Rabbit Costal Chondrocytes Research Articles

Chemical Composition and Bioactivity of Polypeptide from Velvet Antlers of Cervus nippon Temminck

In order to study the isolation and purification of an active peptide component from velvet antler of sika deer(Cervus nippon Temminck),confirm the primary structure of the novel peptide and compare the sequence and the activity with those of velvet antler of red deer(Cervus elaphus Linaeus),the velvet antler polypeptide was isolated and purified by ion exchange chromatography,gel filtration and RP-HPLC,which showed a single peak in HPLC chromatography and a single band in SDS-PAGE.The molecular weight was measured by MALDI-TOF MS.This polypeptide consists of a single linear chain of 32 amino acid residues VLSATDKTNVLAAWGKVGGNAPAFGAEALERM.The experiment results of bioactivity showed that the polypeptide had stimulant effects on the growth of rat epithelial cells,rabbit costal chondrocytes and rat NIH3T3 cell line,respectively in vitro.The straight chain of 32 amino acid residues of the velvet antler polypeptides of Cervus nippon Temminck was consisted with that of Cervus elaphus Linaeus,but they make a difference of 5,8,11 and 30 amino acid.There was no effect on stimulating proliferation of cells.

Role of mechanical-stress inducible protein Hcs24/CTGF/CCN2 in cartilage growth and regeneration: Mechanical stress induces expression of Hcs24/CTGF/CCN2 in a human chondrocytic cell line HCS-2/8, rabbit costal chondrocytes and meniscus tissue cells

Mechanical stress plays an important role in the cartilage metabolism. The aim of this study is to determine the influence of mechanical load magnitude and frequency on cartilage metabolism in terms of the expression of hypertrophic chondrocyte-specific gene product 24/connective tissue growth factor/CCN family 2 (Hcs24/CTGF/CCN2), as an essential mediator of extracellular matrix (ECM) production. When a human chondrocytic cell line, HCS-2/8 was exposed to uni-axial cyclic mechanical force (6% elongation, 10 times/min) only for 30 min, the expression level of Hcs24/CTGF/CCN2 (CCN2) increased, and c-Jun N-terminal protein kinase (JNK) was activated. These findings suggest that stretch-induced CCN2 may be mediated by the JNK pathway. When HCS-2/8 cells were subjected to cyclic tension force at 15 kPa, 30 cycles/min, which has been reported to be a degradation force for HCS-2/8 cells, the expressions of CCN2 and aggrecan were inhibited, and such expressions remained unchanged in rabbit hyaline costal cartilage cells. However, these expressions increased in rabbit meniscus tissue cells. These findings suggest that the sensitivity of mechanical stretch may be different depending on the type of cells. Furthermore, CCN2 was co-localized with aggrecan in this meniscus tissue region exposed to mechanical stress in vivo. These findings suggest that CCN2 induced by mechanical stress may therefore play some role in meniscus growth and regeneration.

Differential regulation of IGF-binding proteins in rabbit costal chondrocytes by IGF-I and dexamethasone.

Cartilage is a primary target tissue for the IGFs. The mitogenic activity of these peptides is regulated by a family of high-affinity IGF-binding proteins (IGFBP-1 to -6). We characterized the IGFBPs produced by cultured chondrocytes derived from rib cartilage of prepubertal rabbits. Culture medium, which had been conditioned by these cells for 48 h showed bands of 22 kDa, 24 kDa and a 31/32 kDa doublet by Western ligand blotting with [(125)I]IGF-II. When the cells were grown in the presence of increasing amounts of IGF-I or IGF-II, the 31/32 kDa doublet increased in intensity (reaching a plateau of about 11-fold stimulation between 2 and 10 nM IGF-I). The 22 kDa and 24 kDa bands increased only slightly while a 26 kDa band became faintly visible. By Western immunoblotting the 31/32 kDa doublet was identified as IGFBP-5. An IGF-I analog with reduced affinity for IGFBPs, Long-R3 IGF-I, also induced IGFBP-5, while insulin was less effective (2.2-fold stimulation at 10 nM). IGF-I protected IGFBP-5 against proteolytic degradation by conditioned medium. IGF-I also enhanced the level of IGFBP-5 mRNA. LY294002, a specific inhibitor of the intracellular signaling molecule phosphatidylinositol 3-kinase, inhibited stimulation of IGFBP-5 by IGF-I. Dexamethasone suppressed IGFBP-5 (by 70% at 20 nM) but, at the same time, a 39/41 kDa doublet (presumably IGFBP-3) was induced. IGFBP-5 has been shown in several cell types to enhance the mitogenic activity of IGF-I. IGFBP-3 generally acts as a growth inhibitor. Therefore, the differential effects of dexamethasone on these regulatory proteins could account, at least in part, for the growth-arresting effect of this glucocorticoid.

