1 Institute of Biology, National Center for Scientific Research "Demokritos," 15310 Agia Paraskevi, Athens, Greece; 2 Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; and 3 Department of Pediatrics, University of Minnesota Medical School, Minneapolis, Minnesota 55455
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ABSTRACT |
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In cultured human
glomerular epithelial cells (HGEC), 25 mM glucose resulted in decreased
expression of 3-,
2-, and
1-integrins and increased expression of
5- and
v
3-integrins. This
change was accompanied by decreased binding of HGEC to type IV
collagen. In the presence of normal (5 mM) glucose concentration, cell
binding to type IV collagen was primarily mediated by
2
1- and
5
1-integrins, as indicated by experiments
in which cell adhesion to type IV collagen was competed by specific
anti-integrin monoclonal antibodies. In the presence of high (25 mM)
glucose, the upregulated
5- and
v
3-integrins were mainly involved in cell
binding to type IV collagen. Furthermore, high glucose decreased
expression of matrix metalloproteinase-2 (MMP-2), a collagenase
regulated in part by
3
1-integrin, as
suggested by the use of ligand-mimicking antibodies against these
integrins, which resulted in release of increased amounts of MMP-2 in
the culture medium. Finally, tissue inhibitor of metalloproteinase-2,
the specific inhibitor of MMP-2, was upregulated in high glucose and
could contribute to matrix accumulation. These changes could help
explain basement membrane thickening in diabetes.
matrixins; tissue inhibitors of metalloproteinases; signaling; diabetes
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INTRODUCTION |
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THE GLOMERULAR BASEMENT MEMBRANE (GBM) underlying glomerular epithelial cells, an important component of the kidney permselective barrier, is thickened in diabetic nephropathy (DN). GBM thickening could be due to increased deposition (synthesis and accumulation) of the extracellular matrix (ECM) macromolecules such as collagen, fibronectin, laminin, and proteoglycans, which could be explained by an imbalance between matrix synthesis and degradation. Matrix synthesis and degradation are regulated in part by cell-matrix interactions (7, 39).
Interactions of cells with matrix molecules are primarily mediated by the integrin superfamily receptors (14, 15). Most integrins recognize more than one ECM protein, such as collagens, fibronectin, and laminin (33), and, on binding, they transduce signals to the cell interior via mechanisms such as protein phosphorylation (6). For example, tyrosine kinase pp125FAK [focal adhesion kinase (FAK)] becomes phosphorylated and activated after ligand-induced integrin clustering. Integrin ligation regulates cell functions such as adhesion, migration, anchorage-dependent growth, and gene expression (6, 11). FAK may function as a key mediator for these events by integrating signals from integrins.
One function attributed to integrins is regulation of the expression of matrix metalloproteinases (MMPs)/matrixins, zinc-dependent endopeptidases linked to the degradation and remodeling of ECM (24, 25, 34, 35, 45). Examples are gelatinases A and B [72-kDa gelatinase (MMP-2) and 92-kDa gelatinase (MMP-9), respectively], which degrade collagen types IV, V, VII, and IX, gelatin, elastin, and fibronectin (27, 28). Gelatinases are synthesized and secreted as inactive forms (pro-MMPs), and their matrix-degrading activities are regulated by activators and inhibitors. Most cells produce and secrete specific tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, which preferentially bind to MMP-9 and MMP-2, respectively, thus regulating their matrix-degrading activity (27, 28).
To elucidate mechanisms leading to GBM thickening in DN, we have used
as a model T-SV40 immortalized human glomerular epithelial cells (HGEC)
to study whether increased glucose concentrations affected
integrin-mediated interactions of these cells with type IV collagen,
thus contributing to differential expression of several factors
controlling ECM synthesis and degradation. T-SV40-immortalized HGEC
express differentiation markers on the surface of primary glomerular
epithelial cells, interact with type IV collagen, and are therefore
similar to their primary counterparts (8, 22). The
integrin profile of HGEC was examined in normal (5 mM) and high (25 mM)
glucose, and quantitative changes in integrin expression were observed
in the presence of increased glucose concentrations. These changes were
accompanied by modulation of integrin-mediated interactions of HGEC
with type IV collagen, a predominant component of the GBM. Furthermore,
high glucose altered the expression and production of proteins involved
in matrix degradation. In our experiments, the expression of MMP-2 was
regulated in part by 3
1-integrin, which,
on ligation, enhanced FAK phosphorylation and resulted in
upregulation of MMP-2.
Our findings suggest that increased glucose concentrations altered normal matrix-related cell functions of HGEC and resulted in differential gene expression, possibly contributing to matrix accumulation. The observed changes could help explain the thickening of the GBM in DN.
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MATERIALS AND METHODS |
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Cell culture.
