Abstract
The appearance of neonatal mice that develop in the absence of parathyroid hormone- related peptide (PTHrP) established that the peptide is necessary for normal development and growth of the skeleton. The shortening of appendicular bones in knockout mice is explained by an effect of PTHrP on endochondral bone growth where it functions to inhibit the hypertrophy of chondrocytes in the epiphyseal growth plate. Whether or not PTHrP influences intramembranous ossification is less clear. The puipose of this study was to examine the expression of PTHrP and its receptor (PTH1R) by osteogenic cells involved in intramembranous ossification in vitro, and to determine the effect of exogenous PTHrP on this process. Cells isolated from calvariae of neonatal rats were treated continuously or intermittently with PTHrP (1-36). PTHrP and PTH1R were localized by immunohistochemistry and alkaline phosphatase (AP) was detected by enzyme histochemistry. Staining was visualized by confocal microscopy. The number and size of bone nodules and cell colonies were determined at 3-21 days of treatment.The effects of PTHrP on proliferation, apoptosis and gene expression were measured using BrdU incorporation, TUNEL staining and real time RT-PCR respectively. Results showed that PTHrP and PTH1R were co-localized in cell colonies as early as day 3 while AP was not detected until day 6. During the course of bone nodule development PTHrP and its receptor were co-localized in pre-osteoblasts, osteoblasts, and osteocytes. By comparison, the AP activity exhibited by pre-osteoblasts and osteoblasts disappeared in osteocytes. Both continuous and intermittent exposure to PTHrP (1-36) caused inhibition in bone nodule formation and reduced nodule size. The inhibitory effect was reversible. The effect of PTHrP on bone formation was mediated by stimulation of adenyl cyclase /PKA pathway. Treatment with PTHrP (1-36) had no effect on AP activity, cell proliferation, apoptosis, or the level of AP, COLL I and osteopontin mRNAs. However, bone sialoprotein (BSP) mRNA was reduced by > 90% compared to control. These results suggest that PTHrP functions as an autocrine factor for cells in the osteoblast lineage involved in intramembranous ossification. Since PTHrP inhibited bone nodule formation and reduced BSP gene expression and protein, we conclude that PTHrP regulates bone mineralization by controlling BSP production.