Cultured Chick Embryo Chondrocytes Research Articles

STUDI PERTUMBUHAN DAN PERKEMBANGAN KONDROSIT EMBRIO AYAM DALAM KULTUR DENGAN ASAM BORAT

The teratogenisity mechanism of boric acid is less known. There are contradictory observation on the effect of boric acid on the fetus malformation. An observation reported that boric acid is bound to the riboflavin and this complex causes a riboflavin deficiency on fetus. However, the other observation suggested that the present of riboflavin and boric acid had no decresed malformation on fetus, but increased malformation. It is giving an idea that the one of teratogenisity mechanism of boric acid may the biological mechanism to the target cells. The goal of this study was to evaluate the effect of boric acid on the growth and development of cultured chick embryo chondrocytes. Chondrocytes were dispersed from sternal cartilage of 12-d chick embryos (Gallus gallus). Randomized completely design was used to arrange this treatment with five replicates for each treatment. The dosage of boric acid were 10, 20, 40, 60, 80, and 100 μg/ml. The cultures were observed on 1, 3, 5, 7, 9 day after boric acid treatment, including: the cell proliferation, cell adhesion, cell attachment and colony formation. The data were analyzed descriptively. Result of this experiment showed that on high dose (80 and 100 μg/ml) cell adhesion was not formed, therefore the colony was not formed, and formed vacuola in the cell. This study indicated that boric acid on the high dose can inhibit the cell proliferation, cell adhesion, cell attachment and colony formation.

Purification of chondroitin 6-sulfotransferase secreted from cultured chick embryo chondrocytes.

Chondroitin 6-sulfotransferase, which transfers sulfate from 3'-phosphoadenylyl sulfate to position 6 of N-acetylgalactosamine in chondroitin, was purified 1,430-fold to apparent homogeneity with a 22% yield from the serum-free culture medium of chick embryo chondrocytes by affinity chromatography on heparin-Sepharose CL-6B, wheat germ agglutinin-agarose, and 3',5'-ADP-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single broad protein band with an apparent molecular weight of 75,000. Since the purified enzyme has an apparent molecular weight of 160,000 as judged by gel chromatography on Superose 12, the active form of chondroitin 6-sulfotransferase may be a dimer. The purified enzyme transferred sulfate to chondroitin, chondroitin sulfate, and corneal keratan sulfate. Chondroitin sulfate E from squid cartilage, dermatan, sulfate, and heparan sulfate hardly served as acceptors of the sulfotransferase. The sulfated product derived from keratan sulfate was degraded by keratanase but not by chondroitinase ABC.

Heat-shock response in cultured chick embryo chondrocytes. Osteonectin is a secreted heat-shock protein.

We investigated the induction of specific protein expression by heat shock in dedifferentiated and hypertrophic chick embryo chondrocytes in a culture system that allows 'in vitro' differentiation of cartilage cells [Castagnola, P., Moro, G., Descalzi-Cancedda, F. and Cancedda, R. (1986) J. Cell. Biol. 102, 2310-2317]. As control, we used cultures of embryonic fibroblasts from the whole body and from the skin. In the cell lysates of all cultures we identified four major heat-shock proteins (HSP), with a molecular size corresponding to HSP families previously described (HSP 90, HSP 70, HSP 47 and HSP 26). Some of these proteins were constantly induced when the temperature was raised, others were expressed in a more variable manner. Differences also existed in the relative amount of the HSP synthesized by the four cultures. When we specifically investigated HSP species released into the culture medium, we observed a 43-45 kDa protein constantly expressed and secreted in large amount by the cells. On the basis of its biochemical characteristic and its precipitation by specific antibodies, this protein has been identified as osteonectin (SPARC, BM-40).

Secretion of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase from cultured chick embryo chondrocytes

We found that chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase were released into the culture medium from the cultured chick embryo chondrocytes. Since the release of the sulfotransferases was observed not to only in serum-supplemented medium but also in serum-free medium, the released sulfotransferases were unlikely to be derived from serum. Addition of ascorbate to the serum-free medium supported the continuous release of the sulfotransferases. Monensin, which is known to cause dilatation of the Golgi apparatus and to inhibit sulfation of proteoglycan, was found to affect the release of the sulfotransferases. In the presence of 10 −6 M monensin, chondroitin 6-sulfotransferase activity in the cell layer was decreased to less than one tenth of the control, and the rate of the release of the activity became much smaller than the control after the initial rapid release. The activity of chondroitin 4-sulfotransferase was also affected by monensin, but the reduction of the chondroitn 4-sulfotransferase activity in the cell layer was not so great as the reduction of chondroitin 6-sulfotransferase activity. Unlike to the microsomal sulfotransferases, both chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase released into the culture medium were retained in the soluble fraction after centrifugation of 100 000 × g for 60 min, and were not activated by detergent. pH optimum and requirements for sulfhydryl compounds of the released sulfotransferases were similar to those observed previously in the chondroitin sulfotransferases from chick embryo cartilage and from cultured chick embryo chondrocytes. These results suggest that chondroitin sulfotransferases, which are localized in the Golgi apparatus, may be secreted to the extracellular space in a soluble form under the culture conditions.

