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• W2037871279 abstract "A culture system was developed to analyze the relationship between proteoglycans and growth factors during corneal injury. Specifically, the effects of transforming growth factor β-1 (TGF-β1) and fetal calf serum on proteoglycan synthesis in corneal fibroblasts were examined. Glycosaminoglycan synthesis and sulfation were determined using selective polysaccharidases. Proteoglycan core proteins were analyzed using gel electrophoresis and Western blotting. Cells cultured in 10% dialyzed fetal calf serum exhibited decreased synthesis of more highly sulfated chondroitin sulfate and heparan sulfate compared with cells cultured in 1% dialyzed fetal calf serum. The amount and sulfation of the glycosaminoglycans was not significantly influenced by TGF-β1. The major proteoglycan species secreted into the media were decorin and perlecan. Decorin was glycanated with chondroitin sulfate. Perlecan was linked to either chondroitin sulfate, heparan sulfate, or both chondroitin sulfate and heparan sulfate. Decorin synthesis was reduced by either TGF-β1 or serum. At early time points, both TGF-β1 and serum induced substantial increases in perlecan bearing chondroitin sulfate and/or heparan sulfate chains. In contrast, after extended periods in culture, the amount of perlecan bearing heparan sulfate chains was unaffected by TGF-β1 and decreased by serum. The levels of perlecan bearing chondroitin sulfate chains were elevated with TGF-β1 treatment and were decreased with serum. Because both decorin and perlecan bind growth factors and are proposed to modulate their activity, changes in the expression of either of these proteoglycans could substantially affect the cellular response to injury. A culture system was developed to analyze the relationship between proteoglycans and growth factors during corneal injury. Specifically, the effects of transforming growth factor β-1 (TGF-β1) and fetal calf serum on proteoglycan synthesis in corneal fibroblasts were examined. Glycosaminoglycan synthesis and sulfation were determined using selective polysaccharidases. Proteoglycan core proteins were analyzed using gel electrophoresis and Western blotting. Cells cultured in 10% dialyzed fetal calf serum exhibited decreased synthesis of more highly sulfated chondroitin sulfate and heparan sulfate compared with cells cultured in 1% dialyzed fetal calf serum. The amount and sulfation of the glycosaminoglycans was not significantly influenced by TGF-β1. The major proteoglycan species secreted into the media were decorin and perlecan. Decorin was glycanated with chondroitin sulfate. Perlecan was linked to either chondroitin sulfate, heparan sulfate, or both chondroitin sulfate and heparan sulfate. Decorin synthesis was reduced by either TGF-β1 or serum. At early time points, both TGF-β1 and serum induced substantial increases in perlecan bearing chondroitin sulfate and/or heparan sulfate chains. In contrast, after extended periods in culture, the amount of perlecan bearing heparan sulfate chains was unaffected by TGF-β1 and decreased by serum. The levels of perlecan bearing chondroitin sulfate chains were elevated with TGF-β1 treatment and were decreased with serum. Because both decorin and perlecan bind growth factors and are proposed to modulate their activity, changes in the expression of either of these proteoglycans could substantially affect the cellular response to injury. The extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; CS, chondroitin sulfate; CSPG, chondroitin sulfate proteoglycan; FCS, fetal calf serum; dFCS, dialyzed FCS; DMEM, Dulbecco's modified Eagle's medium; GAG, glycosaminoglycan; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; KS, keratan sulfate; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TGF-β, transforming growth factor β of the corneal stroma is synthesized and maintained by keratocytes. The matrix is primarily composed of collagen fibrils stacked in orderly lamellae surrounded by proteoglycans. The organization of proteoglycans and collagen fibrils in the stroma may be responsible for the optical and structural properties of the tissue (1Trinkaus-Randall