Motomi Enomoto

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 [Nle8,-Nle18, Tyr34] 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.

Cartilage-derived anti-tumor factor (CATF) inhibits the proliferation of endothelial cells in culture.

Previous studies showed that a cartilage-derived anti-tumor factor (CATF), which was extracted from bovine cartilage with 1 M guanidine hydrochloride, inhibited the growth of solid sarcoma 180, but not the growth of ascites tumors of sarcoma 180 [Suzuki, F. et al. Jpn. J. Bone Metab., 2, 231-235 (1984)]. This study showed that CATF inhibited the proliferation and DNA synthesis of endothelial cells isolated from bovine pulmonary artery, but not the proliferations of sarcoma 180, L1210 and L cells. These results suggest that CATF has anti-angiogenic properties, thereby inhibiting the growth of solid tumors.

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

SummaryTumor necrosis factor α (TNFα) 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 TNFα produced putative “cartilage-specific” proteoglycans identified by density gradient centrifugation under dissociative conditions. Although TNFα decreased the synthesis of the proteoglycans, it did not change their monomeric size, which is a marker of cartilage phenotypes. Moreover, TNFα did not affect the responsiveness to parathyroid hormone, insulin-like growth factor I, or transforming growth factor β, which is known to stimulate GAG synthesis in cultured chondrocytes. TNFα 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 TNFα 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, TNFα promotes the proliferation of chondrocytes while suppressing their further differentiation at the stage of synthesis of cartilage-specific proteoglycans.

Establishment from mouse growth cartilage of clonal cell lines with responsiveness to parathyroid hormone, alkaline phosphatase activity, and ability to produce an endothelial cell growth inhibitor

SummaryThree clonal cell lines with differences in responsiveness to parathyroid hormone (PTH), alkaline phosphatase activity, and ability to produce an endothelial cell growth inhibitor(s) during more than 3 years, more than 58 passages, in culture were established from growth cartilage (GC) of mouse ribs. In sparse, cultures the three clonal cell lines, MGC/T1.4, MGC/T1.17, and MGC/T1.18, all showed fibroblast-like morphology. However, as they became confluent, MGC/T1.4 cells became polygonal and then multilayered. MGC/T1.18 cells also became polygonal, but showed contact inhibition. MGC/T1.17 cells remained fibroblastic in confluent cultures and formed nodules when cultured for more than 7 days after they became confluent. These nodules calcified in the presence of β-glycerophosphate. Glycosaminoglycan (GAG) synthesis in the parent uncloned line, MGC/T1 cells, at early passages was about 50–75% of that of primary cultures of mouse GC cells. The GAG syntheses in the three clonal lines were much lower than that of primary cultures of GC cells Moreover, the sizes of proteoglycan monomers synthesized by these cells were not the same as that of cartilage-specific proteoglycan. The three clonal lines mainly synthesized type I collagen. PTH increased the intracellular cyclic AMP level in MGC/T1, MGC/T1.4, T1.17, and T1.18 cells: their maximal levels, observed after 2 minutes, were, respectively, about 160, 150, 70, and 200 times that of controls. The activity of alkaline phosphatase in MGC/T1.17 cells was higher than that in primary cultures of mouse GC cells, whereas those in MGC/T1 and T1.4 cells were comparable with that of GC cells, and that in MGC/T1.18 was lower. The three clonal lines, and especially MGC/T1.4, secreted a heat-stable, nondializable, growth inhibitor(s) of endothelial cells into the culture medium. Because of their different properties, these cell lines should be useful for studies on endochondral ossification, the actions of PTH on skeletal cells, and anti-angiogenesis factors.

Cartilage-derived anti-tumor factor (CATF) - Partial purification and correlation of inhibitory activity against tumor growth with anti-angiogenic activity

Cartilage-derived anti-tumor factor (CATF) inhibits the proliferation and DNA synthesis of bovine pulmonary artery endothelial (BPAE) cells in culture (Takigawa, M.et al. Cell. Biol. Inn. Rep., 9, 619–625, 1985). In the present study, we partially purified CATF by monitoring inhibition of DNA synthesis in BPAE cells and tested the effects of the purified materials on the growth of solid tumors and tumor-induced angiogenesis. Crude CATF (CATF20–300k), the fraction of 20 k to 300 k daltons, separated by ultrafiltration was further separated into three fractions by ultrafiltration. The fraction of 100 k to 300 k daltons (CATF100–300k) caused slightly more inhibition than CATF20–300k of DNA synthesis in BPAE cells. On the other hand, the fraction of 20 k to 50 k daltons had only a slight effect, and the fraction of 50 k to 100 k had even less effect on DNA synthesis in BPAE cells. CATF100–300k caused slightly more inhibition than CATF20–300k of the growth of solid tumors of B16 melanoma, while the fraction of 20 k to 100 k daltons did not inhibit the growth of tumors at all. CATF100–300k also inhibited B16 melanoma-induced angiogenesis in chick embryo chorioallantoic membranes (CAM), whereas the fraction of 20 k to 100 k daltons had little effect on the angiogenesis. CATF100–300k was further purified by DEAE-Sepharose CL-6B chromatography. The main peak with activity on DNA synthesis in BPAE cells was eluted with 0.3 to 0.35 M NaCl at pH 8.0. The activity of this peak on DNA synthesis in BPAE cells was about 70 fold that of CATF100–300k. The purified CATF also inhibited the growth of B16 melanoma and B16 melanoma-induced angiogenesis in CAM. On the other hand, the inactive fraction on DNA synthesis in BPAE cells obtained by DEAE-Sepharose chromatography was also inactive in inhibiting the growth of B16 melanoma and B16 melanoma-induced angiogenesis in CAM. These findings strongly suggest that CATF is an anionic macromolecule(s) and has anti-angiogenic activity, thereby inhibiting the growth of solid tumors.

Parathyroid Hormone-Responsive Clonal Cell Lines from Mouse Growth Cartilage

The major target organs for parathyroid hormone (PTH) are the bone and kidney. PTH regulates or influences metabolism of calcium and phosphate in these tissues and its action is presumably mediated by 3’, 5’-cyclic AMP (cAMP) (Rosenblatt, 1982). For investigation of the role of cAMP in mediating the action of PTH, several PTH-responsive clonal cell lines have been established from bone (Aubin et al., 1982; Nakatani et al., 1984) and osteosarcoma (Majeska et al., 1980; Partridge et al., 1983). However, no PTH-responsive cell line has yet been established from cartilage, which is another target organ for PTH (Takigawa et al., 1979; 1980; Kawashima et al., 1980). We have shown that PTH increases the intracellular cAMP level in rabbit costal chondrocytes grown in primary culture (Takigawa et al., 1981) but that the cells lose the abilities to proliferate and to respond to PTH during successive passages (Takigawa et al., 1987). This paper reports the establishment from mouse growth cartilage (GC) of a cell line, and derivative clonal lines that respond to PTH.