Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France; and * Service d'Anatomie Pathologique Générale, Hôpitaux Universitaires, Hôpital de Hautepierre, 67098 Strasbourg Cedex, France
Skin wound healing depends on cell migration and extracellular matrix remodeling. Both processes, which are necessary for reepithelization and restoration of the underlying connective tissue, are believed to involve the action of extracellular proteinases. We screened cDNA libraries and we found that six matrix metalloproteinase genes were highly expressed during rat skin wound healing. They were namely those of stromelysin 1, stromelysin 3, collagenase 3, gelatinase A (GelA), gelatinase B, and membrane type-1 matrix metalloproteinase (MT1-MMP). The expression kinetics of these MMP genes, the tissue distribution of their transcripts, the results of cotransfection experiments in COS-1 cells, and zymographic analyses performed using microdissected rat wound tissues support the possibility that during cutaneous wound healing pro-GelA and pro-gelatinase B are activated by MT1-MMP and stromelysin 1, respectively. Since MT1-MMP has been demonstrated to be a membrane-associated protein (Sato, H., T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, and M. Seiki. 1994. Nature (Lond.). 370: 61-65), our finding that GelA and MT1-MMP transcripts were expressed in stromal cells exhibiting a similar tissue distribution suggests that MT1-MMP activates pro-GelA at the stromal cell surface. This possibility is further supported by our observation that the processing of proGelA to its mature form correlated to the detection of MT1-MMP in cell membranes of rat fibroblasts expressing the MT1-MMP and GelA genes. These observations, together with the detection of high levels of the mature GelA form in the granulation tissue but not in the regenerating epidermis, suggest that MT1-MMP and GelA contribute to the restoration of connective tissue during rat skin wound healing.
SKIN wound healing is a complex process characterized
by reepithelization and restoration of the underlying connective tissue. During this process, keratinocytes, endothelial cells, fibroblasts, and inflammatory
cells proliferate and/or migrate to the site of injury, interacting with each other and with extracellular matrices
(ECMs)1 (Gailit and Clark, 1994 The migration of cells and the remodeling of tissues during wound healing require the controlled degradation of
the ECM and the activation or release of growth factors
(Vassalli and Saurat, 1996 When the present study was initiated, ten human MMPs
were known (Col1-3, stromelysin1-3 [ST1-3], GelA, GelB,
matrilysin, and the macrophage metalloelastase; reviewed in
Birkedal-Hansen, 1995 MT1-MMP is the first member of a new MMP subgroup
characterized by its localization at the cell surface, where it
is anchored through a transmembrane domain located at
the COOH-terminal portion of the molecule. Three additional MT-MMPs (MT2-MMP, Will and Hinzmann, 1995 In the present study, we show that MT1-MMP and GelA
RNAs are both expressed in stromal cells of rat skin
wounds, and that high levels of the mature GelA form are
detected in the wound stroma, but not in the regenerating
epithelium. These findings suggest that during rat skin
wound healing, GelA is activated at the stromal cell surface by MT1-MMP and contributes to the remodeling
processes implicated in the restoration of the connective tissue.
Animals
Pathogen- and virus-free 10-wk-old female Wistar rats were obtained
from IFFA-CREDO (L'Arbresle, France). Animals were maintained under the guidelines of the Institut de Génétique et de Biologie Moléculaire
et Cellulaire (IGBMC). Rats were fed with "D 04" (Usine Alimentation
Rationnelle, Epinay-sur-Orge, France) and tap water ad libitum. Rat dorsal skin was shaved and cleaned with isopropyl alcohol under anesthesia,
and the skin was linearly incised (3-cm long) up to the level of the subcutaneous adipose tissue. Incisions were made on either side of the midline using a #10 sterile surgical scalpel. A sterile dressing was then placed circumferentially around the trunk in order to protect against infection. On days
1, 3, 5, 7, 10, and 14 after wounding, the entire dorsal skin encompassing
healing wounds was excised. For RNA and protein extractions, wound tissues were immediately frozen in liquid nitrogen. For RNA in situ hybridization and histopathological examination, the samples were embedded in
OCT compound (Miles, Elkhart, IN), and frozen in liquid nitrogen. Adequate sampling was ensured by obtaining wound specimen from at least
three separate animals for each time point.
Cell Lines
COS-1 cells (Amer. Type Culture Collection, Rockville, MD), and RAT1
and FR3T3 rat fibroblasts (Matrisian et al., 1985 RNA Extraction
Tissues were homogenized in 4 M guanidinium thiocyanate (Merck, Darmstadt, Germany). Total RNA was purified by ultracentrifugation (SW60
rotor, Beckman Instrs., Fullerton, CA, 35,000 rpm) through a 2.5-ml cushion of 5.7 M cesium chloride. Cells in culture were washed with PBS, and
RNA was extracted using the single-step method of Chomczynski and
Sacchi (1987) Construction of a Rat Skin Wound cDNA Library and
Cloning of cDNAs Encoding MT1-MMP
Poly(A)+RNA from rat skin wounds was purified using the Pharmacia purification kit (Uppsala, Sweden). The RNA thus isolated was used to construct a cDNA library with the Uni-ZAPTMXR Vector kit (Stratagene, La
Jolla, CA). This library was screened with size-selected (~350-550 bp) RTPCR products. For the RT-PCR experiments, poly(A)+RNA from rat
skin wounds was reverse-transcribed using an oligo (dT) primer, and the
resulting cDNA was amplified using a set of degenerate primers corresponding to the cysteine-switch and zinc-binding domains of rat MMPs
(5'-C(CT)N(AC)GNTGTGGN(AG)(AT)NCCNGA, and 5 Cloning of cDNAs Encoding Rat TIMP2 and TIMP3
Rat tissue inhibitor of metalloproteinase type-2 (TIMP2) (Santoro et al., 1994 RNA Analyses
For Northern blot analysis, 10 µg of total RNA from rat tissues was electrophoresed on formaldehyde-agarose (1%) gels and transferred to Hybond-N membranes (Amersham, Buckinghamshire, UK), according to the
supplier's instructions. Hybridizations were carried out with 32P-labeled
cDNA probes in 50% formamide at 42°C. The cDNA probes used for
these hybridizations were identical to those used for the differential
screening of the skin wound cDNA library. An additional probe used for
hybridization was rat MT1-MMP cDNA (nucleotides 911-2410).
