Constitutive myc expression impairs hypertrophy and calcification in cartilage.

The myc oncogene is expressed by proliferating quail embryo chondrocytes (QEC) grown as adherent cells and is repressed in QEC maintained in suspension culture. To investigate the interference of myc expression during chondrocyte differentiation, QEC were infected with a retrovirus carrying the v-myc oncogene (QEC-v-myc). Uninfected or helper virus-infected QEC were used as control. In adherent culture, QEC-v-myc displayed a chondrocytic phenotype and synthesized type II collagen and Ch21 protein, while control chondrocytes synthesized type I and type II collagen with no Ch21 protein detected as long as the attachment to the plastic was kept. In suspension culture, QEC-v-myc readily aggregated and within 1 week the cell aggregates released small single cells; still they secreted only type II collagen and Ch21 protein. In the same conditions control cell aggregates released hypertrophic chondrocytes producing type II and type X collagens and Ch21 protein. In the appropriate culture conditions, QEC-v-myc reconstituted a tissue defined as nonhypertrophic, noncalcifying cartilage by the high cellularity, the low levels of alkaline phosphatase enzymatic activity, and the absence of type X collagen synthesis and of calcium deposition. We conclude that the constitutive expression of the v-myc oncogene keeps chondrocytes in stage I (active proliferation and synthesis of type II collagen) and prevents these cells from reconstituting hypertrophic calcifying cartilage.     

Retinoic acid, hemin and hexamethylen bisacetamide interference with "in vitro" differentiation of chick embryo chondrocytes.

We have investigated the effect of all-trans Retinoic acid, and of substances (Hemine and Hexamethylene bisacetamide) which interfere with "in vitro" differentiation of mesenchyme derived cell lineages on the expression of specific markers of hyperthrophy in "in vitro" differentiating chick embryo chondrocytes. (Castagnola P., et al., 1986). Continuous treatment of chondrogenic cells in conditions allowing differentiation "in vitro" with Retinoic acid resulted in persistence of type I collagen synthesis and in lack of type X collagen and Ch 21 protein expression. Hemin treated cells secreted a reduced amount of type X collagen. HMBA treatment inhibited type X collagen expression and caused reduction of the ratio between type II collagen and Ch 21 synthesized. The data indicate an independent regulation of these markers during chondrocyte differentiation.     

Developmentally regulated synthesis of a low molecular weight protein (Ch 21) by differentiating chondrocytes

When transferred to suspension culture on agarose-coated dishes, dedifferentiated chick embryo chondrocytes resume the chondrocyte phenotype and continue their maturation to hypertrophic chondrocytes (Castagnola, P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102:2310-2317). In this paper we report the identification, purification, and characterization of a low molecular weight protein, named Ch 21, expressed and secreted by in vitro differentiating chondrocytes at a late stage of development. This protein is detectable in the cells after a short pulse labeling and is directly secreted in the culture medium. The Ch 21 protein has a peculiar resistance to limited pepsin digestion; nevertheless it is not collagenous in nature as revealed by its unaltered mobility when isolated from cells grown in the presence of alpha-alpha' dipyridyl, its resistance to bacterial collagenase, and its amino acid composition. By metabolic labeling of tissue slices and by immunohistochemistry, we show that in the chick embryo tibia the Ch 21 protein first appears at the boundary of the cone of hypertrophic cartilage and in the newly formed bone between the 6 and 10 d of embryo development and localizes in calcifying hypertrophic cartilage thereafter. The Ch 21 protein synthesized by the cultured chondrocytes is closely related and possibly identical to a 21K transformation- sensitive protein associated to the cell substratum of chick embryo fibroblasts.     

Synthesis and secretion of Ch 21 protein in embryonic chick skeletal tissues

We reported the identification, purification and characterization of a low molecular weight protein (Ch 21) expressed in vitro by differentiating chondrocytes at a late stage of development and observed in vivo in the growth plate region of the long bones at the border between hypertrophic cartilage and newly formed bone (Descalzi Cancedda, F., P. Manduca, C. Tacchetti, P. Fossa, R. Quarto, R. Cancedda, J. Cell Biol. 107, 2455-2463 (1988]. In this article, the synthesis and location of Ch 21 protein in the chick embryo tibia at late stage of development were further investigated. Ch 21 was observed in the cartilage matrix surrounding marrow cavities and in the prearticular outer layer by immunolocalization. In addition, the timing of Ch 21 appearance during the tibia development and its distribution in the growth plate region was better defined. We first observed presence of Ch 21 in the perichondral mid-diaphyseal sleeve of 7-day-old tibia. Ch 21 antibodies stained also the newly formed bone. Synthesis and secretion in the culture medium of Ch 21 protein was observed when bone fragments or cultured osteoblasts isolated from 19-day-old embryo tibiae were labeled in vitro. A search for the presence of Ch 21 in the chick embryo sternum was performed. The synthesis of Ch 21, both in the presumptive calcification cranial portion and in the permanent cartilaginous caudal portion of the sternum, was shown by metabolic labeling of tissue slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Ch21 protein, developmentally regulated in chick embryo, belongs to the superfamily of lipophilic molecule carrier proteins

