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• W37041364 abstract "We have previously shown that glycosaminoglycan (GAG) storage in animal models of the mucopolysaccharidoses (MPS) leads to inflammation and apoptosis within cartilage. We have now extended these findings to synovial tissue and further explored the mechanism underlying GAG-mediated disease. Analysis of MPS rats, cats, and/or dogs revealed that MPS synovial fibroblasts and fluid displayed elevated expression of numerous inflammatory molecules, including several proteins important for lipopolysaccharide signaling (eg, Toll-like receptor 4 and lipoprotein-binding protein). The expression of tumor necrosis factor, in particular, was elevated up to 50-fold, leading to up-regulation of the osteoclast survival factor, receptor activator of nuclear factor-κB ligand, and the appearance of multinucleated osteoclast-like cells in the MPS bone marrow. Treatment of normal synovial fibroblasts with GAGs also led to production of the prosurvival lipid sphingosine-1-phosphate, resulting in enhanced cell proliferation, consistent with the hyperplastic synovial tissue observed in MPS patients. In contrast, GAG treatment of normal chondrocytes led to production of the proapoptotic lipid ceramide, confirming the enhanced cell death we had previously observed in MPS cartilage. These findings have important implications for the pathogenesis and treatment of MPS and have further defined the mechanism of GAG-stimulated disease. We have previously shown that glycosaminoglycan (GAG) storage in animal models of the mucopolysaccharidoses (MPS) leads to inflammation and apoptosis within cartilage. We have now extended these findings to synovial tissue and further explored the mechanism underlying GAG-mediated disease. Analysis of MPS rats, cats, and/or dogs revealed that MPS synovial fibroblasts and fluid displayed elevated expression of numerous inflammatory molecules, including several proteins important for lipopolysaccharide signaling (eg, Toll-like receptor 4 and lipoprotein-binding protein). The expression of tumor necrosis factor, in particular, was elevated up to 50-fold, leading to up-regulation of the osteoclast survival factor, receptor activator of nuclear factor-κB ligand, and the appearance of multinucleated osteoclast-like cells in the MPS bone marrow. Treatment of normal synovial fibroblasts with GAGs also led to production of the prosurvival lipid sphingosine-1-phosphate, resulting in enhanced cell proliferation, consistent with the hyperplastic synovial tissue observed in MPS patients. In contrast, GAG treatment of normal chondrocytes led to production of the proapoptotic lipid ceramide, confirming the enhanced cell death we had previously observed in MPS cartilage. These findings have important implications for the pathogenesis and treatment of MPS and have further defined the mechanism of GAG-stimulated disease. The mucopolysaccharidoses (MPS) are a group of 11 distinct lysosomal storage disorders attributable to defective catabolism of glycosaminoglycans (GAGs), leading to severe joint and bone disease.1Neufeld EF Muenzer J The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3421-3452Google Scholar Our previous studies in MPS animal models showed that inflammation is a critical aspect of these disorders, secondary to GAG accumulation.2Simonaro CM Haskins ME Schuchman EH Articular chondrocytes from animals with a dermatan sulfate storage disease undergo a high rate of apoptosis and release nitric oxide and inflammatory cytokines: a possible mechanism underlying degenerative joint disease in the mucopolysaccharidoses.Lab Invest. 2001; 81: 1319-1328Crossref PubMed Scopus (118) Google Scholar, 3Simonaro CM D'Angelo M Haskins ME Schuchman EH Joint and bone disease in mucopolysaccharidoses VI and VII: identification of new therapeutic targets and biomarkers using animal models.Pediatr Res. 2005; 57: 701-707Crossref PubMed Scopus (132) Google Scholar As part of this inflammatory cascade, tumor necrosis factor (TNF-α) and other inflammatory cytokines [eg, interleukin (IL-1β)] are released from chondrocytes, resulting in apoptosis. In addition, matrix metalloproteinases (MMPs) are released, contributing to the joint and bone destruction. Lipopolysaccharide (LPS), a molecule that is