Immune interferon suppresses levels of procollagen mRNA and type II collagen synthesis in cultured human articular and costal chondrocytes

Cultured human articular and costal chondrocytes were used as a model system to examine the effects of recombinant gamma-interferon (IFN-gamma) on synthesis of procollagens, the steady state levels of types I and II procollagen mRNAs, and the expression of major histocompatibility complex class II (Ia-like) antigens on the cell surface. Adult articular chondrocytes synthesized mainly type II collagen during weeks 1-3 of primary culture, whereas types I and III collagens were also produced after longer incubation and predominated after the first subculture. Juvenile costal chondrocytes synthesized no detectable alpha 2(I) collagen chains until after week 1 of primary culture; type II collagen was the predominant species even after weeks of culture. The relative amounts of types I and II collagens synthesized were reflected in the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. In articular chondrocytes, the levels of alpha 1(I) procollagen mRNA were disproportionately low (alpha 1(I)/alpha 2(I) less than 1.0) compared with costal chondrocytes (alpha 1 (I)/alpha 2(I) approximately 2). Recombinant IFN-gamma (0.1-100 units/ml) inhibited synthesis of type II as well as types I and III collagens associated with suppression of the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. IFN-gamma suppressed the levels of alpha 1(I) and alpha 1(II) procollagen mRNAs to a greater extent than alpha 2(I) procollagen mRNA in articular but not in costal chondrocytes. Human leukocyte interferon (IFN-alpha) at 1000 units/ml suppressed collagen synthesis and procollagen mRNA levels to a similar extent as IFN-gamma at 1.0 unit/ml. In addition, IFN-gamma but not IFN-alpha induced the expression of HLA-DR antigens on intact cells. The lymphokine IFN-gamma could, therefore, have a role in suppressing cartilage matrix synthesis in vivo under conditions in which the chondrocytes are in proximity to T lymphocytes and their products.     

Methylation of type II and type I collagen genes in differentiated and dedifferentiated chondrocytes

The methyl-sensitive restriction endonucleases HpaII and HhaI as well as the methyl-insensitive enzyme MspI were used to examine the methylation status of the pro-alpha 1(II) collagen gene of cartilage. Five different cell types with varying abilities to express type II collagen were studied. Chick embryo chondrocytes express type II collagen, while 5-bromodeoxyuridine-treated chondrocytes, retinoic acid-treated chondrocytes, chick embryo fibroblasts, and erythrocytes do not synthesize type II collagen. Both cDNA and genomic probes for the pro-alpha 1(II) collagen gene were used, covering the complete 3' end of the gene and its flanking sequences. The pro-alpha 1(II) collagen DNA was undermethylated in chondrocytes, compared to either fibroblasts or erythrocytes. However, the methylation of the 5-bromodeoxyuridine-treated and retinoic acid-treated chondrocytes was identical to that of control chondrocytes. The methylation pattern of two regions of the gene of the pro-alpha 2(I) collagen chain was identical in all cell types tested, whether or not the gene was expressed. Our results indicate that genes for these collagen chains differ in their methylation pattern. The type II collagen gene shows reduced methylation in expressing cartilage, but does not acquire an increase in methylation in "dedifferentiated" chondrocytes. The changes in DNA methylation that occur during cell differentiation do not appear to be sufficient to explain gene activation and deactivation.     

Effect of relaxin on the phenotype of collagens synthesized by cultured rabbit chondrocytes

The effect of porcine relaxin on rabbit articular and growth plate chondrocytes in primary culture was investigated by measurement of total collagen production and analysis of the phenotypes of newly synthesized collagen chains. A 24-h treatment of monolayer articular and multilayer growth plate chondrocytes with 2 micrograms per ml relaxin had no effect on total DNA and did not significantly modify the amount of [3H]proline-labelled collagen chains secreted by the cells. However, polyacrylamide gel electrophoresis demonstrated relevant modifications in relaxin treated chondrocytes. A significant increase was observed in the proportion of type III collagen and in the intensity of the band corresponding to alpha 2I chains. Two-dimensional peptide mapping of CNBr-cleaved molecules indicated that the band that was identified as alpha 1II on monodimensional gels contained a significant proportion of alpha 1I collagen chains, as demonstrated by the presence of alpha 1I cyanogen bromide-digested peptides. The intensity of this band was increased by relaxin treatment. Furthermore, total RNA analysis by slot blot and Northern blot techniques showed a dose-dependent stimulation of alpha 1I and alpha 1III mRNA levels after incubation with increased relaxin concentrations, but no change in the amount of alpha 1II mRNA. These results suggested that when added to cartilage cells in vitro, relaxin modulated the expression of type I, type II and type III collagen genes by amplifying the dedifferentiation process.     

SV40-immortalization of rabbit articular chondrocytes: alteration of differentiated functions.