V. Lanza R.P. Langer R. Chick W.L. Principles of Tissue Engineering. Academic Press, Inc, San Diego, CA1997: 386-388Google Scholar). The corneal stroma contains two major classes of proteoglycans, one possessing keratan sulfate side chains and the other possessing chondroitin/dermatan sulfate side chains (2Axelsson I. Heinegard D. Biochem. J. 1978; 169: 517-530Crossref PubMed Scopus (53) Google Scholar, 3Hassell J.R. Newsome D.A. Hascall V.C. J. Biol. Chem. 1979; 254: 12346-12354Abstract Full Text PDF PubMed Google Scholar, 4Gregory J.D. Coster L. Damle S.P. J. Biol. Chem. 1982; 257: 6965-6970Abstract Full Text PDF PubMed Google Scholar, 5Funderburgh J.L. Conrad G.W. J. Biol. Chem. 1990; 265: 8297-8303Abstract Full Text PDF PubMed Google Scholar). Three corneal keratan sulfate proteoglycans, lumican, keratocan, and mimecan, have been cloned and sequenced (6Blochberger T.C. Vergnes J.-P. Hempel J. Hassell J.R. J. Biol. Chem. 1992; 267: 347-352Abstract Full Text PDF PubMed Google Scholar, 7Funderburgh J.L. Funderburgh M.L. Brown S.J. Vergnes J.-P. Hassell J.R. Mann M.M. Conrad G.W. J. Biol. Chem. 1993; 268: 11874-11880Abstract Full Text PDF PubMed Google Scholar, 8Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Funderburgh J.L. Corpuz L.M. Roth M.R. Funderburgh M.L. Tasheva E.S. Conrad G.W. J. Biol. Chem. 1997; 272: 28089-28095Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The gene for the corneal chondroitin/dermatan sulfate proteoglycan protein core has been cloned from chick corneas and identified as decorin (10Li W. Vergnes J.-P. Cornuet P.K. Hassell J.R. Arch. Biochem. Biophys. 1992; 296: 190-197Crossref PubMed Scopus (94) Google Scholar). The deduced amino acid sequences of decorin, lumican, and keratocan identify them as members of a group of small leucine-rich proteoglycans (5Funderburgh J.L. Conrad G.W. J. Biol. Chem. 1990; 265: 8297-8303Abstract Full Text PDF PubMed Google Scholar, 8Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). The structural and biochemical properties of ECM molecules in the corneal stroma are altered upon injury. Corneal wounds contain collagen fibrils with abnormally large diameter and irregular interspacing (11Schwarz W. Keyserling D.G. Virchows Arch. 1969; 347: 115-128Crossref Scopus (5) Google Scholar,12Cintron C. Hassinger L.C. Kublin C.L. Cannon D.J. J. Ultrastruct. Tissue Res. 1978; 65: 13-22Crossref PubMed Scopus (77) Google Scholar). Disruption of the fibrillar organization of collagen fibrils in corneal wounds is thought to be attributed, in part, to alterations in the proportion and chemical characteristics of specific proteoglycans. Injured corneas contain unusually large chondroitin/dermatan sulfate proteoglycans possessing glycosaminoglycan (GAG) side chains with higher than normal sulfation and increased amounts of iduronic acid (13Hassell J.R. Cintron C. Kublin C. Newsome D.A. Arch. Biochem. Biophys. 1983; 222: 362-369Crossref PubMed Scopus (179) Google Scholar). Keratan sulfate (KS) chains in corneal scars have increased size and lower sulfation (14Funderburgh J.L. Cintron C. Covington H.I. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1988; 29: 1116-1124PubMed Google Scholar, 15Funderburgh J.L. Chandler J.W. Invest. Ophthalmol. Visual Sci. 1989; 30: 435-442PubMed Google Scholar). The ratio of chondroitin/dermatan sulfate to keratan sulfate has been shown to increase after wounding, and heparan sulfate (HS) has been detected in corneal scars (13Hassell J.R. Cintron C. Kublin C. Newsome D.A. Arch. Biochem. Biophys. 1983; 222: 362-369Crossref PubMed Scopus (179) Google Scholar, 16Brown C.T. Applebaum E. Banwatt R. Trinkaus-Randall V. J. Cell. Biochem. 1995; 59: 57-68Crossref PubMed Scopus (47) Google Scholar, 17Cintron C. Gregory J.D. Damle S.P. Kublin C.L. Ophthalmol. Visual Sci. 1990; 31: 1975-1981PubMed Google Scholar). Interestingly, both transforming growth factor-β (TGF-β) and basic fibroblast growth factor are detected transiently in corneal wounds coincident with the expression of heparan sulfate proteoglycans (HSPGs) (16Brown C.T. Applebaum E. Banwatt R. Trinkaus-Randall V. J. Cell. Biochem. 1995; 59: 57-68Crossref PubMed Scopus (47) Google Scholar, 18Trinkaus-Randall V. Nugent M.A. J. Controlled Release. 