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Institute of Nephrology, The University of Wales College of Medicine, Heath Park, Cardiff, Wales, United Kingdom.
Correspondence to Dr. John Martin, Institute of Nephrology, The University of Wales College of Medicine, Heath Park, Cardiff CF11 4XN, Wales, United Kingdom. Phone: 0044 2920748446; Fax: 0044 2920748470; E-mail: MartinJ1{at}cf.ac.uk
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The principal enzymes implicated in the remodeling of ECM are the matrix metalloproteinases (MMP), a group of enzymes that, together, are able to degrade all of the constituents of the ECM. Metalloproteinase production by glomerular cells is now well documented (3,4,5). In particular, human glomerular mesangial cells (HMC) can secrete MMP2 and MMP9, gelatinases that degrade the major constituent of the GBM type IV collagen. These enzymes are zinc-dependent proteinases that are active in the neutral pH of the extracellular environment, and their final activity is carefully controlled at several levels: by the regulation of transcription, the rate of secretion, and the activation of the enzymes. The MMP are secreted as a proenzyme or latent form, and the activation of these enzymes is due to the proteolytic removal of the propeptide exposing the active site (6). The activation of MMP2 seems to be through a different mechanism to that described for the other secreted MMP, and it now seems that the newly described membrane type metalloproteinases, the MT-MMP, are involved in the activation of this enzyme (7). In addition, the active enzymes can be inhibited on a one-to-one basis by their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMP) of which TIMP I and TIMP II are the best described. Thus, the modification of MMP activity at any one of these steps can affect the turnover of the glomerular matrix.
The regulation of the rate of secretion of these enzymes by cytokines has received a great deal of attention (8,9,10). Less well understood, however, is the effect that matrix itself may have on the degree and kinetics of its own turnover through the control of MMP activity. Changes in the status and composition of the ECM have been shown to affect the function of a variety of different cells and to modulate the synthesis and release of MMP in other systems (11,12,13,14). Thus, any change in the composition of the mesangial matrix or in the degradation of the constituent proteins that cause the release of soluble protein fragments would be expected to have profound effects on the normal activity of these HMC. This study examines the interaction of HMC with matrix proteins and determines the effect of these proteins on the secretion and activation of the MMP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Culture and Identification
Human glomeruli were obtained by the serial sieving of the normal regions
of human kidney recovered at nephrectomy. HMC were maintained in RPMI-1640
containing 20% vol/vol fetal calf serum (FCS). Cells in low passage number
(between passages 1 and 5) were used in all experiments. Before experimental
procedures, HMC were growth-arrested for 48 h by culture in medium containing
0.2% (wt/vol) lactalbumin hydrolysate (Difco Laboratories, Detroit, MI) in the
absence of serum (4). The cells
were confirmed to be mesangial cells by morphology and the use of
immunohistochemistry as described
(9). Cells showed positive
staining for intracellular myosin fibrils and were negative for factor VIII
and cytokeratin. The effect of all subsequent treatments on cell viability was
assessed by 3-[4,5-dimethylthiazole-2-yl]-2,4 diphenyltatrazolium bromide
assay (10).
Cell Adhesion to Defined ECM Proteins
Ninety-six-well plates were precoated with the following matrix proteins
overnight at 4°C: collagen IV, gelatin IV, collagen 1, gelatin I,
fibronectin, vitronectin, and laminin (10 µg/ml in phosphate-buffered
saline [PBS]). Then they were washed with PBS and blocked with PBS/BSA (1
mg/ml) before use. Controls consisted of wells treated with BSA (1 mg/ml) in
PBS. HMC were removed from confluent cultures by the use of
ethylenediaminetetraacetate (EDTA; 2 mM in PBS) alone, washed in RPMI, added
to the wells at a concentration of up to 6 x 104/ml in RPMI
BSA (1 mg/ml), and allowed to adhere for 1 h at 37°C in an atmosphere of
5% CO2. Nonadhered cells were removed by washing with PBS
containing 2 mM MgCl2. Adhered cells were fixed with
paraformaldehyde (3% vol/vol) for 15 min and stained with a solution of
crystal violet (0.5% wt/vol) for 3 min. The cells were then washed with PBS,
dried, and solubilized with SDS (1% wt/vol), and the absorbance at 540 nm was
determined on a Dynatech MR5000 platereader (Billinghurst, UK).
