Metabolic effects of forskolin in chick chondrocytes

The effects of forskolin on parameters of energy metabolism and proteoglycan synthesis have been investigated in chick embryo sternal chondrocyte cultures. After 8 h exposure to 100 microM forskolin, ATP levels and oxygen consumption were unaltered. Protein synthesis was unaffected up to 50 microM forskolin and protein degradation was unaffected by forskolin up to 100 microM. In contrast, incorporation of the proteoglycan precursors, 35SO4 and [3H]glucosamine, was more sensitive to forskolin. Inhibition was linear with dose between 10 and 100 microM, reaching 70% at 100 microM. Incorporation of 35SO4 into glycosaminoglycan chains initiated on an artificial beta-xyloside acceptor was inhibited in the same manner. cAMP accumulation was maximal at 10 microM forskolin, a concentration which did not alter proteoglycan synthesis. We conclude that a major, acute effect of forskolin in these short-term experiments is inhibition of proteoglycan synthesis in a cAMP-independent manner.     

Effect of glucocorticoid on glycosaminoglycans synthesis in cultured mouse embryonic palatal mesenchymal cells

The effect of glucocorticoids (GC) on the synthesis of glycosaminoglycans (GAGs) in mesenchymal cells obtained from palatal shelves of mouse embryos was studied. Both hyaluronic acid (HA) synthesis and sulfated GAG synthesis were inhibited with triamcinolone acetonide (TA) in a concentration-dependent manner. However, the degree of inhibition of synthesis with TA was greater for HA than for the sulfated GAGs. To determine whether the inhibitory effects of TA on HA and sulfated GAGs synthesis were mediated through GC receptors, we performed experiments using progesterone, vitamin B6, and molybdate. The inhibitory effect of TA was antagonized by all three. The results obtained in the present study suggest that the inhibition of synthesis of HA and sulfated GAGs with GC is mediated through GC receptors and is involved in cleft palate induction by GC.     

The effect of ascorbate on embryonic chick sternal chondrocytes cultured in agarose

Primary cultures of embryonic chick sternal chondrocytes were embedded in a three-dimensional matrix of 1% solid agarose which was overlaid with nutrient media. The chondrocytes divided and formed nests of spherically shaped cells which were surrounded by an extensive extracellular matrix containing high molecular weight proteoglycans. Using light and electron microscopy, condensation of proteoglycan was observed pericellularly, often forming septa between cells of a nest, and as part of the outer boundary of the cell nest. No cross-striated collagen fibers were observed in the extracellular matrix although proteoglycan appeared to decorate a network of fine strands. Upon the addition of ascorbate to the nutrient media high molecular weight proteoglycans were synthesized, but there was a marked decrease in the synthesis of proteoglycans after a 10 day exposure to ascorbate. Morphologically, the decrease in proteoglycan synthesis was manifested in the discontinuous arrangement of the pericellular matrix as well as the diffuse form of the cell-nest boundary. Both of these structures were clearly defined in control cultures and were enriched in proteoglycan as demonstrated by ruthenium red staining. This study demonstrates that embryonic chondrocytes remain differentiated when cultured in solid agarose for a period of up to 15 days. They continue to synthesize their tissue specific macromolecules and are phenotypically stable when exposed to ascorbate for extended periods of time.     

Extracellular glycosaminoglycans (GAG) released by chick embryonic fibroblasts

Summary Administration of Concanavalin A (Con A) to cultured skin fibroblasts derived from chick embryos at two developmental stages produce variations in the relative concentration of individual glycosaminoglycan (GAG) secreted by the cells. This effect is different: at 7 days (increase of hyaluronic acid and dermatan sulphate and decrease of chondroitin sulphate) and at 14 days (dermatan sulphate is not detectable).All the cells bind the Con A specifically, but a different pattern of agglutination is present in fibroblasts of the two embryonic ages. Since Con A is well known to bind carbohydrate-containing surface proteins, the result suggests that the release of GAG by chick embryonic fibroblasts can be modulated by cell surface receptors.     

