Glucose-induced changes in integrins and matrix-relatedfunctions in cultured hum - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:35 |只看该作者 |倒序浏览 |打印

作者：Paraskevi V.Kitsiou, Athina K.Tzinia, William G.Stetler-Stevenson, Alfred F.Michael, Wei-WeiFan, BingZhou,  Effie C.Tsilibary作者单位：1 Institute of Biology, National Center forScientific Research “Demokritos,“ 15310 Agia Paraskevi, Athens,Greece; Laboratory of Pathology, NationalCancer Institute, National Institutes of Health, Bethesda, Maryland20892; and Department of Pediatrics, University ofMinnesota Medical School
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      【摘要】. L: H; r. |. {( g, C! D2 P2 D
   In cultured humanglomerular epithelial cells (HGEC), 25 mM glucose resulted in decreasedexpression of 3 -, 2 -, and 1 -integrins and increased expression of 5 - and v 3 -integrins. Thischange was accompanied by decreased binding of HGEC to type IVcollagen. In the presence of normal (5 mM) glucose concentration, cellbinding to type IV collagen was primarily mediated by 2 1 - and 5 1 -integrins, as indicated by experimentsin which cell adhesion to type IV collagen was competed by specificanti-integrin monoclonal antibodies. In the presence of high (25 mM)glucose, the upregulated 5 - and v 3 -integrins were mainly involved in cellbinding to type IV collagen. Furthermore, high glucose decreasedexpression of matrix metalloproteinase-2 (MMP-2), a collagenaseregulated in part by 3 1 -integrin, assuggested by the use of ligand-mimicking antibodies against theseintegrins, which resulted in release of increased amounts of MMP-2 inthe culture medium. Finally, tissue inhibitor of metalloproteinase-2,the specific inhibitor of MMP-2, was upregulated in high glucose andcould contribute to matrix accumulation. These changes could helpexplain basement membrane thickening in diabetes.
      【关键词】 matrixins tissue inhibitors of metalloproteinases signaling diabetes
   INTRODUCTION

THE GLOMERULAR BASEMENT MEMBRANE (GBM) underlying glomerular epithelial cells,an important component of the kidney permselective barrier, isthickened in diabetic nephropathy (DN). GBM thickening could be due toincreased deposition (synthesis and accumulation) of the extracellularmatrix (ECM) macromolecules such as collagen, fibronectin, laminin, andproteoglycans, which could be explained by an imbalance between matrixsynthesis and degradation. Matrix synthesis and degradation areregulated in part by cell-matrix interactions ( 7, 39 ).! \% t/ s( `+ T! i* s& P% F

Interactions of cells with matrix molecules are primarily mediated bythe integrin superfamily receptors ( 14, 15 ). Most integrins recognize more than one ECM protein, such as collagens, fibronectin, and laminin ( 33 ), and, on binding, theytransduce signals to the cell interior via mechanisms such as proteinphosphorylation ( 6 ). For example, tyrosine kinasepp125 FAK [focal adhesion kinase (FAK)] becomesphosphorylated 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 integratingsignals from integrins.
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One function attributed to integrins is regulation of the expression ofmatrix 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 ). Gelatinasesare synthesized and secreted as inactive forms (pro-MMPs), and theirmatrix-degrading activities are regulated by activators and inhibitors.Most cells produce and secrete specific tissue inhibitors ofmetalloproteinases, TIMP-1 and TIMP-2, which preferentially bind toMMP-9 and MMP-2, respectively, thus regulating their matrix-degradingactivity ( 27, 28 ).3 `  y5 i9 i' \! D, |3 Q7 h: G- E

