Renal Fibrosis
2001; Elsevier BV; Volume: 159; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)62518-7
ISSN1525-2191
AutoresMichael Zeisberg, Gary D. Bonner, Yohei Maeshima, Pablo Colorado, G. Müller, Frank Strutz, Raghu Kalluri,
Tópico(s)Renal Diseases and Glomerulopathies
ResumoType IV collagen is a major component of basement membranes and it provides structural and functional support to various cell types. Type IV collagen exists in a highly complex suprastructure form and recent studies implicate that protomer (the trimeric building unit of type IV collagen) assembly is mediated by the NC1 domain present in the C-terminus of each collagen α-chain polypeptide. Here we show that type IV collagen contributes to the maintenance of the epithelial phenotype of proximal tubular epithelial cells, whereas type I collagen promotes epithelial-to-mesenchymal transdifferentiation (EMT). In addition, the recombinant human α1NC1 domain inhibits assembly of type IV collagen NC1 hexamers and potentially disrupts the deposition of type IV collagen, facilitating EMT in vitro. Inhibition of type IV collagen assembly by the α1NC1 domain up-regulates the production of transforming growth factor-β1 in proximal tubular epithelial cells, an inducer of EMT. These results strongly suggest that basement membrane architecture is pivotal for the maintenance of epithelial phenotype and that changes in basement membrane architecture potentially lead to up-regulation of transforming growth factor-β1, which contributes to EMT during renal fibrosis. Type IV collagen is a major component of basement membranes and it provides structural and functional support to various cell types. Type IV collagen exists in a highly complex suprastructure form and recent studies implicate that protomer (the trimeric building unit of type IV collagen) assembly is mediated by the NC1 domain present in the C-terminus of each collagen α-chain polypeptide. Here we show that type IV collagen contributes to the maintenance of the epithelial phenotype of proximal tubular epithelial cells, whereas type I collagen promotes epithelial-to-mesenchymal transdifferentiation (EMT). In addition, the recombinant human α1NC1 domain inhibits assembly of type IV collagen NC1 hexamers and potentially disrupts the deposition of type IV collagen, facilitating EMT in vitro. Inhibition of type IV collagen assembly by the α1NC1 domain up-regulates the production of transforming growth factor-β1 in proximal tubular epithelial cells, an inducer of EMT. These results strongly suggest that basement membrane architecture is pivotal for the maintenance of epithelial phenotype and that changes in basement membrane architecture potentially lead to up-regulation of transforming growth factor-β1, which contributes to EMT during renal fibrosis. Basement membranes are present throughout the human body. In contrast to interstitial extracellular matrix, it is a highly organized structure that consists primarily of laminin, nidogen, and collagen type IV, which as the most abundant matrix molecule serves as a scaffold for the basement membrane proteins.1Yurchenco PD O'Rear JJ Basal lamina assembly.Curr Opin Cell Biol. 1994; 6: 674-681Crossref PubMed Scopus (264) Google Scholar Type IV collagen includes six genetically distinct isoforms named α1(IV) through α6(IV).2Hudson BG Reeders ST Tryggvason K Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis.J Biol Chem. 1993; 268: 26033-26036Abstract Full Text PDF PubMed Google Scholar These isoforms organize themselves into a unique network that provides basement membrane specificity and inequality.2Hudson BG Reeders ST Tryggvason K Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis.J Biol Chem. 1993; 268: 26033-26036Abstract Full Text PDF PubMed Google Scholar Assembly of type IV collagen is initiated by the formation of protomers (trimers).1Yurchenco PD O'Rear JJ Basal lamina assembly.Curr Opin Cell Biol. 1994; 6: 674-681Crossref PubMed Scopus (264) Google Scholar, 3Timpl R Oberbaumer I von der Mark H Bode W Wick G Weber S Engel J Structure and biology of the globular domain of basement membrane type IV collagen.Ann NY Acad Sci. 1985; 460: 58-72Crossref PubMed Scopus (48) Google Scholar Three α chains come together