Effects of viral transformation on synthesis and secretion of collagen and fibronectin-like molecules by embryonic chick chondrocytes in culture

Monolayer cultures of chondrocytes isolated from 11-day-old chick embryo vertebral cartilage were transformed by Rous sarcoma virus, and the effects of transformation on synthesis and secretion of extracellular proteins by these cells were studied. Transformation resulted in decreased synthesis of type II collagen which did not appear to be due to underhydroxylation of collagenous protein but to a decrease in the total amount synthesized. Carboxymethyl-cellulose chromatography and polyacrylamide disc gel electrophoresis failed to demonstrate any alpha 2 chains as a result of the transformation, suggesting that conversion of type II to type I collagen did not occur. In contrast to the decrease in collagen synthesis, synthesis of a molecule with biochemical characteristics similar to fibronectin increased markedly in virally transformed cultures. Although there were no significant differences in the amount of fibronectin-like molecules in the cell layers of normal and transformed chondrocytes, a marked increase of these molecules in the culture media of the transformed cells was demonstrated. These findings were confirmed by experiments with temperature-sensitive mutants of the virus.     

Line-restricted hemoglobin synthesis in chick embryonic erythrocytes

The presence of embryonic hemoglobin in early definitive erythrocytes was checked by indirect immunofluorescence assay, using specific antibodies raised against embryonic Hb P. As positive control we used anti-Hb A which reacted with the alpha A chain shared by the minor embryonic Hb E and the adult Hb A. The assay was performed using blood smears from embryos between 6 and 15 days of incubation and yolk sac sections from embryos between 4 and 6 days. Hb P was never detected in the definitive line in circulating erythrocytes or in maturing erythroblasts still sequestered in the blood islands of the yolk sac. The expression of the 'specific' embryonic genes is thus restricted to the primitive line (as the 'specific' adult beta gene is restricted to the definitive line), and the hemoglobin switch is the result of the progressive substitution of the primitive line by the definitive one.     

Stimulation of sulfated-proteoglycan synthesis by forskolin in monolayer cultures of rabbit articular chondrocytes

Forskolin, a plant cardiotonic diterpene, stimulated proteoglycan biosynthesis by chondrocytes in monolayer culture. The quantitative increase in proteoglycans was dependent on the concentration of forskolin, but was relatively independent of the presence of serum. At forskolin concentrations that stimulated proteoglycan synthesis, a significant stimulation of adenylate cyclase and cAMP was also measured. The quantitative increase in proteoglycans was characterized, qualitatively, by an increased deposition of newly synthesized proteoglycan in the cell-associated fraction. An analysis of the most dense proteoglycans (fraction dA1) in the cell-associated fraction showed that more of the proteoglycans eluted in the void volume of a Sepharose CL-2B column, indicating that an increased amount of proteoglycan aggregate was synthesized in forskolin-treated cultures. The proteoglycan monomer dA1D1 secreted into the culture medium of forskolin-stimulated cultures overlapped in hydrodynamic size with that of control cultures, although cultures stimulated with forskolin and phosphodiesterase inhibitors produced even larger proteoglycans. The hydrodynamic size of 35SO4 and 3H-glucosamine-labelled glycosaminoglycans isolated from the dA1D1 fraction of the culture medium was greater in forskolin-treated chondrocytes, especially from those in which phosphodiesterase inhibitors had been added. These results indicated that forskolin, a direct activator of chondrocyte adenylate cyclase mimicked the effects of cAMP analogues on chondrocyte proteoglycan synthesis previously reported. These results implicate activation of adenylate cyclase as a regulatory event in the biosynthesis of cartilage proteoglycans, and more specifically in the production of hydrodynamically larger glycosaminoglycans.     

