© 1997 American Heart Association, Inc.
Articles |
cAMP Involvement in the Expression of MMP-2 and MT-MMP1 Metalloproteinases in Human Endothelial Cells
From the Istituto di Ricerche Farmacologiche Mario Negri, "G. Paone" Environmental Health Center, Department of Vascular Medicine and Pharmacology, Consorzio Mario Negri Sud, S. Maria Imbaro, Italy.
Correspondence to Dr Francesca Peracchia, "G. Paone" Environmental Health Center, Consorzio Mario Negri Sud, Via Nazionale, 66030 S. Maria Imbaro, Italy. E-mail peracchia{at}cmns.mnegri.it
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Abstract |
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Abstract Matrix metalloproteinases (MMPs) are a multigene family of enzymes secreted by a variety of cells, including human umbilical vein endothelial cells (HUVECs). Because metalloproteinases are potentially destructive agents, their production is tightly controlled at several levels. Rather little is known about the presence and regulation of MMPs in endothelial cells. In this study, we investigated the expression and regulation of MMP-2 and membrane type-matrix metalloproteinase (MT-MMP1), a membrane metalloproteinase strictly related to MMP-2 activation. Zymographic analysis of conditioned medium (CM) of HUVECs showed the presence of gelatinolytic activity mainly at 72 and 64 and 62 kD. The 64- and 62-kD bands, respectively, represent the intermediate and the completely active forms of MMP-2. When HUVECs were treated with forskolin (FK) (100 and 25 µmol/l), there was a decrease in the appearance of the 64 to 62 kDa doublet, suggesting an inhibition of the fully activated form of MMP-2. FK raises intracellular cAMP in HUVECs. The same data were obtained using dibutyryl-cAMP. Northern analysis revealed that the expression of MMP-2 increased slightly after treatment with FK, in contrast with gelatin zymography results. Taking into consideration the mechanism of activation of MMP-2, we tested the hypothesis that this compound could modulate MT-MMP1. As expected, FK was able to decrease MT-MMP1 expression. These data correlate with experiments using membranes of FK-treated HUVECs and incubated with control CM. Zymography revealed that when CM was incubated with membranes prepared from FK-treated HUVECs, there was a decrease in the appearance of the 64-kDa band, suggesting that the expression of MT-MMP1 was negatively modified. These results correlate with the MT-MMP1 protein level, negatively modified after FK treatment.
Key Words: metalloproteinases • endothelial cells • cAMP
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Introduction |
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The extracellular matrix is a complex and dynamic meshwork of proteins and proteoglycans that not only provides structural support to cellular systems, but also has a profound influence on many biologic activities. These include basic processes such as cell proliferation and differentiation, as well as cell adhesion and angiogenesis. It is therefore conceivable that agents modifying the ECM have the potential to affect a wide variety of normal and pathologic processes.1
An initial step in the process of angiogenesis is the degradation of matrix proteins on the basement membrane of endothelial cells. After matrix remodeling, endothelial cells are able to migrate, proliferate, and form vessels.2 It is generally assumed that plasminogen activators, plasmin, and MMPs play an important role in cell migration and angiogenesis.3 MMPs, a multigene family of metal-dependent enzymes, are classified on the basis of their substrate specificity and include interstitial collagenase (type I collagenase), a 72-kD gelatinase (type IV collagenase, MMP-2), stromelysin (MMP-3), neutrophil collagenase (MMP-8), and a 92-kD gelatinase (type V collagenase, MMP-9).4 The activity of these enzymes is modulated by the fact that they are produced in a latent form that requires proteolytic processing for activation to occur.5–8 MMP-2 is unique among MMPs in that it cannot be activated after treatment with exogenous proteinases such as plasmin, elastase, and cathepsin G,9 which are identified as putative physiologic activators of some members of this family. It has been reported that MMP-2 is activated on the surface of fibroblasts10,11 and tumor cell lines treated with concanavalin A.12 Thus, the activator on the cell surface responsible for MMP-2 activation has been characterized as MT-MMP1.12,13 A second way in which the activity of MMPs is regulated is based on the presence and activity of specific inhibitors: TIMP-18,14 and TIMP-2.15,16 The interaction of MMPs with their specific inhibitors determines the net activity of secreted enzymes.
Many of these MMPs and their inhibitors can be induced or enhanced on stimulation of the cells with inflammatory mediators or phorbol ester (PMA),17 an activator of protein kinase C.11,18 In endothelial cells, which represent a biologic barrier between circulating blood and the ECM, PMA activation of progelatinase A or MMP-2 is a cell membrane event that is mediated in part through a protein kinase C-dependent mechanism and is accompanied by increased synthesis of MT-MMP.19,20 In this study, we have investigated the expression and regulation of MMP-2 and MT-MMP1 on stimulation of HUVECs with FK and dibutyryl-3'5'-cAMP, cAMP-elevating agents.
