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.     

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.     

Effects of various tumor promoters on expression of cartilage phenotypes in rabbit costal chondrocytes in culture

12-O-Tetradecanoylphorbol-13-acetate (TPA), a skin tumor-promoting phorbol ester, and teleocidin and aplysiatoxin, which are potent tumor promoters in mouse skin but are chemically unrelated to phorbol esters, induced change of cultured rabbit costal chondrocytes from a polygonal to a fibroblastic shape and inhibited glycosaminoglycan (GAG) synthesis and metachromatic matrix formation in these cells. The potencies of teleocidin and aplysiatoxin to inhibit GAG synthesis were almost the same as that of TPA. On the other hand, Tween 60 and cantharidin, weak mouse skin tumor promoters, phenobarbital, a liver tumor promoter, and saccharin, a bladder tumor promoter, had no effect on the morphology or GAG synthesis of cultured chondrocytes. Like TPA, teleocidin and aplysiatoxin increased DNA and RNA syntheses of chondrocytes. Parathyroid hormone (PTH) and dibutyryl cyclic AMP reversed the morphological and histochemical changes caused by a 4-day treatment with teleocidin or aplysiatoxin as well as with TPA, reversal being apparent after 2 days. PTH increased intracellular cyclic AMP after 2 min in chondrocytes pretreated with teleocidin or aplysiatoxin as well as with TPA. PTH also increased ornithine decarboxylase [ODC; EC 4.1.1.17] activity in these chondrocytes after 4 h. These results show that retention of responsiveness to PTH is a typical characteristic of chondrocytes dedifferentiated by treatment with TPA-type tumor promoters such as TPA, teleocidin and aplysiatoxin. The results also suggest that ODC induction mediated by elevation of cyclic AMP plays an important role in re-differentiation of teleocidin- and aplysiatoxin-treated chondrocytes.     

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.     

Differentiation and de-differentiation of cultured chondrocytes: increase in monomeric size of 'cartilage-specific' proteoglycans by dibutyryl cyclic AMP and complete inhibition of their synthesis by retinoic acid

Dibutyryl cyclic AMP (DBcAMP) induced an increase in the monomeric size of 'cartilage-specific' proteoglycans (PG-I) in rabbit costal chondrocytes in culture. This increase in size was due to an increase in the average molecular weight of the glycosaminoglycan (GAG) chains. In contrast, retinoic acid completely inhibited the synthesis of PG-I. However, the synthesis and monomeric size of 'ubiquitous' proteoglycans (PG-II) were little affected by these agents. These results suggested that modulation of the differentiated state of chondrocytes is closely related to not only the synthesis of 'cartilage-specific' proteoglycans but also their monomeric size.     

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.     

Prostaglandin stimulation of adenosine 3',5'-monophosphate accumulation in cultured chondrocytes in the presence or absence of parathyroid hormone

The effect of prostaglandin analogues on the cyclic AMP level in cultured chondrocytes were examined. Prostaglandin E1 at 0.4 to 30 microM, increased the intracellular concentration of cyclic AMP in chondrocytes. Its effect was rapid, being evident within 1 min and reaching a maximum in 10 to 20 min. The maximum level was sustained until 30 min after its addition and then decreased gradually. Prostaglandin D2 and E2 also increased the cyclic AMP level in chondrocytes, but they had less effect than prostaglandin E1. Prostaglandin A1 had no effect on the nucleotide level in chondrocytes, although they markedly increased the level in fibroblasts. The time course of stimulation of cyclic AMP accumulation in chondrocytes by prostaglandin E1, D2 or E2 was quite different from that by parathyroid hormone (PTH): the effect of prostaglandin was slower and more sustained than that of PTH. PTH potentiated the effect of prostaglandin E1, E2, or D2 on the cyclic AMP level in chondrocytes and that the combined effects of prostaglandin and PTH were more than additive. Addition of an inhibitor of cyclic nucleotide phosphodiesterase with prostaglandin, PTH or both produced a synergistic effect on the accumulation of cyclic AMP in the chondrocytes. These findings suggest that prostaglandin E1, E2, and D2 increase the synthesis of cyclic AMP and that the combined effect of the prostaglandins and PTH on the cyclic AMP level in chondrocytes is partly attributed to the synergistic synthesis of cyclic AMP in the cells.     

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.     