Developmental changes in glycosaminoglycans,collagen and collagenase activity in embryonic chick skin

In order to study remodelling of connective tissue during development, changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin at various stages have been studied.Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20).At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis.Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

Biosynthesis of glycosaminoglycans by the separated tissues of the embryonic chick cornea

Corneal tissues (epithelium, endothelium, and stroma) were isolated from chick embryos at 14, 17, and 20 days of incubation and immediately labeled in vitro with d-[6-³H]glucosamine and H₂³⁵SO₄. Amount of label incorporated into each type of glycosaminoglycan or into glycopeptides was determined by specific degradative techniques, in conjunction with gel filtration chromatography. Results suggested that corneal epithelium synthesized little, if any, corneal keratan sulfates, but that corneal endothelium may have synthesized small amounts of corneal keratan sulfates. Nearly all corneal keratan sulfates were derived from the stroma. Corneal heparan sulfates appeared to be derived predominantly from corneal epithelium at later stages of development. Corneal endothelium contributed large proportions of the hyaluronic acids of the cornea. Only epithelium produced a large proportion of sulfated glycoproteins. In addition, epithelium synthesized a large proportion of a sulfated, high molecular weight polysaccharide which was resistant to treatments degrading known types of glycosaminoglycans. Each corneal tissue may not only affect corneal morphogenesis directly by contributing a unique spectrum of glycosylated proteins to the extracellular matrix, but also may regulate the extracellular matrix composition indirectly by modulating the biosynthetic activities of the other corneal tissues.     

Quantitative analysis of collagen expression in embryonic chick chondrocytes having different developmental fates

A quantitative determination of collagen expression was carried out in cultured chondrocytes obtained from a tissue that undergoes endochondral bone replacement (ventral vertebra) and one that does not (caudal sterna). The "short chain" collagen, type X is only expressed in the former while the other "short chain" collagen type IX, was primarily expressed in the latter. These two tissues also differ in that vertebral chondrocytes express moderate levels of both type I procollagen mRNAs which were translated into full length procollagen chains both in vivo and in vitro, while caudal sternal chondrocytes did not. The percent of collagen synthesis was about 50% in both cell types, but sternal cells expressed twice as much collagen as vertebral cells even though type II procollagen was more efficiently processed to alpha-chains in vertebral chondrocytes than in sternal chondrocytes. The number of type II procollagen mRNA molecules/cell was found to be about 2300 in vertebral chondrocytes and about 8000 in sternal cells, in good agreement with the results reported by Kravis and Upholt (Kravis, D., and Upholt, W. B. (1985) Dev. Biol. 108, 164-172). There were about 630 copies of type I procollagen mRNAs with an alpha 1/alpha 2 ratio of 1.6 in vertebral chondrocytes compared with 5100 copies and an alpha 1/alpha 2 ratio of 2.2 in osteoblasts, and less than 40 copies in sternal cells. Since the rate of type I collagen chain synthesis was 50 times greater in osteoblasts than in vertebral cells, type I procollagen mRNAs were about six times less efficiently translated in vertebral cells than in osteoblasts. The type I mRNAs in vertebral chondrocytes were polyadenylated and had 5' ends that were identical in osteoblasts, fibroblasts, and myoblasts. Moreover, type I mRNAs isolated from vertebral chondrocytes were translated into full length preprocollagen chains in vitro in rabbit reticulocyte lysates. Thus, chondrocytes isolated from cartilage tissues with different developmental fates differed quantitatively and qualitatively in total collagen synthesis, procollagen processing, and distribution of collagen types.     

Developmental changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin.

In order to study remodeling of connective tissue during development, changes in glycosaminoglycan, collagen and collagenase activity in embryonic chick skin at various stages have been studied. Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20). At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum, indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis. Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

The nature and origin of the glycosaminoglycans of the embryonic chick vitreous body

