(Received for publication, June 4, 1996, and in revised form, November 7, 1996)
From the Matrix metalloproteinases (MMPs) produced by rat
smooth muscle cells (SMCs) were investigated. SMCs expressed three
kinds of membrane-type MMP, MT1-MMP, MT2-MMP, and MT3-MMP, and the
MT-MMP expression was stimulated by the presence of serum. MT3-MMP was characterized further by cloning its cDNA. A rat MT3-MMP cDNA encoding 607 amino acids and a cDNA for its transmembrane
domainless variant MT3-MMP-del were cloned from a rat SMC cDNA
library; a human MT3-MMP cDNA was cloned from a fetal brain
cDNA library. Human brain MT3-MMP was similar but not identical to
the previously reported human placenta MT3-MMP (94.4% homology). When
the MT3-MMP cDNA was expressed in COS-7 cells, endogenous
progelatinase A was processed to the mature form. The transfection of
rat MT3-MMP-del efficiently converted progelatinase A to the
intermediate form but not to the mature one, indicating that the
transmembrane domain is important for the complete processing of
progelatinase A to maturation. Both MT3-MMP-del and MT3-MMP hydrolyzed
gelatin and casein, indicating their broad substrate specificity.
Results of experiments with a synthetic MMP inhibitor suggested that
MT3-MMP-del and MT3-MMP are rapidly degraded immediately after
maturation. The present study suggests that multiple forms of MMPs
including MT3-MMP are involved in the matrix remodeling of blood
vessels.
Matrix metalloproteinases (MMPs)1 play
an essential role in tissue remodeling under various physiological and
pathological conditions such as morphogenesis, angiogenesis, tissue
repair, arthritis, and tumor invasion (for review, see Refs. 1-3). For example, migrating cells such as invasive tumor cells and inflammatory leukocytes utilize these enzymes to degrade extracellular matrix proteins present in basement membranes and connective tissues as
physical barriers. Among the MMP family, gelatinase A (MMP-2) and
gelatinase B (MMP-9) are critical in the invasion of tumor cells and
other cells into basement membranes because of their strong activity
against type IV collagen, the major component of basement membranes
(4-7). Most MMPs are secreted in a latent form (pro-MMP) and activated
by serine proteinases or some activated MMPs. The activities of
activated MMPs are regulated by natural inhibitors called tissue
inhibitors of metalloproteinases (TIMPs) (1-3).
In tumor tissues, gelatinase A is often present on tumor cell surfaces
(8, 9), although its mRNA is mainly expressed by surrounding
stromal cells (10-12). Many studies have shown that progelatinase A is
activated by a metalloproteinase bound to cell membrane (13-17). Using
reverse transcription-polymerase chain reaction (RT-PCR), Sato and
co-workers (18, 19) recently identified a novel membrane-type MMP,
named MT-MMP (MT1-MMP), which is responsible for the activation of
progelatinase A on the cell surface. MT1-MMP mRNA is expressed at a
high level in various cancer tissues compared with corresponding normal
tissues or benign tumors (18, 20). Therefore, MT1-MMP is believed to
play a key role in the spatially regulated proteolysis by invasive
tumor cells. Very recently, three other MT-MMPs, MT2-MMP (21), MT3-MMP
(22), and MT4-MMP (23), have been identified. However, at present,
these new MT-MMPs are relatively poorly characterized compared with
MT1-MMP.
Like invasive tumor cells, vascular smooth muscle cells (SMCs) are
known to migrate and proliferate under certain pathological conditions
such as intimal hyperplasia after arterial injury (24), vascular
grafting (25), and atherosclerosis (26). These processes are presumed
to require partial degradation of the vascular basement membrane and
extracellular matrix surrounding the cells (27, 28). However, less is
known about MMP species involved in these processes and their roles.
Some previous studies have shown that SMCs produce interstitial
collagenase (29, 30), gelatinases A and B (31, 32), and stromelysin
(32). In the present study, we analyzed MMP, especially MT-MMP
mRNAs expressed in rat vascular SMCs and normal tissues, using the
RT-PCR method and Northern analysis. In addition, the cDNA for two
forms of MT3-MMPs with and without the transmembrane domain were
cloned, and their enzymatic functions were investigated.
cDNA libraries constructed from human
placenta and fetal brain and plasmid vector pBluescript SK(+) were
purchased from Stratagene (La Jolla, CA). [ Vascular SMCs were isolated from rat thoracic
aorta by the explant method of Chamley-Cambell (34). SMCs were grown in
Dulbecco's modified Eagle's medium (Nissui, Tokyo, Japan)
supplemented with 10% fetal calf serum, 2 mM glutamine,
and 30 µg/ml gentamycin under humidified 5% CO2
conditions. SMCs were repeatedly subcultured by trypsinization and used
for experiments between the 5th and 20th passages. COS-7 cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum and 2 mM glutamine.