To elucidate mechanisms leading to GBM thickening in DN, we have usedas a model T-SV40 immortalized human glomerular epithelial cells (HGEC)to study whether increased glucose concentrations affectedintegrin-mediated interactions of these cells with type IV collagen,thus contributing to differential expression of several factorscontrolling ECM synthesis and degradation. T-SV40-immortalized HGECexpress differentiation markers on the surface of primary glomerularepithelial cells, interact with type IV collagen, and are thereforesimilar to their primary counterparts ( 8, 22 ). Theintegrin profile of HGEC was examined in normal (5 mM) and high (25 mM)glucose, and quantitative changes in integrin expression were observedin the presence of increased glucose concentrations. These changes wereaccompanied by modulation of integrin-mediated interactions of HGECwith type IV collagen, a predominant component of the GBM. Furthermore,high glucose altered the expression and production of proteins involvedin matrix degradation. In our experiments, the expression of MMP-2 wasregulated in part by 3 1 -integrin, which,on ligation, enhanced FAK phosphorylation and resulted inupregulation of MMP-2.6 n( }1 f* g( C, K! {

Our findings suggest that increased glucose concentrations alterednormal matrix-related cell functions of HGEC and resulted indifferential gene expression, possibly contributing to matrix accumulation. The observed changes could help explain the thickening ofthe GBM in DN.. g' @. }+ A: ]
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MATERIALS AND METHODS

Cell culture. HGEC ( 8, 22 ) were cultured at 37°C in media composed ofDMEM-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 cultureflasks for passaging or use in experiments by treatment with 0.05%trypsin in 1 mM EDTA. For experiments, cells were cultured for at leastthree passages. For zymography and Western blotting analysis ofconditioned media, cells (2 × 10 6 ) were cultured for48 h. The medium was then replaced with 10 ml of serum-freemedium, and cells were incubated for 24 h. At the end of the 24-hincubation period, the conditioned media were collected, supplementedto 1 mM Na 2 EDTA and 0.02% sodium azide, and stored at 20°C for further use.& t: w9 v/ W' v4 n

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-FAKantibody specific for human pp125 FAK andantiphosphotyrosine MAb (clone 4G10) were obtained from Upstate Biotechnology. Peroxidase-conjugated goat anti-rabbit immunoglobulins and sheep anti-mouse immunoglobulins were purchased from Amersham.1 i2 f! m. I- _
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Culture of cells with MAbs. Cells were detached from confluent monolayer cultures, resuspended inculture medium (3 × 10 6 cells/ml), preincubated withMAb at 50 µg/ml each for 30 min at 37°C, and then diluted 10-foldto a final concentration of 5 µg/ml each or preincubated withoutantibody and cultured for 24 h. The medium was then replaced withserum-free medium with or without 5 µg/ml of each MAb, and cells wereincubated for 24 h. At the end of the 24-h incubation period, theconditioned media were collected for zymography, and the cells wereharvested and counted.' o( g; L/ d* b# C

Isolation of type IV collagen. Type IV collagen was isolated from the Engelbreth-Holm-Swarm tumorsystem using previously described techniques ( 19, 29 ). Protein concentration was determined using the method of Waddell ( 44 ).' {1 |, Y5 w' z. d# _7 f

Cell adhesion assay. Microtiter plates (96-well; Microlon 600, Greiner) were coated with 50 µl of increasing concentrations of type IV collagen (0.3-200µg/ml PBS) and allowed to evaporate to dryness at 29°C. Theremaining 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 usedfor experiments. HGEC were grown in 5 or 25 mM D -glucose inT-25 flasks until 75-80% confluency was reached and weremetabolically labeled for 18 h with 0.15 mCi of[ 35 S]methionine (Amersham) per flask. Cells were washedtwice with DMEM, and 50 µl (5,000 cells) of cell suspension inbinding buffer (DMEM, 2 mg/ml BSA, and 25 mM HEPES, pH 7.5) were addedto 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, thefollowing criteria were selected to achieve optimal antibody effect:half-maximal binding of HGEC to type IV collagen by using 5 µg/mltype IV collagen and a short-term assay (30 min). After plates werecoated with 50 µl of type IV collagen at 5 µg/ml, MAb to integrinsubunit or MAb to HLA was added to each well (100 µl/well) at a finalconcentration of 10 µg/ml followed immediately by 50 µl of cellsuspension in binding buffer (5,000 cells/well). Cells were allowed toadhere for 30 min at 37°C and processed as for the adhesion assays.The concentration of antibodies used in inhibition assays was above thesaturating concentration as determined by flow cytometry. The data werenormalized by expressing the binding in the absence of antibody asmaximal (100%), and adhesion in the presence of antibodies is shown aspercentage of binding in the absence of antibody. All assays wereperformed a minimum of three times in hexaplicate for each experimental condition.: a$ Z/ {: O3 s, r8 i/ Z+ X

Zymography. Gelatin zymography was performed as previously described( 3 ). Briefly, aliquots of each sample of conditioned mediawere concentrated and subjected to SDS-PAGE under nonreducingconditions in 10% polyacrylamide gels containing 0.1% gelatin. Thevolume of conditioned medium loaded per lane was adjusted according to the cell number obtained at harvest. After electrophoresis, the gel waswashed three times for 30 min each with 50 mMTris · HCl, pH 7.5, 5 mM CaCl 2, 1 µM ZnCl 2, 2.5% Triton X-100, and 0.02% NaN 3 at room temperature, incubated in the same buffer without Triton X-100for 48 h at 37°C, stained with Coomassie brilliant blue R-250for 3 h, and destained in water.0 E* @4 H. o4 O" T/ D1 m

Total protein extraction. Cells were lysed in a buffer containing 1% Triton X-100, 1 mMCaCl 2, 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. Insolublematerial was removed by centrifugation, and the supernatant was storedat 20°C. Protein estimation was performed by the method of Bradford (Pierce)./ d+ r" B, G, `

Electrophoresis and immunoblotting. Electrophoresis in the presence of SDS was performed on 7.5% or 10%polyacrylamide gels under reducing or nonreducing conditions. Theresolved proteins were subsequently electrotransferred to Hybond-ECLnitrocellulose membrane (Amersham). Blots were saturated for 2 hat 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 immunoglobulinsor sheep anti-mouse immunoglobulins and detection of bound peroxidaseactivity were carried out as described for the enhancedchemiluminescence blotting detection system (Amersham).

Immunoprecipitation of FAK. Confluent HGEC cultured in 5 mM D -glucose were serumstarved for 24 h before detachment with trypsin. Cells were washedtwice 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 × 10 6 cells wereincubated in suspension at 37°C for 90 min with or without 10 µg/mlof each anti- 3 - and anti- 1 -integrin MAb.At the end of the incubation period, cells were washed twice with coldPBS and lysed in a modified RIPA buffer containing 50 mM Tris · HCl, pH 7.4, 1% NP-40, 0.25% sodiumdeoxycholate, 150 mM NaCl, 1 mM Na 2 EDTA, 1 mMphenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM sodiumpyrophosphate, 50 mM NaF, and a cocktail of protease inhibitors. Then500 µg of protein from each cell lysate were incubated overnight at4°C with 2 µg of a rabbit polyclonal antibody specific for humanpp125 FAK. Immune complexes were precipitated for 2 hat 4°C with protein A-Sepharose and washed three times in ice-coldmodified RIPA buffer. Immune complexes were extracted into boilingLaemmli sample buffer containing 10% -mercaptoethanol,electrophoresed on 7.5% polyacrylamide gels, and electrotransferred tonitrocellulose membrane. A monoclonal antiphosphotyrosine antibody wasused to analyze Western blots for phosphotyrosine-containing proteins.The immunoblots were stripped in Re-Blot Plus Western blot strippingsolution (catalog no. 2500, Chemicon) as recommended by themanufacturer, and then the proteins were immunoblotted with anti-FAKantibody to determine whether equal amounts of FAK were loaded per lane.' ]. g, ], ~0 q

Statistical analysis. Values are means ± SD. In the assays of adhesion and inhibitionof adhesion, the means of groups were compared using one-way ANOVA withpost hoc testing using the Newman-Keuls test as appropriate. Resultsfrom images of Western blots and zymograms were analyzed usingStudent's t -test and one-way ANOVA. Both tests gave similar results. P significant.
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RESULTS. W, O5 r* u, W3 I! [+ t5 x3 P

Expression of integrins by cultured HGEC in 5 and 25 mM glucose. Integrins may play a role in altered matrix synthesis and degradationin diabetic conditions. Therefore, we examined and compared theexpression of the main integrin subunits of HGEC grown in 5 and 25 mM glucose.7 D) _9 n" L3 D$ ~& V0 P4 ^

Western blot analysis (Fig. 1 )demonstrated that 3 -, 1 -, and 2 -integrins were decreased by 30-35%, whereas theprotein levels of 5 -, v -, and 3 -integrins were increased by 90%, 60%, and 30%,respectively, in HGEC grown in 25 mM glucose compared with cells grownin 5 mM glucose. These data were also confirmed by flow cytometry (datanot shown). The observed effects were specifically due to D -glucose and not to osmotic effect, because Western blotanalysis of cells grown in 25 mM L -glucose revealed nochange in the expression of integrin subunits compared with cells grownin 5 mM D -glucose. Representative blots of the main 3 - and 1 -integrins are shown in Fig. 1 C.$ n! v+ w% p, V4 l3 P! u* Q. s
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Fig. 1. Western blot analysis of integrin expression in humanglomerular epithelial cells (HGEC) cultured in low and high glucose. A : total protein was extracted from cells cultured in 5 mM(L) or 25 mM (H) D -glucose. Total protein (20 µg) wasanalyzed on 7.5% SDS-PAGE under nonreducing conditions andimmunoblotted with the appropriate dilution of polyclonal antibodiesagainst integrin subunits. Blots were stripped and reprobed withantitubulin antibody to verify protein loads, to which all quantitativedata were normalized. B : quantification of protein contentof each integrin subunit by scanning densitometry. Value of eachcontrol (open bar) is set at 100%. Values are means ± SD of 3 independent experiments. * P t -test and 1-way ANOVA). C : Western blot of 3 - and 1 -integrin expression in HGECcultured in 5 mM D -glucose (L) or 25 mM L -glucose (H).3 P* P* h3 L1 w# B- s

HGEC adhesion to type IV collagen in the presence of 5 and 25 mMglucose. The previous experiment indicated that the presence of increasedglucose concentrations resulted in up- or downregulation of differentintegrin subunits of HGEC. This change could be accompanied by changesin cell adhesion to the GBM and its individual components. Therefore,we examined integrin-mediated adhesion of HGEC to type IV collagen bysolid-phase binding assays. Type IV collagen binding of HGEC grown in25 mM glucose was decreased by ~15-45% compared with cellsgrown in 5 mM glucose, depending on the concentration of type IVcollagen used (Fig. 2 ). The observeddifferences were statistically significant ( P of type IV collagen.1 N( `% A& a% U; k1 u) }+ Z0 j
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Fig. 2. Adhesion of HGEC to solid-phase type IV collagen.[ 35 S]methionine-labeled HGEC cultured in media containing5 or 25 mM D -glucose were seeded in 96-well plates (5,000 cells/well) coated with increasing concentrations of type IV collagenand allowed to adhere for 45 min at 37°C. Nonadherent cells werewashed off, adherent cells were lysed, and radioactivity wasquantitated. Bound counts are expressed as percentage of total countsto give percent adhesion. Nonspecific adhesion of cells to BSA, whichwas P
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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 thepresence of various inhibiting anti-integrin monoclonal antibodies wasexamined. The extent of inhibition of adhesion varied depending on thetype of antibody used against different integrin subunits and glucoseconcentration. More specifically, anti- 1 -integrin antibodies (P5D2) almost completely inhibited the adhesion of HGEC totype IV collagen in 5 and 25 mM glucose (Fig. 3 ). In 5 mM glucose, antibodies Lm609 andP1D6 against the v 3 - and 5 -integrins, respectively, resulted in ~14% and~36% inhibition of maximal adhesion (adhesion in 5 mM glucose in theabsence of antibody), respectively (Fig. 3 A ), 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. 3 B ). In 5 mM glucose, antibody P1E6against the 2 -subunit resulted in 40% inhibition ofmaximal adhesion (Fig. 3 A ), whereas in 25 mM glucose, only~13% inhibition of maximal adhesion was observed (Fig. 3 B ). MAb P1B5 against 3 -integrin caused HGECaggregation and was not effective in blocking adhesion. However, usingthe F(ab) fragment of anti- 3 -integrin MAb (P1B5), wepreviously documented that 3 -integrin participated inthe binding of HGEC to type IV collagen ( 22 ). There was noinhibition of adhesion by MAbs against HLA (negative control).

