β2-microglobulin modified with advanced glycation end products modulates collagen synthesis by human fibroblasts
1998; Elsevier BV; Volume: 53; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1755.1998.00882.x
ISSN1523-1755
AutoresWilliam F. Owen, Fan Fan Hou, Robert O. Stuart, Jonathan Kay, Joshua A. Boyce, Glenn M. Chertow, Ann Marie Schmidt,
Tópico(s)Chronic Kidney Disease and Diabetes
Resumoβ2-microglobulin modified with advanced glycation end products modulates collagen synthesis by human fibroblasts. β2-microglobulin amyloidosis (Aβ2m) is a serious complication for patients undergoing long-term dialysis. β2-microglobulin modified with advanced glycation end products (β2m-AGE) is a major component of the amyloid in Aβ2m. It is not completely understood whether β2m-AGE plays an active role in the pathogenesis of Aβ2m, or if its presence is a secondary event of the disease. β2-microglobulin amyloid is mainly located in tendon and osteo-articular structures that are rich in collagen, and local fibroblasts constitute the principal cell population in the synthesis and metabolism of collagen. Recent identification of AGE binding proteins on human fibroblasts lead to the hypothesis that the fibroblast may be a target for the biological action of β2m-AGE. The present study demonstrated that two human fibroblast cell lines exhibited a decrease in procollagen type I mRNA and type I collagen synthesis after exposure to β2m-AGE for 72 hours. Similar results were observed using AGE-modified albumin. Antibody against the RAGE, the receptor for AGE, attenuated this decrease in synthesis, indicating that the response was partially mediated by RAGE. In addition, antibody against epidermal growth factor (EGF) attenuated the decrease in type I procollagen mRNA and type I collagen induced by β2m-AGE, suggesting that EGF acts as an intermediate factor. These findings support the hypothesis that β2m-AGE actively participates in connective tissue and bone remodeling via a pathway involving fibroblast RAGE, and at least one interposed mediator, the growth factor EGF. β2-microglobulin modified with advanced glycation end products modulates collagen synthesis by human fibroblasts. β2-microglobulin amyloidosis (Aβ2m) is a serious complication for patients undergoing long-term dialysis. β2-microglobulin modified with advanced glycation end products (β2m-AGE) is a major component of the amyloid in Aβ2m. It is not completely understood whether β2m-AGE plays an active role in the pathogenesis of Aβ2m, or if its presence is a secondary event of the disease. β2-microglobulin amyloid is mainly located in tendon and osteo-articular structures that are rich in collagen, and local fibroblasts constitute the principal cell population in the synthesis and metabolism of collagen. Recent identification of AGE binding proteins on human fibroblasts lead to the hypothesis that the fibroblast may be a target for the biological action of β2m-AGE. The present study demonstrated that two human fibroblast cell lines exhibited a decrease in procollagen type I mRNA and type I collagen synthesis after exposure to β2m-AGE for 72 hours. Similar results were observed using AGE-modified albumin. Antibody against the RAGE, the receptor for AGE, attenuated this decrease in synthesis, indicating that the response was partially mediated by RAGE. In addition, antibody against epidermal growth factor (EGF) attenuated the decrease in type I procollagen mRNA and type I collagen induced by β2m-AGE, suggesting that EGF acts as an intermediate factor. These findings support the hypothesis that β2m-AGE actively participates in connective tissue and bone remodeling via a pathway involving fibroblast RAGE, and at least one interposed mediator, the growth factor EGF. Β2-microglobulin amyloidosis Β2-microglobulin Β2-microglobulin-advanced glycation end products bovine serum albumin Dulbecco's modified Eagle's medium epidermal growth factor fetal calf serum human serum albumin interleukin phosphate buffered saline receptor for advanced glycation end products tumor necrosis factor-α. β2-microglobulin amyloidosis (Aβ2m) is a progressive and incapacitating comorbid condition affecting patients with chronic renal failure1.Kay J. Beta 2-microglobulin amyloidosis.Amyloid Int J Exp Clin Invest. 