Sulfation of chondroitin oligosaccharides in vitro. Analysis of sulfation ratios.

Microsomal preparations from cultured chick embryo chondrocytes were incubated with 3'-phosphoadenosine 5'-phosphosulfate and oligosaccharides prepared from chondroitin. Rates of 4- and 6-sulfation were measured at pH 6 and 8 in the presence of MnCl2 and Brij 58. Ratios of the overall 6-sulfation to 4-sulfation rates ranged from 40-200 at pH 8 and from 6-35 at pH 6, depending upon the composition of the assay mixture. When saturating concentrations of 3'-phosphoadenosine 5'-phosphosulfate and the oligosaccharide acceptors were used, the resulting products were mixtures of monosulfated oligosaccharides. The compositions of the mixtures formed from oligosaccharides with degrees of polymerization from 4-12 at pH 6 and 8 were analyzed. Sulfate substituents were found at all N-acetyl-D-galactosamine (GalNAc) residues in the acceptors but were not evenly distributed along the oligosaccharide chains. For oligosaccharides with nonreducing terminal D-glucuronic acid (GlcUA) residues, sulfation at the nonreducing terminal GlcUA----GalNAc occurred exclusively at the C6 of the GalNAc residue. However, for oligosaccharides with nonreducing terminal GalNAc residues the rate of 6-sulfation of the nonreducing terminal GalNAc was markedly reduced and was similar to the rate of 4-sulfation at the same position. The rates of sulfation at the reducing ends of the oligosaccharides were relatively high for the shorter oligosaccharide acceptors but decreased with increasing length of the acceptor, suggesting that the sulfotransferases recognized primarily the GalNAc residues in the nonreducing terminal regions.

Deposition of type X collagen in the cartilage extracellular matrix.

In cultured chick embryo chondrocytes, type X collagen is preferentially deposited in the extracellular matrix, the ratio between type II and type X collagen being about 5 times higher in the culture medium than in the cell layer. When the newly synthesized collagens deposited in slices from the epiphyseal cartilage of 17-day-old embryo tibiae were isolated, type X collagen was always the major species. In agreement with this result the mRNA for type X collagen was the predominant mRNA species purified from the same tissue. When the total collagen (unlabeled) deposited in the epiphyseal cartilage was analyzed, it was observed that type X collagen represented only 1/15 of the type II collagen recovered in the same preparation. The possible explanations for these differences are discussed.

Cytoskeleton and adhesion patterns of cultured chick embryo chondrocytes during cell spreading and Rous sarcoma virus transformation

The cytoskeleton and the adhesion complex of chick embryo chondrocytes maintained in vitro have been studied by fluorescence and interference reflection microscopy during the process of cell spreading. The pattern of actin-containing microfilaments and the distribution of vinculin speckles on adhesion plaques have been found to change as a function of the culture time. Newly plated chondrocytes adhere to the substratum mostly around a peripheral ring-like region and show a complex tridimensional array of microfilaments. When chondrocytes flatten, they develop stress fibres and show a diffuse system of vinculin-containing adhesion plaques scattered over the entire ventral side of the cells. Upon infection with Rous sarcoma virus (RSV) chondrocytes display one or more actin-containing ruffles located on the dorsal side similar to the ‘actin flowers’ earlier described in other cell types. These structures have been found to accumulate vinculin too. In chondrocytes infected with two t d-t s mutants of RSV, ‘actin flowers’ have been found to persist at the restrictive temperature. At this temperature, however, in the majority of cells, stress fibres and adhesion plaques reappear.

A unique low molecular weight collagen secreted by cultured chick embryo chondrocytes.

A unique low molecular weight collagen secreted by cultured chick embryo chondrocytes.

Formation of matrix vesicles by cultured chick embryo chondrocytes.