Ch21, a developmentally regulated low molecular weight protein observed in chick embryo skeletal tissues, is expressed "in vitro" by differentiating chondrocytes at a late stage of development. Here we report the complete amino acid sequence of the protein. 86% of the total amino acid sequence was deduced by sequences of 17 high performance liquid chromatography-separated proteolytic fragments and 33 amino acid residues at the amino-terminal end of protein purified from spent culture medium of hypertrophic chondrocytes. Furthermore we isolated by molecular cloning the corresponding cDNA and determined its nucleotide sequence. By combining protein and nucleotide sequence data we determined the primary structure of the entire Ch21. It consists of 158 amino acids and has a molecular mass of 18.065 kDa. Computer-assisted analysis showed that the Ch21 belongs to the superfamily of low molecular weight proteins sharing a basic framework for binding and transport of small hydrophobic molecules.     

Characterization and primary structure of a second thioredoxin from the green alga, Chlamydomonas reinhardtii

A second thioredoxin, Ch1, distinct from the one recently reported [Decottignies, P., Schmitter, J.M., Jacquot, J. P., Dutka, S., Picaud, A. & Gadal, P. (1990) Arch, Biochem. Biophys. 280, 112-121] has been purified from the green alga, Chlamydomonas reinhardtii, and its functional and structural properties investigated. Its activity in various enzymatic assays has been compared with the activities of different plant thioredoxins (Ch2 from C. reinhardtii and spinach m and f). Ch1 cannot serve as a substrate for Escherichia coli thioredoxin reductase, but can be reduced by spinach ferredoxin-thioredoxin reductase. It is less efficient than its spinach counterpart in the activation of corn leaf NADP-dependent malate dehydrogenase by light or dithiothreitol, and it only activates spinach fructose-1,6-bisphosphatase at very high concentrations. The complete primary structure of C. reinhardtii thioredoxin Ch1 was determined by automated Edman degradation of the intact protein and of peptides derived from trypsin, chymotrypsin and Staphylococcus aureus V8 protease digestions. When needed, peptide masses were verified by plasma desorption mass spectrometry. Ch1 consists of a polypeptide of 111 amino acids (11634 Da) and contains the well-conserved active site sequence Trp-Cys-Gly-Pro-Cys. Compared to thioredoxins from other sources, the algal thioredoxin Ch1 displays few sequence similarities with all the thioredoxins sequenced so far. Preliminary evidence indicates that Ch1 may be an h-type thioredoxin.     

Multiple signals induce endoplasmic reticulum stress in both primary and immortalized chondrocytes resulting in loss of differentiation, impaired cell growth, and apoptosis

The endoplasmic reticulum is the site of synthesis and folding of secretory proteins and is sensitive to changes in the internal and external environment of the cell. Both physiological and pathological conditions may perturb the function of the endoplasmic reticulum, resulting in endoplasmic reticulum stress. The chondrocyte is the only resident cell found in cartilage and is responsible for synthesis and turnover of the abundant extracellular matrix and may be sensitive to endoplasmic reticulum stress. Here we report that glucose withdrawal, tunicamycin, and thapsigargin induce up-regulation of GADD153 and caspase-12, two markers of endoplasmic reticulum stress, in both primary chondrocytes and a chondrocyte cell line. Other agents such as interleukin-1beta or tumor necrosis factor alpha induced a minimal or no induction of GADD153, respectively. The endoplasmic reticulum stress resulted in decreased chondrocyte growth based on cell counts, up-regulation of p21, and decreased PCNA expression. In addition, perturbation of endoplasmic reticulum function resulted in decreased accumulation of an Alcian Blue positive matrix by chondrocytes and decreased expression of type II collagen at the protein level. Further, quantitative real-time PCR was used to demonstrate a down-regulation of steady state mRNA levels coding for aggrecan, collagen II, and link protein in chondrocytes exposed to endoplasmic reticulum stress-inducing conditions. Ultimately, endoplasmic reticulum stress resulted in chondrocyte apoptosis, as evidenced by DNA fragmentation and annexin V staining. These findings have potentially important implications regarding consequences of endoplasmic reticulum stress in cartilage biology.     

