From the Department of Biology, University of Liege,
B-4000 Liege, Belgium, ¶ Biocenter and Department of Biochemistry,
University of Oulu, FIN-90570 Oulu, Finland, and the
Division of
Matrix Biology, Department of Medical Biochemistry and Biophysics,
Karolinska Institute, S-171 77 Stockholm, Sweden
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ABSTRACT |
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Knowledge about the regulation of cell
lineage-specific expression of extracellular matrix metalloproteinases
is limited. In the present work, the murine matrix metalloproteinase 9 (MMP-9) gene was shown to contain 13 exons, and the 2.8-kilobase pair upstream region was found to contain several common promoter elements including a TATA box-like motif, three GC boxes, four AP-1-like binding
sites, an AP-2 site, and three PEA3 consensus sequences that may be
important for basic activity of the gene. In order to identify
cell-specific regulatory elements, constructs containing varying
lengths of the upstream region in front of a LacZ reporter gene were made and studied for expression in transgenic mice generated by microinjection into fertilized oocytes. Analyses of the mice revealed that the presence of sequences between Mammalian extracellular matrix metalloproteinases
(MMPs)1 form a family
of related enzymes that are capable of degrading various components of
the connective tissue (1-3). These proteases are either secreted or
membrane-bound and are produced as latent enzymes. They have a
conserved Zn2+ binding catalytic site and can be inhibited
by specific tissue inhibitors of metalloproteinases (4-7). In
vitro studies imply diverse substrates and functions for these
enzymes in vivo. Several genetically distinct enzymes have
been identified. Based on in vitro substrate specificities,
they are placed in different categories: interstitial collagenases that
degrade fibrillar collagens (8-10); the stromelysins (11-13) with
activity against several noncollagenous proteins and collagens with
interrupted triple helices; matrilysin that degrades fibronectin,
laminin, casein, gelatin, and proteoglycans (2); macrophage
metalloelastase, which degrades elastin (15, 16); MMP-2 and MMP-9,
which cleave type IV collagen and gelatin (17-21), respectively; and
finally a recently described group of the membrane type of matrix
metalloproteinases that can activate other metalloproteinases and also
degrade matrix proteins (22-24).
The metalloproteinases are believed to have an important role in normal
turnover of extracellular matrix and in remodeling of tissues.
Furthermore, they have been shown to be highly expressed in areas of
inflammation and tumor invasion. However, the specific roles of the
various metalloproteinases in vivo as well as regulation of
their genes are still largely unexplored. MMPs 2 and 9, which form a
distinct subgroup of MMPs, based on their primary structure and
substrate specificity, have been shown to have high activity against
gelatin, and they also degrade type IV, V, and VII collagens. However,
they do not show high activity against type I collagen, proteoglycan,
or laminin. Despite the apparently identical substrate specificity,
their temporal and spatial expression in vivo varies extensively, indicating that the two enzymes are required for different
purposes. This is also emphasized by the different expression patterns
of the two genes (21). Thus, MMP-2 is primarily expressed in stromal
fibroblast-like cells during mouse development (25), while its
expression is insignificant in the stroma of adult mice. This
suggests that MMP-2 has a major role in the remodeling of the stromal
compartment, in addition to the proposed role in the turnover of
basement membrane type IV collagen. In fact, it is possible that the
enzyme mainly functions as a stromal gelatinase by removing
gelatin derived from degraded fibrillar collagens. In invasive tumors,
intense expression of MMP-2 is observed in stromal cells adjacent to
the tumor front, not in the tumor cells themselves (26-29).
MMP-9 has a completely different expression pattern. By in
situ hybridization analysis, its expression has been shown to be almost completely confined to osteoclasts at the site of bone formation
during mouse development (30). This is the only known example of a
highly osteoclast-specific proteinase, which indicates that the enzyme
is important for the turnover of bone matrix, possibly as a gelatinase
required for the removal of denatured collagen fragments (gelatin)
generated by interstitial collagenases. MMP-9 expression has also been
localized to keratinocytes of healing skin wound (31), macrophages (27,
28, 29, 32), and trophoblasts of the implanting embryo (33, 34).