1998; 53: 205-214Crossref PubMed Scopus (18) Google Scholar). TGF-β has been implicated as a regulatory agent in numerous cellular and physiological processes, including proteoglycan expression (19O'Kane S. Ferguson M.W.J. Int. J. Biochem. Cell Biol. 1997; 29: 63-78Crossref PubMed Scopus (573) Google Scholar). This influence appears to be at the level of core protein synthesis and GAG chain elongation (20Bassols A. Massague J. J. Biol. Chem. 1988; 263: 3039-3045Abstract Full Text PDF PubMed Google Scholar). TGF-β has been detected in corneal wounds and in corneal fibroblast cultures, suggesting that it plays a role in regulating the synthesis of stromal ECM components (16Brown C.T. Applebaum E. Banwatt R. Trinkaus-Randall V. J. Cell. Biochem. 1995; 59: 57-68Crossref PubMed Scopus (47) Google Scholar, 21Hayashi K. Frangieh G. Wolf G. Kenyon K.R. Invest. Ophthalmol. Visual Sci. 1989; 30: 239-247PubMed Google Scholar, 22Wilson S.E. He Y.-G. Lloyd S.A. Invest. Ophthalmol. Visual Sci. 1992; 33: 1756-1765PubMed Google Scholar). Although TGF-β has been detected in vivo and in vitro, the relationship between TGF-β and proteoglycan expression by corneal fibroblasts has not been fully elucidated. Cultured corneal fibroblasts synthesize proteoglycans remarkably similar to those in wounded corneas. Early reports indicated cultures of rabbit corneal fibroblasts produce mainly chondroitin sulfate (CS) and HS, with only low levels of KS (23Yue B.Y.J.T. Baum J.L. Silbert J.E. Biochem. J. 1976; 158: 567-573Crossref PubMed Scopus (58) Google Scholar, 24Conrad G.W. Hamilton C. Haynes E. J. Biol. Chem. 1977; 252: 6861-6870Abstract Full Text PDF PubMed Google Scholar, 25Dahl I.-M.S. Coster L. Exp. Eye. Res. 1978; 27: 175-190Crossref PubMed Scopus (31) Google Scholar). Hassell et al.(26Hassell J.R. Schrecengost P.K. Rada J.A. SundarRaj N. Sossi G. Thoft R.A. Invest. Ophthalmol. Visual Sci. 1992; 33: 547-557PubMed Google Scholar) reported human corneal fibroblasts in culture synthesize substantial amounts of decorin and perlecan (basement membrane HSPG). Schrecengost et al. (27Schrecengost P.K. Blochberger T.C. Hassell J.R. Arch. Biochem. Biophys. 1992; 292: 54-61Crossref PubMed Scopus (19) Google Scholar) reported reduced levels of a keratan sulfate proteoglycan containing truncated unsulfated keratan chains in chick corneal fibroblasts. Funderburgh et al. (28Funderburgh J.L. Funderburgh M.L. Mann M.M. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 31431-31436Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) reported that bovine corneal fibroblasts in culture synthesize keratan sulfate proteoglycans with shorter KS chains and lower sulfation compared with those in normal corneas. The altered properties of KS and the increase in chondroitin sulfate proteoglycan (CSPG) and HSPG synthesis suggests that the conditions of cell culture may recapitulate some of the aspects of injury. To date, most studies of corneal proteoglycans produced in vitro have been based upon biochemical analysis of GAG chains, with only limited analysis of the protein cores. Additionally, each of these studies employed different culture techniques, making the results difficult to compare. We developed a culture system to examine the regulation of proteoglycan synthesis by corneal fibroblasts during injury. The major proteoglycans synthesized by corneal fibroblasts were characterized and identified after culture in a defined environment. Specifically, we evaluated the effects of TGF-β1 and serum on the synthesis of specific GAGs and protein cores. We found that corneal fibroblasts synthesized predominantly CS and HS, with only trace amounts of an unsulfated form of keratan. The major proteoglycan species secreted into the medium were decorin and perlecan, and proteoglycan synthesis was mediated by TGF-β1 and serum. This model will allow us to systematically examine the relationship between specific growth factors and proteoglycan expression using a defined culture system. Chondroitinase ABC (protease-free), keratanase II, chondroitin sulfate B, keratan sulfate, and the mouse monoclonal antibody 3G10 directed against unsaturated uronic acid residues arising