Download Expression of collagenolytic/gelatinolytic metalloproteinases

Investigative Ophthalmology & Visual Science. Vol. 31. No. 9. September 1990
Copyright © Association for Research in Vision and Ophthalmology
Expression of Collogenolytic/Gelatinolytic
Metalloproreinases by Normal Cornea
M. Elizaberh Fini and Marie T. Girard
Members of the gelatinasc subclass of the matrix metalloprotcinasc family have the capacity to
degrade denatured collagens of all types and native types IV, V, and VII collagens. The authors
identified the metalloprotcinasc species of the gclatinasc class produced by the cells of rabbit cornea!
tissue. Two different molecular forms of gclatinasc, visualized as enzymatic activities, that undergo
electrophoresis with different mobilities on gelatin zymograms arc synthesized by corncal cells in
serum-free organ culture. The enzyme species that has the slower mobility is biochemically and
immunologically related to a gelatinasc synthesized by macrophages and ncutrophils which has been
called both type IV and type V collagenasc. The second gelatinase species is related to a second
enzyme, the product of a different gene, which has also been called type IV collagenasc. The elcctrophorctic mobilities of these enzymes on polyacrylamide gels indicate the inactive proenzyme forms.
The authors refer to these enzymes as 92-kilodalton (kD) gelatinasc and 72-kD gclatinasc based on
their electrophoretic mobilities under sulfhydryl-rcducing conditions. In primary cell culture, corneal
epithelial cells were found to synthesize predominantly the 92-kD gclatinasc species whereas the
72-kD gelatinase is synthesized mostly by stromal fibroblasts. However, each cell type can produce
small amounts of the other enzyme. The 72-kD gelatinase, mostly in the proenzyme form, can be
extracted from the normal corncal stroma without culturing, but expression of 92-kD gelatinase can
only be detected in cell or organ culture. The substrate specificities of these enzymes suggests that
they may be of central importance in the degradation of the epithelial basement membrane and in
formation of the epithelial defect that precedes corneal ulccration. Invest Ophthalmol Vis Sci
31:1779-1788, 1990
It is currently accepted that a tissue stroma is
maintained by a delicate balance between processes
that lead to extracellular matrix (ECM) deposition or
degradation. In a number of tissue disorders, including rheumatoid arthritis, atherosclerosis, and recessive epidermolysis bullosa. the normal controls of
degradative activity appear to be lost, leading to
pathologic tissue destruction.1 Corneal ulceration can
also be considered as a disorder of tissue maintenance. Its physical manifestation, corneal "melting,"
results from loss of the structural components of the
corneal stroma. That this loss is due to an excess of
specific, ECM-degrading enzymatic activities within
the cornea has been clearly demonstrated.2"8 The best
characterized of the degradative proteinascs found in
the cornea during ulceration is collagenase, the only
known enzyme that can cleave the native, type I collagen helix at the neutral pH of the extracellular
From the Eye Research Institute and the Departments of Ophthalmology, and Anatomy and Cell Biology, Harvard Medical
School. Boston. Massachusetts.
Submitted for publication: January 4. 1990: accepted March 22.
1990.
Reprint requests: M. Elizabeth Fini. PhD, Eye Research Institute. 20 Staniford Street. Boston. MA 021 14.
space.9 Type I collagen is an important structural
component of the corneal stroma, and a precise arrangement of type I collagen fibrils is essential for
stromal clarity and good vision. This fact assigns type
I collagenase to an important role in corneal ulceration.10
The type I collagen-degrading enzyme is the only
collagenase that has been identified in cornea thus
far, but type I collagen is not the only collagen type
found in cornea. Type V collagen also appears to be
an important component of corneal stroma, and it is
thought to copolymerize in fibrils with type I." In
addition, the epithelial basement membrane of the
cornea contains collagcnous components different
from those found in the stroma, including type VII
and type IV collagens.12 Since in animal models for
alkali burn, the epithelial basement membrane disappears just before the onset of stromal ulceration,1314 enzymes must exist that can degrade the
collagenous components of this structure.
