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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 9, 2001 as doi:10.1096/fj.00-0871fje. |
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Division of Vascular Surgery, Department of Surgery and
* Division of Medical Genetics, Department of Medicine, University of Washington School of Medicine, Seattle, Washington 98195-6410, USA
2Correspondence: Division of Vascular Surgery Department of Surgery, University of Washington School of Medicine, HSB BB442; Box 356410. 1959 NE Pacific St., Seattle, WA 98195-6410, USA. E-mail: clowes{at}u.washington.ed
SPECIFIC AIMS
Matrix metalloproteinases (MMPs) contribute to the progression of vascular diseases such as atherosclerosis, restenosis, and aortic aneurysms. MMPs are expressed by vascular smooth muscle cells (SMCs) and degrade matrix macromolecules such as collagens, fibronectin, and elastin. We have studied the membrane-type MMPs (MT-MMPs), a membrane-bound subgroup of MMPs expressed by SMCs and have reported that MT1- and MT3-MMPs are expressed by activated SMCs. MT-MMPs are involved in matrix degradation through the activation of MMP-2 (gelatinase A, type IV collagenase) or directly. However, their specific functions in SMCs are still unknown. In this report, we investigate MT-MMP functions using an adenoviral system to overexpress MT1- and MT3-MMPs in cultured baboon SMCs.
PRINCIPAL FINDINGS
1. MMP-2-activating activity and degradation of MT-MMP and
their inhibition by synthetic and natural inhibitors
Rat MT1-MMP cDNA in both sense and antisense orientations (AdMT1S
and AdMT1AS, respectively) and human MT3-MMP cDNA (AdMT3) in a sense
orientation were expressed in baboon SMCs by an adenoviral system. To
analyze the MMP-2-activating activity of MT-MMPs expressed by the
system, conditioned media (CMs) were analyzed by gelatin zymography. In
AdMT1S-infected cells (multiplicity of infection (MOI) of
3 x102), endogenous 66 kDa proMMP-2 was
efficiently converted to the 57 kDa active and 59 kDa intermediate
forms (Fig. 1A
). Activation was inhibited by both the natural and
synthetic MMP inhibitors TIMP-2 and BB94, and their effect was dose
dependent (Fig. 1A
, C
). AdMT1AS infection (MOI
3x102) did not affect the pattern of activation
(not shown). AdMT3 infection (MOI 3x102) also
promoted MMP-2 activation, although the pattern was different from that
of AdMT1. The 59 kDa band on gelatin zymograms was more intense than
the 57 kDa band (Fig. 1A
). This difference was conserved in
all conditions tested. The activation of MMP-2 by MT3-MMP was partially
inhibited by BB94 and TIMP-2.
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When total cell lysates were analyzed by immunoblotting, MT1-MMP
protein (60 kDa) was increased after AdMT1S infection. On the other
hand, AdM1AS had no effect on the basal protein expression of MT1- or
MT3-MMP (not shown). The MT1-MMP protein accumulation increased when
BB94 or TIMP-2 was added to the culture (Fig. 1B
, D
).
MT3-MMP (65 kDa) was not detected in AdMT3-infected cells except when
BB94 was added to the culture. This suggests that MT3-MMP is more
rapidly degraded than MT1-MMP. Even at 200 nM, TIMP-2 weakly inhibited
the degradation of MT3-MMP. These results show that these two MT-MMPs
differentially respond to MMP inhibitors and that MT3-MMP is resistant
to synthetic and natural inhibitors. Activated MMP-2 is unlikely to be
responsible for the degradation of MT-MMPs, because the pattern of MT1-
and MT3-MMP protein accumulation in the presence or absence of BB94 was
similar in an MMP-2-negative gastric carcinoma cell line, MKN-45 (data
not shown).
To investigate possible interactions between MT1- and MT3-MMP, we coinfected SMCs with MT1-MMP and MT3-MMP. Increasing amounts of AdMT3 were coinfected with AdMT1. The protein expression of MT1-MMP examined by immunoblotting was decreased in a dose-dependent manner (data not shown). This change was inhibited by BB94 addition. These and other data suggest that MT1-MMP could be degraded by MT3-MMP, presumably on the cell surface.
