McCauley L K, Koh A J, Beecher C A, Cui Y, Decker J D, Franceschi R T
Department of Periodontics/Prevention/Geriatrics, University of Michigan School of Dentistry, Ann Arbor, USA.
J Bone Miner Res. 1995 Aug;10(8):1243-55. doi: 10.1002/jbmr.5650100815.
TGF beta has opposing effects on osteoblasts which are thought to be differentiation stage dependent; however, little is known concerning the effects of TGF beta 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 TGF beta 1 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 beta-glycerophosphate. The first group served as a vehicle control and the remaining five groups received a single 48 h TGF beta 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/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 TGF beta 1 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 TGF beta 1 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 TGF beta 1 was administered later in the culture period. These studies indicate that expression of the PTH/PTHrP receptor increases with osteoblastic differentiation and suggest that TGF beta 1 inhibits osteoblastic maturation with more persistent effects found in less differentiated osteoblastic cells.
转化生长因子β（TGF-β）对成骨细胞具有相反的作用，这种作用被认为取决于分化阶段；然而，关于TGF-β对不同成熟阶段成骨细胞特性的影响却知之甚少。本研究的目的是描述甲状旁腺激素/甲状旁腺激素相关蛋白（PTH/PTHrP）受体在体外正常成骨细胞分化过程中的mRNA表达模式，并评估TGF-β1在成骨细胞分化不同阶段对PTH/PTHrP受体和骨钙素（OCN）稳态mRNA的影响。将MC3T3-E1前成骨细胞低密度接种，并用抗坏血酸和β-甘油磷酸诱导分化。第一组作为溶剂对照，其余五组每周间隔48小时接受一次TGF-β1（3.0 ng/ml）脉冲处理，持续30天。每周收集细胞培养物，并进行以下评估：通过Northern分析检测PTH/PTHrP受体和OCN mRNA的稳态水平、钙和磷水平、通过Von Kossa染色检测骨结节、碱性磷酸酶水平和羟脯氨酸水平。第1组（对照）样本呈现正常的增殖、细胞外基质沉积和矿化模式。在收集期内，PTH/PTHrP受体和OCN mRNA表达分别增加了8倍和10倍。当在细胞快速增殖的最初48小时内给予TGF-β1（第2组）时，在所有时间点PTH/PTHrP受体表达均受到持续抑制，OCN mRNA表达显著降低。当在成骨细胞分化后期给予TGF-β1时（第3 - 6组），PTH/PTHrP受体和OCN表达也下调；然而，这些影响并不持久。此外，第2组培养物中完全没有骨结节形成，而第3 - 6组由于在培养后期给予TGF-β1，骨结节形成逐渐增加。这些研究表明，PTH/PTHrP受体的表达随着成骨细胞分化而增加，并提示TGF-β1抑制成骨细胞成熟，在分化程度较低的成骨细胞中发现其作用更持久。
