Characterization of proteoglycans synthesized by rabbit articular chondrocytes in response to transforming growth factor-beta (TGF-beta)

The effect of transforming growth factor-beta (TGF-beta, 1 ng/ml) on proteoglycan synthesis by rabbit articular chondrocytes in culture was studied in the presence of fetal bovine serum. Exposure of confluent cells for 24 h to the factor resulted in a marked increase of 35S-labeled sulfate incorporation in the newly synthesized proteoglycans (PG), as estimated by glycosaminoglycan (GAG) radioactivity (+58%). The onset was observed 6 h after addition of the factor but was significant after 12 h. TGF-beta also enhanced the uptake of [35S]sulfate by chondrocytes, but had no effect on the release of PG by these cells. The effect of TGF-beta on the distribution of PG between the medium and the cell layer was shown to be dependent on the serum concentration in the medium: the relative proportion of cell-layer associated GAG of TGF-beta-treated cells decreased with increasing concentration of fetal bovine serum. The proportion of aggregated PG, the hydrodynamic size of PG monomers and GAG chains were not modified by TGF-beta, but the relative distribution of disaccharides 6- and 4-sulfate in GAG chains was altered by the factor: the proportion of chondroitin 6-sulfate (C6S) was decreased while that of chondroitin 4-sulfate (C4S) was augmented in presence of TGF-beta, leading to a decrease of the ratio C6S/C4S (-11 to -22%, P less than 0.01). The present study indicates that TGF-beta promotes the synthesis of a modified extracellular matrix in cultured articular chondrocytes. This mechanism could be relevant to some aspects of cartilage repair in osteoarticular diseases.     

The synthesis of cartilage collagen by rabbit and human chondrocytes in primary cell culture

Summary This report describes a method for preparing primary cell cultures of differentiated rabbit sternal and human vertebral cartilage cells. These cell cultures were shown to synthesize primarily α1 chains, which is taken to mean that at least 82% of the collagen produced is cartilage specific collagen (type II). This work was supported in part by grant HD-05505 from NIH.     

Production of hydrogen peroxide by rabbit articular chondrocytes. Enhancement by cytokines

Recent evidence suggests that reactive oxygen intermediates may play a role in the etiology of cartilage matrix degradation in arthritis. We have previously established that normal articular chondrocytes can functionally act as macrophages. These functions include expression of class II MHC Ag, presentation of Ag and induction of mixed and autologous lymphocyte stimulation. Inasmuch as the production of reactive oxygen intermediates is a hallmark of macrophage activity during inflammatory response, we were interested in examining the ability of normal articular chondrocytes to produce reactive oxygen intermediates. Using the trapped indicator 2',7'-dichlorofluorescin diacetate (DCFH-DA), we measured the levels of intracellular hydrogen peroxide within normal rabbit articular chondrocytes. We found that Concanavalin A induces chondrocytes to rapidly oxidize 2',7'-dichlorofluorescin diacetate to a highly fluorescent dichlorofluorescin in a dose- and time-dependent manner. Fluorescent dichlorofluorescin oxidation by chondrocytes was inhibited by the addition of catalase, an enzyme that detoxifies hydrogen peroxide. Exposure of rabbit chondrocytes to either IFN-gamma or TNF primed the chondrocytes to produce significantly greater amounts of hydrogen peroxide with or without further stimulation. Using scopoletin oxidation as a measure of the release of hydrogen peroxide, we confirmed that chondrocytes released this reactive oxygen intermediate after adherence to serum coated culture plates. Rabbit articular chondrocytes produced and released greater amounts of hydrogen peroxide than pulmonary alveolar macrophages, a well characterized macrophage cell type. These observations suggest that chondrocytes are an important source of reactive oxygen intermediates. Furthermore, the production of reactive oxygen intermediates by chondrocytes may be an important mechanism by which chondrocytes induce structural and functional alterations in cartilage matrix observed during arthritis.     

