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Unlabelled: PAR-2 is expressed by osteoblasts and activated by proteases present during inflammation. PAR-2 activation inhibited osteoclast differentiation induced by hormones and cytokines in mouse bone marrow cultures and may protect bone from uncontrolled resorption., Introduction: Protease-activated receptor-2 (PAR-2), which is expressed by osteoblasts, is activated specifically by a small number of proteases, including mast cell tryptase and factor Xa. PAR-2 is also activated by a peptide (RAP) that corresponds to the "tethered ligand" created by cleavage of the receptor's extracellular domain. The effect of activating PAR-2 on osteoclast differentiation was investigated., Materials and Methods: Mouse bone marrow cultures have been used to investigate the effect of PAR-2 activation on osteoclast differentiation induced by parathyroid hormone (PTH), 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], and interleukin-11 (IL-11). Expression of PAR-2 by mouse bone marrow, mouse bone marrow stromal cell-enriched cultures, and the RAW264.7 osteoclastogenic cell line was demonstrated by RT-PCR., Results: RAP was shown to inhibit osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11. Semiquantitative RT-PCR was used to investigate expression of mediators of osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11 in mouse bone marrow cultures and primary calvarial osteoblast cultures treated simultaneously with RAP. In bone marrow and osteoblast cultures treated with PTH, 1,25(OH)2D3, or IL-11, RAP inhibited expression of RANKL and significantly suppressed the ratio of RANKL:osteoprotegerin expression. Activation of PAR-2 led to reduced expression of prostaglandin G/H synthase-2 in bone marrow cultures treated with PTH, 1,25(OH)2D3, or IL-11. RAP inhibited PTH- or 1,25(OH)2D3-induced expression of IL-6 in bone marrow cultures. RAP had no effect on osteoclast differentiation in RANKL-treated RAW264.7 cells., Conclusion: These observations indicate that PAR-2 activation inhibits osteoclast differentiation by acting on cells of the osteoblast lineage to modulate multiple mediators of the effects of PTH, 1,25(OH)2D3, and IL-11. Therefore, the role of PAR-2 in bone may be to protect it from uncontrolled resorption by limiting levels of osteoclast differentiation.

We examined the effect of calcitonin in cultured mouse calvarial bones after prestimulation with different activators of adenylyl cyclase. Calcitonin (100 ng/ml), added after 48 h of culture, inhibited bone resorption (assessed as release of 45Ca from prelabeled bones cultured for 96-144 h) stimulated with parathyroid hormone (PTH, 10 nM; 0-144 h) or the adenylyl cyclase stimulator forskolin (2 microM; 0-144 h). However, no effect of calcitonin was demonstrated when bone resorption was prestimulated with the adenylyl cyclase stimulator cholera toxin, at and above 1 ng/ml, at any time point studied. In contrast, two other types of inhibitors of bone resorption in vitro, the carbonic anhydrase inhibitor acetazolamide (10 microM) and the aminobisphosphonate AHPrBP (10 microM), significantly inhibited cholera toxin-stimulated bone resorption. No cyclic AMP response to calcitonin was seen after preculture for 48 h with cholera toxin (0.1-100 ng/ml), although bones precultured in basic medium, in the absence or presence of forskolin, were still able to respond to calcitonin with elevation of cyclic AMP. Binding studies with [125I]calcitonin demonstrated that the preculture with cholera toxin did not affect the binding of calcitonin to the receptor. In summary, our data show that cholera toxin pretreatment makes calvarial bones insensitive to calcitonin-induced inhibition of bone resorption as a result of an interaction with cholera toxin at the level of calcitonin receptor-linked signal transduction. We suggest that the interaction, distal to the calcitonin receptor, is caused by the irreversible activation of Gs produced by cholera toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of gamma-interferon (gamma-IFN) on bone resorption and cyclic AMP formation stimulated by parathyroid hormone (PTH), forskolin, and cholera toxin have been studied in cultured neonatal mouse calvarial bones. Bone resorption was assessed by the release of 45Ca from prelabeled mouse calvarial bone fragments. Cyclic AMP formation was quantified by analyzing the amount of the nucleotide in calvarial bone tissue. gamma-IFN completely blocked the 45Ca release response to forskolin and cholera toxin in 96 h cultures. In contrast, the 45Ca release response to PTH was only partially inhibited, an effect that was seen over a