Demonstration of endothelin (ET) receptors on cultured rabbit chondrocytes and stimulation of DNA synthesis and calcium influx by ET-1 via its receptors

Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with 125 I-ET-1, two major bands of 43 kDa and 46 kDa were separated by SDS-PAGE. Scatchard analysis demonstrated two classes of ET receptors with K d values of 1 × 10 -10 M and 5 × 10 -9 M. The numbers of high- and low- affinity receptors were 1 × 10 4 and 2 × 10 5 per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-β1, IL-1β, RA and EGF. ET-1 stimulated DNA synthesis in cultured rabbit chondrocytes. ET-1 also stimulated calcium incorporation through the cell membrane of chondrocytes. These findings indicate that ET-1 has a physiological effect on chondrocytes via its receptors on the cells.

Histatin as a Synergistic Stimulator with Epidermal Growth Factor of Rabbit Chondrocyte Proliferation

Histatin 5 dose-dependently increased DNA synthesis in rabbit costal chondrocytes in culture. The level of DNA synthesis was stimulated by histatin 5 to about 4 times that of the control. The combination of histatin 5 and epidermal growth factor (EGF) increased the DNA synthesis to about 40 times that of the control while EGF alone stimulated it 15-fold, indicating synergistic stimulation by both factors. Enzyme-linked immunosorbent assay using anti-histatin 5 demonstrated that human serum contained histatin-like substances. These findings suggest that histatins play an important role in chondrocyte proliferation, presumably as a physiological modulator of the action of EGF.

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-1β, 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.

Effects of various growth factors on a chondrocyte differentiation model.

We developed a new 4-dimensional differentiation model of growth-cartilage cells (temporally and spatially regulated). composed of a high-density suspension culture of rabbit costal chondrocytes. In this system, fibroblast growth factor (FGF) or transforming growth factor-beta (TGF beta) stimulated proliferation and matrix synthesis, but reversibly inhibited terminal differentiation-induction of alkaline phosphatase (Alpase) activity, type X collagen synthesis and subsequent calcification. When parathyroid hormone (PTH) was added to the differentiation model, both the induction of Alpase and calcium uptake were reversibly suppressed. In contrast, calcitonin (CT) stimulated Alpase activity and calcium uptake dose-dependently. Bone morphogenetic protein (BMP) strongly stimulated DNA synthesis of chondrocytes in the presence of FGF and induced rapid maturation of chondrocytes at a growing stage. Moreover, BMP stimulated Alpase activity in multilayered chondrocytes at the calcifying stage.

Effects of various growth and differentiation factors on expression of parathyroid hormone receptors on rabbit costal chondrocytes in culture.

In our preliminary report we demonstrated PTH receptors on rabbit costal chondrocytes in culture. In the present study regulation of expression of PTH receptors of the chondrocytes by various growth and differentiation factors and its relation to the synthesis of glycosaminoglycan (GAG), a differentiated phenotype of chondrocytes, were investigated. Treatment with retinoic acid decreased GAG synthesis in cultured chondrocytes and the number of PTH receptors, measured by binding of [125I]-[Nle8,18,Tyr34]bovine PTH-(1-34) amide to the cells. However, the affinity of the receptors did not change. The decrease in the number of PTH receptors was dose and time dependent and parallel to the decrease in GAG synthesis. When chondrocytes that had been treated with retinoic acid were cultured in the absence of retinoic acid for 3 days, both their GAG synthesis and their number of PTH receptors were restored. Epidermal growth factor and fibroblast growth factor also decreased both the number of PTH receptors and GAG synthesis in the cells. However, these treatments did not change their affinity. Treatment with insulin-like growth factor-I, (Bu)2cAMP, and transforming growth factor-beta resulted in increases in GAG synthesis as well as in the number of PTH receptors without any change in their affinity. In addition, the PTH-stimulated cAMP level in chondrocytes pretreated with retinoic acid, epidermal growth factor, and fibroblast growth factor was lower than that in control cells. On the other hand, the PTH-stimulated cAMP level in chondrocytes pretreated with insulin-like growth factor-I and transforming growth factor-beta was higher than that in control cells. These observations suggest that the increase in the number of PTH receptors on chondrocytes is closely related to expression of the differentiated phenotype of chondrocytes and that the number of the receptors is a good marker of the differentiated phenotype of chondrocytes.