Mitochondrial uptake of rhodamine 123 by rabbit articular chondrocytes

Rhodamine 123 was used to stain and analyze by flow cytometry the mitochondria of rabbit articular chondrocytes. Stationary primary cultures and exponentially growing subcultures were compared to enzymatically released chondrocytes from cartilage. The increase in mitochondrial fluorescence, when chondrocytes are transferred from cartilage to culture environment, is suggestive of some change in chondrocyte adaptation and/or differentiation in these conditions.     

Effects of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on rabbit articular chondrocytes in culture

Investigations were carried out to assess the effects of disodium 1-hydroxyethane-1,1-diphosphonate and disodium dichloromethanediphosphonate (compounds containing a P-C-P bond) on isolated rabbit articular chondrocytes in culture. Studies on growth behaviour showed that both diphosphonates displayed inhibitory actions, dichloromethanediphosphonate producing the larger effect. Both compounds inhibited the uptake of 2-deoxy-d-glucose, dichloromethanediphosphonate once more being the more potent of the two. The uptake of alpha-aminoisobutyrate was considerably increased by chondrocytes treated with dichloromethanediphosphonate, whereas 1-hydroxyethane-1,1-diphosphonate showed no effects. The biosynthesis of sulphated extracellular macromolecules secreted by the cells into the pericellular space as well as into the growth medium was greatly increased by dichloromethanediphosphonate but not by 1-hydroxyethane-1,1-diphosphonate. The stimulatory effect was dose-dependent. Short-term exposure of already confluent cells to dichloromethanediphosphonate as opposed to growing the cells to confluence in the presence of the diphosphonate revealed that the stimulatory effects were already evident after 24h, indicating that cell division is not necessarily required to produce the observed effects. The increment in proteoglycan synthesis was still evident with cells that were exposed continuously to the diphosphonate in primary as well as secondary culture. Pulse-chase experiments together with studies on the enzyme arylsulphatase revealed that the appearance of increased amounts of proteoglycans was the result of a stimulation in synthesis and not due to an inhibition in turnover.     

Subculture of rabbit articular chondrocytes within a collagen gel: growth and analysis of differentiation

Primary cultures of rabbit articular chondrocytes have been subcultured within three-dimensional (3D) collagen gels. Under these conditions, the cells remained viable and divided, but with a lower proliferation rate than that observed in control monolayer cultures. Flow cytometric analysis of progression of the cells into the cell cycle has confirmed and extended these findings. Also the cellular volume was decreased in 3D-culture, being in the same range as thein vivo size of cartilage cells. Specific staining for proteoglycans and type II collagen immunolocalization on sections of gels showed the expression of differentiated phenotypes and revealed the accumulation of these matrix components in the immediate surroundings of the cells. The use of Ultroser G (a serum substitute) improved the conditions for 3D- culture of rabbit articular chondrocytes.     

Proteoglycan aggregate formation by articular chondrocytes. Decrease in link-protein synthesis during culture.

The synthesis of link-stabilized proteoglycan aggregates by rabbit articular chondrocytes was investigated by [35S]sulphate labelling of primary monolayer cultures maintained for up to 21 days. (1) At all culture times the cells secreted a high-molecular-weight cartilage-type proteoglycan monomer of which 75%-80% formed aggregates with hyaluronic acid. (2) At 2 days of culture all of the aggregates were in link-stabilized form, but by 21 days only 5% were link-stabilized, as shown by displacement of monomers from the aggregate by hyaluronic acid oligosaccharides. (3) The addition of purified link protein to 21-day culture medium increased the proportion of link-stable aggregate from 5% to 70%. (4) Analysis of [3H]serine-labelled proteoglycan aggregates in the medium showed a marked decrease with culture time in the ratio of 3H-labelled link protein to 3H-labelled core protein present. The results suggest that the secretion of proteoglycan monomers and link protein by articular chondrocytes changes independently during prolonged monolayer culture.     

