Characterization of acetylcholinesterase expression and secretion during osteoblast differentiation.

Although best known for its role in cholinergic signalling, a substantial body of evidence suggests that acetylcholinesterase (AChE) has multiple biological functions. Previously, we and others identified AChE expression in areas of bone that lacked expression of other neuronal proteins. More specifically, we identified AChE expression at sites of new bone formation suggesting a role for AChE as a bone matrix protein. We have now characterised AChE expression, secretion and adhesive function in osteoblasts. Using Western blot analysis, we identified expression of two AChE species in osteoblastic cells, a major species of 68 kDa and less abundant species of approximately 55 kDa. AChE colocalised with the Golgi apparatus in osteoblastic cells and was identified in osteoblast-conditioned medium. Further analyses revealed differentiation-dependent secretion by osteoblasts, with AChE secretion levels corresponding with alkaline phosphatase activity. AChE expression by osteoblastic cells was also found to be regulated by mechanical strain both in vitro and in vivo. Finally, we investigated the possibility of a functional role for AChE in osteoblast adhesion. Using specific inhibitors, blockade of sites thought to be responsible for AChE adhesive properties caused a concentration-dependent decrease in osteoblastic cell adhesion, suggesting that AChE is involved in regulating cell-matrix interactions in bone.

Mechanical loading: biphasic osteocyte survival and targeting of osteoclasts for bone destruction in rat cortical bone.

Bone is removed or replaced in defined locations by targeting osteoclasts and osteoblasts in response to its local history of mechanical loading. There is increasing evidence that osteocytes modulate this targeting by their apoptosis, which is associated with locally increased bone resorption. To investigate the role of osteocytes in the control of loading-related modeling or remodeling, we studied the effects on osteocyte viability of short periods of mechanical loading applied to the ulnae of rats. Loading, which produced peak compressive strains of -0.003 or -0.004, was associated with a 78% reduction in the resorption surface at the midshaft. The same loading regimen resulted in a 40% relative reduction in osteocyte apoptosis at the same site 3 days after loading compared with the contralateral side (P = 0.01). The proportion of osteocytes that were apoptotic was inversely related to the estimated local strain (P < 0.02). In contrast, a single short period of loading resulting in strains of -0.008 engendered both tissue microdamage and subsequent bone remodeling and was associated with an eightfold increase in the proportion of apoptotic osteocytes (P = 0.02) at 7 days. This increase in osteocyte apoptosis was transient and preceded both intracortical remodeling and death of half of the osteocytes (P < 0.01). The data suggest that osteocytes might use their U-shaped survival response to strain as a mechanism to influence bone remodeling. We hypothesize that this relationship reflects a causal mechanism by which osteocyte apoptosis regulates bone's structural architecture.