Type I collagenase is only one member of a family
of enzymes with closely related structures, the matrix
metalloproteinases13 (MMPs). Members of this family have a common requirement for zinc as a cofactor
and calcium for stability; they show optimal activity
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Seprember 1990
at the neutral pH of the extracellular space. Each
enzyme is secreted by cells as an inactive species, the
zymogen or proenzyme form. Activation in vitro
usually results in a decrease in molecular weight due
to proteolytic cleavage from the N-terminal of the
protein, and similar activation mechanisms may
occur in vivo within the extracellular space.16 The
MMPs can be grouped into three subfamilies based
on specificity toward different components of the
ECM. Collagenases degrade native type I, II, or III
collagens,10 and stromelysins specifically cleave proteoglycans and fibronectin and laminin.17 Enzymes
of the third subfamily, gelatinases, have activity
against denatured collagen molecules (gelatin) and
native type IV, V, and VII collagens.18 Two different
gelatinase species have been well characterized. The
lower-molecular-weight species (around 72 kilodaltons [kD]), also called type IV collagenase, is produced by a number of different normal cell types and
is expressed by transformed epithelial cells and
tumors.1920 A higher-molecular-wcight form (90-100
kD), originally characterized as the product of polymorphonuclear leukocytes and monocytes/macrophages and called type V collagenase,21 is now known
to be produced by various cell types18 and has also
been called type IV collagenase.22 Sequencing studies
establish that the two gelatinase species are products
of separate genes.1922
We report the identification and characterization
of gelatinases of the MMP family that can be synthesized and secreted by corneal cells in organ and primary culture. Epithelial cells produced predominantly the high-molecular-weight form of progelatinase (92 kD) whereas stromal fibroblasts synthesize
mostly the lower-molecular-weight progelatinase (72
kD). The 72-kD progelatinase can be extracted from
the stroma without culturing, whereas 92-kD progelatinase expression appears to be stimulated in organ
culture or cell culture. We discuss the roles that these
enzymes might play in corneal health and disease.
Materials and Methods
Corneal Organ and Cell Culture
Rabbit corneas were obtained from New Zealand
White rabbits (2.5 kg) killed by intravenous injection
of sodium pentobarbital just before harvesting the
tissues. Procedures were performed according to the
ARVO Resolution on the Use of Animals in Research. Corneal buttons (9 mm) were prepared by
trephination, and the endothelial layer of tissue was
stripped mechanically. Epithelial/stromal buttons
were dissected into four equal pieces and then cultured in serum-free medium consisting of Minimal
Essential Medium (GIBCO, Grand Island, NY) sup-
Vol. 31
plemented with antibiotics/antimycotics. Corneal epithelial cell/stromal fibroblast isolation and plating in
culture were performed essentially as described.23 Epithelial cells and stromal fibroblasts were plated at
desired densities in 16-mm diameter wells of 24-well
cluster dishes. Cell adherence and spreading was facilitated by inclusion of 10% calf scrum (Hyclone,
Logan, UT) in the medium, but after 16-24 hr, cells
were changed to serum-free medium for experimentation. This was done to avoid the possible influence
of serum cytokincs on proteinase expression and because the large quantities of albumin in serum interfere with electrophoretic resolution of enzymatic species. Traces of serum proteins were removed by
washing cultures three times with Hank's balanced
salt solution (HBSS; GIBCO, Grand Island, NY) before addition of fresh medium. In preliminary experiments, lactalbumin hydrolysate was added to
serum-free culture medium as described.24 However,
we observed that this agent could up-regulate collagenase expression in primary stromal cell cultures.
Thus, all experiments were done using medium without this additive. Cells remained completely viable
during 72 hr of culturing as evidenced by continued
adherence to the culture dish and continued incorporation of "S-mcthionine into protein. When required, cells were treated with cytochalasin B (CB;
Aldrich, Milwaukee, WI) at 5 /zg/ml as recommended.23
In some experiments, primary epithelial cell cultures were established by preparing epithelial sheets
using Dispase II (Boehringer Mannheim, Indianapolis, IN).25 Corneal epithelial/stromal buttons were
placed in 2 mg/ml of Dispase II prepared in scrumfree medium and incubated for I hr in a humidified
atmosphere of 5% CO2 in air at 37°C. Full-thickness
epithelial sheets were gently peeled back with forceps,
rinsed in HBSS, and placed in 16-mm cell culture
wells. Sheets were cultured for 3 days in medium
containing 10% calf serum. After this time, cells had
attached to the dish and grown out of the epithelial
sheet to cover the bottom of the culture well.
Stromal fibroblasts were passaged by subculturing
as described.26 These cultures were also changed to
serum-free medium before beginning an experiment
and, when necessary, treated with CB as above.
Labeling of Cell-Culture Proteins,
Gel Electrophoresis, and Immunoprecipitation
35
S-methionine (Amersham, Arlington Heights,
IL), with a specific activity > 1000 Ci/mmol, was
added to serum-free culture medium at 80 mCi/ml
for biosynthetic labeling of proteins. Cell cultures
were pulse-labeled for 4 hr. After this time, medium
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COLLAGENASES / GELATINASES SYNTHESIZED DY CORNEA / Fini ond Girord
containing labeled, secreted cell proteins was collected. Samples of 10-15 i*\ were diluted 2:5 in sample buffer, reduced by addition of/3-mercaptoethanol
to 5%, and underwent electrophoresis on 8% sodium
dodecyl sulfate (SDS) gels27 prepared using a 19:1
stock of acrylamide to £/.v-acrylamide (InstaPAGE;
IBI, New Haven, CT). Gels were dried and autoradiographed to display labeled proteins. Relative
amounts of individual proteins were quantitatcd by
densitomctry. In selected cases the identities of gel
bands that corresponded to procollagenase or prostromelysin were verified by immunoprecipitation
using sheep anti-rabbit antisera (gifts of C. BrinckerholT, Dartmouth).26 To ensure that the proteins arrived in the culture medium by secretion rather than
cell lysis, the protein profile in the culture medium
was compared with that which could be extracted
from the remaining cell layers with 0.1% Triton
X-100 (Fisher Scientific, Pitlsburg, PA). The two
protein profiles were different.
