The mitogenic effect of parathyroid hormone is associated with E2F-dependent activation of cyclin-dependent kinase 1 (cdc2) in osteoblast precursors.
Injections of parathyroid hormone (PTH) have been reported to stimulate skeletal accretion through increased bone formation in several species, and osteoblast proliferation is a critical component of bone formation. However, the biological mechanisms of PTH-stimulated bone cell proliferation are largely unknown. In this study, we demonstrated that PTH stimulates proliferation of the osteoblast precursor cell line, TE-85, in association with increasing cdc2 protein levels and its kinase activity. cdc2 antisense oligonucleotides blocked PTH-induced DNA synthesis and cell cycle progression. Analysis of the time course of PTH-stimulated cdc2 message levels demonstrated that cdc2 mRNA levels were increased 1.5- to 4-fold between 3-18 h following release from cell synchronization. Transfections of TE-85 cells with a series of cdc2 promoter-luciferase deletion constructs revealed PTH stimulation of the cdc2 promoter. Promoter constructs containing a mutation in the E2F binding site were not stimulated by PTH. Gel mobility shift assays demonstrated increased free E2F levels in TE-85 nuclear extracts in response to PTH. Furthermore, the ratios of hyperphosphorylated to hypophosphorylated forms of Rb protein were increased by PTH treatment. These results demonstrate that PTH-stimulated cdc2 expression was associated with TE-85 cell proliferation and that the mechanism of stimulating cdc2 gene expression involved increasing the levels of free E2F.
Onishi
T
T
Renal Division, Barnes-Jewish Hospital/Washington University, St. Louis, Missouri 63110, U.S.A.
Zhang
W
W
Cao
X
X
Hruska
K
K
D016203:CDC2 Protein Kinase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D050684:E2F Transcription Factors; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008934:Mitogens / Q000494:pharmacology*; D009154:Mutation / Q000187:drug effects / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects*; D012516:Osteosarcoma; D010281:Parathyroid Hormone / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000096:biosynthesis*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D008934:Mitogens; D016376:Oligonucleotides, Antisense; D010281:Parathyroid Hormone; D012333:RNA, Messenger; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D004247:DNA; D016203:CDC2 Protein Kinase