Stimulation of Osteoprotegerin Ligand and Inhibition of Osteoprotegerin Production by Glucocorticoids in Human Osteoblastic Lineage Cells: Potential Paracrine Mechanisms of Glucocorticoid-Induced Osteoporosis

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Abstract
Osteoporosis is a serious complication of systemic glucocorticoid use. However, while glucocorticoids increase bone resorption in vitro and in vivo, the mechanism(s) of this effect are at present unclear. Recent studies have identified the osteoprotegerin (OPG) ligand (OPG-L) as the final effector of 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 os- teoblastic lineage cells using Northern analysis, RT-PCR, and ELISA. Dexamethasone inhibited constitutive OPG messenger RNA (mRNA) steady-state levels by 70 -90% in primary (MS) and immortalized stromal cells (hMS), primary trabecular osteoblasts (hOB), immor- talized fetal osteoblasts (hFOB), and osteosarcoma cells (MG-63). In hFOB cells, dexamethasone inhibited constitutive OPG mRNA steady-state levels in a dose- and time-dependent fashion by 90%, and also suppressed cytokine-stimulated OPG mRNA steady-state levels. Dexamethasone-induced inhibition of OPG mRNA levels was not affected by the protein synthesis inhibitor, cycloheximide, and was shown to be due to inhibition of OPG gene transcription using a nuclear run-on assay. Moreover, dexamethasone also dose depen- dently (10210 M-1027 M) inhibited constitutive OPG protein concen- trations in the conditioned medium of hFOB cells from 2.59 6 0.02 ng/ml (control) to 0.30 6 0.01 ng/ml (88% inhibition; P , 0.001 by ANOVA). Concurrently, dexamethasone stimulated OPG-L mRNA steady-state levels in MS and hFOB cells by 2- and 4-fold, respec- tively. Treatment of murine marrow cultures with conditioned me- dium harvested from dexamethasone-treated MG-63 cells increased tartrate-resistant acid phosphatase (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 (1028 M) promoted osteoclast formation in vitro, as assessed by a 2.5-fold increase of TRAP activity in cell lysates (P , 0.001) and the appearance of TRAP-positive multinucleated cells. Our data are thus consistent with the hypothesis that glucocor- ticoids promote osteoclastogenesis by inhibiting OPG and concur- rently stimulating OPG-L production by osteoblastic lineage cells, thereby enhancing bone resorption. (Endocrinology 140: 4382- 4389, 1999)

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