Overexpression of α1β1 integrin directly affects rat mesangial cell behavior

Kidney International, Vol. 58 (2000), pp. 1088–1097

Overexpression of a1b1 integrin directly affects rat mesangialcell behavior

SHOJI KAGAMI, SHUZI KONDO, MAKI URUSHIHARA, KLEMENS LOSTER, WERNER REUTTER,TAKAHIKO SAIJO, AKIKO KITAMURA, SHOKO KOBAYASHI, and YASUHIRO KURODA

Department of Pediatrics, School of Medicine, University of Tokushima, Tokushima, Japan, and Institute fur Molekularbiologieund Biochemi, Freie Universitat Berlin, Berlin-Dahlem, Germany

Overexpression of a1b1 integrin directly affects rat mesangial Glomerular mesangium, which is composed of mesan-cell behavior. gial cells (MCs) and mesangial extracellular matrix (ECM),

Background. Glomerular mesangial cell (MC) proliferation, is the target of injuries with immunologic causes (im-hypertrophy, and abnormal matrix remodeling characterizedmune complex, T cell, or antineutrophil cytoplasmic anti-by increased expression of fibronectin, laminin and collagenbody-mediated) and nonimmunological causes (diabetes,type IV, and neoexpression of collagen I and III are the main

biological features of progressive glomerulonephritis (GN). Es- hyperlipidemia, amyloidosis, and systemic hypertension).pecially, persistent pathological matrix remodeling may lead to It also displays prominent morphological changes suchglomerular scar formation (glomerular scarring). We reported as mesangial expansion and sclerosis [1, 2]. Under theserecently that a1b1 integrin, a major collagen receptor for MCs,

pathological settings, there are sequential and/or simul-may be a potential adhesion molecule for MC-mediated patho-taneous changes in MC phenotypes, including initial MClogical collagen matrix remodeling in GN.

Methods. To address further the direct role of a1b1 integrin hypertrophy or proliferation, transformation into a myo-in MC behavior, such as cell growth and matrix remodeling, fibroblast-like cell [a-smooth muscle actin positivea1b1 integrin was overexpressed in MCs by transfecting an

(a-SMA1)] and finally MC-mediated pathological ECMexpression vector containing a full-length rat a1 integrin cDNA.remodeling (sclerosis) [1–4]. Thus, the investigation ofFlow cytometry and immunoprecipitation analysis were ap-

plied for selection of transfectants with a stable expression of the molecular and cellular mechanisms responsible forthe a1 integrin subunit. The effect of a1b1 integrin overexpres- these MC phenotypes may help us to understand thesion on MC biology was examined with a 3H-thymidine incor- pathogenesis of progressive glomerular sclerosis.poration assay, flow cytometric analysis of cell size and DNA

b1-Integrins, cell surface heterodimeric receptors, con-content, Western blot analysis of a cyclin-dependent-kinasesisting of the noncovalently-associated common b1 sub-inhibitor, p27Kip1, a-smooth muscle actin expression, and a colla-

gen gel contraction assay. unit and several types of a subunits, mainly mediateResults. The a1 transfectants displayed a dramatic inhibition the interaction between cells and extracellular matrix

of 3H-thymidine incorporation as compared with the mock (ECM), and play a critical role in many aspects of celltransfectants. Increased expression of the a1 subunit inverselyphenotype such as growth, ECM protein synthesis, ECMcorrelated with cell cycle progression and paralleled the expres-assembly, and differentiation, thereby contributing tosion of p27Kip1 and a-smooth muscle actin, as well as the cell size

in MCs. In addition, the a1-transfectants were able to enhance embryonic development, tissue homeostasis, and woundcollagen matrix reorganization effectively. healing [5–7]. Of the b1-integrins, the a1b1 integrin, a

Conclusion. These results indicate that MC-a1b1 integrin ex- collagen/laminin receptor, has recently been found to bepression is a critical determinant of MC phenotypes, includinga marker of smooth muscle phenotype [8–10]. It has alsocell growth, cell size, and collagen matrix remodeling ability,

and thereby contributes to scar matrix remodeling (sclerosis) been shown to be involved in collagen matrix reorganiza-in GN. tion [10–14]. In addition, this integrin has been reported

to be expressed on myofibroblast-like cells seen in woundhealing and fibrosis of several organs [8, 10–13]. We haverecently shown that increased expression of a1b1 integrinin phenotypically changed MCs (a-SMA1) may be in-Key words: cell growth, hypertrophy, collagen matrix, glomerulosclero-

sis, progressive renal disease, scarring. volved in abnormal ECM remodeling in rat and humanglomerulonephritis (GN) [12, 13]. Additionally, by colla-Received for publication August 25, 1999gen gel contraction assay, we demonstrated an in vitroand in revised form February 22, 2000