through associations among their noncollagenous (NC) domains followed by folding of the collagenous domains into triple helices.4Yurchenco PD Assembly of basement membranes.Ann NY Acad Sci. 1990; 580: 195-213Crossref PubMed Scopus (59) Google Scholar Each protomer is associated with another protomer by its NC1 domain to form interlocking hexamers.5Kalluri R Cosgrove D Assembly of type IV collagen. Insights from alpha3(IV) collagen-deficient mice.J Biol Chem. 2000; 275: 12719-12724Crossref PubMed Scopus (59) Google Scholar Along with lateral association of the collagenous triple helices and covalent binding of 7S domains, association of the α-chain of NC1 domain is essential to allow the formation of the protomeric network that serves as a network scaffold for other basement membrane proteins.6Siebold B Deutzmann R Kuhn K The arrangement of intra- and intermolecular disulfide bonds in the carboxyterminal, non-collagenous aggregation and cross-linking domain of basement-membrane type IV collagen.Eur J Biochem. 1988; 176: 617-624Crossref PubMed Scopus (97) Google Scholar With six different α-chains known at present, 56 different combinations of triple-helical protomers are possible. In Engelberth Holm Swarm-sarcoma-derived type IV collagen, the most abundant protomers are preferentially those that contain only α1 and α2 chains in a 2:1 ratio, stressing that the NC1 domain of the α1 chain (α1NC1) has a central role in assembly of type IV collagen.7Timpl R Structure and biological activity of basement membrane proteins.Eur J Biochem. 1989; 180: 487-502Crossref PubMed Scopus (908) Google Scholar In human kidney, as in mouse, differences in type IV collagen composition may hint to specific roles of specialized basement membranes.7Timpl R Structure and biological activity of basement membrane proteins.Eur J Biochem. 1989; 180: 487-502Crossref PubMed Scopus (908) Google Scholar In adults, human renal tubular basement membrane (TBM) that surrounds proximal tubules, consists exclusively of α1/α2 protomers, whereas distal tubules also contain some α3 chain.8Miner JH Renal basement membrane components.Kidney Int. 1999; 56: 2016-2024Crossref PubMed Scopus (295) Google Scholar, 9Van Vliet AI Van Alderwegen IE Baelde HJ de Heer E Killen PD Kalluri RK Bruijn JA Bergijk EC Differential expression of collagen type IV alpha-chains in the tubulointerstitial compartment in experimental chronic serum sickness nephritis.J Pathol. 1999; 189: 279-287Crossref PubMed Scopus (7) Google Scholar In the glomerular basement membrane the type IV collagen network involves the α3, α4, and α5 chains.8Miner JH Renal basement membrane components.Kidney Int. 1999; 56: 2016-2024Crossref PubMed Scopus (295) Google Scholar Type IV collagen binds various cells via surface receptors such as integrins, which is suggestive of its capacity to modulate specific cell behavior.10Hynes RO Integrins: versatility, modulation, and signaling in cell adhesion.Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9503) Google Scholar Renal interstitial fibrogenesis, as the common pathway in progressive chronic renal disease, is traditionally characterized by an increasing number of interstitial fibroblasts that mediate excessive deposition of interstitial matrix components leading to tubular atrophy.11Eddy AA Molecular insights into renal interstitial fibrosis.J Am Soc Nephrol. 1996; 7: 2495-2508Crossref PubMed Google Scholar, 12Nath KA Tubulointerstitial changes as a major determinant in the progression of renal damage.Am J Kidney Dis. 1992; 20: 1-17PubMed Scopus (894) Google Scholar, 13Strutz F Muller GA On the progression of chronic renal disease.Nephron. 1995; 69: 371-379Crossref PubMed Scopus (68) Google Scholar, 14Fogo AB Pathology of progressive nephropathies.Curr Opin Nephrol Hypertens. 2000; 9: 241-246Crossref PubMed Scopus (13) Google Scholar, 15Alpers CE Pichler R Johnson RJ Phenotypic features of cortical interstitial cells potentially important in fibrosis.Kidney Int. 1996; 54: S28-S31Google Scholar, 16Alpers CE The evolving contribution of renal pathology to understanding interstitial nephritis.Ren Fail. 1998; 20: 763-771Crossref PubMed Scopus (3) Google Scholar Recent observations stress a pivotal role of tubular epithelial cells as mediators of renal scarring.17Becker GJ Hewitson TD The role of tubulointerstitial injury in chronic renal failure.Curr Opin Nephrol Hypertens. 