from heparinase digestion of heparan sulfate were purchased from Seikagaku America, Inc. (Ijamsville, MD). Endo-β-galactosidase was purchased from Boehringer Mannheim. Heparan sulfate, heparinase I, heparinase III, phenylmethylsulfonyl fluoride, benzamidine,N-ethylmaleimide, and peroxidase-conjugated donkey anti-sheep IgG antibodies were from Sigma. Q-Sepharose came from Pharmacia Biotech Inc. (Uppsala, Sweden). Peroxidase-conjugated donkey anti-rat IgG antibodies were purchased from Amersham Pharmacia Biotech. Ultrapure urea, sodium chloride, Tween-20, Tris-HCl, bovine serum albumin, and EDTA were obtained from American Bioanalytical (Natick, MA). TGF-β1 was obtained from R & D Systems (Minneapolis, MN). All cell culture reagents were purchased from Life Technologies, Inc. The mouse monoclonal antibody A7L6 directed against perlecan was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). The polyclonal sheep antiserum directed against rabbit corneal decorin was a generous gift from Dr. Charles Cintron (Schepen Eye Research Institute, Boston, MA). The rabbit polyclonal antibody R36 that binds unsaturated uronic acid residues resulting from chondroitinase ABC treatment was a generous gift from Dr. John Couchman (University of Alabama, Birmingham, AL). Corneas were excised from whole rabbit eyes purchased from Pel Freeze (Rogers, AR), and the epithelium and endothelium were removed as described previously (29Trinkaus-Randall V. Capecchi J. Sammon L. Gibbons D. Leibowitz H.M. Franzblau C. Invest. Ophthalmol. Visual Sci. 1990; 31: 1321-1326PubMed Google Scholar). The corneas were washed two times with Dulbecco's modified Eagle's medium (DMEM) containing 1000 units/ml penicillin, 1.0 mg/ml streptomycin sulfate, and 20 units/ml nystatin. The corneas were minced with a sterile razor blade and subsequently digested with collagenase A (1.5 mg/ml) in DMEM containing 200 units/ml penicillin, 200 μg/ml streptomycin, and 100 units/ml nystatin for 2–3 h with agitation at 37 °C. The digests were centrifuged at 1840 × g for 10 min, and the cells were suspended in DMEM supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 100 units/ml nystatin, nonessential amino acids, and 10% fetal calf serum (FCS). Cell were plated in 75-mm vented tissue culture flasks, and cultures were maintained in DMEM supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 100 units/ml nystatin, nonessential amino acids and 4% fetal calf serum. The cultures achieved confluency after 7–10 days, at which time cells were passaged 1:4 and cultured in 4% FCS for 3 days. All experiments were performed on confluent fibroblast cultures that had been passaged once. The synthesis of sulfated glycosaminoglycans was followed by metabolically radiolabeling corneal fibroblasts with [3H]glucosamine (18 μCi/ml) and/or [35S]sulfate (36 μCi/ml). Proteoglycan core proteins were metabolically labeled with [35S]cysteine/methionine (50 μCi/ml). Corneal fibroblasts in first passage were cultured until confluent (3 days) in 4% FCS. Upon confluence, corneal fibroblasts were treated as indicated in the figure legends. Radioisotopes were added immediately after the addition of TGF-β1. After the designated radiolabeling period, the medium was collected and immediately combined with two volumes of 10 m urea containing 50 mm Tris-HCl, 10 mm EDTA, pH 7.0. Cell monolayers were washed with phosphate buffered saline (pH 7.4) and ECM proteins were isolated by gently scraping cell monolayers in 1.0m urea, 50 mm Tris-HCl, 50 mm EDTA, pH 7.0. The resulting suspension was centrifuged (5520 ×g) for 10 min, and the supernatant was collected and defined as the ECM fraction. The cell pellet was extracted with TUT (8m urea, 50 mm Tris-HCl, 0.1% Triton X-100, pH 7.0). The extract was clarified by centrifugation, and the supernatant was collected and defined as the cell fraction (30Nugent M.A. Edelman E.R. J. Biol. Chem. 1992; 267: 21256-21264Abstract Full Text PDF PubMed Google Scholar). Cell number was determined by measuring acid phosphatase activity on a replicate set of cultures (31Yang T.T. Sinai P. Kain S.R. Anal. Biochem. 