1990 Fiscal Year Final Research Report Summary
Purification of Receptors to Parathyroid Hormone from Cultured Chondrocytes and the Mechanism of its Signal Transduction
| Project/Area Number |
01571016
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional basic dentistry
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| Research Institution | Osaka University |
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Principal Investigator |
ASADA Akira Osaka University Faculty of Dentistry, Dep. of Biochemistry, Assitant Professor, 歯学部, 講師 (50028734)
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| Co-Investigator(Kenkyū-buntansha) |
TAKIGAWA Masaharu Osaka University Faculty of Dentistry, Dep. of Biochemistry, Assitant Professor, 歯学部, 講師 (20112063)
SUSZUKI Fujio Osaka University Faculty of Dentistry, Dep of Biochemistry, Professor, 歯学部, 教授 (40028717)
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| Project Period (FY) |
1989 – 1990
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| Keywords | Parathyroid hormone / chondrocytes / cultured cells / receptors / established cell line / cell differentiation / growth factors / differentiation factors |
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| Research Abstract |
1) Parathyroid Hormone (PTH) receptors on cultured rabbit costal chondrocytes were demonstrated using HPLC-purified, radio-iodinated [Nle^<8,>-Nle^<18>, Tyr^<34>]bovine PTH- (1-34) amide. Both Growth Cartilage (GC) cells and resting cartilage 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. The number of receptors per cell was approximately 40,000-90,000 on GC cells. After crosslinking the receptors on these cells with the radioligand, One, major^<125>l-labeled band of 76 kDa was separated by SDS-PAGE.
2) Treatment with retinoic acid, epidermal growth factor and fibroblast growth factor of rabbit costal chondrocytes decreased both proteoglycan synthesis and the number PTH receptors while treatment with insulin-like growth factor-I, dibutyryl cyclic AMP and transforming growth factor-beta resulted in increases in proteoglycan synthesis as well as in the number of PTH receptors. However, their affinity did no
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t change by these treatments. The PTH-stimulated cyclic AMP level in chondrocytes pretreated with retinoic acid, epidermal growth factor and fibroblast growth factor was lower than that in control cells while the PTH-stimulated cvclic AMP level in chondrocytes pretreated with insulin-like growth factor-I and transforming growth factor-beta was higher tnan that in control cells. These observations suggest that the increase in the number of PTH receptors on chondrocytes is closely related with expression of the differentiated phenotype of chondrocytes and that the number of the receptors is a good marker of the differentiated phenotype of chondrocytes.
3) Three clonal cell lines with responsiveness to PTH during more than 3 years, more than 58 passages in culture were established from growth cartilage of mouse ribs. Among the three clonal cell lines, a cell line, named MGC/T1.18, showed the highest responsiveness to PTH : The hormone increased intacellular cAMP level in the cells to 200 times that of control. Less
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