Formation of matrix vesicles by cultured chick embryo chondrocytes.

Shedding of hyaluronate from the cell surface of Rous sarcoma virus-transformed chondrocytes.

Transformation of cultured chick embryo chondrocytes with Rous sarcoma virus gives rise to increased incorporation of isotopic precursors into hyaluronate and decreased incorporation into chondroitin 6-sulfate. Chemical measurements of these glycosaminoglycans showed corresponding changes. Comparison of the kinetics of production of glycosaminoglycan by normal and Rous sarcoma virus-transformed chondrocytes demonstrated (i) that the rate of accumulation in the medium was similar in both cultures, and (ii) that approximately 50% of total glycosaminoglycan produced by the normal chondrocytes, but only 10% of that from the transformed cells, accumulated in the cell layer. Prelabel-chase experiments indicated that cell surface-associated hyaluronate, as measured by release from the cell layer by trypsin treatment, was shed rapidly into the medium and accounted for all of the hyaluronate which accumulated in the medium. Thus we conclude (i) that accumulation of cell surface-associated glycosaminoglycan is dramatically reduced in Rous sarcoma virus-transformed chondrocytes, and (ii) that hyaluronate produced by the transformed chondrocytes is first deposited in the cell-associated extracellular compartment and then rapidly shed into the medium, rather than being secreted directly into the medium.

Ultrastructural analysis of the effect of ascorbic acid on secretion and assembly of extracellular matrix by cultured chick embryo chondrocytes

Secondary cultures of differentiated sternal chondrocytes synthesize collagen (α1:α2 = 32.3 suggesting Type II collagen) and proteoglycan granules, but the morphology of the extracellular matrix (ECM) deposited around these cells is quite different from that produced by chondrocytes in vivo . The ECM of cultured chondrocytes is sparse and consists of a few 15-nm-diameter fibers interconnecting numerous oblong granules. This ECM is completely removed by treatment with testicular hyaluronidase. In contrast, the ECM of chondrocytes in vivo is predominantly fibrillar, with bundles of individual 15-nm-diameter fibers studded with granules. Treatment of this ECM with hyaluronidase reduces only the size of the granules, occasionally splaying larger fibers into smaller 5-nm-diameter components. Intracellular examination of cultured chondrocytes shows large, distended cisternae of rough endoplasmic reticulum (RER) filled with flocculent material. Since the enzymes prolyl and lysyl hydroxylases, located on RER, are known to be dependent on ascorbic acid for their activity, cultured cells were treated repeatedly with 50 μ g/ml ascorbic acid for 8 hr to 3 days in an attempt to relieve the impairment in post-translational matrix processing. Even after an 8-hr treatment with ascorbic acid, cultured chondrocytes show reduction of cisternal swelling, synthesis of predominantly α1 chains (α1:α2 = 50.5 suggesting Type II collagen), and deposition of ECM which is similar in morphology and in resistance to hyaluronidase as the ECM made by chondrocytes in vivo . It is concluded that ascorbic acid is not only necessary for normal collagen secretion, but also for proper deposition of ECM.

The effect of ascorbic acid on growth and synthesis of matrix components by cultured chick embryo chondrocytes.

AbstractFifty μg/ml ascorbic acid was found to accelerate the growth of logarithmically growing chick embryo sternal chondrocytes in cell culture. Differences in the number of cells between control and ascorbate‐treated cultures were apparent by the fourth day of treatment when a 58% increase in the number of cells and a 35% increase in protein per culture were observed in ascorbate‐treated cultures. Under these conditions, the cell size and protein content per cell was reduced in ascorbate‐treated cultures.Collagen synthesis, which was measured by the amount of collagenase‐sensitive counts released from 3H‐proline labeled protein, was increased by 116% over controls after 24 hours of ascorbate treatment. Observations with scanning and transmission electron microscopy are consistent with the idea that ascorbate has a much greater effect on collagen secretion and demonstrate an ascorbate‐mediated change in matrix morphology.35S incorporation into hyaluronidase‐sensitive or cetylpyridinium chloride precipitates was accelerated by 57% by 24 hours of ascorbate treatment whereas two, four and six days of treatment caused decreases in this parameter of 19%, 57% and 35%, respectively. This effect was dependent on cell density. 35S incorporation probably reflects synthesis of new glycosaminoglycan similar results were observed when either 35S or 14C‐glucose incorporation was monitored. An effect of ascorbate on both collagen and glycosaminoglycan synthesis is consistent with the possibility that collagen and glycosaminoglycan synthesis can be coregulated under some circumstances.