RANKL synthesized by articular chondrocytes contributes to juxta-articular bone loss in chronic arthritis

Introduction

The receptor activator nuclear factor-kappaB ligand (RANKL) diffuses from articular cartilage to subchondral bone. However, the role of chondrocyte-synthesized RANKL in rheumatoid arthritis-associated juxta-articular bone loss has not yet been explored. This study aimed to determine whether RANKL produced by chondrocytes induces osteoclastogenesis and juxta-articular bone loss associated with chronic arthritis.

Methods

Chronic antigen-induced arthritis (AIA) was induced in New Zealand (NZ) rabbits. Osteoarthritis (OA) and control groups were simultaneously studied. Dual X-ray absorptiometry of subchondral knee bone was performed before sacrifice. Histological analysis and protein expression of RANKL and osteoprotegerin (OPG) were evaluated in joint tissues. Co-cultures of human OA articular chondrocytes with peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with macrophage-colony stimulating factor (M-CSF) and prostaglandin E₂ (PGE₂), then further stained with tartrate-resistant acid phosphatase.

Results

Subchondral bone loss was confirmed in AIA rabbits when compared with controls. The expression of RANKL, OPG and RANKL/OPG ratio in cartilage were increased in AIA compared to control animals, although this pattern was not seen in synovium. Furthermore, RANKL expression and RANKL/OPG ratio were inversely related to subchondral bone mineral density. RANKL expression was observed throughout all cartilage zones of rabbits and was specially increased in the calcified cartilage of AIA animals. Co-cultures demonstrated that PGE₂-stimulated human chondrocytes, which produce RANKL, also induce osteoclasts differentiation from PBMCs.

Conclusions

Chondrocyte-synthesized RANKL may contribute to the development of juxta-articular osteoporosis associated with chronic arthritis, by enhancing osteoclastogenesis. These results point out a new mechanism of bone loss in patients with rheumatoid arthritis.     

The effects of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on collagen synthesis by rabbit articular chondrocytes and rat bone cells.

Investigations were performed to assess the effects of dichloromethanediphosphonate on the synthesis of collagen by (1) isolated rabbit articular chondrocytes, (2) isolated rat calvaria bone cells and (3) bone explants from rats treated with the diphosphonates. The studies showed that dichloromethanediphosphonate, but not 1-hydroxyethane-1,1-diphosphonate, causes articular chondrocytes to increase net collagen biosynthesis, both when measured as 3H-labelled or as non-radioactive material, in a dose-related fashion. The increment in collagen synthesis was still evident with cells that were exposed continuously to the diphosphonate in primary as well as secondary culture; however, it declined with cells in tertiary culture and was absent after the fourth subculture. The type of collagen was not affected by the diphosphonate. The synthesis of collagen by bone cells was likewise increased with dichloromethanediphosphonate. No effects were detected with 1-hydroxyethane-1,1-diphosphonate was tested. Finally, when calvaria and tibiae from diphosphonate-treated rats were cultured in vitro, the positive effect of dichloromethanediphosphonate on collagen synthesis was also evident. 1-Hydroxyethane-1,1-diphosphonate, on the other hand, decreased the incorporation of [3H]proline into the collagen of calvaria and osseous tibial shafts and showed no effect on the collagen synthesis of the cartilaginous tibial heads.     

Fine structure of rabbit articular chondrocytes in tissue culture during logarithmic and confluent stages of growth

Ultrastructural changes in cultured articular cartilage chondrocytes from rabbit, during two growth phases were examined by transmission and scanning electron microscopy. Cells in logarithmic growth are characterized by an abundance of intracellular lipoid bodies, little development of rough endoplasmic reticulum (RER), and few cytoplasmic microfilaments. As the cells reach confluency there is a concomitant development of RER, organization and abundance of microfilaments, loss of lipoid bodies, and increase in the number of mitochondria. The fine structure of cultured chondrocytes is very similar to that of rabbit cartilage cells in situ, in that numerous lipoid bodies and microfilaments are prominent features in both cases.     

