Regulation of gene transcription and proliferation by parathyroid hormone is blocked in mutant osteoblastic cells resistant to cyclic AMP.

We employed a cyclic AMP-resistant subclone of UMR 106-01 osteoblastic osteosarcoma cells (UMR 4-7) with a regulated, dominant-negative mutation of cyclic AMP-dependent protein kinase (PK-A), to examine the mechanism(s) whereby parathyroid hormone (PTH) regulates growth of these cells. Expression of a transiently transfected CAT reporter gene controlled by the cAMP response element of the rat somatostatin gene ('SST-CAT') was used to monitor PK-A activation in intact cells. Agonist-stimulated SST-CAT expression was specific for agents known to activate adenylate cyclase, required an intact cAMP response element and was specifically blocked following induction of the mutant cAMP-resistant phenotype in UMR 4-7 cells.

Protein kinase A-dependent inhibition of alkaline phosphatase release by SaOS-2 human osteoblastic cells: studies in new mutant cell lines that express a cyclic AMP-resistant phenotype.

We have established mutant SaOS-2 cell lines that express a cyclic AMP (cAMP)-resistant phenotype to investigate the regulation and functional importance of orthophosphoric-monoester phosphohydrolase alkaline optimum (ALPase) in the action of parathyroid hormone (PTH). Cells were stably transfected with a plasmid that directs the synthesis of a mutant form of the type I regulatory subunit of protein kinase A (PKA) under the control of the metallothionein promotor. There was no significant difference between parental SaOS-2 cells and the mutant lines in the affinity or number of receptors for 125I-Nle8,18Tyr34bPTH1-34NH2, either in the absence or presence of Zn2+.

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent and cAMP-independent regulation of parathyroid hormone receptors on UMR 106-01 osteoblastic osteosarcoma cells.

The osteoblast-like cells, UMR 106-01, express PTH receptors that are coupled to adenylate cyclase. Recently, we reported the isolation of a UMR 106-01 subclone, UMR 4-7, that is stably transfected with a Zn(++)-inducible mutant of the regulatory subunit of protein kinase A. Incubation of UMR 4-7 cells with Zn++ renders the cells unresponsive to cAMP agonists.

Inhibition of parathyroid hormone responsiveness in clonal osteoblastic cells expressing a mutant form of 3',5'-cyclic adenosine monophosphate-dependent protein kinase.

PTH activates multiple acute intracellular signals within responsive target cells, but the importance of cAMP vs. other second messenger signals in mediating different biological responses to PTH is not known. To address these questions, we developed a genetic approach to block activation of the cAMP-dependent protein kinase (PK-A) in PTH-responsive cell lines.