Accepted for publication March 23, 2000 model of collagenous tissue remodeling that the tworepresentative fibrogenic growth factors in GN, trans- 2000 by the International Society of Nephrology

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Kagami et al: Overexpression of a1b1 integrin in rat MCs 1089

forming growth factor-b (TGF-b) and platelet-derived CA, USA), normal rabbit IgG (R&D Systems, Minneap-olis, MN, USA), and normal hamster IgG (Southerngrowth factor-BB (PDGF-BB) stimulate collagen matrix

reorganization by cultured MCs via different mecha- Biotechnology Associates, Inc., Birmingham, AL, USA)were used as control antibodies for experiments.nisms [14]. The ability of TGF-b to stimulate gel contrac-

tion is dependent on increased a1b1 integrin expression,Cell culturewhereas PDGF-BB enhancement of gel contraction has

been revealed to be dependent on increased a1b1 integ- Rat MCs from isolated glomeruli of four- to five-week-old Sprague-Dawley rats were cultured as previouslyrin-mediated MC migratory activity [14]. These observa-

tions suggest the putative role of MC-a1b1 integrin in the described [15].An MC clone (A9C) was obtained by limiting dilutioncollagen matrix and scar mesangial ECM remodeling

following glomerular injury. However, the relationship from the cultured MCs, and it was maintained in Dulbec-co’s modified Eagle’s medium (DMEM; Sigma Chemicalbetween a1b1 integrin expression and MC phenotype

such as growth, ECM remodeling, and a-SMA expres- Co.) supplemented with 100 U/mL penicillin, 100 mg/mLstreptomycin, 0.1 U/mL insulin, 25 mmol/L HEPESsion in GN, still remains unclear.

To examine the contribution of a1b1 integrin expres- buffer, containing 10% fetal bovine serum (FBS; GIBCOBRL, Orland Island, NY, USA; growth medium) at 378Csion to the MC phenotype in GN more directly, we

overexpressed a collagen/laminin receptor, a1b1 integrin, in a 5% CO2 incubator. This MC clone was used for thetransfection study. An African Green monkey kidneyon MCs using an expression vector containing a1 integrin

cDNA. We found that overexpression of a1b1 integrin is cell line, COS-7 cells (American Type Culture Collec-tion, Rockville, MD, USA), was grown in DMEM sup-associated with growth inhibition, hypertrophy, a-SMA

expression, and the enhanced ability of MCs to perform plemented with 10% FBS.collagen matrix remodeling.

pHbAPr-1-a1 construct formation, transfection, andestablishment of stable transfectants

METHODSThe full-length cDNA for the rat a1-integrin subunit

Extracellular matrix proteins and antibodies [19] was ligated into the eukaryotic expression vector,pHbAPr-1-neo [20], in a sense orientation (pHbAPr-Fibronectin was purified from human plasma by affin-

ity chromatography on gelatin-Sepharose 4B (Pharmacia 1-a1). This allowed for the expression of the a1 subunitunder the control of the b-actin promoter. An MC clone,LKB Biotechnology Inc., Uppsala, Sweden) [15]. Rat

tail collagen type I (collagen I) was obtained from Col- A9C, was transfected with either pHbAPr-1-a1 or thevector alone (pHbAPr-1-neo) using calcium phosphatelaborative Biomedical Products (Bedford, MA, USA).

The purity of these matrix proteins was verified by so- transfection methods. Stable transfectants were selected inthe presence of a neomycin analogue G418 (0.56 mg/mL;dium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE). Monoclonal mouse antirat a1 subunit an- Sigma Chemical Co.), and expression of the a1-subunittranscript and the corresponding polypeptide was con-tibody (mAb, 33.4) and polyclonal rabbit antirat b1 sub-

unit IgG (pAb anti-b1) were produced and characterized firmed by flow cytometry analysis and immunoprecipita-tion analysis, respectively. To confirm whether the MCas previously described [16]. Monoclonal hamster anti-

rat a2 subunit antibody (Ha1/29) [17] and monoclonal clone (A9C), a1- and mock transfectants retain in vivoMC characteristics such as being desmin and Thy-1 anti-mouse anti-mouse a5 subunit antibody (HMa5-1) [18]

were obtained from Pharmingen (San Diego, CA, USA) gen positive [21], they were grown on multichamber glassslides and fixed at 2208C in acetone. Cells were incu-and Showa Electric (Tokyo, Japan), respectively. Rabbit

antidesmin antiserum and mouse monoclonal anti–Thy-1 bated with either an antidesmin antibody or anti-mouseThy-1 antigen antibody followed by incubation with anantibody were obtained from Sigma Chemical Co. (St.

