Yutaka Fukuma

Suppression of RANKL‐dependent heme oxygenase‐1 is required for high mobility group box 1 release and osteoclastogenesis

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. J. Cell. Biochem. 113: 486–498, 2012.

Deltamethrin inhibits osteoclast differentiation via regulation of heme oxygenase-1 and NFATc1

Deltamethrin is a widely used pyrethroid pesticide. Although the cytotoxicity of deltamethrin has been reported, especially in neuronal cells, there is no information concerning the effects of deltamethrin on osteoclasts (OCLs). In this study, we showed that deltamethrin inhibited OCL differentiation in vitro. The effects of deltamethrin on OCL differentiation by receptor activator of nuclear factor kappa-B ligand (RANKL) were investigated in bone marrow-derived macrophages (BMMs) or the murine monocytic cell line RAW-D. Treatment with deltamethrin inhibited OCL formation and bone resorption and up-regulated expression of heme oxygenase-1 (HO-1), an anti-oxidative stress enzyme. Deltamethrin also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a master regulator for OCL differentiation, and concomitantly reduced the expression levels of Src and cathepsin K, which are transcriptionally regulated by NFATc1. The effects of deltamethrin on intracellular signaling during the OCL differentiation of BMMs indicated that deltamethrin-treated OCLs displayed impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, Jun N-terminal kinase, and Akt, and slightly delayed phosphorylation of inhibitor of nuclear factor kappa B alpha (IκBα) compared with untreated OCLs. Thus, deltamethrin possibly affects bone metabolism by inhibiting OCL differentiation.

Genetic backgrounds and redox conditions influence morphological characteristics and cell differentiation of osteoclasts in mice

Osteoclasts (OCLs) are multinucleated giant cells and are formed by the fusion of mononuclear progenitors of monocyte/macrophage lineage. It is known that macrophages derived from different genetic backgrounds exhibit quite distinct characteristics of immune responses. However, it is unknown whether OCLs from different genetic backgrounds show distinct characteristics. In this study, we showed that bone-marrow macrophages (BMMs) derived from C57BL/6, BALB/c and ddY mice exhibited considerably distinct morphological characteristics and cell differentiation into OCLs. The differentiation of BMMs into OCLs was comparatively quicker in the C57BL/6 and ddY mice, while that of BALB/c mice was rather slow. Morphologically, ddY OCLs showed a giant cell with a round shape, C57BL/6 OCLs were of a moderate size with many protrusions and BALB/c OCLs had the smallest size with fewer nuclei. The intracellular signaling of differentiation and expression levels of marker proteins of OCLs were different in the respective strains. Treatment of BMMs from the three different strains with the reducing agent N-acetylcysteine (NAC) or with the oxidation agent hydrogen peroxide (H2O2) induced changes in the shape and sizes of the cells and caused distinct patterns of cell differentiation and survival. Thus, genetic backgrounds and redox conditions regulate the morphological characteristics and cell differentiation of OCLs.

Inhibitory effects of tert-butylhydroquinone on osteoclast differentiation via up-regulation of heme oxygenase-1 and down-regulation of HMGB1 release and NFATc1 expression

Osteoclasts (OCLs) are multinucleated bone‐resorbing cells that are differentiated by receptor activator of nuclear factor kappa‐B ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF). Our recent studies have shown that heme‐oxygenase‐1 (HO‐1), a stress‐induced cytoprotective enzyme, plays an important role in OCL differentiation, although the pharmacological significance of this effect remains unknown. In this study, we investigated the effects of tert‐butylhydroquinone (tBHQ), a pharmacological HO‐1 inducer, on in vitro differentiation of bone marrow‐derived macrophages (BMMs) or murine monocytic cell line RAW‐D into OCLs. tBHQ inhibited the formation and the bone‐resorbing activity of OCLs. Moreover, tBHQ treatment decreased the expression of nuclear factor of activated T cells cytoplasmic‐1 (NFATc1), a master regulator of OCL differentiation, and of OCL markers transcriptionally regulated by NFATc1, such as Src and cathepsin K. In addition, tBHQ impaired phosphorylation of extracellular signal‐regulated kinase, p38 mitogen‐activated protein kinase (MAPK), Jun N‐terminal kinase, Akt, and inhibitor of nuclear factor kappa B alpha (IκBα). Finally, we show that tBHQ inhibited the release of high mobility group box 1 (HMGB1), a recently identified activator of OCL differentiation. Thus, tBHQ inhibits OCL differentiation through the HO‐1/HMGB1 pathways. Copyright