Interactions between Steroid Hormones and Insulin-Like Growth Factor-I in Rabbit Chondrocytes

The mechanism of action of steroid hormones on skeletal growth is not understood in detail. We examined the interactions of steroid hormones and insulin-like growth factor-I (IGF-I) during DNA and sulfated proteoglycan synthesis in rabbit costal chondrocytes. Progesterone at 0.05 nM stimulated the incorporation of [3H]thymidine into DNA by 30% above the control level in confluent cultures, but neither testosterone nor 17 beta-estradiol stimulated DNA synthesis. None of the hormones affected [3H]thymidine incorporation stimulated by IGF-I when chondrocytes were incubated with one of the hormones and IGF-I simultaneously. In contrast, when confluent cultures were incubated with one of the sex steroids for 24 h before the addition of IGF-I, stimulation of DNA synthesis by the growth factor was enhanced about 45% above the control value by 0.5 nM progesterone, 50% by 0.5 nM testosterone, and 80% by 50 nM 17 beta-estradiol. The effects of IGF-I on proteoglycan synthesis, as judged by the incorporation of [35S]sulfate, were stimulated by treatment with progesterone or testosterone. Dexamethasone at physiological concentrations inhibited chondrocyte DNA synthesis in confluent cultures to 10% of the control level. At 50 nM, dexamethasone suppressed IGF-I induction of DNA synthesis by 60%. This suppression was greater when dexamethasone was added before IGF-I than when the additions were simultaneous. When chondrocytes were treated with hydrocortisone or dexamethasone for 24 h before the addition of IGF-I, the glucocorticoids synergistically accelerated proteoglycan synthesis mediated by IGF-I. These findings suggest that steroid hormones have priming effects on the biological action of IGF-I in cartilage metabolism.

A Case of Dwarfism with Severely Reduced Activity of Growth Hormone-Binding Protein

We presented a 16-year-old boy with severe growth retardation and markedly decreased levels of growth hormone-binding protein (GHBP) in plasma, which was shown to correspond to the extracellular composition of hepatic GH receptor and suggested to reflect tissue concentration of the receptor. His height was 92.5 cm (-13.5 SD), the weight 9.6kg (-5.8 SD) and Tanner stage was I. His bone age was 3.5 years old at 16 years of age. Karyotype was 46,XY and thyroid function was normal. SM-C levels, determined by Nichols RIA using unextracted plasma, were within the low normal range, 0.67/0.68U/ml. In contrast, using a method of acid-ethanol extraction, IGF-I and IGF-II levels were definitely low, 29ng/ml (normal 88-240) and 165ng/ml (374-804) respectively. GH responses in various provocation tests, including insulin, arginine and GRF, were within normal. Basal GH levels were 20 +/- 12ng/ml and urinary GH excretion rates 217 +/- 85pg/mg. Cr, which were elevated compared to age-matched control. Molecular size of his circulating GH was similar to control subjects. The biological activities of GH, evaluated by radioreceptor assay and Nb2 cell bioassay, were proportional to the immunoactivities of GH. SM bioactivities, which were determined by the stimulatory effects on DNA synthesis of rabbit costal chondrocytes and human fibroblasts, were apparently reduced. Electrophoretic patterns of IGF-binding protein was similar to those of GH deficient cases. Daily administration of hGH (4U/day) for 5 days resulted in a poor response of SM-C production (before 0.68, after 0.77U/ml). GHBP activities were definitely low by gel-filtration, immunoprecipitation and charcoal methods, as seen in Laron dwarfism which is defined as a syndrome of congenital GH receptor defects. These results indicate that the tissue content of GH receptor in this case was quantitatively reduced and as a result, he showed a resistance to endogenous and exogenous GH. It remains to be elucidated whether the GH receptor defect in our case is derived from a genetic origin or an acquired condition.