2000; 9: 133-138Crossref PubMed Scopus (138) Google Scholar, 18D'Agati V Appel GB Renal pathology of human immunodeficiency virus infection.Semin Nephrol. 1998; 18: 406-421PubMed Google Scholar Tubular epithelial cells function as a source of fibrogenic growth factors and chemokines in the initiation of fibrogenesis, contribute to tubular atrophy by undergoing apoptosis, and potentially contribute to increased numbers of interstitial fibroblasts by epithelial-mesenchymal transdifferentiation (EMT).19Muller GA Zeisberg M Strutz F The importance of tubulointerstitial damage in progressive renal disease.Nephrol Dial Transplant. 2000; 15: 76-77Crossref PubMed Scopus (51) Google Scholar EMT is defined as the acquisition of phenotypic as well as functional properties of mesenchymal fibroblasts by epithelial cells.20Hay ED Zuk A Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced.Am J Kidney Dis. 1995; 26: 678-690Abstract Full Text PDF PubMed Scopus (362) Google Scholar It occurs in development, carcinogenesis, and chronic diseases in different organs.21Birchmeier W Birchmeier C Epithelial-mesenchymal transitions in development and tumor progression.EXS. 1995; 74: 1-15PubMed Google Scholar, 22Wallner EI Yang Q Peterson DR Wada J Kanwar YS Relevance of extracellular matrix, its receptors, and cell adhesion molecules in mammalian nephrogenesis.Am J Physiol. 1998; 275: F467-F477PubMed Google Scholar, 23Kanwar YS Carone FA Kumar A Wada J Ota K Wallner EI Role of extracellular matrix, growth factors and proto-oncogenes in metanephric development.Kidney Int. 1997; 52: 589-606Crossref PubMed Scopus (70) Google Scholar EMT is increasingly being considered as a possible mechanism leading to renal fibrogenesis.24Okada H Danoff TM Kalluri R Neilson EG Early role of Fsp1 in epithelial-mesenchymal transformation.Am J Physiol. 1997; 273: F563-F574PubMed Google Scholar, 25Remuzzi G Bertani T Pathophysiology of progressive nephropathies.N Engl J Med. 1998; 339: 1448-1456Crossref PubMed Scopus (1164) Google Scholar, 26Strutz F Muller GA Transdifferentiation comes of age.Nephrol Dial Transplant. 2000; 15: 1729-1731Crossref PubMed Scopus (56) Google Scholar The current concept of EMT postulates a mechanism in which tubular epithelial cells become activated by exogenous stimuli, followed by a loss of contact with neighboring cells and basement membrane.20Hay ED Zuk A Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced.Am J Kidney Dis. 1995; 26: 678-690Abstract Full Text PDF PubMed Scopus (362) Google Scholar, 27Hay ED An overview of epithelio-mesenchymal transformation.Acta Anat. 1995; 154: 8-20Crossref PubMed Scopus (1287) Google Scholar After initiation of EMT, cells move through their basement membrane into the interstitial matrix where they become detectable as fibroblasts/myofibroblasts.20Hay ED Zuk A Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced.Am J Kidney Dis. 1995; 26: 678-690Abstract Full Text PDF PubMed Scopus (362) Google Scholar Thus, in this hypothetical model of EMT, the epithelial phenotype is clearly associated with TBM microenvironment whereas mesenchymal phenotype is associated with interstitial microenvironment. Therefore, in the present study, we investigated the role of type IV collagen composition, assembly, and integrity on the phenotype of proximal tubular epithelial cells in vitro. Our studies suggest that disruption of TBM leads to increased expression of transforming growth factor (TGF)-β1 by mouse proximal tubular epithelial cells (MCT). Alterations of cell-matrix interactions potentially facilitate EMT and contribute to fibroblast population in the renal interstitium. Recombinant human TGF-β1, human epithelial growth factor (EGF), and the neutralizing polyclonal goat antibodies to TGF-β and EGF were purchased from R&D Systems (Minneapolis, MN). Mouse monoclonal antibody to vimentin was obtained from Boehringer Mannheim (Mannheim, Germany). Rabbit polyclonal antibody to cytokeratin, fluorescein isothiocyanate-labeled F(ab′) goat anti-rabbit IgG, fluorescein isothiocyanate-conjugated anti-mouse