Biochem. Cell Biol. 82: 597–601 (2004) doi: 10.1139/O04-051 © 2004 NRC Canada

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Glomerular mesangial cell adhesion to fibrinogenis mediated by �v�3 integrin

Edgar G. Fischer

Abstract: The biological behavior of glomerular mesangial cells is thought to play a critical role in human and experimentalforms of mesangioproliferative glomerulonephritis. In these diseases, mesangial cells proliferate and produce increasedamounts of extracellular matrix proteins, which can lead to glomerulosclerosis and end-stage renal disease. Mesangialcells interact with extracellular matrix proteins through integrin-mediated cell adhesion. Fibrinogen as a plasma-derivedprotein is known to be deposited in the mesangium of kidneys affected by mesangioproliferative glomerulonephritis.The adhesive interactions between fibrinogen and mesangial cells, however, have not been reported. Results in thiswork show that mesangial cells adhere to immobilized fibrinogen in an integrin-dependent fashion. This process wasinhibited by the αvβ3-selective peptide cyclo-RGDFV and the monoclonal anti-β3 integrin chain antibody F11. Ca2+

ions are a known strong inhibitor of the fibrinogen-αvβ3 interaction, and mesangial cell adhesion did not occur whenCa2+ was the only divalent cation present. Therefore, mesangial cell adhesion to fibrinogen is mediated by αvβ3integrin, and divalent cations have a fundamental role in regulating this process.

Key words: glomerular mesangial cells, adhesion, extracellular matrix, fibrinogen, integrins, αvβ3.

Résumé : Le comportement biologique des cellules mésangiales glomérulaires pourrait jouer un rôle important dans lesformes humaines et expérimentales de glomérulonéphrite mésangioproliférante. Dans ces maladies, les cellules mésangialesprolifèrent et produisent des quantités accrues de protéines de la matrice extracellulaire, ce qui peut résulter englomérulosclérose et en maladie rénale en phase terminale. Les cellules mésangiales interagissent avec les protéines dela matrice extracellulaire par un phénomène d’adhésion dépendant des intégrines. Le fibrinogène, en tant que protéineplasmatique, est reconnu pour se déposer dans la zone mésangiale des reins affectés par la glomérulonéphritemésangioproliférante. Cependant, les interactions adhésives entre le fibrinogène et les cellules mésangiales n’ont pasété documentées. Les résultats de ce travail montrent que les cellules mésangiales adhèrent au fibrinogène immobiliséde façon dépendante des intégrines. Ce processus est inhibé par le peptide cyclo-RGDF dérivé spécifiquement de αvβ3ainsi que par l’anticorps monoclonal F11 dirigé contre la chaîne β3. Alors que les ions Ca2+ sont reconnus pour inhiberles interactions entre le fibrinogène et αvβ3, les cellules mésangiales n’adhèrent pas lorsque le Ca2+ est le seul cationdivalent présent dans le milieu. Par conséquent, l’adhésion des cellules mésangiales au fibrinogène est dépendante del’intégrine αvβ3 et les cations divalents ont un rôle fondamental à jouer dans la régulation de ce phénomène.

Mots clés : cellules glomérulaires mésangiales, adhésion, matrice extracellulaire, fibrinogène, intégrines, αvβ3.

[Traduit par la Rédaction] Fischer 601

Introduction

Glomerular mesangial cells are thought to play a criticalrole in the pathogenesis of human and experimental mesangio-proliferative glomerulonephritis. In humans, this group ofdiseases includes IgA nephropathy, membranoproliferativeglomerulonephritis, and various forms of lupus nephritis. Inthe pathogenesis of these diseases, glomerular injury leads to

activation of mesangial cells and cell proliferation with consecu-tive hypercellularity. Activated mesangial cells also produceand deposit increased amounts of extracellular matrix proteins.Both glomerular hypercellularity and matrix expansion arekey steps in the development of glomerulosclerosis andend-stage renal disease (Couser 1990; Floege et al. 1993;Kreidberg and Symons 2000; Sterzel and Rupprecht 1997).

Mesangial cells adhere to proteins in their extracellular

Received 9 December 2003. Revision received 8 April 2004. Accepted 26 April 2004. Published on the NRC Research Press Website at http://bcb.nrc.ca on 12 August 2004.

Abbreviations: BSA, bovine serum albumin; ECM, extracellular matrix; FB, fibrinogen; FN, fibronectin; VN, vitronectin;GRGDSP, H-Gly-Arg-Gly-Asp-Ser-Pro-OH; GRADSP, H-Gly-Arg-Ala-Asp-Ser-Pro-OH; cyclo-RGDFV, cyclo-(Arg-Gly-Asp-D-Phe-Val).

E.G. Fischer.1 Department of Pathology, University of New Mexico School of Medicine, Health Sciences Center, BMSB #335,915 Camino de Salud, Albuquerque, NM 87131, USA. (e-mail: efischer@salud.unm.edu).

