Faculty Profiles - TAKIGAWA Masaharu

Papers - TAKIGAWA Masaharu

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Establishment of an in vitro cell derived from human angiosarcoma Reviewed

T Hisa, S Taniguchi, K Kakudo, M Ichihashi, T Takashima, Y Kato, R Hayakawa, M Takigawa

BULLETIN DU CANCER 83 ( 7 ) 589 - 591 1996.7

Language：English Publishing type：Research paper (scientific journal) Publisher：EDITIONS SCIENTIFIQUES ELSEVIER

We have succeeded in the establishment of a human endothelial cell line derived from a human angiosarcoma. These cells grew as monolayers with a <<cobble stone>> morphology. The cells produced endothelin and showed Weibel-Palade bodies in their cytoplasm. These findings show that the cells have specific differenciated functions, and might be useful for both fundamental endothelial cell biology and biological investigation of angiosarcoma.

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Vitamin D inhibits endothelial cell migration Reviewed

T Hisa, S Taniguchi, D Tsuruta, Y Hirachi, S Ishizuka, M Takigawa

ARCHIVES OF DERMATOLOGICAL RESEARCH 288 ( 5-6 ) 262 - 263 1996.5

Mouse Meckel's cartilage chondrocytes evoke bone-like matrix and further transform into osteocyte-like cells in culture Reviewed

K Ishizeki, M Takigawa, T Nawa, F Suzuki

ANATOMICAL RECORD 245 ( 1 ) 25 - 35 1996.5

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

Background: we reported that when Meckel's cartilage was transplanted ectopically, chondrocytes transformed into osteocyte-like cells accompanying the extracellular calcified matrix. However, we could not determine whether the osteocyte-like cells were derived from host tissues or from Meckel's cartilage itself. Therefore, we examined whether the Meckel's cartilage chondrocytes, which have a retrogressive ultimate fate, are capable of inducing the observed calcification and further transform into osteocyte-like cells in culture.
Methods: Meckelian chondrocytes isolated enzymatically were plated at a low density and grown in alpha-MEM containing 10% FBS at 37 degrees C under 5% CO2 in air for up to 4 weeks.
Results: Chondrocytes were fibroblast-like cells early in culture, but gradually transformed from polygonal cells into typical chondrocytes showing metachromasia with toluidine blue staining. After an additional week of culture, the chondrocytes transformed from large to small round cells accompanying nodule formations. Small round cells multiple-layered actively, and showed more intense alkaline phosphatase (ALPase) activity. Immunostaining identified type II collagen in the extracellular matrix at 2 weeks of culture, and type I collagen and osteocalcin were later synthesized by round cells. von Kossa's reaction showed extensive precipitation of calcification throughout the flocculent materials. Ultrastructural analysis showed that the cells surrounded by calcified matrix strongly resembled osteocytes.
Conclusions: The present study suggested that the Meckel's cartilage chondrocytes can express the osteocyte-like phenotype in vitro during synthesis of bone-type marker proteins such as osteocalcin or type I collagen. (C) 1996 Wiley-Liss, Inc.

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Neurotrophin-3 increases the DNA-binding activities of several transcription factors in a mouse osteoblastic cell line Reviewed

E Iwata, T Nakanishi, N Ogawa, K Ohyama, T Murakami, M Takigawa

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1311 ( 2 ) 85 - 92 1996.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

In the mouse osteoblastic cell line MC3T3-E1, the signaling responses of several DNA-binding proteins induced by the treatment of neurotropin-3 were examined using electrophoretic mobility shift assay. Neurotrophin-3 increased binding activities in nuclear extracts of MC3T3-E1 cells to TPA-responsive element (TRE), cyclic AMP-responsive element (CRE) and serum-responsive element (SRE), but not binding activity in the nuclear extracts to c-Myc binding DNA element. Competition experiments revealed that the binding activity to TRE in the nuclear extracts of neurotrophin-3-treated MC3T3-E1 cells was entirely inhibited by the both unlabeled TRE and CRE probes. On the other hand, the binding activity to CRE was abolished by the unlabeled CRE probe but not by the same amount of unlabeled TRE probe. Moreover, immunodepletion/supershift assay using antibodies directed to Fos, Jun and CREB proteins, showed that the binding activities to TRE and CRE in the nuclear extracts were derived in part from these proteins.

