Effects of differentiation and transforming growth factor β1 on PTH/PTHrP receptor mRNA levels in MC3T3-E1 cells

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Abstract
TGFβ has opposing effects on osteoblasts which are thought to be differentiation stage dependent; however, little is known concerning the effects of TGFβ on osteoblastic characteristics at different stages of maturation. The purpose of this study was to characterize the pattern of mRNA expression for the PTH/PTHrP receptor during normal osteoblastic differentiation in vitro, and evaluate the effects of TGFβ1 on PTH/PTHrP receptor and osteocalcin (OCN) steady-state mRNA at different stages of osteoblastic differentiation. MC3T3-E1 preosteoblasts were plated at low density and induced to differentiate with ascorbic acid and β-glycerophosphate. The first group served as a vehicle control and the remaining five groups received a single 48 h TGFβ1 (3.0 ng/ml)-pulse staggered on a weekly basis for 30 days. Cell cultures were harvested weekly and evaluated for: steady-state PTH/PTHrP receptor and OCN mRNA levels via northern analysis, calcium and phosphorous levels, bone nodules via Von Kossa staining, alkaline phosphatase enzyme levels, and hydroxyproline levels. Group 1 (control) samples followed a normal pattern of proliferation, extracellular matrix deposition, and mineralization. PTH/PTHrP receptor and OCN mRNA expression increased 8-fold and 10-fold respectively, over the collection periods. When TGFβ1 was administered during the first 48 h period (group 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-regulation of PTH/PTHrP receptor and OCN expression when TGFβ1 was administered later during osteoblast differentiation (groups 3–6); however, these effects were not persistent. In addition there was a total lack of bone nodule formation in group two cultures, whereas groups 3–6 had increasing bone nodule formation because the TGFβ1 was administered later in the culture period. These studies indicate that expression of the PTH/PTHrP receptor increases with osteoblastic differentiation and suggest that TGFβ1 inhibits osteoblastic maturation with more persistent effects found in less differentiated osteoblastic cells.
Funding Information
  • National Institutes of Health (DK46919 (LKM), DK35317, DE10412 (RTF))
  • American Cancer Society
  • American Federation for Aging Research

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