Stimulation by glucocorticoids of the differentiated phenotype of chondrocytes and the proliferation of rabbit costal chondrocytes in culture

Hydrocortisone stimulated glycosaminoglycan (GAG) synthesis, a characteristic of the cartilage phenotype, of rabbit costal chondrocytes in confluent quiescent culture, as judged by the incorporations of [35S]sulfate and [3H]glucosamine. Hydrocortisone also stimulated incorporation of [3H]serine into proteoglycan. The stimulation of GAG synthesis by hydrocortisone was dose-dependent and maximal at a physiological concentration of 10(-7) M. Hydrocortisone also stimulated GAG synthesis in cultures in the log-phase of growth. In this case, its maximal effect was observed at a concentration of 10(-6) M. The magnitude of the increase of GAG synthesis in response to hydrocortisone was larger in confluent culture than in log-phase cultures. Hydrocortisone stimulated DNA synthesis dose-dependently, and its effect was observable at a physiological concentration. However, no stimulation of DNA synthesis by hydrocortisone was observed in serum-free medium, in contrast to that of GAG synthesis. Hydrocortisone also increased protein synthesis and the cell number. Dexamethasone also stimulated the syntheses of both GAG and DNA. These results show that glucocorticoids stimulated both the differentiated phenotype of chondrocytes and the proliferation of rabbit costal chondrocytes in culture. Moreover, the effect of glucocorticoids was primarily on the differentiated phenotype of chondrocytes and its effect on proliferation was permissive.     

Cytoskeleton and differentiation: effects of cytochalasin B and colchicine on expression of the differentiated phenotype of rabbit costal chondrocytes in culture

Cytochalasin B changed the shape of cultured rabbit costal chondrocytes from polygonal to nearly spherical and stimulated glycosaminoglycan synthesis, which is a differentiated phenotype of chondrocytes, whereas colchicine changed them from polygonal to flattened and inhibited glycosaminoglycan synthesis. These morphological changes occurred parallel with the changes in glycosaminoglycan synthesis. Induction of ornithine decarboxylase by parathyroid hormone, which is a good marker of differentiated chondrocytes, was markedly potentiated in the spherical cells which had been pretreated with cytochalasin B, whereas pretreatment with colchicine inhibited the induction of the enzyme. Both cytochalasin B and colchicine inhibited DNA synthesis. The inhibitions were observed after the appearance of changes in the morphology of the cells and glycosaminoglycan synthesis. These findings suggest that intactness of microtubules and disruption of microfilaments are involved in regulating the expression of the differentiated phenotype of chondrocytes in culture.     

Comparison of inhibition by a tumor promoter (12-O-tetradecanoylphorbol-13-acetate) of expression of the differentiated phenotype of chondrocytes in rabbit costal chondrocytes in culture with inhibition by retinoic acid