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Novel FNR homologues identified in four representative oral facultative anaerobes: Capnocytophaga ochracea, Capnocytophaga sputigena, Haemophilus aphrophilus, and Actinobacillus actinomycetemcomitans Reviewed

T Hattori, K Takahashi, T Nakanishi, H Ohta, K Fukui, S Taniguchi, M Takigawa

FEMS MICROBIOLOGY LETTERS 137 ( 2-3 ) 213 - 220 1996.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Based upon DNA sequence data and positive immunochemical reactivity of expressed protein, novel homologues of the FNR family were identified in four representative oral facultative anaerobes: Capnocytophaga ochracea, Capnocytophaga sputigena, Haemophilus aphrophilus, and Actinobacillus actinomycetemcomitans. The similarity to E. coli FNR and to HlyX (itself 71% similar to E. coli FNR, while regulating expression of hemolysin operon in Actinobacillus pleuropneumoniae) was estimated from the deduced partial amino acid sequence to be, in the above order of tested species, 98, 98, 86, and 85%, and 75, 75, 88, and 88%, respectively. The phylogenetic relatedness indicates a rather closer link of HlyX to the FNR homologues from both pathogens, H. aphrophilus and A. actinomycetemcomitans. The possibility that the A. actinomycetemcomitans FNR homologue functions as a redox-sensing transcriptional factor to regulate, in addition to anaerobic respiration, microaerobic expression of the leukotoxin operon (Itx gene) is suggested.

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Expression of c-fos gene inhibits proteoglycan synthesis in transfected chondrocyte Reviewed

M Tsuji, S Funahashi, M Takigawa, M Seiki, K Fujii, T Yoshida

FEBS LETTERS 381 ( 3 ) 222 - 226 1996.3

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

The effect of expression of c-fos gene on proteoglycan synthesis, one of the important markers of cartilage metabolism, was examined by introducing the c-fos DIVA into HCS 2/8 chondrocytes. The [S-35]sulfate incorporation into proteoglycan was decreased in the c-fos transfectants expressing exogenous c-fos mRNA, when compared to a control transfectant. A significant increase in transcription of MMP-3 with the suppressed transcription of aggrecan and TIMP-1 were also observed in the c-fos transfectants, Moreover, analysis of the effect of AP-1 proteins on the collagenase and TIMP-1 promoters in gastric carcinoma KKLS cells revealed that c-Fos combined with any of the Jun-related proteins failed to stimulate the TIMP-1 promoter, though collagenase promoter was effectively activated by any Fos/Jun-related protein heterocomplex, These findings indicate that the c-fos expression may govern the cartilage metabolism and hence may play an important role in the pathogenesis of joint destruction in arthritis.

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Meckel's cartilage chondrocytes in organ culture synthesize bone-type proteins accompanying osteocytic phenotype expression Reviewed

K Ishizeki, M Takigawa, Y Harada, F Suzuki, T Nawa

ANATOMY AND EMBRYOLOGY 193 ( 1 ) 61 - 71 1996.1

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER VERLAG

We examined whether Meckel's cartilage of embryonic mice, 17 days in utero, undergo the cellular transformation into the osteocyte-like phenotype under organ culture conditions. Explants were grown by our original pithole method modified Trowell-type cultures for up to 4 weeks at 37 degrees C under 5% CO2 in air. Specimens were examined using histological procedures including immunostaining and electron microscopy. In addition, the effects of beta-glycerophosphate on matrix calcification were also examined in cultures with or without beta-glycerophosphate. Addition of beta-glycerophosphate induced calcification at a higher level, but calcium mineral deposition occurred regardless of the addition of beta-glycerophosphate to the culture medium. Light and electron microscopic analyses showed that freshly isolated chondrocytes prior to cell culture had typical hypertrophic morphology, but shortly after commencement of culture, they showed morphological modifications. The cells showing chondrocytic phenotypes became basophilic elliptical cells, and eventually transformed into flattened osteocyte-like cells. Bone-like features for cellular elements were characterized by spindle-shaped cells with elongated processes accompanying bone-specific thick-banded collagen fibrils. Immunostaining showed that at 2 weeks in culture, type I and type II collagens coexisted in the matrix, but subsequently type II collagen synthesis ceased and was replaced by type I collagen synthesis. Immunofluorescent labeling for osteocalcin was noted first in the peripheral cells by 1 week, but at 3 weeks this reaction spread to the central zone in explants. Alkaline phosphatase activity (ALPase) was expressed on the cells in the central zone prior to calcium mineral deposition as shown by von Kossa's reaction at 3 weeks in culture. These results showed that Meckel's cartilage chondrocytes in organ culture synthesize bone-type proteins accompanying osteocytic phenotype expression.