HGEC (8, 22) were cultured at 37°C in media composed of
DMEM-Ham's F-12 containing 1% FCS, 15 mM HEPES, 2 mM glutamine, ITS
(5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml sodium selenite), 50 nM dexamethasone, antibiotics, and 5 or 25 mM
D-glucose. Cells were released from their tissue culture
flasks for passaging or use in experiments by treatment with 0.05%
trypsin in 1 mM EDTA. For experiments, cells were cultured for at least
three passages. For zymography and Western blotting analysis of
conditioned media, cells (2 × 106) were cultured for
48 h. The medium was then replaced with 10 ml of serum-free
medium, and cells were incubated for 24 h. At the end of the 24-h
incubation period, the conditioned media were collected, supplemented
to 1 mM Na2EDTA and 0.02% sodium azide, and stored at
20°C for further use.
Antibodies.
Rabbit anti-human polyclonal antibodies to integrin subunits,
collagenase MMP-9, and TIMP-1 and TIMP-2, as well as monoclonal antibodies (MAbs) against 2 (P1E6)-,
3
(P1B5)-,
5 (P1D6)-,
v
3
(Lm609)-, and
1 (P5D2)-integrin subunits, were obtained from Chemicon International. Polyclonal antibody Ab45 against collagenase MMP-2 was provided by Dr. Stetler-Stevenson. Anti-human leukocyte antigen (HLA) MAb (W6/32) was used as negative control (catalog no. HB95, American Type Culture Collection, Rockville, MD).
Antitubulin MAb was purchased from Sigma. Rabbit polyclonal anti-FAK
antibody specific for human pp125FAK and
antiphosphotyrosine MAb (clone 4G10) were obtained from Upstate Biotechnology. Peroxidase-conjugated goat anti-rabbit immunoglobulins and sheep anti-mouse immunoglobulins were purchased from Amersham.
Culture of cells with MAbs. Cells were detached from confluent monolayer cultures, resuspended in culture medium (3 × 106 cells/ml), preincubated with MAb at 50 µg/ml each for 30 min at 37°C, and then diluted 10-fold to a final concentration of 5 µg/ml each or preincubated without antibody and cultured for 24 h. The medium was then replaced with serum-free medium with or without 5 µg/ml of each MAb, and cells were incubated for 24 h. At the end of the 24-h incubation period, the conditioned media were collected for zymography, and the cells were harvested and counted.
Isolation of type IV collagen. Type IV collagen was isolated from the Engelbreth-Holm-Swarm tumor system using previously described techniques (19, 29). Protein concentration was determined using the method of Waddell (44).
Cell adhesion assay. Microtiter plates (96-well; Microlon 600, Greiner) were coated with 50 µl of increasing concentrations of type IV collagen (0.3-200 µg/ml PBS) and allowed to evaporate to dryness at 29°C. The remaining reactive sites were blocked with 0.2% BSA in PBS for 2 h at 37°C. Plates were then washed once with PBS and immediately used for experiments. HGEC were grown in 5 or 25 mM D-glucose in T-25 flasks until 75-80% confluency was reached and were metabolically labeled for 18 h with 0.15 mCi of [35S]methionine (Amersham) per flask. Cells were washed twice with DMEM, and 50 µl (5,000 cells) of cell suspension in binding buffer (DMEM, 2 mg/ml BSA, and 25 mM HEPES, pH 7.5) were added to each well and allowed to adhere for 45 min at 37°C. The wells were then washed three times with binding buffer to remove nonadherent cells, and lysis buffer (0.5 N NaOH and 1% SDS) was added to each well. The lysate was transferred to scintillation vials and counted. For inhibition of cell adhesion to type IV collagen, cells were processed as for the adhesion assay. In competition experiments, the following criteria were selected to achieve optimal antibody effect: half-maximal binding of HGEC to type IV collagen by using 5 µg/ml type IV collagen and a short-term assay (30 min). After plates were coated with 50 µl of type IV collagen at 5 µg/ml, MAb to integrin subunit or MAb to HLA was added to each well (100 µl/well) at a final concentration of 10 µg/ml followed immediately by 50 µl of cell suspension in binding buffer (5,000 cells/well). Cells were allowed to adhere for 30 min at 37°C and processed as for the adhesion assays. The concentration of antibodies used in inhibition assays was above the saturating concentration as determined by flow cytometry. The data were normalized by expressing the binding in the absence of antibody as maximal (100%), and adhesion in the presence of antibodies is shown as percentage of binding in the absence of antibody. All assays were performed a minimum of three times in hexaplicate for each experimental condition.
Zymography. Gelatin zymography was performed as previously described (3). Briefly, aliquots of each sample of conditioned media were concentrated and subjected to SDS-PAGE under nonreducing conditions in 10% polyacrylamide gels containing 0.1% gelatin. The volume of conditioned medium loaded per lane was adjusted according to the cell number obtained at harvest. After electrophoresis, the gel was washed three times for 30 min each with 50 mM Tris · HCl, pH 7.5, 5 mM CaCl2, 1 µM ZnCl2, 2.5% Triton X-100, and 0.02% NaN3 at room temperature, incubated in the same buffer without Triton X-100 for 48 h at 37°C, stained with Coomassie brilliant blue R-250 for 3 h, and destained in water.