Both retinoids and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit expression of the differentiated phenotype by rabbit costal chondrocytes in culture, as judged by morphological changes and decreased sulfation of glycosaminoglycans (GAG). However, the inhibition of the differentiated phenotype of chondrocytes in TPA-treated cells is restored by parathyroid hormone (PTH), while the inhibition by retinoids is not [Takigawa et al. (1982) Mol. Cell. Biochem. 42, 145-153; Takigawa et al. (1983) Cell Differ. 13, 283-291]. In the present study, we examined the difference between TPA-treated chondrocytes and retinoic acid-treated chondrocytes to determine the mechanism of the restoration of the differentiated phenotype in de-differentiated cells treated with TPA. PTH increased the activity of ornithine decarboxylase [ODC; EC 4.1.1.17], a rate limiting enzyme of polyamine biosynthesis, and proteoglycan synthesis in chondrocytes pretreated with TPA as well as in normal chondrocytes. The maximal stimulations of ODC activity and GAG synthesis were observed 4 h and 24-36 h, respectively, after addition of PTH. The dose-response curve for ODC induction by PTH was parallel to that of PTH-stimulated proteoglycan synthesis both in TPA-treated chondrocytes and in normal chondrocytes. PTH also increased the intracellular cyclic AMP level after 2 min in TPA-treated cells as in normal cells. Addition of dibutyryl cyclic AMP (DBcAMP) induced ODC and restored proteoglycan synthesis in TPA-treated cells. The dose-response curve for induction of ODC by DBcAMP was parallel to that of DBcAMP-stimulated proteoglycan synthesis in both TPA-treated chondrocytes and normal chondrocytes. On the other hand, the increases by PTH in the intracellular cyclic AMP level, ODC activity, and proteoglycan synthesis were inhibited in chondrocytes pretreated with a combination of TPA and retinoic acid as well as in those pretreated with retinoic acid alone. TPA stimulated the syntheses of DNA and RNA in chondrocytes but did not increase the cyclic AMP level or ODC activity. PTH and DBcAMP inhibited the syntheses of DNA and RNA both in TPA-treated cells and in normal cells. These results suggest that ODC induction mediated by elevation of cyclic AMP plays an important role in re-differentiation of de-differentiated cells pretreated with these agents.     

Restoration by parathyroid hormone and dibutyryl cyclic AMP of expression of the differentiated phenotype of chondrocytes inhibited by a tumor promoter, 12-0-tetradecanoylphorbol-13-acetate

12-0-Tetradecanoylphorbol-13-acetate (TPA) inhibited expression of the differentiated phenotype of chondrocytes in rabbit costal chondrocytes in culture. TPA transformed typical polygonal chondrocytes into multilayered, fibroblastic cells and also inhibited the rate of [35S]sulfate incorporation into glycosaminoglycan (GAG), a differentiated phenotype of chondrocytes. These changes were apparent within 24 h and reached a plateau at 48 h after the addition of TPA. Phorbol didecanoate and phorbol dibenzoate also inhibited sulfation of GAG, even though the effect was weaker than that of TPA. Phorbol diacetate and 4-0-methyl TPA did not inhibit sulfation of GAG. Addition of parathyroid hormone (PTH) or dibutyryl cyclic AMP simultaneously with TPA overcame the inhibition caused by TPA. PTH and dibutyryl cyclic AMP also reversed the inhibition and stimulated expression of the differentiated phenotype of chondrocytes even in de-differentiated cells which had been pretreated for 3 days with TPA. These findings suggest that cyclic AMP plays an important role in the restoration of the differentiated phenotype of chondrocytes in TPA-treated chondrocytes, and that the TPA-treated cells retain some of the differentiated phenotype of the original cells, such as responsiveness to PTH.     

Restoration by cyclic AMP of the differentiated phenotype of chondrocytes from de-differentiated cells pretreated with retinoids

Summary Parathyroid hormone (PTH) increases the cyclic AMP level in rabbit costal chondrocytes in culture. PTH, dibutyryl cyclic AMP (DBcAMP), and 8-bromo cyclic AMP (8-Br cAMP) induce ornithine decarboxylase (ODC) and expression of the differentiated phenotype of chondrocytes in this cell system. On the other hand, retinoids inhibit expression of the differentiated phenotype of chondrocytes. In the present study, the effects of PTH, DBcAMP, and 8-Br cAMP on rabbit costal chondrocytes pretreated with retinoids were examined.PTH did not increase the cellular cyclic AMP level in de-differentiated cells that had been pretreated with retinyl acetate or retinoic acid for three days, but it did increase the cyclic AMP level four days after removal of retinoids. PTH did not stimulate ODC activity or expression of the differentiated phenotype of chondrocytes in the de-differentiated state. On the other hand, DBcAMP or 8-Br cAMP stimulated expression of the differentiated phenotype of chondrocytes even in de-differentiated cells, as judged by morphological and bistological changes of the cells and increase in glycosaminoglycan synthesis. Cyclic AMP analogues also induced ODC in these cells.     