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Influence of hydrostatic pressure on expression of heat shock protein 70 and matrix synthesis in chondrocytes Reviewed

K Takahashi, T Kubo, Y Arai, Y Hirasawa, J Imanishi, K Kobayashi, M Takigawa

HIGH PRESSURE BIOSCIENCE AND BIOTECHNOLOGY 13 79 - 82 1996

Language：English Publishing type：Research paper (international conference proceedings) Publisher：ELSEVIER SCIENCE PUBL B V

To investigate the influence of hydrostatic pressure (HP) on the expressions of heat shock protein 70 (HSP70), and to know the relationship between HSP70 expression and matrix synthesis, we subjected chondrocyte-like cells to HP. HSP70 were enhanced after exposure to 10 to 50 MPa of HP. S-35 sulfate incorporation into the cultured cells increased after exposure to 5 MPa of HP and decreased after 50 MPa of HP.

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Specific serum antibodies against membranous proteins of a human immortal chondrocytic cell line (HCS-2/8) in rheumatoid arthritis and their relationship to the natural history of this disease Reviewed

Akira Sakawa, Yasutaka Yutani, Kentaro Inui, Akira Shimazu, Yoshiki Yamano, Akira Kinosita, Fujio Suzuki, Takako Hattori, Masaharu Takigawa

Journal of Bone and Mineral Metabolism 14 ( 3 ) 146 - 152 1996

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Japan

An immortal human chondrocytic cell line (HCS-2/8) derived from a chondrosarcoma was used as a source of human antigens to find humoral antibodies to cell surface proteins of human chondrocytes in sera from patients with rheumatoid arthritis (RA). Membrane fractions prepared from the cell line were subjected to Western blot analysis using RA and normal sera as probes. RA sera recognized about a dozen bands, but three of these bands, with molecular weights of 105 kDa, 68 kDa, and 47 kDa, were found to be specific for the RA sera (P &lt
0.05). These bands disappeared following V8 protease digestion, indicating that they were proteins. Among patients with 4 years or more of RA disease activity, reactivity against 105-kDa and 68-kDa proteins was relatively high in those whose joints showed a high degree of erosion. We suspect that levels of these two antibodies are suggestive of changes associated with the natural course of RA.

DOI： 10.1007/BF02239482

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Interleukin-1 induces Matrix degradation and release of basic fibroblast growth factor in clutured rabbit articular chondrocytes via nitric oxide production : A possible mechanism of pathological neovascularization in arthritis.

Tamura, T, Makanishi, T, Kimura, Y, Hattori, T, Sasaki, K, Norimatsu, H, Takahashi, K, Takigawa, M, Nitric oxide, mediates interleukin, induced matrix degradation, basic fibroblast growth factor release in cultured rabbit articular chondrocytes, A possible mechanism of, pathological neovascularization in arthritis

Endocrinology 137 ( 9 ) 3729 - 3737 1996

APOPTOSIS IN NORMAL SKIN

T HISA, S TANIGUCHI, H KOBAYASHI, Y SHIGENAGA, S NOMURA, M TAKIGAWA

ACTA DERMATO-VENEREOLOGICA 75 ( 5 ) 412 - 413 1995.9

PURIFICATION OF AN ANGIOGENESIS INHIBITOR FROM CULTURE-MEDIUM CONDITIONED BY A HUMAN CHONDROSARCOMA-DERIVED CHONDROCYTIC CELL-LINE, HCS-2/8 Reviewed

Y OHBA, Y GOTO, Y KIMURA, F SUZUKI, T HISA, K TAKAHASHI, M TAKIGAWA

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1245 ( 1 ) 1 - 8 1995.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

We previously reported that a novel human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, produced an anti-tumor angiogenesis factor and secreted it into the culture medium [Takigawa et al.: Anticancer Res., 10, 311-316, 1990]. In the present study, we purified the inhibitor by monitoring gelatinase inhibitory activity from the conditioned medium (CM) of HCS-2/8 cells. By a simple three-step procedure, gel filtration chromatography, ion-exchange chromatography, and reverse-phase HPLC, 200 mu g of the inhibitor was obtained from 6 liters of CM with 239-fold enrichment. Purified inhibitor, named hCHIAMP (human chondrocyte-derived inhibitor of angiogenesis and metalloproteinase activity), showed a single protein band with a molecular mass (M(r)) of 24 000 (24K) under reducing conditions and M(r) 22K under nonreducing conditions on SDS-PAGE. On reverse-zymography, purified hCHIAMP showed a single band of 22K M(r) under nonreducing conditions. Its NH2-terminal amino acid sequence determined up to the 11th amino acid residue was identical with that of the tissue inhibitor of metalloproteinases-2 (TIMP-2). On Western blotting, anti-human TIMP-2 antibody cross-reacted with hCHIAMP. hCHIAMP at a dose of as little as 0.45 mu g significantly inhibited angiogenesis in the yolk sac of chick embryos induced by 0.25 mu mol of spermine. Because HCS-2/8 is a clonal cell line, these findings clearly showed for the first time that chondrocytes themselves produce a potent inhibitor of angiogenesis, which is also an inhibitor of gelatinase. The findings also indicate that hCHIAMP is a TIMP-2-like molecule. Because the HCS-2/8 cells are an immortal cell line and of human origin, hCHIAMP could be useful for therapy of angiogenic diseases including solid tumors.

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DEMONSTRATION OF ENDOTHELIN (ET) RECEPTORS ON CULTURED RABBIT CHONDROCYTES AND STIMULATION OF DNA-SYNTHESIS AND CALCIUM INFLUX BY ET-1 VIA ITS RECEPTORS Reviewed

A KINOSHITA, T TAMURA, C AOKI, T NAKANISHI, S SOBUE, F SUZUKI, K TAKAHASHI, M TAKIGAWA

CELL BIOLOGY INTERNATIONAL 19 ( 8 ) 647 - 654 1995.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS (LONDON) LTD

Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with I-125-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 x 10(-10) M and 5 x 10(-9) M. The numbers of high- and low- affinity receptors were 1 x 10(4) and 2 x 10(5) per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-beta 1, IL-1 beta 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.

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Expression of trkC in a mouse osteoblastic cell line and its response to neurotrophin-3. Reviewed

Nakanishi T, Ohyama K, Aoki C, Kudo A, Hattori T, Takahashi K, Taniguchi S, Takigawa M

Biochemical and biophysical research communications 203 ( 2 ) 1268 - 1274 1994.9

CHANGES IN PARATHYROID-HORMONE RECEPTORS DURING CHONDROCYTE CYTODIFFERENTIATION Reviewed

M IWAMOTO, A JIKKO, H MURAKAMI, A SHIMAZU, K NAKASHIMA, M IWAMOTO, M TAKIGAWA, H BABA, F SUZUKI, Y KATO

JOURNAL OF BIOLOGICAL CHEMISTRY 269 ( 25 ) 17245 - 17251 1994.6

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

The purpose of this study was to investigate the relationship between changes in parathyroid hormone (PTH) receptor levels and chondrocyte maturation during endochondral ossification. Chondrocytes were isolated from the growth plate of rabbit ribs and maintained in the presence of 10% serum in mass cultures. Treatment with PTH-(1-84) and a PTH-(1-34) fragment suppressed the increases in alkaline phosphatase activity and in type X collagen and 1 alpha,25-dihydroxyvitamin D-3 receptor levels and abolished Ca-45 incorporation into mineral, all of which occurred in parallel untreated cultures in the hypertrophic (terminal) stage. These effects of PTH were observed at low concentrations (10(-10) to 10(-9) M) and within 24-48 h of treatment. PTH-(1-84) and PTH-(1-34) also increased [S-36]sulfate incorporation into newly synthesized proteoglycans. In contrast, the middle and carboxyl-terminal fragments of PTH tested had little effect on proteoglycan synthesis or terminal differentiation. The binding of I-125-PTH-(1-34) to cells in the growth plate was greater than that to cells in liver, skin, muscle, brain, or kidney. When the correlation between binding levels and stage of maturation was examined, we found that I-125-PTH-(134) binding to its 72-kDa receptor was low in resting and proliferating chondrocytes, increased 10 fold in matrix-forming chondrocytes, and thereafter decreased in hypertrophic chondrocytes both in vitro and in situ. Scatchard analysis revealed that the changes in PTH binding were due to changes in the number, and not in the affinity, of the receptor. The changes in PTH-(1-34) binding paralleled those in [S-35]sulfate incorporation into proteoglycans. These findings suggest that stage dependent increases in PTH/PTH-related peptide receptor levels localize the hormone stimulation of proteoglycan synthesis and inhibition of precocious hypertrophy in the matrix-forming zone of growth plates.

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PROTOONCOGENE EXPRESSION IN A HUMAN CHONDROSARCOMA CELL-LINE - HCS-2/8 Reviewed