IgG, alkaline-phosphatase-conjugated anti-rabbit IgG, and alkaline phosphatase-labeled anti-mouse IgG were purchased from Sigma (St. Louis, MO). Polyclonal rabbit antibody to type IV collagen was purchased from ICN (Aurora, OH). Polyclonal rabbit antibody to FSP-1 was generated as described elsewhere.28Strutz F Okada H Lo CW Danoff T Carone RL Tomaszewski JE Neilson EG Identification and characterization of a fibroblast marker: FSP1.J Cell Biol. 1995; 130: 393-405Crossref PubMed Scopus (917) Google Scholar Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium were obtained from Gibco BRL Ltd. (Paisley, UK), fetal calf serum was purchased from BioWhittaker (Walkersville, MD), rat tail collagen type I and type IV collagen were obtained from Becton Dickinson (Franklin Lakes, NJ). Murine renal cell lines were established previously and had been cloned several times.24Okada H Danoff TM Kalluri R Neilson EG Early role of Fsp1 in epithelial-mesenchymal transformation.Am J Physiol. 1997; 273: F563-F574PubMed Google Scholar, 28Strutz F Okada H Lo CW Danoff T Carone RL Tomaszewski JE Neilson EG Identification and characterization of a fibroblast marker: FSP1.J Cell Biol. 1995; 130: 393-405Crossref PubMed Scopus (917) Google Scholar, 29Haverty TP Kelly CJ Hines WH Amenta PS Watanabe M Harper RA Kefalides NA Neilson EG Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis.J Cell Biol. 1988; 107: 1359-1368Crossref PubMed Scopus (302) Google Scholar, 30Alvarez RJ Sun MJ Haverty TP Iozzo RV Myers JC Neilson EG Biosynthetic and proliferative characteristics of tubulointerstitial fibroblasts probed with paracrine cytokines.Kidney Int. 1992; 41: 14-23Crossref PubMed Scopus (108) Google Scholar They were grown in recommended conditions:24Okada H Danoff TM Kalluri R Neilson EG Early role of Fsp1 in epithelial-mesenchymal transformation.Am J Physiol. 1997; 273: F563-F574PubMed Google Scholar MCTs and tubulointerstitial fibroblasts were cultured in DMEM supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. In EMT experiments the medium was replaced with serum-free K1 medium (1:1 Ham's F12/DMEM with 5 μg/ml transferrin, 5 μg/ml insulin, and 5 × 10−8 mol/L hydrocortisone) containing cytokines or collagen domains.31Taub N Livingston D The development of serum-free hormone-supplemented media for primary kidney cultures and their use in examining renal functions.Ann NY Acad Sci. 1981; 372: 406-421Crossref PubMed Scopus (19) Google Scholar Homogeneity, cell surface markers, and phenotype characteristics have been documented extensively for both MCT cells and tubulointerstitial fibroblast cells.24Okada H Danoff TM Kalluri R Neilson EG Early role of Fsp1 in epithelial-mesenchymal transformation.Am J Physiol. 1997; 273: F563-F574PubMed Google Scholar, 28Strutz F Okada H Lo CW Danoff T Carone RL Tomaszewski JE Neilson EG Identification and characterization of a fibroblast marker: FSP1.J Cell Biol. 1995; 130: 393-405Crossref PubMed Scopus (917) Google Scholar A pDS plasmid containing α1NC1 domain cDNA was used to add a leader signal sequence in frame into the pcDNA3.1 (Invitrogen, Carlsbad, CA) eukaryotic expression vector by polymerase chain reaction amplification. The leader sequence from the 5′ end of the full-length α1 type IV collagen chain was cloned 5′ to the cDNA of α1NC1 domain to enable protein secretion into the culture medium. The α1NC1 domain was confirmed as described previously.32Colorado PC Torre A Kamphaus G Maeshima Y Hopfer H Takahashi K Volk R Zamborsky ED Herman S Sarkar PK Ericksen MB Dhanabal M Simons M Post M Kufe DW Weichselbaum RR Sukhatme VP Kalluri R Anti-angiogenic cues from vascular basement membrane collagen.Cancer Res. 2000; 60: 2520-2526PubMed Google Scholar The α1NC1 domain containing plasmid and control plasmid were used to transfect 293 human embryonic kidney cells using the calcium chloride method. Transfected clones were selected by Geneticin (Life Technologies, Inc., Gaithersburg, MD) antibiotic treatment. The cells were passed for 3 weeks in the presence of the antibiotic until no cell death was evident. Clones were expanded into T-225 flasks and grown until confluent. The supernatant was collected and concentrated using an Amicon (Beverly, MA) concentrator. The concentrated