Identification of the pentapeptide constituting a dominant epitope common to all eukaryotic heat shock protein 90 molecular chaperones

Abstract We previously reported that, in human heat shock protein (Hsp) 90 (hHsp90), there are 4 highly immunogenic sites, designated sites Ia, Ib, Ic, and II. This study was performed to further characterize their epitopes and to identify the epitope that is potentially common to all members of the Hsp90 family. Panning of a bacterial library carrying randomized dodecapeptides revealed that Glu251-Ser-X-Asp254 constituted site Ia and Pro295-Ile-Trp-Thr-Arg299, site Ic. Site II (Asp701-Pro717) was composed of several epitopes. When 19 anti-hHsp90 monoclonal antibodies (mAbs) were subjected to immunoblotting against recombinant forms of 7 Hsp90-family members, 2 mAbs (K41110 and K41116C) that recognized site Ic bound to yeast Hsp90 with affinity identical to that for hHsp90, and 1 mAb (K3729) that recognized Glu222-Ala231 of hHsp90β could bind to human 94-kDa glucose-regulated protein (Grp94), an endoplasmic reticulum paralog of Hsp90. Among the 5 amino acids constituting site Ic, Trp297 and Pro295 were essential for recognition by all anti–site-Ic mAbs, and Arg299 was important for most of them. The necessity of Ile296, Thr298, and Arg299, which are replaced by Leu, Met/Leu, and Lys, respectively, in some eukaryotic Hsp90, was dependent on the mAbs, and K41110 and K41116C could react with Hsp90s carrying these substitutions. From these data taken together, we propose that the pentapeptide Pro295-Ile-Trp-Thr-Arg299 of hHsp90 functions as an immunodominant epitope common to all eukaryotic Hsp90.

Cafestol has a weaker inhibitory effect on osteoclastogenesis than kahweol and promotes osteoblast differentiation

Bone homeostasis is regulated by a balance between osteoclast (OCL)-mediated bone resorption and osteoblast (OBL)-mediated bone formation. Thus, developing a compound that simultaneously inhibits OCL function and promotes OBL function would be useful as a new medical therapy for bone diseases. Here, we examined the effects of cafestol, a coffee diterpene, on the differentiation of OCLs and OBLs. Cafestol prevented OCL formation in a dose-dependent manner and suppressed the bone-resorbing activity of OCLs. Interestingly, the viability of OCLs treated with 10-50 µM cafestol was significantly higher than that of untreated cells. At the molecular level, cafestol markedly decreased RANKL-induced phosphorylation of extracellular signal-regulated kinase (Erk) and inhibitor of nuclear factor kappa B alpha (IκBα). Compared to kahweol, another coffee-specific diterpene, the inhibitory effects of cafestol were milder on OCL differentiation, and cafestol and kahweol showed different characteristics in induction of the phase ΙΙ antioxidant enzymes and sensitivities in nuclear factor-erythroid 2-related factor 2 (Nrf2)-deficient BMMs. In addition to inhibiting OCLs, cafestol enhanced the differentiation of osteoblastic cells by increasing the mRNA levels of differentiation markers. Thus, cafestol inhibits OCL differentiation and promotes OBL differentiation, suggesting that cafestol may be a novel agent for bone diseases.

Dual Effects of Liquiritigenin on the Proliferation of Bone Cells: Promotion of Osteoblast Differentiation and Inhibition of Osteoclast Differentiation.