Total protein extraction.
Cells were lysed in a buffer containing 1% Triton X-100, 1 mM
CaCl2, a cocktail of protease inhibitors (catalog no.
P8340, Sigma), 1 mM phenylmethylsulfonyl fluoride, and 1 mM
N-ethylmaleimide in PBS for 30 min at 4°C. Insoluble
material was removed by centrifugation, and the supernatant was stored
at 20°C. Protein estimation was performed by the method of Bradford (Pierce).
Electrophoresis and immunoblotting. Electrophoresis in the presence of SDS was performed on 7.5% or 10% polyacrylamide gels under reducing or nonreducing conditions. The resolved proteins were subsequently electrotransferred to Hybond-ECL nitrocellulose membrane (Amersham). Blots were saturated for 2 h at room temperature with 5% nonfat milk in Tris-buffered saline-0.1% Tween 20 and incubated overnight at 4°C with the appropriate dilutions of polyclonal antibodies in the same buffer without Tween 20. Incubations with peroxidase-conjugated goat anti-rabbit immunoglobulins or sheep anti-mouse immunoglobulins and detection of bound peroxidase activity were carried out as described for the enhanced chemiluminescence blotting detection system (Amersham).
Immunoprecipitation of FAK.
Confluent HGEC cultured in 5 mM D-glucose were serum
starved for 24 h before detachment with trypsin. Cells were washed
twice with DMEM, suspended in DMEM containing 25 mM HEPES and 2 mg/ml BSA, and incubated in suspension at 37°C for 45 min to allow kinases to become quiescent. Then 3 × 106 cells were
incubated in suspension at 37°C for 90 min with or without 10 µg/ml
of each anti-3- and anti-
1-integrin MAb.
At the end of the incubation period, cells were washed twice with cold
PBS and lysed in a modified RIPA buffer containing 50 mM Tris · HCl, pH 7.4, 1% NP-40, 0.25% sodium
deoxycholate, 150 mM NaCl, 1 mM Na2EDTA, 1 mM
phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM sodium
pyrophosphate, 50 mM NaF, and a cocktail of protease inhibitors. Then
500 µg of protein from each cell lysate were incubated overnight at
4°C with 2 µg of a rabbit polyclonal antibody specific for human
pp125FAK. Immune complexes were precipitated for 2 h
at 4°C with protein A-Sepharose and washed three times in ice-cold
modified RIPA buffer. Immune complexes were extracted into boiling
Laemmli sample buffer containing 10%
-mercaptoethanol,
electrophoresed on 7.5% polyacrylamide gels, and electrotransferred to
nitrocellulose membrane. A monoclonal antiphosphotyrosine antibody was
used to analyze Western blots for phosphotyrosine-containing proteins.
The immunoblots were stripped in Re-Blot Plus Western blot stripping
solution (catalog no. 2500, Chemicon) as recommended by the
manufacturer, and then the proteins were immunoblotted with anti-FAK
antibody to determine whether equal amounts of FAK were loaded per lane.
Statistical analysis. Values are means ± SD. In the assays of adhesion and inhibition of adhesion, the means of groups were compared using one-way ANOVA with post hoc testing using the Newman-Keuls test as appropriate. Results from images of Western blots and zymograms were analyzed using Student's t-test and one-way ANOVA. Both tests gave similar results. P < 0.05 was considered statistically significant.
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RESULTS |
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Expression of integrins by cultured HGEC in 5 and 25 mM glucose. Integrins may play a role in altered matrix synthesis and degradation in diabetic conditions. Therefore, we examined and compared the expression of the main integrin subunits of HGEC grown in 5 and 25 mM glucose.
Western blot analysis (Fig. 1) demonstrated that
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HGEC adhesion to type IV collagen in the presence of 5 and 25 mM
glucose.
The previous experiment indicated that the presence of increased
glucose concentrations resulted in up- or downregulation of different
integrin subunits of HGEC. This change could be accompanied by changes
in cell adhesion to the GBM and its individual components. Therefore,
we examined integrin-mediated adhesion of HGEC to type IV collagen by
solid-phase binding assays. Type IV collagen binding of HGEC grown in
25 mM glucose was decreased by ~15-45% compared with cells
grown in 5 mM glucose, depending on the concentration of type IV
collagen used (Fig. 2). The observed
differences were statistically significant (P < 0.05)
in all but the highest concentration of type IV collagen.
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Integrins mediating the binding of HGEC to type IV collagen in 5 and 25 mM glucose.
