ARTICLE

Matrix Metalloproteinase Activity Correlates With

Blastema Formation in the Regenerating MRL
Mouse Ear Hole Model
Dmitri Gourevitch, Lise Clark, Pan Chen, Alexander Seitz, Stefan J. Samulewicz, and Ellen Heber-Katz
*
The MRL mouse was proposed as a model of mammalian regeneration because it can close ear holes completely with
the restoration of normal tissue. This regeneration process involves the formation of a blastema during healing, the
re-appearance of cartilage and hair follicles, and healing without scarring. Such a process requires extensive tissue
remodeling. To characterize differences in ear wounding responses between regenerating and nonregenerating mice,
we examined and compared the extracellular matrix remodeling and the matrix metalloproteinase (MMP) and tissue
inhibitor of metalloproteinase (TIMP) response in the MRL and C57BL/6 mouse strains after injury. We found a
correlation between the MRLs ability to break down the basement membrane, form a blastema, and close ear hole
wounds and an inammatory response with neutrophils and macrophages seen in the ear after injury. These cells
were positive for MMP-2 and MMP-9 as well as TIMP-2 and TIMP-3. Clear differences between the MRL and B6
response to injury were seen that could explain the differences in healing and blastema formation in the MRL and lack
of it in the B6 mice. This nding was further supported by enzyme activity as determined by gelatin zymography.
Developmental Dynamics 226:377387, 2003. 2003 Wiley-Liss, Inc.
Key words: collagen type I and IV; MMPs; MRL mice; regeneration; TIMPs
Received 15 August 2002; Accepted 7 November 2002
INTRODUCTION
The biological response to traumatic
injury in higher organisms can be
subdivided into two general classes:
regenerative and nonregenerative
types of wound healing (Gross, 1996;
Stocum, 1996). In the mammalian
adult, the healing process is normally
accomplished by the replacement
of mature cells, but not organs,
and the formation of scar tissue
(Clark, 1996). In studying through-
and-through ear hole wounds and
their closure, we found both pheno-
typic (Clark et al., 1998; Heber-Katz,
1999; Samulewicz et al., 2002) and
genotypic (McBrearty et al., 1998;
Blankenhorn et al., in press) differ-
ences in the wound healing re-
sponse of MRL vs. C57BL/6 (B6) adult
mice. Healing in the MRL appeared
fetal-like (Hopkinson-Woolley et al.,
1994; Armstrong and Ferguson, 1995)
with the formation of a blastema,
scarless healing, and with the re-
placement of lost tissue such as car-
tilage by functionally and architec-
turally normal tissue. This nding was
not true for the B6 strain, which
scarred at the margins of the wound
with minimal hole closure. Thus, this
experimental model is regenerative
rather than repair and is one more of
the rare examples of regeneration
among mammals including the re-
placement of antlers (Goss, 1970),
digits (Douglas, 1972; Illingworth,
1974; Borgens, 1982; Neueld, 1989),
and closure of rabbit ear-holes (Goss
and Grimes, 1975).
The breakdown of the extracellu-
lar matrix (ECM) is a critical event in
wound healing (Clark, 1996; Werb,
1997; Murphy and Gavrilovic, 1999).
Matrix metalloproteinases (MMPs)
are part of a general protease sys-
tem that includes serine and metal-
lo- and cysteine proteinases and
play a key role in ECM breakdown
and in a variety of normal and
pathologic processes (Matrisan,
The Wistar Institute, Philadelphia, Pennsylvania
Grant sponsor: NIH; Grant numbers: AI42395; AI07518; DE13359.
*Correspondence to: Ellen Heber-Katz, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104.
E-mail: heberkatz@wistar.upenn.edu
DOI 10.1002/dvdy.10243
DEVELOPMENTAL DYNAMICS 226:377387, 2003
2003 Wiley-Liss, Inc.
1992; Parks and Mecham, 1998).
They are involved in development
(Hay, 1984; Chin and Werb, 1997) as
well as in regenerative processes
(Grillo et al., 1968; Yang and Bryant,
1994; Stocum, 1995; Chernoff et al.,
2000).
