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Α1ß1 Integrin-Mediated Collagen Matrix Remodeling by Rat

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Α1ß1 Integrin-Mediated Collagen Matrix Remodeling by Rat J Am Soc Nephrol 10: 779–789, 1999 a1b1 Integrin-Mediated Collagen Matrix Remodeling by Rat Mesangial Cells Is Differentially Regulated by Transforming Growth Factor-b and Platelet-Derived Growth Factor-BB SHOJI KAGAMI,* SHUJI KONDO,* KLEMENS LOSTER,¨ † WERNER REUTTER,† TAKASHI KUHARA,* KOJI YASUTOMO,* and YASUHIRO KURODA* *Department of Pediatrics, School of Medicine, University of Tokushima, Tokushima, Japan; and †Institut fu¨r Molekularbiologie und Biochemie, Freie Universitat Berlin, Berlin-Dahlem, Germany. Abstract. Pathologic remodeling of mesangial matrix after glo- mRNA analysis experiments demonstrated that TGF-b, but not merular injury is the central biologic feature of glomerular PDGF-BB, increases the expression of a1b1 integrin in mes- scarring (sclerosis). Transforming growth factor-b (TGF-b) angial cells cultured on plastic surface and in collagen gels. and platelet-derived growth factor (PDGF)-BB have been im- The upregulation of a1b1 integrin expression by TGF-b cor- plicated in the development of glomerular scarring in rat and related with increases in gel contraction and collagen-depen- human glomerulonephritis. To clarify molecular and cellular dent adhesion but not migration of mesangial cells. On the mechanisms involved in abnormal mesangial remodeling, this other hand, PDGF-BB enhanced MC-mediated gel contraction study focused on the role of a1b1 integrin, a collagen/laminin and migration without affecting cell adhesion to collagen I. receptor, in rat mesangial cells, using collagen gel contraction Growth factor-induced collagen-dependent adhesion, migra- as an experimental model of in vivo collagen matrix remodel- tion, and gel contraction were significantly attenuated by in- ing and scar formation. In addition, the influence of TGF-b and cubation with anti-a1, b1 subunit antibodies. Thus, these data PDGF-BB on mesangial cell (MC)-mediated collagen gel con- indicate that a1b1 integrin-mediated collagen matrix remod- traction in association with the a1b1 integrin expression was eling can be modulated by TGF-b and PDGF-BB via different evaluated. Integrin function blocking studies using anti-a1, b1 mechanisms. a1b1 integrin-mediated mesangial matrix remod- subunit antibodies indicated that MC-a1b1 integrin is essen- eling induced by TGF-b or PDGF-BB may be a pathogenic tially required not only for collagen-dependent adhesion/mi- mechanism leading to glomerular scarring. gration, but also for gel contraction. Protein synthesis and Abnormal mesangial extracellular matrix (ECM) remodeling associated a and b subunits, mainly mediate cell/ECM inter- after glomerular injury is characterized by increased expression actions. b1 integrins may play a critical role in cell prolifera- of ECM components such as fibronectin, laminin, and collagen tion, migration, differentiation, and matrix assembly, and thus type IV (collagen IV) and/or neoexpression of collagen I and participate in tissue development and remodeling after injury (7). III. In addition, it is the central biologic feature of progressive Recently, transforming growth factor-b (TGF-b) and plate- glomerular diseases leading to glomerular dysfunction and let-derived growth factor (PDGF) have been recognized to be scarring (sclerosis) (1–4). One current concept emerging from key mediators in the development of glomerular scarring in molecular cell biologic studies in progressive kidney diseases experimental and human kidney diseases (1–4). In this context, is that this pathologic mesangial ECM remodeling is caused by b1 integrins are effective biologic targets to modulate various uncontrolled interaction between mesangial cells, ECM, and growth factor actions (8,9). In fact, our previous experimental growth factors (5,6). studies using a rat model of acute glomerulonephritis (GN) and b A large body of studies demonstrated that 1 integrins, a mesangial cell (MC) culture system suggested that enhanced family of cell surface receptors consisting of noncovalently expression of glomerular TGF-b concomitantly increases the expression of b1 integrins (a1b1, a5b1 integrins) and ECM components (fibronectin, collagen I, IV) and thereby contrib- Received March 16, 1998. Accepted September 24, 1998. utes to pathologic mesangial ECM accumulation (2,10). In This work was presented in part at the 29th annual meeting of the American Society of Nephrology, November 3 to 6, 1996, and has appeared in abstract addition, our immunohistologic study of human GN found form (J Am Soc Nephrol 7: A2550, 1996). significant changes of glomerular b1 integrins in IgA nephrop- Correspondence to Dr. Shoji Kagami, Department of Pediatrics, School of athy and lupus GN that paralleled both the severity of the Medicine, University of Tokushima, Kuramoto-cho-3-chome, Tokushima 770- glomerular lesions such as ECM deposition and cellularity, and 8503, Japan. Phone: 011 81 886 31 3111; Fax: 011 81 886 31 8697; E-mail: b b [email protected] glomerular synthesis of TGF- (11). However, 1 integrin- mediated