Louis, MO, USA) and Pharmingen, respectively. Fluo- appropriate FITC-conjugated second antibody (donkeyanti-rabbit IgG antibody or donkey anti-mouse IgG anti-rescein isothiocyanate (FITC)-conjugated donkey poly-

clonal antibody against either mouse, hamster, or rabbit body). All experiments using transfectants were per-formed after a two-day incubation in serum-free DMEMIgG was purchased from Jackson ImmunoResearch Lab-

oratories Inc. (West Grove, PA, USA). Mouse mono- to arrest cell growth as previously described [14].clonal anti–a-SMA antibody (1 A4; Sigma Chemical

Flow cytometry analysisCo.), horseradish peroxidase (HRP)-conjugated goatanti-mouse IgG (Bio-Rad Laboratories, Hercules, CA, Flow cytometry analysis was performed to examine

the cell surface expression of the rat a1 integrin subunitUSA) and HRP-conjugated monoclonal mouse anti-p27Kip1 antibody (Transduction Laboratories, Lexington, in transfectants [22]. Growth-arrested transfectants were

incubated with growth medium for two days and wereMA, USA) were used for Western blot analysis. Mousemyeloma IgG1 (Zymed Laboratories Inc., San Francisco, then detached from culture plates (Nunc Inc., Naperville,

Kagami et al: Overexpression of a1b1 integrin in rat MCs1090

IL, USA) by treatment with 0.05% trypsin and 0.02% the a1 integrin subunit for 16 hours at 48C. Immunecomplexes were recovered by binding to protein A-Seph-ethylenediaminetetraacetic acid (EDTA). The cells were

then incubated with the anti-rat a1 integrin subunit mAb arose. Beads were washed with a lysis buffer, and immu-noprecipitates were analyzed by 4 to 12% SDS-PAGE33.4 (IgG1) in phosphate-buffered saline (PBS) containing

0.2% FBS and 0.1% sodium azide (wash buffer) for 30 under nonreducing conditions as described previously[15]. Integrin bands were quantitated by densitometericminutes at 48C. After washing, the cells were incubated

with FITC-conjugated donkey anti-mouse IgG for 30 scanning using an LKB ultroScan XL apparatus (Phar-macia LKB Biotechnology Inc.).minutes at 48C and were analyzed for fluorescence on a

FACStar PLUS Flow Cytometer (Becton Dickinson, SanGrowth assayJose, CA, USA). For control samples, the primary anti-

Growth-arrested transfectants were diluted to 1 3 105bodies were replaced with a mouse myeloma IgG1. Ex-cells/mL in growth medium, and 0.3 mL/well was seededpression of the integrin subunits, a2, a5, and b1, was simi-into 24-well culture plates (Costar, Cambridge, MA,larly analyzed using the antibodies Ha1/29, HMa5-1, andUSA). Some wells were coated with either collagen I orpAb anti-b1, respectively. For flow cytometric analysisfibronectin (20 mg/mL, respectively) at 378C followed byof DNA content, 106 cells were stained with a 100 mg/mLblocking in 3% bovine serum albumin (BSA) in PBS.solution of propidium iodide (Sigma Chemical Co.) inThe cells were pulsed with 0.5 mCi/well 3H-thymidine0.1% Triton X-100–PBS containing 50 mg/mL RNase A,(Dupon NEN Research Product, Boston, MA, USA)and the percentages of the cells in the G0/G1, S, andduring the last 12 hours of a 48-hour culture. Cells wereG2/M phases were determined by flow cytometry [23, 24].washed once with PBS, twice with ice-cold 5% trichloro-The relative cell size for 104 cells in each sample was de-acetic acid to remove the unincorporated 3H-thymidine,termined by quantitation of forward light scattering [24].solubilized in 0.25 N NaOH in 0.1% SDS, and neutral-

Western blot analysis ized. Aliquots of samples were added to scintillation fluidand were counted (Beckman Instruments, Inc., Fuller-Growth-arrested transfectants were incubated in

growth medium for two days. Total proteins were then ton, CA, USA).extracted from these transfectants with a lysis buffer [100