Fig. 3. Monoclonal antibody (MAb) inhibition of HGEC adhesion totype IV collagen in solid-phase binding assays.[ 35 S]methionine-labeled HGEC cultured in media containing5 mM ( A ) or 25 mM ( B ) D -glucose wereseeded in 96-well plates (5,000 cells/well) coated with 50 µl of typeIV collagen (5 µg/ml) and allowed to adhere for 30 min at 37°C.Anti-integrin MAbs were added to the wells at a final concentration of10 µg/ml before seeding with cells. Adhesion in the absence of MAband in the presence of anti-HLA MAb (W6/32) was used as positive andnegative control, respectively. Data were normalized by expressingbinding in the absence of antibody as maximal (100%), and adhesion inthe presence of antibodies was shown as percentage of binding in theabsence of antibody. Values are means ± SD of 6 replicates andwere analyzed using 1-way ANOVA and Newman-Keuls test.* P* W& Y' I+ P. w7 n6 |
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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 HGECgrown in 5 or 25 mM glucose with gelatin zymography. Enzymatic activitywas detected at two major bands corresponding to 92/88-kDa (MMP-9) and72/68-kDa (MMP-2) collagenases (Fig. 4 A ). Densitometric analysisindicated 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 mMglucose. There were no significant changes in the total amount of the92/88-kDa form of MMP-9 (Fig. 4 B ).* \* }( E% h2 q, A% V5 h: M0 E4 Z! B
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Fig. 4. Gelatinase activity of matrix metalloproteinases (MMP)-2and MMP-9 secreted by HGEC. A : aliquots of serum-freemedia conditioned by cells cultured in 5 mM (L) or 25 mM (H) D -glucose were analyzed on 10% polyacrylamide gelcontaining 0.l% gelatin under nonreducing conditions. Volume ofconditioned medium loaded per lane was adjusted according to the cellnumber obtained on harvest. Collagenolytic activity was detected asclear bands after incubation in enzyme buffer and staining withCoomassie brilliant blue. B : quantification of the 72/68-kDaform of MMP-2 and the 92/88-kDa form of MMP-9 using scanningdensitometry. Value of each control (open bar) is set at 100%. Valuesare means ± SD of 3 independent experiments. * P t -test and 1-way ANOVA)." J! w4 c1 Y7 J! E) s

We then examined and compared the protein levels of MMPs expressed byHGEC grown in 5 and 25 mM glucose. Western blotting of conditionedmedia from HGEC grown in 25 mM glucose demonstrated an ~70% decreasein MMP-2 total protein levels (bands corresponding to 72-kDa proenzymeand 68-kDa active form of MMP-2) compared with media from control cellsgrown in 5 mM glucose (Fig. 5, A and B ). Additionally, the latent(92 kDa) and activated (88 kDa) forms of MMP-9 were detected in mediafrom HGEC, but protein levels were not affected by high-glucosetreatment (Fig. 5, A and B ).
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Fig. 5. Effect of glucose on collagenases and collagenase inhibitorsexpressed by HGEC. A : serum-free media conditioned by HGEC,previously cultured in 5 mM (L) or 25 mM (H) D -glucose,were collected. Aliquots of each sample were concentrated and subjectedto 10% SDS-PAGE under reducing conditions. Volume of conditionedmedium loaded per lane was adjusted according to the cell numberobtained on harvest. Electrophoretically transferred proteins wereimmunoblotted using primary antibodies against MMP-2 and MMP-9( A ) and tissue inhibitors of metalloproteinases (TIMP)-1 andTIMP-2 ( C ). B and D : results fromWestern blots analyzed by scanning densitometry. Value of each control(open bar) is set at 100%. Values are means ± SD of 3 independent experiments. * P t -test and 1-way ANOVA).$ A2 C, W0 m+ |( K. C

