Proc. Nat. Acad. Sci. USA Vol. 71, No. 10, pp. 4047-4051, October 1974 Stimulation of Synthesis of Free Chondroitin Sulfate Chains by ,B-D-Xylosides in Cultured Cells (glycosaminoglycans/cartilage cells/mesenchyme cells/gligl cells/hepatoma) NANCY B. SCHWARTZ, LEONARDO GALLIGANI, PEI-LEE HO, AND ALBERT DORFMAN Department of Pediatrics and Biochemistry, Joseph P. Kennedy, Jr., Mental Retardation Research Center, Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60637 Contributed by Albert Dorfman, July 29, 1974 ABSTRACT Previous studies have shown that D-xylose types that normally synthesize only minimal amounts of this partially overcomes the puromycin inhibition of chon- sulfated glycosaminoglycan. droitin sulfate synthesis in cultured chick embryo chon- drocytes. Likewise, Dxylose stimulates chondroitin sulfate MATERIA.LS AND METHODS synthesis by limb bud ipesenchyme cells previously treated with BrdU or limb bud cartilage cells treated with puro- Materials. F-12 powdered medium was obtained from North mycin. The studies reported here show thatp-nitrophenyl- American Biologicals, Inc. Modified Eagle's medium (10), _i-D-xylopyranoside and 4-methyl-umbelliferyl-fl-D-xylo- pyranoside cause a much greater stimulation than does fetal calf serum, horse serum, 2.5% lyophilized trypsin, D-xylose and are active at much lower concentrations. In 0.25% (w/v) trypsin-EDTA solution, and Hank's balanced contrast to D-xylose, the xylosides strikingly stimulate salt solution (calcium- and magnesium-free) were purchased chondroitin sulfate synthesis in predifferentiated mesen- from Grand Island Biologicals Co. D-Xylose was obtained chyme cells. The xylosides stimulate synthesis ofchondroi- tin sulfate by rat glial cell tumor cells (RC-6), a mouse from Pfanstiehl; p-nitrophenyl-j3-D-xylopyranoside, p-nitro- neuroblastoma (C1300, NB41A), and two strains of cul- phenyl-a-D-xylopyranoside, and 4-methylumbelliferyl-f3-D- tured rat hepatoma cells (HTC, H4). These results indicate xylopyranoside from Koch-Light Laboratories, Ltd., that certain types of nonconnective tissue cells contain the 4-methylumbelliferyl-,-D-galactopyranoside from Pierce enzymic machinery for synthesis of chondroitin sulfate Chemical Co., p-nitro- which is normally not utilized because of limited synthesis p-nitrophenyl-,3-D-galactopyranoside, of core protein and/or xylosyltransferase. The ,-xylosides phenyl-a-D-galactopyranoside, and p-nitrophenyl-N-acetyl- may be used as a probe of the capacity of various cell types #-D-galactosamine from Sigma Chemical Co. Puromycin to synthesize sulfated glycosaminoglycans. dihydrochloride was purchased from Nutritional Biochemicals Co. and 5-bromo-2'-deoxyuridine (BrdU) from Sigma. The Sulfated glycosaminoglycans in animal tissues are bound radioactive precursors, H285SO4 (carrier-free, 43 Ci/mng) and covalently to protein by way of a galactosyl-galactosyl- sodium [U-qIjacetate (100 Ci/mol), were obtained from xylosyl linkage to the hydroxyl groups of serine (1). Bio- New England Nuclear Corp. Chondroitinase ABC, prepared synthesis of these proteoglycans requires formation of core by Seikagaku Kogyo Co., Ltd., was purchased from Miles protein followed by stepwise addition of the individual mono- Laboratories, Inc. Streptococcal hyaluronidase (Varidase) saccharides from uridine diphosphosugars by a series of gly- was obtained from Lederle Laboratories. Carrier chondroitin- cosyltransferases (2-5). Presumably the same enzymes 4-sulfate was donated by Dr. J. A. Cifonelli, and twice- catalyze the transfer of the individual monosaccharides to crystallized papain was a gift from Dr. Lennart Rod6n. acceptors in vitro (3-6). In minced cartilage, Brett and Robinson (7) showed that Cell Culture. Cultures of chondrocytes from sternal cartilage high concentrations of D-xylose reversed the inhibition by of 15-day chick embryos were established according to pro- puromycin of chondroitin sulfate synthesis. Most likely, cedures described by Cahn et al. (11). Cells were plated at an xylose acted as an initiator of polysaccharide chain formation, initial density of 2 to 5 X 105 cells on 60-mm Falcon tissue circumventing the requirement for core protein and xylosyl- culture dishes in F-12 medium with 7% horse serum and 10% transferase. These results were confirmed and extended in the fetal calf serum (v/v). After the cells were allowed to attach studies of Levitt and Dorfman (8), who additionally showed to the dishes (about 3 days), subsequent growth was main- that high concentrations of D-xylose increased chondroitin tained by feeding the cells at 2-day intervals with modified sulfate synthesis in progeny of BrdU-treated limb bud cells. Eagle's medium containing 7% horse serum and 10% fetal More recently, Okayama et al. (9) observed that p-nitro- calf serum. When appropriate, BrdU was added (32 uAM) phenyl-#-D-xyloside acted as an exogenous substrate for the from the sixth to the tenth day of culture after plating. cell-free