Zymography
Zymography was done by the method of Birkedahl-Hansen and Taylor.28 With this technique, proteolytic species are separated on the basis of molecular size by electrophoresis through an SDS-polyacrylamide gel within which a substrate for the enzyme of
interest is copolymerized. Subsequently, the position
of each enzyme in the gel is visualized by its ability to
degrade the substrate. Often, proenzyme species and
proteolytically activated species can both be visualized; most proenzymes can be activated without a
reduction in molecular size as a result of the change
in their protein structure produced by the SDS in the
gel. The SDS-gels (11%) were prepared using a 37.5:1
stock of acrylamide to £/.v-acrylamide (Boehringer
Mannheim), and gelatin was included in the gel at a
concentration of 0.1% (from bovine skin; Sigma, St.
Louis, MO). Samples of crude cell-conditioned medium were diluted 2:5 in gel sample buffer before
loading on a gel. After eleclrophoresis of the samples,
the gel was shaken in a 2.5% solution of Triton X-100
for 1 hr, to remove SDS and then incubated in reaction buffer (50 mM Tris, pH 7.5, 10 mM CaCI2)
overnight at 37°C. The positions of enzymatic species
could be easily identified, after the gel was stained
with Coomassie brilliant blue (Sigma), as clear bands
in the stained gelatin background.
Extraction of Gclatinases from Tissue
For direct extraction of gelatinases from tissue, the
endothelial layer of the cornea was manually removed, and the epithelium was scraped from the
cornea with a scalpel blade. Epithelial or stromal tis-
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sues were frozen in liquid nitrogen and placed in separate tubes. Soluble proteins were extracted from
corncal tissue with a solution of 2.0% SDS. Extracts
were diluted 2:5 with sample buffer and underwent
electrophoresis on standard zymogiams for analysis
of gelatinolytic enzymes present.
Immunoprecipitation of Enzymatic Activities and
Western Blotting
Identity of gclatinases was confirmed using immunoprecipitation and Western blotting.29 A monoclonal antibody specific for human type IV collagenase (generously donated by L. Liolta and W. SlellerStevenson,20 NIH) was used for Western blotting.
Samples were prepared for auloradiography, with or
without reduction. Dog antiserum directed against
type V collagcnase from human macrophages (a gift
of Dr. K. Hasty,21 University of Tennessee) was used
for immunoprecipitation analysis. Antigen-antibody
complexes were precipitated with Protein-A-Sepharose CL-4B beads (#P-3391; Sigma). The beads were
then resuspended in Laemmli sample buffer, and the
slurry was loaded on a gelatin zymogram, without
heating or addition of /3-mercaptoethanol, for analysis of immunoprecipitated gelatinolytic species.
Results
Organ-Cultured Corncal Epithelial/Stromal Buttons
Synthesize and Secrete Two Different
Gelatinolytic MMPs
Gelatin zymography identified gelatin-degrading
enzymes produced by corneal cells. Two enzymatic
species with distinctly different molecular weights
could be visualized on gels displaying samples of
conditioned medium from corneal organ cultures
(Fig. 1, left). The more slowly migrating species were
found at an apparent molecular weight of 92 kD with
respect to reduced protein standards. A second gelatinolytic species could be visualized, migrating at an
apparent molecular weight of 65 kD. Each of the
enzymatic activities could be further resolved into
two closely spaced zymogram bands. Two subspecies
were always resolved in the 92-kD species. However,
the 65-kD subspecies found in organ culture medium
seemed to be characteristic of a given rabbit rather
than some unknown variable in the culture conditions since all quarters from an individual corneal
button released the same 65 kD subspecies (Fig. 1,
right). This observation suggests that the two 65-kD
subspecies represent allelic variants.
Visual inspection of the degree of substrate clearing
produced by the samples indicated that each enzyme
species accumulated in the organ culture medium
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Fig. 1. Gelatinolytic spe0 24 hrs V>0 48 hrs ^ 0 72 hrs ^1 2 fold dilutions
cies produced by rabbit cor65kD subspecies
neal epithelial/stromal buttons in organ culture. {Left)
Epithelial/stromal buttons
were quartered and each
section incubated in 350 ^1
of serum-free medium in
wells of a 24-well cluster
plate. Quadruplicate cultures were terminated at 24, 48, and 72 hr. A 10 p\ sample from each culture well was tested by zymography for accumulated gelatinases.