Total RNA was extracted from SMCs
by the acid-guanidinium phenol chloroform method (35) and reverse
transcribed in the presence of random hexanucleotides. Synthesized
cDNA was amplified by PCR. A degenerated primer set for PCR was
designed from two highly conserved sequences of known MMPs, the
cysteine switch (PRCGVPD) and the Zn2+ binding site
(AAHELGH). Taking account of the codon usage in rat (36), two 20-mer
oligonucleotides, CCI C/AGI TGT GGI GTI CCT/A GA for the sense
primer (cysteine switch) and TG ICC IAG CTC ATG IGC AGC for the
antisense primer (Zn2+ binding site) were synthesized. PCR
was performed with 35 cycles of heat denaturation at 94 °C for 1-2
min, annealing at 50 °C for 2 min, and polymerization at 72 °C
for 3 min. Resultant amplified DNA fragments were electrophoretically
separated and cloned into a pUC118 plasmid vector (Takara Shuzo).
Poly(A)+ RNA was
isolated from total RNA of rat SMCs using oligo(dT)-coupled latex beads
(OligoTex dT; Takara Shuzo). A cDNA library that contained 3.2 × 106 recombinant clones was constructed from 5 µg of
the rat SMC poly(A)+ RNA by the method of Gubler and
Hoffman (37), using a ZAP-cDNA cloning kit (Stratagene).
cDNA
libraries were screened by the standard plaque hybridization method.
Digoxigenin-labeled probes were prepared by PCR using
digoxigenin-11-dUTP (Boehringer Mannheim, Germany), heat denatured, and
used for hybridization. The hybridized probe molecules on the
transferred membranes were visualized by the Dig-ELISA (enzyme
linked-immunosorbent assay) non-radioisotopic nucleic acid detection
system (Boehringer Mannheim) with an anti-digoxigenin antibody
conjugated with alkaline phosphatase. Alternatively, a
32P-labeled probe was used to screen a cDNA library
from human fetal brain with the sequence of rat MT3-MMP. From the
isolated clones, insert cDNAs were subcloned to an appropriate
plasmid vector.
DNA sequences were determined by the
non-radioisotopic dideoxynucleotide chain termination method with a
LI-COR model 4000L DNA sequencer (Lincoln, NE). Determined DNA
sequences were analyzed by DNASIS software (Hitachi Software
Engineering, Kanagawa, Japan) and Genetyx Mac software (Software
Development Co. Ltd., Tokyo).
Total RNAs were electrophoresed on
1% agarose-formaldehyde gels and transferred onto nylon membranes by
capillary elution. The nylon membranes were hybridized with
32P-labeled probes and washed by the standard method. The
hybridized signals were visualized by autoradiography or a bio-imaging
analyzer BAS-2000II (Fuji Film, Tokyo).
Both
full-length cDNAs of human MT3-MMP and rat MT3-MMP-del, a
transmembrane domainless variant of MT3-MMP, were inserted into a
mammalian expression vector pGM for transient expression. The
recombinant plasmids were transfected into COS-7 cells by the
standard lipofection method. To obtain serum-free conditioned medium
(CM), the transfected cells were incubated in serum-free medium for 2 days. The resultant CM was collected, dialyzed against H2O,
lyophilized, and dissolved in a small volume of 10 mM
Tris-HCl (pH 7.5) buffer containing 0.05% Brij-35. To analyze
intracellular enzymes, the transfected cells were directly dissolved in
sodium dodecyl sulfate-sample buffer and used.
Zymography was carried out on 7.5 and 10%
polyacrylamide gels containing 1 mg/ml gelatin or casein, as described
before (7, 38).