MMP-2 and MMP-9 (known as
type IV and V collagenases or the
72-kDa gelatinase A and 92-kDa
gelatinase B, respectively) are se-
creted by several cells during ECM
remodeling (Parks, 1999; Steffensen
et al., 2001). The level of MMP-2
and MMP-9 expression is up-regu-
lated in the wound stroma due to
migratory broblasts. These cells
have the potential to cleave and
degrade type IV collagen in the
basement membrane and collag-
ens type I and III in the ECM. MMPs
are not expressed, however, by
stationary tissue broblasts (Hakki-
nen et al., 2000). Neutrophils
(Schwartz et al., 1998) and macro-
phages inltrating through vessel
walls and vascular basement
membrane are key producers of
MMPs in wound healing with these
molecules also being important in
phagocytosis (Yong et al., 1998).
The MMPs are secreted as inactive
zymogens (pro-form) and activated
by proteolytic cleavage of their N-
terminus (Parks and Mecham, 1998).
This process is carried out by the pro-
tease MT1-MMP (Okada et al., 1997).
A family of specic endogenous tis-
sue inhibitors of MMP (TIMP) con-
trol MMP cleavage leading to
enzymatic activity (Woessner, 1991;
Baker and Leaper, 2000). This family
has four members: TIMP-1, TIMP-2,
TIMP-3, and TIMP-4. TIMP-1 is a strong
inhibitor of all MMPs, can bind to
MMP-9, and be secreted from the
cell as a complex (Clark, 1996).
TIMP-2 preferentially interacts with
MMP-2 (Boone et al., 1990; Howard
et al., 1991) and TIMP-3 with MMP-9
(Suomela et al., 2001). In both of
these cases, TIMP interactions block
MMP catalytic activity but have no
effect against other types of protein-
ases. In addition, TIMPs have been
reported to have an ability to pro-
mote growth in a variety of human
and bovine cell types (Hayakawa et
al., 1992).
In the studies presented here, we
have shown the disappearance of
the ECM and basement membrane
in the healing MRL ear wounds be-
fore blastema formation and have
examined MMP-2 and MMP-9 ex-
pression, activity, and cellular loca-
tion during ear hole closure. We
have similarly examined TIMP-2 and
TIMP-3 expression. We have shown
that there are clear differences be-
tween the MRL and B6 response to
injury which could explain the differ-
ences in healing and blastema for-
mation in the MRL and lack of it in
the B6.
RESULTS
Breakdown of the ECM After
Ear Punching
Ear hole punches were made in the
ear pinnae. At various times after in-
jury, ears were removed, xed in 10%
formalin, sectioned through the ear
hole, and stained with hematoxylin
and eosin. Because eosin binds to
protein and is uorescent, changes
in protein levels and distribution in
the ECM and basement membrane
can be visualized.
The rate of ear hole closure in the
MRL accelerates starting at approx-
imately day 5 (Clark et al., 1998; He-
ber-Katz, 1999). As seen in Figure
1AH, epiuorescence microscopy
showed ECM and basement mem-
brane buildup and breakdown that
was different between the MRL and
B6 healing ear. The amount of uo-
rescent material was greater on day
4 in the B6 (Fig. 1A) than the MRL
(Fig. 1B) at this same time point, al-
though in both cases, the basement
membrane dividing the epidermis
and dermis is clear. By day 5, much
of the thick ECM uorescence is
gone in the B6 (Fig. 1C), although
the basement membrane is still
present. On the other hand, the
basement membrane in the MRL
can no longer be seen (Fig. 1D). On
days 6 and 7, the B6 still shows a
strong basement membrane (Fig.
1E,G), whereas the MRL has little or
none (Fig. 1F,H). This is an intriguing
result which might be accounted for
by the action of proteases.
Protease Expression During
Healing
Our initial studies examined RNA ex-
tracted from tissue around the cir-
cumference of the healing ear hole
from the B6 and MRL mouse on days
0 and 5 after wounding. Microarray
analysis (Clontech arrays) indicated
that MMP-2 was up-regulated in the
MRL healing ear hole tissue com-
pared with the B6 on day 5 after
injury and TIMP-3, an inhibitor of
MMP-9, was lower in the MRL ear
hole tissue compared with B6 on
both days 0 and 5 (data not shown),
suggesting a role for MMP-9 as well
as for MMP-2. Because MMP-2 and
MMP-9 have similar functions and
have been shown to work in combi-
nation in many diverse systems
(Mackay et al., 1990; Matsubara et
al., 1991; Fini et al., 1995; Trengove et
al., 1999; Itoh et al., 2002), we fo-
cused on both MMP-2 and MMP-9
expression during healing.