MC action in abnormal mesangial ECM build-up 1046-6673/1004-0779$03.00/0 Journal of the American Society of Nephrology after injury remains obscure. Copyright © 1999 by the American Society of Nephrology Many types of cells including MC are able to reorganize 780 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 779–789, 1999 collagen fibers and to perform gel contraction when cultured in Japan). A rabbit polyclonal antiserum (AB1936) reactive with the three-dimensional collagen I gels (12–17). This system has cytoplasmic domains of a2 subunit was purchased from Chemicon been extensively used as a model of ECM remodeling similar International (Temecula, CA) (26). Function-blocking monoclonal a to wound healing and pathologic scarring (12–14). Gel con- mouse anti-rat 1 subunit antibody (mAb 33.4) (IgG1) and polyclonal b b traction involves multiple cellular properties including forma- rabbit anti-rat 1 subunit IgG (pAb anti- 1) were produced and characterized as described previously (27). Each antibody is directed tion of contract sites to collagen fibers, reorganization of the to the extracellular domain of b1ora1 integrin subunit, respectively, network formed by collagen fibrils, and cell migration within and can disrupt MC adhesion to ECM proteins with ligand specificity collagen lattice (14–17). Several studies have clearly docu- (10). A mouse myeloma IgG1(Zymed Laboratories, San Francisco, mented that the collagen and laminin receptors—the a1b1 and CA), normal rabbit serum, and rabbit IgG (R&D Systems, Minneap- a2b1 integrins—are necessary components in the process of olis, MN) were used as control antibodies. collagen matrix reorganization and collagen gel contraction (18–24). Of the two, the a1b1 integrin was identified as the Immunohistochemistry major collagen binding receptor of rat glomerular mesangial Immunofluorescence (IF) analysis was performed to examine the cells in vivo (2,24). expression of a1, a2, b1 integrin subunits in mesangial cells cultured To investigate the potential role of a1b1 integrin in abnor- on plastic or in gels, respectively. Briefly, mesangial cells cultured on mal mesangial matrix remodeling observed in progressive glo- multichamber slides (Nunc, Naperville, IL) were washed with cold merular diseases, we employed an in vitro collagen gel system phosphate-buffered saline (PBS), then fixed and permeabilized with using isolated rat mesangial cells and focused on how this 2% paraformaldehyde, 0.1% Triton X-100 for 10 min. Cells were b a a integrin contributes to the contraction process. Additional ex- incubated with a rabbit antiserum against 1, 1, or 2 subunit for 1 h periments have been performed to study the effect of two at 37°C, followed by FITC-coupled donkey anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for1hat representative fibrogenic growth factors, TGF-b and PDGF- a b a b 37°C. After washing, the slides were coverslipped and examined BB, on MC- 1 1 integrin expression and on 1 1 integrin- under a fluorescence microscope. For IF examination of collagen I gel mediated collagen gel contraction. cultures, the gels were removed from the 24-well plates after culture This report shows that a1b1 integrin is required for collag- for 24 h and snap-frozen in OCT embedding compound (Miles, Inc., en-dependent MC adhesion, migration, and gel contraction. Elkhart, IN). Cryostat sections (5 mm) were placed on albumin-coated The ability of TGF-b to stimulate gel contraction was found to slides, then fixed and stained as described above. Some sections were be dependent on increased a1b1 integrin synthesis, whereas double-stained with a combination of monoclonal mouse anti-rat a1 PDGF-BB enhancement of gel contraction and cell migration subunit antibody (mAb) 33.4 and polyclonal rabbit anti-rat b1 subunit was revealed to be dependent on a1b1 integrin activity. IgG (pAb anti-b1)for1hat37°C, followed by a combination of FITC-coupled donkey anti-mouse IgG antibody and tetramethyl-rho- damine isothiocyanate-coupled donkey anti-rabbit IgG antibody Materials and Methods (Jackson ImmunoResearch Laboratories) for1hat37°C. Cell Culture Rat mesangial cells were obtained from intact glomeruli of 4- to 35S-Methionine Labeling of Mesangial 6-wk-old Sprague Dawley rats and characterized according to pub- Cells and Immunoprecipitation lished methods (25). Mesangial cells were used between passages 5 Mesangial cells on monolayer or in gel culture were metabolically and 9 and were maintained in RPMI 1640 (Sigma Chemical Co., St. 35 labeled with S-methionine for 18 h in methionine-free medium. Louis, MO) supplemented with 18% fetal bovine serum (Life Tech- After metabolic labeling, collagen gels were immersed in 25 mM nologies, Grand Island, NY), 100 U/ml penicillin, 100 mg/ml strep- Tris-HCl, pH 7.4, containing 100 mM N-octyl-b-D-glycopyranoside tomycin, 0.1 U/ml insulin, and 25 mM Hepes buffer at 37°C in a 5% (Sigma), 150 mM NaCl, 1 mM CaCl , and 1 mM MgCl (lysis CO incubator. All experiments were performed after a 2-d incubation 2 2 2 buffer), minced into fine pieces, repeatedly aspirated into syringe, and in serum-free RPMI 1640 to reach mesangial cells quiescence (25).
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