The long-term objective of this project is to define the effects of 1,25-Dihydroxyvitamin D and glucocorticoids on the cAMP pathway for parathyroid hormone (PTH) action on the osteoblast in culture, from the point of PTH binding to cellular functional responses that can be understood at the tissue level. These studies relate to understanding the mechanisms of glucocorticosteroid-induced osteoporosis, more effective and rational, use of vitamin D metabolites in this disease, and the possibility of devising other therapeutic agents which also antagonize the skeletal effects of glucocorticoids. The rationale for the approach originated with our observations that 1,25(OH)2D completely antagonizes the effect of glucocorticoid to increase the cAMP response of cell culture models of the osteoblast to PTH . Important new data show that these effects are mediated predominantly at the level of PTH-R regulation and the effects are transmitted to the next step in the cAMP pathway, the protein kinase. We have characterized PTH stimulation of mRNA for the tissue plasminogen activator (tPA). This enzyme is believed to play a crucial role in the osteoblast's ability to signal the need for bone resorption. Three hypotheses will be tested in the continuation of the project. (1) 1,25D and GC have opposing regulatory effects on transduction of the PTH signal into functional responses in normal osteoblasts. (2) As a consequence of modulating PKA activity, 1,25D and GC alter effects of PTH cyclase catalytic subunit in order to accommodate the changes in PTH receptor number that they induce. For hypothesis (1) we shall: (a) determine the effects of 1,25D and GC on the dose-response relationship for PTH stimulation of tPA mRNA; (b) demonstrate that PTH acts to increase transcription of tPA mRNA; (c) identify the individual cells expressing tPA mRNA in response to PTH as osteoblasts by showing they contain alkaline phosphatase and do not synthesize type III collagen. For hypothesis (2) we shall determine, in ROS 17/2.8 cells, the effects of 1,25D and GC on (a) the dose-response for PTH activation of protein phosphatase inhibitor-1 and (b) the dose-response for PTH activation of the high affinity (Type III) cAMP phosphodiesterase (PDE). For hypothesis (3) we shall use both ROS 17/2.8 and UMR106 cells to (a) measure the effect of 1,25D and GC on the amount and turnover number of the adenylate cyclase catalytic subunit and its relationship to PTH receptor number and (b) demonstrate that the amount of immunoreactive Gs (a) protein solubilized from membranes is in excess relative to PTH receptor number and adenylate cyclase. Routine procedures include primary culture of osteoblasts, radioimmunoassay of cAMP, adenylate cyclase assay, PTH radioreceptor assay, electrophoresis and DNA hybridization analysis of mRNA. In situ hybridization of mRNA in cultures will be performed with the collaboration of a local expert in this technique. Adenylate cyclase catalytic subunit will be measured by [3H] forskolin binding, Gs protein by RIA; serine protein phosphatase and cAMP phosphodiesterase assays will be performed after chromatographic separation.