We have previously shown that no cell-specific expression and
regulation of the MMP-9 gene could be achieved through in
vitro experiments using transiently transfected cells (35).
Therefore, to examine the regulation of the MMP-9 gene and, in
particular, the regulatory mechanisms of its tissue expression, we have
cloned and characterized the mouse gene and studied its regulation
using transgenic mice. In this study, we demonstrated that the minimum element(s) required for expression in osteoclasts and migrating keratinocytes reside in the 5'-flanking region of the gene between 2.7 and 7.7 kb upstream of the transcription start site.
Isolation and Characterization of the Mouse MMP-9
Gene--
Mouse genomic libraries cloned in the cosmid pWE15
(Stratagene, catalog no. 95303) and DNA Sequencing--
The nucleotide sequence was determined by
the dideoxynucleotide chain termination procedure (36) using Sequenase
or TAQuence DNA sequencing kits (U.S. Biochemical Corp.) and M13
universal primers or specific oligonucleotide primers. Both strands of
the gene and its promoter were sequenced following subcloning into pBluescript II SK+/ Primer Extension--
Total RNA from 7-day-old mouse skull was
isolated by the acid guanidium thiocyanate/phenol chloroform extraction
method (37). Primer extension was performed by hybridizing 20 µg of
total RNA with an antisense nucleotide primer annealing at positions
117-144 in the cDNA (Ref. 30, Fig. 1). The primer was
end-labeled by [ Plasmid Constructs--
Promoter-LacZ reporter gene
constructs were created using the pKK2480 vector (kindly provided by
Mikkel Rohde, University of Copenhagen, Denmark), which contains a
multiple cloning site immediately upstream of the LacZ gene.
Different length segments of the 5'-flanking region as well as the
5'-end of the MMP-9 gene containing the first exon and intron were
excised with appropriate restriction enzymes and used for the
construction of MMP-9/LacZ fusion genes.
Generation and Analysis of Transgenic Mice--
Transgenic
animals harboring promoter-reporter gene constructs were generated by
injection of the linearized LacZ fusion constructs into
pronuclei of fertilized mouse oocytes C57BL/6 × DBA/2 F1 (39).
Microinjected eggs (15-20) were then transferred into the oviduct of
pseudopregnant NMRI mice, and the mice were allowed to develop to term.
At 3 weeks of age, tail DNA was isolated (40), and transgenic animals
were identified by polymerase chain reaction analysis using two
internal primers of the LacZ gene (41). Mouse embryos from
positive mice were recovered at different time points and fixed for
2 h or overnight at 4 °C in 2% paraformaldehyde, 0.2%
glutaraldehyde in PBS. They were stained with
5-bromo-4-chloro-3-indolyl- Histologic and Immunohistochemical Analyses--
X-gal-stained
mouse embryos and other tissues were rinsed several times in PBS,
dehydrated, and embedded in paraffin. Sections of 5-8 µm were
stained either by hematoxylin and eosin (43) or with safranin. Sections
were stained for 2-5 min in 0.2% safranin, 1% acetic acid,
differentiated for 1-5 s in 95% ethanol, 2-5 s in absolute ethanol,
and mounted from xylene.
Immunohistochemical staining of paraffin sections (5-10 µm) from
skin wounds was carried out by using either the ABComplex horseradish
peroxidase kit (DAKO) or the TSA kit (NEN Life Science Products).
Deparaffinized sections were treated 5 min. with 0.4% pepsin in 0.01 M HCl at 37 °C. Endogenous peroxidase activity was
quenched by incubation for 20 min in 3% H2O2,
and sections were then incubated with the antiserum raised against
cytokeratin (rabbit anti-cytokeratin (Pan), Zymed
Laboratories Inc.) for 1.5 h at room temperature. or MMP-9
(rabbit polyclonal antibody kindly provided by P. Carmeliet) overnight
at room temperature. For the anti-cytokeratin immunostaining, the
sections were washed in PBS, and subsequently, biotinylated Swine
anti-rabbit IgG (Boehringer Mannheim) (1:400 dilution) was applied for
30 min at room temperature. After phosphate-buffered saline washes, a
20-min incubation with ABComplex/horseradish peroxidase was carried out
(DAKO Code K355). Peroxidase activity was revealed by incubation with
the chromogen substrate 3,3-diaminobenzidine tetrahydrochloride.