Two-fold serial dilutions were made ofthe last ofthe 72-hr samples. Thefirstsample in the dilution series is undiluted, the second is 2-fold, the
third 4-fold, etc. {Right) When samples were electrophoresed at lower amperage, 65 kD gelatinolytic species could be resolved into two
different subspecies. {Top) The first two gel lanes show 65 kD gelatinase activity produced by culturing cornea! sections from a single rabbit.
The second two lanes show a more rapidly migrating enzyme species produced by corneal organ cultures obtained from a different rabbit.
{Bottom) Thefirsttwo gel lanes show a single 65 kD gelatinase activity produced by culturing corneal sections from a third rabbit. The second
two lanes show that corneal organ cultures obtained from a fourth rabbit produce two subspecies of 65 kD gelatinase activity.
over a 3-day time course in culture (Fig. 1, left). Comparison of the degree of substrate clearing produced
by samples harvested at each time point was made
with a series of twofold dilutions of the last of the
72-hour samples shown in Figure 1. This enabled us
to make a rough estimation ofthe rate of gelatinase
accumulation in culture. The 65-kD species increased only slightly (two- to fourfold) over time. In
contrast, time in organ culture appeared to stimulate
accumulation ofthe 92-kD species. After 24 hours of
culturing, the enzyme activity could just barely be
detected in the conditioned culture medium; however, activity was easily detected after 72 hours (an
increase of about 16-fold). Accumulation of both enzyme species could be inhibited by treating the cultures with the protein synthesis inhibitor cycloheximide (data not shown), indicating that new synthesis
and secretion is a factor determining accumulation of
the gelatinases when corneal buttons are placed in
organ culture.
Characterization of the Gelatinolytic Species
Several criteria were used to characterize the gelatinolytic species. First, zymography was done under
conditions that were optimal for members of the
MMP class of enzymes (neutral pH in the presence of
Ca+2). Second, the enzymatic activity for both proteinase species could be inhibited by incubation of
the zymogram with 10 mM ethylenediaminetetraacetic acid or 10 mM 1,10-phenanthroline, both
MMP inhibitors, but not by phenylmethylsulfonyl
fluoride, a serine proteinase inhibitor (data not
shown). These data are consistent with membership
ofthe gelatinolytic species in the MMP family.
The relationship of the gelatinolytic species with
known members ofthe MMP family was investigated
by using specific antisera. The apparent molecular
size ofthe 92-kD gelatinase suggested that it might be
related to the high-molecular-weight gelatinase, initially identified as the product of macrophages and
neutrophils.2' Monospecific, polyclonal antiserum
raised to human macrophage gelatinase was used for
immunoprecipitation analysis of conditioned medium taken from corneal epithelial/stromal buttons
cultured for 3 days. Precipitated proteins underwent
electrophoresis on a gelatin zymogram. Figure 2 (top,
left) shows that the macrophage gelatinase-specific
antibody could immunoprecipitate the 92-kD gelatinolytic species; both subspecies were precipitated.
This experiment demonstrates the close relationship
between corneal 92-kD gelatinase and macrophage
gelatinase. The apparent molecular size of this enzyme suggests that it is the inactive, proenzyme form.
The macrophage antibody did not immunoprecipitate the 65-kD species. These results show that the
65-kD gelatinolytic species represents an independent enzymatic species and is not a degradation product of the 92-kD species.
