In an expanded time course, collagenase expression was
analyzed at one, four and eight weeks after injury. To
reduce unnecessary manipulation of the corneal tissue, the
Table 1. Spatial expression of collagenase in repairing
corneal stromas up to seven days after injury
Days
Location
1
Repair
Repair-adjacent
Peripheral
Repair
Repair-adjacent
Peripheral
Repair
Repair-adjacent
Peripheral
Repair
Repair-adjacent
Peripheral
3
5
7
Optical density volume
996
115
ND
676
267
83
1772
280
454
3830
874
100
Conditioned media were harvested from epithelial and stromal explants
isolated from control (4) and repairing (4) corneas. Equal volumes of
pooled samples were immunoprecipitated with collagenase antiserum.
Electrophoresed proteins were visualized by autoradiography.
Immunoreactive collagenase could be detected only in conditioned
medium from stromal explants; procollagenase levels produced by this
tissue layer were quantified by densitometry and are summarized here.
ND, not detectable.
Table 2. Distribution of collagenase in repairing corneal
stromas up to eight weeks after injury
Weeks
Location
1
Repair
Repair-adjacent
Peripheral
Repair
Repair-adjacent
Peripheral
Repair
Repair-adjacent
Peripheral
4
8
Optical density volume
2923
590
440
3979
888
73
939
84
39
Pooled samples of conditioned medium from combined
epithelial/stromal explants were prepared at one (3 corneas), four (2
corneas) and eight weeks (3 corneas) after injury. The amount of
procollagenase was quantified by immunoprecipitation analyses, as
explained in Table 1.
epithelium was not separated from the stroma for this experiment. Collagenase synthesis was detected in explants from
injured corneas over the 8-week time course (Table 2). As
in the previous experiment, highest levels were measured
in the area of repair; lowest levels in the peripheral cornea.
The peak level of collagenase expression occurred at 4
weeks after injury. Again, no collagenase synthesis was
detectable in conditioned medium from control tissues.
The final portion of this study determined whether collagenase is still synthesized and secreted during long-term
remodeling of corneal repair tissue. Collagenase expression
by epithelial/stromal explants from corneas injured 9
months earlier was analyzed. As shown in Fig. 2, collagenase synthesis was still detectable in samples from the
repair tissue. However, enzyme synthesis could not be
found in samples from either the repair-adjacent or peripheral cornea (data not shown).
Characterization of cells synthesizing collagenase
and stromelysin in repair tissue
Cells which produce collagenase and stromelysin in repair-
Collagenase synthesis during tissue remodeling 1005
kDa
Fig. 2. Collagenase in corneas nine months
after injury. Pooled conditioned media
from epithelial/stroma explants isolated
from corneas (2) injured nine months
earlier were immunoprecipitated with
either non-immune serum (N) or
collagenase antiserum (C). The arrow
marks the immunoprecipitated protein of
the appropriate size to be procollagenase.
Size standards are indicated in kilodaltons
(kDa).
ing corneas were characterized by immunofluorescence and
in situ hybridization analyses. To increase the sensitivity of
our detection methods, cells were first removed from their
matrices and allowed to attach and spread on glass slides
in tissue culture overnight. For immunofluorescence studies, cells were then treated with monensin to block N-linked
glycosylation. This treatment causes collagenase and
stromelysin, which are normally secreted immediately after
synthesis, to accumulate in the Golgi apparatus. Following
this treatment, cells were double-stained with collagenase
and vimentin antiserum; vimentin is an intermediate filament which is a marker for corneal fibroblasts, but not for
corneal epithelial cells (Risen et al., 1987).
Immunoreactive collagenase was localized by fluorescence microscopy to a specific perinuclear region, corresponding to the location of the Golgi apparatus, in cells isolated from the stromal layer of corneas which had been
undergoing repair for one week (Fig. 3G). In contrast,
immunoreactive collagenase was not detectable in stromal
cells from control corneas (Fig. 3C). Stromal cells from all
areas were vimentin-positive with a fibroblastic phenotype
(Fig. 3D and H), whether or not they were collagenase-positive. One difference between cells from the repair tissue
and those from the undamaged areas of repairing corneas
was that collagenase-positive cells from the repair tissue
were much larger (compare Fig. 3D and H).
An examination of the percentage of collagenase-positive cells in each region demonstrated that a large number
of the cells isolated from the stromal layer of repair tissue
contained intracellular collagenase. Although many cells
isolated from stroma outside of the repairing area were also
collagenase-positive, the percentage of these cells appeared
to decrease with increasing distance from the repairing area.
