M. P. Bos

Expression of the parathyroid hormone receptor and correlation with other osteoblastic parameters in fetal rat osteoblasts

Primary fetal rat calvarial cell cultures were examined for the expression of different osteoblastic parameters at the single cell level and in the whole population. The presence of the parathyroid hormone (PTH) receptor was studied by employing receptor autoradiography. After 3 days of culture, 10% of the cells expressed the PTH receptor. Immunolocalization of osteocalcin in 3-day-old cell cultures was found to be strongly correlated with the presence of the PTH receptor. Alkaline phosphatase (APase) localization in 3-day-old cultures correlated with only 69% of the PTH receptor expressing cells. Our results show that in 3-day-old rat calvarial cell cultures, only about 10% of the cells show markers of osteoblastic differentiation. The presence of the PTH receptor is strongly correlated with the presence of osteocalcin, but less with the presence of APase, indicating that it is the mature osteoblast that expresses the PTH receptor. After 7 days of culture, most receptor labeling, APase, and osteocalcin expression was found in multilayered areas of cells (nodules).

Role of protein kinase C (PKC) in bone resorption: Effect of the specific PKC inhibitor 1-alkyl-2-methylglycerol

The specific inhibitor of protein kinase C, 1-O-alkyl-2-O-methylglycerol (AMG), was studied for its effect on bone resorption, measured as 45Ca-release, in fetal mouse calvariae. AMG (1 to 50 microM) had no effect on basal bone resorption. AMG inhibited parathyroid hormone (40 nM) induced bone resorption in a dose-dependent manner. Resorption induced by 1,25 (OH)2-vitamin D3 (10 nM) or prostaglandin E2 (5 microM) was also inhibited by AMG. The release of beta-glucuronidase activity paralleled the course of the 45Ca-release. The production of interleukin 6, induced by parathyroid hormone, in fetal rat calvarial osteoblasts was not affected by AMG. AMG (1 to 50 microM) had no cytotoxic effects on cells or calvariae. From these results it is concluded that protein kinase C may have an important role in the regulation of bone resorption.

Independent and interrelated regulation of ornithine decarboxylase by calcium and cAMP in fetal rat osteoblasts

The present study was undertaken to determine in fetal rat osteoblasts whether and how the intracellular messengers calcium and cAMP are involved in stimulation of ornithine decarboxylase (ODC) activity. For that purpose we used different drugs affecting [Ca2+]i and cAMP concentration. A23187 stimulates ODC activity in a biphasic way, with maximal stimulation at 100 nM A23187. At that concentration no stimulation of cAMP production was observed. Basal and A23187-stimulated (100 nM) ODC activity were inhibited by EGTA and trifluoperazine. Forskolin stimulated dose-dependently both ODC activity and cAMP production. Besides these effects forskolin (1 and 10 microM) increased the [Ca2+]i via an increased calcium influx. Addition of La3+, verapamil or EGTA, but not of trifluoperazine, significantly inhibited the forskolin-stimulated (10 microM) ODC activity. When forskolin (100 nM and 1 microM) was added together with 1 microM A23187, a synergistic stimulation of ODC activity was observed. These results implicate that calcium is involved in basal ODC activity, and that ODC activity can be stimulated via (1) a cAMP-independent calcium pathway, and (2) a calcium-dependent, cAMP pathway. It is proposed that ODC activity can be stimulated via interaction between calcium and cAMP.

The effects of MC903 on 1,25-(OH)2D3 receptor binding, 24-hydroxylase activity and in vitro bone resorption.

MC903, a new vitamin D analog has been shown to exert potent effects on cell proliferation and differentiation, while in vivo a decreased activity on calcium metabolism has been observed. In the osteoblast-like cell line UMR-106, MC903 displaces tritiated 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) from its receptor at least as efficiently as 1,25-(OH)2D3. The effect of MC903 on 1,25-(OH)2D3 receptor up-regulation in UMR-106 cells and on bone resorption in fetal mouse radii/ulnae was comparable to that of 1,25-(OH)2D3. MC903 was about 50% less effective in inducing 24-hydroxylase activity and the subsequent C24-side chain oxidation of 25-(OH)D3 compared to 1,25-(OH)2D3. Ketoconazole did not potentiate MC903-induced 1,25-(OH)2D3 receptor up-regulation as was found with 1,25-(OH)2D3 which suggests that the C24-oxidation plays a minor role in the inactivation of MC903. Nevertheless, the comparable effects of MC903 and 1,25-(OH)2D3 on in vitro bone resorption indicate that the lower effectivity of MC903 on bone calcium mobilization in vivo has to be due to a higher metabolic clearance rate.

Down-regulation and differential restoration of cAMP responses upon transient phorbol ester treatment of primary osteoblastic cells.

We studied cAMP responses induced by parathyroid hormone (PTH), prostaglandin E2 (PGE2) and forskolin in foetal rat calvariae-derived osteoblastic cells after 24 h treatment with a protein kinase C (PKC) activating phorbol ester. After this treatment, meant to down-regulate PKC activity, all tested cAMP responses were attenuated and were indeed accompanied by a decline in PKC activity. PTH receptor affinity was not altered and PTH receptor number was only slightly lowered after 24 h phorbol ester treatment. These results indicate that modulation of the cAMP responses by 24 h PMA treatment was mainly caused by a general impairment of adenylyl cyclase activity. Removal of the phorbol ester and subsequent culture for 2 days rendered the cells hyper-responsive to PTH: the PTH-induced cAMP response was 2 to 3 times higher than in control cells. Again no change in binding affinity of the PTH receptor was observed and receptor number was just 10% lower than in control cells. The PGE2- and forskolin-induced cAMP responses were not higher than normal. So, transient phorbol ester treatment leads to a differential, agonist-dependent restoration of the cAMP signalling system.