Glycosaminoglycans of the embryonic chicken vitreous were characterized and then were used as markers to establish which tissues synthesize the vitreous humor during development. The glycosaminoglycans are predominantly chondroitin sulfates by several criteria. They are resistant to streptomyces hyaluronidase, an enzyme which degrades only hyaluronate, and are digested by testicular hyaluronidase and chondroitinase AC, enzymes which degrade hyaluronate plus chondroitin 4- and 6-sulfates. On electrophoresis on cellulose acetate in 0.15 M phosphate buffer, pH 6.7, the vitreous glycosaminoglycans migrate slightly slower than authentic chondroitin sulfate, but, in 0.1 N HCl, they migrate very close to chondroitin sulfate standards. Finally, the disaccharides produced by digestion of these radioactively labeled glycosaminoglycans with chondroitinases AC and ABC were identified as Δdi-4S and Δdi-6S, as expected for chondroitin 4- and 6-sulfate. By using incorporation of radioactive precursors into chondroitin sulfates in vitro, we than determined which tissues synthesize the vitreous humor in the developing eye. Late in development, on Day 12–13, the isolated vitreous is very active in chondroitin sulfate synthesis, while the neural retina, the lens, and the pecten are less active and produce a high proportion of enzyme-resistant GAG. The eye tissues isolated from embryos labeled in ovo retain similar amounts and types of glycosaminoglycans, indicating that cells within the vitreous synthesize the vitreous humor glycosaminoglycans at this time. Earlier in development, from Days 6 to 8, the isolated vitreous incorporates very low levels of radioactivity into GAG, but the neural retina incorporates high levels of radioactivity into chondroitin sulfate. When the embryos are labeled in ovo and the same tissues are isolated following incorporation, the vitreous retains more radioactive chondroitin sulfate than does the neural retina. Thus, the vitreous humour glycosaminoglycan is initially synthesized by the neural retina and is secreted into the vitreous space.     

Matrices containing glycosaminoglycans in the developing anterior chambers of chick and Xenopus embryonic eyes.

The fibrous matrix present in the anterior chamber of the chick eye on the 4th day of development has been shown by autoradiography and histochemistry to contain chondroitin sulfate, protein, neutral polysaccharides, and possibly hyaluronic acid (HA). Its synthesis, probably by the mesenchyme of the angle of the eye, is completed by around 10 days of development. In the scanning electron microscope (SEM) it can be seen that, although the matrix thins as the eye grows, it does not disappear until the 15th day. The development of the Xenopus cornea is described; this animal has a matrix in its anterior chamber from soon after the formation of the inner cornea (stage 41) until metamorphosis 7 weeks later. In the SEM, this material appears as a dense, featureless aggregate rather than as a matrix of thick fibres; in the transmission electron microscope, it is seen to be a network of fine filaments containing small dark-staining granules. Histochemistry shows that it contains HA, protein, and neutral polysaccharides. The morphological evidence is compatible with the matrix being made by the inner cornea. The probable major role of the matrix is to separate lens from cornea in establishing the anterior chamber. In the chick embryo, at least, the matrix is also likely to help stabilise the endothelium during its formation.     

DNA synthesis in the embryonic chick central nervous system

Summary DNA synthesis has been studied in chick embryos age between 2 and 10 days, using labelling with tritiated thymidine and stripping film autoradiography. The observations made earlier in the literature on a premitotic migration of the nuclei in the neural epithelium have been verified. In young stages (before day 7) peripherally migrated cells do not synthesize DNA, but after day 7 such a synthesis occurs. In spite of this, few mitoses are seen. The interpretation of these facts is discussed.The costs of this investigation were defrayed by grants from the Swedish Medical Research Council, the Medical Faculty of Lund, and the Royal Physiographic Society.     

Glycosaminoglycan synthesis by the early embryonic chick heart

Glycosaminoglycans of embryonic chick hearts labeled in situ were characterized by means of labeled precursor incorporation, electrophoretic mobility, sensitivity to testicular hyaluronidase, elution characteristics from CPC-cellulose columns, and hexosamine content. During the initial period of overt cardiac muscle differentiation (approximately stage 10) chondroitin sulfates are not detectable but an undersulfated component is present. Chondroitin sulfate synthesis appears shortly after overt muscle differentiation. Hyaluronate is present both during and after overt myocardial differentiation. Although epimerization of ³H-glucosamine-derived labeled UDP-N-acetyl-d-glucosamine occurs (determined by recovery of incorporated labeled galactosamine), label does not appear in chondroitin sulfate. ³H-Glucosamine is thus a relatively specific precursor for unsulfated glycosaminoglycans, a fact that we exploited in demonstrating their distribution radioautographically. Glycosaminoglycan synthesis was also examined in hearts labeled (a) in isolated organ culture, (b) in situ but exposed directly to the medium by removal of the splanchnopleure. In both cases fully sulfated chondroitin sulfate and chondroitin are not synthesized. Hearts make only hyaluronate and undersulfated chondroitin sulfate.