Catabolic effects of muramyl dipeptide on rabbit chondrocytes

Muramyl dipeptide, an essential structure for the diverse biologic activities of bacterial cell wall peptidoglycan, inhibited the synthesis of glycosaminoglycan/proteoglycan in cultured rabbit costal chondrocytes in a dose-dependent manner. Muramyl dipeptide, as well as lipopolysaccharide and interleukin-1 alpha, also enhanced the release of 35S-sulfate-prelabeled glycosaminoglycan/proteoglycan from the cell layer, which seems to reflect, at least partially, the increasing degradation of glycosaminoglycan/proteoglycan. Five synthetic analogs of muramyl dipeptide known to be adjuvant active or adjuvant inactive were tested for their potential to inhibit synthesis of glycosaminoglycan/proteoglycan and to enhance the release of glycosaminoglycan/proteoglycan in chondrocytes. The structural dependence of these synthetic analogs on chondrocytes was found to parallel that of immunoadjuvant activity. These results suggest that muramyl dipeptide is a potent mediator of catabolism in chondrocytes.

Effects of tumor necrosis factor α on proliferation and expression of differentiated phenotypes in rabbit costal chondrocytes in culture

Tumor necrosis factor alpha (TNF alpha) decreased the synthesis of glycosaminoglycan (GAG) in rabbit costal chondrocytes in culture, but did not stimulate the release of GAG from cell layers. Like chondrocytes cultured in control medium, chondrocytes cultured in the presence of TNF alpha produced putative "cartilage-specific" proteoglycans identified by density gradient centrifugation under dissociative conditions. Although TNF alpha decreased the synthesis of the proteoglycans, it did not change their monomeric size, which is a marker of cartilage phenotypes. Moreover, TNF alpha did not affect the responsiveness to parathyroid hormone, insulin-like growth factor I, or transforming growth factor beta, which is known to stimulate GAG synthesis in cultured chondrocytes. TNF alpha decreased the alkaline phosphatase activity in the chondrocytes dose dependently. On the other hand, it stimulated their DNA synthesis slightly, but significantly. The stimulatory effect of TNF alpha on DNA synthesis was potentiated by fibroblast growth factor, epidermal growth factor, and fetal bovine serum. These findings suggest that in the presence of hormones and growth factors, TNF alpha promotes the proliferation of chondrocytes while suppressing their further differentiation at the stage of synthesis of cartilage-specific proteoglycans.

Stimulation by concanavalin A of cartilage-matrix proteoglycan synthesis in chondrocyte cultures.

The effect of concanavalin A on proteoglycan synthesis by rabbit costal and articular chondrocytes was examined. Chondrocytes were seeded at low density and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of concanavalin A to the culture medium induced a morphologic alteration of the fibroblastic cells to spherical chondrocytes and increased by 3- to 4-fold incorporation of [35S]sulfate and [3H]glucosamine into large chondroitin sulfate proteoglycan that was characteristically found in cartilage. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, as chemical analyses showed a 4-fold increase in the accumulation of macromolecules containing hexuronic acid in concanavalin A-maintained cultures. Furthermore, the effect of concanavalin A on [35S]sulfate incorporation into proteoglycans was greater than that of various growth factors or hormones. However, concanavalin A had smaller effects on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant glycosaminoglycans. Since other lectins tested, such as wheat germ agglutinin, lentil lectin, and phytohemagglutinin, had little effect on [35S]sulfate incorporation into proteoglycans, the concanavalin A action on chondrocytes seems specific. Although concanavalin A decreased [3H]thymidine incorporation in chondrocytes, the stimulation of proteoglycan synthesis could be observed in chondrocytes exposed to the inhibitor of DNA synthesis, cytosine arabinoside. These results indicate that concanavalin A is a potent modulator of proteoglycan synthesis by chondrocytes.

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 Culture1

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.

Demonstration of receptors for parathyroid hormone on cultured rabbit costal chondrocytes

Parathyroid hormone (PTH) receptors on cultured rabbit costal chondrocytes were demonstrated using HPLC-purified, radioiodinated [Nle 8,-Nle 18,Tyr 34]bovine PTH-(1–34)amide. PTH binding was found to be specific for PTH agonists and antagonists and dependent on the time and temperature of incubation. Both growth cartilage (GC) cells and resting cartilage (RC) cells were shown to have a single class of saturable, high affinity PTH binding sites with a dissociation constant of 0.6–0.7 nM. However, the numbers of receptors per cell were approximately 49,000 on GC cells and 19,000 on RC cells. After crosslinking the receptors on these cells with the radioligand, one, major 125I-labeled band of 76 kDa was separated by SDS-PAGE.

