PTH/PTHrP receptor is temporally regulated during osteoblast differentiation and is associated with collagen synthesis.
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.
McCauley
L K
LK
University of Michigan, School of Dentistry, Department of Periodontics/Prevention/Geriatrics, Ann Arbor 48109-1078, USA.
Koh
A J
AJ
Beecher
C A
CA
Cui
Y
Y
Rosol
T J
TJ
Franceschi
R T
RT
D016475:3T3 Cells; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001667:Binding, Competitive; D002454:Cell Differentiation; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis*; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D005109:Extracellular Matrix / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D005994:Glycerophosphates / Q000494:pharmacology; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D005994:Glycerophosphates; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D012333:RNA, Messenger; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; C002195:3,4-dehydroproline; D003094:Collagen; D004247:DNA; D011392:Proline; D000242:Cyclic AMP; D001205:Ascorbic Acid; C031463:beta-glycerophosphoric acid