supernatant was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and enzyme-linked immunosorbent assay (ELISA) with anti-α1NC1 domain and anti-FLAG antibodies. The α1NC1 domain-containing supernatant was subjected to affinity chromatography using α1NC1 domain-specific antibodies.32Colorado PC Torre A Kamphaus G Maeshima Y Hopfer H Takahashi K Volk R Zamborsky ED Herman S Sarkar PK Ericksen MB Dhanabal M Simons M Post M Kufe DW Weichselbaum RR Sukhatme VP Kalluri R Anti-angiogenic cues from vascular basement membrane collagen.Cancer Res. 2000; 60: 2520-2526PubMed Google Scholar Renal cortex NC1 hexamer was prepared as described elsewhere.33Gunwar S Ballester F Kalluri R Timoneda J Chonko AM Edwards SJ Noelken ME Hudson BG Glomerular basement membrane. Identification of dimeric subunits of the noncollagenous domain (hexamer) of collagen IV and the Goodpasture antigen.J Biol Chem. 1991; 266: 15318-15324Abstract Full Text PDF PubMed Google Scholar After purification, native hexamers from the kidney cortex were subjected to treatment with 50 mmol/L formic acid followed by incubation for 10 minutes at room temperature to disassemble protein to monomers. Tris, 1 mol/L (pH 7.5), was added and incubation was extended for an additional 12-hour period at room temperature for reassembly of hexamers. For Western blot analysis proteins were transferred to trans-blot nitrocellulose membranes (Bio-Rad, Hercules, CA) and then probed with antibodies for 1 hour at room temperature. After extensive washes, bands were visualized using reagents for enhanced chemiluminescence (ECL Western blotting kit, Amersham, Arlington Heights, IL). For detection of incorporated FLAG-α1NC1 domain in hexamers, native hexamers were allowed to reassemble in presence of FLAG-α1NC1 domain and were run on a nondenaturing gel. The band corresponding to hexamers was cut out and protein was extracted. For detection protein was electrophoresed in denaturing polyacrylamide gel. FLAG-α1NC1 domain was detected by anti-FLAG antibody. For analysis of tissue culture supernatant, medium was removed and concentrated 10-fold using Centricon YM10 centrifugal filter devices (Millipore, Bedford, MA). ELISAs were performed as described previously.24Okada H Danoff TM Kalluri R Neilson EG Early role of Fsp1 in epithelial-mesenchymal transformation.Am J Physiol. 1997; 273: F563-F574PubMed Google Scholar Cells (5 × 104 cells/well) were plated in 12-well plates and grown in DMEM with 10% fetal calf serum for 6 hours. Then the medium was replaced with serum-free K1 media containing cytokines, antibodies, or type IV collagen domains. After 24, 48, and 72 hours cells were harvested by trypsin/ethylenediaminetetraacetic acid, spun down, and resuspended in phosphate-buffered saline (PBS). The number of cells was counted using a hemocytometer. Cells (5 × 104) were pelleted, then lysed in 500 μl of 6 mol/L guanidine hydrochloride, pH 7.5. Microtiter ELISA plates (96-well) were coated in quadruplicate with 150 μl of cell lysate and were then incubated overnight at room temperature. After coating, the plates were washed with 0.15 mol/L NaCl 0.05% Tween-20 washing solution and blocked with 2% bovine serum albumin and 0.1% Tween-20 in PBS for 30 minutes at room temperature. The wells were incubated with 1:500 dilution of anti-FSP-1 or 1:200 dilution of anti-cytokeratin antibodies in the incubation buffer. After incubation with the primary antibody for 1 hour at room temperature, the plates were washed three times with washing solution and then incubated with alkaline phosphatase (ALP)-conjugated secondary antibody diluted 1:1000 in incubation buffer. Finally the plates were washed thoroughly and disodium p-nitrophenyl phosphate (5 μg/ml) was added. After color development the absorbance was measured with an ELISA plate reader at 450 nm. The effect of α1NC1 domain on EMT was visualized by immunofluorescent staining of FSP-1 and vimentin after incubation for 48 hours as described above. At the end of incubation cells were washed twice with PBS and fixed with ethanol/acetic acid (50:50, v/v) at 4°C. The cells were subsequently washed again and incubated for 2 hours with a 1:40 dilution of antibody to FSP-1 or 1:8 dilution of antibody to vimentin. Cells