The apparent molecular size ofthe 65-kD gelatinolytic species suggested that it might be related to the
enzyme known as type IV collagenase,20 the product
of a different gene from the higher-molecular-weight
gelatinase species.19 Antiserum raised against purified
human type IV collagenase was used to probe a western blot of conditioned medium from corneal buttons that were cultured for 3 days. The samples were
reduced by treatment with /3-mercaptoethanol before
electrophoresis. A 72-kD protein from the culture
medium was bound by the antiserum (Fig. 2? top,
right). A similar 72-kD protein was found in the culture medium conditioned by fourth-passage corneal
fibroblasts. The proenzyme form of type IV collagenase is known to undergo electrophoresis at 72 kD
when reduced, but it shifts to a lower apparent molecular weight when run under the unreduced condi-
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COLLAGENA5ES / GELATINASES SYNTHESIZED DY CORNEA / Fini and Girord
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2
3
-65
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Fig. 2. Immuno-identification of gelatinolytic species produced
by cornea! organ cultures. (Top left) Immunoprecipitation analysis
of 92 kD gelatinolytic activity. Lane 1: Immunoprecipitation analysis, using dog preimmune serum, of a culture medium sample
from corneal organ cultures. No gelatinolytic activity was precipitated. Lane 2: Gelatinases in crude culture medium sample used for
immunoprecipitation in lane 1. The expected gelatinolytic activities are visible at 92 and 65 kD. Lane 3: Immunoprecipitation
analysis of the sample shown in lane 2 using dog antiserum raised
against 92 KD gelatinase (type V collagenase) purified from human
macrophages. (Top right) Western blot probed with rabbit antiserum raised against 72 kD gelatinase (type IV collagenase). Lane 4:
Crude culture medium sample from corneal organ cultures, These
cultures contain a 50 kD protein that cross-reacts with the antibody. However, a 72 kD protein, which is the appropriate size to be
type IV collagenase, is also apparent. Lane 5: Crude sample from
passaged stromal fibroblast culture. Note that the 50 kD crossreacting protein present in organ cultures is not found in cell cultures. The antibody binds only the 72 kD protein. (Bottom) Western blot probed with rabbit antiserum raised against 72 kD gelatinase (type IV collagenase). Lane 1: Size standard. Lanes 2 and 3:
Crude samples from passaged stromal fibroblast culture. The sample was reduced before electrophoresis. Lane 4: Size standard.
Lanes 5 and 6: Crude samples from passaged stromal fibroblast
culture. The samples were not reduced before electrophoresis.
These samples were run in gel lanes distant from the reduced size
standards to prevent reduction that might occur by diffusion of
j3-mercaptoethanol during electrophoresis. Note that the protein
bound by the antibody has shifted to an electrophoretic mobility
appropriate for a protein of 65 kD.
tions required for zymography.19'20 We found that
this is also true of the 72-kD protein from fibroblast
conditioned medium; when the sample was not reduced before electrophoresis, the type IV collagenase
antibody bound a protein of 65 kD (Fig. 2, bottom).
Thus the 65-kD gelatinolytic species seen on zymograms must be produced by an enzyme that is the
same as, or closely related to, type IV collagenase.
Because of its apparent molecular size on reducing
gels, this enzyme will be referred to as 72-kD gelatinase in the rest of this paper.
Cell Origin of the Gelatinolytic MMPs
To identify the corneal cell layer that produces
each gelatinase, corneal stromal and epithelial cells
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were isolated and cultured individually. The tissue
layers of epithelial/stromal buttons were split by
treatment with trypsin. The epithelial cells were then
scraped off the stromata and concentrated by centrifugation. Subsequent treatment of the stromata with
bacterial collagenase freed the fibroblasts from the
stromal matrix, and these cells were also concentrated by centrifugation. Both cell types were plated
in culture using medium containing 10% serum to
facilitate attachment of cells to the culture dish, but
on the next day this medium was replaced with fresh,
serum-free medium. After 24 hr, the cell-conditioned
medium was collected for analysis by gelatin zymography.
Figures 3 and 4 show representative examples of
gelatin zymograms done using conditioned medium
from cells prepared and cultured as described above.
Both 92- and 72-kD gelatinases (their sizes appropriate for the proenzyme form) were found in conditioned medium from primary epithelial cells or primary fibroblasts. However, the relative levels of the
gelatinase species differed in the two types of culture.
Epithelial cell cultures produced predominantly
92-kD gelatinase, whereas fibroblasts cultures produced mostly the 72-kD gelatinase. Conditioned medium from stromal fibroblasts that were subcultured
once before plating for this experiment produced a
ratio of gelatinase species similar to that produced by
primary cultures. With further passage, however, we
found that it becomes more and more difficult to
detect constitutive production of 92-kD gelatinase by
stromal fibroblasts. In our hands, epithelial cells do
not passage well and are rapidly overgrown by fibroblasts with culture passage. This suggests that the
barely detectable levels of 92-kD gelatinase present in
Con
Fig. 3. Gelatinases produced by corneal epithelial cells in primary culture. Samples of culture medium from epithelial cells
plated in wells of a 24-well cluster plate and cultured in serum-free
medium for 72 hr. (Left) Medium from four different untreated
cultures (right) medium from four different CB treated cultures.