1997; 4: 187-211Google Scholar, 2.Drueke T. Touam M. Zingraff J. Dialysis-associated amyloidosis.Adv Renal Repl Ther. 1995; 2: 24-39Google Scholar, 3.Gejyo F. Homma N. Arakawa M. Long-term complications of dialysis: Pathogenic factors with special reference to amyloidosis.Kidney Int. 1993; 43: S78-S82Google Scholar. Amyloid deposits are predominantly found in tendons, synovium, and bone, resulting in shoulder periarthritis, hand flexor tenosynovitis, carpal tunnel syndrome, destructive spondyloarthropathy, and cystic bone lesions1.Kay J. Beta 2-microglobulin amyloidosis.Amyloid Int J Exp Clin Invest. 1997; 4: 187-211Google Scholar, 2.Drueke T. Touam M. Zingraff J. Dialysis-associated amyloidosis.Adv Renal Repl Ther. 1995; 2: 24-39Google Scholar, 3.Gejyo F. Homma N. Arakawa M. Long-term complications of dialysis: Pathogenic factors with special reference to amyloidosis.Kidney Int. 1993; 43: S78-S82Google Scholar. Recent biochemical and immunochemical studies have demonstrated that β2-microglobulin modified with advanced glycation end products (β2m-AGE) is a major component of the amyloid fibrils in Aβ2m4.Miyata T. Oda O. Inagi R. Iida Y. Araki N. Yamada N. β2-microglobulin modified with advanced glycation end products is a major component of hemodialysis-associated amyloidosis.J Clin Invest. 1993; 92: 1243-1252Crossref PubMed Scopus (398) Google Scholar. However, it is not completely understood whether β2m-AGE actively contributes to osteo-articular destruction or are innocent bystanders accumulating during the course of the disease5.Sprague S.M. Is β2-microglobulin a mediator of bone disease?.Kidney Int. 1995; 47: 1-6Abstract Full Text PDF PubMed Scopus (23) Google Scholar. Supporting the hypothesis that β2m-AGE is an indirect provocateur is the observation that it stimulates production of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 by macrophages6.Miyata T. Inagi R. Iida Y. Sato M. Yamada N. Oda O. Maeda K. Seo H. Involvement of β2-microglobulin modified with advanced glycation end products in the pathogenesis of hemodialysis-associated amyloidosis.J Clin Invest. 1994; 93: 521-528Crossref PubMed Scopus (339) Google Scholar,7.Iida Y. Miyata T. Inagi R. Sugiyama S. Maeda K. Beta 2-microglobulin modified with advanced glycation end products induces interleukin-6 from human macrophages: Role in the pathogenesis of hemodialysis-associated amyloidosis.Biochem Biophys Res Commun. 1994; 201: 1233-1241Google Scholar. Indeed, recent studies have suggested that enhanced interaction of β2m-AGE with macrophage AGE receptor (RAGE) results in increased macrophage migration and activation, as measured by increased production of TNF-α mRNA and protein8.Miyata T. Hori O. Zhang J.H. Yan S.D. Ferran L. Iida Y. Schmidt A.M. The receptor for advanced glycation end products (RAGE) is a central mediator of the interaction of AGE-β2-microglobulin with human mononuclear phagocytes via an oxidant-sensitive pathway.J Clin Invest. 1996; 98: 1088-1094Crossref PubMed Scopus (265) Google Scholar, 9.Schmidt A.M. Vianna M. Gerlach M. Brett J. Ryanj Kao J. Esposito C. Hegarty H. Hurley W. Clauss M. Wang F. Pan Y.C. Tsang T.C. Stern D. Isolation and characterization of binding proteins for advanced glycosylation end products from lung tissue which are present on the endothelial cell surface.J Biol Chem. 1992; 267: 14987-14997Abstract Full Text PDF PubMed Google Scholar, 10.Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C. Elliston K. Stern D. Shaw A. Cloning and expression of RAGE: A cell surface receptor for advanced glycosylation end products of proteins.J Biol Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar. Tendon and osteo-articular tissues, the preferential sites of β2m amyloid deposition, are rich in collagen. In normal life processes and during inflammation, fibroblasts and related mesenchymal cells are the principal site for collagen synthesis and metabolism. Previous identification of AGE binding proteins on human fibroblasts11.Kirstein M. van Deventer S. Vlassara H. Advanced glycosylation end product (AGE) binding to its specific receptor stimulates increase in EGF and EGF receptor mRNAs: Role in tissue remodeling.J Cell Biochem. 1990; 14E (abstract): 76Google Scholar lead us to postulate that fibroblasts may be a direct target for the agonist actions of β2m-AGE. Pursuant to this hypothesis, we evaluated the capacity of β2m-AGE to affect collagen synthesis by human skin fibroblasts. Reported herein is that β2m-AGE decreased synthesis of type I collagen in a dose- and time-dependent manner. The inhibition of this effect by antibody against RAGE suggested a receptor-mediated effect. These findings support a novel agonist role for β2m-AGE that may adversely influence repair and remodeling of connective tissues in Aβ2m. A normal human, non-fetal skin fibroblast cell line (GM05757A) was obtained from NIGMS Human Genetic Mutant Cell Repository (Camden, NJ, USA). The cells were grown to confluence at 37°C in 95% air and 5% CO2 in Dulbecco's modified Eagle medium; Nutrient Mixture F-12 (DMEM/F-12) contains L-glutamine (GIBCO Life Technologies, Grand Island, NY, USA) with 10% (vol/vol) fetal calf serum (FCS; GIBCO), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, St. Louis, MO, USA). Confluent cultures of fibroblasts were harvested by incubation with 0.1% trypsin/0.02% EDTA (Sigma) and subcultured at a 1:3 split ratio. Cells between passages 7 to 10 were harvested for experiments by incubation with 0.1% trypsin/0.02% EDTA for three minutes at 37°C to 167°C, followed by extensive washing in DMEM/F-12 and resuspension in DMEM/F-12 containing 10% FCS at a concentration of 106 cells/ml. Since phenotypic modulation is a concern with cell passage, we compared the response to β2m-AGE between two strains of human skin fibroblast (GM05757A and GM03377C) and between different passages (passages 4, 8, and 12). Culture conditions and harvesting procedures were as described earlier. Advanced glycation end product-modified proteins were prepared in vitro as previously described12.Vlassara H. Brownlee M. Cerami A. High-affinity-receptor-mediated uptake and degradation of glucose-modified proteins: A potential mechanism for the removal of senescent macromolecules.Proc Natl Acad Sci USA. 1985; 82: 5588-5592Crossref PubMed Scopus (284) Google Scholar,13.Hou F.F. Chertow G.M. Kay J. Boyce J. Lazarus J.M. Braatz J.A. Owen Jr, W.F. The interaction between β2-microglobulin and advanced glycation end products in the development of dialysis related-amyloidosis.Kidney Int. 1997; 51: 1514-1519Abstract Full Text PDF PubMed Scopus (58) Google Scholar. Briefly, 1.75 mg/ml of purified normal human β2m (Cortex Biochem, San Leandro, CA, USA), normal human serum albumin (HSA; Sigma), or bovine serum albumin (BSA; Sigma) were incubated at 37°C for 30 days with 100 mm D-glucose in 100 mm phosphate buffer containing 200 U/ml penicillin, 70 μg/ml gentamicin, and 1.5 mm PMSF (Sigma). Samples