The immunosuppressant rapamycin, alone or with transforming growth factor-β, 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 β (TGF-β) receptors, and TGF-β enhances osteoclastogenesis induced by RANKL, we also examined potential synergistic effects of rapamycin and TGF-β₁. 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-β₁(1 ng/ml) increased TRAP⁺MNC 3.1- and 6.9-fold as compared with rapamycin or TGF-β₁ alone, respectively, and enhanced CTR mRNA expression induced by TGF-β₁ 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-β by 3-fold, compared with rapamycin alone. These data thus indicate that rapamycin, alone or in synergy with TGF-β, directly enhances osteoclastogenesis and may affect bone metabolism in vivo after long-term use.