J ZHU, HO PAN, F SUZUKI, M TAKIGAWA

JAPANESE JOURNAL OF CANCER RESEARCH 85 ( 4 ) 364 - 371 1994.4

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE CANCER ASSOCIATION

HCS-2/8 is a stable human chondrosarcoma cell line with many chondrocytic characteristics and has the capacity to form chondrosarcomas in nude mice. The cells display both biochemically and morphologically definable changes in sparse, subconfluent, confluent and over-confluent phases of in vitro culture. Such features of HCS-2/8 cells may reflect the processes of both proliferation and differentiation of chondrocytes in vivo. We examined the correlations of these changes of HCS-2/8 cells with their transcript levels of 21 proto-oncogenes by Northern analysis. We found no detectable transcripts of 9 proto-oncogenes (c-sis, c-met, c-src, c-lyn, c=fgr, c-ros, c-pim, Blym and N-myc), but detected transcripts of 12 other proto-oncogenes (int-2, erbB, c-abl, c-raf-l, c=fyn, K-ras, H-ras, c-mos, c-myc, c-myb, c-fos, and c-jun). In the over-confluent phase, the levels of c=fos and c-raf-1 were increased several dozen times and about 5 times, respectively, while the level of c-abl was about 1/5th of that in the sparse, subconfluent and confluent phases of culture. The level of int 2 increased about 10-fold in the confluent and overconfluent phases of in vitro culture. The transcript levels of c-mos and K-ras were high in the sparse phase, low in the subconfluent and confluent phases and high in the over-confluent phase. The levels of the other 6 proto-oncogenes in HCS-2/8 cells were constant in all phases of in vitro culture.

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CONFORMATION DEPENDENCE OF INTEGRIN-TYPE-II COLLAGEN-BINDING - INABILITY OF COLLAGEN PEPTIDES TO SUPPORT ALPHA(2)BETA(1) BINDING, AND MEDIATION OF ADHESION TO DENATURED COLLAGEN BY A NOVEL ALPHA(5)BETA(1)-FIBRONECTIN BRIDGE Reviewed

DS TUCKWELL, S AYAD, ME GRANT, M TAKIGAWA, MJ HUMPHRIES

JOURNAL OF CELL SCIENCE 107 993 - 1005 1994.4

Language：English Publishing type：Research paper (scientific journal) Publisher：COMPANY OF BIOLOGISTS LTD

The mechanism of interaction of chondrocytic cells with cartilage-specific type II collagen has been examined using HCS-2/8 human chondrosarcoma cells as a model system. By the criteria of specific collagen secretion and integrin expression profile, HCS-2/8 have a similar differentiated phenotype to normal chondrocytes and are therefore a good model system. HCS-2/8 cells were able to attach and spread on both native and heat-denatured pepsinised type II collagen, and assays using denatured cyanogen bromide fragments apparently localised the major cell binding site to the CB10 fragment. However, when they were used as soluble inhibitors, cyanogen bromide fragments were found to block adhesion to denatured collagen, but had no effect on either attachment or spreading on the native molecule. The inability of cyanogen bromide fragments to reproduce the cell binding site of native collagen demonstrated a strict dependence on collagen conformation. This was also reflected in the receptors that were employed by HCS-2/8 cells for binding to type II collagen: binding to native collagen was mediated by the integrin alpha(2) beta(1) while binding to denatured collagen was mediated by a novel alpha(5) beta(1)-fibronectin bridge. The identification of this bridge adds to the mechanisms by which cells can bind to denatured collagens. The previously characterised KDGEA active site peptide from type I collagen was found to be inactive as an inhibitor of type II collagen-mediated adhesion. The implications of these findings for the strategies used to identify adhesive active sites within collagens are discussed. In particular, these data suggest that, unlike other integrin ligands, a synthetic peptide-based approach is not suitable for the identification of collagen active sites.

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HISTATIN AS A SYNERGISTIC STIMULATOR WITH EPIDERMAL GROWTH-FACTOR OF RABBIT CHONDROCYTE PROLIFERATION Reviewed

Y MURAKAMI, H NAGATA, S SHIZUKUISHI, K NAKASHIMA, T OKAWA, M TAKIGAWA, A TSUNEMITSU

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 198 ( 1 ) 274 - 280 1994.1

DEMONSTRATION OF RECEPTORS FOR EPIDERMAL GROWTH-FACTOR ON CULTURED RABBIT CHONDROCYTES AND REGULATION OF THEIR EXPRESSION BY VARIOUS GROWTH AND DIFFERENTIATION FACTORS Reviewed

A KINOSHITA, M TAKIGAWA, F SUZUKI

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 183 ( 1 ) 14 - 20 1992.2

EFFECTS OF VARIOUS GROWTH AND DIFFERENTIATION FACTORS ON EXPRESSION OF PARATHYROID-HORMONE RECEPTORS ON RABBIT COSTAL CHONDROCYTES IN CULTURE Reviewed

M TAKIGAWA, A KINOSHITA, M ENOMOTO, A ASADA, F SUZUKI

ENDOCRINOLOGY 129 ( 2 ) 868 - 876 1991.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ENDOCRINE SOC

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 [I-125]-[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.

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