Determination of Gelatinolytic Activity
To determine the effect of matrix proteins on the secretion of
gelatinolytic activity by the cells, we growth-arrested confluent wells of
mesangial cells by culture for 48 h in serum-free medium supplemented with
0.2% (wt/vol) lactalbumin hydrolysate. Extracellular matrix proteins (10
µg/ml) were added in solution in the above medium, and the incubation was
continued for an additional 72 h. The conditioned medium (CM) was then
collected and subjected to zymography. Gelatinolytic activity was determined
by subjecting a sample of CM to SDS-PAGE using either a 7.5% gel or a 5 to 12%
gradient gel, incorporating gelatin (1 mg/ml) under nonreducing conditions, as
described previously (4). To
determine net gelatinolytic activity (not inhibited by TIMP) in the
supernatants, we assayed samples of CM for their potential to degrade
14C-labeled gelatin as described previously
(4,5).
Purification of Latent MMP2
Gelatinolytic activity was semipurified by passing the CM containing latent
MMP2 down a gelatin agarose affinity column (Sigma), equilibrated with 50 mM
Tris (pH 7.4), containing 0.5 M NaCl, 0.05% Brij wt/vol, and 10 mM EDTA, and
then washed in the same buffer. Activity was eluted with 50 mM Tris,
containing 1 M NaCl 0.05% (vol/vol) Brij, 10 mM EDTA, and 5% dimethyl
sulfoxide (vol/vol). The enzyme was then dialysed into 50 mM Tris (pH 7.4),
0.5M NaCl, 0.05% Brij, 5 mM CaCl2, and stored at -20°C until
use.
Detection of Cell-Associated MT1-MMP Protein
HMC were lysed in SDS-PAGE sample buffer, and whole-cell lysates were
separated on 7.5% SDS-PAGE gels. After electrophoretic transfer to
nitrocellulose and blocking in 5% skimmed milk in PBS, 0.5% Tween 20, the
blots were incubated with 10 µg/ml anti-MT1-MMP antibody (TCS Biologicals
Ltd., Bodolph Claydon, UK) in PBS, 0.5% Tween 20, 1% BSA overnight. Primary
antibody binding was visualized by enhanced chemiluminescence (Amersham
Pharmacia Biotech UK, Ltd., Little Chalfont, UK).
Preparation of Mesangial Cell Membranes
HMC were scraped off flasks and pelleted by centrifugation in PBS
containing a cocktail of proteinase inhibitors (pepstatin A, aprotinin and
leupeptin at 10 µg/ml, phenylmethylsulfonyl fluoride at 0.5 µM; Sigma).
Cells were resuspended in hypotonic buffer (10 mM Tris [pH 7.2] containing 1
mM MgCl2 plus proteinase inhibitor cocktail), mixed with 4 volumes
of sucrose buffer (0.25 M sucrose dissolved in hypotonic buffer) and
homogenized in this buffer using a Dounce pestle.
The homogenate was centrifuged at 500 x g for 5 min to remove debris. A membrane-enriched fragment was prepared by centrifuging the supernatant at 25,000 x g for 30 min at 4°C, and the resulting pellet was resuspended in PBS containing 1 mM phenylmethylsulfonyl fluoride. The protein content of the membrane samples was determined by the method of Bradford (16). Membranes were stored at -70°C until use.
To determine the activating properties of the membrane preparations, latent MMP2 was added to membrane fractions (10 µg protein) and the mixture was incubated for 24 h at 37°C. The degree of MMP activation was then determined by gelatin zymography as described.
