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J. Biol. Chem., Vol. 277, Issue 3, 2065-2072, January 18, 2002
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From the New England Eye Center, Tufts University School of
Medicine, and the Tufts Center for Vision Research, Boston,
Massachusetts 02111, the
Received for publication, August 9, 2001, and in revised form, October 28, 2001
We studied the role of the matrix
metalloproteinase gelatinase B (gelB; MMP-9) in epithelial regeneration
using the gelB-deficient mouse. We report the novel finding that, in
contrast to other MMPs expressed at the front of the advancing
epithelial sheet in wounds of cornea, skin, or trachea,
gelB acts to inhibit the rate of wound closure. We determined this to
be due to control of cell replication, a novel capacity for MMPs not
previously described. We also found that gelB delays the inflammatory
response. Acceleration of these processes in gelB-deficient mice is
correlated with a delay in signal transduction through Smad2, a
transcription factor that inhibits cell proliferation, and in
accumulation of epithelial-associated interleukin-1 Epithelial tissues surface all organs of the body exposed to the
external environment, acting as a protective barrier. Unlike most organ
stromal tissues, epithelia can regenerate completely following injury.
Epithelial regeneration involves multiple distinct processes, including
the concerted migration of cells as a sheet to resurface the wound bed,
mitosis to replace cell numbers, re-stratification of the sheet into a
multilayered structure, and restoration of stable adhesive interaction
with the underlying tissue stroma (1-6). These processes must be
temporally coordinated with one another, and with the protective
inflammatory response, which also occurs in response to injury. This is
thought to involve an orderly progression and synergism of cytokines
and growth factors within the wound environment (7-15). Despite many
recent advances, mechanisms for integrating and orchestrating the
multiple processes involved in epithelial regeneration are still poorly understood.
The matrix metalloproteinases (MMPs)1
are a family of zinc endopeptidases that
act as key effectors and regulators of tissue remodeling in vertebrates
(16). Molecular substrates for the MMPs include all classes of
extracellular matrix proteins and molecules that organize
tissues such as the cadherins (17, 18). MMPs are also reactive against
signaling molecules such as cytokines and growth factors, controlling
their activity and bioavailability (18). While MMP activity is
regulated at multiple levels, gene expression constitutes the major
control mechanism (19). Induced expression of an array of MMPs occurs
as part of the tissue response to injury (20, 21). Inappropriate
expression or overexpression of MMPs contributes to the pathophysiology
of diverse disorders occurring across all organ systems (22), including
healing disorders (20, 23-25).
Gelatinase B (gelB; MMP-9) is an MMP that catalyzes cleavage of
denatured collagens of all types and native basement membrane components (26, 16). In addition, recent work has identified fibrin(ogen) (27), The corneal epithelium can be considered a prototype for a stratified
epithelium similar to that surfacing skin, lung, and other organs.
Moreover, the cornea offers an ideal model for evaluating mechanisms of
tissue repair and regeneration because the avascularity of this organ
reduces the number of interacting tissues. In this study we have
investigated specific roles for gelB in regeneration of the corneal
epithelium, utilizing a strain of mice made genetically deficient for
gelB.
Mice and Surgical Procedures--
GelB-deficient mice
(GelB Quantification of Re-epithelialization Rate--
Eyes were
enucleated between 10 and 20 h after surgery and tethered to
paraffin by pinning through the extraocular muscles. The remaining
epithelial defects were delineated by application of Richardson's
stain to the ocular surface. Images were projected at a fixed distance
and the circumference of the area stained by blue dye was traced in
pencil. The area was then quantified using NIH image software by an
observer unaware of the mouse genotype.
To quantify healing in skin wounds, animals were anesthetized and then
the outside perimeter was traced with a fine point marker onto a
sterile slide directly applied to the wound site, after removing the
old dressing and before applying a new one. The wound shape was traced
immediately after surgery and every 2 days from the third day
afterward. The image of the slide with the wound tracing was captured
through a computerized video camera with Pax-it! Software (version
1.83, Imaging Systems for Science and Industry). The area of wound
tracing on slide was quantified using computer image analysis software (Optimus).
Organ Culture--
Eyes were enucleated immediately following
surgery, tethered to paraffin beds in the individual wells of 24-well
plates, and cultured in serum-free medium as described (32). For
"rescue" experiments, purified recombinant human gelB proenzyme
(Chemicon, Temecula, CA) was added to culture medium at the beginning
of an experiment. In other experiments, organ cultures were treated with Ilomastat, a synthetic peptide hydroxamic acid analogue of the
transition state of zinc metalloproteinases (39). A peptide analogue
lacking the active hydroxamic acid group was used as a control (both
agents from AMS Scientific, Pleasant Hill, CA).
Zymography--
Epithelium from the repairing areas of corneas
was debrided with a surgical scalpel, placed in 200 µl of ice-cold
200 Tris-HCl, pH 7.5, and pulverized with a Pellet Pestle
(Kimble-Knotes, NJ). Insoluble material was removed by centrifugation
at 14,000 × g for 10 min at 4 °C. The soluble
proteins were precipitated using cold acetone and protein concentration
was determined with the Bio-Rad reagent. Gelatin zymography was
performed according to our standard methods (31, 32).