Bone is constantly controlled by a balance between osteoblastic bone formation and osteoclastic bone resorption. Liquiritigenin is a plant‐derived flavonoid and has various pharmacological effects, such as antioxidative, antitumor, and antiinflammatory effects. Here, we show that liquiritigenin has dual effects on the proliferation of bone cells, regarding the promotion of osteoblast differentiation and the inhibition of osteoclast differentiation. Liquiritigenin‐treated murine osteoblastic MC3T3‐E1 cells showed an increased alkaline phosphatase activity and enhanced phosphorylation of Smad1/5 compared with untreated cells. Moreover, liquiritigenin inhibited osteoclast differentiation, its bone‐resorption activity through slightly decreased the phosphorylation of extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase, and inhibitor of nuclear factor kappa Bα; however, the phosphorylation of Akt and p38 slightly increased in bone marrow‐derived osteoclasts. The expression levels of the osteoclast marker proteins nuclear factor of activated T‐cell cytoplasmic‐1, Src, and cathepsin K diminished. These results suggest that liquiritigenin may be useful as a therapeutic and/or preventive agent for osteoporosis or inflammatory bone diseases. Copyright

Cobalt protoporphyrin represses osteoclastogenesis through blocking multiple signaling pathways

Cobalt protoporphyrin (CoPP) is a metallo-protoporphyrin that works as a powerful inducer of heme oxigenase-1 (HO-1) in various tissues and cells. Our recent studies have demonstrated that induction of HO-1 by several reagents inhibited differentiation and activation of osteoclasts (OCLs), which are multinucleated bone resorbing cells. However, the effects of CoPP on osteoclastogenesis remain to be elucidated. In this study, we report that CoPP inhibits receptor activator of nuclear factor κB ligand (RANKL)-induced OCL formation in a dose dependent manner. Importantly, CoPP had little cytotoxicity, but rather enhanced cell proliferation of OCLs. CoPP suppressed the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1) as well as those of OCLs markers such as Src and cathepsin K, which are transcriptionally regulated by NFATc1 in mature OCLs. Western blot analyses also showed that CoPP abolished RANKL-stimulated phosphorylation of several major signaling pathways such as IκB, Akt, ERK, JNK and p38 MAPKs in OCL precursor cells. Thus, our results show that CoPP represses osteoclastogenesis through blocking multiple signaling pathways.

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

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 in vivo without severe gross abnormalities. In addition, Keap1‐deficient macrophages were unable to differentiate into osteoclasts in vitro via 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 Mafb, 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 Irf8 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. FASEB J. 31, 4011–4022 (2017). www.fasebj.org—Sakai, Eiko, Morita, Masanobu, Ohuchi, Masahiro, Kido, Mizuho A., Fukuma, Yutaka, Nishishita, Kazuhisa, Okamoto, Kuniaki, Itoh, Ken, Yamamoto, Masayuki, Tsukuba, Takayuki, 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. FASEB J. 31, 4011–4022 (2017)

Sanguiin H-6, a constituent of Rubus parvifolius L., inhibits receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis and bone resorption in vitro and prevents tumor necrosis factor-α-induced osteoclast formation in vivo.

BACKGROUND Osteoclasts are multinucleated bone-resorbing cells that differentiate in response to receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). Enhanced osteoclastogenesis contributes to bone diseases, such as osteoporosis and rheumatoid arthritis. Rubus parvifolius L. is traditionally used as an herbal medicine for rheumatism; however, its detailed chemical composition and the molecular mechanisms responsible for its biological action have not been elucidated. PURPOSE To investigate the mechanisms by which R. parvifolius L. extract and its major constituent sanguiin H-6, inhibit osteoclastogenesis and bone resorption. METHODS Cell proliferation, cell differentiation, and bone resorption were detected in vitro. Inhibition of signaling pathways, marker protein expression, and protein nuclear translocation were evaluated by western blot analysis. Tumor necrosis factor-α (TNF-α)-mediated osteoclastogenesis was examined in vivo. RESULTS R. parvifolius L. extract inhibited the bone-resorption activity of osteoclasts. In addition, sanguiin H-6 markedly inhibited RANKL-induced osteoclast differentiation and bone resorption, reduced reactive oxygen species production, and inhibited the phosphorylation of inhibitor of NF-κB alpha (IκBα) and p38 mitogen-activated protein kinase. Sanguiin H-6 also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), cathepsin K, and c-Src. Moreover, sanguiin H-6 inhibited the nuclear translocation of NFATc1, c-Fos, and NF-κB in vitro, as well as TNF-α-mediated osteoclastogenesis in vivo. CONCLUSIONS Our data revealed that R. parvifolius L. has anti-bone resorption activity and suggest that its constituent, sanguiin H-6, can potentially be used for the prevention and treatment of bone diseases associated with excessive osteoclast formation and subsequent bone destruction.