Abstract
Parathyroid hormone (PTH) plays a major role in bone remodeling and has the ability to increase bone mass if administered daily. In vitro, PTH inhibits the growth of osteoblastic cell lines, arresting them in G(1) phase. Here, we demonstrate that PTH regulates the expression of at least three genes to achieve the following: inducing expression of MAPK phosphatase 1 (MKP-1) and p21(Cip1) and decreasing expression of cyclin D1 at both mRNA and protein levels. The induction of MKP-1 causes the dephosphorylation of extracellular signal-regulated kinase and therefore the decrease in cyclin D1. Overexpression of MKP-1 arrests UMR cells in G(1) phase. The mechanisms involved in PTH regulation of these genes were studied. Most importantly, PTH administration produces similar effects on expression of these genes in rat femoral metaphyseal primary spongiosa. Analyses of p21(Cip1) expression levels in bone indicate that repeated daily PTH injections make the osteoblast more sensitive to successive PTH treatments, and this might be an important feature for the anabolic functions of PTH. In summary, our data suggest that one mechanism for PTH to exert its anabolic effect is to arrest the cell cycle progression of the osteoblast and hence increase its differentiation.
Highlights
The adult human skeleton is continuously resorbed and renewed by the actions of osteoclasts and osteoblasts
Cells at different stages were treated with rPTH-(1–34) (10Ϫ8 M) for different time periods, and the level of Mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) mRNA was measured by real time RT-PCR
There are contradictory reports regarding whether Parathyroid hormone (PTH) stimulates or inhibits osteoblast cell proliferation
Summary
Chemicals—Synthetic human PTH-(1–38), human PTH-(1–34), human PTH-(1–31), and human PTH-(13–34) were purchased from Bachem (Torrance, CA). Cells were seeded in 100-mm dishes at a density of 1.2 ϫ 104 cells/cm overnight and switched to serum-free minimal essential medium for 1 day before the addition of PTH. Rat primary calvarial osteoblastic cells were obtained and cultured as described previously [22]. Before PTH treatment, these cells were serum starved for 1 day. In Vivo Injection of PTH into Rats—Sprague-Dawley rats (4-week-old male, about 75 g, and 3-month old female, about 250 g) were purchased from Hilltop (Scottdale, PA). Each analysis was performed three to four times with independent sets of cells or tissues from PTH treatment to RT-PCR to obtain mean values and S.E. shown in the figures. For femoral samples and primary osteoblastic cells, glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. Immunoblotting—Preparation of cell lysates and Western blot analyses were performed as described previously [24]
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