MMP mRNAs expressed in cultured rat SMCs were screened
by RT-PCR with a degenerated primer set, which had been designed from the two highly conserved sequences of known MMPs, the Zn2+
binding active site and the regulatory site cysteine switch. Total RNA
purified from SMCs was reverse transcribed, and the products were
subjected to PCR. Agarose gel electrophoresis of the RT-PCR products
reproducibly detected amplified DNA fragments of 750, 520, 450, and 400 bp (Fig. 1). Each DNA fragment was isolated, cloned into
a plasmid vector, and sequenced. The sequence analysis showed that the
amplified fragments of 750 and 520 bp were nonspecific amplification
products. The 400-bp fragment contained two different sequences known
as rat stromelysin 1 (39) and rat collagenase, the latter of which had
recently been identified as the homolog of human collagenase 3 (40)
(data not shown). On the other hand, three similar but distinct
sequences were identified from the electrophoretically single 450-bp
fragment. One of the sequences was identical to that of rat membrane
type 1 MMP (MT1-MMP) reported by Okada et al. (20). The
others were highly homologous to human MT2-MMP and MT3-MMP,
respectively, which had been reported recently by different
groups (21, 22). Thus the two sequences were regarded as rat
homologs of MT2-MMP and MT3-MMP. These results indicate that rat
SMC expresses three types of MT-MMPs simultaneously.
When the CM of cultured SMCs was analyzed by gelatin zymography, a high
level of progelatinase A and a low level of its activated form were
detected (data not shown).
To date, no normal
cells producing MT3-MMP have been reported, and this enzyme has been
characterized poorly compared with MT1-MMP. Therefore, we attempted to
clone rat MT3-MMP cDNA. A rat SMC cDNA library was screened
with the 450-bp MT3-MMP cDNA fragment as a probe, and three
positively hybridized clones were isolated. These clones contained an
insert cDNA of approximately 3.5 kilobase pairs with a typical
poly(A)+ tail and a polyadenylation signal. Therefore, the
longest clone, termed pratMT3-MMPc1, was sequenced completely on both
strands. This MT3-MMP cDNA was 3,536 bp in length and encompassed a
long open reading frame that started from ATG at nucleotide position 432 and stopped at position 2,255. The open reading frame contained the
sequence identical to the originally identified RT-PCR product of 450 bp. The MT3-MMP cDNA encodes a protein of 607 amino acids, and its
molecular weight is calculated to be 69,622. The deduced amino acid
sequence of the MT3-MMP contains a carboxyl-terminal hydrophobic
stretch reported as a transmembrane sequence of MT3-MMP (see Fig.
3).
In addition to the MT3-MMP cDNA clone, another MT3-MMP cDNA was
cloned from the same cDNA library of rat SMC. This cDNA
differed from the above cDNA in two nucleotides at positions 2070 and 2071 as follows: CCA A TGA in the second cDNA
and CCA AT GA in the first cDNA. These nucleotide
addition and replacement in the second clone produce the termination
signal TGA just before the transmembrane domain, and hence the cDNA
encodes a putative transmembrane domainless MT3-MMP variant,
MT3-MMP-del, which is shorter than the normal MT3-MMP by 60 amino acids
(see Fig. 3). When the presence of the two sequences in cDNAs from
cultured SMC and the testis and brain of normal rats was examined by
RT-PCR, only the MT3-MMP sequence with the putative transmembrane
domain was obtained, suggesting that the MT3-MMP-del was either a very
minor variant or an artificial product resulting from the misreading of
reverse transcriptase in the construction of the cDNA library. When
the rat SMC cDNA library was screened with the rat MT3-MMP probe,
three weakly hybridized clones were also obtained. Analysis of their
partial nucleotide sequences indicated that they corresponded to rat
MT1-MMP. One of the clones, termed pratMT1-MMP, was used as a probe for Northern blotting analysis of rat MT1-MMP.
To examine the presence of MT3-MMP-del in humans, we also attempted to
clone human MT3-MMP. Because Northern blotting analysis showed that
MT3-MMP mRNA was most predominantly expressed in human fetal brain,
the cDNA library from the tissue was screened with the rat cDNA
as the probe. From eight positively hybridized clones, a cDNA of
2,052 bp containing a whole open reading frame was obtained. As shown
in Fig. 3, this open reading frame encodes 607 amino acids, in
accordance with rat MT3-MMP, and the predicted amino acid sequence
contains the putative transmembrane domain and is 98.0% homologous to
that of rat MT3-MMP. All of the seven other clones had the same
sequence. The predicted primary structure of the human brain MT3-MMP is
similar but not identical in both amino acid sequence and number of
total amino acids to that of the MT3-MMP cloned from human placenta by
Takino et al. (22): 33 of the amino acid residues differ,
including the insertion of 3 amino acids in the brain MT3-MMP (94.4%
homology). The differences are localized in two regions (residues
273-289 and 501-527). The amino acid sequences in these regions of
human brain MT3-MMP are identical to those of rat MT3-MMP (Fig.