Reverse Transcription-PCR
Confluent wells of mesangial cells were growth-arrested as described and
cultured in the presence of fibronectin for an additional 6 h. Total RNA was
extracted using RNA Isolator (Genosys Ltd., Cambridge, UK) according to the
manufacturer's instructions. cDNA was prepared by the reverse transcription of
1 µg of RNA using random primers, and the equivalent of 0.05 µg was
amplified by PCR using the following primers specific for MT1-MMP, MT2-MMP,
MT3-MMP, and 
actin (Genosys Ltd., Cambridge, UK):
actin
Northern Blot Analysis
Total RNA (up to 10 µg) was run on a denaturing agarose gel and
transferred by vacuum blotting onto a Hybond nylon membrane (Amersham). mRNA
for MT-MMP was detected by hybridization with a 32P-labeled probe
prepared from PCR products and detected as described previously
(9).
Analysis of TIMP I and TIMP II
TIMP I and TIMP II were measured by enzyme-linked immunosorbent assay on
suitably diluted samples using reagents generously supplied by Dr. A. J. P.
Docherty (Celltech Ltd., Slough, UK) essentially as described previously
(10,17).
Standards of TIMP I and TIMP II were prepared using purified TIMP kindly
supplied by Prof. T. E. Cawston (Department of Medicine [Rheumatology],
University of Newcastle, UK) in the range of 0.15 to 150 ng/ml for TIMP 1 and
in the range of 0.15 to 300 ng/ml for TIMP II.
Statistical Analyses
Results are expressed as mean ± SEM of n experiments. All
data were analyzed using the Wilcoxon signed rank test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Matrix Protein Stimulation of MMP Release
The addition of soluble matrix proteins to HMC resulted in the selective
modulation of MMP release, detected by zymography. This release, however, was
not directly associated with the degree of HMC binding that each protein
supported. Figure 2
demonstrates that of the matrix proteins added, collagen I and fibronectin
stimulated an increase in the amount of latent MMP2 present in the CM, and, in
a similar manner to collagen IV, they also induced its conversion to a lower
molecular weight band (66 kD) on the zymogram. In addition, fibronectin
induced the secretion and activation of MMP9 seen as bands of lysis at 92 and
95 kD. The effect of fibronectin was dose-dependent with maximal activation of
MMP2 and induction of MMP9 occurring above 10 µg/ml
(Figure 3). The increase in MMP
protein levels was paralleled by an increase in the mRNA (as shown by reverse
transcription-PCR [RT-PCR]) for MMP2 and the induction of the mRNA for MMP9
(data not shown). This effect was mimicked by incubation with anti-CD49e
monoclonal antibody specific for the 5 integrin chain (Immunotech,
Birmingham, UK; Figure 4),
suggesting that the response to fibronectin was mediated through this
integrin.
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Changes in the amount of MMP detected were not related to changes in
proliferation, and there was no difference in cell number in response to
co-culture with any of the ECM proteins under the conditions used (data not
shown). Furthermore, analysis (Cytosets Antibody Pair kits, Biosource
International, Nivelle, Belgium) of the CM from stimulated HMC demonstrated
that there were no detectable levels of cytokines previously described as
potential promoters of MMP secretion (interleukin-6 [IL-6] and IL-8 below 15
pg/ml, IL-1ß and tumor necrosis factor- below 10 pg/ml,
transforming growth factor-ß below 5 pg/ml; data not shown).
MMP Activation by HMC
MMP2 is now believed to be activated in vivo by the action of the
membrane bound metalloproteinases, the MT-MMP. To determine whether this might
be occurring in this instance, we incubated latent MMP2 with cell membranes
isolated from HMC cultured with different concentrations of fibronectin. HMC
membranes were shown to activate latent MMP2, and this activity was markedly
increased in the membranes of cells that had been incubated in the presence of
fibronectin or FCS (Figure 5).
