To define the molecular mechanism(s) by which interleukin (IL)-4 reversibly inhibits formation of osteoclasts (OCs) from bone marrow macrophages (BMMs), we examined the capacity of this T cell-derived cytokine to impact signals known to modulate osteoclastogenesis, which include those initiated by macrophage colonystimulating factor (M-CSF), receptor for activation of NF-B ligand (RANKL), tumor necrosis factor (TNF), and IL-1. We find that although pretreatment of BMMs with IL-4 does not alter M-CSF signaling, it reversibly blocks RANKL-dependent activation of the NF-B, JNK, p38, and ERK signals. IL-4 also selectively inhibits TNF signaling, while enhancing that of IL-1. Contrary to previous reports, we find that MEK inhibitors dose-dependently inhibit OC differentiation. To identify more proximal signals mediating inhibition of OC formation by IL-4, we used mice lacking STAT6 or SHIP1, two adapter proteins that bind the IL-4 receptor. IL-4 fails to inhibit RANKL/M-CSF-induced osteoclastogenesis by BMMs derived from STAT6-, but not SHIP1-, knockout mice. Consistent with this observation, the inhibitory effects of IL-4 on RANKL-induced NF-B and mitogen-activated protein kinase activation are STAT6-dependent. We conclude that IL-4 reversibly arrests osteoclastogenesis in a STAT6-dependent manner by 1) preventing IB phosphorylation and thus NF-B activation, and 2) blockade of the JNK, p38, and ERK mitogen-activated protein kinase pathways.

SUMMARY Osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B (RANK) are members of the TNFR superfamily that regulate osteoclast formation and function by competing for RANK ligand (RANKL). RANKL promotes osteoclast development through RANK activation, while OPG inhibits this process by sequestering RANKL. For comparison, we solved crystal structures of RANKL with RANK, and RANKL with OPG. Complementary biochemical and functional studies reveal that the monomeric cytokine-binding region of OPG binds RANKL with ~500 fold higher affinity than RANK, and inhibits RANKL-stimulated osteoclastogenesis ~150 times more effectively, in part because the binding cleft of RANKL makes unique contacts with OPG. Several side chains as well as the C-D and D-E loops of RANKL occupy different orientations when bound to OPG versus RANK. High affinity OPG binding requires a 90s-loop Phe residue that is mutated in juvenile Paget’s disease. These results suggest cytokine plasticity may help to fine tune specific TNF-family cytokine/receptor pair selectivity.

Highly active antiretroviral therapy (HAART), which includes HIV protease inhibitors (PIs), has been associated with bone demineralization. To determine if this complication reflects accelerated resorptive activity, we studied the impact of two common HIV PIs, ritonavir and indinavir, on osteoclast formation and function. Surprisingly, we find that ritonavir, but not indinavir, inhibits osteoclast differentiation in a reversible manner and also abrogates bone resorption by disrupting the osteoclast cytoskeleton, without affecting cell number. Ritonavir given in vivo completely blunts parathyroid hormone-induced osteoclastogenesis in mice, which confirms that the drug is bone sparing. In keeping with its antiresorptive properties, ritonavir impairs receptor activator of nuclear factor κB ligand-induced (RANKL-induced) activation of NF-κB and Akt signaling pathways, both critical to osteoclast formation and function. In particular, ritonavir is found to inhibit RANKL-induced Akt signaling by disrupting the recruitment of TNF receptor-associated factor 6/c-Src complex to lipid rafts. Thus, ritonavir may represent a bone-sparing PI capable of preventing development of osteopenia in patients currently on HAART. IntroductionThroughout life, bone remodeling occurs by a finely orchestrated process of osteoclastic resorption and osteoblastic formation. When intact, this process ensures maintenance of skeletal integrity and calcium homeostasis. In all circumstances bone loss occurs when the activity of the resorptive cell exceeds that of its anabolic counterpart. For example, postmenopausal osteoporosis is caused by an absolute increase of osteoclasts and osteoblasts, with the former activity outpacing that of the latter (1). Senile (type II) osteoporosis, in contrast, is a low-turnover disease, but once again bone resorptive activity exceeds that of matrix deposition and calcification (1). Therefore, one approach to dissecting the cause of bone loss in a specific clinical circumstance is to examine the direct effects of various drugs on generation and activity of osteoclasts and osteoblasts.Osteoclasts are multinucleated cells generated by the fusion of mononuclear progenitors of the monocyte/macrophage family (2). The pathway involved in osteoclast differentiation and activation requires two key elements: receptor activator of nuclear factor κB (RANK), found in osteoclasts and their precursors, and RANK ligand (RANKL), produced by osteoblasts and stromal cells in the bone marrow (2, 3). In addition, M-CSF is required for survival and proliferation of osteoclast precursors. Ligation of RANKL to RANK on macrophages prompts selective intracellular signals that eventuate in the assumption of the osteoclast phenotype (4).