incubated in an identical manner in the absence of glucose were used as controls. After incubation, all samples were dialyzed against phosphate buffer (pH 7.4). AGE-modified proteins were characterized by ELISA using anti-KLH-AGE antibody at 1:2500 dilution (gift of Dr. John W. Baynes, University of South Carolina, Columbia, SC, USA) and fluorospectrometry as described4.Miyata T. Oda O. Inagi R. Iida Y. Araki N. Yamada N. β2-microglobulin modified with advanced glycation end products is a major component of hemodialysis-associated amyloidosis.J Clin Invest. 1993; 92: 1243-1252Crossref PubMed Scopus (398) Google Scholar, 13.Hou F.F. Chertow G.M. Kay J. Boyce J. Lazarus J.M. Braatz J.A. Owen Jr, W.F. The interaction between β2-microglobulin and advanced glycation end products in the development of dialysis related-amyloidosis.Kidney Int. 1997; 51: 1514-1519Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 14.Araki N. Ueno N. Chakrabarti B. Morino Y. Horiuchi S. Immunochemical evidence for the presence of advanced glycation end products in human lens proteins and its positive correlation with aging.J Biol Chem. 1992; 267: 10211-10214Abstract Full Text PDF PubMed Google Scholar. The AGE content was quantitated by fluorospectrometry. For β2m-AGE, it was 26.4 units/mg protein; for HSA-AGE 26.1 units/mg protein; for BSA-AGE 26.0 units/mg protein; and for β2m, HSA and BSA controls 0.9 units/mg protein. The endotoxin content in all samples was measured by Limulus amoebocyte lysate assay (E-toxate, Sigma) and found to be < 0.2 ng/ml. The presence of AGE receptors (RAGE) on fibroblasts was assessed by immunoblot analysis as described15.Schmidt A.M. Yan S.D. Brett J. Mora R. Nowygrod R. Stern D. Regulation of human mononuclear phagocyte migration by cell surface-binding protein for advanced glycation end products.J Clin Invest. 1993; 92: 2155-2168Crossref Scopus (264) Google Scholar. Briefly, 108 dispersed fibroblasts were solubilized in Tris-HCl (20 mm, pH 7.4), NaCl (0.1 m), octyl-β-glucoside (1%), and PMSF (1 mm), and the lysate eluted through a 10 ml hydroxylapatite column (IBF Biotechnics, Columbia, MD, USA). The column was washed with Tris-buffered saline (Tris, 20 mm, pH 7.4; NaCl, 0.1 m) containing octyl-β-glucoside (0.1%), and the protein extract eluted in the same buffer with the NaCl concentration increased to 1.0 m. The membrane-enriched extract was subjected to SDS-PAGE (10%) followed by Western blotting using the Blotto procedure16.Johnson D. Gautsch J. Sportsman J. Elder J. Improved technique utilizing non-fat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose.Gene Anal Tech. 1984; 1: 3-8Crossref Scopus (1159) Google Scholar. After incubation with 45 μg/ml of rabbit anti-human RAGE IgG8.Miyata T. Hori O. Zhang J.H. Yan S.D. Ferran L. Iida Y. Schmidt A.M. The receptor for advanced glycation end products (RAGE) is a central mediator of the interaction of AGE-β2-microglobulin with human mononuclear phagocytes via an oxidant-sensitive pathway.J Clin Invest. 1996; 98: 1088-1094Crossref PubMed Scopus (265) Google Scholar or nonimmune rabbit IgG (Sigma) for 60 minutes at room temperature, sites of IgG binding were visualized by chemiluminescence using peroxidase-conjugated secondary antibody (Amersham Corp., Arlington Heights, IL, USA). Fibroblasts were allowed to adhere to 12-mm coverslips for 24 hours at 37°C. The cells were washed with PBS (pH 7.4) and fixed in 2% paraformaldehyde for 15 minutes at 4°C. The coverslips were incubated wtih rabbit anti-human RAGE antibody (33.5 μg/ml) for 45 minutes at 37°C, washed with PBS (pH 7.4), and incubated with FITC-conjugated goat anti-rabbit IgG (Sigma) at 1:1000 dilution for 45 minutes at 37°C. Nonimmune rabbit IgG replaced anti-RAGE IgG at