Rabbit Costal Chondrocytes Research Articles

Cartilage-derived factor (CDF): II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [ 35S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [ 35S]sulfate incorporation but also [ 3H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [ 3H]uridine incorporation into rabbit chondrocytes and significantly enhanced [ 3H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit 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.

Cartilage-derived factor (CDF): I. Stimulation of proteoglycan synthesis in rat and rabbit costal chondrocytes in culture

A protease-sensitive factor was extracted from fetal bovine cartilage with 1 M guanidine hydrochloride and partially purified by gel filtration on Bio-Gel A 0.5 m and CM-Sephadex column chromatography. This cartilage-derived factor (CDF) stimulated proteoglycan synthesis in rat and rabbit costal chondrocytes in culture, as shown by increased incorporation of 35SO 4 −2, [ 3H]-glucosamine and [ 3H]serine into material precipitated with cetylpyridinium chloride. In addition, CDF stimulated the synthesis of sulfated glycosaminoglycans in a dose-dependent manner. These findings suggest that CDF is involved in the control of chondrogenesis.

Polyamine and differentiation: induction of ornithine decarboxylase by parathyroid hormone is a good marker of differentiated chondrocytes.

The activity of ornithine decarboxylase (OD-Case:L-ornithine carboxy-lyase, EC 4.1.1.17) in rabbit costal chondrocytes in culture increased markedly after addition of parathyroid hormone (PTH), reaching a maximum 4 to 5 hr after PTH addition. The increase in ODCase activity was followed by increase in the intracellular concentrations of polyamines, especially putrescine, which increased in 6 hr to about 3-fold that of untreated cultures. The induction of ODCase by PTH was not observed in L, 3T3, HeLa, buffalo rat liver, or BHK cells. Retinyl acetate and retinoic acid both inhibited expression of the differentiated phenotype of chondrocytes by rabbit costal chondrocytes in culture within 3 days after their addition, as judged by morphological change and decrease in sulfate incorporation into glycosaminoglycans but did not inhibit cell proliferation. PTH could not induce an increase in ODCase in de-differentiated cells that had been pretreated with retinyl acetate or retinoic acid for 3 days. but 4 days after removal of the retinoids, these de-differentiated cells regained the ability to synthesize ODCase in response to PTH. These facts suggest that the induction of ODCase and the formation of putrescine by PTH are good markers of the differentiated phenotype of cultured chondrocytes.

Induction by parathyroid hormone of ornithine decarboxylase in rabbit costal chondrocytes in culture.

The activity of ornithine decarboxylase (ODC) in rabbit costal chondrocytes in culture markedly increased after addition of parathyroid hormone (PTH), reaching a maximum 4 to 5 h after PTH addition. The effect of PTH was dose-dependent both in the presence and absence of fetal calf serum. Addition of cycloheximide or actinomycin D prevented the induction of ODC by PTH. Calcitonin, 1,25-dihydroxyvitamin D3, and vitamin A had no stimulatory effect on the activity of ODC, and did not inhibit the induction of ODC by PTH, when added with PTH.

Demonstration of somatomedin activity of "multiplication-stimulating activity" in rabbit costal chondrocytes in culture.

Multiplication-stimulating activity (MSA), a substance obtained from conditioned medium of Buffalo rat liver cells, stimulated replication of rabbit costal chondrocytes in culture and their DNA synthesis, sulfation of glycosaminoglycans, protein synthesis, and collagen synthesis. These stimulatory effects of MSA were dose-dependent in serum-free medium, indicating that MSA has intrinsic somatomedin activity. Even after several successive passages, cultured chondrocytes were more responsive to MSA than other organ- and cell-culture systems reported. Therefore, cultured rabbit costal chondrocytes proved a good in vitro system for analysis of somatomedin actions.