kt-5720 and Osteosarcoma

PTH is a major systemic regulator of bone metabolism and plays an important role in both bone formation and resorption. PTH either inhibits or stimulates osteoblastic cell proliferation depending on the model that is studied. We analyzed the cell cycle of the UMR-106 cell line, a relatively differentiated osteoblastic osteogenic sarcoma line in which PTH is known to inhibit proliferation but the mechanism of action is unknown. PTH decreased the proportion of cells in S phase and increased the number of G1 phase cells. We examined the effect of PTH on the regulators of the G1 phase cyclin-dependent kinases and found that PTH increased p27Kip1, but not p21Cip1, levels. This effect was mimicked by 8-bromo-cAMP, but not by phorbol 12-myristate 13-acetate. The protein kinase A inhibitor KT5720 abolished the effect of PTH on the increase in p27Kip1 expression. PTH increased CDK2-associated p27Kip1 without affecting the levels of CDK2. CDK2 activity was down-regulated by both PTH and 8-bromo-cAMP treatment. These data suggest that PTH blocks entry of cells into S phase and inhibits cell proliferation as the consequence of an increase in p27Kip1, which is mediated through the protein kinase A pathway. The inhibition of G1 cyclin-dependent kinases by p27Kip1 could cause a reduction of phosphorylation of key substrates and inactivation of transcription factors essential for entry into S phase. The inhibition of cell cycle progression through PKA-mediated p27Kip1 induction might play an important role in PTH-induced differentiation of osteoblasts.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Carbazoles; Cell Cycle Proteins; Cell Differentiation; Cell Line; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Enzyme Inhibitors; G1 Phase; Gene Expression; Indoles; Microtubule-Associated Proteins; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Pyrroles; Rats; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tumor Suppressor Proteins

The pericellular proteoglycan biglycan is among the major secretory products of osteoblasts and articular chondrocytes but the regulatory agents and signal transduction pathways that ultimately lead to alterations in biglycan gene expression are poorly defined. We report here on the transcriptional up-regulation of biglycan in MG-63 osteosarcoma cells by agents that increase intracellular cAMP levels. Transfection of these cells with biglycan promoter luciferase reporter fusion genes and subsequent treatment with forskolin or the cAMP analog 8-Bromo-cAMP resulted in an up to 3.8-fold stimulation of biglycan promoter activity. This effect could be prevented with the compound KT5720, a specific inhibitor of the cAMP-dependent protein kinase. Up-regulation of transcription is also reflected at the level of mRNA expression, since biglycan mRNA steady state levels in MG-63 cells increased approximately 2-fold after 24 hours of forskolin treatment. These data suggest that elevated levels of intracellular cAMP increase transcription from the biglycan promoter in bone cells and implicate for the first time the cAMP/protein kinase A signal transduction pathway in the regulation of biglycan gene expression.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Biglycan; Carbazoles; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Indoles; Osteosarcoma; Plasmids; Promoter Regions, Genetic; Proteoglycans; Pyrroles; RNA, Messenger; Sequence Deletion; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transfection; Tumor Cells, Cultured