The differentiation of osteoclasts is regulated by several essential cytokines, such as receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor. Recently, high mobility group box 1 (HMGB1), a chromatin protein, also has been identified as one of these osteoclast differentiation cytokines. However, the molecular mechanisms that control HMGB1 release from osteoclast precursor cells are not known. Here, we report that RANKL-induced suppression of heme oxygenase-1 (HO-1), a heme-degrading enzyme, promotes HMGB1 release during osteoclastogenesis. In contrast, induction of HO-1 with hemin or curcumin in bone marrow-derived macrophages or RAW-D murine osteoclast precursor cells inhibited osteoclastogenesis and suppressed HMGB1 release. Since an inhibitor for p38 mitogen-activated protein kinase (MAPK) prevented the RANKL-mediated HO-1 suppression and extracellular release of HMGB1, these effects were p38 MAPK-dependent. Moreover, suppression of HO-1 in RAW-D cells by RNA interference promoted the activation of caspase-3 and HMGB1 release, whereas overexpression of HO-1 inhibited caspase-3 activation as well as HMGB1 release. Furthermore, these effects were regulated by redox conditions since antioxidant N-acetylcysteine abolished the HO-1/HMGB1/caspase-3 axis. These results suggest that RANKL-dependent HO-1 suppression leads to caspase-3 activation and HMGB1 release during osteoclastogenesis.

Abstract. Osteoclasts (OCLs) are multinucleated bone resorbing cells whose differentiation is regulated by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colonystimulating factor (M-CSF). It is known that inflammatory cytokines and oxidative stress stimulate differentiation of OCLs. Here we evaluated the effects of kahweol, a coffee-specific diterpene, which has been reported to possess anti-oxidant and anti-inflammatory properties, on the differentiation of bone marrow-derived macrophages (BMMs) or murine monocytic cell line RAW-D cells into OCLs. Kahweol dose-dependently inhibited the formation of tartrate-resistant acid phosphatase staining-positive OCLs from both BMMs and RAW-D cells. In addition, kahweol prevented the bone resorbing activity of OCLs. Kahweol completely abolished RANKL-stimulated phosphorylation of extracellular signal-regulated kinase and impaired phosphorylation of Akt. Moreover, the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator for OCL differentiation; and OCL markers transcriptionally regulated by NFATc1 such as Src and cathepsin K were down-regulated by kahweol treatment. As one of the molecular mechanisms for the inhibitory effects of kahweol, we also showed that kahweol up-regulated heme oxygenase-1 and inhibited high mobility group box 1 release. Thus, kahweol in coffee is a useful constituent for inhibition of OCL differentiation.

Abstract. Osteoclasts (OCLs) are multinucleated bone-resorbing cells that are differentiated by stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor. We recently demonstrated that regulation of heme-oxygenase 1 (HO-1), a stress-induced cytoprotective enzyme, also functions in OCL differentiation. In this study, we investigated effects of fisetin, a natural bioactive flavonoid that has been reported to induce HO-1 expression, on the differentiation of macrophages into OCLs. Fisetin inhibited the formation of OCLs in a dose-dependent manner and suppressed the bone-resorbing activity of OCLs. Moreover, fisetin-treated OCLs showed markedly decreased phosphorylation of extracellular signal-regulated kinase, Akt, and Jun N-terminal kinase, but fisetin did not inhibit p38 phosphorylation. Fisetin up-regulated mRNA expression of phase II antioxidant enzymes including HO-1 and interfered with RANKL-mediated reactive oxygen species (ROS) production. Studies with RNA interference showed that suppression of NF-E2-related factor 2 (Nrf2), a key transcription factor for phase II antioxidant enzymes, rescued fisetin-mediated inhibition of OCL differentiation. Furthermore, fisetin significantly decreased RANKL-induced nuclear translocation of cFos and nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a transcription factor critical for osteoclastogenic gene regulation. Therefore, fisetin inhibits OCL differentiation through blocking RANKLmediated ROS production by Nrf2-mediated up-regulation of phase II antioxidant enzymes.

Kelch-like ECH-associated protein 1 (Keap1) binds to nuclear factor E2 p45-related factor 2 (Nrf2), a transcription factor for antioxidant enzymes, to suppress Nrf2 activation. The role of oxidative stress in many diseases supports the possibility that processes that are associated with Nrf2 activation might offer therapeutic potential. Nrf2 deficiency induces osteoclastogenesis, which is responsible for bone loss, by activating receptor activator of NF-κB ligand (RANKL)-mediated signaling; however, the effects of Keap1 deficiency remain unclear. By using Keap1-deficient newborn mice, we observed that talus and calcaneus bone formation was partially retarded and that osteoclast number was reduced without severe gross abnormalities. In addition, Keap1-deficient macrophages were unable to differentiate into osteoclasts attenuation of RANKL-mediated signaling and expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), a key transcription factor that is involved in osteoclastogenesis. Furthermore, Keap1 deficiency up-regulated the expression of , a negative regulator of NFATc1. RANKL-induced mitochondrial gene expression is required for down-regulation of IFN regulatory factor 8 (IRF-8), a negative transcriptional regulator of NFATc1. Our results indicate that Keap1 deficiency down-regulated peroxisome proliferator-activated receptor-γ coactivator 1β and mitochondrial gene expression and up-regulated expression. These results suggest that the Keap1/Nrf2 axis plays a critical role in NFATc1 expression and osteoclastogenic progression.-Sakai, E., Morita, M., Ohuchi, M., Kido, M. A., Fukuma, Y., Nishishita, K., Okamoto, K., Itoh, K., Yamamoto, M., Tsukuba, T. Effects of deficiency of Kelch-like ECH-associated protein 1 on skeletal organization: a mechanism for diminished nuclear factor of activated T cells cytoplasmic 1 during osteoclastogenesis.

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