Sections were counterstained with hematoxylin and eosin. For the MMP-9
immunostaining, peroxidase swine anti-rabbit IgG (DAKO) was applied and
followed by tyramide signal amplification (TSA kit; NEN Life Science
Products). AEC chromogen substrate (DAKO) was used to detect peroxidase
activity. Sections were counterstained with hematoxylin.
In Situ Hybridization--
A mouse MMP-9 cDNA fragment of
324 bp containing SmaI and EcoRI restriction
sites from the M92KD-2 cDNA clone (bases 1915-2239) (30) was
subcloned into pSP64 ans pSP65 plasmid vectors (Promega). The pSP64
(sense) and pSP65 (antisense) plasmids vectors were linearized with
EcoRI and BamHI restriction enzymes,
respectively, and the 35S-uridine 5'-triphosphate (1000 mCi/nmol, Amersham Pharmacia Biotech) labeled RNA probes were
transcribed using a transcription kit (Promega). The labeled probes
were precipitated with ethanol, dissolved in hybridization buffer, and
used at 50,000-60,000 cpm/µl. The in situ hybridization
was carried out according to Wilkinson and Green (44). Prior to
hybridization the mouse embryos were stained with X-gal, embedded in
paraffin, and sectioned. The specimens were pretreated as described
before (45) and hybridized with the probe at 5O °C for 16 h.
After washing under high stringency conditions, the sections were dried
and dipped in an autoradiographic emulsion (nitro blue tetrazolium-2;
Eastman Kodak Co.), exposed for 14 days at 4 °C. After development
of the sections, they were stained with hematoxylin and mounted.
Structure of the Gene and 5'-Flanking Region--
Screening of the
genomic libraries yielded several clones, one of which (cosmid MGC-1
(about 34 kilobases)), contained the entire 7.7-kilobase gene (Fig.
1), as well as 3 and 23 kilobases of the
5'- and 3'-end flanking regions, respectively. Another
The initiation site for transcription as determined by primer extension
revealed a double start site located 19 and 20 bp upstream of the
translated sequence (Fig. 2). Sequencing
of about 2800 base pairs of the 5'-flanking region revealed several
common promoter elements (Fig. 1). There is a TATA box-like motif TTAAA at positions Generation of Transgenic Mice--
In order to explore the
regulatory mechanisms of the MMP-9 gene, we generated transgenic mice
by microinjection of different promoter-reporter gene constructs into
fertilized oocytes. A total of six constructs containing different
portions of the 5'-end of the gene and the Expression of MMP-9 Promoter/LacZ Reporter Constructs in Transgenic
Mice--
Transgenic mice were first generated with constructs
containing the minimum promoter, 645-LacZ, and a longer one,
2700-LacZ, containing 2.7 kb of the upstream region.
However, mice and mouse embryos made with these constructs did not
yield any expression of the LacZ gene in cells that normally
express MMP-9 in transgenic embryos or adult tissues. Ectopic
expression could be observed in some lines with both constructs, but
its pattern was neither uniform nor repeatable in four founder lines
analyzed (data not shown). The addition of the 5'-end of the MMP-9
gene, including intron 1, to constructs 645-LacZ and
2700-LacZ (i.e. the 5'-untranslated region and
the first exon and intron) did not alter the expression pattern.
Consequently, it could be concluded that the first intron does not
contain cis-acting elements conferring tissue-specific expression of
the endogenous MMP-9 gene.