Effects of parathyroid hormone and cyclic AMP analogues on the activity of ornithine decarboxylase and expression of the differentiated phenotype of chondrocytes in culture

Parathyroid hormone (PTH) greatly increased the level of adenosine 3', 5' cyclic monophosphate (cAMP) in rabbit costal chondrocytes in culture 2 minutes after its addition. PTH, as well as N6 O2' dibutyryl adenosine 3', 5' cyclic monophosphate (DBcAMP) and 8 Bromo adenosine 3', 5' cyclic monophosphate (8 Br-cAMP) induced ornithine decarboxylase (ODC; L-ornithine carboxylyase; EC 4.1.1.17), which reached a maximum 4 hours after their addition. Neither cAMP, N6 O2' dibutyryl guanosine 3', 5' cyclic monophosphate (DBcGMP), nor sodium butyrate increased the activity of the enzyme. PTH had no effect on DNA synthesis, while DBcAMP and 8 Br-cAMP decreased DNA synthesis. Expression of the differentiated phenotype of chondrocytes in culture was also induced by PTH, DBcAMP, and 8 Br-cAMP, but not by cAMP, DBcGMP, or sodium butyrate, as judged by morphological change. Glycosaminoglycan synthesis, a characteristic of the cartilage phenotype, began to increase 8 hours after addition of PTH or DBcAMP, reaching a plateau 32 hours after their addition. These findings suggest that PTH induces increase of ODC activity and expression of the differentiated phenotype of chondrocytes through increase of cAMP and that induction of OCD is closely related to expression of the differentiated phenotype of chondrocytes.     

Effect of retinoic acid on proliferation and differentiation of cultured chondrocytes in terminal differentiation

We obtained terminally differentiated chondrocytes in monolayer culture from chick embryonal growth plates, and examined the effect of retinoic acid on these cells. The cells treated with retinoic acid ceased type X collagen synthesis and showed decreased calcium incorporation into cell layers. Retinoic acid tended to stimulate proliferation of the cultured chondrocytes. It also increased DNA accumulation dose-dependently in the range from 1 nM to 1 microM. DNA synthesis in the growth phase and confluency was stimulated within 10 h after addition of 0.1 microM retinoic acid. [3H]Retinoic acid binding, which was inhibited by simultaneous addition of excess unlabeled retinoic acid, was detected in both the cytosolic and nuclear fractions of the chondrocytes. The retinoic acid binding capacity of the nuclear fraction was increased by pretreating the cells with retinoic acid. These results indicate that retinoic acid binds to both the cytosolic and nuclear fractions of cultured chondrocytes, and induces their proliferation and dedifferentiation.     

Influence of cytochalasin d-induced changes in cell shape on proteoglycan synthesis by cultured articular chondrocytes

There is growing evidence that cell shape regulates both proliferation and differentiated gene expression in a variety of cell types. We have explored the relationship between the morphology of articular chondrocytes in culture and the amount and type of proteoglycan they synthesize, using cytochalasin D to induce reversible cell rounding. When chondrocytes were prevented from spreading or when spread cells were induced to round up, 35SO4 incorporation into proteoglycan was stimulated. Incorporation into the cell layer was stimulated more than into the medium. When the cells were allowed to respread by removing cytochalasin D, proteoglycan synthesis returned to control levels. Cytochalasin D-induced stimulation of 35SO4 incorporation reflected an increase in core protein synthesis rather than lengthening of glycosaminoglycan chains, because [3H]serine incorporation into core protein was also stimulated. The observed stimulation of proteoglycan synthesis was not due to an overall stimulation of protein synthesis, to inhibition of DNA synthesis, or to accumulation of cells in one phase of the cell cycle. Cytochalasin D-treatment of cells in suspension caused no further stimulation of 35SO4 incorporation, suggesting that the observed effects were due to cell rounding rather than exposure to cytochalasin D per se; nevertheless, we cannot completely rule out other, nonspecific, effects of the drug. Fibroblasts and chondrocytes that had been passaged to stimulate dedifferentiation did not incorporate more 35SO4 when treated with cytochalasin D, suggesting that increased proteoglycan synthesis in response to rounding may itself be a differentiated property of chondrocytes.     

Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture

Collagen phenotypes were determined for rabbit articular chondrocytes in cartilage slices and first through fifth monolayer cultures. During the first 24 hr of slice culture, chondrocytes exhibited the following collagen phenotype: 96% type II, 3% X₂Y and 1% type III. In primary monolayer culture, no other types of collagen were added to this differentiated chondrocyte phenotype; however, the synthesis per cell of each of the expressed collagens was stimulated. By the fifth day of primary culture, X₂Y synthesis increased 10 fold, and by the eighth day, a further 4 fold. In contrast, the synthesis of collagen types II and III showed no change by the fifth day, but increased 7 fold by the eighth day. These results suggest independent regulation of X₂Y in this situation. In a separate experiment, first through fifth cultures were studied. The synthesis per cell of type II collagen declined steadily and essentially ceased by the fifth culture, indicating the loss of differentiated function by these chondrocyte progeny. The loss of type II synthesis was not quantitatively replaced by the synthesis of type I trimer and type I collagen which was first detected in the third culture. While these qualitative changes in phenotype occurred, the stimulated rate of type III collagen synthesis did not change and that of X₂Y declined only slightly. Thus the termination of type II synthesis did not significantly alter the synthesis of the other collagens produced by differentiated chondrocytes. The final “de-differentiated” phenotype was 41% type I, 25% X₂Y, 20% type I trimer, 13% type III and 1% type II.     

Effects of polyamines on DNA synthesis using various subcellular DNA polymerases extracted from normal rat liver, tumour-bearing rat liver, and tumour cells

The effects of polyamines on DNA synthesis in vitro using various subcellular DNA polymerase fractions from normal and tumour-bearing rat livers, and tumour cells were investigated. When nuclear and mitochondrial DNA polymerase fractions were used, DNA synthesis on activated DNA was increased 3.5-8-fold by the addition of 20 mM putrescine or cadaverine. However, DNA synthesis was not stimulated by the addition of spermidine or spermine at any concentration tested. In contrast, DNA synthesis using the cytoplasmic DNA polymerase fraction was not stimulated at various concentrations of any of the four polyamines tested. The stimulatory effects of putrescine and cadaverine were absent when nuclear fractions from tumour-bearing rat liver or from tumour cells were used. In addition, in vitro DNA synthesis was not stimulated by 20 mM putrescine or cadaverine when nuclear extracts from the livers of rats administered putrescine subcutaneously were used. The specific activities of DNA polymerases extracted from tumour cells and tumour-bearing rat liver were already fully stimulated. These results suggest that DNA polymerases in tumour cells and tumour-bearing liver cells are stimulated by trapped putrescine produced in tumour cells and are thus no longer activated by exogenous putrescine.     

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.     

Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels

The differentiated phenotype of rabbit articular chondrocytes consists primarily of type II collagen and cartilage-specific proteoglycan. During serial monolayer culture this phenotype is lost and replaced by a complex collagen phenotype consisting predominately of type I collagen and a low level of proteoglycan synthesis. Such dedifferentiated chondrocytes reexpress the differentiated phenotype during suspension culture in firm gels of 0.5% low T_m agarose. Approximately 80% of the cells survive this transition from the flattened morphology of anchorage-dependent culture to the spherical morphology of anchorage-independent culture and then deposit characteristic proteoglycan matrix domains. The rates of proteoglycan and collagen synthesis return to those of primary chondrocytes. Using SDS-polyacrylamide gel electrophoresis of intact collagen chains and two-dimensional cyanogen bromide peptide mapping, we demonstrated a complete return to the differentiated collagen phenotype. These results emphasize the primary role of cell shape in the modulation of the chondrocyte phenotype and demonstrate a reversible system for the study of gene expression.     