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12
3 4
5 6 7 8
»!\ ."111
RFl
RF°
Fig- 4. MMPs secreted by untreated and CB-treated cultures of
primary and passaged stromal fibroblasts. Duplicate cultures of
primary corneal stromal fibroblasts, orfirst-passagecorneal fibroblasts plated in wells of a 24-well cluster plate were left untreated or
treated with CB (5 fig/ml) for 24 hr in serum-free medium. At 20
hr, 15S-methionine was added to pulse label the newly synthesized
and secreted proteins. At 24 hr all samples were harvested and
MMP expression was assayed by zymography or by autoradiography. (Top, left and center) Gelatin zymogram demonstrating 92
and 72 kD progelatinase expression. (Bottom, left and center) Autoradiograms demonstrating procollagenase and prostromelysin expression from the same samples as used for zymography. (Lanes 1,
2)first-passagefibroblasts(RFl) untreated; (lanes 3, 4) first-passage
fibroblasts treated with CB; (lanes 5; 6) primaryfibroblasts(RF°),
untreated; (lanes 7, 8) primary fibroblasts treated with CB. (Bottom, right) Autoradiogram showing immunoprecipitation analysis
of a sample of culture medium containing pulse-labelled secreted
proteins. (Lane 9) proteins immunoprecipitated with nonimmune
serum. (Lane 10) proteins immunoprecipitated with sheep antiserum raised to rabbit collagenase. (Lane 11) proteins immunoprecipitated with sheep antiserum raised to rabbit stromelysin.
conditioned medium from primary cultures might be
due to contamination with low levels of epithelial
cells. Likewise, the low levels of constitutive 72-kD
gelatinase produced by epithelial cells might originate
from contaminating stromal fibroblasts. We are unable to exclude these possibilities entirely. However,
we further attempted to confirm the purity of our
cultures for each of the cell types.
To substantiate epithelial cell purity further, we
prepared epithelial cells by a more stringent method.
The tissue layers of epithelial/stromal buttons were
split using the enzyme preparation, Dispase II, instead of trypsin. Dispase treatment leaves the epithelial sheet intact and does not break the bonds between
individual cells.25 After enzyme treatment, the epi-
Vol. 01
thelial sheet was peeled from the stroma and lifted
gently out of the medium with forceps. This method
of purifying epithelium effectively separates it from
any contaminating fibroblasts that might be released
from the stromata during enzyme treatment and
which would subsequently copurify with the epithelial cells when they are concentrated by centrifugation. Pieces of epithelial sheet isolated in this way
were placed in culture (with 10% serum) for several
days until cells spread from the sheet and attached to
the culture well; then the cultures were changed to
serum-free medium. After 24 hr, secreted proteins
were assayed by zymography. Cultures established in
this way secreted both the 92- and the 72-kD gelatinase species in approximately the same ratios as cultures established by using trypsin to separate the epithelial layers (data not shown).
Pure primary cultures of stromalfibroblasts,unlike
passaged cultures, do not secrete collagenase constitutively, and cannot be induced to secrete this enzyme in response to treatment with CB. However,
even a low level of contamination of primary stromal
fibroblasts by epithelial cells can provide permissive
conditions for CB induction of collagenase expression in primary fibroblast cultures. This is thought to
be because epithelial cells produce a cytokine that is
essential for induction.30 Thus, as a way to test the
purity of fibroblast cultures further, we determined
whether collagenase expression could be induced by
CB. In the experiment shown in Figure 4, equal numbers of primary fibroblasts or fibroblasts that had
been subcultured a single time were plated in medium containing 10% serum in the wells of a 24-well
cluster plate. The next day, the medium was changed
to serum-free, and CB was included in half of the
culture wells at 5 mg/ml. 35S-methionine was added
to the culture medium after 20 hr and newly synthesized cell proteins were pulse-labeled for the next 4
hr. At the end of this time, crude medium was collected and analyzed by gel electrophoresis. The passaged cells constitutively synthesized and secreted a
53- and 51 -kD protein into the culture medium, both
of which were induced in response to CB treatment.
Immunoprecipitation analysis (Fig. 4) demonstrated
that the 53-kD protein was collagenase and the 51-kD
protein was stromelysin; the molecular sizes of these
proteins indicate the proenzyme form. In addition, a
57-kD protein, which appeared after CB treatment,
bound collagenase antibody; this protein is the appropriate size to be the glycosylated form of procollagenase.26 The CB treatment induced expression of
procollagenase proteins by sixfold and prostromelysin by fourfold as determined by densitometry. In
contrast to the passaged cell cultures, primary cells
made no detectable procollagenase or prostromelysin
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constitutively and could not be induced to make the
enzymes in response to CB. These data suggest that
our primary fibroblast cultures are free of contaminating epithelial cells.
The CB also had no obvious effect on gelatinase
accumulation in culture medium from primary epithelial cells (Fig. 3) or in medium from primary stromal fibroblast cultures (Fig. 4). A small decrease in
72-kD gelatinase expression could be visualized after
CB treatment in the experiment shown in Figure 3. A
small increase in accumulation of 92-kD gelatinase,
but not 72-kD gelatinase, in conditioned medium
from passaged stromal fibroblasts could be seen after
treatment with CB in the experiment shown in Figure
4. These were two of the largest changes we found
after many repetitions of these cell treatments. Generally, CB treatment caused little or no apparent
change in gelatinase accumulation in cell culture medium.