2). The homology of the human brain MT3-MMP to other
human MMPs is as follows: MT1-MMP (54.3%), gelatinase A (43.3%) (6),
gelatinase B (41.6%), stromelysin (39.7%) (41), stromelysin 3 (36.0%) (42), and matrilysin (39.4%) (43).
Using the same method as for MT3-MMP, we also cloned a cDNA for
MT2-MMP from a human placental cDNA library. The sequence of human
MT2-MMP cDNA was identical to that of MT2-MMP reported by Will and
Hinzmann (21).
Expression of three MT-MMPs in rat SMCs and rat tissues
was examined by Northern blotting analysis. Fig. 3 shows
the expression of three MT-MMP mRNAs in SMCs cultured in the
presence or absence of 10% fetal calf serum. The three MT-MMP genes
were expressed predominantly in SMCs in the presence of serum,
indicating that their expression was up-regulated by serum factors.
Expression of three MT-MMP mRNAs in eight tissues from normal rats
is shown in Fig. 4. MT3-MMP mRNA was strongly
detectable in the lung and brain, weakly detectable in the spleen and
liver, but undetectable in the heart, skeletal muscle, and kidney. In
the testis, a smaller sized transcript was observed. This seemed to be
the product of the MT3-MMP gene that had been alternatively spliced or
that suffered some other modification. It should be noted that there is
a big difference in the size of mRNA between rat MT3-MMP (3.5 kilobases) and human MT3-MMP (12 kilobases) (22), although their
protein products contain the same number of amino acid residues.
MT2-MMP mRNA was most abundant in the lung and detectable in the
brain, liver, skeletal muscle, and kidney but undetectable in the
heart, spleen and testis. MT1-MMP mRNA was most abundant in the
lung and liver and detectable in the other tissues tested except for the heart.
To verify enzymatic
activity, the cDNAs for human brain-derived MT3-MMP and rat
SMC-derived MT3-MMP-del were individually transfected into COS-7 cells
by the lipofection method. As shown in Fig.
5A, the CM of COS-7 cells transfected with
human brain MT3-MMP cDNA showed weak but clear bands of the
intermediate form (59 kDa) and active form (57 kDa) of gelatinase A in
addition to an intense band of progelatinase A (66 kDa), although the
cells transfected with the control vector showed no band at 59 or 57 kDa. On the other hand, the transfection of rat MT3-MMP-del cDNA produced a much higher level of the intermediate form and a trace amount of the active form. The less efficient conversion of the intermediate form to the active form seems due to the poor
intermolecular autolytic activation of the intermediate form in
solution. These results indicate that MT3-MMP is able to process
progelatinase A regardless of the presence or absence of the
transmembrane sequence, but the sequence is important for the complete
activation of progelatinase A. On the other hand, activation of
progelatinase B (92 kDa) was not seen in any transfectants (Fig.
5A).
The gelatin zymography of the CM of the MT3-MMP-del transfectants
specifically showed faint and broad gelatinolytic activity at 45-50
kDa in addition to the activities due to gelatinases A and B (Fig.
5A). The broad activity appeared to consist of a major
45-kDa and a minor 50-kDa band and was more prominent in the casein
zymography (Fig. 5B). The proteolytic activity of 45-50 kDa
was Ca2+-dependent, and its electrophoretic
mobility was hardly affected by the treatment with
p-aminophenylmercuric acetate (data not shown), indicating
that it has already been activated or can not be activated by
p-aminophenylmercuric acetate. The molecular size (45-50
kDa) of the unidentified enzyme is compatible with the predicted size
of MT3-MMP-del. In other experiments, recombinant rat MT3-MMP-del
expressed in Escherichia coli also degraded gelatin and
casein in zymography and was resistant to activation by
p-aminophenylmercuric acetate (data not shown). Taken
together, we considered that the activity of 45-50 kDa was due to the
secreted MT3-MMP-del expressed transiently in the cultured cells.