1996; 241: 103-108Crossref PubMed Scopus (148) Google Scholar). Total radiolabeled protein present in [35S]cysteine/methionine labeled samples was determined by performing trichloroacetic acid precipitation on aliquots of medium and cell fractions prior to proteoglycan purification and quantitating the radioactivity in a liquid scintillation counter (27Schrecengost P.K. Blochberger T.C. Hassell J.R. Arch. Biochem. Biophys. 1992; 292: 54-61Crossref PubMed Scopus (19) Google Scholar). Medium, cell, or ECM fractions were mixed with 1.0 ml of a 70% Q-Sepharose suspension and rocked for 45 min. The slurries were poured into 5.0-ml disposable minicolumns (Pierce), and the unbound fractions were discarded. The columns were washed with 25 column volumes of TUE (8 m urea, 50 mm Tris-HCl, 50 mm EDTA, pH 7.0) and subsequently washed with 25 column volumes TUE containing 0.2m NaCl. Columns were eluted with 7 column volumes of TUE containing 1.5 m NaCl. Salt fractions were exhaustively dialyzed against Milli-Q water, using membranes with a molecular weight cutoff of 25,000 (Spectropore, Laguna Hills, CA), and lyophilized. Samples were resuspended in 2 mm sodium phosphate, 30 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 10 mm N-ethylmaleimide, pH 7.4. Selective polysaccharidases were used to identify and quantitate GAGs and proteoglycan core proteins. Digestion conditions were optimized for time, temperature, concentration, and pH. Enzymes were routinely tested for activity and specificity using highly purified GAG standards (Seikagaku, Tokyo, Japan) and the dimethyl methylene blue assay (32Farndale R.W. Buttle D.J . Barrett A.J. Biochim. Biophys. Acta. 1986; 883: 173-177Crossref PubMed Scopus (2907) Google Scholar). Purified proteoglycans were subjected to digestion for 3 h at 37 °C in 40 mm Tris-HCl. The pH of the digest was adjusted to the optimum for each enzyme: chondroitinase ABC (1.0 unit/ml, pH 8.0), a mixture of heparinase I and heparinase III (10 and 20 units/ml respectively, pH 7.3), and a mixture of keratanase II and endo-β-galactosidase (both enzymes 0.01 unit/ml, pH 5.9). Specific GAGs were quantitated by measuring the low molecular weight digestion products released after polysaccharidase treatment. Purified GAGs co-radiolabeled with [3H]glucosamine and [35S]SO4 were treated with chondroitinase ABC, a mixture of heparinase I and heparinase III, a mixture of keratanase II and endo-β-galactosidase, or control with buffer lacking enzyme. Digests were subjected to ultrafiltration (Microcon, Millipore) to separate GAG digestion products from intact proteoglycans. The radioactivity in the filtrate was determined using liquid scintillation. A 10,000 molecular weight cut-off filter was used to recover CS and KS digestion products, and a 30,000 molecular weight cut-off filter was used to recover HS digestion products. The radioactivity in the filtrate from the undigested control was subtracted from the enzyme treated samples. In experiments with a large number of samples, fractions containing [35S]SO4-GAGs were analyzed, without any prior purification. [35S]Glycosaminoglycans in medium, cell, and ECM fractions were quantitated by dot-blotting samples onto cationic nylon filters as described previously (30Nugent M.A. Edelman E.R. J. Biol. Chem. 1992; 267: 21256-21264Abstract Full Text PDF PubMed Google Scholar). Briefly, filters (Zeta-probe; Bio-Rad) were prehydrated in TBS (50 mmTris-HCl, 0.15 m NaCl, pH 8.0). The filter was then placed into a Bio-Dot apparatus (Bio-Rad) and washed once by drawing TBS through each well with vacuum. Samples (100 μl) were applied to each well and pulled through under vacuum and wells were washed with 0.6 ml of TUT. The filter was washed twice with TBS followed by two additional washes with Milli-Q water (Millipore, Bedford MA). The washed filter was briefly immersed in 95% ethanol, and the area of the filter containing each sample was removed and counted using liquid scintillation. Heparan sulfate and CS were determined by treating a replicate filter with nitrous acid, which selectively degrades HS chains. After sample application, washed filters were treated twice with fresh nitrous acid (0.48 m sodium nitrite combined with 3.6 macetic acid) for 90 min followed by a wash with TBS containing 0.65m NaCl. The