1,25-Dihydroxyvitamin D reduces parathyroid hormone receptor number in ROS 17/2.8 cells and prevents the glucocorticoid-induced increase in these receptors: Relationship to adenylate cyclase activation

Abstract

We have previously shown that 1,25-dihydroxyvitamin D [1,25-(OH)₂D₃] and glucocorticoid modulate adenylate cyclase activation by PTH in osteoblast-like cells. Here we examine whether steroid effects on PTH receptor density explain the modulation of PTH action. Receptor assays were performed on late logarithmicphase monolayers of ROS 17/2.8 cells using human PTH-like peptide (hPLP) as radioligand. K_d and receptor density were computed from competition of tracer amounts of [¹²⁵I-Tyr³⁶]hPLP-(1–36) with unlabeled hPLP-(1–36) (0.1–30 nM). Steroid treatment had little or no effect on affinity for ligand. Pretreating cells with 10 nM 1,25-(OH)₂D₃ for 48 h decreased PTH receptor number to 17% of control values. Treating cells with 10 nM of the glucocorticoid triamcinolone acetonide (TRM) increased receptor number 10-fold, but simultaneous treatment with 1,25-(OH)₂D₃ (10 nM) completely prevented this receptor increase. Steroid effects required 13–18 h of treatment. Dose-response relationships for steroid modulation, determined from binding at 0.17 nM radioligand, indicated an EC₅₀ of 0.3 nM for glucocorticoid augmentation of PTH receptor number and 0.02 nM for 1,25-(OH)₂D₃ reduction of receptor number in the presence or absence of the maximum TRM effect. The initial rate of cAMP production by receptor-saturating concentrations of PTH was 11,500 molecules per receptor per minute in untreated cells, comparable to reported turnover numbers for mammalian adenylate cyclase. Control experiments were validated measuring cAMP in intact cells as an indicator of adenylate cyclase activity. Cyclic AMP production was reduced 63% by 1,25-(OH)₂D₃ (10 nM) treatment. Glucocorticoid (10 nM) enhanced cAMP production twofold but reduced cAMP generation per receptor by 80%. We conclude that 1,25-(OH)₂D₃ reduction of PTH receptor number modulates ROS cell responses to PTH and prevents glucocorticoid-enhanced PTH action. The data also suggest that other components of the adenylate cyclase complex limit amplification of the PTH signal when PTH receptor number is elevated by glucocorticoid treatment.