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 E 1 or prostaglandin I 2. 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.

Combined effects of somatomedin-like growth factors with fibroblast growth factor or epidermal growth factor in DNA synthesis in rabbit chondrocytes.

The somatomedin-like growth factors cartilage-derived factor (CDF) and multiplication-stimulating activity (MSA) stimulate DNA synthesis and proliferation of rabbit costal chondrocytes under serum-free conditions. Previously, we suggested that CDF and MSA act on chondrocytes in an early G1 phase to stimulate DNA synthesis. CDF and MSA have synergistic effects with epidermal growth factor (EGF) or fibroblast growth factor (FGF) in stimulating DNA synthesis of the cells. The mode of combined action of CDF or MSA with EGF or FGF in chondrocytes was studied by sequential treatments with these agents. EGF or FGF had synergistic effects with CDF or MSA in stimulating DNA synthesis, even when added 10 h after the latter. Synergism was also observed in cells pretreated with CDF or MSA; That is, the cultures were treated for 5 h with CDF or MSA and then washed, and treated with FGF or EGF. However, when CDF or MSA was added more than 5 h after EGF or FGF, no synergism of effects was observed. These findings suggest that the cultured chondrocytes become activated to interact with FGF or EGF for commitment to DNA synthesis when they are exposed to somatomedin-like growth factors at an early stage in the G1 phase. Thus chondrocytes are under a different mechanism of growth control from fibroblastic cells.

Effect of vanadate on cartilage-matrix proteoglycan synthesis in rabbit costal chondrocyte cultures.

The effect of vanadate on proteoglycan synthesis by cultured rabbit costal chondrocytes was examined. Rabbit chondrocytes were seeded at low densities and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of 4 microM vanadate to the culture medium induced a morphologic differentiation of the fibroblastic cells to spherical chondrocytes, and increased by two- to threefold incorporation of [35S]sulfate and [3H]glucosamine into large, chondroitin sulfate proteoglycans. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, in that chemical analyses showed increases in the accumulation of macromolecules containing hexuronic acid and hexosamine in vanadate-maintained cultures. However, vanadate had only a marginal effect on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant material. These results provide evidence that vanadate selectively stimulates the synthesis of proteoglycans characteristically found in cartilage by rabbit costal chondrocyte cultures.

Effects of Various Tumor Promoters on Expression of Cartilage Phenotypes in Rabbit Costal Chondrocytes in Culture1

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.

Stimulation of DNA synthesis in quiescent rabbit chondrocytes in culture by limited exposure to somatomedin-like growth factors.

Cartilage-derived factor (CDF), extracted from fetal bovine cartilage, and multiplication-stimulating activity (MSA) stimulated DNA synthesis in quiescent rabbit costal chondrocytes in culture under serum-free conditions. As described previously, when added in the presence of fibroblast growth factor (FGF) or epidermal growth factor (EGF) a somatomedin-like growth factor, CDF or MSA, synergistically stimulated DNA synthesis in the cultured chondrocytes. The present study showed that exposure of the cells to MSA or CDF for only the initial 5 h was sufficient for transmission of their full stimulatory effect. Furthermore, the limited exposure did not alter the time course of stimulation of DNA synthesis: [3H]thymidine incorporation into DNA began to increase after 16 h and reached a maximum after 24 h. In contrast to the somatomedin-like growth factors, FGF and EGF were required continuously in the culture medium during traverse of the entire G1 phase for stimulation of DNA synthesis, and the mitogenic effects of FGF and EGF in cultured chondrocytes were stronger than those of CDF and MSA. Synergistic stimulation of DNA synthesis by CDF or MSA in the presence of FGF or EGF could be observed as long as FGF or EGF was continuously present, even when CDF or MSA was withdrawn after the first 5 h of culture. These findings suggest that, in contrast to FGF and EGF, somatomedin-like growth factors affect an early distinct stage in the G1 phase of chondrocytes.