difference between the radioactivity measured on the non-acid-treated and acid-treated filters was defined as HS. The amount of radioactivity remaining after nitrous acid treatment was defined as CS. Proteoglycan core proteins were identified and quantified using selective polysaccharidases in conjunction with SDS-PAGE and/or Western blotting. Proteoglycans radiolabeled with [35S]cysteine/methionine were digested with either chondroitinase ABC, heparinases I and III, or a mixture of chondroitinase ABC and heparinases I and III. Digests were run on 5 or 10% SDS-PAGE gels under reducing conditions (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), and loading was normalized to total radioactive protein present in fractions prior to purification (trichloroacetic acid precipitation). Gels were either processed for autoradiography or electrophoretically transferred to polyvinylidene difluoride membrane (Millipore) using a semi-dry transfer apparatus (integrated separations systems) in 25 mm Tris, 192 mm glycine, 20% methanol. The membranes were blocked in 5% bovine serum albumin in TBS-T buffer (10 mm Tris, 100 mm NaCl, 0.1% Tween 20, pH 7.2) and were incubated with either mouse monoclonal anti-perlecan (2 μg/ml), sheep polyclonal anti-rabbit corneal decorin (1:9000), mouse anti-HS stub (1:1000), or rabbit anti-CS stub in 5% bovine serum albumin in TBS-T at room temperature for 1 h. Blots were washed with 1% bovine serum albumin in TBS-T and incubated with appropriate secondary antibodies coupled to horseradish peroxidase (1:3000) for 1 h at room temperature. Proteins were visualized using chemiluminescence (NEN Life Science Products). To distinguish between chemiluminescence and radioactivity, a piece of transparent plastic was placed between the membrane and the film, which was demonstrated to block >95% of the radioactivity. After probing, the blots were treated with 10% sodium salicylate in methanol and dried. Radiolabeled proteins were detected using autoradiography. Confluent corneal fibroblasts were cultured for 96 h in either 1 or 10% dialyzed fetal calf serum with or without daily treatments with TGF-β1 (1 ng/ml). Cellular viability studies were performed, and no significant differences were observed after 6 days of culture in 0, 1, or 10% FCS (data not shown). To evaluate GAG synthesis and sulfation, cells were metabolically labeled with both [35S]SO4 and [3H]glucosamine. Glycosaminoglycans in medium, cell, and ECM fractions were purified using anion exchange chromatography on Q-Sepharose. The chromatographic conditions were optimized to separate highly charged proteoglycans from weakly charged glycoproteins and hyaluronic acid (16Brown C.T. Applebaum E. Banwatt R. Trinkaus-Randall V. J. Cell. Biochem. 1995; 59: 57-68Crossref PubMed Scopus (47) Google Scholar). Specific GAGs were quantitated by selective digestion with polysaccharidases. Chondroitin sulfate was determined using chondroitinase ABC. Glycosaminoglycans susceptible to chondroitinase ABC are referred to as CS because this enzyme does not distinguish between polymers containing iduronate and glucuronate (34Ernst S. Langer R. Cooney C.L. Sasisekharan R. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 387-444Crossref PubMed Scopus (352) Google Scholar). Keratan sulfate was determined using a mixture of keratanase II and endo-β-galactosidase, and HS was determined using a mixture of heparinases I and III. Sulfation was defined as the ratio of polysaccharidase-sensitive [35S]SO4 to polysaccharidase-sensitive [3H]glucosamine. Tables Iand II summarize the data obtained by enzymatic analysis of purified GAGs.Table I[3H]Glucosamine incorporation into GAGs by corneal fibroblasts cultured in 1 or 10% dFCS with or without TGF-β1 for 96 hFraction[dFCS]Chondroitin/dermatan sulfateaData obtained using chondroitinase ABC.Keratan sulfatebData obtained using a mixture of keratanase II and endo-β-galactosidase.Heparan sulfatecData obtained using a mixture of heparinases I and III.