Depression by hyaluronic acid of glycosaminoglycan synthesis by cultured chick embryo chondrocytes

The effects of hyaluronic acid on the expression of differentiation by pure populations of cultured chick embryo chondrocytes are studied. In medium containing serum, hyaluronic acid has little effect on sulfate incorporation. In the absence of serum, 200 μg/ml of hyaluronic acid inhibits sulfate incorporation into hyaluronidase-sensitive material associated with the cell layer by as much as 50%. This effect is seen by 6 hr and is progressive. Hyaluronic acid has no effect on leucine or thymidine incorporation or sulfate uptake, matrix turnover, or collagen synthesis under these same conditions. It is shown that hyaluronic acid inhibits chondroitin sulfate synthesis and the accumulation of newly synthesized proteoglycan in the cell layer. These effects are not produced by other glycosaminoglycans, such as chondroitin sulfate, keratan sulfate, or heparan sulfate, or by monosaccharides, but oligosaccharides derived from hyaluronic acid mimic the effect of hyaluronic acid. These data indicate that specific glycosaminoglycans, such as hyaluronic acid, can affect the synthesis and accumulation of other glycosaminoglycans and should be considered as possible regulators of specific cellular activities.

Stimulation of synthesis of free chondroitin sulfate chains by beta-D-xylosides in cultured cells.

Previous studies have shown that D-xylose partially overcomes the puromycin inhibition of chondroitin sulfate synthesis in cultured chick embryo chondrocytes. Likewise, D-xylose stimulates chondroitin sulfate synthesis by limb bud mesenchyme cells previously treated with BrdU or limb bud cartilage cells treated with puromycin. The studies reported here show that p-nitrophenyl-beta-D-xylopyranoside and 4-methyl-umbelliferyl-beta-D-xylopyranoside cause a much greater stimulation than does D-xylose and are active at much lower concentrations. In contrast to D-xylose, the xylosides strikingly stimulate chondroitin sulfate synthesis in predifferentiated mesenchyme cells. The xylosides stimulate synthesis of chondroitin sulfate by rat glial cell tumor cells (RC-6), a mouse neuroblastoma (C1300, NB41A), and two strains of cultured rat hepatoma cells (HTC, H(4)). These results indicate that certain types of nonconnective tissue cells contain the enzymic machinery for synthesis of chondroitin sulfate which is normally not utilized because of limited synthesis of core protein and/or xylosyltransferase. The beta-xylosides may be used as a probe of the capacity of various cell types to synthesize sulfated glycosaminoglycans.

The selective inhibition of mucopolysaccharide synthesis by vitamin A treatment of cultured chick embryo chondrocytes.

The effect of vitamin A on the synthesis of acid mucopolysaccharides has been studiedin vitro in cell cultures of chick embryo sternal chondrocytes. Vitamin A produces a concentration-dependent inhibition of acid mucopolysaccharide synthesis. the inhibitory effect of vitamin A on acid mucopolysaccharide synthesis is not the result of a general cytotoxicity, since, under the conditions studied here, while there is over a 50% inhibition of mucopolysaccharide synthesis, the incorporation of leucine into acid insoluble material or the synthesis of collagen is unaltered. Lysosomal activation does not appear to be involved in the inhibition of sulfate incorporation, since E-amino caproic acid (a protease inhibitor) and chloroquine (a lysosomal membrane stabilizer) do not reverse this inhibition. In addition, enhanced polysaccharide breakdown is too small to account for vitamin A-inhibited sulfate incorporation. The effect of vitamin A is not readily reversed and chondrocytes become fibroblastic after a 24 hour treatment with 3 μg/ml of vitamin A. Because the incorporation of glucose into polysaccharides is inhibited faster than glucosamine or sulfate, it is suggested that vitamin A acts by an unknown mechanism to inhibit a step early in the pathway for mucopolysaccharide biosynthesis.