Due to the lack of expression with the constructs described above, we
made another one, 7700-LacZ, containing 7.7 kb of the upstream sequence and generated transgenic mice. In contrast, to the
shorter constructs, mouse embryos harboring 7700-LacZ
revealed expression of the LacZ gene in bones of
14.5-16.5-day-old embryos. For example, at embryonic day 15.5, distinct expression could be observed in the scapula, long bones of
fore and hind limbs, ribs, and the lower jaw (Fig.
4). Additionally, expression was observed
in hair follicles in different mouse lines made with this construct.
Construct 7700ExIn-LacZ, containing additionally the first
exon and intron, yielded similar expression pattern in bones as
7700-LacZ when analyzed in whole X-gal-stained embryos, with
the exception that no expression was present in hair follicles (Fig.
4).
The Localization of 7700-LacZ Expression to Migrating
Keratinocytes--
MMP-9 has been shown to be expressed in cultured
keratinocytes (31), but our previous in situ hybridization
studies in developing embryos and adult mice did not reveal expression
of the gene in normally developing epithelia (30). However, we have
shown by in situ hybridization that the MMP-9 gene is
expressed in migrating keratinocytes of healing skin wounds, indicating
a role for the enzyme in the repair process (28). To examine if any of
the MMP-9 promoter-LacZ gene constructs are expressed in
epithelial cells, we analyzed tissues from mice made transgenic with
the different constructs for expression of
We then analyzed if keratinocytes of healing skin wounds expressed the
constructs. Incision wounds of about 1 cm were introduced into the
dorsal skin and sutured with a couple of stitches to bring the wound
edges together. When pieces of whole recovering wounds were stained
with X-gal 1-7 days later, expression of LacZ could be
followed in mice harboring constructs containing 7.7 kb of the
5'-flanking region of the MMP-9 gene (Fig.
6, A-G). When the surface of
a wound was stained with X-gal, one could macroscopically observe cells
expressing It is well established that some of the members of the large
family of MMPs exhibit highly restricted temporal and spatial expression patterns in vitro, indicating tissue-specific
roles for these enzymes in extracellular matrix turnover. However,
there are currently no reports on the regulatory mechanisms
driving these cell lineage-specific expression patterns. The
present study provides the gene structure and sequence of the promotor
region of mouse MMP-9 as well as evidence that cis-regulatory
element(s) necessary for the most of the cell-specific expression
patterns of MMP-9 reside in a region between 2722 and 7745 base pairs
upstream of the transcription initiation site.
The mouse gene was shown to be 7.7 kilobases and to contain 13 exons as
previously shown for the human, mouse, and rabbit genes (21, 48, 49).
The present study revealed the presence of two potential transcription
initiation sites at positions 19 and 20 upstream of the ATG translation
initiator codon. One of those sites corresponds to the site mapped for
the human gene (21), but they differ slightly from that reported by
Masure et al. (48). Sequencing of the upstream region
revealed high sequence conservation between The present results from the studies with transgenic mice demonstrated
that the 2722-base pair 5' upstream region does not confer
cell-specific expression in vivo. However, some of the numerous transcription factor consensus binding motifs contained within
this sequence are probably essential for the basic activity of the
MMP-9 promoter, as reported in several studies. Thus, Yokoo and
Kitamura (54) showed in transiently transfected glomerular mesangial
cells that AP-1 activation is essential for the induction of MMP-9 by
interleukin-1, which is also mediated through NF- The experiments with promoter/LacZ reporter gene constructs
in transgenic mice showed that expression of the MMP-9 gene in osteoclasts and migrating keratinocytes requires the region between At the cellular level, both the 7700-LacZ and
7700ExIn-LacZ constructs were shown to yield highly specific
expression in osteoclasts of developing bone, cells that normally
strongly express MMP-9 (30). Coexpression of The results of the present study represent an initial step in
identifying the exact mechanisms of cell lineage-specific expression of
an MMP, which has a highly cell-specific expression pattern and
presumably specific function. Further work will be aimed at narrowing
down the region necessary for this expression and finally the actual
nucleotide sequences responsible for the activities. It will also be
particularly interesting to find out whether a single or separate
elements direct expression to osteoclasts and macrophages that are both
derived from monocytes as well as epithelial keratinocytes that appear
to express the MMP-9 gene only during migration.