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environment. This occurs through integrins, which are cell-matrix adhesion molecules expressed on the cell surface(Kreidberg and Symons 2000; Sterzel and Rupprecht 1997).Integrins are a family of heterodimeric cell-surface receptorscomposed of an α and a β subunit (Damsky and Werb 1992;Felding-Habermann and Cheresh 1993; Hynes 1992). Integrinsbind specific peptide sequences, including the Arg–Gly–Asp(RGD) sequence in extracellular matrix proteins, to promotecell adhesion to the extracellular environment. Conversely,cells adherent to matrix proteins receive signals from theirextracellular milieu. This process is also mediated by integrinsand regulates cellular functions like proliferation and matrixproduction (Damsky and Werb 1992; Felding-Habermannand Cheresh 1993; Hynes 1992; Kreidberg and Symons 2000;Sterzel and Rupprecht 1997).

Many studies have reported which members of the integrinfamily are expressed by mesangial cells in vitro; these includeα1-, αv-, α8-, β1-, and β3-integrin chains (Hafdi et al. 1997;Pröls et al. 1998; Sterk et al. 1998; Tsai et al. 1995). Integrinexpression has also been studied in vivo in the normal anddiseased kidneys (Cosio et al. 1990; Kanahara et al. 1994;Shikata et al. 1995; Sterk et al. 1998). Expression of β1 andαvβ3 integrins is increased in IgA nephropathy, and expressionof fibronectin and vitronectin is also increased in the mesangialmatrix (Kanahara et al. 1994; Shikata et al. 1995).

Plasma-derived fibrinogen (FB) is deposited in the mes-angium in various forms of human proliferative glomeru-lonephritis, including lupus nephritis (Colasanti et al. 1987),presumably because of extravasation from injured capillaries.FB was also found in rat kidneys when mesangioproliferativeglomerulonephritis was experimentally induced by anti-Thy

1.1 antibodies (Floege et al. 1993). However, the role of FBin the adhesion of mesangial cells in vitro has not beendetermined. Therefore, this report analyzes the interaction ofrat mesangial cells with immobilized FB, and addresses thehypothesis that mesangial cells adhere to FB via the αvβ3integrin.

Materials and methods

Vitronectin (VN), FB, and radioimmunoassay-grade bovineserum albumin (BSA) were purchased from Sigma–Aldrich(St. Louis, Mo.); fibronectin (FN) was from Invitrogen LifeTechnologies (Carlsbad, Calif.). The synthetic peptidesGRGDSP, GRADSP, and cyclo-RGDFV were from Calbiochem(San Diego, Calif.). The monoclonal antibody F11 againstthe rat β3-integrin chain was from PharMingen (San Diego,Calif.). Mesangial cells from rat glomeruli were cultured inaccordance with the methods described by Lang et al. (2000).The mesangial origin of the cells was confirmed by positiveimmunofluorescence staining with antibodies against Thy 1.1and α smooth muscle actin (DAKO Diagnostika, Hamburg,Germany), and the lack of staining for factor VIII antigen(DAKO) and cytokeratins 5 and 8 (Progen Biotechnik,Heidelberg, Germany). Cells were cultured in Dulbecco’smodified Eagle’s medium (DMEM), supplemented with 10%heat-inactivated fetal calf serum (Invitrogen Life Technol-ogies), 50 U penicillin/mL, 50 µg streptomycin/mL, 2 mmolglutamine/L, and 5 µg insulin/mL, in a 95% air : 5% CO2humidified atmosphere at 37 °C (Lang et al. 2000). Cellsbetween passages 5 and 15 were used. Cell-adhesion assayswere performed in nontissue-culture-treated 96-well plates

Fig. 1. (A) Adhesion of rat mesangial cells to the extracellular matrix proteins fibronectin, vitronectin, and fibrinogen. Plastic wellswere coated with the proteins, and cells in suspension were added and allowed to adhere for 30 min. Adherent cells were quantifiedusing an optical density (O.D.) plate reader, as described in Materials and methods. (B) Adhesion of rat mesangial cells to fibrinogenin the presence of the peptide integrin antagonists GRGDSP and cyclo-RGDFV, the control peptide GRADSP, and the monoclonalantibody F11 against the β3-integrin chain. Peptide and antibody solutions were added to plastic wells before the cell suspension.

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(Costar, Cambridge, Mass.), in accordance with methods de-scribed by Fischer et al. (1999). Plates were coated with FN,VN, or FB for 1 h at 37 °C, washed, blocked with 1% heat-denatured BSA for 1 h at 37 °C, and washed again. Cellswere split the day before the assay to obtain healthy log-phasecultures. Cells were harvested with 0.025% trypsin and 20 mmolEDTA/L, washed twice, and resuspended in assay medium(DMEM with 1% BSA, 20 mmol HEPES/L, pH 7.4). Asuspension of 75 000 cells in 50 µL was added to each well.For inhibition assays, peptide or antibody solutions wereadded to each well before the cells were added. Cells wereallowed to adhere for 30 min at 37 °C. Nonadherent cellswere removed with two gentle washes of assay medium, andwells were aspirated dry. Cells adherent to the bottom of thewells were quantified by assaying for lysosomal hexosami-nidase activity; 50 µL per well of 5 mmol p-nitrophenyl-N-acetyl-β-D-glucosaminide/L was added to 50 mmol Na2HPO4/NaH2PO4 buffer/L, pH 5.4, containing 0.5% Triton X-100.After adding 10 mmol EDTA/L to 0.5 mol NaOH/L, plates

were read in an ELISA plate reader at 405 nm. Adhesionassays in the presence of a single divalent cation were per-formed in an assay buffer (concentrations were as follows(mmol/L): HEPES, 20; NaCl, 137; KCl, 2.7; glucose, 2; 1%BSA; pH 7.4) with the addition of one of the following(mmol/L): Ca2+, 1; Mg2+, 1; or Mn2+, 0.01. Assays werecarried out in triplicate. Data shown are representative ofone of at least three experiments yielding similar results.