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Methods |
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Cells
HUVECs were isolated from the umbilical cord vein as described.21 Cells were grown on 1.5% gelatin (Merck) in medium M199 (Gibco) with 20% heat-inactivated fetal calf serum (Seromed), penicillin/streptomycin, 50 mg/mL of endothelial cell growth factor, crude extract isolated from bovine hypothalmus,22 and 50 mg/mL of heparin (from porcine intestinal mucosa; Sigma Chemical Co) in 5% CO2 at 37°C. Cells were used within six in vitro passages (splitting ratio,1:3).
Cell Stimulation
For experiments, CM from confluent cultures of HUVECs was
obtained by incubating the cells in 10 cm2 dishes
for 6 and 12 hours with stimuli. Before stimulation, cells were washed
twice with serum-free medium M199 and then incubated with 1.5 mL of
serum-free M199 with 20 mmol/L HEPES supplemented with
0.1% human serum albumin (Sigma Chemical Co), 5 U/mL of
heparin, 150 mg/mL of endothelial cell growth
factor, and penicillin/streptomycin. The CM was centrifuged to
remove floating cells, and samples were frozen at -20°C until used.
At the end of the incubation time, cells were detached by brief
exposure to trypsin (Gibco), cell number was evaluated by counting, and
viability was assessed by trypan blue (Gibco) exclusion. No difference
in cell number was observed between control and treated cells (control,
22.3x104±5.4; 100 µmol/L FK,
22.3x104±3.6; 25 µmol/l FK,
24.0x104±1.7). Cell viability always exceeded
90%. The stimuli used were FK (Calbiochem) and dibutyryl-3'5'-cAMP
(Sigma Chemical Co).
Substrate Gel Analysis
Gelatinolytic activity of secreted MMPs was
analyzed by zymography on gelatin-containing
polyacrylamide gels.17 The samples,
normalized for protein content, were applied to 10%
polyacrylamide gels copolymerized with 1 mg/mL of
gelatin. After electrophoresis, the gels were washed three times for 30
minutes. in 50 mmol/L Tris-HCl, pH 8, containing 5
mmol/L CaCl2, 1 µmol/L
ZnCl2, and 2.5% Triton X-100 to remove SDS,
followed by three 10-minute washes in 50 mmol/L Tris-HCl,
pH 8, containing 5 mmol/L CaCl2, and
incubated overnight in the same buffer at 37°C. The gels were stained
with Coomassie Brilliant Blue R-250.
RNA Analysis
Total cellular RNA was isolated by the guanidine isothiocyanate
method.23 Ten micrograms of total RNA were
analyzed by electrophoresis through 1% agarose formaldehyde
gels. To evaluate the quality and quantity of nucleic acid loaded, RNA
samples were added with ethidium bromide, and after the run, the gels
were observed by an ultraviolet source. After electrophoresis, the RNA
was transferred by capillary blotting to a nytran membrane. Filters
were prehybridized in 50% formamide, 5x SSC, 0.1 mol/L sodium
phosphate, 1x Denhardt's solution. and 250 mg/mL of
denaturated salmon sperm. Hybridization was performed at 42°C using
32P-labeled cDNA probes: MMP-2 (1591-bp
EcoRI-BamHI fragment), which is a partial cDNA
cloned from 
gt11 human placental cDNA library (Clontech) of 1582-bp
Kpn I /Kpn I fragment cloned into the
Kpn I site of pGEM4Z (Promega) (kindly provided by Dr
Galloway from British Biotech Pharmaceutical Limited); TIMP-2 (675-bp
HindIII-EcoRI fragment)24;
GAPDH (1469-bp BamHI-Pst I fragment);and MT-MMP1
(1242 -bp EcoRI-Hind III fragment) (kindly
provided by Dr H. Sato).12
cAMP Levels
HUVECs were grown in completed M199 to confluency on 96-well
plates. Cells were washed twice and incubated in 0.1 mL of serum-free
M199 containing 0.5 mmol/L 3-isobutyl-1-methylxanthine
(Sigma Chemical Co) in the presence of FK. Incubation was run for 1
hour at 37°C. The reaction was terminated by aspiration of the medium
followed by the addition of 0.1 mL of cold absolute ethanol. After
overnight freezing at -20°C, the ethanol supernatants were dried,
and the intracellular cAMP levels were measured using a
radioimmunoassay (125I) kit (Amersham). Results
are expressed as picomoles of cAMP per 106
cells.