This effect was dose-dependent and became apparent at 10 µg/ml fibronectin
(Figure 6).
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To ascertain whether this result could be due to an increase in the amount of the MT-MMP synthesized by the HMC, we examined the levels of mRNA for MT1-MMP, MT2-MMP, and MT3-MMP relative to actin by RT-PCR (Figure 7) and Northern blotting (Figure 8). The mRNA for all three MT-MMP was detected in HMC. There was, however, no increase in the levels of mRNA. Densitometric analysis of these figures confirmed that there was no increase in the amount of mRNA for these MT-MMP relative to actin, confirming that there was no increased expression of these potential MMP2 activators. Analysis of whole-cell lysates by SDS-PAGE and immunoblotting, however, demonstrated an increase in the ratio of the lower molecular weight form of MT1-MMP over the larger form with increasing concentrations of fibronectin (Figure 9).
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Effect of Exogenous Fibronectin Added to HMC on TIMP Release
The consequence of the addition of fibronectin to HMC on the secretion of
the inhibitors of MMP was examined by enzyme-linked immunosorbent assay. There
was no statistically significant effect on the secretion of either TIMP I or
TIMP II in response to fibronectin (Figure
10), although there was a trend toward an increase in TIMP II
secretion with increasing concentrations of fibronectin in some experiments.
Whether this trend reflected a real change in the total gelatinolytic activity
released by HMC was investigated in a radiometric assay of net activity in the
CM of stimulated cells
(4,5).
In response to 10 µg/ml fibronectin or 4 µg/ml anti-5 integrin
antibody, however, there was a 1.5-fold increase or a 3.6-fold increase in
activity, respectively, over unstimulated cells, suggesting that the observed
trends in TIMP II expression were not sufficient to inhibit the increased
gelatinolytic activity released from the cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of MMP2 occurs through a different mechanism to that for the other MMP. The enzyme is activated in vitro by incubation with mercuric salts such as p-amino phenyl mercuric acetate. It has also been reported that MMP2 may be activated by other proteinases, such as plasmin (22), or by oxygen radicals. Recently, another activation mechanism involving MT-MMP has been described. Five MT-MMP have now been described (23,24,25), at least two of which (MT1-MMP and MT2-MMP) are capable of activating latent MMP2. MMP2 is secreted by cells in an inactive form and is activated on the cell surface as part of a complex involving the MT-MMP and TIMP II. It is now accepted that the MT1-MMP binds to TIMP II and that this complex acts as a receptor for MMP2 by binding to the carboxyl end of the molecule (7,26,27). MT1-MMP then proteolytically cleaves the propeptide of the MMP2, activating the latent enzyme. However, if TIMP II is present in excess, it will function as a specific inhibitor by binding through its N terminal domain. Therefore the presence of TIMP II in the extracellular milieu can serve either to assist in the activation of the gelatinase or to inhibit the active enzyme in a 1:1 manner. Such a role for this inhibitor would seem to ensure that the MMP2 will be active locally but be inhibited before it can degrade matrix proteins more distal from the cell surface. In vivo, other mechanisms may also be involved in MMP activation. For example, urokinase plasminogen activator induces the activation of MT1-MMP in HMC (28). In addition, the activation of MMP9 that was observed after the addition of fibronectin to HMC may be secondary to the production of activated MMP2 as active MMP2 has been demonstrated to activate the latent MMP9 in vitro (29) and this enzyme is known to have a different activation mechanism to MMP2.