Furthermore, expression of TIMPs was determined by Western blotting inconditioned media from HGEC grown in 5 and 25 mM glucose. TIMP-2, asingle 22-kDa immunoreactive band, was increased by 2.5-fold inhigh-glucose media compared with control media. In contrast, the 28-kDaTIMP-1 immunoreactive band underwent a modest decrease (~30%)compared with the control (Fig. 5, C and D ).$ s1 v, j- C, K4 Z6 w& p
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Effect of ligation of 3 1 - and v 3 -integrins on MMP-2 expression in 5 and25 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-glucoseconditions. HGEC grown in 5 or 25 mM glucose were cultured in theabsence 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 thesame experiment.3 {5 m4 n7 g! N5 n& F
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The presence of MMP-2 in conditioned media was examined by gelatinzymography. In HGEC grown in 5 mM glucose, ligation of 3 1 -integrin with anti- 3 -and anti- 1 -integrin MAbs simultaneously resulted in a~2.5-fold increase in the amount of secreted MMP-2 compared withuntreated cells (Fig. 6, A and B ), whereas ligation ofintegrin subunits with anti- 3 - oranti- 1 -integrin MAb caused a modest increase(40-45%) in MMP-2 secretion compared with control untreated cells(Fig. 6, C and D ). This observation suggested acooperative effect of the two subunits. In comparison, ligation of v 3 -integrin, another abundant integrindimer of HGEC, with specificanti- v 3 -integrin MAbs, had no effect onthe amount of secreted MMP-2 (Fig. 6, A and B ).Moreover, ligation of 5 - or 2 -integrinwith specific anti-integrin MAbs had no effect on the secretion ofMMP-2 (data not shown).3 U, F: m0 v$ N$ K& g5 J, {

Fig. 6. Effects of anti-integrin MAbs on MMP-2 expression andsecretion in HGEC. Cells were cultured in 5 or 25 mM D -glucose medium with or without 5 µg/ml of eachanti-integrin MAb for 24 h at 37°C. Medium was then replacedwith serum-free medium with or without 5 µg/ml of each MAb, and cellswere cultured for 24 h. A and C : aliquots ofeach sample of conditioned media were analyzed by gelatin zymography(volume of conditioned medium loaded per lane was adjusted according tocell number obtained on harvest). B and D :results from gelatin zymography were analyzed by scanning densitometry.Values are means ± SD of 3 independent experiments.* P t -test and 1-wayANOVA)., U4 E  J  Y  O( u
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When ligation of 3 1 -integrin wasperformed by specific antibodies in HGEC grown in 25 mM glucose, theamount of MMP-2 released in the media was substantially less than incells grown in 5 mM glucose in the presence of the same antibodies(Fig. 6, A and B ). In 25 mM glucose, the amountof MMP-2 in HGEC media in the presence of anti- 3 - andanti- 1 -integrin antibodies was similar to the amountsecreted by control cells, which were grown in 5 mM glucose in theabsence of antibodies. Again, treatment of HGEC grown in 25 mM glucosewith anti- 3 - or anti- 1 -integrin antibody alone caused a modest increase (30-35%) in MMP-2 levels compared with cells grown in 25 mM glucose in the absence of antibodies (Fig. 6, C and D ).

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 thatmediates regulation of the expression of MMPs by integrins. Therefore,we examined tyrosine phosphorylation of pp125 FAK in control(untreated) HGEC and HGEC treated with MAbs against 3 -and 1 -integrins simultaneously (Fig. 7 ). In cells treated withanti- 3 - and anti- 1 -integrin MAbs, therewas a ~30% increase in phosphotyrosine of pp125 FAK compared with control untreated cells. To ensure equal amounts ofprotein loading, blots were stripped and reprobed with polyclonal anti-FAK antibodies.
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Fig. 7. Effect of 3 1 -integrinligation on focal adhesion kinase (FAK) tyrosine phosphorylation inHGEC. Cells cultured in 5 mM D -glucose were incubated insuspension at 37°C for 90 min with or without 10 µg/ml of eachanti- 3 -integrin (P1B5) andanti- 1 -integrin (P5D2) MAbs, lysed, andimmunoprecipitated with an anti-FAK antibody. A :immunoprecipitates analyzed for tyrosine phosphorylation byantiphosphotyrosine (anti-pTyr) immunoblotting. B :immunoblot stripped and reprobed with anti-FAK antibody to determinewhether equal amounts of FAK were loaded per lane. C :results of densitometric analysis on protein and phosphorylationsignals. Phosphorylation signal in untreated cells has been expressedas 100%. Relative percentage of FAK phosphorylation in cells treatedwith MAbs was calculated using the following equation: (phosphorylationsignal in treated cells/phosphorylation signal in untreated cells) × (protein signal in untreated cells/protein signal in treatedcells) × 100. Values are means ± SD from 3 experiments.* P t -test and 1-wayANOVA).! G4 Q; I. M) ?