structurally similar to GAGs, stimulates a signaling pathway that is pivotal to the pathogenesis of many chronic inflammatory diseases, including rheumatoid arthritis (RA).4De Vries-Bouwstra JK, Goekoop-Ruiterman YP, Wesoly J, Hulsmans HJ, de Craen AJ, Breedveld FC, Dijkmans BA, Allaart CF, Huizinga TW: Ex vivo IL1 receptor antagonist production upon LPS stimulation is associated with development of RA and with greater progression of joint damage. Ann Rheum Dis 2007, Jan 12:[Epub ahead of print]Google Scholar LPS signaling occurs through Toll-like receptor 4 (TLR4) and can result in the release of TNF-α and other proinflammatory cytokines. Based on these observations, we put forth a hypothesis suggesting that GAG accumulation in MPS connective tissues induces TNF-α release and inflammation through stimulation of the LPS signaling pathway.3Simonaro CM D'Angelo M Haskins ME Schuchman EH Joint and bone disease in mucopolysaccharidoses VI and VII: identification of new therapeutic targets and biomarkers using animal models.Pediatr Res. 2005; 57: 701-707Crossref PubMed Scopus (132) Google Scholar Notably, although TNF-α promotes apoptosis in cartilage,5Goldring SR Pathogenesis of bone erosions in rheumatoid arthritis.Curr Opin Rheumatol. 2002; 4: 406-410Crossref Scopus (67) Google Scholar this cytokine has a proliferative effect in synovial cells.6Hemmings BA Akt signaling: linking membrane events to life and death decisions.Science. 1997; 275: 628-630Crossref PubMed Scopus (436) Google Scholar This would be consistent with the hyperplasia observed in MPS synovial tissue.7Auclair D Hein LK Hopwood JJ Byers S Intra-articular enzyme administration for joint disease in feline mucopolysaccharidosis VI: enzyme dose and interval.Pediatr Res. 2006; 59: 538-543Crossref PubMed Scopus (34) Google Scholar, 8Pastores GM Meere PA Musculoskeletal complications associated with lysosomal storage disorders: Gaucher disease and Hurler-Scheie syndrome (mucopolysaccharidosis type I).Curr Opin Rheumatol. 2005; 17: 70-78Crossref PubMed Scopus (62) Google Scholar Abnormal GAG metabolism has been observed in several common autoimmune diseases, including RA, scleroderma, systemic lupus erythematosus, and others.9Parildar Z Uslu R Tanyalcin T Doganavsargil E Kutay F The urinary excretion of glycosaminoglycans and heparan sulphate in lupus nephritis.Clin Rheumatol. 2002; 21: 284-288Crossref PubMed Scopus (6) Google Scholar, 10Edward M Fitzgerald L Thind C Leman J Burden AD Cutaneous mucinosis associated with dermatomyositis and nephrogenic fibrosing dermopathy: fibroblast hyaluronan synthesis and the effect of patient serum.Br J Dermatol. 2007; 156: 473-479Crossref PubMed Scopus (37) Google Scholar For example, patients with RA have elevated concentrations of GAGs in blood and synovial fluid, and the destruction of joints in these patients correlates positively with high GAG levels in synovial fluid. Injection of GAGs into normal mice also induces arthritis, tendosynovitis, dermatitis, and other pathological conditions, and it has been suggested that GAGs stimulate expansion of inflammatory cells, provoking autoimmune dysfunction.11Wang JY Roehrl MH Glycosaminoglycans are a potential cause of rheumatoid arthritis.Proc Natl Acad Sci USA. 2002; 99: 14362-14367Crossref PubMed Scopus (83) Google Scholar Thus, the aim of the current study was to characterize further the mechanism of GAG-mediated lesions in the MPS disorders, with the long-term goal of using these findings to develop novel treatment strategies for these and other GAG-mediated diseases. Gene and protein expression analysis was performed on synovial fibroblasts obtained from rats with one MPS type (MPS VI, Maroteaux-Lamy disease), revealing a markedly abnormal, proinflammatory expression pattern. Of note, several molecules important for LPS signaling were elevated in the MPS cells [eg, LPS-binding protein (LBP), TLR4, CD14, MyD88]. We also investigated how GAG storage influenced the levels of two important signaling lipids, ceramide and sphingosine-1-phosphate (S1P), known mediators of LPS activation. GAG treatment of chondrocytes led to elevation of the proapoptotic lipid, ceramide, consistent with enhanced apoptosis in these cells.2Simonaro CM Haskins ME Schuchman EH Articular chondrocytes from animals with a dermatan sulfate storage disease undergo a high rate of apoptosis and release nitric oxide and inflammatory cytokines: a possible mechanism underlying degenerative joint disease in the mucopolysaccharidoses.Lab Invest. 