Regulation and enzymatic basis of bone resorption by human osteoclasts.

Although much has been learned recently of the mechanisms that regulate osteoclastic differentiation, much less is known of the means through which their resorptive activity is controlled. This is especially so for human osteoclasts. We have recently developed an assay that allows us to measure resorptive activity while minimizing confounding effects on differentiation by optimizing osteoclastogenesis, so that measurable resorption occurs over a short period, and by relating resorption in each culture during the test period to the resorption that had occurred in the same culture in a prior control period.

A novel assay for analysis of the regulation of the function of human osteoclasts.

Background: Very little is known of the regulation of the function of human osteoclasts, largely due to the virtual impossibility of obtaining human osteoclasts ex vivo. It has recently become possible to generate human osteoclasts in vitro, by incubation of peripheral blood mononuclear cells (PBMCs) in macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL). However, the assays at present available do not distinguish clearly between the distinct effects of agents on differentiation and function.

Secretion of tartrate-resistant acid phosphatase by osteoclasts correlates with resorptive behavior.

There have been dramatic advances recently in our understanding of the regulation of osteoclastic differentiation. However, much less is known of the mechanisms responsible for the induction and modulation of resorptive behavior. We have developed a strategy whereby osteoclasts can be generated in vitro and released into suspension in a fully-functional state.

Murine osteoclast formation and function: differential regulation by humoral agents.

Although much has been learned recently of the mechanisms that regulate osteoclastic differentiation, much less is known of the means through which their resorptive activity is controlled. We have developed an assay that allows us to measure resorptive activity while minimizing the confounding effects of the test agent on differentiation. In this assay, murine osteoclasts were harvested from plastic substrates and sedimented onto bone slices in MEM with 10% fetal calf serum.

Hydrogen peroxide is essential for estrogen-deficiency bone loss and osteoclast formation.

We recently found that estrogen deficiency leads to a lowering of thiol antioxidant defenses in rodent bone. Moreover, administration of agents that increase the concentration in bone of glutathione, the main intracellular antioxidant, prevented estrogen-deficiency bone loss, whereas depletion of glutathione by buthionine sulfoximine administration provoked substantial bone loss. To analyze further the mechanism by which antioxidant defenses modulate bone loss, we have now compared expression of the known antioxidant enzymes in osteoclasts.

Thioredoxin-1 mediates osteoclast stimulation by reactive oxygen species.

We found that the antioxidant protein thioredoxin-1 (Trx) is more highly expressed in osteoclasts than in macrophages. Moreover, transfection of RAW 264.7 (RAW) cells with a Trx-expression construct resulted in a dramatic increase in their capacity for osteoclast formation.

A crucial role for thiol antioxidants in estrogen-deficiency bone loss.

The mechanisms through which estrogen prevents bone loss are uncertain. Elsewhere, estrogen exerts beneficial actions by suppression of reactive oxygen species (ROS). ROS stimulate osteoclasts, the cells that resorb bone.

TNFalpha potently activates osteoclasts, through a direct action independent of and strongly synergistic with RANKL.

TNFalpha is pivotal to the pathogenesis of inflammatory and possibly postmenopausal osteolysis. Much recent work has clarified mechanisms by which TNFalpha promotes osteoclastogenesis, but the means by which it activates osteoclasts to resorb bone remain uncertain. We found that very low concentrations of TNFalpha promoted actin ring formation, which correlates with functional activation in osteoclasts, both in osteoclasts formed in vitro and extracted from newborn rats.