Light Microscopy--
Methacrylate cross-sections (6 µm) were
stained with hematoxylin and eosin. Relative thickness of the corneal
epithelial and stromal layers in gelB-deficient mice and normal mice
was compared by examining 10 sections taken through the center of each
cornea from 10 different mice of each strain. Measurements were made using a micrometer viewed in the light microscope field alongside the
tissue section. Significance of differences between strains was
assessed using the Student's t test.
Cryostat sections (6 µm) were processed for indirect
immunofluorescent localization as described previously (31). The
primary antibodies used were a rat polyclonal antibody against the
inflammatory cell marker, mouse Mac-1 (CD11b/CD18 or
Confocal microscopy was performed on mouse corneas fixed with 1%
paraformaldehyde in phosphate-buffered saline. Whole corneas were then
stained en bloc with rhodamine-conjugated phalloidin (Molecular Probes, Eugene, OR) to identify actin filaments. Corneas were mounted epithelial side down onto 25-µm thick, 60-mm diameter mylar Petri dishes (Bachofer, Hamburg, Germany) and scanned using a
confocal microscope (Confocal Laser Scanning Microscopy, Leica, Deerfield, IL) equipped with a Leica Fluorovert microscope,
argon/krypton laser and 568/590 excitation/detection filters.
Three-dimensional data sets were collected and transferred to a Silicon
Graphics work station (Personal Iris 4D-35G), and maximum intensity
volume renderings were made using the ANALYZE software program (Mayo Medical Adventure Inc., Rochester, MN).
BrdU Labeling and Detection--
Central corneal
debridement (1.5-mm) wounds were made in the right eyes of the mice and
allowed to heal for 16 h in vivo. Two hours before the
mice were sacrificed, BrdU (Sigma) dissolved in phosphate-buffered
saline was injected subcutaneously in the dorsal subscapular region at
100 µg/g body weight. Mice were sacrificed and the eyes removed
immediately and frozen for cryostat sectioning. Cross-sections were
immunohistochemically stained with a monoclonal antibody against BrdU
(Becton Dickinson, San Jose, CA). The primary antibody was detected by
avidin-biotin complex immunoperoxidase technique using an ABC Elite Kit
(Vector Laboratories, Burlingame, CA) following the manufacturer's
directions. Color was developed with 3',3'-diaminobenzidine
peroxidase substrate (Sigma).
Western Blotting--
Eyes were frozen immediately after the
mice were sacrificed by immersion in liquid nitrogen to prevent Smad
activation during sample preparations. The entire corneal epithelium
was then removed by gentle scraping with a scalpel. The pooled tissues
from 10 eyes were lysed in a buffer containing 50 mM
Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml each aprotinin, leupeptin,
pepstatin, 1 mM Na3 VO4, 10 mM NaF. Equal amounts of protein (10 µg/sample) were
separated by 10% SDS-PAGE, transferred onto polyvinylidene difluoride
membranes and probed with specific primary antibody reagents.
Immunodetection of primary antibody binding was accomplished by
incubation with horseradish peroxidase-conjugated secondary antibody
followed by chemiluminescence (PerkinElmer Life Sciences) and exposure
to x-ray film. Rabbit anti-Smad3 and anti-phospho-Smad2 were purchased
from Upstate Biotechnology (Lake Placid, NY), and monoclonal anti-Smad2
(clone 18) was purchased from Transduction Laboratories (San Diego,
CA). A goat polyclonal antibody raised against recombinant mouse
IL-1 GelB-deficient Mice Have Normal Corneas--
Mice homozygous for a
targeted mutation that inactivates the gene for gelB show delayed
growth of long bones due to impaired vascularization of the growth
plate, but ultimately they attain normal adulthood (38). Routine gross
examination of adult eyes revealed no obvious anatomical differences
between gelB-deficient homozygotes and their normal counterparts. Eyes
of the gelB-deficient mice were of normal size and lenses were clear.
Corneas were clear and avascular, with no evidence of malformation or
inflammation by gross or histological analysis (data not shown). No
statistically significant difference was observed between strains in
stromal or epithelial layer thickness, or in number of epithelial
layers (data not shown). These observations indicate that a deficiency of gelB does not affect the formation and maintenance of normal corneal structure.
Increased Rate of Corneal Re-epithelialization in GelB-deficient
Mice--
To investigate the effects of gelB deficiency on
regeneration of the corneal epithelium following injury, we used an
epithelial debridement model (42, 32) adapted for the mouse in which the epithelium is surgically removed to the level of the basement membrane within a circular region of 1.5-mm diameter. We collected epithelial tissue from the repairing portions of normal and
gelB-deficient corneas at a time when wounds were partially closed, and
analyzed tissue extracts by gelatin zymography (Fig.
1A). A gelatinase of a size
appropriate to be the gelB proenzyme was detectable in extracts derived
from littermate control mice, but was absent from extracts derived from
gelB-deficient mice. In contrast, a gelatinase of the size appropriate
to be the gelatinase A (gelA; MMP-2) proenzyme was detectable in
extracts from both gelB-deficient mice and their normal counterparts.
The intensity of the gelA band was essentially the same in both mouse
lines, indicating that gelB deficiency does not result in compensatory
up-regulation of this related enzyme.