DISCUSSION
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We have provided evidence that increased glucose concentrationsmodulate integrin expression and integrin-related functions of culturedT-SV40-immortalized HGEC, which were shown to be similar to theirprimary counterparts ( 8, 22 ). The observed effects werespecifically due to D -glucose and not to osmotic effect. Weand others previously showed that L -glucose, ribitol, ormannitol had no effect ( 2, 30 ). We previously reportedthat 3 1 is the main integrin present athigh density on the surface of ~97% of HGEC grown in the presence of5 mM glucose ( 22 ). This dimer can mediate the binding ofHGEC 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 asignificant decrease in 3 -, 2 -, and 1 -protein levels with a concomitant increase of 5 -, v -, and 3 -subunitscompared with control cells, which were grown in 5 mM glucose. Severaldifferent cell types were also documented to respond to increasedglucose 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 ), humandiabetic kidneys ( 4, 16 ), and human glomerular diseases( 12, 18 ). In sections from renal tissues, podocytes fromshort- and long-term diabetic rats had decreased expression of 3 1 -integrin when examined withimmunoelectron microscopy; this decrease was thought to represent anearly event that precedes the onset of DN ( 31, 32 ).Therefore, the reported findings in podocytes in diabetic conditions insitu corroborate our observations related to the effects of highglucose in cultured HGEC, insofar as the expression of 3 1 -integrin was concerned. The mechanismsby which hyperglycemia alter the expression of integrins are notclearly understood. In kidneys, transforming growth factor- 1(TGF- 1) has been reported to be a strong regulator of the expressionof integrins ( 38 ). TGF- 1 has been demonstrated tosuppress the expression of 3 -integrin in glomeruli fromnephrotic rats ( 18 ), and an overexpression of TGF- 1 hasalso been reported to occur in diabetic rat kidneys as early as2-3 days after induction of hyperglycemia ( 37 ).Therefore, one mechanism of altered integrin expression could involveoverexpression of TGF- 1.

Furthermore, our data provide evidence that glucose-induced changes inintegrin expression were accompanied by altered binding to GBMcomponents, such as type IV collagen. Type IV collagen was selected forthe study of interactions with HGEC, because it is the predominantglycoprotein of the GBM ( 8, 22 ). We observed that highglucose decreased the number of HGEC bound to solid-phase type IVcollagen. This binding was mediated in part by different integrinsdepending on glucose concentration. For example, there was substantialinhibition of HGEC binding to type IV collagen in the presence ofanti- 2 - and anti- 5 -integrin MAbs in 5 mMglucose (~40% and 35%, respectively), whereas there was nosignificant 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 forcell binding to fibronectin ( 15, 33 ). However, primary andimmortalized HGEC express higher levels of 5 -integrinthan 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 wassignificant in the presence of anti- v 3 -and anti- 5 -integrin MAbs (60% and 50%, respectively),whereas antibodies against 2 -integrin did not blockadhesion, indicating that there was a partial glucose-dependent switchof integrin subunits that bind HGEC to type IV collagen. Anti- 1 -integrin MAb resulted in nearly completeinhibition of adhesion to type IV collagen in either glucoseconcentration, as expected. The data indicate that 1 isa predominant integrin; indeed, by fluorescein-activated cell sorteranalysis, it is present at a high density on the surface of 84% ofprimary HGEC and 97% of immortalized HGEC ( 22 ). Thissubunit associates with different -subunits, thus serving formultiple binding events to matrix components.