Cell lines were established from rabbit articular chondrocytes following transfection with a plasmid encoding SV40 early function genes. This resulted in cell immortalization (130 passages have been completed for the oldest cell line) with acquisition of characteristics of partial transformation such as reduced serum requirements for normal and clonal growth. The immortalized chondrocytes, called SVRAC, did not form multilayer foci when maintained in postconfluent culture. Their ability to form colonies in soft agar was not increased in comparison with normal chondrocytes, but they were weakly tumorigenic in nude mice. SVRAC lost the ability to synthesize type II collagen and Alcian blue-stainable matrix, which are markers of the differentiated chondrocyte phenotype, and synthesized predominantly type I collagen. Studies of collagen gene expression showed that pro alpha 1 (II) mRNA was undetectable, whereas pro alpha 1 (I) collagen mRNA was expressed even in late passage cultures. Unlike normal dedifferentiated chondrocytes, SVRAC were unable to re-express the differentiated phenotype in response to tridimensional culture or microfilament depolymerization. Cell lines obtained from chondrocytes transfected either in primary culture or just after release of cells from cartilage displayed the same behaviour. Thus SV40 early genes were able to immortalize rabbit articular chondrocytes, but the resulting cell lines displayed an apparently irreversibly dedifferentiated phenotype. These cell lines can be used as models to identify regulatory pathways that are required for the maintenance or reexpression of differentiated function in chondrocytes.     

Modulation of sulfated proteoglycan synthesis and collagen gene expression by chondrocytes grown in the presence of bFGF alone or combined with IGF1

Prepubertal rabbit epiphyseal chondrocytes were grown in high density primary culture for 3 d. They were then incubated for 3 additional d in serum-free culture medium to which bFGF (1-50 ng/ml) was added. During the last 24 h incubation period, either IGF1 (1-80 ng/ml) or Insulin (1-5 micrograms/ml) was added to the culture medium. Chondrocyte DNA was significantly augmented with the increasing concentration of bFGF used, thus confirming its mitogenic effect on chondrocytes. On the other hand, bFGF was also shown to modulate the phenotypic expression of the chondrocytes. The 35S-sulfate incorporation into newly synthesized proteoglycans by the cultured cells decreased in a dose-dependent manner with bFGF concentration used. In addition, chondrocyte collagen gene expression was also shown to be modulated by bFGF. Total RNA extracted from the cultured cells was analyzed by dot blot and Northern blot with cDNA probes encoding for alpha 1 II and alpha 1 I procollagen chains. A significant lower level of type II collagen mRNA, the marker of chondrocytic phenotype, was observed when cells were grown in the presence of bFGF while the level of type I mRNA remained unchanged. When IGF1 or a high concentration of insulin was added to the cells during the last 24 h of incubation with bFGF, sulfated proteoglycan synthesis, as well as collagen type II mRNA level, were significantly stimulated when compared with chondrocytes incubated with bFGF alone. In conclusion, in the present experimental conditions, bFGF appears to be a growth promoting agent for chondrocytes in vitro with dedifferentiating action on chondrocyte phenotype. IGF1 or insulin used at a high concentration can prevent the dedifferentiating effect of bFGF without inhibiting its stimulating effect on chondrocyte DNA synthesis.     

Human meniscus cells express hypoxia inducible factor-1alpha and increased SOX9 in response to low oxygen tension in cell aggregate culture

In previous work we demonstrated that the matrix-forming phenotype of cultured human cells from whole meniscus was enhanced by hypoxia (5% oxygen). Because the meniscus contains an inner region that is devoid of vasculature and an outer vascular region, here we investigate, by gene expression analysis, the separate responses of cells isolated from the inner and outer meniscus to lowered oxygen, and compared it with the response of articular chondrocytes. In aggregate culture of outer meniscus cells, hypoxia (5% oxygen) increased the expression of type II collagen and SOX9 (Sry-related HMG box-9), and decreased the expression of type I collagen. In contrast, with inner meniscus cells, there was no increase in SOX9, but type II collagen and type I collagen increased. The articular chondrocytes exhibited little response to 5% oxygen in aggregate culture, with no significant differences in the expression of these matrix genes and SOX9. In both aggregate cultures of outer and inner meniscus cells, but not in chondrocytes, there was increased expression of collagen prolyl 4-hydroxylase (P4H)alpha(I) in response to 5% oxygen, and this hypoxia-induced expression of P4H alpha(I) was blocked in monolayer cultures of meniscus cells by the hypoxia-inducible factor (HIF)-1alpha inhibitor (YC-1). In fresh tissue from the outer and inner meniscus, the levels of expression of the HIF-1alpha gene and downstream target genes (namely, those encoding P4H alpha(I) and HIF prolyl 4-hydroxylase) were significantly higher in the inner meniscus than in the outer meniscus. Thus, this study revealed that inner meniscus cells were less responsive to 5% oxygen tension than were outer meniscus cells, and they were both more sensitive than articular chondrocytes from a similar joint. These results suggest that the vasculature and greater oxygen tension in the outer meniscus may help to suppress cartilage-like matrix formation.     

Loss of alpha I type I collagen gene expression in rat clonal bone cell lines is accompanied by DNA methylation

Four clonal cell lines subcloned from a clonal population of fetal rat calvaria cells show a loss of type I collagen synthesis. Northern blot analysis showed that the level of alpha 1(I) collagen mRNA expression in each of the clonal populations parallels the level of collagen protein expression in each of these cell lines. The methylation pattern of the collagen gene in these clonal cell lines was determined using the restriction endonucleases MspI and HpaII. It was found that the loss in collagen type I expression correlated positively with the degree of methylation of alpha 1(I) procollagen genes, indicating that methylation of CpG may be an important mechanism of collagen gene regulation.     