Membrane Preparation
HUVECs were treated with FK, washed with phosphate-buffered
saline, scraped, and centrifuged at 1000g for 10
minutes. Cells were resuspended in 25 mmol/L Tris-HCl (pH
7.4) containing 8.5% sucrose, 50 mmol/L NaCl, and protease
inhibitors and homogenized in a Dounce
homogenizer. The homogenate was
centrifuged at 3000g for 10 minutes in a
refrigerated centrifuge, and the resulting supernatant was
centrifuged at 100 000g for 2 hours. The pellet was
resuspended in 25 mmol/L Tris and 50 mmol/L
NaCl (pH 7.4) containing inhibitors, separated further on a
discontinuous sucrose gradient (20%, 30%, 50%, and 60% sucrose in
water), and centrifuged at 100 000g for 2 hours at
4°C. The plasma membrane-enriched fraction appearing as a visible
band at the 30%/50% sucrose interface was collected, pelleted at
100 000g for 2 hours, and stored at -80°C. Extraction of
plasma membranes with nonionic detergents and activation of MMP-2 were
performed as described.25
Electronic autoradiography for mRNA blots quantitation was performed by an Instant Imager apparatus (Packard).
Western Blotting
Control and treated cells were treated with 1% SDS, and the
extract was centrifuged to remove particulate material. The
supernatant was concentrated and subjected to
SDS-polyacrylamide gel electrophoresis.
Immunoblotting was performed using affinity-purified
rabbit antibodies directed against the 114-1F2 peptide of MT-MMP1
(REVPYAYIREGHEK), which recognizes MT-MMP1 in the plasma membranes of
transfected cells,12 and visualized using an ECL
detection system (Amersham).
Statistical Analysis
Student's t test was used, and differences were
considered statistically significant when P<.05.
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Results |
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Analysis of serum-free CM of stimulated HUVECs by zymography showed the presence of gelatinolytic activities mainly at 72, 64 to 62, and to a lesser extent at 55 kD (as a faint doublet). These activities were not observed in the presence of 1,10-phenantroline, demonstrating that these gelatinolytic activities were bona fide MMPs (data not shown).
The 64- and 62-kD bands, respectively, represent intermediate
and fully activated forms of MMP-2,25
both present in CM of control cells (Fig 1
, lane 1). When cells were treated with
FK (100 and 25 µmol/L), there was a decrease (in a
dose-dependent manner) in the appearance of bands at 64 to 62 kD,
suggesting an inhibition of the fully activated form of MMP-2
(Fig 1, lanes 2 and 3), compared with control CM. FK did not reduce the
gelatinolytic activity; in the absence of cells
(data not shown) FK raises intracellular cAMP by activating
adenylylcyclase (Fig 2). To verify
whether the observed effect was really cAMP-dependent, we used
dibutyryl-cAMP, a cAMP analogue (Fig 3).
Even in these experiments, when cells were treated with dibutyryl-cAMP
(0.5 mmol/L), there was a decrease in the appearance of 64-
to 62-kD (lane 2) bands, confirming the role of cAMP in the inhibition
of the fully activated form of MMP-2 compared with control
(lane 1) and control ethanol (lane 3). Northern blot analysis
after 6 hours of treatment (Fig 4)
revealed that FK, at both 100 and 25 µmol/l, did not
appear to have a substantial effect on the expression of MMP-2 (lanes 2
and 3, respectively). This is in contrast with gelatin zymography
results, in which the 64- to 62-kD bands (Fig 1) decreased, although to
a different degree, after treatment with FK. The same results were
obtained for MMP-2 mRNA expression at both 12 and 24 hours (data not
shown). It is apparent that FK is able to modulate a mechanism that
controls the active form of MMP-2.
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Taking into consideration the mechanism of activation of MMP-2, we
performed experiments to verify the hypothesis that the treatment was
able to modulate the expression of MT-MMP1 responsible, in turn, for
the regulation of the appearance on gelatin zymography of 64- to 62-kD
bands. As expected (Fig 5), after 6 hours
of treatment, FK, at both 100 and 25 µmol/L, was able to
decrease the MT-MMP1 gene expression (lanes 2 and 3, respectively). The
negative effect of FK on MT-MMP1 expression was long-lasting, remaining
detectable even at 24 hours (data not shown). Under the same
experimental conditions, we tested the filters with TIMP-2 cDNA, to
verify if this compound was able to regulate TIMP-2 expression. As
shown in Fig 5, no significant modulation was observed at 6 hours,
while a slight increase in TIMP-2 expression was observed after 24
hours of treatment with FK. To have a biologic comparison of MT-MMP1
mRNA results, we tested the hypothesis that MT-MMP1 is also
downregulated at the membrane level. For this purpose we treated HUVECs
with FK and after 12 hours membranes were prepared and incubated with
control CM (Fig 6, lane 1). As shown in
Fig 5, in CM incubated with HUVEC membranes treated with FK, there was
a decrease in the appearance of the 64-kD band (lane 3),
representing the intermediate of MMP-2 activated by
MT-MMP1, compared with CM incubated with control membranes (lane 2),
confirming the inhibitory role of FK on MT-MMP1 expression.