the same concentration as a negative control. The cells were observed using phase contrast and fluorescence microscopy, then photographed (Nikon, Labophot-2, Japan). Fibroblasts were plated onto 75 cm2 flasks in DMEM/F-12 with 10% FCS and grown to confluence. The monolayers were washed three times with Hanks balanced salt solution without divalent cations (HBSS-) to remove traces of serum. For analysis of dose-dependence, the monolayers were cultured for 72 hours with incremental concentrations of β2m-AGE or unmodified β2m in serum-free DMEM/F-12 with 0.2% lactalbumin (DMEM-LH; Gibco Life Technologies, Grand Island, NY, USA). To evaluate the time-dependence of the response, cells were incubated with 50 μg/ml β2m-AGE or unmodified β2m for different durations. Negative controls were incubated with DMEM-LH alone, and plates incubated with 60 ng/ml of recombinant human IL-1β (Genzyme, Cambridge, MA, USA) served as positive controls. To compare the effect of various AGE-modified proteins, fibroblasts were incubated with 50 μg/ml of β2m-AGE, HSA-AGE, BSA-AGE, or their unmodified forms for 72 hours. At the endpoints of incubation, the supernatants were collected for quantitation of type I collagen. The corresponding monolayers were washed and trypsinized. The cells were counted, and Trypan blue dye exclusion was performed to test cell viability. Greater than 98% of the cells were Trypan blue excluding in all experiments. Then cells were lysed in 1 ml TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OH, USA). The lysate was collected, and phase separated by the addition of 0.2 ml of chloroform. The aqueous phase was used for total RNA isolation, and the interphase and organic phases were used for protein analysis as described17.Chomczynski P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples.Bio Tech. 1993; 15: 532-537Google Scholar. For steady-state mRNA analysis, 5 to 10 μg of total RNA from representative samples were electrophoresed on a 1.2% agarose/6% formaldehyde gel and transferred onto a nylon membrane18.Colige A. Lambert C.A. Nusgens B.V. Lapiere C.M. Effect of cell-cell and cell-matrix interactions on the response of fibroblasts to epidermal growth factor in vitro.Biochem J. 1992; 285: 215-221Crossref PubMed Google Scholar. For slot-blotting, 5 to 8 μg of total RNA was denatured at 68°C for 15 minutes in 20% formaldehyde and 6× SSC, serially diluted at the desired concentration, and slotted onto a nylon membrane using the BIO-DOT SF apparatus (BIO-RAD, Hercules, CA, USA). The filters were baked at 80°C and prehybridized6.Miyata T. Inagi R. Iida Y. Sato M. Yamada N. Oda O. Maeda K. Seo H. Involvement of β2-microglobulin modified with advanced glycation end products in the pathogenesis of hemodialysis-associated amyloidosis.J Clin Invest. 1994; 93: 521-528Crossref PubMed Scopus (339) Google Scholar. After the denatured [32P] labeled probe for human type I collagen α1 (106 cpm/ml; ATCC, Rockville, MD, USA) was added directly to the prehybridization fluid, hybridization was performed overnight at 42°C. The filters were washed and exposed to Kodak X-Omat. The densitometric scanning of the slot-blot autoradiograms was performed using a densitometer (Molecular Dynamics, Sunnyvale, CA, USA). The results were corrected by the amount of RNA loaded and expressed as density unit/μg RNA. Type I collagen in the cell supernatants and in the extracts of the cell layer were quantitated by ELISA using the ELISA Starter Kit (Pierce, Rockford, IL, USA). Ninety-six-well polystyrene plates were coated with monoclonal anti-human type I collagen antibody (ICN Biomedicals, Inc., Aurora, OH, USA) at 10 μg/ml by incubation for 12 hours at 4°C. Each well was washed three times with washing buffer, and