Enzymatic Activity by Using
Zymography
Zymography is an electrophoretic
technique that allows the separa-
tion of molecules by their molecular
weights and their ability to break
down given substrates that are in-
corporated into the running gel. This
method requires the use of nonde-
naturing conditions for the display of
enzymatic activity.
Protein extracted from the healing
tissue of the ear holes on days 0, 1, 3,
5, and 7 after ear punching was ex-
amined for both MMP-2 and MMP-9
by using a gelatin (denatured colla-
gen) substrate. Zymograms showed
multiple enzymatic bands consistent
with the pro-form (92105 kDa) and
the active form (8789 kDa) of
MMP-9 (Fig. 2A). On day 0, the pro-
form of MMP-9 is seen in both strains.
After injury, the MRL expressed more
pro- and active form of MMP-9 than
B6 at all time points.
Proteolytic bands for MMP-2 were
seen on gelatin substrate gels at 72
kDa (pro-form) and 6668 kDa (ac-
tive form). Pro- and active forms of
MMP-2 were seen on day 0 in both
strains. After injury, MMP-2 activity
was signicantly less than MMP-9 ac-
tivity in general; however, the MRL
378 GOUREVITCH ET AL.
Fig. 1. Eosin staining and epiuorescence showing the extracellular matrix. Ear punches were made in the center of the ear pinnae,
creating 2-mm through-and-through wounds. Sections were made through the holes and stained with hematoxylin and eosin. Epiuo-
rescence using a broad-spectrum uorescein isothiocyanate lter was used to generate the pictures seen. Sections from B6 and MRL ears,
respectively, on day 4 (A,B), day 5 (C,D), day 6 (E,F), and day 7 (G,H) can be seen at a 20magnication. The presence of the basement
membrane between the epidermis and dermis can be seen (white arrowheads) as well as its absence (black arrowheads).
PROTEASE ACTIVITY IN THE MRL MOUSE EAR 379
expressed a higher level of the ac-
tive form of MMP-2. Relative levels
of activity as determined by the
amount of protein loaded, and the
zymogram band density (see Fig.
2A) can be seen (Fig. 2D). Similar
results were seen by using collagen
type IV as a substrate (Fig. 2C), al-
though only MMP-9specic bands
could be seen. Other bands were
also seen on gelatin gels, and in all
cases, MRL expressed higher levels
than B6. Such bands were found at
approximately 115 to 130 kDa (data
not shown). With higher concentra-
tions of protein run on a gelatin sub-
strate, bands were also seen be-
tween 30 and 45 kDa (Fig. 2C).
Cellular Localization of
Proteases
The same time points as those
above were used to detect specic
molecules in parafn-embedded tis-
sue sections. Antibodies shown to be
specic for MMP-2 (Fig. 3A,B) and
MMP-9 (data not shown) as well as
antibodies to TIMP-2 (Fig. 3C,D) and
TIMP-3 (data not shown) were used.
Little staining in normal ear sections
was seen. After wounding, stained
cells were found near the wound site
with a dramatic increase in cell
number 1 to 3 days after wounding
and then a continual decrease until
day 7 after wounding (Figs. 3, 4). The
staining was localized mainly to a
population of small cells. As can be
seen in Figure 4, the number of cells
expressing MMP-2 was lower in the
B6 than in the MRL at all time points,
although the expression of TIMP-2
was nearly identical. MMP-9posi-
tive cells were greater in number in
the MRL, and TIMP-3positive cells
were greater in the B6.
The data in Figure 4 are re-ex-
pressed in Figure 5 to allow a direct
comparison of the number of MMP-
and TIMP-positive cells in each strain.
In the B6 ears, the ratio of MMP-2 to
TIMP-2positive cells and MMP-9 to
TIMP-3positive cells was less than 1
from days 1 to 7 after wounding. On
the other hand, in the MRL, the ratios
of MMP-2 to TIMP-2positive cells
and MMP-9 to TIMP-3positive cells
were always greater than 1 from
days 1 to 7. If these ratios (Table 1)
represent functional TIMP and MMP
activity in wounds, then this nding
would indicate that the MRL should
have signicantly more active form
of MMP compared with the B6 (see
Fig. 2D). This would explain the more
rapid breakdown of the ECM and
basement membrane observed in
the MRL.