The MT-MMP1 protein was identified by immunoblotting,
as a 63-kD protein, using polyclonal antibodies, and as shown in Fig 7, there was a decreased intensity at 63
kD after FK treatment (lane 2) compared with control cells (lane
1).
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Discussion |
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Endothelial cells, which form the inner coating of blood vessels, normally exhibit one of the lowest turnover rates of any cell type in the body.26 However, new blood vessel growth occurs during wound healing and in serious complications of many disease states, including diabetic retinopathy and malignancies. Cell translocation during both normal and pathologic processes of tissue remodeling requires spatially regulated degradation of ECM macromolecules. Two proteolytic systems are apparently involved in this process: (1) plasminogen activator of the urokinase type, which is sequestered on the cell surface and activated via interaction with its receptor27 and (2) secreted metalloproteinases that initiate degradation of collagens and proteoglycans.28,29 All members of the latter group of enzymes are secreted as zymogens and require activation in the extracellular space. Their activation can be accomplished by a proteolytic cascade initiated by serine proteases, such as plasmin, by processing the amino terminus of the enzyme,8,16 followed by further processing the amino terminus by autoproteolysis or with the involvement of other related metalloproteinases, such as stromelysin.5
MMP-2 is believed to play an important role in the degradation of subendothelial basement membrane, initiating the process of angiogenesis.30,31 In this respect, the control of MMP-2 activity may be a determinant step for the formation of new functional blood vessels. Changes in the levels of this enzyme activity in endothelial cells may be involved in both the early degradation or the late reconstruction of the basement membrane, underlying the new capillaries. Recently,12,25,32 a cell membrane-dependent mechanism of activation has been described, which would be specific for MMP-2 gelatinase: a plasma membrane fraction prepared from tumor cells treated with concanavalin A was able to activate MMP-2, and the reaction was sensitive to chelating agents and TIMP-2.13,32 Thus, this MMP-2 activator is a member of the MMPs family called MT-MMP1.12 It has been demonstrated that MT-MMP1 mRNA transcript is expressed in normal vascular endothelial cells33 and that its expression is significantly increased after PMA treatment.19,20 In this article we report the expression and regulation of MMP-2, MT-MMP1s and TIMP-2 in HECs. We have shown that FK, a cAMP agonist, inhibits MT-MMP1 expression in HECs.
An increase in cAMP has been described to inhibit gene transcription34 and appears to involve an inhibitory phosphorylation of one or more of the transcription factors necessary for gene expression. On the other hand, MMPs can be suppressed by cAMP.35,36 It has been reported that increased cAMP levels were associated with a reduction in collagenase mRNA levels in a synoviocyte cell line, and its inhibitory effect is exerted on the promoter region of the collagenase gene.37 Our results and results from other authors on MMP-2 mRNA expression38 being slightly upregulated after FK treatment could be explained by the fact that the first exon of MMP-2 gene contains the AP-2 binding site.39 The AP-2 protein appears to mediate transcription activation through protein kinase C as well as cAMP-dependent protein kinase A. Zymography results, in which the 64- to 62-kD bands were markedly reduced after FK treatment correlate well with the decreased expression and activity of MT-MMP1 on HUVECs. It has been reported that cAMP-elevating agents inhibit transendothelial migration of T cells40 by modifications in the function of T cells and endothelial permeability. Moreover, an intracellular increase in cAMP on HUVECs impairs cytoskeleton organization and the formation of clusters of b3 and b1 integrin receptors; the latter recognize and bind different components of ECM without affecting cell attachment, enhancing even cell adhesion and strongly inhibiting cell motility.41 Cell motility plays a central role in the process of new vessel formation, which takes place after degradation of matrix proteins by endothelial-secreted metalloproteinases.
In conclusion, our results show that cAMP agonists could contribute to vascular integrity, preventing activation of MMP-2 gelatinase by decreasing the expression of MT-MMP1 on the endothelial cell surface.
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Acknowledgments |
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This work was supported by the Italian National Research Council (Convenzione C.N.R.—Consorzio Mario Negri Sud, 1995). The authors wish to thank Dr H. Sato (Department of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, Japan) for providing MT-MMP1 cDNA, and R. Bertazzi for expert assistance in the preparation of figures. The Gustavus A. Pfeiffer Memorial Library staff and P. Di Nardo helped prepare the manuscript.
Received December 5, 1996; accepted July 2, 1997.
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