For this experiment, adhesion of HGEC to type IV collagen in the
presence of various inhibiting anti-integrin monoclonal antibodies was
examined. The extent of inhibition of adhesion varied depending on the
type of antibody used against different integrin subunits and glucose
concentration. More specifically, anti-1-integrin antibodies (P5D2) almost completely inhibited the adhesion of HGEC to
type IV collagen in 5 and 25 mM glucose (Fig.
3). In 5 mM glucose, antibodies Lm609 and
P1D6 against the
v
3- and
5-integrins, respectively, resulted in ~14% and
~36% inhibition of maximal adhesion (adhesion in 5 mM glucose in the
absence of antibody), respectively (Fig. 3A), whereas in 25 mM glucose, ~60% and ~55% inhibition of maximal adhesion
(adhesion in 25 mM glucose in the absence of antibody), respectively,
was observed (Fig. 3B). In 5 mM glucose, antibody P1E6
against the
2-subunit resulted in 40% inhibition of
maximal adhesion (Fig. 3A), whereas in 25 mM glucose, only
~13% inhibition of maximal adhesion was observed (Fig.
3B). MAb P1B5 against
3-integrin caused HGEC
aggregation and was not effective in blocking adhesion. However, using
the F(ab) fragment of anti-
3-integrin MAb (P1B5), we
previously documented that
3-integrin participated in
the binding of HGEC to type IV collagen (22). There was no
inhibition of adhesion by MAbs against HLA (negative control).
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Expression of matrixins (MMP-2 and MMP-9) and their inhibitors
(TIMP-1 and TIMP-2) in 5 and 25 mM glucose.
We first compared proteolytic activities of conditioned media from HGEC
grown in 5 or 25 mM glucose with gelatin zymography. Enzymatic activity
was detected at two major bands corresponding to 92/88-kDa (MMP-9) and
72/68-kDa (MMP-2) collagenases (Fig. 4A). Densitometric analysis
indicated that media from HGEC grown in 25 mM glucose contained 70%
less of the 72/68-kDa form of MMP-2 than media from cells grown in 5 mM
glucose. There were no significant changes in the total amount of the
92/88-kDa form of MMP-9 (Fig. 4B).
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Effect of ligation of 3
1- and
v
3-integrins on MMP-2 expression in 5 and
25 mM glucose.
This experiment was performed to examine the possible role of
3
1, the main integrin dimer in HGEC
(22), in the expression of MMPs in low- and high-glucose
conditions. HGEC grown in 5 or 25 mM glucose were cultured in the
absence or presence of MAbs against
3- and
1-integrins (see MATERIALS AND METHODS).
Alternatively, antibodies against
v
3-,
5-, and
2-integrins were used for the
same experiment.
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Phosphorylation of FAK in HGEC treated with MAbs against
3- and
1-integrins.
FAK has been described to be a component of the signaling pathway that
mediates regulation of the expression of MMPs by integrins. Therefore,
we examined tyrosine phosphorylation of pp125FAK in control
(untreated) HGEC and HGEC treated with MAbs against
3-
and
1-integrins simultaneously (Fig.
7). In cells treated with
anti-
3- and anti-
1-integrin MAbs, there
was a ~30% increase in phosphotyrosine of pp125FAK
compared with control untreated cells. To ensure equal amounts of
protein loading, blots were stripped and reprobed with polyclonal anti-FAK antibodies.
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DISCUSSION |
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We have provided evidence that increased glucose concentrations
modulate integrin expression and integrin-related functions of cultured
T-SV40-immortalized HGEC, which were shown to be similar to their
primary counterparts (8, 22). The observed effects were
specifically due to D-glucose and not to osmotic effect. We
and others previously showed that L-glucose, ribitol, or
mannitol had no effect (2, 30). We previously reported
that 3
1 is the main integrin present at
high density on the surface of ~97% of HGEC grown in the presence of
5 mM glucose (22). This dimer can mediate the binding of
HGEC to collagen and laminin components of the GBM (22,
40). In addition, HGEC expressed
5-,
2-, and
v
3-integrins,
which have been described to mediate cell binding to collagens,
fibronectin, and fibrinogen (15).
In HGEC cultured in the presence of 25 mM glucose, we observed a
significant decrease in 3-,
2-, and
1-protein levels with a concomitant increase of
5-,
v-, and
3-subunits
compared with control cells, which were grown in 5 mM glucose. Several
different cell types were also documented to respond to increased
glucose concentrations by changing the expression of their integrins. For example, modulation of integrin expression was shown in primary human mesangial cells grown in 25 mM glucose (36), human
diabetic kidneys (4, 16), and human glomerular diseases
(12, 18). In sections from renal tissues, podocytes from
short- and long-term diabetic rats had decreased expression of
3
1-integrin when examined with
immunoelectron microscopy; this decrease was thought to represent an
early event that precedes the onset of DN (31, 32).