Physiological role of vitamin A in growth cartilage cells: low concentrations of retinoic acid strongly promote the proliferation of rabbit costal growth cartilage cells in culture

We have demonstrated that high concentrations of retinoic acid (RA) inhibit expression of the differentiated phenotypes of rabbit costal chondrocytes in culture [M. Takigawa et al. (1980) Proc. Natl. Acad. Sci. U.S. 77, 1481-1485]. In this study we examined the effects of low concentrations of RA on rabbit costal chondrocytes cultured in medium containing vitamin A-deficient serum. In vitamin A-deficient medium, chondrocytes isolated from growth cartilage (GC) proliferated only very slowly, and RA strongly stimulated their proliferation. This stimulatory effect was observable at a concentration of 10(-10) M RA and maximal at a concentration of 10(-8) M. RA at 10(-8) M did not change GC cells from a typical polygonal shape to fibroblast-like cells or inhibit their synthesis of type II collagen. Moreover, RA-treated cells did not synthesize type I collagen. RA inhibited glycosaminoglycan (GAG) synthesis by the cells dose-dependently, but did not change the distribution profile of proteoglycan monomers as determined by glycerol gradient centrifugation. The inhibitory action of RA on GAG synthesis was reversible: after removal of RA from the culture, the rate of GAG synthesis increased within 2 days. In contrast, resting cartilage (RC) cells proliferated well in vitamin A-deficient medium without addition of RA, and RA (10(-8) M) stimulated their proliferation only slightly. Furthermore, the inhibitory effect of RA on GAG synthesis in RC cells was much weaker than that in GC cells. These observations suggest a physiological role of RA in cartilage in stimulating the proliferation of GC cells without causing drastic change in their differentiated phenotypes.     

Modulation of collagen synthesis in normal and osteoarthritic cartilage

In osteoarthritic cartilage, chondrocytes are able to present heterogeneous cellular reactions with expression and synthesis of the (pro)collagen types characteristic of prechondrocytes (type IIA), hypertrophic chondrocytes (type X), as well as differentiated (types IIB, IX, XI, VI) and dedifferentiated (types I, III) chondrocytes. The expression of type IIA procollagen in human osteoarthritic cartilage support the assumption that OA chondrocytes reverse their phenotype towards a chondroprogenitor phenotype. Recently, we have shown that dedifferentiation of mouse chondrocytes induced by subculture was associated with the alternative splicing of type II procollagen pre-mRNA with a switch from the IIB to the IIA form. In this context, we demonstrated that BMP-2 favours expression of type IIB whereas TGF-beta1 potentiates expression of type IIA induced by subculture. These data reveal the specific capability of BMP-2 to reverse the program of chondrocyte dedifferentiation. This interesting feature needs to be tested with human chondrocytes since cell amplification is required for the currently used autologous chondrocyte transplantation.     

Serum-induced cytosolic calcium movements and mitogenesis in cultured preosseous chondrocytes

The differentiation of preosseous chondrocytes begins with the proliferation of resting cells and results in the expression of the hypertrophic phenotype. The effect of fetal calf serum on chondrocyte mitogenesis and intracellular Ca2+ concentration was studied in resting and hypertrophic cells in primary culture. Resting chondrocytes respond to the growth stimulus with immediate release of Ca2+ from intracellular stores and with opening of the plasma membrane Ca2+ channels. These events may be related to the elevated [3H]thymidine incorporation observed after serum exposure. In contrast, in hypertrophic chondrocytes the lower rate of DNA synthesis seems to be coupled with a lower activity of the Ca2+ signaling mechanism and, probably, with reduced intracellular calcium stores. It is proposed that expression of the Ca2+ signaling mechanism may be modulated during the differentiation of preosseous chondrocytes.     