To investigate
autolytic processing of the MT3-MMPs with and without the transmembrane
domain, a synthetic inhibitor of MMPs, KB8301, was used. When 10 µM KB8301 was added to the culture of human MT3-MMP and
rat MT3-MMP-del transfectants, the activation of progelatinase A was
blocked completely in both transfectants (Fig.
6A). In addition, the treatment with KB8301
markedly increased the gelatinolytic activity of 45-50 kDa in the CM
of MT3-TM-del transfectants, suggesting that KB8301 inhibited the
autolytic degradation of the secreted MT3-MMP-del. When the cell
lysates of the transfectants were analyzed by casein zymography, a
caseinolytic activity of 62 kDa was detectable in the MT3-MMP
transfectants (Fig. 6B). The treatment of the MT3-MMP
transfectants with KB8301 produced an additional caseinolytic activity
of 55 kDa. The 62- and 55-kDa activities seemed to correspond to the
proform and the mature form of membrane-bound MT3-MMP, respectively.
The cell lysate of the MT3-MMP-del transfectants showed a major 55-kDa activity and a minor 50-kDa activity regardless of the KB8301 treatment. It seemed likely that the 55-, 50-, and 45-kDa bands in the
MT3-MMP-del transfectants corresponded to the proform, intermediate
form, and the mature form of MT3-MMP-del, respectively. These results
strongly suggest that both MT3-MMP and MT3-MMP-del are degraded rapidly
after intracellular activation.
The present study showed that rat SMCs express at least three
kinds of MT-MMPs, MT1-MMP, MT2-MMP, and MT3-MMP, in addition to
gelatinase A, collagenase 3, and stromelysin 1. We cloned cDNAs for
rat MT3-MMP, its transmembrane domainless variant MT3-MMP-del, and
human MT3-MMP. Human MT3-MMP cDNA was cloned previously by Takino
et al. (22) from a human placental cDNA library as a membrane-bound activator of progelatinase A. Human brain MT3-MMP in
this study was similar to the previously reported MT3-MMP but differed
in 33 amino acid residues including the insertion of 3 amino acids
(94.4% homology). These differences between human brain MT3-MMP and
human placenta MT3-MMP were completely conserved between rat SMC and
human brain MT3-MMPs. In addition, the sequences of eight positively
hybridized clones obtained from the fetal brain cDNA library
matched with the brain MT3-MMP but not with the reported placenta
MT3-MMP. Therefore, it is concluded that the MT3-MMP that is expressed
mainly in the brain and SMCs has the structure determined in this
study. At present it is not clear whether the structural differences
between the two human MT3-MMPs are the result of some experimental
error, gene multiplicity, or microheterogeneity of a single gene.
Similarly, little is known about rat MT3-MMP-del, which lacks the
transmembrane domain. Such MT-MMP variants have not been reported
elsewhere. Despite repeated cDNA cloning, we could not obtain this
variant cDNA. Therefore, MT3-MMP-del seems to be a very minor
variant of rat MT3-MMP or an artificial product resulting from the
misreading of the reverse transcriptase. However, MT3-MMP-del may prove
a useful tool for understanding the enzymatic activity of MT3-MMP.
MT1-MMP and MT3-MMP activate progelatinase A when their cDNAs are
transfected into progelatinase A-producing cells (18, 22). MT2-MMP also
has membrane-dependent progelatinase-A-activating activity (our
unpublished data). Cao et al. (44) reported that MT1-MMP
lacking the COOH-terminal transmembrane domain could neither generate
the active form of gelatinase A nor hydrolyze other substrates. However, recent studies showed that recombinant MT1-MMP from which the
membrane-spanning domain had been truncated was able to activate progelatinase A and to degrade gelatin, casein, and some extracellular matrix proteins such as fibronectin or vitronectin (45-47). They also
showed that soluble MT1-MMP expressed in E. coli was
activated by autolysis or partial digestion with trypsin but not by
p-aminophenylmercuric acetate treatment. In the present
study, we showed that rat MT3-MMP-del processed progelatinase A to the
intermediate form even more efficiently than human brain MT3-MMP when
the respective cDNA was transiently expressed in COS-7 cells. The
efficient processing of progelatinase A by MT3-MMP-del seems due to the
high availability of progelatinase A in culture medium. However, the
complete activation of progelatinase A hardly occurred with
MT3-MMP-del. The conversion of the partially activated gelatinase A to
the mature form is known to involve intermolecular autolytic processing
(48). Apparently the membrane-bound MT3-MMP is more suited than the
soluble enzyme to this process because the intermediate form of
gelatinase A can be concentrated on the cell surface in the former
enzyme. In the case of MT3-MMP-del, TIMP-2 may quickly bind to the
partially activated gelatinase A in solution, blocking the autolytic
processing. These results suggest that the membrane binding of MT3-MMP
is not essential for the initial processing of progelatinase A but
important for the autolytic conversion of the intermediate form to the
mature form.