Cell adhesion assaymmol/L Tris-HCl, pH 7.4, containing 150 mmol/L NaCl,Adhesion studies of transfectants were performed as1% Triton X 100, 0.5% sodium deoxycholate, 0.1% SDS,

previously described [15]. Briefly, 96-well flat bottom1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1plates (Becton Dicknson Labware, Lincoln Park, NJ,mmol/L EDTA, 1 mmol/L NaVO3, and 10 mg/mL leu-USA) were coated overnight at 48C with either collagenpeptin]. Samples (10 mg of total protein) were resolvedI or fibronectin (10 mg/mL, respectively) and were thenby SDS-PAGE, transferred to a polyvinylpyrrolidoneblocked with 10 mg/mL of BSA for one hour at 378C.membrane (Micron Separations Inc., Westboro, MA,Growth-arrested transfectants were incubated in growthUSA), and probed either with the HRP-conjugated mousemedium for two days and were then detached, washed,anti-p27Kip1 monoclonal antibody or with the anti–a-and pelleted. Cells were suspended in RPMI 1640 con-SMA monoclonal antibody, followed by the HRP-conju-taining 1 mmol/L MgCl2 and 1 mmol/L CaCl2 plated ontogated rabbit anti-mouse IgG. Bound protein was detectedthe ECM-coated wells at 104 cells/well and incubated atby enhanced chemiluminescence (Amersham Corp., Ar-378C for 50 minutes. Cell adhesion was evaluated bylington Heights, IL, USA).staining adherent cells with 0.1% crystal violet, solubiliz-

35S-methionine labeling of transfectants ing cells with 0.2% Triton X-100 in PBS and reading theand immunoprecipitation absorbance at 630 nm with a Corona Microplate Reader

Growth-arrested transfectants were incubated in growth (Corona Electric Co., Ibaragi, Japan). This procedure ofmedium for two days. Then they were metabolically la- quantitation was demonstrated to give values propor-beled with 35S-methionine for 18 hours in a methionine- tional to the number of adherent cells [15]. When thefree medium. After metabolic labeling, the cells were effect of the anti-a1 subunit mAb 33.4 or anti-b1 subunitlyzed in a 25 mmol/L Tris-HCl, pH 7.4, containing 100 antibody (pAb anti-b1) on cell adhesion was assessed,mmol/L n-octyl-b-D-glycopyranoside (Sigma Chemical antibodies were incubated with the transfectants for 30Co.), 150 mmol/L NaCl, 1 mmol/L CaCl2, and 1 mmol/L minutes at 208C prior to adding them to ECM protein-MgCl2 (lysis buffer). Radiolabeled protein in the cell ly- containing wells.sates was counted, and a standard amount of radioactivity

Collagen gel contraction assaywas used in the immunoprecipitation analysis. The lysatesupernatants (5 3 106 cpm of 35S-labeled proteins/mL), Collagen gel assays were performed to examine the

ability of the transfectants to reorganize and contractprecleared by incubation with normal mouse serum andprotein A-Sepharose (Pharmacia LKB Biotechnology three-dimensional collagen I gel [14]. Growth-arrested

transfectants were incubated in growth medium for twoInc.), were immunoprecipitated with mAb 33.4 against


days and were then harvested and suspended at a cellconcentration of 3 3 105 cells/mL in 0.5 mL of 1.25 3RPMI 1640. The cell suspension was mixed with 0.5 mLof collagen I solution [59% 1.25 3 RPMI 1640, 40% rattail collagen I (3.75 mg/mL), 1% 0.2 N NaOH]. Collagen/cell suspensions (500 mL each) were incubated in 24-well plates (Costar) at 378C to polymerize the collagen.At selected time points, the diameter of the hydratedgels was measured with an inverted microscope.

Statistical analysisData were collected from three independent experi-

ments of duplicate or triplicate wells. Data are presentedas mean 6 SD. A value of P , 0.05 was consideredsignificant (Student’s t-test).

RESULTSGeneration and characterization of a1-transfectants inthe rat MC clone, A9C