Stimulation of cartilage-matrix proteoglycan synthesis by morphologically transformed chondrocytes grown in the presence of fibroblast growth factor and transforming growth factor-beta

The effects of transforming growth factor-beta (TGF-beta) on the synthesis of cartilage-matrix proteoglycan by cultured rabbit chondrocytes were examined. Rabbit chondrocytes were seeded at low density and exposed to a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with 0.5% fetal bovine serum, 1% bovine serum albumin, 50 micrograms/ml ascorbic acid, and 2 x 10(-7) M hydrocortisone (Medium A). Various combinations of TGF-beta, insulin-like growth factor-I (IGF-I), and fibroblast growth factor (FGF) were also added to Medium A, and the chondrocytes were grown to confluency. Chondrocytes grown with TGF-beta or FGF alone became flat or fibroblastic, those grown with FGF and TGF-beta became very elongated and formed distinct foci, and those grown with FGF and IGF-I showed the spherical configuration characteristic of overtly differentiated chondrocytes. Nevertheless, the incorporation of 3H with glucosamine into the large, chondroitin sulfate proteoglycan synthesized by cultures with FGF and TGF-beta was similar to that in cells grown with FGF and IGF-I and five times that in cells cultured with FGF alone. The increases in incorporation of 3H reflected real increases in proteoglycan synthesis, because chemical analyses showed an increase in the accumulation of macromolecules containing uronic acid in cultures with FGF and TGF-beta or with FGF and IGF-I. However, FGF in combination with either TGF-beta or IGF-I had little effect on the incorporation of 3H into small proteoglycans or hyaluronic acid. These results indicate that chondrocytes morphologically transformed with TGF-beta and FGF fully express the differentiated proteoglycan phenotype rather than the transformed glycosaminoglycan phenotype.     

Changes in polyamine metabolism in WI38 cells stimulated to proliferate.

Quiescent confluent monolayers of WI38 human diploid fibroblasts were stimulated to proliferate by replacement of the exhausted medium with fresh medium containing 10% fetal calf serum. The cellular content of the polyamines, putrescine, spermidine, and spermine was studied at various intervals after the nutritional change. The putrescine content increased during the pre-replicative phase of the cell cycle, whereas the content of spermidine and spermine did not increase until after the initiation of DNA synthesis. By varying the composition of the stimulating medium it was possible to alter the percentage of cells that were stimulated to proliferate. Measurement of the cellular polyamine content and ³H-thymidine (³H-TdR) incorporation into DNA at the time of the maximal rate of DNA synthesis showed that the magnitude of putrescine accumulation depended on the percentage of cells that were stimulated to proliferate. These results indicate that there may be a connection between polyamine synthesis and subsequent DNA replication.     

Effect of polyamines on the activity of malarial alpha-like DNA polymerase

DNA polymerase from the malarial parasite Plasmodium falciparum required Mg2+ for activity, Putrescine (1 mM) caused a twofold increase in enzyme activity in the presence of a suboptimal concentration of MgCl2 (2 mM). Spermidine (1.5-2.0 mM) or spermine (0.1-0.3 mM) increased the activity of malarial DNA polymerase, in the presence of 2 mM MgCl2, by factors of 6 and 3-5, respectively. The activity of DNA polymerase from calf thymus or from NIH 3T3 cells transformed by the ras oncogene were not stimulated by these polyamines to the same extent. These findings suggest that in malaria-infected erythrocytes, polyamines, at physiological concentrations, serve as a cofactor for the parasitic alpha-like DNA polymerase. Malarial parasites grown in cultured human erythrocytes did not synthesize DNA after treatment with alpha-difluoromethylornithine, which caused polyamine depletion in the infected cells. DNA synthesis was resumed after adding putrescine to the polyamine-depleted cultures. DNA synthesis was also initiated when actinomycin D was added along with putrescine to polyamine-depleted cells. It thus appears that polyamines are essential for the translation of the DNA polymerase mRNA and that polyamines play an important role in regulating the cell cycle of the malarial parasite.