PTH/PTHrP receptor is temporally regulated during osteoblast differentiation and is associated with collagen synthesis
| Autor: | L K, McCauley, A J, Koh, C A, Beecher, Y, Cui, T J, Rosol, R T, Franceschi |
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| Rok vydání: | 1996 |
| Předmět: |
Osteoblasts
Proline Parathyroid Hormone-Related Protein Proteins Cell Differentiation 3T3 Cells Ascorbic Acid DNA Binding Competitive Extracellular Matrix Rats Mice Gene Expression Regulation Parathyroid Hormone Glycerophosphates Cyclic AMP Animals Receptors Parathyroid Hormone Collagen RNA Messenger Cells Cultured Receptor Parathyroid Hormone Type 1 |
| Zdroj: | Journal of cellular biochemistry. 61(4) |
| ISSN: | 0730-2312 |
| Popis: | The temporal sequence of PTH/PTHrP receptor mRNA, binding, biologic activity, and its dependence on matrix synthesis was determined using MC3T3-E1 preosteoblast-like cells and primary rat calvarial cells in vitro. Osteoblastic cells were induced to differentiate and form mineralized nodules with the addition of ascorbic acid and beta-glycerophosphate, and samples were collected from 0-26 days of culture. DNA levels as determined by fluorometric analysis increased 12- and 17-fold during the collection period for both MC3T3-E1 and primary calvarial cells respectively. Steady state mRNA levels for the PTH/PTHrP receptor as determined by northern blot analysis, were initially low for both cell types, peaked at day 4 and 5 for MC3T3-E1 and primary calvarial cells respectively, and declined thereafter. Competition binding curves were performed during differentiation using 125I-PTHrP. The numbers of receptors per microgram DNA were greatest at days 3 and 5 for MC3T3-E1 and primary calvarial cells respectively. The biologic activity of the receptor was evaluated by stimulating the cells with 10 nM PTHrP and determining cAMP levels via a binding protein assay. The PTHrP-stimulated cAMP levels increased 5-fold to peak values at day 5 for MC3T3-E1! cells and 6-fold to peak values at day 4 for the primary calvarial cells. Ascorbic acid was required for maximal development of a PTH-dependent cAMP response since ascorbic acid-treated MC3T3-E1 cells had twice the PTH-stimulated cAMP levels as non-treated cells. When the collagen synthesis inhibitor 3,4-dehydroproline was administered to MC3T3-E1 cultures prior to differentiation, there was a subsequent diminution of the PTH/PTHrP receptor mRNA gene expression and numbers of receptors per cell; however, if administered after the initiation of matrix synthesis there was no reduction in PTH/PTHrP receptor mRNA. These findings indicate that the PTH/PTHrP receptor is associated temporally at the level of mRNA, protein, and biologic activity, with a differentiating, matrix-producing osteoblastic cell in vitro. |
| Databáze: | OpenAIRE |
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