were then washed again and incubated for 1 hour at room temperature with fluorescein isothiocyanate-conjugated secondary antibody. Staining was analyzed by fluorescence microscopy. This assay was performed as described previously with minor modifications.34Maeshima Y Manfredi M Reimer C Holthaus KA Hopfer H Chandamuri BR Kharbanda S Kalluri R Identification of the anti-angiogenic site within vascular basement membrane derived tumstatin.J Biol Chem. 2001; 276: 15240-15248Crossref PubMed Scopus (209) Google Scholar Ninety-six-well plates were coated with 10 μg/ml of type IV collagen, type I collagen, or fibronectin in PBS or with 10% w/v bovine serum albumin/PBS as negative control at 37°C overnight. Before seeding cells, wells were blocked with 10% bovine serum albumin/PBS for 2 hours at 37°C. MCT cells were maintained in K1 medium or K1 medium that contained 3 ng/ml TGF-β1 and 10 ng/ml EGF for 48 hours in T75 flasks as described. Cells were harvested by trypsin, and 4000 cells per well were seeded in 100 μl of K1 medium. After 30 minutes, medium was removed, wells were washed once, and cells were incubated again for 1 hour at 37°C. The number of attached cells was determined with methylene blue staining. MCT cells were grown for 6 hours in DMEM containing 10% fetal calf serum before the medium was replaced with serum-free medium or with medium containing growth factors or α1NC1 domains. Total cellular RNA was extracted using Trizol reagent (Gibco BRL Ltd.) according to instructions of the manufacturer. RNA concentrations were determined by absorbance at 260 nm and samples were stored at −80°C before use. Northern blot analysis was performed as described previously with minor modifications.35Rocco MV Chen Y Goldfarb S Ziyadeh FN Elevated glucose stimulates TGF-beta gene expression and bioactivity in proximal tubule.Kidney Int. 1992; 41: 107-114Crossref PubMed Scopus (290) Google Scholar Forty μg of total RNA were electrophoresed on a 1.2% agarose gel containing 2.2 mol/L formaldehyde using 1×× MOPS, pH 7.0, as the running buffer. RNA was transferred to a nylon membrane by capillary transfer for 12 hours using 10× standard saline citrate as the transfer buffer. Blots were UV-crosslinked. TGF-β1 cDNA probes were linearized and labeled with [γ32p]-ATP by random labeling according to instructions of the manufacturer (Boehringer Mannheim,). Hybridizations were performed overnight at 68°C after 1 hour of prehybridization with hybridization buffer (0.5 mol/L NaPo4, 7% w/v SDS, 1% w/v bovine serum albumin, 1 mmol/L ethylenediaminetetraacetic acid). Washing steps were performed three times using a solution containing 0.25 mol/L Na2HPO4 at 68°C. After washing, autoradiograms were obtained. All blots were stripped and reprobed with isotope-labeled GAPDH cDNA as a control for equal loading and transfer. Quantitative analysis was performed relative to the GAPDH band using a densitometer and quantitation software (Bio-Rad). All values are expressed as mean ± SEM unless specified. Analysis of variance was used to determine statistical differences between groups using Sigma-Stat software (Jandel Scientific, San Rafael, CA). Further analysis was performed using t-test with Bonferroni correction to identify significant differences. A level of P < 0.05 was considered statistically significant. Tubular epithelial cells typically adhere tightly to the TBM, whereas interstitial fibroblasts are surrounded by type I collagen in vivo.36Zuk A Hay ED Expression of beta 1 integrins changes during transformation of avian lens epithelium to mesenchyme in collagen gels.Dev Dyn. 1994; 201: 378-393Crossref PubMed Scopus (87) Google Scholar To test whether the microenvironment has specific effects on tubular cells, MCTs were exposed to culture dishes that were coated with either type I collagen or with type IV collagen composed exclusively of the α1 and α2 chains. In cell attachment assays, untreated MCT cells adhered better to type IV collagen (Figure 1A, left), whereas after induction of EMT with TGF-β1 and EGF, cells adhered preferably to type I collagen (Figure 1A, right) and appeared to display a more spindle-shaped morphology (Figure 1B). Cultivation on type I collagen resulted in an increase in the