72-kD Progelatinase is a Normal Component
of Corneal Stromata In Situ
Tissue injury produced by dissection when isolating corneal tissue might be the stimulus that induces
expression of corneal gelatinases in organ culture.
Thus, it was important to learn whether the gelatinases could be found in normal, uninjured corneas.
The three tissue layers of cornea were manually dissected from freshly prepared corneal buttons, and
Fig. 5.72 kD progelatinase can be extracted from normal corneal
stroma. Gelatinolytic activities recovered from SDS-extracted corneal tissues. (Lane 1) extract from epithelial sheet. (Lane 2) extract
from corneal stroma; (lane 3) gelatinases from culture medium of
passaged stromalfibroblasts:92 = 92 kD progelatinase, 72 p = 72
kD progelatinase, 72 a = gelatinase activity found in culture medium after storage. The size of this species suggests that it is the
activated form of 72 kD progelatinase. Note that both proenzyme
and activated forms of 72 kD gelatinase can be extracted from
normal stroma, but no detectable 92 kD progelatinase can be extracted from either tissue.
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stromal and epithelial tissues were extracted with
2.0% SDS. A sample of the solubilized material underwent electrophoresis on a gelatin zymogram. Figure 5 shows that stroma contained easily detectable
levels of 72-kD progelatinase. In addition, a small
amount of a gelatinolytic species, about 8 kD less in
molecular weight, was also visible. This is an appropriate size to be the activated form of 72-kD gelatinase.19 No detectable enzymatic activity could be extracted from epithelium.
Discussion
This is the first report identifying the MMP species
of the gelatinase class synthesized by corneal cells.
Two different enzymes, visualized as 92- and 65 kD
on gelatin zymograms, are synthesized by corneal epithelial/stromal buttons in serum-free organ culture.
We showed that these enzymes have properties similar to members of the MMP family; their apparent
molecular sizes on zymograms suggested that they
might be identified as specific members of the gelatinase subclass of this enzyme family. Immunologic
analysis further substantiated our identification. The
larger of these enzymes species is biochemically and
antigenically related to the gelatinase synthesized by
macrophages and neutrophils;21 this enzyme has also
been called both type IV collagenase19 and type V
collagenase.21 The second gelatinase species is related
to the product of a different gene, but it has also been
called type IV collagenase.20 Each of these enzymes is
known to have the common capacity to degrade types
IV, V, and VII collagen and denatured collagen.18 A
nomenclature for the increasingly numerous
members of the MMP family has not yet been established and accepted by investigators in the field (although a system has been proposed).31 Thus, for the
time being, we chose to refer to the corneal gelatinases as 92-kD gelatinase and 72-kD gelatinase to emphasize that they are different enzyme species which
are the products of different genes, but that they have
similar, if not identical specificities against ECM substrates. The molecular size of each enzyme is that
observed on reducing gels. We showed that the 65-kD
gelatinase visualized by gelatin zymography shifts to
72 kD when reduced. In primary cell culture studies,
we found that corneal cells show tissue-specific differences in the enzyme species that they produce.
Corneal stromalfibroblastssynthesize predominantly
the 72-kD gelatinase whereas epithelial cells synthesize mostly 92-kD gelatinase. However, each cell type
appears to produce small amounts of the other enzyme. The apparent molecular size of each enzyme
species on polyacrylamide gels is indicative of the
inactive proenzyme form.
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Expression of 72-kD gelatinase in organ culture
seems to be a simple continuation of expression in
vivo since this enzyme could be extracted from the
corneal stroma even without culturing. The finding
that cells from normal, uninjured corneas express a
collagenolytic MMP is surprising. It is known that the
MMP, type I collagenase, is not expressed by the cells
of uninjured cornea.23 In fact, it is generally recognized that the presence of collagenase in a tissue is
correlated with the events of matrix turnover.32 Yet
there seems to be very little matrix turnover in normal corneal stroma which is an unusually static tissue.33 Despite its uniqueness, however, the presence
of 72-kD gelatinase in the stromal tissue is not inconsistent with the lack of matrix degradation since most
of the gelatinolytic activity isolated has a molecular
weight appropriate for the inactive, proenzyme form
of the enzyme. Some of the enzyme isolated from the
tissue had a molecular weight consistent with activation. However, the collagenascs are known to be sensitive to freeze-thawing,16 and this or other steps during the isolation procedure could have caused this
change in molecular weight.
