EFFECTS OF DIFFERENTIATION AND TRANSFORMING GROWTH-FACTOR BETA(1) ON PTH PTHRP RECEPTOR MESSENGER-RNA LEVELS IN MC3T3-E1 CELLS/

TGF beta has opposing effects on osteoblasts which are thought to be d ifferentiation stage dependent; however, little is known concerning th e effects of TGF beta on osteoblastic characteristics at different sta ges of maturation, The purpose of this study was to characterize the p attern of mRNA expression for the PTH/PTHrP receptor during normal ost eoblastic differentiation in vitro, and evaluate the effects of TGF be ta(1) on PTH/PTHrP receptor and osteocalcin (OCN) steady-state mRNA at different stages of osteoblastic differentiation, MC3T3-E1 preosteobl asts were plated at low density and induced to differentiate with asco rbic acid and beta-glycerophosphate, The first group served as a vehic le control and the remaining five groups received a single 48 h TGF be ta(1) (3.0 ng/ml)-pulse staggered on a weekly basis for 30 days, Cell cultures were harvested weekly and evaluated for: steady-state PTH/PTH rP receptor and OCN mRNA levels via northern analysis, calcium and pho sphorous levels, bone nodules via Von Kossa staining, alkaline phospha tase enzyme levels, and hydroxyproline levels, Group 1 (control) sampl es followed a normal pattern of proliferation, extracellular matrix de position, and mineralization. PTH/PTHrP receptor and OCN mRNA expressi on increased 8-fold and 10-fold respectively, over the collection peri ods, When TGF beta(1) was administered during the first 48 h period (g roup 2) while cells were rapidly proliferating, there was a persistent inhibition of PTH/PTHrP receptor expression and a striking reduction in OCN mRNA expression at all time points. There was also a down-regul ation of PTH/PTHrP receptor and OCN expression when TGF beta(1) was ad ministered later during osteoblast differentiation (groups 3-6); howev er, these effects were not persistent, In addition there was a total l ack of bone nodule formation in group two cultures, whereas groups 3-6 had increasing bone nodule formation because the TGF beta(1) was admi nistered later in the culture period, These studies indicate that expr ession of the PTH/PTHrP receptor increases with osteoblastic different iation and suggest that TGF beta(1) inhibits osteoblastic maturation w ith more persistent effects found in less differentiated osteoblastic cells.