2722 and
7745 allowed for expression in osteoclasts and migrating keratinocytes, i.e. cells that have been shown to normally express the
enzyme in vivo. The results represent the first in
vivo demonstration of the location of cell-specific control
elements in a matrix metalloproteinase gene and show that element(s)
regulating most cell-specific activities of 92-kDa type collagenase are
located in the
2722 to
7745 base pair region.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
Fix phage (Stratagene, catalog
no. 46309) were screened using a human MMP-9 cDNA probe (pHG1, Ref. 21) labeled with 32P by random priming. Hybridization was
performed in 5× SSC, 5× Denhardt's solution, and 0.1% SDS overnight
at 42 °C, after which the filters were washed at a final
concentration of 0.1× SSC and 0.1% SDS at 42 °C. The clones were
isolated and purified utilizing standard procedures and mapped using
restriction endonucleases.
(Stratagene).
-32P]ATP using T4 polynucleotide
kinase (38). The reverse transcription reaction was carried out under
standard conditions, and the primer-extended products were run on a
sequencing gel along with sequencing reactions from the mouse MMP-9
gene using the same oligonucleotide as in the primer extension assay.
-galactopyronoside (X-gal) as described
by Behringer et al. (42).
RESULTS
phage clone
CM-1 contained about 11,000 bp of the 5'-flanking region. Sequencing of
exons revealed that the murine gene contains 13 exons, which correspond
in size to those of the human gene (Fig. 1, Table
I). The only significant differences are
exons 9 and 13 which, respectively, contain 54 and 15 base pairs more than in the human gene. Furthermore, introns 1, 2, 3, and 12 in the
mouse gene are about half the size of corresponding human introns
(21).
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Fig. 1.
Structure of the mouse MMP-9 gene and
sequence of the 5'-end. Top, the entire gene and
flanking regions were contained in the MGC-1 cosmid clone, which
reached 23 kilobases downstream from the last exon. The CM-1 clone
further provided about 11 kilobases of the 5'-flanking region. The
restriction map was obtained using NotI (N),
XhoI (X), BamHI (B),
EcoRI (E), and HindIII (H).
Middle, the exons of the gene are depicted by
boxes, numbered from the 5'-end, and the introns and
flanking sequences are shown by a solid line. Scale in
kilobases is shown. Bottom, nucleotide sequence of the
5'-end flanking region. The bent arrow indicates the
transcription initiation site as determined by primer extension. The
numbering of nucleotides starts at the transcription initiation site.
The TATA motif, GC boxes, AP-1-like, AP2, polyoma virus enhancer
A-binding protein-3, and NF-
B binding consensus sequences are
boxed. Alternating CA-rich sequences are
underlined.
Exon-intron junctions of the mouse gene for MMP-9
30 to
25 but no CCAAT box. There are three GC boxes that may serve as binding sites for the transcription factor Sp1 (46)
(located at positions
62 to
57,
451 to
446, and
598 to
589). Four AP-1-like binding sites were also identified (
50 to
44,
88 to
80,
472 to
465, and
1080 to
1072). Two of those
correspond to similar sequences in the human gene, but sites corresponding to the first one (
50 to
44) and the most upstream one
have not been reported in the human gene. Several conserved sequence
elements with similarity to the polyoma virus enhancer A-binding
protein-3 sites (47) were found in the 5'-flanking sequence (
365 to
360,
479 to
474,
658 to
653, and
901 to
896), as well as
in the first intron (Fig. 1). One consensus sequence (5'-CCCCAGGC-3')
for AP-2 (
590 to
483), several microsatellite segments of
alternating CA residues, as well as one NF-
B motif (
527 to
519)
were also present. A putative tumor growth factor-
1-inhibitory element found in the human gene was absent in the murine promoter. During the course of this work, characterizations of the MMP-9 gene
from mouse (48) and rabbit (49) were published.
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Fig. 2.