What might be the role of 72-kD progelatinase in
normal tissue not undergoing remodeling? It may be
pertinent that the gelatinases are unique among the
MMPs in their capacity to bind to their substrates
even when in the inactive proenzyme form.1922 The
corneal stroma contains a substrate for 72-kD gelatinase; stromal collagen fibrils, at least in the chick, are
composed of copolymerized type I and V collagens.12
Perhaps 72-kD progelatinase might serve a surveillance function; bound to its type V substrate in the
stroma, it would be perfectly situated to facilitate removal of denatured collagens when needed and when
conditions were appropriate for its conversion to an
active enzyme. In addition, again considering the
unique binding properties of the proenzyme, it might
have a novel, nonenzymatic role, possibly participating in assembly of any new collagen fibrils when required.
No detectable 92-kD gelatinase could be extracted
from stroma, consistent with our observation in primary culture that stromal fibroblasts produce very
little of this enzyme. Extracts from normal corneal
epithelium demonstrated no gelatinase activity of
any type. Again, this is consistent with our organ-culturing experiments; after 1 day of culture, the enzyme
was essentially undetcctable in culture medium. With
increased time in organ culture, however, expression
of 92-kD gelatinase was stimulated. Expression of
this enzyme was even more easily detectable in primary epithelial cell culture. Wounding of the corneal
epithelium during its removal from the animal for
organ culture or damage to the epithelial sheet that
Vol. 31
occurs when cells are separated for primary culture
might initiate expression of 92-kD gelatinase. Perhaps wounding of the epithelial sheet might also stimulate expression of 92-kD gelatinase in vivo. Considering its capacity for degradation of denatured collagens of all types, it seems reasonable to propose a
role for 92-kD gelatinase in facilitating removal of
damaged collagens after wounding to clear the path
for resurfacing of the cornea by the epithelial sheet.
In the rabbit, small amounts of type IV collagen are
deposited under corneal epithelial cells when they
migrate over a wound surface devoid of basement
membrane;34 this material gradually disappears with
time after wounding. Our discovery of the 92-kD gelatinase produced by corneal epithelial cells suggests
the enzyme that might be responsible for removal of
the type IV collagen. Both types IV and VII collagen,
another substrate for gelatinases,35 are generally recognized to be important components of epithelial
basement membranes. This suggests that epithelial
gelatinase might also participate in remodeling of the
basement membrane of corneal epithelium after
wounding. Surprisingly, however, little or no type IV
collagen has been localized to this structure in the
rabbit,34 guinea pig, or chick,36 and only a small
amount of type IV collagen has been detected in the
basement membrane of the human cornea.13 This is
curious since the basement membrane of the cornea
is similar in ultrastructure to other basement membranes. Possibly, in the cornea, most type IV collagen
is replaced by a functional homologue—maybe type
VII collagen, which is unusually abundant in corneal
basement membrane.37 Type VII collagen forms the
anchoring fibrils linking the basement membrane to
the underlying connective tissue in the cornea,37 as in
other squamous epithelial tissues.
Although controlled expression or activation of
gelatinases might facilitate homeostatic processes,
overexprcssion or inappropriate activation of these
enzymes could have detrimental effects. Gelatin-degrading and type IV collagen-degrading activities
have been detected in cells taken from keratoconus
corneas.3839 A gelatin-degrading activity was also observed in cultures begun from ulcerating corneas.40
Clinicians have long observed that persistent or recurrent epithelial defects almost always precede stromal
ulceration.41 Adhesion of corneal epithelium to the
stromal layer is thought to be mediated through components of the epithelial basement membrane. It is
generally recognized that even when epithelial defects
are present, stromal ulceration does not occur until
after the epithelial basement membrane dissapears.10
We identified here, for the first time to our knowledge, enzymes produced by epithelial cells and stromal fibroblasts that have the capacity to degrade the
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COLLAGENASES / GELATINASES SYNTHESIZED DY CORNEA / Fini ond Girord
collagenous components of basement membrane. We
propose that the gelatinases play an important role
during development of the epithelial defect which
leads to corneal ulceration. The factors that control
expression and activation of the gelatinases by corneal epithelial cells and stromal fibroblasts will be
important to determine.
Key words: collagenase, gelatinasc, metalloproteinasc basement membrane, cornea
Note added in proof: After acceptance of this manuscript, we became aware of a publication describing a 65 kD gelatinolytic metalloproteinasc produced by cultured fibroblasts from corneal stromia (Kcnney ct al., Biochem Biophys Res Commun 161:353,
1989). This enzyme is probably identical to the one that we have
identified as 72 kD gelatinase.
13.
14.
15.
16.
17.
18.
Acknowledgments
19.
The authors thank Constance Brinckerhoff, PhD, Karen
Hasty, PhD, William Stetler-Stevcnson, PhD, and Lance
Liotta, PhD, for providing antisera. They also thank Jerome Gross, MD, Irene Kuter, MD, and Hideake Nagase,
PhD, for helpful discussions and Johnna Corin for her excellent technical assistance. Portions of this work were previously presented in abstract form.42
20.
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