We were not able to obtain enough cells from the repair
tissue for a quantitative analysis. However, such an analysis was possible for cells from areas of stroma outside of
the repair tissue (Fig. 4B and D). A more thorough examination of randomly-selected, non-overlapping fields of
cells from repair-adjacent and peripheral stroma showed
that a higher percentage of cells contained intracellular collagenase in the repair-adjacent (172 positive cells out of
216 cells; 80%) than in the peripheral stroma (51 positive
cells out of 132 cells; 39%).
Immunoreactive collagenase was not detectable in cells
isolated from the repair epithelium (Fig. 3E), nor in epithelia isolated from the repair-adjacent (Fig. 4A) or repairperipheral (Fig. 4C) regions of repairing corneas, nor from
control corneas (Fig. 3A). Cells from control corneas
(Fig. 3B) or the repair-adjacent and peripheral regions of
repairing corneas displayed a vimentin-negative, epithelial
phenotype. Two cell phenotypes were apparent in the
epithelium isolated from the repair tissue. One (Fig. 3F)
was vimentin-negative; the other (data not shown) was
vimentin-positive. The significance of these two phenotypes
is unknown, but neither phenotype contained collagenase
enzyme.
In situ hybridization was used to determine whether
stromelysin synthesis in repairing tissue might be controlled
by accumulation of new mRNA (Fig. 5). Cultured epithelial and stromal cells, isolated from corneas seven days after
injury, were hybridized with a 35S-labeled, rabbit
stromelysin-specific RNA probe. Labeling above background was observed only over stromal cells from repairing corneas. Labeled cells were observed at a frequency of
64% (472 positive cells out of 740 cells) in the repairing
stroma, 82% (508 positive cells out of 618 cells) in the
repair-adjacent stroma, and 38% (215 positive cells out of
566 cells) in the peripheral stroma. Positive cells had a
fibroblastic phenotype.
Presence of neutral caseinases in the repair
tissue
To ascertain whether our cell culture studies accurately
reflect events occurring in vivo, we utilized the technique
of zymography to visualize collagenase and stromelysin in
extracts of tissue fragments harvested one week after injury
and snap-frozen. Thawed samples were electrophoresed on
SDS gels containing casein, since casein is a good substrate
for stromelysin (Chin et al., 1985). As shown in Fig. 6,
caseinolytic activity was found only in stromal samples
from corneas undergoing repair. Two caseinases were
observed in the repairing stroma. One had a molecular mass
of 88 kDa. The other (at arrow) had an approximate molecular mass of 50 kDa. The size of the smaller caseinolytic
enzyme is appropriate for the inactive, proenzyme form of
either collagenase or stromelysin; this enzyme coelectrophoresed with prostromelysin produced by cultures of stromal fibroblasts (data not shown). No caseinolytic activity
could be detected in the epithelium harvested from repairing corneas. These results are consistent with the timing
and localization of stromelysin expression as determined
from our explant and cell culture studies, and suggest that
our data accurately reflect events occurring in vivo.
DISCUSSION
Following penetrating keratectomy, the repairing stroma,
which consists of a haphazard meshwork of cells and fib-
1006 M. T. Girard and others
Fig. 3. Dual indirect immunofluorescence analysis of cells from repairing epithelium and stroma. Epithelial and stromal cells were
harvested from control (8) and repairing (8) corneas one week after injury. Cells were probed with anti-collagenase (as seen in Left
Panels) and anti-vimentin (as seen in Right Panels) primary antibodies. Localization of bound collagenase antibody was visualized with
FITC-conjugated secondary antibody and localization of vimentin antibody with RITC-conjugated secondary antibody. (A and B) central
control epithelium; (C and D) central control stroma; (E and F) repairing epithelium; (G and H) repairing stroma. ×449.
