The immunosuppressant rapamycin, alone or with transforming growth factor-beta, 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 beta (TGF-beta) receptors, and TGF-beta enhances osteoclastogenesis induced by RANKL, we also examined potential synergistic effects of rapamycin and TGF-beta1. 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-beta1 (1 ng/ml) increased TRAP+MNC 3.1- and 6.9-fold as compared with rapamycin or TGF-beta1 alone, respectively, and enhanced CTR mRNA expression induced by TGF-beta1 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-beta by 3-fold, compared with rapamycin alone. These data thus indicate that rapamycin, alone or in synergy with TGF-beta, directly enhances osteoclastogenesis and may affect bone metabolism in vivo after long-term use.
Endocrine Research Unit, Division of Endocrinology and Metabolism, Mayo Clinic and Foundation, Rochester, Minnesota, USA.
C
C
Shui
B L
BL
Riggs
S
S
Khosla
D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004357:Drug Synergism; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D051379:Mice; D009000:Monocytes / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D010010:Osteoclasts / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D053245:RANK Ligand; D012333:RNA, Messenger / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D018003:Receptors, Calcitonin / Q000235:genetics / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology*; D016559:Tacrolimus / Q000494:pharmacology; D000071681:Tartrate-Resistant Acid Phosphatase; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D002352:Carrier Proteins; D004338:Drug Combinations; D007166:Immunosuppressive Agents; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; D018003:Receptors, Calcitonin; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D016212:Transforming Growth Factor beta; D016572:Cyclosporine; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase; D020123:Sirolimus; D016559:Tacrolimus