fibroblast-specific marker FSP-1, whereas cultivation on type IV collagen did not alter the expression of FSP-1 compared to uncoated plates (Figure 1C, left). When EMT was induced with TGF-β1 and EGF, cultivation on type IV collagen reduced levels of FSP-1 expression and thus stabilized the epithelial phenotype, whereas cultivation on type I collagen further increased FSP-1 expression (Figure 1C, right). Type IV collagen exhibits self-assembly in vitro and potentially in vivo.37Oberbaumer I Wiedemann H Timpl R Kuhn K Shape and assembly of type IV procollagen obtained from cell culture.EMBO J. 1982; 1: 805-810Crossref PubMed Scopus (70) Google Scholar, 38Yurchenco PD Ruben GC Basement membrane structure in situ: evidence for lateral associations in the type IV collagen network.J Cell Biol. 1987; 105: 2559-2568Crossref PubMed Scopus (277) Google Scholar, 39Yurchenco PD Ruben GC Type IV collagen lateral associations in the EHS tumor matrix. Comparison with amniotic and in vitro networks.Am J Pathol. 1988; 132: 278-291PubMed Google Scholar, 40Petitclerc E Boutaud A Prestayko A Xu J Sado Y Ninomiya Y Sarras Jr, MP Hudson BG Brooks PC New functions for non-collagenous domains of human collagen type IV. Novel integrin ligands inhibiting angiogenesis and tumor growth in vivo.J Biol Chem. 2000; 275: 8051-8061Crossref PubMed Scopus (277) Google Scholar The initial process of type IV collagen assembly involves six α-chains and their NC1 domains to form NC1 hexamers.5Kalluri R Cosgrove D Assembly of type IV collagen. Insights from alpha3(IV) collagen-deficient mice.J Biol Chem. 2000; 275: 12719-12724Crossref PubMed Scopus (59) Google Scholar, 41Tsilibary EC Reger LA Vogel AM Koliakos GG Anderson SS Charonis AS Alegre JN Furcht LT Identification of a multifunctional, cell-binding peptide sequence from the a1(NC1) of type IV collagen.J Cell Biol. 1990; 111: 1583-1591Crossref PubMed Scopus (45) Google Scholar Type IV collagen network is formed by lateral assembly of collagenous domains and by covalent association of 7S domains.4Yurchenco PD Assembly of basement membranes.Ann NY Acad Sci. 1990; 580: 195-213Crossref PubMed Scopus (59) Google Scholar, 37Oberbaumer I Wiedemann H Timpl R Kuhn K Shape and assembly of type IV procollagen obtained from cell culture.EMBO J. 1982; 1: 805-810Crossref PubMed Scopus (70) Google Scholar, 38Yurchenco PD Ruben GC Basement membrane structure in situ: evidence for lateral associations in the type IV collagen network.J Cell Biol. 1987; 105: 2559-2568Crossref PubMed Scopus (277) Google Scholar We hypothesized that type IV collagen α1NC1 domain, which lacks the collagenous- and 7S domain, will incorporate into hexamers and thus act in a dominant-negative manner on type IV collagen self-assembly involving the collagenous chain to form triple helices.3Timpl R Oberbaumer I von der Mark H Bode W Wick G Weber S Engel J Structure and biology of the globular domain of basement membrane type IV collagen.Ann NY Acad Sci. 1985; 460: 58-72Crossref PubMed Scopus (48) Google Scholar, 42Boutaud A Borza DB Bondar O Gunwar S Netzer KO Singh N Ninomiya Y Sado Y Noelken ME Hudson BG Type IV collagen of the glomerular basement membrane. Evidence that the chain specificity of network assembly is encoded by the noncollagenous NC1 domains.J Biol Chem. 2000; 275: 30716-30724Crossref PubMed Scopus (186) Google Scholar Type IV collagen hexamers, isolated from bovine kidney cortex, were used in hexamer association and dissociation experiments. In the presence of FLAG-tagged α1NC1 domain the reassembled hexamers incorporated the recombinant FLAG-tagged human α1NC1 domain, as determined by nondenaturing gel electrophoresis of the reconstituted hexamers (Figure 2A). To further demonstrate the incorporation of FLAG-α1NC1 domain into the hexameric structure, the hexamer band (arrow in Figure 2A) was excised and eluted from the nondenaturing gel and resolved by SDS-PAGE and immunoblotted with anti-FLAG antibodies. These results show that FLAG-α1NC1 is incorporated in the hexameric structure and detectable as a monomer band in dissociated hexamer in SDS-PAGE (Figure 2B), similar to FLAG-tagged human α1NC1 domain (Figure 2B). Thus FLAG-α1NC1 doma
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