Down-regulation of the receptor for parathyroid hormone (PTH) and PTH-related peptide by transforming growth factor-beta in primary fetal rat osteoblasts.

We studied the effects of transforming growth factor-beta 2 (TGF beta 2) on the level of PTH/PTH-related peptide-(PTHrP) receptor messenger RNA (mRNA), PTHrP binding, and PTH-stimulated cAMP accumulation in cultured osteoblasts derived from fetal rat calvariae (ROB). When ROB were pretreated with TGF beta 2 at concentrations ranging from 1-100 pM for 24 h, dose-dependent decreases in the level of PTH/PTHrP receptor mRNA, PTHrP binding, and PTH-stimulated cAMP accumulation were observed. For the PTH/PTHrP receptor mRNA level and PTH-stimulated cAMP accumulation, the half-maximal effective concentration was approximately 4 pM. For the inhibition of PTHrP binding, the half-maximal effective concentration was much higher. A 50% decrease in both PTH/PTHrP receptor mRNA level and PTH-stimulated cAMP accumulation was obtained when ROB were treated with 100 pM TGF beta 2 for 4 h. A comparable decrease in PTHrP binding was only observed after 24 h of incubation with 100 pM TGF beta 2. Actinomycin D induced a rapid decrease in the PTH/PTHrP receptor mRNA level (70% after 4 h), indicating a half-life for the receptor mRNA of 2-3 h. Under the same conditions, PTHrP binding and PTH-stimulated cAMP accumulation did not change. When ROB were treated with cycloheximide for the same period, only a small decrease in PTHrP binding (20%) was observed, suggesting that PTH/PTHrP receptors do not have a rapid turnover. Cycloheximide also reduced PTH-stimulated cAMP production; after coincubation of cycloheximide with TGF beta 2, this inhibition was smaller than that in ROB cultures treated with TGF beta 2 exclusively. From these observations we conclude that TGF beta 2 induces a decrease in steady state levels of PTH/PTHrP receptor mRNA that results in decreased PTHrP receptor binding. The PTH-stimulated cAMP accumulation is at least to some extent independent of the PTH/PTHrP receptor availability. Furthermore, there is a high turnover of PTH/PTHrP receptor mRNA, whereas turnover of the receptor protein is much slower. Finally, protein synthesis is required for TGF beta 2-induced desensitization of cAMP responsiveness to PTH.