The Immunosuppressant Rapamycin, Alone or with Transforming Growth Factor-b, Enhances Osteoclast Differentiation of RAW264.7 Monocyte-Macrophage Cells in the Presence of RANK-Ligand

Immunosuppressant therapy is known to cause bone loss. Since this may partly result from direct effects on osteoclast development, we investigated whether cyclosporin A (CsA), rapamycin, or FK506 affect osteoclastic differentiation of RAW264.7 monocytic cells induced by RANK-ligand (RANKL). Furthermore, since the rapamycin receptor protein binds transforming growth factor b (TGF-b) receptors, and TGF-b enhances osteoclastogenesis induced by RANKL, we also examined potential synergistic effects of rapamycin and TGF-b1. Rapamycin inhibited cell proliferation and stimulated tartrate-resistant acid phosphatase (TRAP) activity of RAW cells in a dose-dependent manner. At the optimal concentration of 10 ng/ml, it increased the number of TRAP+ multinucleated cells (MNC) more than 20-fold and enhanced the expression of TRAP and calcitonin receptor (CTR) mRNAs 2.1- and 10-fold, respectively. CsA, at 125–2000 ng/ml, similarly inhibited proliferation, but at high doses (1000–2000 ng/ml) it decreased TRAP activity, TRAP+MNC formation, and the expression of TRAP and CTR mRNAs. FK506 had no effect on cell proliferation or TRAP activity at concentrations up to 2000 ng/ml; however, like CsA, 1000 ng/ml FK506 inhibited TRAP+MNC formation and the expression of TRAP and CTR mRNAs. The combination of rapamycin (10 ng/ml) and TGF-b1 (1 ng/ml) increased TRAP+MNC 3.1- and 6.9-fold as compared with rapamycin or TGF-b1 alone, respectively, and enhanced CTR mRNA expression induced by TGF-b1 by 1.9-fold. Rapamycin also increased osteoclastic resorption activity by 6.5-fold compared with control, and this was enhanced further by the addition of TGF-b by 3-fold, compared with rapamycin alone. These data thus indicate that rapamycin, alone or in synergy with TGF-b, directly enhances osteoclastogenesis and may affect bone metabolism in vivo after long-term use.