Fig. 1. Ectopic expression of the rat a1-subunit in COS-7 cells. COS-7At first, to confirm the ability of the expression vector,cells were transfected with an expression vector, pHbAPr-1-a1, or con-

pHbAPr-1-a1, to express rat a1 subunit protein on the trol vector, pHbAPr-1-neo, and were analyzed for cell surface expres-sion of the rat a1-subunit by flow cytometry. The ectopic rat a1-subunitcell surface, the monkey kidney cell line, COS-7 cells,was detected with anti-rat a1-subunit–specific mAb 33.4, which doeswere transfected with the pHbAPr-1-a1 or a controlnot react with the endogenous monkey a1-subunit. The pHbAPr-1-a1-vector, pHbAPr-1-neo, using calcium phosphate trans- transfectants (a1-transfectants; thick line) yielded a bimodal population

fection methods. The transfectants were selected by incu- of high and middle expressors as compared with the pHbAPr-1-neo-transfectants (mock transfectants; thin line).bation with G418 for three weeks, and then cells were

analyzed by flow cytometry using rat a1 subunit-specificmAb 33.4. As shown in Figure 1, the bulk sorts of thepHbAPr-1-a1-transfectants (a1-transfectants) success-

functionally active state of the a1b1 heterodimer. Asfully expressed the rat a1-integrin subunit on the COS-7shown in Figure 4A, the a1 transfectants stronglycell surface at higher levels compared with the control,attached to collagen I (a twofold increase) comparedpHbAPr-1-neo-transfectants (mock transfectants), indi-with the mock transfectants (P , 0.01), while cell attach-cating that this expression vector could induce the ratment of a1 transfectants or mock transfectants to fibro-a1 subunit protein on the monkey COS-7 cell surface.nectin was at the same level. Specificity of adhesion wasThe same procedure was applied to a cultured rat MCdemonstrated by application of the anti-a1 subunit mAbclone, A9C. Three stable a1-transfectants (1pHbAPr-33.4 (100 mg/mL), which almost entirely inhibited the1-a1; 2A1, 2A2, 2A5) or three mock transfectantsadhesion of a1 or mock transfectants to collagen I (90%(1pHbAPr-1-neo; 1B, 2B, 3B) were selected for a1-sub-decrease). On the other hand, this mAb had no effectunit protein expression by flow cytometry and immuno-on the adhesion of either transfectant to fibronectin.precipitation analyses, respectively (Figs. 2 and 3). ThePAb anti-b1 (200 mg/mL) equally inhibited the adhesioneffect of the expression vector, pHbAPr-1-a1, was spe-of the a1 and mock transfectants to either fibronectin orcific for the a1 subunit, since there were no changes incollagen I, indicating that each transfectant used the b1expression of the other a integrin subunits, a2 or a5 (Fig. 2).subunit for cell adhesion to both fibronectin and collagenSince the amount of a subunit is the limiting factor inI. Thus, the a1b1 heterodimer in a1 transfectants functionsintegrin heterodimer assembly [25], the expressed a1 sub-to increase the ability of ligand-specific cell adhesion.unit induced by pHbAPr-1-a1 cDNA synthesizes sub-Either transfectant was able to spread on collagen I andstantial amounts of a1 subunit, resulting in increasedfibronectin. Moreover, consistent with the increased ex-expression of the a1b1 integrin heterodimer on the cellpression of a1b1 integrin, the a1 transfectants were ablesurface as compared with the mock transfectants (Figs.to enhance collagen gel contraction effectively compared2 and 3). Immunofluorescence study showed that thewith that of mock transfectants (P , 0.01; Fig. 4B). BothMC clone (A9C), a1, and mock transfectants retainedthe increased adhesion to collagen I and the efficientin vivo characteristics such as being desmin and Thy-1reorganization of the collagen matrix by a1 transfectantsantigen positive.provided evidence that the newly expressed a1b1 integrinAttachment of a1 transfectants or mock transfectants

to fibronectin and collagen I was assayed to prove the was a functionally active heterodimer.


Fig. 2. Flow cytometry analysis of thetransfectants (2A1, 1B). The transfectantswere stained with either an anti-a1 subunitmonoclonal antibody (mAb), 33.4 (a1), anti-a2

subunit antibody, Ha1/29 (a2), anti-a5 subunitantibody, HMa5-1 (a5), or anti-b1 subunit anti-body (pAb anti-b1), and were followed byincubation with an appropriate FITC-labeledsecondary antibody. The cells were analyzedfor fluorescence on flow cytometer. Back-ground staining of an a1-transfectant, 2A1,with a secondary antibody only is indicated inthe left-hand peak. Symbols are: (thick line)2A1; (thin line) 1B; (dotted line) background.