Further examination of the cell
types expressing these molecules
(Fig. 6A,B) showed that the ratios
shown above held true for small cells
(5.6 microns 0.8; Fig. 3B, upper in-
set). Immunospecic double label-
Fig. 2. Zymography showing proteolytic matrix metalloproteinase (MMP) activity. Zy-
mography was carried out on nondenatured protein from the tissue of healing ear holes
by using gelatin (A,C) and collagen type IV (B) substrates. Samples (6 g of total protein)
were adjusted for protein concentration by OD
750
readings (Lowry protein assays, data not
shown) and conrmed by running equal aliquots of the same samples on the zymography
gels (A) and on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels,
which were then stained with Coomassie blue (not shown). Bands at 97 kDa are likely to be
the pro form of MMP-9, and the bands just below that are the active form of MMP-9. Bands
at 72 kDa are appropriate for the pro form of MMP-2, and the bands below that are the
active form of MMP-2. At higher concentrations of sample (approximately 12 g protein/
sample), bands below MMP-2 can be seen by using a gelatin substrate (C). Lanes in A,B
contain protein from B6 day 0 (lane 1), MRL day 0 (lane 2), B6 day 1 (lane 3), MRL day 1
(lane 4), B6 day 5 (lane 5), and MRL day 5 (lane 6). In C, lanes correspond to B6 (lanes
1,3,5,7,9) and MRL (lanes 2,4,6,8,10) on days 0, 1, 3, 5, and 7. The bands seen in C are lower
than both MMP-2 and MMP-9 and have not been identied. D: Relative activity derived
from A can be seen. This was determined by measuring the density of a given band size
by using ImageQuant 5.1 (Molecular Dynamics) and dividing that by the amount of
protein as determined by OD
750
measurements of the samples loaded.
380 GOUREVITCH ET AL.
ing of these cells found in the ear
after wounding with antiMMP-2
and antiLy-6G showed that approx-
imately 80% of these cells were
Ly-6G positive, a neutrophil-specic
antibody (Fig. 7), supporting the no-
tion that these were inammatory
cells and that almost all of the neu-
trophils were MMP-2 positive. This
nding is consistent with the time of
arrival, the specic morphology, and
the localization in the wound.
A minority population of cells that
were considered large cells (14.4 mi-
crons 2.1; Fig. 3B, lower inset) were
shown to be approximately 30%
mast cells by staining with toluidine
blue, and approximately 70% were
macrophages as stained with F4/80
(data not shown). However, the large
cells made up only approximately
10%of the total cells stained. This large
cell population was both greater in
number in the MRL and was higher in
MMP-2positive cells and lower in
TIMP-2positive cells from days 5 to 7
compared with the B6.
In addition to inltrating cells, anti
MMP-9 antibody stained keratino-
cytes in the epithelial cell layer as
well as broblasts and the ECM
(data not shown). MMP-2 expression
was seen mainly in broblasts and
ECM. MMP-2 (see Fig. 3B, arrows)
and MMP-9 expression is highest at
the apex of the closing wound and
peaks on day 1, similar to the kinetic
pattern seen with the neutrophil inl-
trates.
RNA Expression Levels
RNA from healing ear hole tissue was
extracted and real-time reverse
transcription-polymerase chain re-
action (RT-PCR) was carried out on
these samples. MMP levels were nor-
malized to glyceraldehyde-3-phos-
phate dehydrogenase (GAPDH)
levels for each sample. MMP-9 ex-
pression levels follow that seen in
protein expression levels in the zymo-
grams and also that seen from the
immunohistochemistry and the num-
ber of small cells appearing in the
ear after injury (Fig. 8B). MRL levels
are at most twofold higher than B6
levels. This nding correlates well
with the neutrophils inltrating the
wound site that are synthesizing
MMP-9.
On the other hand, MMP-2 RNA
expression levels are highest in un-
wounded ears and especially in the
MRL (Fig. 8A). This nding follows the
results from the zymograms, which
show the presence of active MMP-2
proteinase in uninjured ears. How-
ever, it does not follow the protein
expression levels in the early inam-
matory cells as determined by im-
munohistochemistry and suggests
that these cells express protein be-
fore they arrive.
DISCUSSION AND CONCLUSION
Vertebrates exhibit several types of
progressively more complex repair
and regenerative processes: wound
repair, tissue regeneration, and re-
Fig. 3. Ear immunohistochemistry. Ears were punched, and sections through the ear hole were analyzed at various time points early after
injury. Antibodies to matrix metalloproteinase (MMP) -2 and MMP-9 tissue inhibitor to metalloproteinase (TIMP) -2, and TIMP-3 were used
and binding visualized through the use of diaminobenzidine. Shown here is a representative ear section at 10 magnication for A and
B and 20 magnication for C and D, with the binding of MMP-2 in B6 (A) and MRL (B) ears and TIMP-2 in B6 (C) and MRL (D) ears, all 1
day after ear hole punching. In B, broblasts and possibly extracellular matrix are positive for MMP-2 (arrowhead) and small (upper inset)
and large (lower inset) inammatory cells (at 100 magnication) are MMP-2positive (arrows).