2. Morphological alteration of SMC-transduced MT-MMPs and the
effect of MMP inhibitors
Significant morphological changes were observed in both MT1- and
MT3-MMPs overexpressing cells. SMCs infected with AdMT1 or AdMT3 at MOI
3 x 102 became rounded and some cells
detached from the culture dishes. Infection with AdMT1AS produced no
effect. These changes were prevented by the addition of BB94 (1 µM)
or inhibited by TIMP-2 (200 nM). These results suggest that the
proteolytic activity of MT-MMPs could be involved in the alteration of
phenotype. The growth of the SMCs was the same after infection with
AdMT1S, AdMT1AS or AdMT3 infection (data not shown).
3. Reduced adhesion and Increased migration of SMC overexpressing
MT-MMPs
The altered morphology of SMCs overexpressing MT-MMPs prompted us
to analyze their adherence and migration. MT1- and
MT3-MMP-overexpressing SMCs showed reduced adhesion to type I collagen
and fibronectin compared with control Adß-gal or AdMT1AS cells (35
and 30% reduction, respectively). Cell migration activity was measured
in a modified Boyden chamber. MT1- and MT3-MMP-transduced cells showed
increased migration through the filter vs. control Adß-gal-transduced
(Fig. 2A
, B
) or AdMT1AS cells (not shown). BB94 inhibited
platelet-derived growth factor (PDGF) -induced migration of the MT1-
and MT3-MMP-expressing cells but not the control cells (Fig. 2C
).
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CONCLUSIONS AND SIGNIFICANCE
Although MT1-MMP is well established as an activating enzyme for MMP-2, MT3-MMP has been studied less. The present study demonstrates that MT3-MMP is less effective in the activation of MMP-2 and that the enzymatic activity of MT3-MMP is resistant to both natural and synthetic inhibitors. This is the first report to establish the functional differences of these two proteinases. Despite these enzymological differences, the overexpression of MT1-MMP and MT3-MMP causes similar changes in SMC phenotype; they become round, less adhesive, and migrate more rapidly.
The degradation of both MT1- and MT3-MMP proteins is prevented by
a hydroxamic inhibitor (Fig. 1)
. MT3-MMP protein was undetectable
without BB94, although the MMP-2-activating activity was easily
detected by zymography. MT3-MMP was degraded more rapidly than MT1-MMP.
Most important, the natural inhibitor TIMP-2 failed to inhibit the
degradation. Consequently, MT3-MMP would likely have a very short
half-life in a physiological setting. The tertiary structure of MT3-MMP
on the cell surface might prevent the inhibitors from accessing the
reactive site. Others have reported that the proteinase activity of
recombinant soluble MT3-MMP expressed in an Escherichia coli
system is completely inhibited by BB94 and TIMP-2. Activated MMP-2
seems not to be involved in the degradation, because BB94 was required
for MT-MMP accumulation in an MMP-2-negative cell line. Thus, all of
the evidence presented here strongly suggests that the BB-94-sensitive
degradation of MT-MMPs could be autodegradation. However, other
BB94-sensitive enzymes might be responsible for the degradation.
Our working model to explain the role of MT-MMPs in the modulation of
SMC functions is illustrated in Fig. 3
. Although we do not completely understand the mechanisms involved in
phenotypic changes in MT-MMP-overexpressing cells, they could
depend directly on the MT-MMPs and not the activation of MMP-2: MT3-MMP
inefficiently activates MMP-2 overexpression of this enzyme and
MT1-MMP produced similar changes. However, it is evident that MMPs,
particularly MMP-2, are involved in altering cell behavior. Activated
MMP-2 can cleave several matrix molecules, such as laminins,
fibronectin, and collagens, and the cleaved molecules possess activity
that can stimulate cell migration. MT-MMPs could trigger such complex
mechanisms directly or through MMP-2 activation. MT-MMPs might be
responsible for the modulation of the activation status of integrins.
We also provided the first evidence that MT3-MMP could degrade MT1-MMP
on the cell surface to down-regulate these functions. Our results
support the hypothesis that MT-MMPs, especially MT3-MMP, not only act
as MMP-2-activating enzymes, but can also degrade matrix directly and
modify the behavior of SMCs.
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FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0871fje ; to cite this
article, use FASEB J. (July 9, 2001)
10.1096/fj.00-0871fje 
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