2001; 81: 1319-1328Crossref PubMed Scopus (118) Google Scholar In contrast, a decrease in ceramide and elevated production of the prosurvival lipid, S1P, was observed in MPS synovial cells, leading to an enhanced proliferation rate and contributing to the hyperplastic MPS synovial membranes. We further studied the effects of elevated TNF-α expression on MPS joint and bone pathogenesis. TNF-α and receptor activator of nuclear factor (NF-κB) ligand (RANKL) stimulate osteoclast differentiation in RA.12Shigeyama Y Pap T Kunzler P Simmen BR Gay RE Gay S Expression of osteoclast differentiation factor in rheumatoid arthritis.Arthritis Rheum. 2000; 43: 2523-2530Crossref PubMed Scopus (203) Google Scholar RANKL is essential for osteoclast differentiation, expressed on T cells and fibroblasts within inflamed synovial tissue, and regulated by proinflammatory cytokines. We found that RANKL expression and activity were markedly elevated in MPS synovial tissues and bone marrow. In addition, MPS bone marrow cultures had tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclast-like cells, consistent with the osteopenia previously observed in these animals.13Nuttall JD Brumfield LK Fazzalari NL Hopwood JJ Byers S Histomorphometric analysis of the tibial growth plate in a feline model of mucopolysaccharidosis type VI.Calcif Tissue Int. 1999; 65: 47-52Crossref PubMed Scopus (42) Google Scholar Lastly, in MPS animals we previously showed that MMPs play a crucial role in the degradation of the articular cartilage.3Simonaro CM D'Angelo M Haskins ME Schuchman EH Joint and bone disease in mucopolysaccharidoses VI and VII: identification of new therapeutic targets and biomarkers using animal models.Pediatr Res. 2005; 57: 701-707Crossref PubMed Scopus (132) Google Scholar This is also likely attributable to elevated TNF-α, because this cytokine has been shown to promote cartilage degradation by stimulating the synthesis and release of MMPs.14Migita K Eguchi K Kawabe Y Ichinose Y Tsukada T Aoyagi T Nakamura H Nagataki S TNF-alpha mediated expression of membrane-type matrix metalloproteinase in rheumatoid synovial fibroblasts.Immunology. 1996; 89: 553-557Crossref PubMed Scopus (81) Google Scholar In the present study we demonstrate that MMP-13 (collagenase 3) and MMP-1 are elevated in MPS synovial membranes, contributing to the pathological remodeling of the extracellular matrix in these diseases. Thus, these studies have provided new insights into the molecular mechanisms leading to MPS joint and bone disease, suggesting new therapeutic strategies that may be used to treat these and other GAG-mediated disorders. In addition, some of the molecules identified in this study may be used as biomarkers to monitor disease progression and the response to therapy, not only for the MPS disorders, but perhaps other GAG-mediated diseases as well. The MPS VI cats and rats and MPS VII dogs have been previously described.15Jezyk PF Haskins ME Patterson DF Mellman WJ Greenstein M Mucopolysaccharidosis in a cat with arylsulfatase B deficiency: a model of Maroteux-Lamy syndrome.Science. 1977; 198: 834-836Crossref PubMed Scopus (93) Google Scholar, 16Haskins ME Desnick RJ DiFerrante N Jezyk PF Patterson DF Beta-glucuronidase deficiency in a dog: a model of human mucopolysaccharidosis VII.Pediatr Res. 1984; 18: 980-998PubMed Google Scholar, 17Yoshida M Noguchi J Ikadai H Takahashi M Nagase S Arylsulfatase B-deficient mucopolysaccharidosis in rats.J Clin Invest. 1993; 91: 1099-1104Crossref PubMed Scopus (62) Google Scholar, 18Haskins M Casal M Ellinwood NM Melniczek J Mazrier H Giger U Animal models for mucopolysaccharidoses and their clinical relevance.Acta Paediatr Suppl. 