Therefore, the reported findings in podocytes in diabetic conditions in
situ corroborate our observations related to the effects of high
glucose in cultured HGEC, insofar as the expression of
3
1-integrin was concerned. The mechanisms
by which hyperglycemia alter the expression of integrins are not
clearly understood. In kidneys, transforming growth factor-
1
(TGF-
1) has been reported to be a strong regulator of the expression
of integrins (38). TGF-
1 has been demonstrated to
suppress the expression of
3-integrin in glomeruli from
nephrotic rats (18), and an overexpression of TGF-
1 has
also been reported to occur in diabetic rat kidneys as early as
2-3 days after induction of hyperglycemia (37).
Therefore, one mechanism of altered integrin expression could involve
overexpression of TGF-
1.
Furthermore, our data provide evidence that glucose-induced changes in
integrin expression were accompanied by altered binding to GBM
components, such as type IV collagen. Type IV collagen was selected for
the study of interactions with HGEC, because it is the predominant
glycoprotein of the GBM (8, 22). We observed that high
glucose decreased the number of HGEC bound to solid-phase type IV
collagen. This binding was mediated in part by different integrins
depending on glucose concentration. For example, there was substantial
inhibition of HGEC binding to type IV collagen in the presence of
anti-2- and anti-
5-integrin MAbs in 5 mM
glucose (~40% and 35%, respectively), whereas there was no
significant inhibition by MAbs against
v
3-integrin. The involvement of
5-integrin in the binding of HGEC to type IV collagen was unexpected, because this integrin was described to mainly serve for
cell binding to fibronectin (15, 33). However, primary and
immortalized HGEC express higher levels of
5-integrin
than the collagen-binding
1- and
2-subunits (22). It is possible, therefore,
that in HGEC the
5-subunit participates in cell binding to type IV collagen.
In high glucose, inhibition of HGEC binding to type IV collagen was
significant in the presence of anti-v
3-
and anti-
5-integrin MAbs (60% and 50%, respectively),
whereas antibodies against
2-integrin did not block
adhesion, indicating that there was a partial glucose-dependent switch
of integrin subunits that bind HGEC to type IV collagen. Anti-
1-integrin MAb resulted in nearly complete
inhibition of adhesion to type IV collagen in either glucose
concentration, as expected. The data indicate that
1 is
a predominant integrin; indeed, by fluorescein-activated cell sorter
analysis, it is present at a high density on the surface of 84% of
primary HGEC and 97% of immortalized HGEC (22). This
subunit associates with different
-subunits, thus serving for
multiple binding events to matrix components.
In summary, different integrins in part mediate HGEC binding to type IV
collagen, depending on glucose concentration. In low glucose,
2- and
5-integrins participated in the
binding, whereas, in high glucose, the
v
3- and
5-subunits were
preferentially used. If similar changes occur in situ, in diabetic
conditions, then altered interactions with matrix components could be
anticipated to alter integrin-mediated signaling and various aspects of
integrin-regulated cell functions, including protein phosphorylation
(20, 21) and gene expression (3, 6, 11, 17).
For example, the expression and/or activation of several MMPs was shown
to be regulated by integrins in different cell types (1, 9, 13,
24, 25, 34, 35, 45, 47). Our experiments indicate that changes
in integrin expression in HGEC grown in 25 mM glucose were accompanied
by changes in the expression and/or activity of MMP-2. In vivo,
1-integrin associates with different
-subunits
serving for multiple binding and specific signaling events. Because
3
1-integrin is the main subunit expressed
by HGEC (22) and, moreover, there was decreased expression
of
3
1-integrin and MMP-2 in 25 mM glucose
compared with the control (5 mM glucose), we examined the hypothesis
that
3
1-integrin was involved in regulation of MMP-2 expression. In HGEC grown in 5 mM glucose, ligation
of the
3
1-integrin dimer with
anti-
3- and anti-
1-integrin MAbs
simultaneously resulted in a 2.5-fold increase in secreted MMP-2
compared with the untreated control, whereas MMP-9 remained unchanged
(data not shown). The effect of
3- or
1-integrin ligation was more modest, resulting in a
40-45% increase of MMP-2 secretion. Antibodies against
v
3-integrin, which was increased in high glucose, had no effect on the expression of MMP-2. Furthermore, ligation of other integrins, such as
5 or
2, with specific anti-integrin MAbs had no effect on the
secretion of MMP-2 (data not shown). Upregulation of MMP-2 by ligation
of
3
1-integrin was also observed in HGEC
cultured in 25 mM glucose, albeit to a lesser extent. Glucose-induced
decrease of the expression of
3
1-integrin
could possibly account for this difference.
Our findings suggest then that
3
1-integrin in part regulates the
expression of MMP-2 in HGEC. Several other reports showed that ligation
of
3
1-integrin with MAbs resulted in
upregulation of MMP-2 production by tumor cells (5, 41,
43) or induced the activated form of MMP-2 and enhanced
pro-MMP-2 secretion by rhabdomyosarcoma cells (23).