For in situ hybridization, tissue sections from rat normal skin and skin
wounds were collected as described above. Sections were cut perpendicularly to the cutaneous incision. 35S-labeled RNA sense and antisense
probes were synthesized from DNA templates identical to those used for
Northern blot analyses, using either the T3 or T7 polymerase. Probe
length was reduced to an average size of 150 nucleotides by limited alkaline hydrolysis before hybridization as previously described (Okada et al.,
1995a Construction of Expression Plasmids for Rat MMPs
and TIMPs
To express rat MT1-MMP in E. coli, a cDNA fragment encoding the putative catalytic domain and the hemopexin-like domain (amino acids 112508) was inserted into the pET15b vector containing an NH2-terminal
6-histidine tag (Novagen, Madison, WI). For expression in COS-1 cells,
cDNA fragments containing the complete open reading frame sequence
of rat MT1-MMP, GelA, GelB, ST1, ST3, TIMP1 (Okada et al., 1994 Transient Transfection into COS-1 Cells
pTL1 expression plasmids for rat MMPs and TIMPs, and the control
pTL1 vector alone were transiently transfected into COS-1 cells using the
calcium phosphate procedure. 5 × 105 COS-1 cells per 10-cm diameter culture dish were grown in DMEM supplemented with 5% FCS for 16 h, and
transfected with 3 µg of each expression plasmid and 12 µg of carrier
DNA (pBluescript vector) in fresh culture medium. After a 1-d incubation, cells were washed with DMEM and incubated in 3 ml of serum-free
DMEM for another day.
Gelatin Zymography
Zymography was performed as previously described (Okada et al., 1995b Normal rat skin epidermis and dermis, and proliferative epidermis or
granulation tissue from rat skin wounds were carefully microdissected under a light microscope from 30-µm thick frozen sections. After microdissection, individual samples were suspended in Laemmli's 2× SDS sample
buffer (40 µl/mg of fresh tissue). After a 30-min incubation at 22°C, the
extracted proteins were analyzed by gelatin zymography as described
above.
Western Blot Analysis
Anti-rat MT1-MMP mAb (1MMP-1C1) was obtained by immunizing Balb/c
mice with recombinant rat MT1-MMP (amino acids 112-508) purified from
E. coli inclusion bodies. The antigen was purified by Ni2+-NTA-Agarose
(Qiagen Inc., Chatsworth, CA) chromatography according to the manufacturer's instruction. The specificity of mAb 1MMP-1C1 for MT1-MMP
was demonstrated by immunocytochemical analysis of COS-1 cells transfected with cDNAs for MT1-MMP, MT2-MMP (Will and Hinzmann,
1995 Subconfluent transfected COS-1 cells, and RAT1 and FR3T3 rat fibroblasts incubated with serum-free DMEM in the absence or presence of 50 µg/ml Con A for 24 h, were scraped and lysed in PBS containing 1% NP40 and 5 mM EDTA (100 µl/ 10-cm diam culture plate). After centrifugation at 4,000 g for 15 min, supernatants corresponding to crude cell membrane extracts were collected and used for Western blot analysis (Okada
et al., 1997 Cloning of Rat MT1-MMP from a Skin Wound
cDNA Library
To investigate the contribution of MMPs to skin wound
healing, we devised a two-step procedure to identify MMP
genes expressed during this process. As a first approach,
screening of a 5-d rat skin wound library constructed in the
The next step was to determine whether other MMPs
were expressed during rat skin wound healing. We constructed a second cDNA library in the Uni-ZAPTMXR
vector (Stratagene, La Jolla, CA) using RNA prepared
from healing wounds on days 3 and 5 after cutaneous incision, which corresponded to the period of concomitant expression of the MMP genes already identified (Fig. 1 A).
For screening this second library, a probe was designed in
such a way that any known or novel members of the MMP
family would be identified. We amplified 3- and 5-d rat
skin wound RNA by RT-PCR, using a set of degenerate
primers derived from nucleotide sequences corresponding to the highly conserved cysteine-switch and zinc-binding
domains of the six rat MMPs known at that time (ST3,
GelA, GelB, ST1, ST2, and Col3) (Fig. 2).