PROTEASE ACTIVITY IN THE MRL MOUSE EAR 381
generation of entire organs (Gross,
1996; Stocum, 1995, 1996). Wound
healing involves inammation, re-
epithelialization, matrix deposition,
and tissue remodeling along with
angiogenesis and the formation of
granular tissue, and all of these pro-
cesses require the concerted action
of various proteolytic enzymes, in-
cluding matrix metalloproteinases
(MMP). It has been shown that MMPs
play an important role in the break-
down of the provisional matrix
formed after wounding by breaking
down several components of the
ECM, including type IV collagen, -
bronectin, laminin, entactin, and
elastin, as well as clarifying the cel-
lular and bril debris that surround
the wound bed (Baker and Leaper,
2000). This helps keratinocytes and
broblasts to migrate through the
basement membrane as well. There
is a signicant amount of literature
showing the role of MMPs in the
breakdown of the ECM in regenera-
tion models. They include the forma-
tion of the newt limb blastema (Grillo
et al., 1968; Dresden and Gross,
1970; Miyazaki et al., 1996) as well as
the axolotl limb regenerate (Yang
and Bryant, 1994). MMPs have been
shown to play a role in tail resorption
during metamorphosis of tadpoles
(Gross, 1966) and are regulated by
thyroid hormone (Oofusa and Yo-
shizato, 1991; Damjanovski et al.,
2000; Jung et al., 2002). After injury,
planaria release MMPs, which are
probably involved in remodeling
and regrowth of their body parts
(Sawada et al., 1999).
All enzymes of the MMP family
have several common character-
istics. They have a highly con-
served zinc-binding region (sequence
-HEXGHXXGLXH) that associates with
Zn
2
upon activation and a TIMP-
binding regulatory domain. Another
domain is the N-terminus (PGCGVPD)
pro-domain that is responsible for
maintaining the enzyme in an inac-
tive (zymogene) form. Among the
family of zinc-containing protein-
ases, different classes can be distin-
guished according to their substrate
specicity. All enzymes of the MMP
family also contain a C-terminal 22-
kDa domain that has structural ho-
mology to hemopexin. This domain is
involved in the modulation of MMP-
Fig. 4. Comparison of positive cells after immunohistochemical staining. Data were
generated from the immunohistochemical staining of ear sections after wounding as seen
in Figure 3. The gures show the number of cells positive for matrix metalloproteinase
(MMP) -2 and MMP-9 staining between MRL and B6 ears. A similar comparison of tissue
inhibitor of metalloproteinase (TIMP) -2 and TIMP-3 staining between MRL and B6 ears is
shown. The area of tissue examined includes the original ear tissue (0.3 mm of cartilage)
plus any new tissue formed.
Fig. 5. A direct comparison of matrix metalloproteinase (MMP) and tissue inhibitor of
metalloproteinase (TIMP) -positive cells. The number of MMP-2 and TIMP-2 positive cells are
directly compared in B6 and MRL ears over a 7-day time period after injury. A similar
comparison of MMP-9 and TIMP-3 in B6 and MRL ears is also shown (see Figs. 3, 4).
382 GOUREVITCH ET AL.
TIMP interaction and determines
substrate specicity (Clark, 1996).
MMP-2 and MMP-9 are the only two
proteases that degrade gelatin (de-
natured collagen), basal lamina col-
lagens (type IV, V, VII, X, XI), and the
proteoglycan core protein. MMP-2
and -9, therefore, can breakdown
the components of the basement
membrane, including laminin, colla-
gen type IV, and bronectin (Yong
et al., 1998).