wide range of PTH concentrations. The inhibitory effect of gamma-IFN was dose dependent, with a threshold for action at 10 U/ml. Forskolin-stimulated 45Ca release could only be inhibited when gamma-IFN was added simultaneously with forskolin; gamma-IFN added to bones prestimulated with forskolin had no effect. The inhibitory effect of gamma-IFN on PTH-stimulated 45Ca release was seen first after a time lag of 48 h. In contrast calcitonin caused an inhibition after only 3 h. PTH and cholera toxin stimulation of radioactive calcium release was also inhibited by gamma-IFN in bones treated with indomethacin. gamma-IFN inhibited forskolin-induced 45Ca release in bones treated with the mitotic inhibitor hydroxyurea. No effect of gamma-IFN on cyclic AMP formation induced by PTH, cholera toxin, or forskolin could be seen. These data show that gamma-IFN inhibits forskolin- and cholera toxin-induced bone resorption by a mechanism unrelated to prostaglandin production or mitotic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone resorption in a modified bone culture system, based on incubation of small fragments from neonatal mouse calvarial bones, has been studied. Four bone fragments were dissected out from each mouse calvaria and were thereafter cultured in CMRL 1066 medium in plastic multiwell dishes. Bone resorption was assessed by 45Ca release from prelabeled bones. The rate of bone resorption in response to parathyroid hormone (PTH) was less in the anterior part of the calvaria compared to the posterior part. After removing the anterior region, four parietal bone fragments that showed identical basal and PTH-stimulated release of 45Ca could be dissected out from each mouse. Excretion of lactate dehydrogenase and beta-glucuronidase was the same in bones cultured submerged or on grids. Uptake of [3H]thymidine in bones cultured submerged was 54% of [3H]thymidine uptake in bones cultured on grids. Dose-response curves, established by using parietal bone fragments, showed that the sensitivity and the magnitude of the increase in 45Ca release seen after stimulation with PTH, prostaglandin E2, and 1 alpha-hydroxyvitamin D3 were the same for bones cultured submerged or on grids. The 45Ca release in response to stimulation with PTH, prostaglandin E2, and 1 alpha-OHD3 was the same in calvarial fragments cultured submerged and those previously obtained with calvarial halves cultured on grids. Thus, even though the rate of DNA synthesis was slower in bones cultured submerged, the rate and the magnitude of resorption were the same in bones cultured on grids or submerged. These data show that it is possible to perform studies on bone resorption with small fragments of neonatal mouse parietal bones.

We have previously found that the adenylate cyclase stimulators forskolin and choleratoxin increase cyclic AMP and transiently inhibit bone resorption in cultured mouse calvaria, suggesting that the compounds, directly or indirectly, may inhibit osteoclast activity. In the present study, forskolin and choleratoxin were investigated for their direct effects on surface area and motility of isolated rabbit osteoclasts, and the effects were compared to those of calcitonin (CT). Osteoclasts were cultured on coverslips for different times in the absence or presence of the compounds. The effect on osteoclast mean area was quantified on fixed and stained osteoclasts, and in addition effects were recorded with time-lapse cinemicrography. The effects of CT (100 mU/ml) on mean area and motility were seen within minutes and were maximal after 10-60 minutes. Forskolin (10-30 mumol/liter) produced a rapid (15-60 minutes) inhibition of motility and decrease in area (contraction) of osteoclasts. Choleratoxin (1 microgram/ml) treatment also resulted in cell contraction and inhibition of motility; however, the response was not seen before 45-60 minutes. The difference in the kinetics of the osteoclast response between forskolin, CT, and choleratoxin is similar to differences in time course for the effect on cyclic AMP in calvarial bones, which we reported earlier. Although cells were incubated continuously with forskolin, choleratoxin, or CT, the effects were transient. Thus, after 7-8 h incubation with CT, 3-4 h treatment with forskolin, or 4-6 h with choleratoxin, the osteoclasts started to recover from contraction and immotility. The effect of forskolin and choleratoxin on the mean surface area of osteoclasts was dose dependent. The present study shows that forskolin and choleratoxin have a direct inhibitory action on osteoclast activity and thus provide further evidence that cyclic AMP is a mediator of the action of CT on bone resorption.