Growth-promoting effects of acidic and basic fibroblast growth factor on rabbit articular chondrocytes aging in culture

Rabbit articular chondrocytes have a limited growth potential in vitro. After four passages in culture, chondrocytes have accomplished more than 50% of their life span. At this stage of culture, they are considered to be senescent-like, since a dramatic decrease in proliferative capacity and enhanced cell size and protein content are observed. These aged cells are, however, still able to respond to fibroblast growth factor (FGF). The addition of either acidic or basic FGF (10 ng/ml) to culture medium permitted an enhanced proliferation. The attenuation of FGF mitogenic activity during aging was not observed for both fractions. Moreover, when treated with acidic or basic FGF, aged chondrocytes had a smaller size and a lower protein content. The acidic FGF was less potent than the basic FGF in delaying the evolution of aged chondrocytes to senescence.     

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.     

The low molecular weight collagen synthesized by chick tibial chondrocytes is deposited in the extracellular matrix both in culture and in vivo.

The low mol. wt. collagen (64 K) synthesized by chick embryo chondrocytes in culture is deposited in the extracellular matrix; its deposition is strictly dependent upon a correct hydroxylation. In vivo the 64 K collagen has been isolated from the cartilage of tibiae obtained from 17-day-old chick embryos. The turnover of this collagen in the extracellular matrix is very rapid: within a few hours it is matured into a 30-K fragment released in the medium. Also this maturation is dependent upon a correct hydroxylation of the molecule. The underhydroxylated form, synthesized in the absence of ascorbic acid or in the presence of alpha-alpha' dipyridyl, is not deposited in the extracellular matrix and is directly secreted as 64 K collagen in the culture medium.     

Synthesis of small proteoglycans substituted with keratan sulfate by rabbit articular chondrocytes

35S-Labeled proteoglycans produced by chondrocytes from immature and mature rabbits were fractionated on associative CsCl gradients. In all cultures, greater than 85% of the incorporated radioactivity was present in the A1 fraction (rho 1.60) as chondroitin sulfate/keratin sulfate-substituted aggregating proteoglycan monomer; the remainder was present in small proteoglycans in the A2, A3, and A4 fractions of low buoyant densities (rho 1.53, 1.45, 1.37, respectively). Detailed glycosaminoglycan analysis of the A2, A3, and A4 fractions showed dermatan sulfate-rich species were present throughout. However, in both immature and mature cultures, 30-45% of the glycosaminoglycans in the A3/A4 combined fractions were present as keratan sulfate, as shown by insensitivity to digestion with chondroitinase ABC, specific digestion with endo-beta-galactosidase, and reactivity with antibody 5D4. Immature and mature chondrocytes synthesized very similar amounts of the low buoyant density keratan sulfate proteoglycan on a per cell basis. Moreover, 51 and 37% of the total keratan sulfate produced by immature and mature chondrocytes, respectively, were present in the low buoyant density proteoglycan. Pulse-chase experiments indicated that the low buoyant density keratan sulfate was not derived from the large aggregating proteoglycan by proteolysis in the extracellular space. The small keratan sulfate proteoglycans appear to be present as a species distinct from the small dermatan sulfate proteoglycans in these cultures in that they can be separated on Q-Sepharose chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent size (40-60 kDa), composition, and heterogeneity of the keratan sulfate proteoglycans suggest that they may be related to the small keratan sulfate proteoglycans of cornea.     

Cathepsin B secretion by rabbit articular chondrocytes: modulation by cycloheximide and glycosaminoglycans

Rabbit articular chondrocytes in monolayer culture are modulated away from their differentiated state and undergo morphological and biochemical changes. One of the characteristics of the modulated state is an abnormally high production of the cysteine endopeptidase cathepsin B. Addition to chondrocyte cultures of the protein biosynthesis inhibitor, cycloheximide, resulted in a concentration-dependent reduction of cathepsin B secretion, which was fully restored after removal of cycloheximide. Glycosaminoglycans added to the culture medium of modulated chondrocytes partially reduced the rate of secretion of cathepsin B, this effect being dependent on their structure, the degree of sulfation, and concentration. The age of the chondrocytes and the duration of the treatment also influenced this response. The switching off of cathepsin B release was apparently best favored by a high concentration of negatively charged sulfate groups attached to a polymeric glycosaminoglycan chain; this simulates the natural environment of the chondrocytes in articular cartilage.     