A time course experiment was performed over the period of wound
closure, and the rate of re-epithelialization was compared between
gelB-deficient mice and normal littermate controls. Fig. 1B
shows representative images of the remaining epithelial defects at a
time when wounds were partially closed. No difference was observed in
the quality of re-epithelialization between mouse strains. Corneas from
both gelB KO mice and their normal counterparts re-epithelialized
without incident, with no gross evidence of inflammation. In contrast,
the rate of re-epithelialization was faster in the gelB-deficient mice.
This difference was apparent as early as 10 h after surgery, and
was highly significant by 20 h (Fig. 1C). All corneas
from both mouse strains remained avascular throughout this experiment,
indicating that cytokines or other factors contributed by the
vasculature could not cause the differing re-epithelialization rates.
The substrate on which the epithelium migrates could influence the rate
of wound resurfacing. Therefore, we repeated the experiment described
above using a PRK model in which a laser is used to ablate
superficial corneal tissue removing the epithelium, the basement
membrane, and a portion of the anterior stroma. To resurface this type
of wound, the epithelial sheet migrates across stromal collagen
fibrils. Again, no difference in the quality of healing was observed
between gelB-deficient mice and their normal counterparts; however,
re-epithelialization was still significantly faster in the
gelB-deficient mice (Fig. 1D).
To learn whether faster wound closure generalizes to other organs, we
examined the rate of healing of an excisional wound in the skin (Fig.
1E). As in cornea, wound closure in skin was found to occur
significantly more rapidly in gelB-deficient mice than in their normal
control counterparts. These results indicate that the findings in
cornea are not tissue-specific.
The GelB-deficient Re-epithelialization Phenotype Is Due to a
Direct Loss-of-function in the Eye and Is Different from the Phenotype
Resulting from Broad Spectrum MMP Deficiency--
To differentiate
between systemic and local factors in the gelB-deficient phenotype, we
repeated the surgical debridement experiment, but eyes were enucleated
following surgery and placed in organ culture for healing. The
difference in the re-epithelialization rate between normal and
gelB-deficient mice was less than when re-epithelialization occurred in
the mouse in situ, but was still significant at 15 h
(p < 0.002; n = 10; data not shown)
and 20 h after surgery (Fig.
2A). These data indicate that
at least some of the effects of gelB deficiency are attributable to
local factors.
To provide evidence that the corneal re-epithelialization phenotype in
the gelB-deficient mice is due to a direct loss-of-function, we
attempted a rescue experiment by adding purified gelB protein to
the corneas of gelB-deficient mice undergoing re-epithelialization in
organ culture. GelB was added in the latent proenzyme form, because
this is the form that predominates in re-epithelializing debridement
wounds (see Fig. 1A). Our expectation was that the exogenously added proenzyme would be converted to an active form through the same extracellular mechanisms utilized for activation of
endogenously produced enzyme during wound repair. At 1 µg/ml of
exogenously added gelB proenzyme, the retardation of
re-epithelialization rate was significant (Fig. 2B). In
other words, exogenous addition of gelB returned the rate of wound
re-epithelialization in the gelB-deficient mouse back toward normal,
consistent with rescue of the deficiency phenotype. These data support
the conclusion that the gelB-deficient re-epithelialization phenotype
results from a direct loss-of-function due to inactivation of the gene for gelB.
Inhibition of broad-spectrum or specific MMP activity has been reported
to inhibit epithelial cell migration in culture (21, 33, 43) and to
retard re-epithelialization of skin wounds in organ culture and
in vivo (21, 44). To learn whether this is also true in our
corneal model, we examined the effects of Ilomastat, a broad spectrum
MMP inhibitor (39, 45) on re-epithelialization in normal mice. While
the Ki for gelatinases determined in test tube assay
is 0.4 nM, inhibition of MMPs in tissues requires much
higher concentrations; we used the dose range determined to be
effective by Chin and Werb (46) in their studies with a mandibular
organ culture system. At an ilomastat dose of 1 µM, the
rate of corneal re-epithelialization was significantly retarded in
comparison to controls treated with the same dose of an inactive analogue lacking the hydroxamic acid group (Fig. 2C). These
data are consistent with a requirement for MMP activity in corneal re-epithelialization, and suggest that gelB must be playing a different
role than other MMPs in this process.
Earlier Inflammatory Cell Infiltration, Increased Deposition of
Provisional Matrix, and Enhanced Rate of Epithelial Cell Replacement in
GelB-deficient Mice--
Tangential optical sectioning by confocal
microscopy on corneal whole mounts stained with rhodamine-phalloidin
revealed no differences between gelB-deficient mice and their normal
counterparts in the overall appearance, or in organization of the actin
filaments, in cells at the advancing epithelial front (Fig.
3, Confocal, arrows).
However, in the course of these experiments we observed the
presence of inflammatory cells in the wound bed of gelB-deficient mice
(Fig. 3, Confocal,
In histologically stained cross-sections, the migrating epithelial
sheet appeared as a monolayer in normal mice but was
multilayered in the gelB-deficient mice (Fig. 3,
H & E, arrows). In addition, an
eosinophilic deposit was apparent in the wound bed and below the
migrating epithelium in some gelB-deficient mice that was not observed
in their normal counterparts (Fig. 3, H & E,
arrowheads). This seemed likely to represent an accumulation of
provisional wound matrix and is discussed further below.