Fig. 3. Immunoprecipitation analysis of a1b1

integrin in mock transfectants (1B, 2B, 3B) anda1-transfectants (2A1, 2A2, 2A5). The transfec-tants were radiolabeled with 35S-methioninefor 18 hours. The cell lysates of a standardamount of radioactivity were immunoprecipi-tated with mAb 33.4, and immunoprecipitateswere resolved by SDS-PAGE under nonre-ducing conditions.

a1 transfectants display reduced cell mitogenicity and Recent in vivo and in vitro studies indicated that cellincreased expression of p27Kip1 proliferation is critically controlled by the level of the

cyclin kinase inhibitor (CKI), p27Kip1, in MCs [26–29]. InThe a1 transfectants grew noticeably more slowly thanaddition, an increase in p27Kip1 expression has been associ-the mock transfectants, as measured by 3H-thymidineated with cell cycle arrest in various circumstances, anduptake (P , 0.01; Fig. 5A). Propidium iodide stainingoverexpression of p27Kip1 leads to G1-phase arrest in allrevealed that the number of cells in the G1 phase wascell lines that have been tested so far [26–29]. Therefore,significantly greater in these transfectants than in the mockwe hypothesized that a1b1 integrin overexpression couldtransfectants (P , 0.01). The clone, 2A1, for example,increase the level of p27Kip1 expression, resulting in partialexhibited almost twice the percentage of total cells in

G1, compared with a mock transfectant, 1B (Fig. 5B). G1 arrest in MCs. The protein expression of p27Kip1 in


Fig. 4. Adhesion to extracellular matrix (ECM) molecules and collagen gel contraction by mock transfectants [(j) 1B, ( ) 2B, ( ) 3B] anda1-transfectants [( ) 2A1, (h) 2A2, ( ) 2A5]. (A) Adhesion of the transfectants to either fibronectin (FN) or collagen type I (COL I) in thepresence of rabbit IgG (200 mg/mL; 1); pAb anti-b1 (200 mg/mL; 2), mouse IgG (100 mg/mL; 3) or mAb 33.4 (100 mg/mL; 4). The transfectantswere allowed to adhere to the wells of 96-well flat bottom plates, which had been coated with FN (10 mg/mL) or COL I (10 mg/mL). Each barrepresents the mean 6 SD of triplicate wells in three separate experiments. (B) Collagen gel contraction by the transfectants. Cells were addedto collagen I solution and poured into 24-well plates. Contraction was allowed to proceed for 24 hours. A representative experiment is shown.

either a1 or mock transfectants was assessed by Western ined whether attachment of a1-transfectants to a specificECM component, either collagen I or fibronectin, is es-blotting. An increase in p27Kip1 expression was seen in

a1 transfectants, as compared with mock transfectants, sential to induce increased p27Kip1 expression. Cell attach-ment to either plastic, collagen I, or fibronectin did notsuggesting that a1b1 integrin expression is linked to a

signaling pathway involved in cell cycle regulation in alter the difference in p27Kip1 expression between the a1

transfectants and mock transfectants (Fig. 6B). Similarly,MCs (Fig. 6A). Furthermore, to investigate the mecha-nism leading to increased p27Kip1 expression, we exam- attachment of the transfectants to either collagen I or


Fig. 5. Analysis of growth rates and DNAcontent in mock transfectants (1B, 2B, 3B)and a1-transfectants (2A1, 2A2, 2A5). (A)Cell mitogenicity of the transfectants wasmeasured by 3H-thymidine uptake in the last12 hours of a two-day culture. Each bar repre-sents the mean 6 SD of triplicate wells in threeseparate experiments. (B) The transfectants(1B, 2A1) cultured for two days were stainedwith propidium iodide and were analyzed byFACS to assess DNA content. The G0/G1, S,and G2/M peaks are noted. For the profile in1B, the percentage of cells in G0/G1, S, andG2/M is 31.3%, 44.4% and 24.3%, respec-tively. For the profile in 2A1, the percentageof cells in G0/G1, S, and G2/M is 62.5%, 27.1%,and 10.4%, respectively. A representative ex-periment is shown.

Fig. 6. Protein expression of p27Kip1 in the mocktransfectants (1B, 2B, 3B) and a1-transfectants(2A1, 2A2, 2A5). (A) The transfectants wereplated on tissue culture plastic for two days.Total proteins were extracted from the cul-tures. The samples were adjusted to a standardcontent (10 mg) and analyzed by Western blot-ting using the anti-p27Kip1 monoclonal anti-body. (B) The transfectants (1B, 2A1) wereplated on either culture plastic (PL), fibronec-tin (FN; 20 mg/mL), or collagen type I (COLI; 20 mg/mL)-coated culture plastic for twodays. Total proteins were extracted from thecultures and analyzed by Western blotting asdescribed in this article. A representative ex-periment is shown.