Determination of the transcription initiation
site. The mRNA start site was localized by primer extension
analysis using poly(A) RNA from mouse skull as described under
"Experimental Procedures." Lanes 1-4,
results from a co-run of the sequencing reactions of cloned genomic DNA
with guanidine (G), adenosine (A), thymidine
(T), and cytosine (C) indicated at the
top. The transcription start site is shown in lane
5.
-galactosidase gene as a
reporter were made (Fig. 3). Three
constructs, 645-LacZ, 2700-LacZ, and
7700-LacZ, contained 0.65, 2.7, and 7.7 kb of the
5'-flanking region, and three constructs, 645ExIn-LacZ,
2700ExIn-LacZ, and 7700ExIn-LacZ, contained
additionally the first exon and intron of the MMP-9 gene in front of
the LacZ gene. The constructs containing exon 1 had a
mutation in the ATG initiator codon for translation (ATG
ATC) to
allow translation of the transcript to start from the ATG methionine
initiator codon in the LacZ gene. Intron 1 was included in
some of the construct, since it has been shown to contain enhancer
elements in other genes such as that for the
1 chain of type I
collagen (50). Three to eight lines of mice were generated with each
construct to ensure that the expression pattern obtained with each
construct was repeatable. Polymerase chain reaction and Southern
analyses were carried out to establish integration of the inserts into
the genome, and histochemical analyses with X-gal provided evidence
about cell-specific expression patterns of the transgene.
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Fig. 3.
Schematic illustration of the MMP-9
promoter-LacZ reporter gene constructs. The
numbers on the constructs are the distance (in base pairs)
from the transcription initiation site (+1). The three lowest
constructs (7700ExIn-LacZ, 2700 ExIn-LacZ, and
645ExIn-LacZ) contain the first exon (141 bp) and intron
(435 bp) of the MMP-9 gene. The asterisk in these constructs
depicts the presence of a point mutation introduced to the ATG codon
(ATG mutated to ATC), so that translation starts from the ATG codon in
the LacZ gene.
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Fig. 4.
Expression of MMP-9
promoter/LacZ constructs in 15.5-day-old transgenic
mouse embryos. Embryos containing 7700-LacZ yield
expression in the scapula, long bones, fore and hind limbs, ribs,
and the lower jaw. Furthermore, strong expression is present in hair
follicles. Embryos transgenic for construct 7700ExIn-LacZ
reveal essentially the same expression pattern as 7700-LacZ,
except that the expression in hair follicles is absent.
2722 to
7745 Upstream Region of the MMP-9 Gene Confers
Expression to Osteoclasts--
Only mice made transgenic with
constructs containing 7.7 kb of the 5'-flanking region of the MMP-9
gene yielded expression of the LacZ gene in bones as shown
in whole embryos in Fig. 4. In order to assess the expression pattern
at the cellular level, we carried out microscopic histochemical
analysis, partially combined with in situ hybridization, to
establish if the LacZ expression corresponds to that of the
endogenous gene. In Fig. 5, A
and B, staining with X-gal shows expression of the transgene
in single cells located at the site of endochondral ossification in the diaphysis of long bones, beneath hypertrophic chondrocytes of the
epiphysis. This result is practically identical to what we have
previously shown for the endogenous gene by in situ
hybridization (30). In that report, we also assigned the endogenous
gene expression specifically to cells that were shown to be osteoclasts
by histochemical staining with tartrate-resistant acid phosphatase. In
order to demonstrate that the cells expressing 7700-LacZ
were indeed osteoclasts, we carried out in situ
hybridization of X-gal-stained tissues, and these experiments showed
the signals to be present in cells positive for the blue color produced
by
-galactosidase (Fig. 5C). These experiments
demonstrated that expression of the 7700-LacZ construct was
confined to osteoclasts in developing bone. Transgenic mice harboring
insert 7700ExInLacZ showed exactly the same expression pattern as construct 7700-LacZ, demonstrating that the
upstream segment
2722 to
7745 includes the cis-regulatory
element(s) required for osteoclast expression.
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Fig. 5.