Demonstration of receptors for epidermal growth factor on cultured rabbit chondrocytes and regulation of their expression by various growth and differentiation factors.

Epidermal growth factor (EGF) receptors were demonstrated on cultured rabbit costal chondrocytes. After crosslinking, the receptors on the cells with 125I-EGF, one major band of 170 KDa was separated by SDS-PAGE. Scatchard analysis demonstrated two classes of EGF receptors with Kd values of 0.3 nM and 1.6 nM. The numbers of high and low affinity receptors were 3,000 and 10,000 per cell, respectively. EGF receptors on chondrocytes were increased by treatment with retinoic acid and interleukin-1 beta, which inhibited proteoglycan synthesis. On the other hand, parathyroid hormone and dibutyryl cyclic AMP, which stimulated proteoglycan synthesis, decreased the number of EGF receptors. Treatments with these agents did not change the affinity of the receptors. These findings suggest that the number of EGF receptors is a negative marker of chondrocyte differentiation.     

Prostaglandin stimulation of adenosine 3′,5′-monophosphate accumulation in cultured chondrocytes in the presence or absence of parathyroid hormone

The effect of prostaglandin analogues on the cycle AMP level in cultured chondrocytes were examined. Prostaglandin E₁ at 0.4 to 30 μM, increased the intracellular concentration of cyclic AMP in chondrocytes. Its effect was rapid, being evident within 1 min and reaching a maximum in 10 to 20 min. The maximum level was sustained until 30 min after its addition and then decreased gradually. Prostaglandin D₂ and E₂ also increased the cyclic AMP level in chondrocytes, but they had less effect than prostaglandin E₁. Prostaglandin A₁ had no effect on the nucleotide level in chondrocytes, although they markedly increased the level in fibroblasts. The time course of stimulation of cyclic AMP accumulation in chondrocytes by prostaglandin E₁, D₂ or E₂ was quite different from that by parathyroid hormone (PTH): the effect of prostaglandin was slower and more sustained than that of PTH. PTH potentiated the effect of prostaglandin E₁, E₂, or D₂ on the cyclic AMP level in chondrocytes and that the combined effects of prostaglandin, PTH or both produced a synergistic effect on the accumulation of cyclic AMP in the chondrocytes. These findings suggest that prostaglandin E₁, E₂, and D₂ increase the synthesis of cyclic AMP and that the combined effect of the prostaglandins and PTH on the cyclic AMP level in chondrocytes is partly attributed to the synergistic synthesis of cyclic AMP in the cells.     

Differential effects of parathyroid hormone and somatomedin-like growth factors on the sizes of proteoglycan monomers and their synthesis in rabbit costal chondrocytes in culture

In the proteoglycans extracted from rabbit costal chondrocytes in culture, two populations of proteoglycans were distinguished by density gradient centrifugation under dissociative conditions. The major component was the faster sedimenting population (proteoglycan I), the putative 'cartilage-specific' proteoglycans, and the minor component was the slower sedimenting population (proteoglycan II). The monomeric size of proteoglycan I was closely related to the differentiation-state of chondrocytes and was a good marker of the differentiated chondrocytes. Treatment of the cultures with parathyroid hormone (PTH) induced an increase in the monomeric size of proteoglycan I. This increase was ascribed to an increase in the molecular size of the glycosaminoglycan chain in proteoglycan I. On the other hand, somatomedin-like growth factors, such as multiplication-stimulating activity (MSA) and cartilage-derived factor (CDF), did not affect the size of proteoglycan I, while they markedly stimulated the synthesis of proteoglycan I. In contrast, treatment with nonsomatomedin growth factors, such as fibroblast growth factor (FGF) and epidermal growth factor (EGF), resulted in not only a decrease in glycosaminoglycan synthesis but also a slight decrease in size of proteoglycan I. However, synthesis and size of proteoglycan II were little affected by these agents. Thus, the present study clearly shows that PTH and somatomedin-like growth factors have differential functions in bringing about the expression of the differentiated phenotype of chondrocytes: PTH influences chain elongation and termination of glycosaminoglycans in proteoglycan I, while somatomedin-like growth factors affect primarily the synthesis and secretion of proteoglycan I.     