Oxidative injury induced by synthetic humic acid polymer and monomer in cultured rabbit articular chondrocytes.

Humic substance has been proposed as one of the causative factors of Kashin-Beck disease (KBD), an endemic osteoarthritic disorder with necrosis of chondrocytes widely prevalent in some regions of China. In order to exclude the complications of natural humic substance, here we prepared phenolic polymers of synthetic humic acid (SHA) by oxidation of phenolic monomer, the protocatechuic acid (PCA). The biological effects of SHA and PCA on primary culture of rabbit articular chondrocytes were investigated. We found that not only SHA but also PCA caused chondrocyte injury, as evidenced by the loss of cell viability measured with methylthiazol tetrazolium (MTT) assay and the increased release of intracellular lactate dehydrogenase (LDH). Both SHA and PCA could result in lipid peroxidation and glutathione (GSH) depletion in chondrocytes, indicating that oxidative stress may be involved in chondrocyte injury. Furthermore, a marked increase in intracellular calcium level ([Ca2+]i) occurred after chondrocytes treated with SHA or PCA. These results suggest that chondrocyte injury elicited by SHA or PCA may be mediated through the occurrence of oxidative stress and the disruption of intracellular Ca2+ homeostasis. Data also suggest that the monomeric phenolic acid may be considered one of the causative factors of KBD in addition to humic substance.     

Cell hypertrophy and type X collagen synthesis in cultured articular chondrocytes

Articular cartilage is a permanent tissue whose cells do not normally take part in the endochondral ossification process. To determine whether articular chondrocytes possess the potential to express traits associated with this process such as cell hypertrophy and type X collagen, chondrocytes were isolated from adult chicken tibial articular cartilage and maintained in long-term suspension cultures. As a positive control in these experiments, we used parallel cultures of chondrocytes from the caudal portion of chick embryo sternum. Both articular and sternal chondrocytes readily proliferated and progressively increased in size with time in culture. Many had undergone hypertrophy by 4-5 weeks. Analysis of medium-released collagenous proteins revealed that both articular and sternal chondrocytes initiated type X collagen synthesis between 3 and 4 weeks of culture; synthesis of this macromolecule increased with further growth. Immunofluorescence analysis of 5-week-old cultures showed that about 15% of articular chondrocytes and 30% of sternal chondrocytes produced type X collagen; strikingly, there appeared to be no obvious relationship between type X collagen production and cell size. The results of this study show that articular chondrocytes from adult chicken tibia possess the ability to express traits associated with endochondral ossification when exposed to a permissive environment. They suggest also that the process of cell hypertrophy and initiation of type X collagen synthesis are independently regulated both in articular and sternal chondrocytes.     

The effect of embryo extract on the types of collagen synthesized by cultured chick chondrocytes.

Growth of embryonic chick chondrocytes in dialyzed embryo extract results in both a change in morphology of the cells toward that of a fibroblast and a change in the type of collagen synthesized from the cartilage-specific Type II collagen (chain composition [α1(II)]₃) to a mixture of Type I collagen (chain composition [α1(I)]₂α2) and the Type I trimer (chain composition [α1(I)]₃). Analyses after 6 days of growth in embryo extract show that the synthesis of only Type I collagen and the Type I trimer can be detected. However, on subculturing the cells to a low density and allowing a period of growth without embryo extract, colonies of chondrocytes reappear and the synthesis of Type II collagen apparently resumes. It is suggested that the observed changes represent a “modulation” in cell behavior, this being expressed not only by the morphological changes but also by changes in cell-specific protein synthesis as demonstrated by the changes in the type of collagen synthesized.