2002; 9: S88-S97Crossref Scopus (36) Google Scholar Affected and control animals were raised under National Institutes of Health and United States Department of Agriculture guidelines for the care and use of animals in research. Rats were maintained at the Mount Sinai School of Medicine, whereas the larger animal models were maintained at the University of Pennsylvania School of Veterinary Medicine. The animals were housed with ad libitum food and water, 12-hour light cycles at 21°C, with 12 to 15 air changes per hour. Euthanasia was performed on cats and dogs using 80 mg/kg of sodium pentobarbital (Veterinary Laboratories, Lenexa, KS) in accordance with the American Veterinary Medical Association guidelines. Euthanasia of rats was performed using carbon dioxide inhalation. Breeding colonies were maintained under Institutional Animal Care and Use Committee-approved protocols, and the diagnosis of affected animals was performed by enzymatic assays and/or by polymerase chain reaction (PCR)-based genotyping methods.19Kunieda T Simonaro CM Yoshida M Ikadai H Levan G Desnick RJ Schuchman EH Mucopolysaccharidosis type VI in rats: isolation of cDNAs encoding arylsulfatase B, chromosomal localization of the gene, and identification of the mutation.Genomics. 1995; 29: 582-587Crossref PubMed Scopus (28) Google Scholar, 20Beratis NG Turner BM Weiss R Hirshhorn K Arylsulfatase B deficiency in Maroteaux Lamy syndrome: cellular studies and carrier identification.Pediatr Res. 1975; 9: 475-480Crossref PubMed Scopus (48) Google Scholar Proximal humeri, distal femurs, and proximal tibias were collected from age-matched normal and MPS animals, and either placed in phosphate-buffered saline (PBS) for the isolation of fibroblast-like synoviocytes (FLS) (see below) or fixed in neutral buffered 10% formalin (Sigma Chemical, St. Louis, MO) for immunohistochemical assessment (see below). For immunohistochemistry, the formalin-fixed bones were further decalcified in 8% formic acid (Sigma Chemical) for 5 days, embedded in paraffin, and sectioned (5 μm). The tissues were obtained from four animals in each age group, ranging from 12 to 20 months in cats and dogs and 9 to 12 months in rats. To establish primary FLS cultures, the synovium was sliced from the underlying cartilage and washed in minimal essential medium (Invitrogen, Carlsbad, CA). The tissue was then minced and digested with 1 mg/ml of collagenase type IV (Sigma Chemical) and DNase (Invitrogen) suspended in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) for 2 hours at 37°C. Cells were isolated by centrifugation (450 × g), and resuspended in RPMI 1640 complete medium supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine, penicillin (100 U/ml), and streptomycin (10 μg/ml), cultured in 100-mm2 plates until confluent, and passed at a density of 1 × 104/cm2. After 24 hours the media was changed to serum-free media, and the cells were either grown at 37°C or stored at −20°C. FLS cultures were generally used for experiments between passages 2 to 6. For RNA isolation the cell pellets were stored at −70°C (see below). To establish primary chondrocyte cultures, the articular cartilage was sliced from the underlying bone and washed in minimal essential medium (Invitrogen). The cartilage was minced and digested with 1% hyaluronidase for 1 hour at 37°C, washed three times with PBS containing 1 mmol/L ethylenediaminetetraacetic acid, and sequentially incubated with 2.5% trypsin (Invitrogen) and 0.01 mol/L ethylenediaminetetraacetic acid for 1 hour at 37°C, followed by further digestion at 37°C for 8 hours with 0.2% collagenase (Sigma) prepared in minimal essential medium containing 10% fetal calf serum. Cells were isolated by centrifugation (450 × g), washed twice, and plated in 96-well plates at a concentration of 7 × 104 cells/well in minimal essential medium containing 10% fetal bovine serum, 1% glutamine, penicillin (150 U/ml), and streptomycin (50 μg/ml). After 24 hours the media were changed to serum-free media, and the cells were either grown at 37°C or stored at −20°C. Samples of synovial fluid were obtained from the joints of normal dogs and cats, MPS VI cats, and MPS VII dogs. Animals were euthanized, and the joints were examined to confirm the lack or presence of gross pathological changes. Samples of synovial fluid were collected from the following joints: stifle (knee), elbow, shoulder, and coxofemoral. After isolation, synovial samples were divided into aliquots and stored frozen at −70°C until analyzed. Synovial membranes from MPS VII and normal dogs also were collected from the stifle after euthanasia, embedded in OCT media (Sakura Finetek, Torrance, CA), and stored at −70°C until analyzed. Chondrocytes and FLSs