The present study also showed that the treatment of the cDNA
transfectants with the synthetic inhibitor KB8301 increased the amount
of MT3-MMP-del in culture medium and MT3-MMP in cell membrane, suggesting that these enzymes are rapidly degraded by autolysis after
maturation. This seems to be an important regulatory mechanism to
prevent excess activity of MT-MMPs because these enzymes, unlike most
other MMPs, are intracellularly activated by furin or a similar serine
proteinase (49).
SMCs are known to migrate and proliferate actively under various
physiological and pathological conditions. Zempo et al. (27) have shown that the activities of gelatinases A and B are increased in
rat carotid artery after balloon catheter injury. In the present study,
the expression of MT-MMPs, especially MT3-MMP, in cultured SMCs was
strongly induced by the presence of serum. These facts strongly suggest
that the three MT-MMPs are also involved in the migration and
proliferation of SMCs under various physiological and pathological
conditions. MT3-MMP-del had weak gelatinolytic and relatively high
caseinolytic activities in the zymography assay. The MT3-MMP with the
transmembrane domain also showed caseinolytic activity in zymography.
These results suggest that MT-MMPs probably hydrolyze various
extracellular matrix proteins and other functional proteins on the cell
surface without depending on the activation of progelatinase A. They
might play an important role in the matrix remodeling of blood vessels
by activating progelatinase A and/or directly degrading matrix
proteins.
We detected only a negligible amount of the active form of gelatinase A
in the culture of rat SMCs, even though all three kinds of MT-MMPs were
expressed. This implies that the progelatinase A processing by MT-MMPs
is negatively regulated by some other factors including TIMP-2 in
cultured SMCs. Northern blotting analysis showed that MT1-MMP was
distributed widely to normal rat tissues, although its expression was
especially high in the lung and liver. The expression of MT3-MMP gene
was more restricted to specific organs such as the lung and brain than
was the expression of MT1-MMP and MT2-MMP genes. This suggests some
specific role of MT3-MMP in these organs.
The nucleotide sequences rat MT3-MMP, rat MT3-MMP-del, and
human MT3-MMP reported in this paper have been deposited in the DDBJ,
EMBL, and GenBankTM DNA data bases with
accession nos. D85509[GenBank], D63886[GenBank], and D85511[GenBank], respectively. We thank Dr. Y. Takebe (NIH, Japan) for
providing the mammalian expression vector pcDL-SR
Division of Cell Biology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Materials
-32P]dCTP
was from Amersham (Backinghamshire, United Kingdom). Rat tissue and
human fetal tissue multiple Northern blots were from Clontech
Laboratories (Palo Alto, CA). Mammalian expression vector pGM, which
had been constructed from pCDL-SR
296 (33), was a kind gift from N. Ohkura (Terumo Research and Development Center, Kanagawa, Japan).
Enzymes for DNA digestion and modification were purchased from Takara
Shuzo (Shiga, Japan) and Toyobo (Osaka, Japan). A synthetic hydroxamic
acid inhibitor for MMPs, KB8301, was a generous gift from Dr. K. Yoshino (Kanebo Institute for Cancer Research, Osaka, Japan).
Screening of MMP mRNAs Expressed in Rat SMCs by
RT-PCR
Fig. 1.
Amplification of MMP mRNAs by
RT-PCR. cDNAs obtained from rat SMCs (lane 1) and
human placenta (lane 2) were used as templates for PCR with
the degenerated primers designed from the two highly conserved
sequences of the MMPs. Amplified DNA fragments were separated by
agarose gel electrophoresis. Arrows indicate four DNA
fragments reproducibly amplified in SMCs (400, 450, 520, and 750 bp in
size).
[View Larger Version of this Image (36K GIF file)]
Fig. 3.