Hypertrophic chondrocytes undergo further differentiation in culture

Conditions have been defined for promoting growth and differentiation of hypertrophic chondrocytes obtained in culture starting from chick embryo tibiae. Hypertrophic chondrocytes, grown in suspension culture as described (Castagnola P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102:2310-2317), when they reached the stage of single cells, were transferred to substrate-dependent culture conditions in the presence of ascorbic acid. Cells showed a change in morphology, became more elongated and flattened, expressed alkaline phosphatase, and eventually mineralized. Type II and X collagen synthesis was halted and replaced by type I collagen synthesis. In addition the cells started to produce and to secrete in large amount a protein with an apparent molecular mass of 82 KD in reducing conditions and 63 KD in unreducing conditions. This protein is soluble in acidic solutions, does not contain collagenous domains, and is glycosylated. The Ch21 protein, a marker of hypertrophic chondrocytes and bone cells, was synthesized throughout the culture. We have defined this additional differentiation stage as an osteoblast-like stage. Calcium deposition in the extracellular matrix occurred regardless of the addition of beta glycerophosphate to the culture medium. Comparable results were obtained both when the cells were plated at low density and when they were already at confluence and maintained in culture without passaging up to 50 d. When retinoic acid was added to the hypertrophic chondrocyte culture between day 1 and day 5 the maturation of the cells to the osteoblast-like stage was highly accelerated. The switch in the collagen secretion was already observed after 2 d and the production of the 63-kD protein after 3 d. Mineralization was observed after 15-20 d.     

The amino terminal sequence of the developmentally regulated Ch21 protein shows homology with amino terminal sequences of low molecular weight proteins binding hydrophobic molecules

Ch21 protein, a developmentally regulated chick embryo protein of 21,000 apparent molecular weight, was purified from culture medium of hypertrophic chondrocytes. The purification method included a DEAE cellulose chromatography column, a CM cellulose chromatography column and a HPLC molecular sieve column. The amino acid sequence of the amino terminal end of the protein was determined. Computer assisted analysis showed significant homology between this sequence and the amino terminal sequences of proteins that belong to the superfamily of the low molecular weight binding proteins sharing a basic framework for the binding and transport of small hydrophobic molecules. Determination of the amino terminal sequence of the chicken retinol binding protein excluded identity between this protein and the Ch21.     

Thyroid hormone, insulin, and glucocorticoids are sufficient to support chondrocyte differentiation to hypertrophy: a serum-free analysis

Chondrocytes from chicken embryo tibia can be maintained in culture as adherent cells in Coon's modified Ham's F-12 medium supplemented with 10% FCS. In this condition, they dedifferentiate, losing type II collagen expression in favor of type I collagen synthesis. Their differentiation to hypertrophy can be obtained by transferring them to suspension culture. Differentiation is evidenced by the shift from type I to type II and type IX collagen synthesis and the following predominant expression of type X collagen, all markers of specific stages of the differentiation process. To identify the factors required for differentiation, we developed a serum-free culture system where only the addition of triiodothyronine (T3; 10(-11) M), insulin (60 ng/ml), and dexamethasone (10(-9) M) to the F-12 medium was sufficient to obtain hypertrophic chondrocytes. In this hormonal context, chondrocytes display the same changes in the pattern of protein synthesis as described above. For proper and complete cell maturation, T3 and insulin concentrations cannot be modified. Insulin cannot be substituted by insulin-like growth factor-I, but dexamethasone concentration can be decreased to 10(-12) M without chondrogenesis being impaired. In the latter case, the expression of type X collagen and its mRNA are inversely proportional to dexamethasone concentration. When ascorbic acid is added to the hormone-supplemented medium, differentiating chondrocytes organize their matrix leading to a cartilage-like structure with hypertrophic chondrocytes embedded in lacunae. However, this structure does not present detectable calcification, at variance with control cultures maintained in FCS. Accordingly, in the presence of the hormone mixture, the differentiating chondrocytes have low levels of alkaline phosphatase activity. This report indicates that T3 and insulin are primary factors involved in the onset and progression of chondrogenesis, while dexamethasone supports cell viability and modulates some differentiated functions.     

Association of EXT1 and EXT2, hereditary multiple exostoses gene products, in Golgi apparatus

We prepared the specific antibodies for EXT1 and EXT2, hereditary multiple exostoses (HME) gene products, and characterized their expression, subcellular localization, and protein association among EXT members. Biochemical analyses indicate that EXT1 and EXT2 can associate and form homo/hetero-oligomers in vivo with or without HME-linked mutations, EXT1 (R340C) and EXT2 (D227N), when exogenously expressed in COS-7 cells. An immunocytochemical analysis showed that both EXT1 and EXT2 localized in Golgi apparatus, irrespective of HME mutations. An immunohistochemical analysis on developing bones further showed that both EXT1 and EXT2 were concomitantly expressed in hypertrophic chondrocytes of forelimb bones from 1-day-old neonatal mouse, but down-regulated in maturing chondrocytes of developing cartilage from 21-day-old mouse. Taken together with the recent finding that EXTs encode for the glycosyltransferase required for the synthesis of heparan sulfate [Lind, T., Tufaro, F., McCormick, C., Lindahl, U., and Lindholt, K. (1998) J. Biol. Chem. 273, 26265-26268], our results implied a molecular basis that a HME-linked mutation found in EXT genes could interfere the physiological function(s) of EXT homo/hetero-oligomers as glycosyltransferases in the developing bones of HME patients.     

Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments

The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin-positive vesicles, and in ricin-negative vesicles (derived from cis-Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins.     

AG-041R, a gastrin/CCK-B antagonist, stimulates chondrocyte proliferation and metabolism in vitro

A newly synthesized compound, AG-041R, 3R-1-(2,2Diethoxyethyl)-3-((4methylphenyl) amino-carbonylmethyl)-3-((4methylphenyl)ureido-indoline-2-one), is a cholecyctokinin-B/gastrin receptor antagonist, but unexpectedly magnified cartilage formation in vivo. Indeed, AG-041R is a potentially effective reagent for the repair of articular cartilage defects. To clarify its effects on chondrocytes, we studied the proliferation, matrix formation, and gene expression of rabbit primary chondrocytes cultured in type I collagen gel composites with AG-041R. Both proliferation and glycosaminoglycan synthesis were stimulated with 1 microM AG-041R, but suppressed with 10 microM. The ratio of the amounts of two chondroitin sulfate isomers, chondroitin-6-sulfate to chondroitin-4-sulfate (an indicator of cartilage maturation), increased with 1 microM but decreased with 10 microM AG-041R. Gene expression analysis showed there was no change in the relative expression levels of chondrocyte markers, Type II collagen and Aggrecan, and osteoblast and adipocyte markers, Type I collagen and PPARgamma, respectively. These findings suggest that adequate concentrations of AG-041R stimulate proliferation of chondrocytes in the matrix, without changing their differentiated characteristics.     

Purification, characterization, and complete amino acid sequence of a thioredoxin from a green alga, Chlamydomonas reinhardtii

Two thioredoxins (named Ch1 and Ch2 in reference to their elution pattern on an anion-exchange column) have been purified to homogeneity from the green alga, Chlamydomonas reinhardtii. In this paper, we described the properties and the sequence of the most abundant form, Ch2. Its activity in various enzymatic assays has been compared with those of Escherichia coli and spinach thioredoxins. C. reinhardtii thioredoxin Ch2 can serve as a substrate for E. coli thioredoxin reductase with a lower efficiency when compared to the homologous system. In the presence of dithiothreitol (DTT), the protein is able to catalyze the reduction of porcine insulin. Thioredoxin Ch2 is as efficient as its spinach counterpart in the DTT or light activation of corn NADP-malate dehydrogenase, but it only activates spinach fructose-1, 6-bisphosphatase at very high concentrations. The complete primary structure of the C. reinhardtii thioredoxin Ch2 was determined by automated Edman degradation of the intact protein and of peptides derived from trypsin, chymotrypsin, clostripain, and SV8 protease digestions. It consists of a polypeptide of 106 amino acids (MW 11,808) and contains the well-conserved active site sequence Trp-Cys-Gly-Pro-Cys. The sequence of the algal thioredoxin Ch2 has been compared to that of thioredoxins from other sources and has the greatest similarity (67%) with the thioredoxin from Anabaena 7119.     

Chondrogenic differentiation in chick embryo osteoblast cultures.

Expression of specific differentiation markers was investigated by histochemistry, immunofluorescence, and biosynthetic studies in osteoblasts outgrown from chips derived from tibia diaphyses of 18-day-old chick embryos. The starting osteoblast population expressed type I collagen and alkaline phosphatase in addition to other bone and cartilage markers as the lipocalin Ch21; the extracellular matrix deposited by these cells was not stainable for cartilage proteoglycans, and mineralization was observed when the culture was maintained in the presence of ascorbic acid, calcium and beta-glycerophosphate. During culture, clones of cells presenting a polygonal chondrocyte morphology and surrounded by an Alcian-positive matrix appeared in the cell population. Type II collagen and type X collagen were synthesized in these areas of chondrogenesis. In addition, chondrocytes isolated from these cultures expressed Ch21 and alkaline phosphatase. Chondrocytes were generated also from homogeneous osteoblast populations derived from a single cloned cell. The coexistence of chondrocytes and osteoblasts was observed during amplification of primary clones as well as in subclones. The data show the existence, within embryonic bone, of cells capable in vitro of both osteogenic and chondrogenic differentiation.