nonspecific binding sites were blocked with blocking buffer. Human type I collagen (Gibco Life Technologies) at incremental concentrations, properly diluted samples were added, and the plates were incubated for 60 minutes at room temperature. After washing the plates, a polyclonal rabbit anti-human type I collagen antibody (ICN Biomedicals, Inc.) at 1:1000 dilution was incubated with the plates for one hour at room temperature. The wells were then washed, incubated with a goat anti-rabbit IgG-peroxidase antibody (Pierce) at 1:5000 dilution, followed by addition of the substrate 2,2′-azino-di-3-ethylbenzthiazoline-6-sulfonic acid. The absorbance at 405 nm was measured on a micro-ELISA plate reader (Titertek Multiskan, Mcc/340). For control studies, blocking buffer alone was used instead of samples, and nonimmune rabbit IgG was substituted for rabbit anti-human type I collagen. The amount of type I collagen measured was corrected for the number of cells harvested. To determine the effect of anti-RAGE IgG on type I collagen synthesis, fibroblasts were plated in six-well plates, grown to confluence, and preincubated with serum-free DMEM-LH containing 50 μg/ml of rabbit anti-human RAGE or nonimmune rabbit IgG for two hours at 37°C. This concentration was based on preliminary experiments demonstrating a plateau in activity at concentrations ≥ 50 μg/ml (data not shown). The monolayers were washed three times with DMEM-LH and then incubated with β2m-AGE (50 μg/ml). After 72 hours incubation, the supernatants were collected, and the total RNA and protein were extracted for analysis. To determine the effect of anti-EGF IgG, confluent fibroblasts in six-well plates were incubated with serum-free DMEM-LH containing 50 μg/ml of β2m-AGE and 50 μg/ml of goat neutralizing antibody for human epidermal growth factor (EGF; R&D System, Minneapolis, MN, USA) or 50 μg/ml of β2m-AGE plus 50 μg/ml of nonimmune goat IgG (Sigma). After 72 hours of incubation, the supernatants were collected and the total RNA and protein were extracted. In separate experiments, fibroblasts were preincubated with goat anti-human EGF neutralizing antibody (50 μg/ml) or nonimmune goat IgG (50 μg/ml) for two hours. The cells were washed with DMEM-LH and incubated for 72 hours with β2m-AGE (50 μg/ml). The supernatants were collected, and the total RNA and protein were extracted. To quantitate IL-1β synthesis from fibroblasts, washed monolayers were incubated with β2m-AGE or HSA-AGE (50 μg/ml) for 12 to 72 hours at 37°C. At the endpoint of incubation, the supernatant was collected, and IL-1β was quantitated by using the Predicta Human IL-1β ELISA Kit (Genzyme) with detection limits of 3.0 pg/ml. To determine the effect of anti-IL-1β, fibroblasts were simultaneously incubated with serum-free DMEM-LH containing 50 μg/ml of β2m-AGE and 25 to 250 μg/ml of rabbit neutralizing antibody for human IL-1β (Genzyme) for 72 hours. Type I procollagen mRNA and type I collagen were analyzed as described above. All experiments were performed in triplicate. Continuous variables, expressed as mean ± sd, were compared using analysis of variance (ANOVA). Multiplicative terms were included to evaluate for interaction among explanatory variables. The Student-Newman-Keuls procedure was used to evaluate pairwise comparisons. Two-tailed P values < 0.05 were considered statistically significant. Statistical analyses were conducted with SAS (The SAS Institute, Cary, NC, USA). To ascertain the presence of RAGE on human fibroblasts, membrane-enriched fractions were generated from human fibroblasts and were analyzed for immunoreactive material using polyclonal anti-human RAGE IgG. Two bands were visible by immunoblot analysis, corresponding to relative molecular masses of 32 and 50 kDa Figure 1, lane 1). Irrelevant antibody was without immunoreactivity Figure 1, lane 2). The presence of two different size immunoreactive RAGE polypeptides in the membrane fraction probably reflects post-translational processing/cleavage, in that multiple molecular mass forms of RAGE are observed in cells transfected with full-length RAGE cDNA10.Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C. Elliston K. Stern D. Shaw A. Cloning and expression of RAGE: A cell surface receptor for advanced glycosylation end products of proteins.J Biol Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar. Immunofluorescence studies demonstrated a cell surface pattern of staining for RAGE. Fibroblasts incubated with anti-human RAGE IgG showed a diffuse pattern of membrane staining Figure 2a) as compared with nonimmune rabbit IgG control Figure 2b).Figure 2Detection of RAGE on human fibroblasts by indirect immunofluorescence. Fibroblasts were stained with rabbit anti-RAGE IgG (lane A) or nonimmune rabbit IgG (lane B) as described in the text. Panels 1: Immunofluorescence. Panels 2: Phase contrast. The experiment was repeated two times with identical findings (bar = 10 μm).View Large Image Figure ViewerDownload (PPT) Type I procollagen mRNA was detectable in cells incubated with medium alone (921 ± 126 density units/μg RNA), and was taken as 100% (the negative controls). After a 72 hour incubation with β2m-AGE (50 μg/ml), type I procollagen mRNA levels decreased to 66 ± 9% Figure 3, lane 4) of that observed with cells incubated in medium alone the negative controls; Figure 3, lane 1). In contrast, cells incubated with β2m showed no significant change in level of mRNA coding for type I procollagen 104 ± 8% of the negative controls; Figure 3, lane 3). β-actin mRNA levels were comparable for fibroblasts incubated with medium alone, β2m, and β2m-AGE Figure 3. Fibroblasts were incubated with IL-1β as a positive control for down-regulation of collagen biosynthesis Figure 3, lane 2). The decrease in type I procollagen mRNA was dependent upon the concentration of β2m-AGE contained in the medium Figure 4a). Steady-state mRNA levels were reduced by 25% upon incubation with 0.5 μg/ml of β2m-AGE and 40% upon incubation with 50 μg/ml. A decrement in steady-state mRNA for type I procollagen was not detectable before 48 hours incubation with β2m-AGE Figure 4b). To determine whether the observed decrease in type I procollagen mRNA was associated with a decrease in the synthesis of collagen, type I collagen levels were quantitated both in the supernatants and in the extracts of the cell layer. In fibroblasts incubated with medium alone, the quantities of type I collagen in the supernatants and in the extracts of the cell layer were 195 ± 22 ng/104 cell and 181 ± 16 ng/104 cell, respectively, and were taken as 100% Figure 5. The decrease in type I collagen levels corresponded to the decrement in steady-state mRNA. The quantities of type I collagen in the supernatants and the extracts of cell layer were reduced by 51% and 67% upon incubation with 50 μg/ml of β2m-AGE. Although the magnitude of the absolute response to β2m-AGE was somewhat variable in different lines and passages (cell line GM05757A had decreased type I procollagen mRNA of 61.0 ± 8.2%, 65.0 ± 7.8%, and 67.1 ± 6.2% at passages 4, 8 and 12; cell line GM03377C had decreased type I procollagen mRNA of 52.8 ± 5.8%, 60.5 ± 6.6%, and 61.2 ± 7.2% at passages 4, 8 and 12, N = 3), the decrease in type I procollagen mRNA expression was a uniform phenomenon. To test the specificity of the effect of β2m-AGE on type I collagen synthesis, fibroblasts were incubated with other AGE-modified proteins. No significant difference was seen in type I procollagen mRNA or