Expression of 7700ExIn-LacZ
in developing bone. In A (4× objective) and
B (10× objective) expression of LacZ is present
in single cells located at the site of endochondral ossification in the
diaphysis (d) of tibia beneath hypertrophic chondrocytes of
the epiphysis (e) shown with safranin and X-gal stain.
C, in situ hybridization and X-gal staining
demonstrate that the 7700ExIn-LacZ transgene and endogenous
MMP-9 are coexpressed in most osteoclasts (arrows).
-galactosidase. In
general, we did not observe expression of the transgene in epithelia of organs such as skin, lung, or gastrointestinal tract with any of the
six constructs made in this study.
-galactosidase at the wound edges (Fig. 6A).
Staining of tissue sections from the wounds demonstrated strong
positive reaction in keratinocytes migrating in under the fibrin clot
covering 2-day-old wound (Fig. 6, B and C). Fig.
6D, showing double staining of sections with X-gal and anti-MMP-9 antibodies, demonstrated that most migrating keratinocytes expressing X-gal also costained with the MMP-9 antibody. Furthermore, scattered cells, presumably macrophages, beneath the wound contained the protein, and some of them also expressed the LacZ
reporter gene. The fact that all cells and their immediate surroundings stained with the MMP-9 antibody but not X-gal may be due to secretion of the MMP-9 enzyme. Identification of
-galactosidase-expressing cells as keratinocytes was carried out by counterstaining with cytokeratin antibodies (Fig. 6, E and F).
Keratinocytes resting on the normal basement membrane adjacent to the
wound did not show any staining reaction. At day 7, the
reepithelialization process was complete. The new epidermis was
thicker, and the presence of fibrotic tissue was apparent, but
expression of
-galactosidase by keratinocytes had ceased (Fig.
6G). In all founder lines, cells at wound edges expressed
-galactosidase.
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Fig. 6.
Expression of 7700ExIn-LacZ
in incision skin wounds. A, X-gal staining of the
surface of a 3-day-old wound reveals positive staining of cells located
at the wound edge. B, in a cross-section of a 2-day old
wound, single cells migrating over the wound beneath the fibrin clot
show expression of -galactosidase. In contrast, keratinocytes
resting on a mature basement membrane on both sides of the wound do not
express the transgene, shown with hematoxylin-eosin and X-gal staining.
C, amplification of the boxed area in A.
D, double staining of a 2-day-old wound with X-gal and
anti-MMP-9 antibodies. Most of the migrating keratinocytes exhibit
codistribution of X-gal and MMP-9, while keratinocytes of the normal
epithelium at the edges of the wound are negative for both. Some cells
in the dermis and their immediate surrounding, presumably macrophages,
show the presence of the MMP-9 protein, and some of those cells also
show reaction with X-gal. E, immunostaining of a 2-day-old
wound with anti-cytokeratin antibodies of a hematoxylin-eosin and
X-gal-stained tissue demonstrates that cells expressing
7700ExIn-LacZ are keratinocytes. F, amplification
of the region boxed in E. G, 7-day-old wound.
Reepithelialization is complete. The new epithelium over the fibrotic
scar tissue is thicker than the adjacent normal epithelium. Expression
of 7700ExIn-LacZ has ceased. Hematoxylin-eosin and X-gal
stains were used.
DISCUSSION
1 and
600 bases in the
mouse and human genes. Both genes have a TATA box-like motif, TTAAA, at about position
30 and lack a CCAAT box. The mouse gene has three GC
boxes and three AP-1-like binding sites, as opposed to one and two in
the human gene, respectively (21). Additionally, the mouse gene has a
fourth AP-1-like motif further upstream. Several CA repeat
microsatellite segments were observed in the 2722-base pair upstream
sequence in the mouse gene, including one located close to the
transcription initiation site as in the human counterpart.
Computational analysis of the 5'-flanking region of the gene revealed
several additional putative binding sites whose functionality and
necessity for MMP-9 gene regulation still remain to be assigned.