Sixty thousand plaques of the ZAP library were screened
with the RT-PCR probe. After dehybridization, the same
membranes were hybridized with the five rat MMP cDNA
probes (ST3, GelA, GelB, ST1, and Col3) previously isolated. Among the clones that were detected selectively by
the RT-PCR probe and not by the MMP cDNA probes,
we identified 10 cDNAs which had been previously described that did not belong to the MMP family, and 11 novel cDNA sequences (data not shown). One of them
was found to correspond to a putative novel member of
the MMP family, and by sequence homology we proposed that it represented the rat homologue of human MT1MMP (Sato et al., 1994 Expression of MMP RNAs during Skin Wound Healing
MT1-MMP RNA was found to be expressed in most normal adult rat tissues (data not shown). However, higher
levels of MT1-MMP RNA were detected during rat skin
wound healing. MT1-MMP RNA expression was maximal
on day 5 after cutaneous incision (Fig. 1 A). This corresponded to a period of intense stromal activity, filling of
granulation tissue in the incisional space, maximal neovascularization, and when collagen fibers were present at the
incision margins where the scar tissue was being formed.
The expression kinetics of GelA RNA was identical to that
of MT1-MMP (Fig. 1 A). However, ST3 RNA was highly
expressed only on day 5 and reached a peak on day 7. The
maximal expression of ST1 RNA was detected on day 1 after cutaneous incision. GelB and Col3 RNAs were also
highly expressed on day 1, and maintained high expression
levels until day 5.
We then examined MMP transcript distribution by in
situ hybridization on normal skin sections and skin wound
sections from days 1-14 after cutaneous incision. Each
wound was examined at its extreme and median portions.
The results of analyses performed on days 3 and 5 are presented in Fig. 3. On day 3, various stages of granulation tissue and immature capillary lumens were observed, together with spurs of epithelial cells migrating from both
edges of wounds beneath the surface scab (Fig. 3 A and
data not shown). MT1-MMP transcripts were detected in
cells of the granulation tissue and those of the superficial
dermis juxtaposed to the proliferative epithelial cell layer
(Fig. 3 B). The transcripts were also observed in stromal
cells surrounding hair follicles and especially in the dermal
papilla. Most cells expressing MT1-MMP transcripts were
of the fibroblastic type, thus likely corresponding to fibroblasts or myofibroblasts. However, some cells were round
or oval, suggesting that other types of stromal cells, including endothelial cells or macrophages, may also express the
MT1-MMP gene. While it is clear that additional studies
are required to better define the precise nature of these
stromal cells, MT1-MMP transcripts could not be detected
in any epithelial cell. The localization of GelA transcripts
was similar to that observed for MT1-MMP, although the
expression levels were higher for GelA than for MT1MMP (Fig. 3 C). ST3 transcripts were undetectable, except in a few stromal cells surrounding hair follicles (Fig.
3 D). ST1 transcripts were detected in some proliferative
epithelial basal cells, and in a few stromal cells juxtaposed
to the proliferative epithelial layer (Fig. 3 E). GelB and
Col3 RNAs were highly expressed in the epithelial basal cell
layer, but not in the dermis (Fig. 3, F and G). On day 5, though granulation tissue was still observed in the median
portion of incision (data not shown), reepithelization had
been completed at the wound extremities, where thickened epidermis, scar tissue and tissue contraction were observed (Fig. 3 H). MT1-MMP, GelA, and ST3 transcripts
were detected in cells of the scar tissue, below the thickened epithelial cell layer (Fig. 3, I-K). The distribution of
MT1-MMP transcripts (Fig. 3 I) was more narrow than
that of GelA (Fig. 3 J), but broader than that of ST3 (Fig.
3 K). MT1-MMP, GelA, and ST3 transcripts were not detected in epithelial cells. ST1 transcripts were not detected
(Fig. 3 L). GelB and Col3 transcripts were intensely expressed in some basal cells of the thickened epidermis, and
in some cells of the hair follicle sheath (Fig. 3, M and N).
MMP Interactions during Skin Wound Healing
Expression vectors encoding rat MT1-MMP, GelA, GelB,
ST1, and ST3, were transiently transfected into COS-1
cells, which do not produce any detectable levels of these
MMPs endogenously (Okada et al., 1995b
The possibility that MT1-MMP interacts with GelA, and
ST1 with GelB, respectively, during rat skin wound healing,
appears consistent both with the expression kinetics of their
RNAs (Fig. 1 A) and their expression patterns in wound
tissues (Fig. 3). Thus, MT1-MMP and GelA transcripts were
detected in stromal cells exhibiting a similar tissue distribution (Fig. 3, B, C, I, and J). In the case of ST1 and GelB,
the expression patterns of their transcripts were not superimposable on day 5 after cutaneous incision (Fig. 3, L and
M). However, ST1 and GelB transcripts were co-expressed in
the proliferative epithelial basal cell layer on day 1 (data not shown) and to some extent on day 3 (Fig. 3, E and F),
when the mature form of GelB was detected in wound tissues (see below). We also note that the GelB and Col3
genes exhibited comparable expression kinetics (Fig. 1 A)
and distribution of transcripts in tissues (Fig. 3, F, G, M,
and N), suggesting that GelB and Col3 could also interact
during rat skin wound healing. However, we could not further evaluate this possibility in the present study, since we
could not obtain any full-length rat Col3 cDNA for protein expression.
Pro-GelA Activation by MT1-MMP Is Regulated by
TIMP2 and TIMP3
Activity of mature MMP forms and activation of proforms
are regulated by their physiological inhibitors, the TIMPs
(Stetler-Stevenson et al., 1996 To test the influence of TIMPs on pro-GelA activation
by MT1-MMP, we performed triple transfection experiments in COS-1 cells. Different TIMPs and pro-GelA/
MT1-MMP expression plasmids were used for these triple
transfections. Pro-GelA activation by MT1-MMP was not
affected by the presence of TIMP1 (Fig. 4 B, lane 3) and only partially prevented by TIMP2 (Fig. 4 B, lane 4). However, TIMP3 was found to completely inhibit pro-GelA activation by MT1-MMP (Fig. 4 B, lane 5). In contrast to the
observations made for GelA and MT1-MMP, pro-GelB
activation by ST1 was similarly prevented by all the three
TIMPs (Fig. 4 B, lanes 7-10). These observations strongly
suggest that TIMP3 may be a physiological inhibitor of proGelA activation by MT1-MMP.