The mechanism by which 3
1-integrin
enhances the expression of MMP-2 remains to be substantiated. It has
been previously documented that a major signaling pathway linking
integrins to the regulation of the expression of MMPs involves FAK,
mitogen-activated protein kinase, and transcription factor AP-1
(21, 42). In our experiments, we observed increased FAK
phosphorylation in HGEC as a response to
3
1-integrin ligation, a finding that
indicates the possible involvement of the above-mentioned pathway in
regulation of the expression of MMP-2 in this cell type. This
hypothesis is also in accord with a recent report showing that integrin
interactions with their ligand can transduce stimulatory signals
(through FAK-Src-type kinases) for MMP-2 and MMP-9 expression in human
T lymphocytes (10). However, there might be alternative or
additional regulatory mechanisms. Whatever the mechanism, the observed
decreased expression of MMP-2 could result in impaired degradation of
basement membrane components.
We additionally observed a substantial increase in the expression of TIMP-2, the specific inhibitor of MMP-2, a finding that suggests further impairment of matrix degradation, leading to matrix accumulation. The modest decrease in TIMP-1, the specific inhibitor of MMP-9, could be compensatory.
In situ, even a small decrease in MMP-2 and/or increase in TIMP-2 protein levels could be anticipated to impair the balance between ECM synthesis and degradation, resulting in matrix accumulation. We and others documented that increased glucose concentrations resulted in altered expression of matrix, MMPs, and TIMPs in cultured mesangial cells (3, 26) and also in situ in kidneys of streptozotocin-diabetic rats at early stages of diabetes in the absence of albuminuria (46).
Collectively, our data indicate that glucose-induced modulation of integrin expression was accompanied by functional changes in HGEC in vitro, which in turn could contribute to altered interactions with type IV collagen, a major component of the GBM and, in addition, decreased degradation of matrix proteins. The combined long-term effect could be microalbuminuria due, at least in part, to altered HGEC binding to the GBM components and matrix accumulation, which, if present in situ, could help explain the thickening of the GBM in DN.
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ACKNOWLEDGEMENTS |
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The authors are indebted to C. Economou and P. Karamessinis for expert assistance with image processing and analysis.
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FOOTNOTES |
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This work was supported by Greek General Secretariat for Research and Technology/PENED-99 Grant 174ED (to E. C. Tsilibary) and National Institute on Aging Grant AI-0708 (to A. F. Michael).
Address for reprint requests and other correspondence: P. V. Kitsiou, Institute of Biology, National Center for Scientific Research "Demokritos," 15310 Agia Paraskevi, Athens, Greece (E-mail: pkit{at}mail.demokritos.gr).
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.
First published December 3, 2002;10.1152/ajprenal.00266.2002
Received 24 July 2002; accepted in final form 1 December 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Agrez, M,
Xinhua G,
Turton J,
Meldrum C,
Niu J,
Antalis T,
and
Howard EW.
The v
6 integrin induces gelatinase B secretion in colon cancer cells.
Int J Cancer
81:
90-97,
1999[ISI][Medline].
2.
Anderson, SS,
Tsilibary EC,
and
Charonis AS.
Nonenzymatic glycosylation-induced modifications of intact bovine kidney tubular basement membrane.
J Clin Invest
92:
3045-3052,
1993[ISI][Medline].
3.
Anderson, SS,
Wu K,
Nagase H,
Stetler-Stevenson W,
Kim Y,
and
Tsilibary EC.
Effect of matrix glycation on expression of type IV collagen, MMP-2, MMP-9 and TIMP-1 by human mesangial cells.
Cell Adhes Commun
4:
89-101,
1996[ISI][Medline].
4.
Chen, HC,
Chen CA,
Guh JY,
Chang JM,
Shin SJ,
and
Lai YH.
Altering expression of 3
1 integrin on podocytes of humans and rats with diabetes.
Life Sci
67:
2345-2353,
2000[ISI][Medline].
5.
Chintala, SK,
Sawaya R,
Gokaslan ZL,
and
Rao JS.
Modulation of matrix metalloproteinase-2 and invasion in human glioma cells by 3
1 integrin.
Cancer Lett
103:
201-208,
1996[ISI][Medline].
6.
Clark, EA,
and
Brugge JS.
Integrins and signal transduction pathways: the road taken.
Science
268:
233-239,
1995[ISI][Medline].
7.
Danne, T,
Spiro MJ,
and
Spiro RG.
Effect of high glucose on type IV collagen production by cultured glomerular epithelial cells, endothelial and mesangial.
Diabetes
42:
170-177,
1993[Abstract].
8.
Delarue, F,
Virone A,
Hagege J,
Lacave R,
Peraldi M,
Adida M,
Rondeau E,
Feunteun J,
and
Sraer J.
Stable cell line of T-SV40 immortalized human glomerular visceral epithelial cells.
Kidney Int
40:
906-909,
1991[ISI][Medline].
