Cyclin D1 as a Target for the Proliferative Effects of PTH and PTHrP in Early Osteoblastic Cells

PTHrP induced a proliferative cyclin D1 activation in low-density osteoblastic cells. The process was PKA and MAPK dependent and involved both AP-1 and CRE sites. In ectopic ossicles generated from implanted bone marrow stromal cells, PTH upregulated cyclin D1 after acute or intermittent anabolic treatment. These data suggest a positive role of PTH and PTHrP in the cell cycle of early osteoblasts. Introduction: The mechanisms underlying the actions of PTH and its related protein (PTHrP) in osteoblast proliferation, differentiation, and bone remodeling remain unclear. The action of PTH or PTHrP on the cell cycle during osteoblast proliferation was studied. Materials and Methods: Mouse calvarial MC3T3-E1 clone 4 cells were synchronized by serum starvation and induced with 100 nM PTHrP for 2–24 h under defined low serum conditions. Western blot, real-time PCR, EMSAs, and promoter/luciferase assays were performed to evaluate cyclin D1 expression. Pharmacological inhibitors were used to determine the relevant signaling pathways. Ectopic ossicles generated from implanted bone marrow stromal cells were treated with acute (a single 8- or 12-h injection) or intermittent anabolic PTH treatment for 7 days, and RNA and histologic analysis were performed. Results: PTHrP upregulated cyclin D1 and CDK1 and decreased p27 expression. Cyclin D1 promoter/luciferase assays showed that the PTHrP regulation involved both activator protein-1 (AP-1) and cyclic AMP response element binding protein (CRE) sites. AP-1 and CRE double mutants completely abolished the PTHrP effect of cyclin D1 transcription. Upregulation of cyclin D1 was found to be protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) dependent in proliferating MC3T3-E1 cells. In vivo expression of cyclin D1 in ectopic ossicles was upregulated after a single 12-h PTH injection or intermittent anabolic PTH treatment for 7 days in early developing ossicles. Conclusions: These data indicate that PTH and PTHrP induce cyclin D1 expression in early osteoblastic cells and their action is developmental stage specific.