Pro-GelA Is Activated by MT1-MMP-Expressing
Rat Fibroblasts
During rat skin wound healing, MT1-MMP and GelA transcripts were specifically found in wound stromal cells (Fig.
3, B, C, I, and J). This finding prompted us to examine
MT1-MMP and GelA gene expression in two different rat
fibroblast cell lines, RAT1 and FR3T3. Both cell lines were
found to express high levels of MT1-MMP and GelA transcripts (Fig. 5 A, lanes 1 and 3). However, the MT1-MMP
protein could not be detected either in crude cell membrane extracts or in cytosolic fractions by Western blot
analysis (Fig. 5 B, lanes 1 and 3, and data not shown). Consistently, media conditioned by these cells contained mainly
pro-GelA, as shown by gelatin zymography (Fig. 5 C,
lanes 3 and 5). Since pro-GelA activation has been demonstrated to be induced by Con A treatment in human fibroblasts (Overall and Sodek, 1990
Detection of High Levels of GelA and GelB Activities in
the Granulation Tissue
To directly evaluate pro-GelA activation during rat skin
wound healing, we analyzed microdissected tissue samples
from frozen sections by gelatin zymography. These samples were collected at different times after cutaneous incision. In wound tissues containing both the stroma and epithelium, protein species corresponding to pro-GelA/GelA
were detected from days 1-14, and pro-GelB or pro-GelB/
GelB were detected from days 1-7 (Fig. 6 A). The level of
pro-GelA was maximal on day 5, when the granulation tissue was most active, and pro-GelB expression was elevated all through from days 1-5. However, the mature
form of GelB was only detected on days 1 and 3, while the
mature form of GelA was observed, along with its corresponding pro-form, in all the tissue samples examined (Fig. 6). When the granulation tissue and the proliferative
epithelial cell layer from healing wounds on days 3 and 5 were analyzed separately, pro-GelA/GelA was almost
exclusively detected in the granulation tissue (Fig. 6 B). ProGelB/GelB was also predominantly detected in the granulation tissue, with only low levels associated with the proliferative epithelial cell layer. These observations indicate that the wound stroma is the predominant site of action
for both GelA and GelB during rat skin wound healing.
MMP Gene Expression during Rat Skin Wound Healing
In the present study, we found that six MMP (ST1, ST3,
GelA, GelB, Col3, MT1-MMP) genes were expressed during rat skin wound healing. However, we could not detect
rat ST2 and matrilysin cDNAs, nor the rat homologue of
human Col1 cDNA in the skin wound cDNA libraries
used for our screening. Furthermore, we could detect neither MT2-MMP nor MT3-MMP RNA in wound tissues by
Northern blot analysis (Okada, A., unpublished results).
Among the six MMP genes expressed during rat skin
wound healing, ST1, GelB, and Col3 were mainly expressed between days 1 and 5 after cutaneous incision,
while the three others, MT1-MMP, GelA, and ST3, continued to be expressed at high levels at least until day 7. Interestingly, the transcripts corresponding to the early
genes were found predominantly in migrating epithelial cells while those corresponding to the late genes were specifically detected in wound stromal cells. However, MT1-MMP
and GelA transcripts were both found in granulation and
scar tissues, while ST3 expression was not detected in
granulation tissue.
Pro-GelB Activation by ST1
Ogata et al. (1992) Pro-GelA Activation by MT1-MMP
Our finding that pro-GelA was processed to its corresponding mature form when rat MT1-MMP and GelA
cDNAs were transiently expressed in COS-1 cells, is consistent with the previous observation of Sato et al. (1994) MT1-MMP as a Stromal Cell Surface Activator
of Pro-GelA
MT1-MMP is believed to correspond to a transmembrane
proteinase whose active site is orientated extracellularly
(Sato et al., 1994 Further studies are required to precisely define the sites
where GelA is functionally active in remodeling tissues,
and to determine whether MT1-MMP and molecules such
as integrin ).
). These processes are achieved
by extracellular proteases, particularly those belonging to
the serine protease and matrix metalloproteinase (MMP)
families. It has been demonstrated that urokinase-deficient
mice develop nonhealing skin ulcerations (Carmeliet and
Collen, 1995
) and that plasminogen-deficient mice have a
marked defect in the closure of skin wounds (Rømer et al.,
1996
). However, the exact contribution of MMPs to wound
healing has not been well established yet. The expression
of gelatinase A (GelA) was localized in the connective tissue and that of gelatinase B (GelB) in the migrating epithelial sheet during human skin wound healing (Salo et al.,
1994
). In healing human burn wounds, interstitial collagenase (Col1) RNA has been detected in epithelial cells, hair follicles, and eccrine sweat structures (Stricklin et al., 1993
). Furthermore, in vitro studies indicate that Col1 may play a role
in the formation of new blood vessels (Fisher et al., 1994
).