Induction of spermidine/spermine N1-acetyltransferase by parathyroid hormone in rabbit costal chondrocytes in culture

Parathyroid hormone (PTH) increased the activity of spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme of polyamine biodegradation, in rabbit costal chondrocytes in culture. The enzyme activity increased in a dose-dependent manner after addition of PTH to the culture, reaching a maximum at 8 h. The increase in the enzyme activity was abolished by cycloheximide or actinomycin D. Dibutyryl cyclic AMP also induced the acetyltransferase to some extent. These results suggest that the induction of spermidine/spermine N1-acetyltransferase by PTH may play some significant role in the expression of the differentiated phenotype of chondrocytes.     

Long acting cAMP analogues enhance sulfate incorporation into matrix proteoglycans and suppress cell division of fetal rat chondrocytes in monolayer culture.

The relationship between replication and the synthesis of matrix sulfated proteoglycans was investigated with fetal rat chondrocytes grown in monolayer culture. The effect of N6 O2' dibutyryl adenosine 3', 5' cyclic monophosphate (DBcAMP), adenosine 3', 5' cyclic monophosphate (cAMP), 8 Bromo adenosine 3', 5' cyclic monophosphate (8 Br-cAMP), sodium butyrate and hydroxyurea was examined. Between 0.05 and 0.5 mM DBcAMP, a dose related inhibition of cell division and stimulation of [35SO=/4] incorporation into matrix proteoglycans was demonstrated. At the higher concentrations of DBcAMP, cell division was completely inhibited and the enhancement of [35SO=/4] incorporation into matrix proteoglycans ranged between 40 and 120% (P less than 0.01). Utilizing 14C-glucosamine and photometric determination of proteoglycans with Alcian Blue, it was demonstrated that the increase in sulfate incorporation reflected enhanced accumulation of extracellular matrix. The effects of DBcAMP were mimicked by 8 Br-cAMP, suggesting they were mediated by the adenylyl cyclase system. cAMP (0.05-0.5 mM), sodium butyrate (0.1-0.5 mM) and hydroxyurea (0.5-5 mM) partially or fully inhibited cell division, but either failed or only slightly enhanced sulfate incorporation. The enhanced sulfated proteoglycan deposition promoted by DBcAMP began 8 to 12 hours after serum stimulation, its onset occurred prior to thymidine incorporation and the effect persisted for 28 hours. Determination of cell volume demonstrated an increase in size of DBcAMP treated chondrocytes between 8 to 12 hours, coincident with the onset of increased sulfate incorporation. These results are consistent with a model where matrix sulfated proteoglycan deposition by chondrocytes is mediated by intracellular cAMP levels and occurs in the G1 phase of the cell cycle.     

Retinoids antagonize the induction of ornithine decarboxylase activity by phorbol esters and phospholipase C in rat tracheal epithelial cells

In this study we examined the action of phorbol esters, several phospholipases and retinoids on the induction of ornithine decarboxylase (ODC) activity in rat tracheal epithelial cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induces ODC activity in these cells in a dose-and time-dependent manner. This induction is inhibited by cycloheximide indicating a requirement for protein synthesis. Tracheal epithelial 2C5 cells contain two binding sites for phorbol esters, one with a high affinity KD,1 = 4.58 nM and one with a low affinity KD,2 = 344.8 nM. The ability of several phorbol esters to induce ODC correlates well with the described efficacy with which they bind to the receptor and is in agreement with the concept that phorbol ester receptors are involved in the induction of ODC. There is strong evidence that the phorbol ester receptor is the protein kinase C for which diacylglycerol is the physiological ligand. Treatment of cells with phospholipase C generates diacylglycerol and induces ODC activity in a dose- and time-dependent manner. Treatment with phospholipase A2 or D has no effect on ODC activity. These results support the concept that activation of protein kinase C is related to the induction of ODC activity. The induction of ODC by TPA as well as by phospholipase C is inhibited by retinoids. Specific cytosolic binding proteins for retinoids might be involved in at least some of the responses to these compounds. To examine whether the binding proteins are involved in the inhibition of ODC we determined the presence of these binding proteins and the structure-activity relationship of retinoids. Both retinol and retinoic acid-binding proteins can be detected in 2C5 cells, their levels are 1.06 and 3.36 pmoles/mg protein, respectively. The ability of several retinoids to inhibit ODC induction correlates well with their binding activity and support a role for these binding proteins in the action of retinoids on ODC induction.     

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.     

