Bone anabolic responses to mechanical load in vitro involve COX-2 and constitutive NOS.

Mechanical stimulation is essential for maintaining the homeostasis and architecture of connective tissues including bone. The purpose of our study was to test the importance of several potential signaling intermediates in the anabolic responses of bone to loads applied with a newly developed micromechanical loading device. Tibial bones excised from 7- to 8-day-old CD-1 mice were cyclically loaded at 1 Hz, 1000 muepsilon (microstrain) at a peak load of 100 mN. DNA and protein synthesis were evaluated by measuring the incorporation of 3H-thymidine and 14C-proline, respectively. The roles of cyclooxygenase (COX) isoforms, nitric oxide synthase (NOS) isoforms, and glutamate receptor-gated Ca2+ channeling were examined by incubating the bones in the presence of each of their specific inhibitors. The results indicate that COX-2 and constitutive NOS are important signaling molecules in the anabolic responses of neonatal tibial bone to the micromechanical load in vitro.

In vitro mechanical and cellular responses of neonatal mouse bones to loading using a novel micromechanical-testing device.

Mechanical stimulation is critical for the maintenance of bone architecture and bone mass. These effects are dependent on the magnitude, duration, and rate of the mechanical stimuli. The goals of the present study were to develop and optimize a micromechanical-testing device for in vitro mechanical stimulation of whole viable bones, and to identify the physical parameters of loading that elicit maximal anabolic responses. The model was the 7-8-day-old neonatal CD-1 mouse tibia. A range of cyclic strain magnitudes [500-7000 microstrain (microstrain)] and frequencies [0.2-30 hertz (Hz)] were applied to the neonatal bones. Incremental cyclic compression tests showed that the bones were nonlinearly viscoelastic. Bone stiffness and hysteresis energy dissipation were dependent on the maximum load magnitude. DNA and protein synthesis were significantly enhanced in bones that were cyclically loaded at 0.5 Hz/1000 microstrain, 0.5 Hz/2000 microstrain, or l Hz/1000 microstrain, compared to nonloaded controls. Anabolic responses were maximal at a peak load of 100 mN at l Hz/1000 microstrain. Autoradiography of the bones loaded under these conditions showed proliferation of cells at periosteal surfaces. Hysteresis energy per cycle was greatest at loads that caused the largest anabolic responses. The parameters of strain and load that elicit optimal effects on the neonatal bones are comparable to those in other systems, validating the use of the instrumentation for studying the mechanisms of the anabolic responses. The findings also suggest that hysteresis energy per cycle may be a determinant of the anabolic response of bones to mechanical stimulation.

Phosphatidylcholine-specific phospholipase C inhibitor, tricyclodecan-9-yl xanthogenate (D609), increases phospholipase D-mediated phosphatidylcholine hydrolysis in UMR-106 osteoblastic osteosarcoma cells.

Our previous studies have shown that parathyroid hormone (PTH) stimulates phosphatidylcholine (PC) hydrolysis by phospholipase D (PLD) and transphosphatidylation in UMR-106 osteoblastic cells. To determine whether phospholipase C (PLC) is also involved in the PTH-mediated PC hydrolysis, we used the inhibitor, tricyclodecan-9-yl xanthogenate (D609), a putatively selective antagonist of this pathway. Consistent with this proposed mechanism, D609 decreased (3)H-phosphocholine in extracts from UMR-106 cells prelabeled with (3)H-choline. Unexpectedly, D609 enhanced PC hydrolysis and transphosphatidylation, suggesting that either there was a compensatory increase in PLD activity when PLC was inhibited, or that D609 directly increased PLD activity. The D609-stimulated increase in PC hydrolysis was rapid, being seen as early as 2 min. The effect of D609 was temperature-sensitive, consistent with an enzymatic mechanism. The D609-stimulated increase in PC hydrolysis was PKC-independent, based upon the lack of effect of down-regulation of PKC by phorbol 12,13-dibutyrate on the response. The studies reveal a novel action of this inhibitor on signaling in osteoblastic cells which might influence downstream responses.

Parathyroid hormone (PTH)-(1-34), [Nle(8,18),Tyr34]PTH-(3-34) amide, PTH-(1-31) amide, and PTH-related peptide-(1-34) stimulate phosphatidylcholine hydrolysis in UMR-106 osteoblastic cells: comparison with effects of phorbol 12,13-dibutyrate.

Studies were performed to determine the effects of PTH and related compounds on phosphatidylcholine (PC) hydrolysis in UMR-106 cells and the pathway by which the PTH effects occurred. The responses were compared with those of phorbol 12,13-dibutyrate (PDBu). Both bovine PTH-(1-34) [bPTH-(1-34)] and PDBu stimulated PC hydrolysis within 10 min. Significant effects were elicited by concentrations of 0.3-1 nM bPTH-(1-34) and 5 nM PDBu. Dose-dependent increases were seen at higher concentrations of both compounds, however, the response to bPTH-(1-34) was reduced at 30 nM. Bovine or human PTH-(1-34) and human PTH-related peptide-(1-34) [hPTHrP-(1-34)] were equipotent in their effects, whereas bovine [Nle(8,18)Tyr34]PTH-(3-34) amide [bPTH-(3-34)] and hPTH-(1-31) amide [hPTH-(1-31)] were less potent than bPTH-(1-34). bPTH-(3-34) did not antagonize the effects of bPTH-(1-34). Down-regulation of protein kinase C isozymes by 24-h treatment with PDBu completely prevented the stimulatory effect of PDBu on PC hydrolysis, but did not significantly affect the stimulatory effect of bPTH-(1-34). Both bPTH-(1-34) and PDBu stimulated transphosphatidylation of PC, indicating a phospholipase D-stimulated mechanism. The results suggest that in the UMR-106 cell line PTH can stimulate activation of PLD by a mechanism other than through protein kinase C.