Bromodeoxyuridine (BrdU) labeling was performed on corneal
cross-sections to identify cells undergoing DNA synthesis (Fig. 3,
BrdU). It is known that epithelial injury stimulates
proliferation of cells distal to the wound edge, while the rate of
proliferation decreases in cells at the migratory front (49, 50).
Consistent with this, BrdU-labeled cells from corneas of both normal
and gelB-deficient mice were concentrated in the peripheral epithelium,
with fewer labeled cells localized to the migratory front (central). In
both locations, however, the number of labeled cells was clearly
greater in the gelB-deficient mice. These data provide evidence that
gelB deficiency enhances the rate of epithelial resurfacing of corneal
wounds by increasing the pressure exerted as a result of cell proliferation.
GelB Deficiency Is Associated with a Delay in Smad2 Signaling and
an Increase in Cell-associated IL-1
Western blot analysis of the regenerating epithelium from
gelB-deficient mice showed the same overall changes in Smad2 and Smad3
as the normal mice (Fig. 4B,
Since the cornea is avascular, the corneal wound is devoid of
platelets, a major source of TGF- Impaired Basement Membrane Zone Remodeling and Accumulation of
Provisional Matrix After Injury in GelB-deficient Mice Compromises
Corneal Transparency--
In normal skin or corneal wounds, a
provisional extracellular matrix composed of fibrin(ogen) and
fibronectin is deposited in the wound bed from the serum or tear film,
and provides a substrate for cell attachment and migration (57-62).
This matrix is resorbed once the wound is resurfaced and is replaced by
new epithelial/stromal anchoring complexes (37, 64). The
eosinophilic deposits apparent in the wound bed of
gelB-deficient mice (see Fig. 3, H & E),
suggested that remodeling in the basement membrane zone
might be defective. We investigated this hypothesis by
immunofluorescence analysis (Fig.
5A). In the unwounded areas of
both gelB-deficient and normal corneas that had been healing for 6 days
following PRK, the anchoring fibril component laminin 5 was present as
a thin linear band between the epithelium and the stroma, and there was
no staining for fibronectin or fibrinogen. This staining pattern had
returned in the wound bed of the normal control mice, consistent with
the timing of basement membrane zone remodeling observed in previous
studies (32, 60). In contrast, the immunoreactive band of laminin-5 was
thick and uneven in the wound bed of gelB-deficient corneas at the
6-day time point, and small amounts of fibronectin were present. Most
strikingly, a large fibrinogen deposit was apparent in the wound bed of
the gelB-deficient mice. These results indicate impaired resorption of
provisional matrix and re-establishment of epithelial/stromal adhesion
complexes in gelB-deficient mice.
Defective remodeling at the epithelial basement membrane zone would
have undesirable consequences for long-term health of the epithelium
and could compromise organ function. In cornea, defective remodeling
might interfere with the transparency necessary for transmittal of
light to the retina of the eye. To investigate this hypothesis, we
followed wound healing after PRK over a 2-week time course experiment.
Representative results are shown in Fig. 5B, and data are
summarized in Table I. Two days post-PRK,
re-epithelialization was complete, and the corneas of normal mice were
completely transparent. A minimal amount of clouding or "haze" was
observed in some of the gelB-deficient mice at this time point, at a
level too low to be recorded by photography. By 6 days post-PRK, some
of the corneas from normal mice exhibited well defined, diffuse haze. However, many of the gelB-deficient mice exhibited haze, of a degree to
obstruct iris detail. By day 14, haze was mostly resolved in the normal
mice, but overall haze was increased in the gelB-deficient mice. This
clouding is very similar to that reported after corneal wound healing
in the plasminogen-deficient mouse (41). At no point was there any
evidence of neovascularization of the avascular corneas at any time
point in any experimental animal; thus the excessive material deposited
could not have been due to vascular differences. These results suggest
that defective resorption of provisional matrix and remodeling at the
basement membrane zone due to the absence of gelB causes corneal
clouding.
MMP expression is induced in cells at the migrating epithelial
front in healing wounds of cornea, skin, or lung, and the combined evidence to date (including data presented here) has supported the
hypothesis that MMPs function to promote epithelial resurfacing by
stimulating cell migration (22, 43, 44). Moreover, there is evidence to
support this role specifically for gelB (33, 65). MMPs, including gelB,
have further been implicated in a larger way in the process of cell
migration involving many other tissues. Again, the roles identified for
MMPs have been consistently facilitative and include the clearing of
extracellular matrix to break down physical barriers, modulation of
adhesive interactions with the extracellular matrix to release cells
and to provide traction for their movement, and exposure of signals
necessary to effect motor function and provide chemoattraction (18).
The results reported here reveal that, while gelB may be involved in
epithelial sheet migration, it is clearly not essential for this
process in the normal in vivo situation. In fact, we show that a deficiency in gelB actually accelerates the rate of normal wound
resurfacing. This is the first time that an MMP has been shown to exert
negative control over cell migration. We show that gelB does this by
inhibiting cell replication in the migrating epithelial sheet. This is
a novel role for an MMP, not previously identified.
Peripheral to the main purpose of the current study, we made the new
and significant observation that Smad2/Smad3 signaling is activated in
the regenerating corneal epithelium. Smad signaling inhibits cell
replication (52, 66), and thus Smad activation seems at first
counterproductive to the requirement for cell replacement in epithelial
regeneration. This can be understood, however, when we take into
account that epithelial regeneration involves several other distinct
processes, including resurfacing of the wound bed, re-stratification
into a multilayered structure, and restoration of stable adhesive
interaction with the underlying stroma. Each of these processes is
associated with a withdrawal from the cell cycle (49, 50), and it is
here that Smad activation may play a role.
Significantly, we found a specific delay in activation of Smad2 but not
Smad3 in the gelB-deficient mice. Smad2 and Smad3 are the major
downstream effectors of TGF- Our findings also indicate that gelB controls the timing of the
inflammatory response in the repairing cornea. Again, this may be
attributed to premature accumulation of IL-1 Our work shows that gelB alters the "instructions" that cells
receive from the microenvironment, thus mediating very specific alterations in intracellular signaling pathways, and fine tuning of the
regenerative process. Not only must cell replication be temporal-coordinated with the other processes involved in epithelial regeneration, but also spatially coordinated. Thus, the progressive change in expression pattern of gelB during epithelial regeneration may
be a key factor in its ability to perform a fine tuning function (31,
32, 36). Expression at the leading edge of the migrating corneal
epithelium may serve to coordinate what is happening at the migrating
front with the cell replication that occurs distal to the front. Later,
when the wound is closed, expression across the entire regenerating
epithelium may be important for the complete resorption of provisional
matrix and restoration of normal epithelial/stromal adhesion.
The accumulation of fibrin(ogen) in the wound bed was a striking aspect
of the gelB-deficient phenotype found in this study and indicates that
gelB acts not only to coordinate but also to effect events involved in
epithelial regeneration. Recent work has shown that pericellular
fibrinolysis by migrating endothelial cells is MMP-mediated (67). As
this article was in preparation, it was reported that fibrin(ogen)
accumulation occurs in the gelB-deficient mouse in a kidney disease
model and that gelB can cleave purified fibrinogen in a test tube assay
(27). Thus gelB may act directly on the temporary fibrin(ogen)
scaffolding. GelB may also effect fibrin(ogen) removal indirectly, for
example, by its ability to proteolytically activate plasminogen
activator, or by its ability to proteolytically inactivate inhibitors
of the plasmin-plasminogen activator cascade, such as PAI-1 (26).
Excessive deposition of fibrin(ogen) due to the absence of gelB
appeared to be a major reason for corneal clouding. Corneal clouding or
haze is an undesirable side effect of the wound healing process after
corrective PRK (68) and can be visually debilitating in 1% of human
patients (69-73). Excessive fibrin(ogen) deposition was also
associated with defective restoration of stable adhesive interactions
with the corneal stroma. Again, this suggests parallels to human
pathology. Chronic wounds of skin and cornea are typically characterized by a hyperplastic epithelium (20, 23, 31, 74), which
fails to migrate properly across the wound bed or form proper
attachments to the underlying stroma. This leads to persistent or
recurrent epithelial defects. While fibrin accumulating in acute wounds
is removed within days, it persists in chronic cutaneous wounds (75,
74). It has been suggested that excessive fibrin deposition may
contribute to pathophysiology, by "trapping" of cytokines
controlling epithelial cell dynamics, and by physically interfering
with the restoration of stable epithelial/stromal adhesion.
Taking the findings of this study into consideration with the results
of previous work, we conclude that a balance of gelB activity must be
struck for health of epithelial tissues; too little activity or too
much activity can both lead to pathology. However, it appears that
timing and location of gelB expression in the microenvironment is also
critical to the overall picture of health or pathology. Understanding
the mechanisms for controlling the pattern of gelB expression will be a
future challenge.
We thank Dr. Jay L. Degen (Children's
Hospital Research Foundation, Cincinnati, OH) for his gift of
fibrin(ogen) antibody.
*
This work was supported by project grants AR42981 and
EY12651 (to M. E. F.), EY07348 (to J. V. J.), and
HL47328 (to R. M. S.), National Eye Institute Center
Grant EY13078 (to M. E. F.), the Massachusetts Lions Eye
Research Fund, Inc. (to M. E. F.), by an unrestricted grant
from Research to Prevent Blindness (to M. E. F., and J. V. J.), the Alan A. and Edith L. Wolff Charitable Trust (to
R. M. S.), and the New England Medical Center Hospitals Research Fund (to M. E. F.).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.
§
Present address: EntreMed, Inc., Rockville, MD 20850.
¶
Present address: Eye Research Institute, Oakland University,
Rochester, MI 48309.
¶¶
Jules and Doris Stein Research to Prevent Blindness
Professor. To whom correspondence should be addressed: New England Eye Center, Tufts University School of Medicine, 750 Washington St., Box
450, Boston, MA 02111. Tel.: 617-636-9027; Fax: 617-636-4594; E-mail:
efini@lifespan.org.
