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 ac tivity, and its dependence on matrix synthesis was determined using MC 3T3-E1 preosteoblast-like cells and primary rat calvarial cells in vit ro. Osteoblastic cells were induced to differentiate and form minerali zed nodules with the addition of ascorbic acid and beta-glycerophospha te, and samples were collected from 0-26 days of culture. DNA levels a s determined by fluorometric analysis increased 12- and 17-fold during the collection period for both MC3T3-E1 and primary calvarial cells r espectively. Steady state mRNA levels for the PTH/PTHrP receptor as de termined by northern blot analysis, were initially low for both cell t ypes, peaked at day 4 and 5 for MC3T3-E1 and primary calvarial cells r espectively, and declined thereafter. Competition binding curves were performed during differentiation using I-125-PTHrP. The numbers of rec eptors per mu g DNA were greatest at days 3 and 5 for MC3T3-E1 and pri mary calvarial cells respectively. The biologic activity of the recept or was evaluated by stimulating the cells with 10 nM PTHrP and determi ning cAMP levels via a binding protein assay. The PTHrP-stimulated cAM P levels increased 5-fold to peak values at day 5 for MC3T3-E1 cells a nd 6-fold to peak values at day 4 for the primary calvarial cells. Asc orbic acid was required for maximal development of a PTH-dependent cAM P response since ascorbic acid-treated MC3T3-E1 cells had twice the PT H-stimulated cAMP levels as non-treated cells. When the collagen synth esis inhibitor 3,4-dehydroproline was administered to MC3T3-E1 culture s prior to differentiation, there was a subsequent diminution of the P TH/PTHrP receptor mRNA gene expression and numbers of receptors per ce ll; however, if administered after the initiation of matrix synthesis there was no reduction in PTH/PTHrP receptor mRNA. These findings indi cate that the PTH/PTHrP receptor is associated temporally at the level of mRNA, protein, and biologic activity, with a differentiating, matr ix-producing osteoblastic cell in vitro. (C) 1996 Wiley-Liss, Inc.