However, one or more of those binding sites have been implicated in
mediating the effects of a diverse set of agents, which include tumor
necrosis factor
, 12-O-tetradecanoyl-phorbol-13-acetate (51), v-Src (52), and Ha-Ras (53). While those studies focused on
determining the transcriptional requirements for MMP-9 induction, they
do not provide any information on regulatory requirements driving cell
lineage-specific expression of the MMP-9 gene.
B stimulation.
Himelstein et al. (55) also showed in cell transfection studies that motifs of the MMP-9 promoter, such as NF-
B, Sp1, Ets,
AP-1, and a retinoblastoma element participate in transcriptional regulation of the MMP-9 expression. Furthermore, Gum et al.
(56) have reported that mutation of the most downstream AP-1 motif practically abolishes the activity of a MMP-9 promoter-driven CAT reporter.
2722 and
7745. Constructs containing this segment yielded strong expression in osteoclasts and in migrating keratinocytes of a healing
wound. The present results also demonstrated that the first intron does
not contain an enhancer, as this intron does in several extracellular
matrix genes (50, 57). However, this intron may be important for
restricting ectopic expression, since the 7700-LacZ
construct yielded ectopic expression in epithelial hair follicle cells,
while mice expressing construct 7700ExIn-LacZ only exhibited
expression in cells normally expressing MMP-9.
-galactoctisidase with
that of the endogenous gene was verified in situ
hybridization analysis of the same tissue sections with an MMP-9 probe
(see Fig. 5C). In addition to the major expression in
osteoclasts, expression of MMP-9 has been shown to occur in migrating
keratinocytes of a healing wound (27). In this study, both constructs
7700-LacZ and 7700ExIn-LacZ were expressed in
corresponding keratinocytes of transgenic mice, and we also showed
extensive codistribution of the MMP-9 protein and LacZ
expression by double staining. Therefore, cis-regulatory element(s) for
this expression pattern must also be present in the sequence between
2722 and
7745. Furthermore, as in the in vivo situation
for the endogenous MMP-9 gene, keratinocytes resting on a normal mature
basement membrane did not express the reporter gene in transgenic mice
or contain the MMP-9 protein, as determined by immunohistochemical
staining. Following complete healing and reepithelialization of the
skin wound at day 7, expression of the reporter gene ceased,
essentially as has been shown for the endogenous gene (27). It has
previously been shown that MMP-9 is expressed in invading trophoblasts
of the implanting embryo (33, 34) as well as by macrophages
infiltrating invasive breast and colon cancers, while the actual cancer
cells do not express the enzyme (29, 58). We have recently shown that
trophoblasts of implanting embryos of mice transgenic for constructs
7700-LacZ and 7700ExIn-LacZ express the reporter
gene, and furthermore, in such transgenic mice macrophages located
around invading exogenous carcinoma cells also express the reporter
gene. This suggests that the
2722 to
7745 segment also contains
element(s) necessary for induction of expression of the gene in both
trophoblasts and macrophages.2
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ACKNOWLEDGEMENT |
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We thank Prof. P. Carmeliet from the K. U. (Leuven, Belgium) for kindly providing the antibody raised against MMP-9.
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FOOTNOTES |
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* This work was supported in part by grants from the Academy of Finland, the Swedish Medical Research Council, the Swedish Cancer Society, Hedlundís Foundation, EU Biomed II Grant BMH4-CT96-0017, the Fonds de la Recherche Scientifique Médicale, the Fonds National de la Recherche Scientifique, the Center Anticancéreux prés l'Université de Liège, and the CGER-Assurances 1996/1999.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors contributed equally to this work.
** To whom correspondence should be addressed: Division of Matrix Biology, Dept. of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden. Tel.: 46-8-728-7720; Fax: 46-8-31-61-65; E-mail: karl.tryggvason{at}mbb.ki.se.
2 C. Munaut, T. Salonurmi, S. Kontusaari, P. Reponen, T. Morita, J.-M. Foidart, and K. Tryggvason, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
MMP, matrix
metalloproteinase;
X-gal, 5-bromo-4-chloro-3-indolyl--galactopyronoside;
kb, kilobase pair(s);
bp, base pair(s).
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REFERENCES |
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