−TGF-β1+TGF-β1−TGF-β1+TGF-β1−TGF-β1+TGF-β1%Medium114,289 ± 89013,863 ± 493129 ± 3174 ± 43126 ± 1803594 ± 166109062 ± 1127479 ± 40252 ± 957 ± 73029 ± 602519 ± 91Cell13076 ± 1672037 ± 58427 ± 1425 ± 141842 ± 1591495 ± 392101234 ± 2111233 ± 6710 ± 24 ± 3648 ± 110661 ± 29ECM1486 ± 117358 ± 372 ± 27 ± 1426 ± 95324 ± 3710170 ± 12186 ± 100 ± 11 ± 0106 ± 14118 ± 2Corneal fibroblasts were cultured for 96 h in 1 or 10% dFCS ± TGF-β1 (1 ng/ml), and GAGs were labeled with [3H]glucosamine. Glycosaminoglycans were purified using anion exchange chromatography on Q-Sepharose. Purified GAGs were digested with selective polysaccharidases. Digests were subjected to ultrafiltration (Microcon, Millipore), and the radioactivity in the filtrate was counted. The radioactivity in the filtrate from an untreated control was subtracted from the enzyme-treated samples. Results are expressed as cpm per 1 × 103 cell ± S.E. (n = 3).a Data obtained using chondroitinase ABC.b Data obtained using a mixture of keratanase II and endo-β-galactosidase.c Data obtained using a mixture of heparinases I and III. Open table in a new tab Table II[35S]SO4 incorporation into GAGs by corneal fibroblasts cultured in 1 or 10% dFCS with or without TGF-β1 for 96 hFraction[dFCS]Chondroitin/dermatan sulfateaData obtained using chondroitinase ABC.Keratan sulfatebData obtained using a mixture of keratanase II and endo-β-galactosidase.Heparan sulfatecData obtained using a mixture of heparinases I and III.−TGF-β1+TGF-β1−TGF-β1+TGF-β1−TGF-β1+TGF-β1%Medium12376 ± 552258 ± 66NDdND, not detected.ND437 ± 11475 ± 18102001 ± 231875 ± 81NDND499 ± 8468 ± 18Cell1419 ± 18241 ± 72NDND167 ± 11135 ± 3710243 ± 43251 ± 16NDND79 ± 1586 ± 3ECM164 ± 1443 ± 4NDND36 ± 628 ± 31033 ± 338 ± 2NDND13 ± 116 ± 1Corneal fibroblasts were cultured for 96 h in 1 or 10% dFCS ± TGF-β1 (1 ng/ml), and GAGs were labeled with [35S]SO4. Glycosaminoglycans were purified using anion exchange chromatography on Q-Sepharose. Purified GAGs were digested with selective polysaccharidases. Digests were subjected to ultrafiltration (Microcon, Millipore), and the radioactivity in the filtrate was counted. The radioactivity in the filtrate from the undigested control was subtracted from the enzyme-treated samples. Results are expressed as cpm per 1 × 103 cell ± S.E. (n = 3).a Data obtained using chondroitinase ABC.b Data obtained using a mixture of keratanase II and endo-β-galactosidase.c Data obtained using a mixture of heparinases I and III.d ND, not detected. Open table in a new tab Corneal fibroblasts were cultured for 96 h in 1 or 10% dFCS ± TGF-β1 (1 ng/ml), and GAGs were labeled with [3H]glucosamine. Glycosaminoglycans were purified using anion exchange chromatography on Q-Sepharose. Purified GAGs were digested with selective polysaccharidases. Digests were subjected to ultrafiltration (Microcon, Millipore), and the radioactivity in the filtrate was counted. The radioactivity in the filtrate from an untreated control was subtracted from the enzyme-treated samples. Results are expressed as cpm per 1 × 103 cell ± S.E. (n = 3). Corneal fibroblasts were cultured for 96 h in 1 or 10% dFCS ± TGF-β1 (1 ng/ml), and GAGs were labeled with [35S]SO4. Glycosaminoglycans were purified using anion exchange chromatography on Q-Sepharose. Purified GAGs were digested with selective polysaccharidases. Digests were subjected to ultrafiltration (Microcon, Millipore), and the radioactivity in the filtrate was counted. The radioactivity in the filtrate from the undigested control was subtracted from the enzyme-treated samples. Results are expressed as cpm per 1 × 103 cell ± S.E. (n = 3). Approximately 75–85% of the [3H]GAGs synthesized during the 96-h incubation period were secreted into the medium with the remaining present in the cell (16–21%) and ECM (2–3%) fractions. Compositional analysis revealed that the majority of the [3H]GAGs synthesized by corneal fibroblast in culture were CS (53–80%) and HS (18–47%), with a trace amount of a nonsulfated form of keratan (<1%). Corneal fibroblasts cultured in 10% dFCS showed an overall reduction in CS synthesis compared with cells cultured in 1% dFCS. This was evident from both [35S]SO4 and [3H]glucosamine incorporation. A similar decrease in HS was observed when cells were cultured in 10% dFCS compared with 1% dFCS. Treatment with TGF-β1 resulted in a reduction in the amount of [35S]SO4 and [3H]glucosamine-labeled CS recov" @default.