Collagenase synthesis during tissue remodeling 1007
Fig. 4. Localization of collagenase in repair-adjacent and repair-peripheral areas of corneas. Cells were prepared and probed as explained
in Fig. 3. Repair-adjacent epithelium (A) and repair-adjacent stroma (B); Repair-peripheral epithelium (C) and repair-peripheral stroma
(D). ×460.
rils, is considerably different in structure and composition
from that of the normal stroma (Cintron et al., 1978). This
lack of matrix order may contribute to the opacity and
mechanical weakness of corneal repair tissue. With time,
these deficiencies are corrected through a prolonged process
of synthesis, degradation and resynthesis. The parallel
layers of collagen lamellae, characteristic of the intact
cornea, reform across the injured region. Electron microscopic studies reveal that, during the remodeling process,
collagen fibril size becomes progressively more regular, and
the stromal fibrils attain a more orderly arrangement (Cintron et al., 1978). It is believed that these gradual changes,
which can take months to years, contribute to the eventual
return of normal corneal transparency marking functional
tissue regeneration.
It seems logical to assume that the degradative phase of
tissue remodeling requires the participation of matrixdegrading enzymes, called matrix metalloproteinases
(MMPs). Together, these enzymes, which are descended
from a common ancestral gene (Brinckerhoff and Fini,
1989; Matrisian, 1990), have the capacity to degrade most
components of the extracellular matrix. The MMPs are produced by both resident cells in a tissue and invading inflammatory cells and have greatest activity at the neutral pH of
the extracellular space. Each MMP is secreted into the
extracellular space as an inactive proenzyme which must
be converted to an active form. Different MMPs have different substrate specificities. Fibroblast collagenase
described in this paper can catalyze degradation of native
types I, II or III collagen. In contrast, fibroblast stromelysin
can specifically cleave proteoglycans as well as fibronectin
and laminin. Stromelysin also appears to play another
important role by converting procollagenase to its fully
active form (Nagase et al., 1992).
Despite the large body of knowledge that has accumulated about the structure and biochemistry of MMPs in
vitro, little is known about how the expression or activity
of these enzymes is regulated during corneal remodeling.
It has been shown that corneas undergoing wound repair
for 1 to 2 weeks elaborate collagenolytic activity which is
not produced by explants from uninjured corneas (Brown
and Weller, 1970). However, it has not yet been established
whether this activity arises from the activation of latent collagenase which was previously synthesized or is the result
of new enzyme synthesis. Clarification of this subtle point
has not been adequately addressed in the literature. In the
present study, we demonstrate that collagenase is synthesized in repairing corneal tissue. In comparison, we found
that normal corneas do not synthesize detectable collagenase. Remodeling corneas synthesize and secrete two pro-
1008 M. T. Girard and others
Fig. 5. In situ hybridization analysis
of stromelysin message in corneal
cells. Epithelial and stromal cells
were prepared from control (3) and
injured (6) corneas. Cells were
hybridized with a rabbit stromelysin
35S-labeled antisense RNA probe
and exposed to autoradiographic
emulsion for 2 weeks. (A and B)
repairing stroma; (C and D) central
control stroma. Phase contrast, A
and C. Dark field, B and D. ×1047.
teins which can be immunoprecipitated with collagenase
antiserum. These proteins, with molecular masses of 57 kDa
and 53 kDa, are the appropriate sizes to be the inactive,
proenzyme forms of glycosylated and unmodified fibroblast collagenase, respectively (Fini and Girard, 1990; Fini
et al., 1987). To our knowledge, these data provide the first
direct evidence for the long-held, but previously unproven,
view - that collagenase biosynthesis is a necessary prerequisite for collagen turnover during early repair process
(Cionni et al., 1986; Davison and Galbavy, 1985, 1986). In
addition, we demonstrate for the first time, that synthesis
of collagenase continues in the repair tissue for as long as
nine months after injury. This is the first evidence that this
enzyme plays a role in the long-term process of tissue
remodelling that leads to stromal regeneration.
In the present study, we also determined which corneal
tissue layer produces collagenase following injury. This has
been a subject of controversy for many years. When epithelium and stroma from recently injured corneas or skin are
separated and cultured individually on collagen gels, epithelial, not stromal, explants lyse the gels (Grillo and Gross,
1967; Slansky et al., 1969; Gnadinger et al., 1969; Brown
and Weller, 1970; Slansky and Dohlman, 1970; Pfister et
al., 1971; Berman et al., 1971; Brown, 1971). Similar
results were obtained in studies on healing skin (Grillo and
Gross, 1967). The results of these explant studies have been
difficult to reconcile with the findings of more recent cell
culture experiments demonstrating that collagenases are
generally produced by fibroblasts, PMNs or macrophages
(Alexander and Werb, 1991), which are cell types found in
the stromal layer of early repair tissue. In the present study,
we provide data that might explain the discrepancy between
cell culture and explant studies. We show that explants from
the stromal layer of corneas undergoing repair do indeed
synthesize and secrete immunoprecipitable collagenase.