particulate galactosyltransferase system. Further- Embryonic chick limb buds were cultured as described more, this compound stimulated 85SO4 incorporation into (12). Briefly, limb buds (stage 23-24) were dissociated in material characterized as protein-free chondroitin sulfate 0.25% trypsin-EDTA and 2.5 X 107 cells were plated on 60- chains in puromycin-treated cartilage slices (9). The present mm plastic culture dishes in F-12 medium with 7% horse report is concerned with the stimulation of chondroitin sulfate serum and 8% fetal calf serum. Cultures either remained synthesis by /#-D-xylopyranosides in cultures of chondrocytes untreated or were exposed to 32-M BrdU for the first 2 days and limb bud cells treated with BrdU and puromycin. In of growth. Subsequently, the BrdU was removed and cells addition, enhancement of chondroitin sulfate synthesis by were maintained for an additional 7 days in medium without P-D-xylopyranosides was demonstrated in four other cell BrdU. Cells were fed every other day. 4047 Downloaded by guest on September 28, 2021 4048 Cell Biology: Schwartz et al. Proc. Nat. Acad. Sci. USA 71 (1974) Rat glial cells (clonal strain C-6) and mouse neuroblastoma TABLE 1. Specificity of effect of xylosides on (C1300 clonal strain NB41A) were a gift of Dr. Gordon Sato. glycosaminoglycan synthesis by chondrocytes* Rat hepatoma cells were obtained from Drs. D. Haggerty and G. Popjak. Trypsinized suspensions of rat glial cells were BrdU plated at a density of 5 X 105 cells per 100-mm plastic tissue Puro- + puro- culture dish. During the following 7 days, cells were fed twice Addition Control mycin BrdU mycin with fetal calf with Eagle's medium supplemented 10% Experiment I serum. Neuroblastoma cells and hepatoma cells were cultured None 36,0O 4,250 2,610 1,220 under the same conditions. p-Nitrophenyl-3-D-xylose 56,500 53,700 44,900 39,000 Glycosaminoglycan Synthesis. For assay of sulfated glycosa- p-Nitrophenyl-a-D-xylose 37,400 2,460 4,400 1,350 minoglycan synthesis, the xylosides and either 5 MuCi/ml of 4-Methylumbelliferyl-p- D-xylose 56,300 N.D. 34,750 31,200 H235SO4 or 10 ,Ci/ml of [3H]acetate were added for 6 hr on 42,400 15,000 21,200 17,700 the last day of culture. Media and cells were collected, and fl-D-Xylose the amount of radioactive glycosaminoglycan was determined Experiment II as described (13) after addition of 2 mg of carrier chondroitin- None 41,000 5,730 4,700 408 4-sulfate. p-Nitrophenyl-IB-D-xylose 94,400 102,000 68,600 38,300 p-Nitrophenyl-,-D- Chondroitinase Treatment. Chondroitinase ABC digestions galactose 35,000 7,220 2,980 605 were performed as described by Saito et al. (14) in 10 mM p-Nitrophenyl-a-D- Tris-acetate buffer (pH 8.0) for 21 hr at 370 with 0.2-0.4 galactose 35,800 1,430 4,400 553 units of enzyme in 0.8 ml of incubation mixture. 4-Methylumbelliferyl-#B-D- galactose 35,500 1,730 2,900 152 Streptococcal Hyaluronidase. Digestion mixtures containing p-Nitrophenyl-3-D-N- 0.15 mg of Lederle Varidase in a total volume of 1.15 ml of acetylgalactosamine 47,600 2,060 2,470 785 10 mM phosphate buffer (pH 6.0) and 0.15 M NaCl (15) were incubated for 18 hr at 37°. On the tenth day of culture, chondrocytes (3 X 106 cells/plate) growing in the presence or absence of 32 MM BrdU were incubated Nitrous Acid Degradation. Samples were treated with 1 with Au Ci/ml of H2,5SO4 plus the indicated compounds at a final volume of 5% sodium nitrite and 1 volume of 33% acetic concentration of 1 mM (except for D-xylose, which was used at a acid at room temperature for 2 hr according to the procedure final concentration of 42 mM) and with or without 10 ,g/ml of of Dische and Borenfreund (16). The mixtures were then puromycin for 6 hr. Glycosaminoglycans were isolated from in pooled cells plus media according to Dorfman and Ho (13). N.D., lyophili v,-d tind dissolved distilled water. not determined. Gel Filtration. Labeled polysaccharide was chromatographed . * Results are expressed as cpm/106 cells. on columns of Sephadex G-50 (0.8 X 100 cm) previously - calibrated with bovine serum albumin and UDP-N-acetyl- -synthesis by cartilage (22). Chondrocytes treated with both galactosamine. Samples were eluted with 0.2 M NaCl in frac- BrdMU and puromycin are likewise stimulated by the f3-xylo- tions of 0.8 ml, and aliquots (0.1 or 0.25 ml) of each fraction sides to produce high levels of glycosaminoglycans. were assayed for radioactivity. Glycosaminoglycan synthesis in control cultures is also Molecular size estimates of chondroitin sulfate chains pro- stimulated by P-xylosides. The magnitude of this effect varied duced by limb bud cells treated with 4-methylumbelliferyl- among different experiments, indicating that certain cultures ,3-D-xyloside are based on elution behavior on a calibrated have a greater potential for chondroitin sulfate chain syn- Sephadex G-200 column (1 X 100 cm) according to Hopwood thesis than is expressed; presumably core protein or xylosyl- and Robinson (17).