from normal and MPS VI rats were cultured for 10 minutes to 2 days in the presence or absence of 100 μg/ml of dermatan sulfate (Sigma). After the incubation period was complete, the cells were collected, centrifuged, and washed with PBS, and total cell lysates were prepared by three cycles of freeze/thaw. Lipids were extracted by mixing 150 μl of the cell lysate with chloroform:methanol (1:2, v/v), and sonicated for 5 minutes. After sonication, 100 μl of 1 mol/L NaCl and 10 μl of concentrated HCl were added, vortexed, and centrifuged at 13,000 × g for 2 minutes. The lower organic phase was transferred to a new tube, dried with a SpeedVac concentrator, and resuspended in 10 μl of ethanol. For S1P analysis, the 10-μl lipid extract in ethanol was added to 20 μl of NDA derivatization reaction mixture (25 mmol/L borate buffer, pH 9.0, containing 1.25 mmol/L each of NDA and NaCN). The reaction mixture was diluted 1:3 with ethanol, centrifuged at 13,000 × g for 5 minutes, and incubated at 50°C for 10 minutes. Thirty μl of the supernatant was transferred to a sampling glass vial and 5 μl was applied onto a high performance liquid chromatography system (Waters, Milford, MA) for analysis. The fluorescent derivatives were monitored using a 474 scanning fluorescence detector (Waters) at an excitation wavelength of 252 nm and an emission wavelength of 483 nm. Quantification of the S1P peak was calculated from a S1P standard calibration curve using the Waters Millennium software. A detailed protocol for this method is currently under preparation (X.H. and E.H.S., manuscript is preparation). Ceramide was quantified from the lipid extracts using the diacylglycerol kinase method.21He X Chen F Gatt S Schuchman EH An enzymatic assay for quantifying sphingomyelin in tissues and plasma from humans and mice with Niemann-Pick disease.Anal Biochem. 2001; 293: 204-211Crossref PubMed Scopus (18) Google Scholar The combined data from triplicate experiments were subjected to a t-test analysis, and the results were considered significant at P < 0.01. Chondrocyte and FLS cultures were maintained under the conditions described above (with or without dermatan sulfate) for 48 hours, and the media were collected for nitrite assays using the Griess reagent.22Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Tannenbaum SR Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids.Anal Biochem. 1982; 126: 131-138Crossref PubMed Scopus (10793) Google Scholar The combined data from triplicate experiments were subjected to a t-test analysis, and the results were considered significant at P < 0.005. Total RNA from normal and MPS VI rat FLS cultures was extracted using the Trizol reagent (Invitrogen) according to the manufacturer's instructions and then treated with a DNA-free DNase treatment and removal kit (Ambion, Austin, TX). Three μg of total RNA were reverse-transcribed using T7-polydT primer and converted into double-stranded cDNA using a one-cycle cDNA synthesis kit (Affymetrix Inc., Santa Clara, CA). The resulting templates were then used for in vitro transcription at 37°C for 16 hours to yield biotin-labeled antisense RNA (GeneChip HT IVT labeling kit, Affymetrix Inc.). The labeled, antisense RNA was chemically fragmented and made into a hybridization cocktail according to the Affymetrix GeneChip protocol, which was then hybridized to the Rat Genome 230 Plus 2 array (Affymetrix Inc.). The array image was generated by a high-resolution GeneChip 7G scanner (Affymetrix Inc.) and then converted into digitized data matrix by the Affymetrix MAS 5.0 algorithm. Complete microarray data have been deposited to Array Express (Accession E-TABM-306; www.ebi.ac.uk/arrayexpress). Bone sections were deparaffinized, rehydrated in a series of descending ethanol concentrations, and digested for 1 hour with testicular hyaluronidase (2 mg/ml, Sigma) in PBS. Immunohistochemistry for TLR4, MIP-1α, and MMP-13 was performed using polyclonal anti-goat TLR4 (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal anti-rat MIP-1α antibodies (Peprotech Inc., Rocky Hill, NJ) and polyclonal anti-goat MMP-13, respectively. Dilutions of the antisera were applied in PBS containing 1% bovine serum albumin, and the sections were incubated overnight at 4°C. After several rinses with PBS, visualization of TLR4 was