fibronectin did not significantly affect the differences in liferation/enlargement, and a-SMA expression in humanand rat experimental GN [3, 12, 13, 30]. Furthermore, aDNA content or 3H-thymidine uptake between a1-trans-

fectants and mock transfectants (data not shown). recent report suggested that b1 integrins participate inthe hypertrophic responses in rat ventricular myocytes

a1-Transfectants display cell hypertrophy and [31]. Therefore, we considered a possible relationshipincreased expression of a-smooth muscle actin among a1b1 integrin expression, cell size, and expression

of a-SMA in MCs. This was done by analyzing a1It has been reported that there is a close associationamong increased a1b1 integrin expression, mesangial pro- transfectants or mock transfectants with flow cytometry,


Fig. 7. Forward light scatter flow cytometry of the transfectants. Thetransfectants [(thin line) 1B; (thick line) 2A1] were incubated in growthmedium for two days, collected, and analyzed for cell size by flow cytome- Fig. 8. Protein expression of a-smooth muscle actin (a-SMA) in thetry. The mean cell size of an a1 transfectant, 2A1, was 1.2-fold larger mock transfectants (1B, 2B, 3B) and a1-transfectants (2A1, 2A2, 2A5).than a mock transfectant, 1A. A representative experiment is shown. The transfectants were plated on tissue culture plastic for two days.

Total proteins were extracted from the cultures. The samples wereadjusted to a standard content (10 mg) and analyzed by Western blottingusing the anti–a-SMA monoclonal antibody. The a-SMA bands in thetransfectants were quantitated by densitometric scanning. A graph ofwhich measures cell sizes, and with Western blotting usingthe mean 6 SD of three independent experiments was made after

the anti–a-SMA antibody, respectively. As shown in Fig- densitometry and is shown below.ure 7, the mean cell size of a1-transfectants was foundto be 115 to 125% compared with mock transfectants.Western blotting of a-SMA showed a 2.2-fold expression

gand ligation is provided by our finding that a1-transfec-of a-SMA in a1 transfectants compared with mocktants showed increased expression of the G1 CKI, p27Kip1.transfectants (P , 0.01; Fig. 8). These observations indi-In general, cell proliferation is known to be controlled bycate that the cellular enlargement of a1-transfectants isa network of proteins among which are cyclin-dependentaccompanied by increased a-SMA expression.kinases (Cdks); their positive regulators, the cyclins; andtheir negative regulators, the CKIs [26, 27]. CKI p27Kip1

DISCUSSIONis thought to control activation of the cell cycle in re-

The present study was undertaken to examine whether sponse to mitogenic stimuli. The quantity of p27Kip1 in-the expression of a1b1 integrin could affect the MC phe- creases in quiescent cells and decreases rapidly afternotype. The main findings of our experiments were that stimulation with specific mitogens [28]. Moreover, consti-transfection of the a1-integrin subunit into MCs inhibits tutive expression of p27Kip1 in cultured cells causes cellproliferation and cell cycle progression and enhances cycle arrest in the G1 phase [29]. Thus, the overexpres-ECM reorganization. Of note, the expression level of

sion of a1b1 integrin seems to be linked constitutively toa1b1 integrin negatively affected the growth of MCs with-

the p27Kip1 pathway of growth arrest in MCs. A similarout a1b1 integrin-specific ligand interaction. Therefore,relationship between a constitutive activation of CKIthe results of this study raise the question of how expres-and integrin overexpression has been reported using b4sion of a1b1 integrin affects the capacity of MCs to per-transfectants [35]. An increase in a6b4 integrin expressionform mesangial remodeling after glomerular injury.induced by b4 subunit overexpression resulted in growthThere have been several similar studies noting that thearrest and constitutive activation of another CKI, p21Cip1,integrin a subunit expression regulates cell proliferation.in a carcinoma cell line, RKO cells without a6b4-specificFor example, overexpression of the a5 integrin subunitligand (laminin) ligation [35].in transformed Chinese hamster ovary cells or human

Comparing the ability of a1 transfectants with mockfibrosarcoma cells suppresses cell mitogenicity [32, 33].transfectants in the contraction of collagen gels providedMore closely related to our experiment, the a1b1 integrinus with another important cell biological finding thathas been implicated in growth suppression of a ras-trans-the expression level of a1b1 integrin is also critical forformed rat hepatocyte cell line, NR4 [34]. The pRep/a1collagen matrix reorganization by MCs. This result istransfected cell line, a1C6, which had threefold more a1compatible with our previous study, which found thatsubunit on the cell surfaces than the control cell line,TGF-b–stimulated gel contraction is dependent on the7C9, showed a reduced ability to grow in an anchorage-increased expression of a1b1 integrin by TGF-b [14]. Inindependent manner [34].general, a collagen contraction assay is an accepted inA probable mechanism for a1b1 integrin-mediated MC

growth inhibition in the absence of integrin-specific li- vitro model of collagen matrix reorganization, condensa-


tion of ECM, and contraction scarring seen in many mesangial expansion of human diabetic nephropathy[30, 43]. Finally, the hypertrophy of MCs was accompa-damaged organs, including the skin, lung, and liver [10,