• W2037871279 created "2016-06-24" @default.
• W2037871279 creator A5007469299 @default.
• W2037871279 creator A5032119599 @default.
• W2037871279 creator A5049909328 @default.
• W2037871279 creator A5074638151 @default.
• W2037871279 date "1999-03-01" @default.
• W2037871279 modified "2023-09-28" @default.
• W2037871279 title "Characterization of Proteoglycans Synthesized by Cultured Corneal Fibroblasts in Response to Transforming Growth Factor β and Fetal Calf Serum" @default.
• W2037871279 cites W10373433 @default.
• W2037871279 cites W137187997 @default.
• W2037871279 cites W1492952392 @default.
• W2037871279 cites W1497340641 @default.
• W2037871279 cites W1504247812 @default.
• W2037871279 cites W1506057274 @default.
• W2037871279 cites W1522060763 @default.
• W2037871279 cites W1530263251 @default.
• W2037871279 cites W1539442102 @default.
• W2037871279 cites W1558969468 @default.
• W2037871279 cites W1585661831 @default.
• W2037871279 cites W1592658968 @default.
• W2037871279 cites W1595945752 @default.
• W2037871279 cites W1601454315 @default.
• W2037871279 cites W1624294703 @default.
• W2037871279 cites W1855999912 @default.
• W2037871279 cites W1965084403 @default.
• W2037871279 cites W1965368544 @default.
• W2037871279 cites W1965517168 @default.
• W2037871279 cites W1968956893 @default.
• W2037871279 cites W1971196496 @default.
• W2037871279 cites W1980617596 @default.
• W2037871279 cites W1981787059 @default.
• W2037871279 cites W1982396402 @default.
• W2037871279 cites W1983302217 @default.
• W2037871279 cites W1996941257 @default.
• W2037871279 cites W1997510203 @default.
• W2037871279 cites W1999320651 @default.
• W2037871279 cites W1999411442 @default.
• W2037871279 cites W2004149782 @default.
• W2037871279 cites W2004687677 @default.
• W2037871279 cites W2011824328 @default.
• W2037871279 cites W2013750233 @default.
• W2037871279 cites W2015167429 @default.
• W2037871279 cites W2016613633 @default.
• W2037871279 cites W2017096535 @default.
• W2037871279 cites W2019301585 @default.
• W2037871279 cites W2022022362 @default.
• W2037871279 cites W2023104790 @default.
• W2037871279 cites W2030213916 @default.
• W2037871279 cites W2032255522 @default.
• W2037871279 cites W2036129260 @default.
• W2037871279 cites W2041345827 @default.
• W2037871279 cites W2043990687 @default.
• W2037871279 cites W2047578760 @default.
• W2037871279 cites W2051900610 @default.
• W2037871279 cites W2053352199 @default.
• W2037871279 cites W2061445074 @default.
• W2037871279 cites W2062119438 @default.
• W2037871279 cites W2082876026 @default.
• W2037871279 cites W2090603283 @default.
• W2037871279 cites W2093498304 @default.
• W2037871279 cites W2100837269 @default.
• W2037871279 cites W2113266466 @default.
• W2037871279 cites W2117901334 @default.
• W2037871279 cites W2120347833 @default.
• W2037871279 cites W2161625600 @default.
• W2037871279 cites W2281393459 @default.
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