Published, JBC Papers in Press, October 31, 2001, DOI 10.1074/jbc.M107611200
The abbreviations used are:
MMP, matrix
metalloproteinase;
BrdU, bromodeoxyuridine;
gelA, gelatinase A;
gelB, gelatinase B;
IL-1, interleukin 1;
PRK, photorefractive keratectomy;
TGF-
Matrix Metalloproteinase Gelatinase B (MMP-9) Coordinates and
Effects Epithelial Regeneration*
§,¶,,
,, Department of Ophthalmology, University
of Texas Southwestern Medical Center, Dallas, Texas 75390, and the
** Division of Dermatology and
§§ Department of Medicine, Washington University
School of Medicine at Barnes-Jewish Hospital,
St. Louis, Missouri 63110
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a cytokine that
inhibits Smad2 signaling and promotes the inflammatory response.
GelB-deficient mice also reveal defects in remodeling of extracellular
matrix at the epithelial basement membrane zone, in
particular, failure to effectively remove the fibrin(ogen) provisional
matrix. We conclude that gelB coordinates and effects multiple events
involved in the process of epithelial regeneration.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-proteinase inhibitor (79),
interleukin-1 (IL-1) (28, 29), and transforming growth factor-
(TGF-) as gelB substrates (30). GelB is not expressed in the normal cornea, skin, or trachea; however, expression is induced (along with a
number of other MMPs) in cells at the front of the migrating epithelial
sheet as it begins to resurface the wound bed following injury (22,
31-33). Inflammatory cells infiltrating the wound bed also produce
gelB (34, 35). GelB expression in the epithelium spreads progressively
distal to the migrating front once wound resurfacing is complete and
persists for several weeks thereafter (32, 36). The timing correlates
with the period during which provisional wound matrix is resorbed and
new structures for epithelial/stromal adhesion are assembled in the
epithelial basement membrane zone (37). Overexpression of gelB is
associated with defective re-epithelialization and a reduction in
adhesion complex integrity (23, 31). These data suggest that gelB plays
a key role in the process of epithelial regeneration.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/) were on the 129SvEv/CD-1 mixed background. A
matched littermate control line (GelB+/+) was on the same
background (38). At intervals throughout this study, genotypes were
verified by Southern blotting. Mice were used for experiments between 6 and 12 weeks of age; gelB-deficient mice were age-matched with
littermate controls. Surgical procedures were performed on anesthetized
mice according to approved institutional protocols. For corneal
debridement surgery, the epithelium within an area demarcated by a
1.5-mm diameter trephine was removed to the basement membrane with the
aid of an algebrush (Storz Ophthalmic). Photorefractive keratectomy
(PRK) was performed on corneas using a Summit Apex Excimer Laser
(Summit, Waltham, MA) on phototherapeutic settings. Tissue was ablated
to a depth of 40-44 µm within a 2.0-mm diameter spot size using 160 pulses. To create a full thickness skin wound, hair was removed with a
depilitory agent. Then the back skin was pulled up to form skin-to-skin
double layers and a 6-mm punch biopsy sterilely used to create two
identical full thickness wounds. The wounds were covered with
transparent Op-Site dressing.
M2 integrin) (Chemicon, Temecula,
CA), a mouse monoclonal antibody directed against human laminin-5 (40),
a sheep polyclonal antiserum raised against human fibronectin
(Chemicon, Temecula, CA), and a rabbit polyclonal antibody against
mouse fibrin(ogen), which binds both the fibrinogen precursor and the
proteolytically cleaved fibrin (41).
(catalog number AF-400-NA) was purchased from R & D Systems
(Minneapolis, MN). Information in the company catalogue states that
this antibody was selected for its ability to neutralize the biological
activity of recombinant mouse IL-1. Based on direct enzyme-linked
immunosorbent assay and Western blot results, this antibody showed less
than 10% cross-reactivity with recombinant human IL-1.
Additionally, indirect enzyme-linked immunosorbent assays this antibody
showed no cross-reactivity with other cytokines tested.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
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Fig. 1.
Increased rate of corneal
re-epithelialization in gelB-deficient mice. Epithelial
debridement or PRK surgery was performed on normal "wild-type"
(WT) and gelB-deficient "knock-out" (KO)
mice. The graphs represent the relative mean areas of
remaining epithelial defects at the indicated time point after surgery.
Statistical analysis was performed using the Student's t
test. A, gelatin zymographic analysis of extracts from
epithelium collected 18 h after debridement surgery. Lanes
1 and 2 and lanes 3 and 4 are
duplicate samples. A rabbit fibroblast standard (Std) was
co-electrophoresed with the mouse samples. Mouse progelB migrates with
an apparent Mr of 105,000 as compared with the
rabbit homolog of 92,000. The absence of the 105,000 zymogram band is
noted in the extracts from the gelB-deficient mouse corneas. The
arrowhead indicates the glycosylated form of 92-kDa gelB.
The arrow indicates mouse pro-gelA, which migrates with an
apparent Mr of 65,000. B,
representative photographic images of stained corneas from
gelB-deficient and normal mice sacrificed at the 15-h time point after
epithelial debridement surgery. The asterisks mark the
central area of the irregularly shaped wound perimeter, defined by the
dark stain. C, the graph represents
data obtained from experiments similar to that in B where
mice were sacrificed after 20 h following epithelial debridement
surgery; p < 0.002 (n = 10). This
difference was reproducible in a subsequent reiteration of the
experiment. D, the graph represents the 20-h time
point after PRK surgery; p < 0.01 (n = 12). E, the graph depicts the rate of wound
closure for 6-mm full-thickness punch biopsies in skin. The remaining
wound size was quantified over a period of 21 days and represented as
the percent of the initial wound size. Asterisk (*) indicates < 0.01 (n = 8 or 9 for each experimental group).
View larger version (25K):
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Fig. 2.
The gelB-deficient re-epithelialization
phenotype is due to a direct loss-of-function in the eye, and is
different from the phenotype resulting from broad-spectrum MMP
deficiency. Epithelial debridement surgery was performed on groups
of normal wild-type (WT) and gelB-deficient knock-out
(KO) mice and then the animals were sacrificed and the eyes
were removed to organ culture for re-epithelialization in
vitro. The graphs represent areas of remaining
epithelial defects at the indicated time point after surgery.
Statistical significance was determined using the Student's
t test. A, organ-cultured corneas at the 20-h
time point; p < 0.02; n = 8 for each
group. B, organ-cultured corneas from gelB-deficient mice
were treated with recombinant human pro-gelB for 18 h.
p < 0.003. n = 6 for each group.
C, organ-cultured corneas from gelB-deficient mice were
treated with 1 µM ilomastat (Ilom) or control
peptide (CP) for 18 h. p < 0.017. n = 6 for each group.
/, arrowhead), that were
not seen in their normal counterparts (Fig. 3, Confocal,
+/+, arrowhead). Absence of gelB does not appear to alter
the capacity of inflammatory cells to respond to a chemotactic
stimulus, extravasate from blood vessels, or accumulate in tissues
(47). However, a deficiency in gelB was shown to delay the resolution
of the contact hypersensitivity response in skin (48). A systematic
investigation of inflammatory cell infiltration by immunofluorescent
microscopy on cross-sections revealed consistent staining with the
Mac-1 marker in corneas from gelB-deficient mice examined prior to
epithelial closure (Fig. 3, Immunofluorescence, Mac-1,
/, arrow), while no staining was observed in normal mice
(Fig. 3, Immunofluorescence, Mac-1, +/+, arrow).
However, shortly after wound closure (24 h post-surgery), corneas from
all normal mice stained for the Mac-1 marker, and the amount of
staining appeared only slightly less than in the gelB-deficient mice
(data not shown).
View larger version (79K):
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Fig. 3.
Earlier inflammatory cell infiltration,
increased deposition of provisional matrix, and enhanced rate of
epithelial cell replacement in gelB-deficient mice.
Epithelial debridement surgery was performed on the corneas of normal
(+/+) and gelB-deficient ( /) mice, and corneas were collected for
analysis just prior to wound closure (16-18 h). Confocal,
tangential optical sections by confocal microscopy through the
epithelium of rhodamine-phalloidin-stained corneal whole mounts, taken
18 h after surgery. Arrows indicate the migrating
epithelial front. The arrowhead indicates invading
inflammatory cells in the wound bed. Immunofluorescence,
frozen cross-sections of corneas taken 16 h after surgery and
stained by indirect immunofluorescence using the inflammatory cell
marker Mac-1. Shown is the central portion of cornea which is still not
re-epithelialized at this time point. Arrowheads indicate
positively stained cells. Corneas were counterstained with Hoechst322
dye (DAPI) which binds the nuclear DNA of all cells.
H & E, corneal cross-sections taken 18 h
after surgery were stained with hematoxylin and eosin and visualized by
light microscopy. The presence of eosinophilic deposits in the
gelB-deficient cornea is indicated by the arrowheads. The
leading edge of the migrating epithelium is indicated by the
arrows. BrdU, corneal cross-sections stained with
BrdU antibody to visualize cells undergoing DNA synthesis.
Arrows indicate positively stained cells.
in the Regenerating Corneal
Epithelium--
GelB can cleave a variety of molecules involved in
cell signaling (18), and thus gelB deficiency might alter the net
signaling information received at the cell surface and cause the
changes observed above. A deficiency in the transcription factor Smad3 was recently shown to enhance the rate of cutaneous wound
re-epithelialization by increasing cell proliferation (51), similar to
our findings here on the gelB-deficient phenotype. Both Smad3 and its
closely related homologue, Smad2, translocate from the cytoplasm to the nucleus once phosphorylated in response to signals received at the cell
surface (52). Immunohistochemical staining of cross-sections through
corneas of normal mice revealed that Smad3 undergoes translocation into
nuclei throughout the regenerating corneal epithelium following debridement surgery (Fig. 4A).
Western blotting revealed that Smad2 and Smad3 activity was clearly
increased in the regenerating epithelium of normal mice (Fig.
4B, +/+). For Smad2, increased activity was demonstrated by
comparison of the levels of total Smad2 and phosphorylated Smad2. For
Smad3, reprobing of the same Western blot revealed a new immunoreactive
band of slower electrophoretic mobility in regenerating epithelium,
consistent with phosphorylation. These findings are consistent with a
role for Smad2 and Smad3 in controlling the rate of epithelial cell
proliferation.
View larger version (48K):
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Fig. 4.
GelB deficiency is associated with a delay in
Smad2 signaling and an increase in epithelial cell-associated
IL-1 in the regenerating corneal
epithelium. A, cross-sections through the uninjured
cornea of from normal wild-type (+/+) mice and through corneas at the
16-h time point after debridement surgery. Sections were immunostained
with antibody to Smad3. Arrows indicate translocation of
Smad3 protein into nuclei in the migrating epithelium. B,
Western blot of cell lysates from uninjured corneal epithelium (0 h),
and from the migrating epithelium of corneas, 8 h (8 h)
and 16 h (16 h) after epithelial abrasion surgery.
Lanes were loaded with equal amounts of protein as determined by BCA
assay, and equality of loading was confirmed by Coomassie Blue staining
on a parallel set of lanes. The blot was probed with antibodies against
Smad3, Smad2, and with an antibody specific for the phosphorylated form
of Smad2 (pSmad2). Immunoreactive proteins are indicated by
an arrow. The putative phosphorylated Smad3 isoform
(pSmad3) is also indicated by an arrow.
C, Western blot of cell lysates from uninjured corneal
epithelium (0 h), and from migrating epithelium collected 8 h (8 h) after epithelial abrasion surgery. The blot was probed with an
antibody to IL-1. The immunoreactive protein is indicated with an
arrow.
/). The amount of the
putative active form of Smad3 was similar in the gelB-deficient mice
and their normal counterparts. However, activation of Smad2 was clearly delayed in the gelB-deficient mice; an increase was not observed until
the 16-h time point, and this increase was less than that seen in
normal mice.
and other cytokine mediators of
wound repair. However, the corneal epithelium synthesizes TGF- , IL-1, and other cytokines (53-55) and is thought to substitute for
platelets in controlling the repair process (56). Western blot analysis
of cell extracts from regenerating epithelium was performed to compare
the levels of cell-associated TGF- and IL-1 at the 8-h time point
(Fig. 4C). No difference in the levels of the major TGF-
isoform produced by corneal epithelium (54) could be detected between
gelB-deficient mice and their normal counterparts (data not shown).
However, the amount of the major corneal IL-1 form, IL-1 (53), was
considerably increased in the gelB-deficient mice (Fig. 4C).
Immunofluorescent localization analysis confirmed this increase, with
greater IL-1 observed within cells throughout the migrating
epithelium (data not shown). Since IL-1 is an antagonist of TGF-
signaling (76, 77), these data are consistent with the reduction in
Smad-2 signaling in gelB-deficient mice. Since IL-1 is an
inflammatory cytokine, itself chemotactic for leukocytes (82), the
increased levels of IL-1 are also consistent with the earlier onset
of inflammatory cell infiltration.
View larger version (47K):
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Fig. 5.
Impaired basement membrane zone remodeling
and accumulation of provisional matrix in gelB-deficient mice
compromises corneal transparency. A, cross-sections
from corneas taken from normal (+/+) and gelB-deficient mice ( /) 6 days after PRK. Indirect immunofluorecence microscopy was used to
identify laminin-5, fibronectin, and fibrin(ogen). The arrow
indicates immunoreactive fibrin(ogen) which accumulates in eyes from
gelB-deficient mice. B, eyes of normal mice (+/+) and
gelB-deficient mice (/) were photographed 2 or 6 days after PRK.
Arrows indicate corneal opacities.
Comparison of corneal clarity in repairing corneas of gelB-deficient
mice and their normal counterparts following photorefractive
keratectomy
0.001 (comparing
normal and gelB-deficient mice using the non-parametric Mann-Whitney U
test).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
signaling; however, there is
accumulating evidence to suggest that these proteins are functionally
different (52, 66). While activity of both are stimulated by TGF-
signaling, they can be differentially regulated as a result of
cross-talk among signaling pathways activated by other extracellular
ligands. The IL-1 signaling pathway interacts with the Smad signaling
pathway in this way; therefore the premature increase in IL-1 levels
observed in this study may translate into a specific delay in Smad-2
signaling (76-78, 80-81). GelB is selectively active against the
IL-1 isoform, although a minor capacity to cleave IL-1 could
still translate into a major effect in a specific microenvironment
in situ. However, GelB could alter the accumulation of
IL-1 by any number of indirect mechanisms including degradation of a
proteinase that degrades IL-1 or degradation of a cytokine that
stimulates expression of IL-1 by epithelial cells (for example, see
Refs. 27, 30, and 79). Therefore, it seems likely that the levels of
IL-1 may be controlled by gelB through the enzymes action against
multiple substrate.
, which is a
chemoattractant for leukocytes (63). In a similar vein, an alteration
in cytokine profiles was identified as the mechanism for delayed
inflammatory cell resolution in a cutaneous chemical hypersensitivity
model applied to the gelB-deficient mice (48). The joint regulation of
wound closure and inflammation by controlling the levels of a single
cytokine, IL-1, could serve to coordinate the timing of these two processes.
ACKNOWLEDGEMENT
FOOTNOTES
These authors contributed equally to the results of this work.
Present address: Parke-Davis Pharmaceutical Research, Ann
Arbor, MI 48103.
ABBREVIATIONS
, transforming growth factor-.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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