The results presented here show
rst that both ECM and basement
membrane breakdown in the heal-
ing ear hole precedes the formation
of the blastema. This timing is true for
the MRL mouse, which goes on to
close its ear hole, to heal without
scarring, and to regenerate lost car-
tilage (Clark et al., 1998; Heber-Katz,
1999). The B6 mouse ear, on the
other hand, displays an intact base-
ment membrane, does not develop
a blastema, and shows scar forma-
tion without ear hole closure (Clark
et al., 1998; Heber-Katz, 1999). Both
the MRL and B6 mouse generate an
inammatory response after wound-
ing with cells that are MMP-2 and
MMP-9positive. These same cells
also express TIMP-2 and TIMP-3. How-
ever, the MRL shows more inltrates
and also more MMP-positive cells.
Perhaps more important, however, is
that the number of TIMP-positive
cells is greater in the B6 than in the
MRL, providing a situation in which
inhibition of the MMPs is more likely in
the B6. That this is in fact the case is
suggested by the zymography of
healing ear tissue showing that
MMP-9 and to a lesser extent MMP-2
in the MRL is far more active. Why
MMPs express at a higher level and
the TIMPs at a lower level in the MRL
is not clear but has been attributed
to the function of both TNF and IL-1
(Ito et al., 1990; Murphy et al., 1994).
It has been reported that MRL mac-
rophages are dysregulated in these
two cytokine responses (Hartwell et
al., 1995; Alleva et al., 1997).
The predominant population of
MMP-positive cells that appear in
the ear after wounding are small
cells, which are mainly neutrophils
and appear early after injury. These
cells appear to be actively synthesiz-
ing MMP-9 but not MMP-2 as seen in
the RT-PCR results (Fig. 8), although
there is clearly MMP-2 protein in
these cells as shown by double-la-
beling experiments (see Fig. 7). This
nding is unusual, as it is generally
thought that neutrophils are not
MMP-2positive. Furthermore, these
cells also make TIMPs.
Because TIMPs are inhibitors of
MMPs and both groups of molecules
are secreted into the ECM, the MMP
to TIMP ratio as measured in the ex-
tracellular wound bed (Ladwig et
al., 2002) has been considered a
proxy for MMP effectiveness. We an-
alyzed our data similarly, but instead
of examining secreted molecules,
we analyzed immunohistochemi-
cally positive cells, and specically
neutrophils. This analysis was consis-
tent with the level of active MMPs
found through zymography. Further-
more, this supports the cell surface
activation of MMPs and their inter-
action with TIMPs (Brooks et al., 1996;
Butler et al., 1998; Steffensen et al.,
1998; Miyamori et al., 2001).
Recent data from our laboratory
shows that not only is ECM break-
down important in the regenerative
response in the MRL ear but also in
the MRL heart (Leferovich et al.,
2001). Thus, hydroxyproline as a
measure of scar formation is in-
creased in the B6 heart where the
scar response is predominant and is
decreased in the MRL heart where
cardiomyocyte proliferation and lit-
tle scarring is seen. In a third model
system, lack of scar formation corre-
lates with axonal regeneration in a
spinal cord injury model in B6 mice
(Seitz et al., 2002). In the case of the
ear hole, we propose that the MRL
healing response is similar to what is
seen in the amphibian limb. Initial
events after wounding resemble
wound repair such as the formation
of the provisional matrix and epithe-
lial migration over the wound site
and is similar in the MRL and B6
mouse (Clark, 1996) as well as the
amphibian (Repesh and Oberpriller,
1978,1980; Stocum, 1995). After this,
differences are seen. In the mouse,
a basement membrane is formed,
whereas in the amphibian, it is not
(Repesh and Oberpriller, 1980). How-
ever, the data shown here suggest
that the basement membrane at
approximately 5 days after injury is
selectively broken down in the MRL
mouse by the two basement mem-
branedegrading MMPs, MMP-2
and -9 (Mackay et al., 1990). This
process should then allow for epithe-
lialmesenchymal interactions lead-
ing to the growth of the blastema as
seen in the amphibian limb (Stocum
and Dearlove, 1972; Globus et al.,
1980; Stocum and Crawford, 1987;
Brockes, 1997; Christensen et al.,
2002) as well as during development
(Sun et al., 2002).
Finally, a genetic analysis of the
wound healing trait described in this
study has shown that ear hole clo-
sure is a highly complex trait with ap-
proximately 20 loci currently de-
scribed (McBrearty et al., 1998;
Masinde et al., 2001; Blankenhorn et
al., in press; Heber-Katz et al., manu-
script submitted for publication). It is
of interest that both MMP-2 (on chro-
mosome 8 at 42 cM) and TIMP-2 (on
chromosome 11 at 72 cM) are both
candidate genes for this wound
healing phenotype.