), while only four rat MMPs (ST1,
Matrisian et al., 1986a
; ST2, Breathnach et al., 1987
; Col3
[According to Knäuper et al. (1996)
, the rat collagenase
identified by Quinn et al. (1990)
should be considered as
the rat counterpart of collagenase 3], Quinn et al., 1990
; GelA, Marti et al., 1993
) had been identified. To gain insight into the contribution of MMPs to wound healing, we designed a two-step procedure for screening cDNA libraries,
which allowed the identification of MMPs expressed highly
during rat skin wound healing. We thus identified six rat
MMP cDNAs, including ST1, ST3, Col3, GelA, GelB, and
a novel member of the MMP family. The latter one was
found to exhibit a high level of homology with a human
MMP cloned by Sato et al. (1994)
, and now known as membrane-type-1 MMP (MT1-MMP; Sato and Seiki, 1996
).
;
MT3-MMP [Although the MT-MMP described by Takino
et al. (1995)
was initially termed MT2-MMP, this MMP
should be now referred as MT3-MMP and that found by
Will and Hinzmann (1995)
as MT2-MMP] Takino et al.,
1995
; MT4-MMP, Puente et al., 1996
) have so far been described. While the function of MT4-MMP is still unknown, the three other MT-MMPs have been shown to be cell surface activators of pro-GelA. MT1-MMP was predominantly detected in malignant cells of lung and gastric carcinomas (Sato et al., 1994
; Nomura et al., 1995
; Tokuraku et al.,
1995
). This has led to the proposition that MT1-MMP activates pro-gelA at the cancer cell surface (Sato and Seiki,
1996
). However, MT1-MMP transcripts were predominantly detected in fibroblastic cells of colon, breast, and
head and neck carcinomas (Okada et al., 1995a
; Heppner
et al., 1996
). These observations suggest that pro-GelA activation by MT1-MMP also occurs at the stromal cell surface,
which is supported by the specific expression of MT1-MMP
in microglial cells of brain tissues (Yamada et al., 1995
). Furthermore, MT1-MMP gene expression has been recently
demonstrated in mesenchymal cells of mouse embryos (Kinoh et al., 1996
), indicating that this MMP could contribute to pro-GelA activation in physiological conditions.
Materials and Methods
) were grown and maintained in DMEM supplemented with 5% or 10% FCS, or 10% calf serum,
respectively. Con A (Sigma Chem. Co., St. Louis, MO) treatment of rat fibroblasts in culture was carried out in serum-free DMEM supplemented
with 50 µg/ml Con A for 24 h (Overall and Sodek, 1990
). After treatment,
conditioned media and cells were harvested for protein analysis and RNA
extraction.
.
-(CT)C(AG)
A(AG)(CT)TC (AG)TGNGC(AT)GC(AC)AC, respectively). Additional
screening was conducted using cDNA probes (see Results) encoding rat
GelA (nucleotides 482-2306; accession number No. U65656), GelB (nucleotides 517-2392), ST1 (nucleotides 418-1771), ST3 (nucleotides 644-1810),
and Col3 (nucleotides 1387-2606). The clones which were only recognized
by the RT-PCR probe, were plaque-purified and used for plasmid rescue.
After cross-hybridization experiments, all independent clones were sequenced from their 5
-terminus. The full nucleotide sequence of rat MT1MMP cDNA was determined on both strands as previously described
(Okada et al., 1995a
). Nucleotide and amino acid sequences were analyzed
using the sequence analysis software of the Wisconsin program package.
)
and TIMP3 (Wu and Moses, 1996
) cDNA fragments containing the complete open reading frame sequence were obtained by PCR amplification
from a previously described skin wound library (Okada et al., 1994
) and a
13.5-d placenta library in the
ZAPII vector, respectively. The PCR
products were cloned into the pBluescript vector and sequenced.
). Frozen sections were fixed with 4% paraformaldehyde, dehydrated, and hybridized with 35S-labeled RNA probes. After washing, autoradiography was performed using NTB2 emulsion (Kodak, Rochester,
NY). Slides were then developed and counterstained with toluidine blue.
),
TIMP2, and TIMP3, were inserted into the pTL1 expression vector as previously described (Okada et al., 1995b
). The resulting plasmids,
pET15bMT1-MMP (MT1-MMP, amino acids 112-508), pTL1MT1-MMP (pro-MT1-MMP), pTL1GelA (pro-GelA), pTL1GelB (pro-GelB),
pTL1ST1 (pro-ST1), pTL1ST3 (pro-ST3), pTL1TIMP1 (TIMP1),
pTL1TIMP2 (TIMP2), and pTL1TIMP3 (TIMP3) were verified by restriction enzyme digestion and DNA sequencing.
),
using conditioned media or rat tissues. 20 µl of each conditioned medium
was mixed with a same volume of Laemmli's 2× SDS sample buffer in the
absence of a reducing agent, and incubated for 30 min at 22°C. The samples were electrophoresed on 8%-polyacrylamide gels containing 0.2%
gelatin. After electrophoresis, gels were soaked in 50 mM Tris-HCl (pH
7.6) containing 2.5% Triton X-100 for 30 min, and incubated with 50 mM
Tris-HCl (pH 7.6) containing 150 mM NaCl, 10 mM CaCl2, and 0.02%
Brij-35, at 37°C for 16 h. Gels were subsequently stained with 0.5% Coomassie brilliant blue R-250. Protein molecular standards were purchased
from Bio-Rad (Hercules, CA).
), or MT3-MMP (Takino et al., 1995
).
). Proteins were separated by electrophoresis performed on
10%-polyacrylamide gels under reducing conditions. The proteins were
then electrophoretically transferred to a nylon membrane (Immobilon-P,
Millipore, Bedford, MA). After treatment with a blocking solution, the
nylon membrane was incubated with anti-rat MT1-MMP mouse mAb
1MMP-1C1, diluted 1/1,000 in PBS containing 2% BSA. The nylon membrane was washed and incubated with peroxidase-conjugated goat anti-
mouse IgG antibody (Jackson ImmunoResearch Laboratory, West
Grove, PA). After washing in PBS containing 0.05% Tween-20, proteins
were detected using a chemiluminescence procedure (Renaissance, DuPont, Boston, MA).
Results
gt10 vector (Okada et al., 1994
) with six human MMP
cDNA probes (ST2, ST3, Col1, GelA, GelB, and matrilysin),
led to the identification of four rat MMP cDNAs (ST1,
ST3, GelA, and GelB) (Okada et al., 1995b
, 1997, and
Okada, A., unpublished results). However, ST2, matrilysin, and the rat homologue of human Col1 were not
found in the library. A nearly full-length cDNA for rat
Col3 (nucleotides 145-2583) was obtained by screening the
library with a partial rat Col3 cDNA fragment (nucleotides
865-1262, Quinn et al., 1990
).
Fig. 1.
Northern blot analysis of MMP and TIMP RNAs during rat skin wound healing. Total RNA (10 µg) from normal skin (lanes
1) and skin wounds on days 1, 3, 5, 7, 10, and 14 after cutaneous incision (lanes 2-7), were electrophoresed, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes for rat MT1-MMP, GelA, ST3, ST1, GelB, Col3 (A); TIMP1, TIMP2, TIMP3 (B).
Blots were reprobed with the 36B4 cDNA used as a loading control (Masiakowski et al., 1982).
[View Larger Version of this Image (42K GIF file)]
Fig. 2.
Amino acid sequence alignments of rat MMPs. Amino acids are represented using the one-letter code. The predicted amino
acid sequences of MT1-MMP, ST3, GelA, GelB, and ST1 were derived from cDNAs cloned from two distinct rat skin wound libraries
(Okada et al., 1994, and the present study). One amino acid residue of ST1 (isoleucine 189) differs from the corresponding residue (threonine) in the sequence reported by Matrisian et al. (1985)
. The sequences of ST2, Col3, and matrilysin were obtained from the PIR-protein data bank. These sequences were aligned using the CLUSTAL program of the Wisconsin program package. Identical amino acid residues in all MMPs are indicated by asterisks below the sequences. The cysteine-switch region (P-/LRCGV/NPD), and the zinc-binding domain (XVAXHXHEL/FGHXL/MGLXHS/T) are in the boxed regions. The two regions selected for designing the degenerated oligoprimers
used for RT-PCR amplification are indicated by arrows above the sequences. The putative transmembrane segment (amino acid 539563) of rat MT1-MMP is overlined.
[View Larger Version of this Image (99K GIF file)]
; Okada et al., 1995a
). Although
three other human MMPs containing a transmembrane
domain have since been described (Will and Hinzmann,
1995
; Takino et al., 1995
; Puente et al., 1996
), the rat MMP
that we have identified exhibits the highest sequence homology with human MT1-MMP until now (data not shown).
Fig. 3.
In situ hybridization of rat MMP RNAs in cutaneous wounds. Serial frozen sections of rat skin wounds on day 3 (A-G), or day 5 (H-N) after cutaneous incision, were hybridized with 35S-labeled antisense RNA probes derived from cDNA templates for rat MT1MMP (B and I), GelA (C and J), ST3 (D and K), ST1 (E and L), GelB (F and M), Col3 (G and N), or stained with hematoxylin and eosin
(A and H). Note that the hair follicle which is observed on sections B and C, is only partially visible on A (arrows). In D and E, arrows
indicate transcripts expressed in stromal cells surrounding a hair follicle and juxtaposed to the proliferative epithelial cell layer, respectively. No signal above background could be detected with the corresponding 35S-labeled sense RNA probes (data not shown). gt, granulation tissue; e, epithelial layer; hf, hair follicle; s, scar tissue. Bar, 100 µm.
[View Larger Version of this Image (107K GIF file)]
, 1997, and data
not shown). The expression plasmids were transfected in
pairs into COS-1 cells. Conditioned media were analyzed
by gelatin zymography for GelA and GelB production. As
shown in Fig. 4 A, pro-GelA was selectively activated by
MT1-MMP, and pro-GelB by ST1 only. Other combinations which could not be analyzed by gelatin zymography,
were investigated by Western blot analyses and immunoprecipitation, using specific antibodies against ST1 (Matrisian et al., 1986b
), ST3 (mAb 5ST-4C10, Santavicca et al.,
1995
), and MT1-MMP (mAb 1MMP-1C1). None of the
other pairs led to activation of the MMP proform (data not
shown). Furthermore, MT1-MMP was found exclusively
in crude cell membrane extracts and could not be detected
in conditioned media (data not shown).
Fig. 4.
Pro-GelA and proGelB activation by MT1MMP and ST1, in the absence or presence of TIMPs.
(A) COS-1 cells were transiently cotransfected with expression plasmids for MMPs
whose transcripts were found to be expressed during rat skin
wound healing (Figs. 1 and
3), according to the following
combinations: pTLlGelA and
pTL1 (lane 1) or pTL1MT1MMP (lane 2) or pTL1ST1
(lane 3) or pTL1ST3 (lane 4)
or pTL1GelB (lane 5);
pTL1GelB and pTL1MT1MMP (lane 6) or pTL1ST1 (lane 7) or pTL1ST3 (lane 8)
or pTL1 (lane 9). (B) COS-1
cells were transiently cotransfected with pTL1GelA and
pTL1 (lane 1), or pTL1GelA
and pTL1MT1-MMP (lanes
2-5), or pTL1GelB and pTL1 (lane 6), or pTL1GelB and
pTL1ST1 (lanes 7-10), in the
absence (lanes 1, 2, 6, and 7)
or presence of one of the
TIMP expression plasmids:
pTL1TIMP1 (lanes 3 and 8),
pTL1TIMP2 (lanes 4 and 9), pTL1TIMP3 (lanes 5 and
10). In A and B, 20 µl of each
conditioned medium was analyzed by gelatin zymography.
[View Larger Version of this Image (51K GIF file)]
). In particular, TIMP2 has
been shown to be both a more efficient inhibitor than
TIMP1 in pro-GelA activation (Atkinson et al., 1995
) and
an active agent in promoting pro-GelA binding to MT1MMP (Strongin et al., 1995
). The effect of TIMP3 on the activation of pro-GelA has not been reported yet. As
shown in Fig. 1 B, TIMP1 RNA was not expressed in normal skin, while TIMP2 and TIMP3 RNAs were weakly expressed. The induction kinetics of TIMP1 RNA after cutaneous incision was comparable to that of ST1, while that of
TIMP2 was identical to that of MT1-MMP and GelA (compare Fig. 1, A and B). However, the induction kinetics of TIMP3 RNA was different from that of the other TIMPs
and the six MMPs examined. TIMP3 kinetics exhibited a
biphasic pattern, with the highest RNA levels on day 1 after cutaneous incision, which corresponds to the onset of
epithelial and mesenchymal cell migrations, and on day 10, when cell migration terminates.
), we then examined whether treatment with con A of these rat fibroblasts was
associated with pro-GelA activation. Although both cell
lines stimulated by Con A showed only a weak increase in
MT1-MMP and GelA transcript levels (Fig. 5 A, lanes 2 and 4), the MT1-MMP protein could now be easily detected by Western blot analysis of crude cell membrane fractions prepared from the Con A-treated cells (Fig. 5 B,
lanes 2 and 4). Furthermore, as shown by gelatin zymography, the mature form of GelA was found to be the major
GelA species represented in media conditioned by Con
A-treated cells (Fig. 5 C, lanes 4 and 6).
Fig. 5.
MT1-MMP gene expression in rat fibroblasts and proGelA activation in the presence of Con A. (A) Northern blot
analysis. 10 µg of total RNA obtained from RAT1 (lanes 1 and 2)
or FR3T3 (lanes 3 and 4) rat fibroblasts cultured in the absence
(lanes 1 and 3) or presence (lanes 2 and 4) of Con A (50 µg/ml)
were used in each lane. Blots were hybridized with 32P-labeled
rat MT1-MMP and GelA cDNA probes. Blots were reprobed with the 36B4 cDNA used as a loading control (Masiakowski et al., 1982). (B) Western blot analysis. Crude cell membrane extracts corresponding to half a culture plate of subconfluent RAT1 (lanes 1 and 2) or FR3T3 (lanes 3 and 4) fibroblasts cultured in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of Con A (50 µg/ml) were analyzed. Proteins were detected using anti-rat
MT1-MMP mouse monoclonal antibody, 1MMP-1C1. (C) Gelatin zymography. 20 µl of medium conditioned by COS-1 cells
transiently transfected with pTL1GelA (lane 1), or pTL1GelA
and pTL1MT1-MMP (lane 2), and 20 µl of medium conditioned
by RAT1 (lanes 3 and 4) and FR3T3 (lanes 5 and 6) rat fibroblasts cultured in the absence (lanes 3 and 5) or presence (lanes 4 and 6) of Con A (50 µg/ml), were used.
[View Larger Version of this Image (19K GIF file)]
Fig. 6.
Gelatinolytic activities during rat skin wound
healing. Tissues were microdissected from cryostat sections, incubated with SDS
sample buffer, and subjected
to gelatin zymography. (A)
Normal skin containing both
epidermis and dermis (lane
1), and healing skin wounds
at days 1, 3, 5, 7, 10, and 14 after cutaneous incision (lanes 2-7). (B) Normal skin
adjacent to the wound tissue
and containing both epidermis and dermis (N) (lane 1),
and granulation tissue (G)
(lanes 2 and 4) or proliferative epithelial layer (E) (lanes 3 and 5) of healing skin wounds at days 3 (lanes 2 and 3) and 5 (lanes 4 and 5)
after cutaneous incision. In A and B, proteins extracted from ~1 mg of fresh tissue were analyzed in each lane.
