Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells: Potential paracrine mechanisms of glucocorticoid-induced osteoporosis

Osteoporosis is a serious complication of systemic glucocorticoid use. Howe ver, while glucocorticoids increase bone resorption in vitro and in vivo, t he mechanism(s) of this effect are at present unclear. Recent studies have identified the osteoprotegerin (OPG) ligand (OPG-L) as the final effector o f osteoclastogenesis, an action that is opposed by the soluble neutralizing receptor, OPG. Thus, we assessed glucocorticoid regulation of OPG and OPG- L in various human osteoblastic lineage cells using Northern analysis, RT-P CR, and ELISA. Dexamethasone inhibited constitutive OPG messenger RNA (mRNA ) steady-state levels by 70-90% in primary (MS) and immortalized stromal ce lls (hMS), primary trabecular osteoblasts (hOB), immortalized fetal osteobl asts (hFOB), and osteosarcoma cells (MG-63). In hFOB cells, dexamethasone i nhibited constitutive OPG mRNA steady-state levels in a dose- and time-depe ndent fashion by 90%, and also suppressed cytokine-stimulated OPG mRNA stea dy-state levels. Dexamethasone-induced inhibition of OPG mRNA levels was no t affected by the protein synthesis inhibitor, cycloheximide, and was shown to be due to inhibition of OPG gene transcription using nuclear run-on ass ay. Moreover, dexamethasone also dose dependently (10(-10) M-10(-7) M) inhi bited constitutive OPG protein concentrations in the conditioned medium of hFOB cells from 2.59 +/- 0.02 ng/ml (control) to 0.30 +/- 0.01 ng/ml (88% i nhibition; P < 0.001 by ANOVA). Concurrently, dexamethasone stimulated OPG- L mRNA steady-state levels in MS and hFOB cells by 2- and 4-fold, respectiv ely. Treatment of murine marrow cultures with conditioned medium harvested from dexamethasone-treated MG-63 cells increased tartrate-resistant acid ph osphatase (TRAP) activity by 54% (P < 0.005) compared with medium harvested from control-treated cells (in the presence of OPG-L and macrophage colony -stimulating factor). Moreover, dexamethasone (10(-8) M) promoted osteoclas t formation in vitro, as assessed by a 2.5-fold increase of TRAP activity i n cell lysates (P < 0.001) and the appearance of TRAP-positive multinucleat ed cells. Our data are thus consistent with the hypothesis that glucocortic oids promote osteoclastogenesis by inhibiting OPG and concurrently stimulat ing OPG-L production by osteoblastic lineage cells, thereby enhancing bone resorption.