In the present study, membrane-associated activity, selectively induced by fibronectin, was implicated in the activation of the secreted MMP2. There was, however, no increase detected in the level of mRNA coding for MT1-MMP, MT2-MMP, or MT3-MMP either by RT-PCR or by Northern blots. This finding suggests that the increase in activity seen in the membrane preparation may be due to a modification of the MT-MMP rather than to an increase in the amount produced by the cell. This is further supported by the demonstration that the ratio of the smaller molecular weight (active) form to the larger (latent) form increases with increasing concentrations of fibronectin. This is in agreement with the work of Stanton et al. (30). These workers demonstrated an increase in the activity of MT1-MMP that was paralleled by a decrease in its molecular weight in the absence of any increase in the amount of mRNA present in HT1080 fibrosarcoma cells after their culture on fibronectin. Only a slight increase in total MT1-MMP protein production was seen, whereas there was a marked increase in the amount of activated MT1-MMP present. Similarly, Preaux et al. (31) recently demonstrated an activation of MMP2 from myofibroblasts cultured on collagen I with no increase in MT1-MMP mRNA. However, Haas et al. (32) demonstrated an increase in the transcription of MT1-MMP by proliferating endothelial cells in a three-dimensional collagen matrix.
There have been other reports of matrix proteins influencing MMP secretion.
For example, it has been shown
(11,33)
that the ligation of the fibronectin receptor induced the gene expression of
both collagenase and stromelysin by fibroblasts. This seemed to be mediated
through the 120-kD cell binding fragment. In addition, Larjava et al.
(34) described how anti-body
binding to ß3 integrin in human keratinocytes induced the
92-kD gelatinase (MMP9), although there was no effect of the extracellular
proteins themselves—possibly because these workers cultured the cells on
these substrates rather than adding them in solution to cells in culture.
Brooks et al. (35)
demonstrated that an anti-integrin vß3 antibody was
able to block human breast cancer growth possibly through an effect on MMP
activity. Furthermore, the use of an antibody to this vitronectin receptor or
the addition of vitronectin itself resulted in the increased secretion of MMP2
by a melanoma cell line (12).
Later work, however, showed that the ligation of integrin
5ß1 seemed to be more important than that of
vß3 for stimulating MMP2 secretion in a different
melanoma cell line (36). The
present study demonstrating the induction of MMP by anti-5 suggests
that in HMC, this receptor is responsible for the observed effects of
fibronectin, although whether other receptors may also be involved cannot be
ruled out.
The variety of responses to matrix binding, mediated by integrins, has been
linked to several different signal transduction pathways. For example, binding
to ß1 integrins induces phospholipase activation in rat
glomerular epithelial cells
(37), which may modulate
proliferation. In addition, Malik and Parsons
(38) demonstrated the
activation of the p70 ribosomal S6 kinase signaling pathway. Fibronectin binds
to the 5 ß1 integrin and, in other systems,
induces the activity of the AP1 transcription factor
(39,40).
It is therefore likely that this is one of the mechanisms operating in the
present system because MMP9 possesses an AP1 binding site in its promoter
(41).
The work described here demonstrates that the interaction of mesangial cells with specific matrix proteins can modulate the pattern of gelatinolytic activity secreted by the cells. Thus, any change in the composition of the matrix in vivo would be expected to result in a change in the proteolytic activity present in the glomerulus. Such a change in matrix organization is often seen accompanying glomerulosclerosis and interstitial nephritis, where it is directly linked with the progression to end-stage disease. This is seen both in animal models (42) and in human renal diseases in which increased amounts of fibronectin are localized to areas of sclerosis within the mesangium (43,44). Thus, it seems that in certain pathogenic situations in which the normal constituents and quantities of the extracellular matrix have been disturbed, there may be a related change in the MMP profile and/or activation that may result either in a resolution of the abnormality or, conceivably, in a progression to further damage. Because the expression of particular integrins and their capacity for binding individual proteins, the intracellular signals generated, and the responses stimulated in the cells all vary between different cell types, it will be important to investigate the detailed integrin-specific mechanisms involved in the current study. An understanding of whether the same mechanisms apply to other glomerular cells, e.g., epithelial cells, will also allow the more detailed investigation of the role of individual cells in disease progression.
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