Receptor activator of nuclear factor-kappa B ligand [RANK ligand (RANK-L)] stimulates mature osteoclasts to resorb bone, a process associated with NF-kappa B activation. RANK-L also prompts macrophages to develop the osteoclast phenotype. Although NF-kappa B is essential for osteoclast differentiation, it is not known whether RANK-L activates this transcription complex in osteoclast precursors. We report that RANK-L rapidly induces NF-kappa B activation in both authentic osteoclast precursors, namely bone marrow macrophages, and RAW 264.7 cells, a murine macrophage line also capable of RANK-L-mediated osteoclastogenesis. Supershift studies reveal the RANK-L-induced DNA binding moiety contains p50/p65, the most common NF-kappa B complex. Subcellular translocation of p50 and p65 subunits is confirmed by Western blots and immunofluorescence analysis. RANK-L activates NF-kappa B in both bone marrow macrophages and RAW 264.7 cells by serine phosphorylation of I kappa B alpha within 5 min, resulting in rapid I kappa B alpha degradation and resynthesis. Attesting to function, RANK-L treatment of RAW 264.7 cells transiently transfected with a plasmid containing NF-kappa B consensus elements linked to luciferase greatly enhances reporter activity. Our data suggest that activation of the NF-kappa B pathway is an integral component of RANK-L-induced osteoclast differentiation.

Highly active antiretroviral therapy (HAART), which includes HIV protease inhibitors (PIs), has been associated with bone demineralization. To determine if this complication reflects accelerated resorptive activity, we studied the impact of two common HIV PIs, ritonavir and indinavir, on osteoclast formation and function. Surprisingly, we find that ritonavir, but not indinavir, inhibits osteoclast differentiation in a reversible manner and also abrogates bone resorption by disrupting the osteoclast cytoskeleton, without affecting cell number. Ritonavir given in vivo completely blunts parathyroid hormone-induced osteoclastogenesis in mice, which confirms that the drug is bone sparing. In keeping with its antiresorptive properties, ritonavir impairs receptor activator of nuclear factor κB ligand-induced (RANKL-induced) activation of NF-κB and Akt signaling pathways, both critical to osteoclast formation and function. In particular, ritonavir is found to inhibit RANKL-induced Akt signaling by disrupting the recruitment of TNF receptor-associated factor 6/c-Src complex to lipid rafts. Thus, ritonavir may represent a bone-sparing PI capable of preventing development of osteopenia in patients currently on HAART. IntroductionThroughout life, bone remodeling occurs by a finely orchestrated process of osteoclastic resorption and osteoblastic formation. When intact, this process ensures maintenance of skeletal integrity and calcium homeostasis. In all circumstances bone loss occurs when the activity of the resorptive cell exceeds that of its anabolic counterpart. For example, postmenopausal osteoporosis is caused by an absolute increase of osteoclasts and osteoblasts, with the former activity outpacing that of the latter (1). Senile (type II) osteoporosis, in contrast, is a low-turnover disease, but once again bone resorptive activity exceeds that of matrix deposition and calcification (1). Therefore, one approach to dissecting the cause of bone loss in a specific clinical circumstance is to examine the direct effects of various drugs on generation and activity of osteoclasts and osteoblasts.Osteoclasts are multinucleated cells generated by the fusion of mononuclear progenitors of the monocyte/macrophage family (2). The pathway involved in osteoclast differentiation and activation requires two key elements: receptor activator of nuclear factor κB (RANK), found in osteoclasts and their precursors, and RANK ligand (RANKL), produced by osteoblasts and stromal cells in the bone marrow (2, 3). In addition, M-CSF is required for survival and proliferation of osteoclast precursors. Ligation of RANKL to RANK on macrophages prompts selective intracellular signals that eventuate in the assumption of the osteoclast phenotype (4).