type I collagen levels between cells treated with β2m-AGE, HSA-AGE and BSA-AGE Figure 6. Unmodified HSA or BSA had no effect on type I collagen synthesis. Preincubation of the human fibroblast cultures with anti-human RAGE IgG prior to addition of β2m-AGE attenuated the β2m-AGE-induced decrease of type I procollagen mRNA levels (Figure 7, lanes 2 and 4, respectively, and Table 1). There was no change in procollagen mRNA levels when fibroblasts were pretreated with nonimmune rabbit IgG (Figure 7, lane 3, and Table 1). Similar effects were observed for type I collagen synthesis by the fibroblast cultures treated with anti-RAGE IgG or nonimmune rabbit IgG, respectively (Table 1).Table 1Effect of anti-RAGE, anti-EGF, and anti-IL-1β on β2m-AGE induced decrease of type I procollagen mRNA and type I collagen synthesisaView Large Image Figure ViewerDownload (PPT) Open table in a new tab The simultaneous addition of anti-EGF IgG to the incubation solution blunted the β2m-AGE-induced decrease in both type I procollagen mRNA and type I collagen levels (Table 1). In contrast, addition of nonimmune goat IgG had no effect on β2m-AGE-induced decrease in type I procollagen mRNA. Instead, if fibroblasts were pretreated with anti-EGF IgG, washed, and followed by incubation with β2m-AGE, no significant change could be seen in type I procollagen mRNA expression, or type I collagen synthesis in comparison to cells exposed to β2m-AGE without pretreatment (data not shown). Neither anti-human RAGE IgG nor anti-human EGF IgG affected type I procollagen mRNA levels or type I collagen synthesis in fibroblasts incubated with medium alone or with β2m (data not shown). Using an ELISA with a limit of detection of 3.0 pg/ml, no IL-1β was detected in the supernatants of fibroblasts cultured for 12 to 72 hours with β2m-AGE (50 μg/ml) or HSA-AGE (50 μg/ml). Furthermore, the addition of anti-IL-1β antibody did not alter the β2m-AGE induced decrease in type I collagen synthesis (Table 1). The amyloidosis of chronic renal failure is unique, consisting of β2m and localizing mainly in bones and synovium1.Kay J. Beta 2-microglobulin amyloidosis.Amyloid Int J Exp Clin Invest. 1997; 4: 187-211Google Scholar, 2.Drueke T. Touam M. Zingraff J. Dialysis-associated amyloidosis.Adv Renal Repl Ther. 1995; 2: 24-39Google Scholar, 3.Gejyo F. Homma N. Arakawa M. Long-term complications of dialysis: Pathogenic factors with special reference to amyloidosis.Kidney Int. 1993; 43: S78-S82Google Scholar. Subchondral bone resorption and growing bone cysts are hallmarks of the clinical presentation of Aβ2m. Among maintenance dialysis patients, the signs of hyperparathyroidism decrease with time, but the size and number of subchondral bone cysts increase. Biopsy of these cysts yields β2m amyloid19.Gielen J.L. van Holsbeeck M.T. Hauglustaine D. Verresen L. Verbeken E. Baert A.L. Meeus L. Vandevoorde P. Michiolsen P. Coral A. Growing bone cysts in long-term hemodialysis.Skel Radiol. 1990; 19: 43-49Crossref PubMed Scopus (32) Google Scholar. Controversy exists about whether β2m deposition plays an active role in inducing bone destruction or if it is only a secondary event of the disease5.Sprague S.M. Is β2-microglobulin a mediator of bone disease?.Kidney Int. 1995; 47: 1-6Abstract Full Text PDF PubMed Scopus (23) Google Scholar. In favor of active participation is the observation that β2m increases collagenase synthesis by cultured rabbit synovial cells, suggesting an autocrine function for β2m20.Brinckerhoff C.E. Mttchell T.I. Karmilowicz M.J. Kluve-Beckerman B. Benson M.D. Autocrine induction of collagenase by serum amyloid A-like and β2-microglobulin-like proteins.Science. 1989; 243: 655-657Crossref
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