Human COL2A1-directed SV40 T antigen expression in transgenic and chimeric mice results in abnormal skeletal development

The ability of SV40 T antigen to cause abnormalities in cartilage development in transgenic mice and chimeras has been tested. The cis- regulatory elements of the COL2A1 gene were used to target expression of SV40 T antigen to differentiating chondrocytes in transgenic mice and chimeras derived from embryonal stem (ES) cells bearing the same transgene. The major phenotypic consequences of transgenic (pAL21) expression are malformed skeleton, disproportionate dwarfism, and perinatal/neonatal death. Expression of T antigen was tissue specific and in the main characteristic of the mouse alpha 1(II) collagen gene. Chondrocyte densities and levels of alpha 1(II) collagen mRNAs were reduced in the transgenic mice. Islands of cells which express cartilage characteristic genes such as type IIB procollagen, long form alpha 1(IX) collagen, alpha 2(XI) collagen, and aggrecan were found in the articular and growth cartilages of pAL21 chimeric fetuses and neonates. But these cells, which were expressing T antigen, were not properly organized into columns of proliferating chondrocytes. Levels of alpha 1(II) collagen mRNA were reduced in these chondrocytes. In addition, these cells did not express type X collagen, a marker for hypertrophic chondrocytes. The skeletal abnormality in pAL21 mice may therefore be due to a retardation of chondrocyte maturation or an impaired ability of chondrocytes to complete terminal differentiation and an associated paucity of some cartilage matrix components.     

Regulation of insulin secretion by energy metabolism in pancreatic B-cell mitochondria. Studies with a non-metabolizable leucine analogue.

This study compares the collagen types present in rabbit ear cartilage with those synthesized by dissociated chondrocytes in cell culture. The cartilage was first extracted with 4M-guanidinium chloride to remove proteoglycans. This step also extracted type I collagen. After pepsin solubilization of the residue, three additional, genetically distinct collagen types could be separated by fractional salt precipitation. On SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis they were identified as type II collagen, (1 alpha, 2 alpha, 3 alpha) collagen and M-collagen fragments, a collagen pattern identical with that found in hyaline cartilage. Types I, II, (1 alpha, 2 alpha, 3 alpha) and M-collagen fragments represent 20, 75, 3.5, and 1% respectively of the total collagen. In frozen sections of ear cartilage, type II collagen was located by immunofluorescence staining in the extracellular matrix, whereas type I collagen was closely associated with the chondrocytes. Within 24h after release from elastic cartilage by enzymic digestion, auricular chondrocytes began to synthesize type III collagen, in addition to the above-mentioned collagens. This was shown after labelling of freshly dissociated chondrocytes with [3H]proline 1 day after plating, fractionation of the pepsin-treated collagens from medium and cell layer by NaCl precipitation, and analysis of the fractions by CM(carboxymethyl)-cellulose chromatography and SDS/polyacrylamide-gel electrophoresis. The 0.8 M-NaCl precipitate of cell-layer extracts consisted predominantly of type II collagen. The 0.8 M-NaCl precipitate obtained from the medium contained type I, II, and III collagen. In the supernatant of the 0.8 M-NaCl precipitation remained, both in the cell extract and medium, predominantly 1 alpha-, 2 alpha-, and 3 alpha-chains and M-collagen fragments. These results indicate that auricular chondrocytes are similar to chondrocytes from hyaline cartilage in that they produce, with the exception of type I collagen, the same collagen types in vivo, but change their cellular phenotype more rapidly after transfer to monolayer culture, as indicated by the prompt onset of type III collagen synthesis.     

Stimulation of type II collagen biosynthesis and secretion in bovine chondrocytes cultured with degraded collagen

The functional integrity of articular cartilage is dependent on the maintenance of the extracellular matrix (ECM), a process which is controlled by chondrocytes. The regulation of ECM biosynthesis is complex and a variety of substances have been found to influence chondrocyte metabolism. In the present study we have investigated the effect of degraded collagen on the formation of type II collagen by mature bovine chondrocytes in a cell culture model. The culture medium was supplemented with collagen hydrolysate (CH) and biosynthesis of type II collagen by chondrocytes was compared to control cells treated with native type I and type II collagen and a collagen-free protein hydrolysate. The quantification of type II collagen by means of an ELISA technique was confirmed by immunocytochemical detection as well as by the incorporation of (14)C-proline in the ECM after a 48 h incubation. Chondrocytes in the control group were maintained in the basal medium for 11 days. The presence of extracellular CH led to a dose-dependent increase in type II collagen secretion. However, native collagens as well as a collagen-free hydrolysate of wheat proteins failed to stimulate the production of type II collagen in chondrocytes. These results clearly indicate a stimulatory effect of degraded collagen on the type II collagen biosynthesis of chondrocytes and suggest a possible feedback mechanism for the regulation of collagen turnover in cartilage tissue.