In summary, different integrins in part mediate HGEC binding to type IVcollagen, depending on glucose concentration. In low glucose, 2 - and 5 -integrins participated in thebinding, whereas, in high glucose, the v 3 - and 5 -subunits werepreferentially used. If similar changes occur in situ, in diabeticconditions, then altered interactions with matrix components could beanticipated to alter integrin-mediated signaling and various aspects ofintegrin-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 shownto be regulated by integrins in different cell types ( 1, 9, 13, 24, 25, 34, 35, 45, 47 ). Our experiments indicate that changesin integrin expression in HGEC grown in 25 mM glucose were accompaniedby changes in the expression and/or activity of MMP-2. In vivo, 1 -integrin associates with different -subunitsserving for multiple binding and specific signaling events. Because 3 1 -integrin is the main subunit expressedby HGEC ( 22 ) and, moreover, there was decreased expressionof 3 1 -integrin and MMP-2 in 25 mM glucosecompared with the control (5 mM glucose), we examined the hypothesisthat 3 1 -integrin was involved in regulation of MMP-2 expression. In HGEC grown in 5 mM glucose, ligationof the 3 1 -integrin dimer withanti- 3 - and anti- 1 -integrin MAbssimultaneously resulted in a 2.5-fold increase in secreted MMP-2compared 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 a40-45% 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 thesecretion of MMP-2 (data not shown). Upregulation of MMP-2 by ligationof 3 1 -integrin was also observed in HGECcultured in 25 mM glucose, albeit to a lesser extent. Glucose-induceddecrease of the expression of 3 1 -integrincould possibly account for this difference.

Our findings suggest then that 3 1 -integrin in part regulates theexpression of MMP-2 in HGEC. Several other reports showed that ligationof 3 1 -integrin with MAbs resulted inupregulation of MMP-2 production by tumor cells ( 5, 41, 43 ) or induced the activated form of MMP-2 and enhancedpro-MMP-2 secretion by rhabdomyosarcoma cells ( 23 ).
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The mechanism by which 3 1 -integrinenhances the expression of MMP-2 remains to be substantiated. It hasbeen previously documented that a major signaling pathway linkingintegrins 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 FAKphosphorylation in HGEC as a response to 3 1 -integrin ligation, a finding thatindicates the possible involvement of the above-mentioned pathway inregulation of the expression of MMP-2 in this cell type. Thishypothesis is also in accord with a recent report showing that integrininteractions with their ligand can transduce stimulatory signals(through FAK-Src-type kinases) for MMP-2 and MMP-9 expression in humanT lymphocytes ( 10 ). However, there might be alternative oradditional regulatory mechanisms. Whatever the mechanism, the observeddecreased expression of MMP-2 could result in impaired degradation ofbasement membrane components.
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We additionally observed a substantial increase in the expression ofTIMP-2, the specific inhibitor of MMP-2, a finding that suggestsfurther impairment of matrix degradation, leading to matrixaccumulation. The modest decrease in TIMP-1, the specific inhibitor ofMMP-9, could be compensatory.# M* u  I' d, X' u; W& F

In situ, even a small decrease in MMP-2 and/or increase in TIMP-2protein levels could be anticipated to impair the balance between ECMsynthesis and degradation, resulting in matrix accumulation. We andothers documented that increased glucose concentrations resulted inaltered expression of matrix, MMPs, and TIMPs in cultured mesangialcells ( 3, 26 ) and also in situ in kidneys ofstreptozotocin-diabetic rats at early stages of diabetes in the absenceof albuminuria ( 46 ).
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Collectively, our data indicate that glucose-induced modulation ofintegrin expression was accompanied by functional changes in HGEC invitro, which in turn could contribute to altered interactions with typeIV collagen, a major component of the GBM and, in addition, decreaseddegradation of matrix proteins. The combined long-term effect could bemicroalbuminuria due, at least in part, to altered HGEC binding to theGBM components and matrix accumulation, which, if present in situ,could help explain the thickening of the GBM in DN.

ACKNOWLEDGEMENTS
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The authors are indebted to C. Economou and P. Karamessinis forexpert assistance with image processing and analysis.) H5 R" C1 S% i. T' e4 O
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