EXPERIMENTAL PROCEDURES
Animals
The MRL/MpJ mice were obtained
from the Jackson Laboratories (Bar
Harbor, ME) and C57BL/6 (B6) were
TABLE 1. MMP/TIMP Ratios
Strain MMP(M)/TIMP(T)
No. of cells positive for MMP/
no. of cells positive for TIMP
Day 1 Day 3 Day 5 Day 7
B6 M-2/T-2 0.26 0.60 0.57 0.50
MRL M-2/T-2 1.09 1.28 1.61 1.35
B6 M-9/T-3 0.60 0.70 0.49 0.22
MRL M-9/T-3 1.52 1.63 2.29 1.66
a
MMP, matrix metelloproteinase; TIMP, tissue inhibitor of metelloproteinase.
PROTEASE ACTIVITY IN THE MRL MOUSE EAR 383
obtained from Taconic Laboratories
(Germantown, NY). Animals were
maintained under standard condi-
tions at the Wistar Institute Animal
Facility (Philadelphia, PA).
Tissue Preparation
Mouse ears were cleaned with iso-
propyl alcohol and punched with a
2.1-mm-diameter surgical punch.
Punches were made in each ear on
the midline. On days 1, 3, 5, and 7
after wounding, the entire area en-
compassing the healing wounds
was excised. For protein sample ex-
traction to be used in the zymogra-
phy analysis, wound tissues were im-
mediately frozen in liquid nitrogen.
For the histopathologic examination,
the samples were incubated over-
night in Prefer xative (Anatech, Ltd.,
Battle Creek, MI) and embedded in
parafn, and 5-micron-thick sections
were cut. Samples were collected
from at least three separate animals
for each time point/strain.
Immunohistochemistry
For general pathologic analysis, the
Prefer-xed and parafn-embed-
ded ear sections were stained with
hematoxylin (Surgipath, catalog no.
C.I. 75290) and eosin (Surgipath, cat-
alog no. C.I. 45380) with a 5-min and
1-min exposure, respectively. For the
staining of mast cells, a 0.5% (pH 5)
solution of toluidine blue-O (Hart-
man-Leddon, catalog no. 364) was
used for 20 min followed by a 10-min
wash with H
2
O.
For immunohistologic staining, we
used rabbit anti-human antibodies
to MMP-2 (Sigma, catalog no.
M6302), MMP-9 (Sigma, catalog no.
M5302), TIMP-2 (Sigma, catalog no.
T8062), and TIMP-3 (Sigma, catalog
no. T7812). As a secondary antibody,
we used a biotinylated goat anti-
rabbit antibody (Jackson Immu-
noResearch, catalog no. 111-065-
144). For labeling neutrophils, a rat
anti-mouse antibody against Ly-6G
and Ly-6C was used (Ly-6G, rat anti-
mouse, PharMingen, catalog no.
553126).
For nonuorescent labeling, the
sections were exposed to ABC by us-
ing the Vectastain Elite ABC Kit (Vec-
tor, catalogno. PK-6100) in PBST (0.1 M
sodium phosphate buffer [pH 7.4],
0.005% Triton X-100, 0.9% NaCl) for 15
min, washed four times with PBST (5
min) and developed with diamino-
benzidine (DAB) using the DAB kit
(Vector, catalog no. SK-4100).
For uorescent double labeling,
donkey anti-rabbit, CyTM3-conju-
gated (Jackson ImmunoResearch,
catalog no. 711-165-152) was used
for antiMMP-2 and goat anti-rat,
uorescein isothiocyanateconju-
gated (Santa Cruz, catalog no.
2011) was used for antiLy-6G.
Protein Assay
A standard Lowry procedure was
used with detection by ultraviolet
light at 750 nm. The concentration of
total protein was determined by us-
ing an Ultrospec 3000 spectropho-
tometer (Pharmacia Biotech) using
reagents from Bio-Rad Laboratories -
D
C
Protein Assay Kit (catalog no. 500-
0116).
Fig. 6. A comparison of matrix metalloproteinase (MMP) and tissue inhibitor of metallo-
proteinase (TIMP) -positive cells relative to cell size. The data are analyzed as in Figure 5
except that analysis is now broken down into small (predominantly neutrophils) and large
(predominantly macrophages) cells. A: MMP-2 and TIMP-2 results. B: MMP-9 and TIMP-3
results.
