Glucocorticoids and 1,25-Dihydroxyvitamin D3 Regulate Parathyroid Hormone Stimulation of Adenosine 3′,5′-Monophosphate-Dependent Protein Kinase in Rat Osteosarcoma Cells*

Publisher Website
Google Scholar
Abstract
Glucocoticoids increase and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] decreases the activity of PTH-responsive adenylate cyclase, altering intracellular cAMP in a rat osteoblast-like cell line (ROS 17/2.8). This study was undertaken to measure the subsequent activation of the cAMP-dependent protein kinase (PKA). Pretreatment of ROS cells for 2 days with the glucocorticoid triamcinolone acetonide (TRM), shifted the dose-response curve for PKA activation by PTH upward compared to the control value. Basal PKA activity was enhanced 50% by TRM, and the PTH concentration required for maximal activation of PKA decreased from 1.0 to 0.05 ng/ml. At the lowest effective PTH concentration (0.05 ng/ml) the mean PKA activity ratio increased to 0.73 in TRM-treated cell compared with 0.45 in untreated cells. Pretreatment with 1,25-(OH)2D3 had opposite effects, shifting the dose-response curve for PKA activation by PTH downward and to the right, decreasing the basal activity ratio from 0.26 to 0.16, and increasing the PTH concentration required for maximal activation to 10 ng/ml. 1,25-(OH)2D3-treated cells stimulated with 0.5-1 ng/ml PTH consistently had lower PKA activity ratios than untreated cells. Simultaneous treatment with 1,25-(OH)2D3 reversed the effect of TRM. There were no differences in total PKA activity (2.57 .+-. 0.09 pmol 32P/min .cntdot. .mu.g protein) between treatment groups, suggesting that TRM and 1,25-(OH)2D3 do not alter the cellular PKA concentration. In control experiments exogenous PKA was added to sonication buffer of PTH-stimulated cells to verify that the TRM and 1,25-(OH)2D3 shifts in PKA activation at low PTH does occur before sonication. cAMP-dependent protein kinase activation was also studied by measuring the progressive occupation of regulatory subunit-binding sites by hormonally stimulated endogenous cAMP. [3H] cAMP binding was expressed as the percent change in bound [3H]cAMP per .mu.g protein compared to that in unstimulated cells not steroid treated. [3H]cAMP binding to all cytosol fractions decreased as PTH increased over the concentration ragne predicted by our PKA activation experiment. TRM treatment shifted the curve for [3H]cAMP binding to regulatory subunit downward and to the left, and 1,25-(OH)2D3 treatment shifted it upward and to the right. In cells treated with both TRM and 1,25-(OH)2D3, the curve was similar to control curve. Sonicating unstimulated cells in buffer containing comparable concentration of added cAMP did not alter [3H]cAMP binding. These and the previous controls suggest that changes in PKA activation at low doses of PKA reflect cellular events occurring before cell disruption. In summary, we have used two independent methods to study activation of cAMP-dependent protein kinase by PTH-stimulated endogenous cAMP in the ROS cell. Control experiments show that the observed activation occurs before cell disruption. Glucocorticoid and 1,25-(OH)2D3 regulation of cAMP production in response to PTH alters the subsequent activation of PKA.

This publication has 20 references indexed in Scilit: