DOWN-REGULATION OF THE RECEPTOR FOR PARATHYROID-HORMONE (PTH) AND PTH-RELATED PEPTIDE BY PTH IN PRIMARY FETAL-RAT OSTEOBLASTS

We studied the effects of parathyroid hormone (PTH) on PTH parathyroid hormone related peptide (PTHrP) receptor mRNA level, PTHrP binding, a nd PTH-stimulated cyclic adenosine monophosphate (cAMP) accumulation i n osteoblasts, derived from fetal rat calvariae (ROB). Cells isolated during 10-70 minutes of collagenase treatment were seeded at a density of 25,000 cells/cm(2) and cultured for 4 days. These cells show a fas t increase in cAMP production after stimulation for 5 minutes with 20 nM bovine parathyroid hormone (1-34) (bPTH(1-34)). When ROB are incuba ted with bPTH(1-34) (0.04-40 nM) for 25 h, a dose-dependent decrease o f the PTH/PTHrP receptor mRNA level, PTHrP binding, and PTH-stimulated cAMP accumulation can be observed. Pretreatment of ROB with a high co ncentration of bPTH(1-34) (40 nM) leads within 15 minutes to a decreas e in PTH-stimulated cAMP accumulation. However, it takes greater than or equal to 3 h before a significant decrease in PTH/PTHrP receptor mR NA level can be observed. Also a significant decrease in PTHrP binding is observed after only 4 h of incubation with bPTH(1-34). Compared wi th bPTH(1-34), pretreatment of ROB with bPTH(3-34) (40 and 100 nM) for 24 h causes smaller decreases in PTH-stimulated cAMP accumulation, PT HrP binding, and in the PTH/PTHrP receptor mRNA level. We investigated the possible involvement of the protein kinase A signaling pathway in the regulation of the PTH/PTHrP receptor mRNA expression. Both forsko lin and (Bu)(2)cAMP decreased PTHrP binding and PTH/PTHrP mRNA levels. These observations suggest that chronic activation of the PKA signali ng pathway may down-regulate PTH/PTHrP receptor expression and thus ho rmone responsiveness in ''normal'' osteoblasts. In short, we found tha t the decrease of the PTH-stimulated cAMP accumulation after long-term pretreatment with bPTH(1-34) is correlated with both PTH/PTHrP recept or mRNA level acid PTHrP binding. These data also suggest that the ini tial desensitization (< 30 minutes) of PTH-stimulated cAMP responsiven ess by pretreatment with a high concentration of bPTH(1-34) (40 nM) is not dependent on the number of available PTH/PTHrP receptors. The pro tein kinase A signaling pathway is involved in the regulation of the P TH/PTHrP receptor, but, regarding the effect of bPTH(3-34), other sign aling systems are also involved.