However, the collagenase is in its proenzyme form. Therefore, it would not have been detected by the gel lysis assay
which was used in the previously reported explant studies
because this assay requires active enzyme.
More difficult to resolve than the lack of collagenolytic
activity in stroma is the localization of collagenolytic activity to the epithelium of repairing corneas and skin. These
data suggest that epithelial cells also synthesize collagenase
in repairing tissue. While epithelial cells are not generally
recognized as cells which synthesize collagenase, a few
reports have demonstrated some exceptions to the rule. For
example, it has been reported that collagenase is synthesized by both skin epithelial cells (keratinocytes) after pas-
Collagenase synthesis during tissue remodeling 1009
kDa
Fig. 6. Representative casein zymogram of tissue extracts from
repairing and control corneas. Proteins were extracted in SDS and
electrophoresed under non-reducing conditions on casein substrate
gels. Lane 1, central epithelium; Lane 2, central stroma; Lane 3,
mid epithelium; Lane 4, mid stroma; Lane 5, peripheral
epithelium; Lane 6, peripheral stroma. The arrow indicates the
location of caseinolytic enzymes with the appropriate,
approximate molecular weight to be either procollagenase or
prostromelysin. Size standards are indicated in kilodaltons (kDa).
sage in culture (Petersen et al., 1987) and some epithelial
tumor cell lines (Lyons et al., 1989). In these studies, collagenase synthesis could have been induced by cell culture
or might reflect the abnormal genetic changes that occur
with oncogenic transformation. However, it is also possible that epithelial cells begin to synthesize collagenase
during tissue repair and remodeling. If this were the case,
we should have been able to detect collagenase synthesis
by epithelial cells; the collagenase synthesized by keratinocytes and epithelial tumor cells is the same gene product as the fibroblast collagenase and would have been
immunoprecipitated by our antibodies. Since we were
unable to detect enzyme synthesis either by immunoprecipitation or immunofluorescence, we must conclude that
cells in the repairing corneal epithelium are not an important source of collagenase during tissue remodeling.
What, then, is the biochemical basis for the elaboration
of collagenolytic activity by epithelial explants from repairing corneas or skin? A number of reports have demonstrated
that, while epithelial cells do not generally produce collagenase enzyme, they can play a regulatory role in collagenolysis. Epithelial cells, including those of cornea and
skin, can produce cytokines that positively or negatively
regulate collagenase synthesis by fibroblasts (JohnsonWint, 1980b, 1988). In addition, these cells can produce
proteinases that participate in converting procollagenase to
the active form (He et al., 1989). Therefore, if any procollagenase-producing repair fibroblasts were cultured along
with the corneal epithelium, the resulting conversion of collagenase to active collagenase would allow its detection by
the gel lysis assay. Importantly, most studies reporting
localization of collagenolytic activity to corneal epithelium
made no attempt to isolate epithelium as a pure tissue; they
simply separated corneas into epithelium/anterior stroma
and stroma/endothelium. In only one case was greater care
taken to separate the two tissues (Brown and Weller, 1970),
but contamination with even a few stromal cells may have
been sufficient to provide a source of collagenase. Thus, it
seems likely that the basis of the reported epithelial localization of collagenolytic activity in these previous studies
actually results from the capacity of the epithelium to regulate collagenolysis, rather than to synthesize collagenase
enzyme.
In the present study, we found that expression of
stromelysin was, like collagenase, induced de novo in the
corneal stroma by injury. This has important implications
for the mechanism of procollagenase activation. The conversion of latent collagenase to its active form is thought
to occur via an enzymatic cascade which results in successive cleavages from the N terminus (Nagase et al., 1992).
The initial cleavages yield collagenase that has only 1425% of full activity, and subsequent cleavage by
stromelysin is necessary for conversion of collagenase to
its fully active form. The major protein form that we identified had a molecular mass of 51 kDa and is the appropriate size to be the inactive, proenzyme form (Fini et al.,
1987; Fini and Girard, 1990). Interestingly, a minor portion
of the synthesized enzyme had a size of 40 kDa which is
appropriate for a proteolytically activated form. This suggests that, while induction of collagenase and stromelysin
synthesis in repair tissue is coordinate, activation of these
enzymes is controlled by different factors.
In addition to its role in collagenase activation,
stromelysin can also degrade the core protein of proteoglycans. In the cornea, two types of proteoglycans are commonly found: the cornea-specific molecule, keratan sulfate
proteoglycan (lumican), and a molecule that is also found
in sclera and skin, dermatan sulfate proteoglycan (decorin).
Maintenance of an appropriate ratio (3:2) of these specific
proteoglycan types is thought to be important for determining the precise degree of corneal hydration needed for
tissue transparency (Bettelheim and Plessy, 1975; Hedbys,
1961). In repair tissue, this ratio is altered, with decorin
becoming a more prominant component. It is likely that this
change contributes to repair tissue opacity (Hassell et al.,
1983). Activated stromelysin, via its capacity to catabolize
proteoglycans, is likely to help reestablish transparency by
returning the proteoglycan ratio to normal during remodeling.
We were able to detect newly synthesized collagenase
and stromelysin in explant cultures from repair tissue within
24 h after injury. This is coincident with stromal cell migration into the injured area and the initiation of cell division
(Weimar, 1960). Collagenase levels increased (1.4-fold)
within the repair tissue between one and four weeks after
injury; this increase is considerably smaller than the
increase in cell number (4.5-fold) which we reported in an
earlier investigation (Cintron and Kublin, 1977). Likewise,
collagenase levels decreased (4.2-fold) between four and
eight weeks after injury, but cell number decreased only
1.8-fold between four weeks and ten weeks. Thus, changes
in the rate of collagenase production by repair tissue over
the eight-week time course cannot be explained simply by
changes in the number of cells in the repair tissue. This
1010 M. T. Girard and others
suggests that the rate of collagenase synthesis per cell is a
secondary factor controlling the total amount of collagenase
produced.
We were surprised to learn that expression of collagenase and stromelysin is not localized specifically to the cells
in the repair tissue or to the cells at the wound edge from
which wound fibroblasts are derived. Instead, our biosynthetic studies demonstrated that synthesis of collagenase
and stromelysin occurred in a gradient across the radius of
the corneal stroma, with highest levels in the repair tissue
and lowest levels at the corneal periphery. Perhaps collagenase expression peripheral to the repair tissue is required
to mediate intercalation of the new collagen fibrils into the
existing stromal lamellae (Davison and Galbavy, 1986). In
areas peripheral to the repair tissue, the gradient of enzyme
expression could be explained, at least in part, by the
number of cells engaged in enzyme synthesis across the
area. In corneas repairing for one week, a higher percentage of cells containing intracellular collagenase were found
in the repair-adjacent stroma (80%) than in the repairperipheral stroma (39%). However in the repair tissue,
fewer of the total cells were synthesizing enzyme than in
the repair-adjacent tissue. In addition, we previously determined that the total cell number in the repair tissue at the
one week time point is less than half that in the undamaged areas (Cintron and Kublin, 1977). Yet despite these
factors, the repair tissue produces considerably more
enzyme (4-5 times) than the adjacent stroma, demonstrating that the rate of synthesis is also a controlling factor in
enzyme production. In situ hybridization revealed that the
pattern of mRNA expression paralleled that of enzyme synthesis; this demonstrates regulation of gene expression at
the level of mRNA accumulation. The gradient of enzyme
expression suggests that factors controlling enzyme expression radiate from the repair tissue. Inductive signals could
be passed directly from one cell to another via gap junctions or could be transmitted by diffusible substances originating from the epithelial or stromal layers of the repair
tissue.
In summary, we have shown that resident stromal fibroblasts synthesize and secrete the MMPs, collagenase and
stromelysin, in the repairing corneal stroma. Our results
help to eliminate confusion regarding the tissue source of
collagenase in repairing cornea. To our knowledge, this
may be the first study which establishes that continued collagenase biosynthesis by resident fibroblasts is a component of long-term tissue remodeling.
Sheep antisera directed against rabbit collagenase and
stromelysin were generously donated by Dr Constance Brinckerhoff, Dartmouth Medical School, Hanover, NH. We thank Dr
Romaine Bruns for advice and help with photography, and Mr
Richard Silverstein for handling and care of the experimental animals. We are also grateful to Dr Jerome Gross for enlightening
discussions. This work was supported by NIH grants EY08408
(M.E.F.) and EY01199 (C.C.), and by an agreement between
Massachusetts General Hospital and the Shiseido Company.
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(Received 16 November 1992 - Accepted 6 January 1993)