[View Larger Version of this Image (28K GIF file)]
Discussion
have demonstrated that human proGelB was processed to its mature form by human ST1
when both enzymes were incubated together in vitro. In
agreement with this observation, we found that rat proGelB was processed to its mature form by rat ST1 when
the two cDNAs corresponding to GelB and ST1 were transiently transfected into COS-1 cells. The expression
kinetics of GelB and ST1 genes, the tissue distribution of
cells expressing their transcripts, and the detection of mature GelB protein in wound tissue from days 1 to 3 after
cutaneous incision all together support the possibility that
pro-GelB is activated by ST1 during rat skin wound healing. The most convincing argument is that while GelB
transcripts and protein were detected at similar high levels from days 1 to 5 after cutaneous incision, the active form
of GelB was only detected from days 1 to 3, consistent
with maximal ST1 gene expression on day 1. Finally, we
observed that pro-GelB/GelB were predominantly detected in wound stroma, although their transcripts were
predominantly expressed in the epithelial basal cell layer. Taken together, these observations support the proposition of Salo et al. (1994)
that GelB plays a role in epithelial
cell migration, assuming that the substrate for GelB is
found in the wound stroma immediately surrounding the
migrating epithelial cells.
made with the human enzymes. However, GelB, ST1, or
ST3 could not activate pro-GelA. The possibility that proGelA activation by MT1-MMP also occurs during rat skin
wound healing is supported by some of our observations.
First, MT1-MMP and GelA transcripts were found to be
expressed by wound stromal cells exhibiting a similar tissue distribution. Second, the expression kinetics after cutaneous incision were identical for both genes. Third, high
levels of the mature form of GelA were observed in the
wound stroma, particularly on day 5 after cutaneous incision when both the MT1-MMP and GelA RNAs attained
their highest expression levels. However, the ratios between the amounts of mature GelA and pro-GelA were
found to be similar in all the wound tissue samples examined, and they did not significantly differ from that observed in normal skin. In this respect, pro-GelA activation by MT1-MMP during rat skin wound healing differs from
that of pro-GelB by ST1, although both MMPs are predominantly found in the wound stroma. Another difference observed between GelA and GelB, is that the cells
expressing GelA transcripts are not found specifically at
the stromal-epithelial interface. The GelA transcripts are
in fact distributed throughout the wound stroma, both in
the granulation tissue and in the scar tissue. This observation suggests that GelA activated by MT1-MMP contributes to the remodeling processes implicated in the restoration of connective tissue.
). Our finding that the MT1-MMP and
GelA genes are coexpressed in stromal cells, suggests that
MT1-MMP activates pro-GelA at the stromal cell surface
during rat skin wound healing. Consistent with this suggestion, we observed that the induction of MT1-MMP by Con
A treatment in two different rat fibroblast cell lines, RAT1
and FR3T3, resulted in pro-GelA activation. We note that similar observations have been made in human skin fibroblasts, where the processing of pro-GelA was coincident
with MT1-MMP expression induced by Con A treatment
(Atkinson et al., 1995
). Furthermore, we have previously
shown that MT1-MMP and GelA transcripts were specifically detected in fibroblast-like cells of breast, colon, and
head and neck carcinomas (Okada et al., 1995a
). More recently, MT1-MMP transcripts and the MT1-MMP protein
have been shown to be expressed by mesenchymal cells
during embryonic development (Kinoh et al., 1996
). It remains to be seen, however, whether GelA acts at the same
cell surface where it has been activated by MT1-MMP, or
on the surface of other cells, or maybe after binding to the
ECM. The possibility that GelA binds to cell surface sites distinct from the extracellular domain of MT1-MMP is supported by a recent observation that active forms of GelA
bind to integrin
v
3 on the surface of angiogenic blood
vessels (Brooks et al., 1996
).
v
3 cooperate in targeting active GelA to the
cell surface. The expression of integrin
v
3 and/or molecules with similar GelA-binding function at the stromal
cell surface during wound healing would be consistent
with our finding that the mature form of GelA is specifically found in the stroma. In this case, by activating proGelA, MT1-MMP would contribute to the migration of
stromal cells and ECM remodeling implicated in the restoration of the connective tissue during wound healing.
Received for publication 14 August 1996 and in revised form 4 January 1997.
Address all correspondence to Paul Basset, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, BP 163, 67404 Illkirch Cedex, France. Tel.: 33 3 88 65 34 25. Fax: 33 3 88 65 32 01. E-Mail: basset{at}igbmc.u-strasbg.frWe thank P. Chambon for his support, J.M. Garnier for helpful discussions, R. Kannan for critical reading, I. Stoll for protein purification, S. Vicaire for DNA sequencing, and G. Duval for animal care. We are grateful to Dr. Matrisian (Vanderbilt University, Nashville, TN) for anti-ST1 antibody, to Dr. Will (InViTek GmbH, Berlin-Buch, Germany) for human MT2-MMP cDNA, and to Dr. Seiki (Kanazawa University, Kanazawa, Japan) for human MT3-MMP cDNA.
This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Centre Hospitalier Universitaire Régional, the BristolMyers Squibb Pharmaceutical Research Institute, the Association pour la Recherche sur le Cancer, the Ligue Nationale Française contre le Cancer and the Comité du Haut-Rhin, the Fondation de France, the Programme Hospitalier de Recherche Clinique 1995, the BIOMED 2 (contract no BMH4CT96-0017) and BIOTECH 2 (contract no ERBBIO4CT96-0464) Programmes, and a grant to P. Chambon from the Fondation Jeantet. A.Okada was a recipient of fellowships from the Ministère des Affaires Etrangères and the Fondation pour la Recherche Médicale Française.
Col1, interstitial collagenase; Col2, neutrophil collagenase; Col3, collagenase 3; ECM, extracellular matrix; GelA and GelB, gelatinase A and B; MMP, matrix metalloproteinase; MT1-MMP, MT2-MMP, MT3-MMP, and MT4-MMP, membrane-type-1, -2, -3, and -4 MMP; ST1, ST2, and ST3, stromelysin 1, 2, and 3; TIMP1, 2, and 3, tissue inhibitor of metalloproteinase type-1, -2, and -3.