9.
DiPersio, M,
Shao M,
Di Costanzo L,
Kreidberg JA,
and
Hynes RO.
Mouse keratinocytes immortalized with large T antigen acquire 3
1 integrin-dependent secretion of MMP-9/gelatinase B.
J Cell Sci
113:
2909-2921,
2000
10.
Esparza, BJ,
Vilardell C,
Calvo J,
Juan M,
Vives J,
Urbano-Marquez A,
Yague J,
and
Cid MC.
Fibronectin upregulates gelatinase B (MMP-9) and induces coordinated expression of gelatinase A (MMP-2) and its activator MT1-MMP (MMP-14) by human T lymphocyte cell lines. A process repressed through RAS/MAP kinase signaling pathways.
Blood
94:
2754-2766,
1999
11.
Giancotti, FG,
and
Ruoslahti E.
Integrin signaling.
Science
285:
1028-1032,
1999
12.
Hafdi, Z,
Lesavre P,
Nejjari M,
Halbwachs-Mecarelli L,
Droz D,
and
Noel LH.
Distribution of v
3,
v
5 integrins and the integrin associated protein IAP (CD47) in human glomerular diseases.
Cell Adhes Commun
7:
441-451,
2000[ISI][Medline].
13.
Hofmann, UB,
Westphall JR,
Van Kraats AA,
Ruiter DJ,
and
Van Muijen GNP
Expression of integrin v
3 correlates with activation of membrane-type matrix metalloproteinase-1 (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) in human melanoma cells in vitro and in vivo.
Int J Cancer
87:
12-19,
2000[ISI][Medline].
14.
Humphries, MJ,
Mould AP,
and
Tuckwell DS.
Dynamic aspects of adhesion receptor functionintegrins both txist and shout.
Bioessays
15:
391-397,
1993[ISI][Medline].
15.
Hynes, R.
Integrins: a family of cell-surface receptors.
Cell
69:
11-25,
1992[ISI][Medline].
16.
Jin, DK,
Fish AJ,
Wayner EA,
Mauer M,
Setty S,
Tsilibary EC,
and
Kim Y.
Distribution of integrin subunits in human diabetic kidneys.
J Am Soc Nephrol
7:
2636-2645,
1996[Abstract].
17.
Juliano, RL,
and
Haskill S.
Signal transduction from the extracellular matrix.
J Cell Biol
120:
577-585,
1993[ISI][Medline].
18.
Kagami, S,
Border WA,
Ruoslahti E,
and
Noble NA.
Coordinated expression of 1 integrins and transforming growth factor-induced matrix proteins in glomerulonephritis.
Lab Invest
69:
68-76,
1993[ISI][Medline].
19.
Kleinman, HK,
McGarvey LM,
Liotta LA,
Gehron-Robey P,
Tryggvason K,
and
Martin GR.
Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma.
Biochemistry
21:
6188-6193,
1982[ISI][Medline].
20.
Kornberg, LJ,
Earp HS,
Turner CE,
Prockop C,
and
Juliano RL.
Signal transduction by integrins: increased protein tyrosine phosphorylation caused by clustering of 1 integrins.
Proc Natl Acad Sci USA
88:
8392-8396,
1991[Abstract].
21.
Krishnamurti, U,
Rondeau E,
Sraer JD,
Michael AF,
and
Tsilibary EC.
Alterations in human glomerular epithelial cells interacting with nonenzymatically glycosylated matrix.
J Biol Chem
272:
27966-27970,
1997
22.
Krishnamurti, U,
Yong C,
Michael A,
Kim Y,
Fan WW,
Weislander J,
Brunmark C,
Rondeau E,
Sraer JD,
Delarue F,
and
Tsilibary EC.
Integrin mediated interactions between primary T-SV40 immortalized human glomerular epithelial cells and type IV collagen.
Lab Invest
74:
650-657,
1996[ISI][Medline].
23.
Kubota, S,
Ito H,
Ishibashi Y,
and
Seyama Y.
Anti-3 integrin antibody induces the activated form of matrix metalloproteinase-2 (MMP-2) with concomitant stimulation of invasion through Matrigel by human rhabdomyosarcoma cells.
Int J Cancer
70:
106-111,
1997[ISI][Medline].
24.
Langholz, O,
Rockel D,
Mauch C,
Kozlowska E,
Bank I,
Krieg T,
and
Eckes B.
Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by 1
1 and
2
1 integrins.
J Cell Biol
131:
1903-1915,
1995[Abstract].
25.
Larjava, H,
Lyons JG,
Salo T,
Makela M,
Koivisto L,
Birkedal-Hansen H,
Akiyama SK,
Yamada K,
and
Heino J.
Anti-integrin antibodies induce type IV collagenase expression in keratinocytes.
J Cell Physiol
157:
190-200,
1993[ISI][Medline].
26.
Leehey, D,
Song RH,
Alavi N,
and
Singh AK.