accomplished using a fluorescent secondary antibody, Cy-3, whereas MMP-13 was incubated with Alexa FluorProbe 488 anti-mouse or Alexa FluorProbe 594 anti-rabbit IgG. Nuclei were stained with a bis-benzimide Hoechst dye (1 μg/μl) for 10 minutes, rinsed, and sections were mounted with an anti-bleaching mounting media. On the other hand, MIP-1α was visualized nonfluorescently using the streptavidin-biotin complex method (Histostain-Plus kit; Zymed, San Francisco, CA). The slides were stained with diaminobenzidine, and counterstained with hematoxylin. All slides (TLR4, MIP-1α and MMP-13) were visualized and photographed with a confocal laser-scanning microscope (Carl Zeiss, Thornwood, NY). Total RNA was extracted from cultured FLSs using the RNeasy mini kit (Qiagen, Valencia, CA), and processed according to the manufacturer's instructions. The RNA was first reverse-transcribed into first-strand complementary DNA (cDNA) using the SuperScript II RT kit (Invitrogen) and random hexanucleotide primers. For cDNA amplification, 1 μg of cDNA, 10× PCR buffer (100 mmol/L Tris-HCl, 500 mmol/L KCl, 25 mmol/L MgCl2), 2.5 mmol/L dNTPs, 20 pmol of sense and anti-sense primers, and recombinant Taq polymerase (Invitrogen) was mixed in a total reaction volume of 25 μl. PCR amplification was performed using primers corresponding to cDNA sequences for GAPDH,23Wang X Manner PA Horner A Shum L Tuan RS Nuckolls GH Regulation of MMP-13 expression by RUNX2 and FGF2 in osteoarthritic cartilage.Osteoarthritis Cartilage. 2004; 12: 963-973Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar MMP-1,24Cheon H Yu SJ Yoo DH Chae IJ Song GG Sohn J Increased expression of proinflammatory cytokines and metalloproteinase-1 by TGF-β1 in synovial fibroblasts from rheumatoid arthritis and normal individuals.Clin Exp Immunol. 2002; 127: 547-552Crossref PubMed Scopus (116) Google Scholar MMP-13,23Wang X Manner PA Horner A Shum L Tuan RS Nuckolls GH Regulation of MMP-13 expression by RUNX2 and FGF2 in osteoarthritic cartilage.Osteoarthritis Cartilage. 2004; 12: 963-973Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar and TNF-α.25Simonaro C Haskins ME Kunieda T Evans SM Visser JW Schuchman E Bone marrow transplantation in newborn rats with mucopolysaccharidosis type VI: biochemical, pathological, and clinical findings.Transplantation. 1997; 63: 1386-1393Crossref PubMed Scopus (20) Google Scholar Changes in gene expression were calculated as the ratio of molecules of the target gene/number of molecules of GAPDH. IL-1β and TNF-α were quantified in normal and MPS FLS cells and media, and synovial fluid, by immunoassays using rat and human Quantikine immunoassay kits (R&D Systems, Minneapolis, MN) according to the manufacturer's protocols. RANKL and MMP-13 activities also were measured using commercial assay kits (Chemicon International, Temecula, CA, and AnaSpec Inc., San Jose, CA, respectively) according to the manufacturers’ protocols. All immuno- and enzymatic assay experiments were performed in triplicate. FLSs and articular chondrocytes from 1-year-old normal and MPS VI rats were grown to subconfluence, washed with cold PBS, and then counted using a hemocytometer. To compare directly the protein levels in MPS versus normal animals, an equal number of cells (7.2 × 105 FLSs and 1.6 × 106 chondrocytes) were used to prepare the cell extracts. Cells were lysed by freeze/thaw in buffer containing 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 2 mmol/L ethylenediaminetetraacetic acid, 1% NP, 1 mmol/L vanadate, 5 mmol/L Naf, and 10 mg/ml aprotinine, pH 7.4. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% or 12% precast Nupage Bis/Tris gels under reducing conditions and MES running buffer (Invitrogen), and transferred onto a nitrocellulose membrane (Amersham Biosciences, Little Chalfont, UK) using a semidry transfer apparatus (Bio-Rad, Hercules, CA) and Nupage-MOPS transfer buffer. For immunoblot analysis, the membranes were blocked with TBS/Tween containing 5% dry milk, and then incubated with polyclonal anti-goat LBP, anti-rabbit TLR4, anti-rabbit MyD88, and anti-rabbit protein kinase C-α (PKC-α) as a loading control (Santa Cruz Biotechnology). The bound antibodies were recognized by secondary antibodies conjugated to horseradish peroxidase. Detection of the antibody complex was