Synthesis of Proteochondroitin Sulfate by Normal, Nanomelic, and 5-Bromodeoxyuridine-Treated Chondrocytes in Cell Culture

Abstract

Chondrocytes grown in cell culture from micromelic chick sterna and 5-bromodeoxyuridinetreated normal cells showed reduced incorporation of sulfate into hyaluronidase-sensitive material as compared to normal chondrocytes. Proteochondroitin sulfate synthesized by normal embryonic chick chondrocytes in cell culture was heterogeneous on chromatography. The major chromatographic component was specifically inhibited in normal chondrocytes treated with 5-bromodeoxyuridine, and this component was absent in nanomelic chondrocytes.In studies of the metabolism of chondrocytes in vitro, attention has been directed to the metabolism of chondroitin sulfate and to the activity of enzymes involved in the formation of uridine nucleotide sugar precursors of this glycosaminoglycan. Chondroitin sulfate, a repeating unit of uronic acid and sulfated N-acetyl-galactosamine, is part of a much larger molecule, proteochondroitin sulfate, which consists of several chondroitin sulfate molecules linked to a protein backbone by neutral sugar linkage regions. We have studied the synthesis of proteochondroitin sulfate. More specifically, the synthesis of proteochondroitin sulfate in normal chondrocytes was compared to that in normal chondrocytes exposed to 5-bromodeoxyuridine (BrdU) and in chondrocytes from embryos homozygous for the recessive gene, nanomelia (1). The metabolism of chondroitin sulfate in the cartilage of these micromelic embryos is defective (2, 3).In this report, we present evidence for chromatographic heterogeneity of proteochondroitin sulfate in normal embryonic chick chondrocytes in cell culture. In addition we show a specific inhibition of the major chromatographic component in normal chondrocytes treated with BrdU and the absence of this component in nanomelic chondrocytes. MATERIALS AND METHODSChondrocytes from sternal chick cartilage were used in all cultures. Normal and nanomelic sterna were dissected from 14-day embryos that resulted from matings between birds heterozygous for the recessive lethal gene, nm (1). Phenotypically normal siblings are designated as normal. After careful removal of the perichondrial layer, the sterna were disassociated by the method of Cahn et al. (4). The cells were counted in a hemocytometer and plated at an initial density of 2 X 105 cells in 60-mm plastic culture plates. The medium used for all cell cultures consisted of 50% Ham's F-10 with twice the vitamin and 4 times the aminoacid concentration, 3385 38% Hank's balanced salt solution, bovine-serum albumin (5 /Ag/ml), and 10% fetal-calf serum. A fraction from embryo extract excluded by chromatography on Sephadex G-25 columns was added to the medium for the first 2 days of culture to increase plating efficiency (5). The medium also contained 50 units/ml each of penicillin and streptomycin. When applicable, BrdU was added to a final concentration of 10 ALM 4 days after initial plating of the cells. The cells were grown for 12 days in an atmosphere of 5% C02-95%7 air.Medium (4 ml per plate) was changed every other day. Appropria...

Terminal differentiation and calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor beta and serum factors.

“…When BrdUrd was added to the system on day 6, no increase in uronic acid content was observed (Table 2), as expected (20,21). Furthermore, neither the induction of alkaline phosphatase nor mineral deposition was noted in the BrdUrd-exposed cultures (Table 2).…”

“…Cultures maintained for [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] days in centrifuge tubes were washed three times with methioninefree MEM and then exposed for 3 hr to 10 ,Ci of L-…”

Defects in the cartilaginous growth plates of brachymorphic mice.

“…These disorders apparently involve greater changes in the proteoglycan portion of the cartilage matrix than in the collagen, although collagen fibers are thicker than normal and are well banded in cho/ cho mice (28). In the case of nanomelic chicks, chondrocytes synthesize greatly reduced levels of cartilage-specific proteoglycan monomer (22) which results in the production of only minimal amounts of extracellular matrix material. In contrast, the alteration in the matrix of brachymorphic cartilage does not appear to affect significantly the alignment of the epiphyseal chondrocytes.…”

In vitro regulation of cartilage matrix assembly by a Mr 54,000 collagen-binding protein.

“…The large chondroitin sulfate proteoglycan ofcartilage and its isolated glycosaminoglycan side chains cause some alterations in the kinetics of type I fibril formation but do not reduce the diameter of the fibrils (33). Further, the diameter of the type II collagen fibrils is not noticeably changed in the cartilage of a chicken mutant that lacks proteoglycan (34 7.2), 20% NaCl, 0.01 M EDTA, 0.1 M 6-aminohexanoic acid, and 5 mM benzamidine HCl to remove serum contaminants and was centrifuged at 25,000 x g for 30 min. The residue was reextracted with 500 ml of 2 M urea/0.05 M Tris-HCl, pH 7.2, containing the protease inhibitors used above.…”

“…Thus, a general feature determining matrix structures may be that collagens interact with other matrix components. In the case of cartilage, mutants lacking chondroitin sulfate proteoglycan have a matrix containing thin collagen fibrils, suggesting that proteoglycan is not essential (34). On the other hand, CBP may well bind to proteoglycan as well as to collagen and could link these apparently independent aggregates into one common structure.…”