Expression of three MT-MMP mRNAs in rat
SMCs. Fifteen µg of total RNA isolated from SMCs cultured in the
presence (+) or absence () of 10% fetal calf serum (FCS)
was subjected to Northern blotting with 32P-labeled rat
MT3-MMP, human MT2-MMP, and rat MT1-MMP cDNAs as probes, as
described under "Experimental Procedures." The same blot was
deprobed and hybridized with a glucose-3-phosphate dehydrogenase (G3PDH) probe as an internal control. Ordinate,
mRNA size in kilobases.
[View Larger Version of this Image (69K GIF file)]
Fig. 2.
Comparison of amino acid sequences of rat
MT3-MMP, human brain MT3-MMP, human placenta MT3-MMP, human MT2-MMP,
and human MT1-MMP. Predicted amino acid sequences of the five
MT-MMPs are aligned to show maximum homology. Amino acid residues
conserved among all the five MMPs are boxed.
Arrows indicate the sequences corresponding to the PCR
primers. A consensus sequence for the cleavage by furin-like serine
proteinases is broken underlined. Clusters of hydrophobic
amino acids in the carboxyl-terminal region of MT-MMPs, which are
thought to be a potential transmembrane domain, are
underlined. The position of the carboxyl-terminal amino acid
(Asp-547) of rat MT3-MMP-del is indicated by an asterisk in
the sequence of rat MT3-MMP.
[View Larger Version of this Image (88K GIF file)]
Fig. 4.
Expression of MT-MMP mRNAs in rat
tissues. The membrane that had been blotted with
poly(A)+ RNAs from eight normal tissues of rat (multiple
tissue Northern blot; Clontech) was probed with 32P-labeled
cDNAs of rat MT3-MMP, human MT2-MMP, and rat MT1-MMP. Ske.
musc., skeletal muscle. Ordinate, mRNA size in
kilobases.
[View Larger Version of this Image (71K GIF file)]
Fig. 5.
Zymographic analysis of conditioned media
from COS-7 cells transfected with human brain MT3-MMP cDNA and with
rat MT3-MMP-del cDNA. The two cDNAs were cloned into the
mammalian expression vector pGM. Human MT3-MMP plasmid (lane
M), rat MT3-MMP-del plasmid (lane D), and control
vector plasmid (lane C) were transfected into COS-7 cells by
the lipofection method. Serum-free CMs of the transfectants were
concentrated and analyzed for secreted proteinases by gelatin
zymography (panel A) and casein zymography (panel
B). Arrowheads indicate the gelatinolytic bands of the proform of gelatinase A at 66 kDa (upper), the intermediate
form at 59 kDa (center), and the mature form at 57 kDa
(lower). An arrow at about 50 kDa in lane
D indicates gelatinolytic activity (panel A) and
caseinolytic activity (panel B) due to secreted MT3-MMP-del.
The CM of the MT3-MMP transfectants showed no band in casein zymography
(data not shown). Ordinate, molecular size in kDa.
[View Larger Version of this Image (91K GIF file)]
Fig. 6.
Effect of a synthetic MMP inhibitor, KB8301,
on MMP activity in CMs and lysates of COS-7 cells transfected with rat
MT3-MMP-del cDNA and with human MT3-MMP cDNA. COS-7 cells
transfected with cDNAs for MT3-MMP (MT3), MT3-MMP-del
(Del), or control plasmid (CT) were incubated for
2 days in serum-free medium with (+) or without () 10 µM KB8301. Panel A, gelatin zymography of CMs. Arrowheads indicate the pro- (upper),
intermediate (center), and mature (lower) forms
of gelatinase A; arrows indicate the 45- and 50-kDa
gelatinolytic activities of MT3-MMP-del. Panel B, casein zymography of cell lysates. Arrowheads indicate the 55- and
50-kDa caseinolytic activities of MT3-MMP-del (Del) and the
62- and 50-kDa activities of MT3-MMP (MT3). CT,
control. Ordinate, molecular size in kDa.
[View Larger Version of this Image (62K GIF file)]
*
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1
The abbreviations used are: MMP(s), matrix
metalloproteinase(s); TIMP(s), tissue inhibitor of
metalloproteinase(s); PCR, polymerase chain reaction; RT, reverse
transcription; MT-MMP, membrane-type MMP; SMC(s), smooth muscle
cell(s); del, domainless; CM, conditioned medium; bp, base
pair(s).
296. We are
grateful to Drs. K. Urakami, N. Ohkura, M. Ito, N. Koshikawa, and S. Higashi for valuable discussions throughout the course of this
work.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.