accomplished using an enhanced chemiluminescence detection reagent (Amersham Biosciences). Approximate molecular masses were determined by comparison with the migration of prestained protein standards (Bio-Rad). To calculate the density of the bands produced the blots were scanned using Image J 1.36b software (National Institutes of Health, Bethesda, MD), and absolute numbers were displayed as ratios. Bone marrow was isolated from normal and MPS VI age-matched rats (four each) as previously described.25Simonaro C Haskins ME Kunieda T Evans SM Visser JW Schuchman E Bone marrow transplantation in newborn rats with mucopolysaccharidosis type VI: biochemical, pathological, and clinical findings.Transplantation. 1997; 63: 1386-1393Crossref PubMed Scopus (20) Google Scholar To identify osteoclasts, bone marrow cells were fixed and stained using a TRAP kit according to the manufacturer's protocol (Kamiya Biomedical Comp, Seattle, WA). Normal and MPS VI FLSs were isolated as previously described from 9-month-old rats. Cells were plated at a density of 5 × 103 and followed for 72 hours. Proliferation was assayed using the CELLTiter96Aqueous One solution and performed according to the manufacturer's protocol (Promega, Madison, WI). The combined data from triplicate experiments were subjected to a t-test analysis, and results were considered significant at P < 0.001. All experiments were independently replicated at least three times. The combined data from the triplicate experiments were subjected to a two-tailed t-test analysis. Graphs represent the mean ± SEM of combined data from the triplicate experiments. RNA was prepared from FLSs of 9-month-old normal and MPS VI rats. Gene expression microarray analysis was performed using the Affymetrix 230 rat gene expression array and analyzed using the Affymetrix MAS 5.0 software. Ninety genes were found to be overexpressed at least fivefold in the MPS synovial cells, and 35 genes were underexpressed. As illustrated in Table 1, the majority of the genes overexpressed in the MPS VI cells were those involved in inflammation and/or immunity. Among the proteins encoded by these genes included numerous chemokines, cytokines, inflammatory cell receptors, inflammatory proteases.Table 1Inflammatory Genes Elevated in MPS Fibroblast-Like Synovial CellsFold changeAccession no.GeneDescription13.6gb:AW434057C1qbComplement component 1, q subcomponent, b12.6gb:AF268593.1β2 integrinIntegrin β2 a subunit11.8gb:AA945737CXCr4CXC chemokine receptor10.6gb:AB000818.1Aif1Allograft inflammatory factor 110.4gb:AF156540.1Mpeg1Macrophage-expressed gene 110.2gb:BI285793KFMSMacrophage CSF receptor precursor9.8gb:M10072.1L-CALeukocyte common antigen9.6gb:U54791.1LCR1Chemokine receptor LCR19.6gb:BE111722FCγrIIIHigh-affinity Ig-ε receptor γ precursor9.2gb:AB003042.1C5r1Complement component 5, receptor 18.2gb:NM_013069.1CD74Invariant polypeptide-associated MHC II antigen8.2gb:U22414.1MIP-1αMacrophage inflammatory protein-1α8.0gb:AB003042.1Bcl2a1BCL2-related protein A18.0gb:AF209406.1NMRKRat NK cell receptor 2B48.0gb:AB046592.1Laptm5Lysosomal-associated protein transmembrane 57.6gb:AB015308.1Gna15GTP binding protein α 157.4gb:J05155.1Plcg2Phospholipase C, γ 27.2gb:AF169636.1PIR-BPaired Ig-like receptor-B7.0gb:AF298656.3CybbEndothelial type gp91-phox6.8gb:AF087943.1CD14CD14 antigen6.8gb:AJ222813.1IL-18Precursor interleukin 186.8gb:NM_017260.1Alox5apArachidonate 5-lipoxygenase activating protein6.8gb:NM_017124.1CD37Leukocyte-specific protein6.2gb:BF282632CD53Leukocyte antigen6.0gb:BF550890CD43Leukosianin5.8gb:AF039033.1Ucp2Uncoupling protein 25.8gb:M64370.1FCγrIIIFc receptor, IgG, low affinity III5.8gb:BF289368LBPLipopolysaccharide-binding protein5.8gb:U06434.1MIP-1βMacrophage inflammatory protein-1 beta5.4gb:AI406496TECProtein tyrosine kinase5.2gb:NM_053963.1MMP-12Matrix metalloproteinase 125.0gb:AF208230.1LST1Leukocyte specific transcript 14.8gb:AF072411.1CD36Fatty acid translocaseCD364.4gb:M64711.1EDN1Endothelin 14.2" @default.
• W37041364 created "2016-06-24" @default.
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• W37041364 date "2008-01-01" @default.
• W37041364 modified "2023-10-11" @default.
• W37041364 title "Mechanism of Glycosaminoglycan-Mediated Bone and Joint Disease" @default.
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