Genetic evidence that a phorbol ester tumor promoter stimulates ornithine decarboxylase activity by a pathway that is independent of cyclic AMP-dependent protein kinases in CHO cells

Ornithine decarboxylase (ODC) inductions by cholera toxin and by the phorbol ester tumor promoter, TPA, were compared in wild-type Chinese hamster ovary (CHO) cells and in mutant cells having altered cyclic AMP-dependent protein kinase activity. The aim of these studies was to determine whether cyclic AMP-dependent protein kinase is involved in these inductions. The time course and the magnitude of ODC inductions by either 100 ng/ml cholera toxin or 100 ng/ml TPA were similar in wild-type cells with a maximum at 3-4 hours after treatment and a return to unstimulated levels by 8 hours. Induction of ODC by cholera toxin was suppressed more than 80% in the four protein kinase mutants studied (10215, 10248, 10260, and 10265), strongly implicating a cyclic AMP-dependent kinase step in the mechanism of induction. Similar results were found with the cyclic AMP analog 8-Br-cyclic AMP and the phosphodiesterase inhibitor, methyl-isobutylxanthine. The induction of ODC by TPA, on the other hand, was only partially inhibited (approximately 50%) in three of four mutants. Lower ODC activity in two mutants stimulated by cholera toxin or TPA whose kinetics were studied in more detail could not be ascribed to a reduced affinity (Km) of ornithine for the enzyme, but appeared to be due to reduced catalytic activity (Vmax) in the extracts. These results suggest that the induction of ODC by TPA proceeds by a mechanism which is only partially dependent on an intact cyclic AMP-dependent protein kinase activity.     

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.     

Enhanced responsiveness to parathyroid hormone and induction of functional differentiation of cultured rabbit costal chondrocytes by a pulsed electromagnetic field

Pulsed electromagnetic fields promote healing of delayed united and ununited fractures by triggering a series of events in fibrocartilage. We examined the effects of a pulsed electromagnetic field (recurrent bursts, 15.4 Hz, of shorter pulses of an average of 2 gauss) on rabbit costal chondrocytes in culture. A pulsed electromagnetic field slightly reduced the intracellular cyclic adenosine 3',5'-monophosphate (cAMP) level in the culture. However, it significantly enhanced cAMP accumulation in response to parathyroid hormone (PTH) to 140% of that induced by PTH in its absence, while it did not affect cAMP accumulation in response to prostaglandin E1 or prostaglandin I2. The effect on cAMP accumulation in response to PTH became evident after exposure of the cultures to the pulsed electromagnetic field for 48 h, and was dependent upon the field strength. cAMP accumulation in response to PTH is followed by induction of ornithine decarboxylase, a good marker of differentiated chondrocytes, after PTH treatment for 4 h. Consistent with the enhanced cAMP accumulation, ornithine decarboxylase activity induced by PTH was also increased by the pulsed electromagnetic field to 170% of that in cells not exposed to a pulsed electromagnetic field. Furthermore, stimulation of glycosaminoglycan synthesis, a differentiated phenotype, in response to PTH was significantly enhanced by a pulsed electromagnetic field. Thus, a pulsed electromagnetic field enhanced a series of events in rabbit costal chondrocytes in response to PTH. These findings show that exposure of chondrocytes to a pulsed electromagnetic field resulted in functional differentiation of the cells.     

Fibroblast growth factor stimulates colony formation of differentiated chondrocytes in soft agar

The effect of fibroblast growth factor (FGF) on the growth of chondrocytes in soft agar was examined. FGF induced colony formation by chick embryo and rabbit chondrocytes. The colony-forming efficiency of FGF-exposed chondrocytes was similar to that of Rous sarcoma virus-transformed chondrocytes (15-20%). Other mitogenic agents tested, such as epidermal growth factor, insulin, insulin-like growth factor-l, and platelet-derived growth factor, induced very low levels of colony formation. The induction of growth in soft agar of chondrocytes by FGF was not due to cells' phenotypic transformation, because chondrocytes grown in soft agar with FGF retained the ability to synthesize cartilage-characteristic proteoglycan. FGF did not induce growth in soft agar of chondrocytes whose phenotypic expression was suppressed by retinoic acid or 5-bromodeoxyuridine. In addition, FGF did not induce growth in soft agar of primary fibroblasts and normal rat kidney (NRK) cells. These results suggest that FGF selectively stimulates growth of differentiated chondrocytes in soft agar.