Results and discussion

First, mesangial cell adhesion to several immobilized extra-cellular matrix proteins was analyzed. Cells adhered to FNor VN, depending on the coating concentration (Fig. 1A),and also to collagen I (data not shown). Likewise, cellsattached to immobilized FB in a concentration-dependentfashion (Fig. 1A). Although adhesion to FN, VN, and collagenI has been reported before (Hafdi et al. 1997; Tsai et al.1995), mesangial cell attachment to FB is a novel finding.

Fig. 2. Adhesion of rat mesangial cells to fibronectin, vitronectin, and fibrinogen in the presence of different divalent cations. Cellswere allowed to adhere to protein-coated plastic wells in the presence of one of the following: Ca2+, Mg2+, or Mn2+.

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Adhesion of mesangial cells to FN has been shown to dependon β1 integrins (Tsai et al. 1995), and adhesion to VN onαvβ3 and αvβ5 integrins (Hafdi et al. 1997). To determinewhich integrin is involved in FB adhesion, the inhibitoryeffect of a cyclic and linear integrin antagonist peptide insolution was tested. In the presence of these peptides, assaysshowed strong and dose-dependent adhesion inhibition bycyclo-RGDFV, and weaker inhibition by the linear GRGDSP(Fig. 1B). The linear control peptide GRADSP was ineffective.As determined from the inhibition profiles (Fig. 1B), theinhibitory activity IC50 of cyclo-RGDFV was approximatelyeight times stronger than that of GRGDSP. Pfaff et al. (1994)reported in detailed biochemical studies that cyclo-RGDFVwas a 10-fold-stronger inhibitor than GRGDS for αvβ3, whereasboth peptides had equal effects on the FN receptor α5β1.Likewise, Bilato et al. (1997) reported that cyclo-RGDFVselectively recognized the VN receptors αvβ3 and αvβ5, buthad no effect on other integrins. Therefore, the current resultslead to the conclusion that mesangial cell adhesion to FB ismediated by one of the VN receptors, either αvβ3 or αvβ5.Inhibition studies with the monoclonal antibody F11 againstthe rat β3-integrin chain revealed strong inhibition of mesangialcell adhesion to FB (Fig. 1B), whereas an irrelevant controlantibody was ineffective (data not shown). These resultsprovide evidence that the αvβ3 integrin is critical to thisadhesion process.

Integrin-mediated cell adhesion depends not only on thepresence of divalent cations in the extracellular milieu, butalso on the specific type of cation present. Many authorshave reported that Ca2+, Mg2+, and Mn2+ regulate integrinactivity in profoundly different ways (Hu et al. 1995; Smithet al. 1994; Suehiro et al. 1997). In these studies, Mn2+

supported binding of FB to αvβ3, whereas Ca2+ was a potentinhibitor of this interaction (Smith et al. 1994; Suehiro et al.1997). To further test the hypothesis that mesangial cellsadhere to FB via the αvβ3 integrin, adhesion assays werecarried out in HEPES buffer with the addition of just onetype of divalent cation (Ca2+, Mg2+, or Mn2+). In controlexperiments, mesangial cells adhered to FN and VN in thepresence of Ca2+, Mg2+, or Mn2+ (Fig. 2). In contrast,mesangial cells did not adhere to FB in the presence of Ca2+,but were able to attach when Mg2+ or Mn2+ was present(Fig. 2). Although cells can adhere to VN through eitherαvβ3 or αvβ5, it has been shown that Ca2+ can facilitateαvβ5- but not αvβ3-mediated adhesion to VN (Hu et al.1995). Furthermore, Ca2+ is a known potent inhibitor of FBbinding to αvβ3 (Smith et al. 1994; Suehiro et al. 1997). Thefailure of cells to attach to FB in the presence of Ca2+

(Fig. 2) provides further evidence that mesangial cell attachmentto FB is mediated by the αvβ3 integrin when Mg2+ or Mn2+

ions are present.In summary, the results presented here demonstrate that

rat mesangial cells adhere to FB. Inhibition studies withRGD peptides show that this attachment involves either theαvβ3 or αvβ5 integrin. Inhibition studies with the monoclonalanti-β3 antibody F11 provide evidence that the β3 chain iscrucial for this process. Finally, differential adhesion to FBin the presence of Mg2+ or Mn2+ but not Ca2+ invalidates therole of αvβ5 in this process, and leads to the conclusion thatmesangial cell attachment to FB occurs via the αvβ3 integrin.

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