Mediation of growth hormone-enhanced expression of the cartilage phenotype in vitro by the availability of the essential amino acid valine

Without increasing cell number, ovine growth hormone was shown to stimulate the incorporation of 25SO 4 by cultured chick embryo chondrocytes into chondroitin sulfate. Since the stimulation of sulfation by growth hormone was abolished when the amino acid concentrations in the medium were doubled, the relationship between amino acids and growth hormone in promoting the synthesis of acid mucopolysaccharides was investigated. Comparison of the incorporation of various labeled amino acids into trichloroacetic acid-soluble and insoluble material revealed that growth hormone promoted the incorporation of only valine into trichloroacetic acid-insoluble material. Furthermore, growth hormone stimulated valine incorporation into both extracellular and intracellular protein, rather than preferentially into extracellular chondromucoprotein. Growth hormone gave a 4-fold stimulation of valine incorporation into collagen without stimulating collagen synthesis. That growth hormone enhances sulfation by stimulating valine availability was further supported by the observations: (a) doubling only the valine concentration in the medium enhanced sulfation; (b) in medium with twice the normal valine concentration, sulfation failed to be further stimulated with the addition of growth hormone; and (c) in medium with all the other amino acids except valine at twice normal concentrations, growth hormone enhanced sulfation. In addition the temporal relationships and synthetic events occurring between growth hormonealtered valine availability and enhanced chondromucoprotein synthesis were studied. It was found that growth hormone-promoted valine incorporation into acid-insoluble material is a rapid effect that can be detected by 10 min after hormone addition and does not require RNA synthesis. Increased valine availability is rapidly reversed after growth hormone removal ( t 1 2 = 10 hr ). On the other hand, growth hormone- and valine-enhanced chondromucoprotein synthesis are slower responses, taking over 24 hr of treatment for a maximal stimulation, and are mediated by RNA synthesis, as indicated by actinomycin D sensitivity. Enhanced chondromucoprotein synthesis is also relatively stable after removal of growth hormone or valine ( t 1 2 = 70 hr ). The evidence suggests that the availability of a single amino acid, valine, plays a regulatory role in the synthesis of a specialized cellular product and that growth hormone acts at some level to alter the availability of this essential amino acid.

The requirement for RNA synthesis in the differentiation of cultured chick embryo chondrocytes.

AbstractSecondary chick embryo sternal chondrocyte colonies, derived from differentiated clones, were treated with 0.005 μg/ml of actinomycin D for 18 hours. While RNA synthesis was inhibited by 70%, both growth and matrix accumulation were inhibited. The incorporation of 3H‐uridine into acid precipitable‐base hydrolyzable material, 35SO4 into hyaluronidase‐sensitive material, and of 3H‐proline into collagenase sensitive and insensitive material was measured in high density cultures treated with various doses of actinomycin D for 18 hours. The inhibition of sulfate incorporation was highly correlated with RNA synthesis and was observable within the first six hours after drug addition. Ethidium bromide and lucanthasone had no effect on sulfate incorporation. On the other hand, collagen synthesis was more resistant to actinomycin D treatment and was not inhibited until non‐collagenous protein synthesis was also inhibited. The results are discussed in terms of the independent regulation of the synthesis of two specialized cell products made by the same cell.

The comparative effects of several mammalian growth hormones on sulfate incorporation into acid mucopolysaccharides by cultured chick embryo chondrocytes.

This study reports the effects of varying concentrations of bovine, ovine, porcine, rat, and human growth hormones on the incorporation of 35SO4 into hyaluronidase-sensitive materials by cell cultures of chick embryo sternal chondrocytes. At 10 μg/ml, ovine or bovine growth hormones stimulated sulfation 20–50% over control cultures. Ovine growth hormone was more effective at lower concentrations than was bovine growth hormone. In contrast, porcine growth hormone stimulated sulfation at low concentrations (0.1 μg/ml) but was inhibitory at higher concentrations. At 10 μg/ml of porcine growth hormone, sulfation was inhibited by 10–18%. Rat growth hormone inhibited sulfate incorporation 8–12% at concentrations greater than 1.0 μg/ml. Human growth hormone or ovine prolactin had no measurable effect on sulfation. The results indicate that avian cartilage can respond in vitro to several mammalian growth hormones, and that the differences in the biological activities of growth hormones parallels differences in th...

Stimulation of sulfate incorporation by growth hormone treatment of cultured chick embryo chondrocytes

The ability of ovine growth hormone to stimulate the incorporation of 35S by cultured chick embryo chondrocytes into hyaluronidase-sensitive material is reported. Growth hormone-stimulated incorporation of sulfate (22–120% over controls) is not mediated by an increase in cell number. It was observed that the stimulatory effect of growth hormone on sulfation requires the presence of a heat-sensitive component of fetal calf serum, which is present in the medium, and is abolished when amino acid concentrations in the culture medium are doubled. Evidence is presented which suggests that avian cartilage, unlike mammalian cartilage, is capable of an in vitro response to growth hormone without mediation by serum sulfation factor.