37, 38]. The process of gel contraction is also similar to nied with increased induction of p27Kip1 in an experimen-tal model of diabetic nephropathy in mice [47], suggestingmesangial matrix remodeling or sclerosis in GN, since

abnormal mesangial reorganization of collagens I, III, the presence of a close link among the a1b1 integrin expres-sion, p27Kip1 and cell hypertrophy accompanying a-SMAand IV, dense collagenous matrix deposition, and disfig-

uring scarring of mesangium are characteristic features expression. However, we need to consider another mech-anism underlying the phenotypic changes in a1-trans-of mesangial sclerosis in progressive glomerular diseases

[1–3, 39]. It has been shown that the expression of MC- fected MCs. Since Chen et al recently demonstrated thatcell adhesion controls cell shape and that cell shape con-a1b1 integrin is associated with the level of mesangial

matrix expansion, accompanied by pathological collagen trols cell phenotype [48], the increased adhesive abilityof a1-transfectants to the collagen matrix may lead todeposition in a rat experimental model of GN [12]. Taken

with the previously mentioned information, the in- changes in spread morphology (shape) and thereby in-duce the phenotypic changes observed here. Therefore,creased expression of a1b1 integrin by MCs observed in

GN seems likely to inhibit MC proliferation and be it is important to clarify the involvement of the shape-dependent mechanism in phenotypic changes of a1 trans-linked to the pathological deposition of the collagen ma-

trix into the mesangial matrix in experimental and hu- fectants in this study.In summary, the present study indicated that the over-man GN.

We should stress here that a1b1 integrin expression expression of a1b1 integrin has a negative effect on MCproliferation, possibly by increasing the level of p27Kip1.may also play an important role in cell hypertrophy phe-

nomena and the increased production of a-SMA. b1 in- In addition, we showed that overexpression of a1b1 integ-rin leads to hypertrophy accompanied with increasedtegrins and their intracellular signaling have been impli-

cated in the cardiac hypertrophy in rats [31, 36]. For a-SMA expression and increased collagen gel contractionability. The diverse effects of a1b1 integrin expressionexample, adenovirus-mediated overexpression of b1 in-

tegrin augmented the myocyte hypertrophy response on MC phenotypes, such as cell growth and matrix re-modeling ability, strongly suggest that the MC-a1b1 integ-without influencing cell mitogenicity [31]. Notably, myo-

cytes isolated from the hypertrophic rat heart showed rin in nephritic glomeruli may function as a critical deter-increased production of a1b1 integrin as well as increased minant of cell phenotype, including growth, a-SMAadhesion to collagen I, in comparison to those from adult expression, and collagen matrix assembly, and therebycontrol myocytes [36]. It was recently shown that angio- contribute to pathological scar matrix remodeling (scle-tensin II, or high glucose-induced cell hypertrophy, de- rosis).pends on the induction of p27Kip1 [40–42]. Therefore, wepropose that the increased expression of a1b1 integrin ACKNOWLEDGMENTSleads to increased induction of p27Kip1 and thereby results This work was supported by grants from the Japan Welfare Ministryin the development of MC hypertrophy, although the (8670894). We are grateful to Dr. L.F. Leichardt (University of Califor-

nia, San Francisco, CA, USA) for providing the rat a1 integrin subunitsignaling pathway via a1b1 integrin expression remainscDNA. We thank Dr. Masanori Kitamura (University College Londonunknown. Until recently, many studies in human and rat Medical School, London, UK) for helpful discussions. Part of this work

GN have shown that increased expression of a-SMA is was published in abstract form at the annual meeting of the AmericanSociety of Nephrology, October 25–28, 1998.associated with MC activation, along with the enhanced

mitogenicity and production of ECM [2]. However, there Reprint requests to Shoji Kagami, M.D., PhD., Department of Pediat-are some studies suggesting that the a-SMA expression rics, School of Medicine, University of Tokushima, Kuramoto-cho-3-

chome, Tokushima 770-8503, Japan.by MC is associated with MC hypertrophy, rather thanE-mail: [email protected] proliferation, in GN [43–46]. Makino et al have

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Overexpression of α1β1 integrin directly affects rat mesangial cell behavior