Decreased degradative enzymes in mesangial cells cultured in high glucose media.
Diabetes
44:
929-935,
1995[Abstract].
27.
Matrisian, LM.
The matrix-degrading metalloproteinases.
Bioessays
14:
455-463,
1992[ISI][Medline].
28.
Nagase, H,
and
Woessner JF.
Matrix metalloproteinases.
J Biol Chem
274:
21491-21494,
1999
29.
Orkin, RW,
Gehron P,
Goodwin EB,
Martin GR,
Valentine T,
and
Swarm RA.
Murine tumor producing a matrix of basement membrane.
J Exp Med
145:
204-220,
1977[Abstract].
30.
Park, SH,
and
Han HJ.
The mechanism of angiotensin II binding downregulation by high glucose in primary renal proximal tubule cells.
Am J Physiol Renal Physiol
282:
F228-F237,
2002
31.
Regoli, M,
and
Bendayan M.
Alterations in the expression of the 3
1 integrin in certain membrane domains of the glomerular epithelial cells (podocytes) in diabetes mellitus.
Diabetologia
40:
15-22,
1997[ISI][Medline].
32.
Regoli, M,
and
Bendayan M.
Expression of 1 integrins in glomerular tissue of streptozotocin-induced diabetic rats.
Biochem Cell Biol
77:
71-78,
1999[ISI][Medline].
33.
Ruoslahti, E.
Integrins.
J Clin Invest
87:
1-5,
1991[ISI][Medline].
34.
Seftor, RE,
Seftor EA,
Gehlsen KR,
Stetler-Stevenson WG,
Brown PD,
Ruoslahti E,
and
Hendrix MG.
Role of the v
3 integrin in human melanoma cell invasion.
Proc Natl Acad Sci USA
89:
1557-1561,
1992[Abstract].
35.
Seftor, RE,
Seftor EA,
Stetler-Stevenson WG,
and
Hendrix MG.
The 72-kDa type IV collagenase is modulated via differential expression of v
3 and
5
1 integrins during human melanoma cell invasion.
Cancer Res
53:
3411-3415,
1993[Abstract].
36.
Setty, S,
Anderson SS,
Wayner EA,
Kim Y,
Clegg DO,
and
Tsilibary EC.
Glucose-induced alteration of integrin expression and function in cultured human mesangial cells.
Cell Adhes Commun
3:
187-200,
1995[ISI][Medline].
37.
Shankland, SJ,
and
Schley JW.
Expression of transforming growth factor-1 during diabetic renal hypertrophy.
Kidney Int
46:
430-442,
1994[ISI][Medline].
38.
Sharma, K,
and
Ziyadeh FN.
The emerging role of transforming growth factor- in kidney diseases.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F829-F842,
1994
39.
Steffes, MW,
Bilous RW,
Sutherland DER,
and
Mauer SM.
Cell and matrix components of the glomerular mesangium in type I diabetes.
Diabetes
41:
679-684,
1992[Abstract].
40.
Sterk, LM,
de Melker AA,
Kramer D,
Kuikman I,
Chand A,
Claessen N,
Weening JJ,
and
Sonnenberg A.
Glomerular extracellular matrix components and integrins.
Cell Adhes Commun
5:
177-192,
1998[ISI][Medline].
41.
Sugiura, T,
and
Berditchevski F.
Function of 3
1-tetraspanin protein complexes in tumor cell invasion. Evidence for the role of the complexes in production of matrix metalloproteinase 2 (MMP-2).
J Cell Biol
146:
1375-1389,
1999
42.
Tremble, P,
Damsky CH,
and
Werb Z.
Components of the nuclear signaling cascade that regulate collagenase gene expression in response to integrin-derived signals.
J Cell Biol
129:
1707-1720,
1995[Abstract].
43.
Tzinia, AK,
Kitsiou PV,
Talamagas AA,
Georgopoulos A,
and
Tsilibary EC.
Effects of collagen IV on neuroblastoma cell matrix-related functions.
Exp Cell Res
274:
169-177,
2002[ISI][Medline].
44.
Waddell, NJ.
A simple ultraviolet spectrophotometric method for the determination of proteins.
Lab Invest
48:
311-314,
1956.
45.
Werb, Z,
Tremble PM,
Behrendtsen O,
Crowley E,
and
Damsky CH.
Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression.
J Cell Biol
109:
877-889,
1989[Abstract].
46.
Wu, K,
Setty S,
Mauer SM,
Killen P,
Nagase H,
Michael AF,
and
Tsilibary EC.
Altered kidney matrix gene expression in early stages of experimental diabetes.
Acta Anat (Basel)
158:
155-165,
1997[Medline].
47.
Xu, J,
and
Clark AF.
A three-dimensional collagen lattice induces protein kinase C- activity: role in
2 integrin and collagenase mRNA expression.
J Cell Biol
136:
473-483,
1997