384 GOUREVITCH ET AL.
RT-PCR
RNA was isolated by homogenizing
the ear tissue in Trizol (Gibco BRL)
following the manufacturers instruc-
tions. RNA was treated briey with
DNase to remove any DNA that co-
puried. RNA concentration was
calculated by OD
260
readings and
samples pooled (1 g total RNA/per
animal). RNA was reverse tran-
scribed in a 20-l of cocktail that in-
cluded 1 U of RNaseIN, 0.2 M
dNTPs, 1 M oligo (dT), and 200 U
of SuperScript reverse transcriptase
(Gibco-BRL, catalog no. 18053-017)
in (1) RT buffer. Control reactions
omitted the RT enzyme. Quantitative
real-time PCR was carried out on a
Lightcycler (Roche) to determine
the amount of MMP-2 message (for
primer sequences, see Table 2, line
1) and MMP-9 message (see Table 2,
line 2) (Wittwer et al., 1997). The re-
action was set up by using the Fast-
start DNA Master Syber Green I Kit
(Roche, catalog no. 3003230) ac-
cording to instructions in a 15-l
cocktail (2 mM). The samples were
then normalized to GAPDH message
(see Table 2, line 3). A period of 10
min at 95C was used to denature
and inactivate the Taq-start anti-
body. Cycling was carried out 45
times by using 95C for 10 sec, 61C
for 10 sec, 72C for 20 sec, and the
acquisition temperature was set at
85C for 10 sec. The readout is based
on the uorescence of intercalated
SYBER Green I dye.
Zymography
Protease activity was determined
through zymography using different
substrate casting gels adapted from
published methods (Hamaguchi et
al., 1995). Briey, healing tissue from
around the circumference of the
ear holes was collected, milled in a
glass-to-glass grinder and liquid ni-
trogen, and the homogenized tissue
mixed with proteinase inhibitor cock-
tail (Complete from Boehringer
Mannheim) in 1 PBST (1 g of tissue
dissolved in 15 l of Complete cock-
tail). The sample extraction contin-
ued on the rocker platform (4C) for
20 hr, after which the sample was
claried by centrifugation. After sed-
imentation, samples were normal-
ized by the Lowry procedure. Those
samples were subjected to electro-
phoresis with copolymerized 10% so-
dium dodecyl sulfatepolyacrylam-
ide gels with 1 mg/ml gelatin (from
swine skin; Sigma, catalog no.
G-2500) and 0.5 mg/ml of collagen
type IV (from human placenta;
Sigma, catalog no. C-5533). The gels
were washed with H
2
O for 1 hr and
Fig. 7. Double uorescent immunohistochemistry. MRL ears were hole punched, and parafn sections were analyzed 1 day after injury.
Cell staining is visualized by using rhodamine (red) labeling of antimatrix metalloproteinase-2 antibody (A,D) and uorescein isothio-
cyanate (green) labeling of antiLy-6G antibody (B,E). The two images were digitally combined to give the images seen in C,F. The
original magnications of the upper panels 20; the lower panels were digitally enlarged.
Fig. 8. RNA expression levels of matrix
metalloproteinase (MMP) -2 (A) and MMP-9
(B) in ear hole tissue. Real time reverse tran-
scriptase-polymerase chain reaction (RT-
PCR) results show expression levels of
MMP-2 and -9 at various time points after
ear hole punching in both MRL (full line)
and B6 (dashed line) ears. The values
shown (y-axis) are relative values obtained
from the Lightcycler and then normalized to
the glyceraldehyde-3-phosphate dehydro-
genase values. All of the results were done
on the same tissue during the same RT-PCR
run. Three ear holes/time point/strain were
used, and samples were pooled.
PROTEASE ACTIVITY IN THE MRL MOUSE EAR 385
incubated in development buffer
(50 M Tris OH pH 7.5, 0.2 M NaCl, 10
M CaCl
2
) for 20 hr at 37C. Gel
staining was performed by using
Coomassie blue for at least 3 hr and
briey destained in destaining solu-
tion (50 ml of methanol, 40 ml of
H
2
O, and 10 ml of acetic acid). Zy-
mography data are shown as color
inverted for better visualization.
ACKNOWLEDGMENTS
We thank the F.M. Kirby Foundation
and The G. Harold and Leila Y.
Mathers Foundation for their very
generous support. E.H.K. and L.C. re-
ceived funding from the NIH, and
D.G. received funding from an NIH
training grant.
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PROTEASE ACTIVITY IN THE MRL MOUSE EAR 387