Influence of Glucocorticoids on Human Osteoclast Generation and Activity

Abstract

Using human peripheral blood mononuclear cells as osteoclast precursors, we showed that dexamethasone stimulated osteoclast generation at a pharmacological concentration but did not affect the life span of human osteoclasts. Dexamethasone also dose-dependently increased signals for osteoclastogenesis.Introduction: Glucocorticoid-induced osteoporosis is a common and serious disease. Glucocorticoids predominantly affect osteoblast proliferation and life span. Much of the bone loss is caused by reduced bone formation, but there is also an element of increased bone resorption. Materials and Methods: Human peripheral blood mononuclear cells were cultured on whale dentine and induced to differentiate to osteoclasts by RANKL and human macrophage-colony stimulating factor (M-CSF). Osteoclast activity was quantified by pit area. RANKL and osteoprotegerin (OPG) expression in osteoblasts were measured by real-time RT-PCR. Results: In the early phase of osteoclast generation (0-16 days), cultures from two different donors showed that dexamethasone at 10 −8 M increased pit area by 2.5-fold, whereas lower concentrations had no effect. At the highest dexamethasone concentration (10 −7 M), pit area was reduced. In 21-day cultures from three other donors, a similar increase was seen with dexamethasone at 10 −8 M. There was, however, no evidence of increased life span of osteoclasts with dexamethasone. In human primary osteoblasts, dexamethasone dosedependently reduced OPG and increased RANKL expression as measured by quantitative real time RT-PCR. Conclusion: These data provide some explanation at a cellular and molecular level for the observed increase in bone resorption seen in patients treated with glucocorticoids and indicate that there are clear direct effects of glucocorticoids on bone resorption in human cell systems that may differ from other species.

Glucocorticoids maintain human osteoclasts in the active mode of their resorption cycle

“…To address this issue, we investigated whether GC affects OC resorption directly in a standard 3-day culture pit assay. At low resolution, toluidine staining did not show any remarkable differences in the extent of resorbed surface, in agreement with an earlier report that also used human OCs, (13) but at higher magnification we found a change in the shape of the resorption pits (Fig. 1A).…”

“…However, there are data indicating that TRACP is rather involved at the end of the resorption phase of the resorption cycle, (26,27) and in this case it makes sense that a prolongation of the resorption phase results in the observations made in this study, that is, a decreased TRACP release, which goes along with the increased levels of the resorption product CTX. Noteworthy, the generation of a high proportion of trenches in the presence of GCs is also supported by pictures from another study performed with human OCs generated from peripheral blood mononucleated cells, (13) but this report does not comment on the morphology of the excavations and mentions only the absence of effect of GCs on total resorption area. Our study is in accordance with this absence of effect on resorption area generated by human OCs, (13,15) but not with others, where less purified mouse or rat OCs were used and where GCs rather decreased total resorption areas.…”

“…Increased OC survival is stressed in several studies, (9)(10)(11) but according to other studies, OC survival is decreased by GCs or not affected. (12)(13)(14) Enhanced OC differentiation also has been proposed, (13,15,16) but others found that OC differentiation was decreased by GCs or not affected. (10,17,18) One would expect that besides survival and differentiation, the resorptive activity of existing OCs is also stimulated by GCs because of the very early increase in bone-resorption markers induced by GCs in patients.…”

“…The procedure was as follows: Bone slices were covered in chloroform/isoamyl alcohol (24:1) for 5 minutes at room temperature, dried on paper, incubated for 15 minutes at room temperature while shaking gently in twofold diluted NaOCl stock (10% to 15% active chloride in stock; Sigma-Aldrich), dried on paper, stained with 0.2% toluidin as described previously, (13,21) and analyzed for resorption depth as aforementioned.…”

“…(12,14) So also did OCs generated from mouse bone marrow cells cultured in the presence of macrophage colonystimulating factor (M-CSF) and receptor-activator of NF-kB ligand (RANKL), (10) whereas human OCs generated from peripheral blood mononuclear cells in the presence of the same cytokines and seeded on bone slices did not respond to high-dose GCs. (13,15) This absence of direct stimulation of the resorptive activity of OCs in vitro would fit an indirect mechanism of action of GCs on OCs. A series of indirect mechanisms have been proposed, (2,8) including elevation in parathyroid hormone (PTH) levels resulting from GC-induced impaired calcium uptake in the gut and the kidney, GC-induced reduction of gonadal hormones, and mediation through OB lineage cells.…”