Dihydroartemisinin represses osteoclastogenesis of bone marrow macrophages through reduced NFATc1 expression and impaired phosphorylation of IκBα.

Osteoclasts are multinucleated bone resorbing cells whose differentiation is regulated by several important signaling pathways. Several lines of evidence indicate that dihydroartemisinin (DHA), an anti-malarial drug, inhibits osteoclast differentiation with little cytotoxicity. However, the detailed inhibitory mechanisms of DHA on osteoclastogenesis from native cells remain to be elucidated. In this study, we investigated the effects of DHA on the differentiation of bone marrow-derived macrophages into osteoclasts. DHA inhibited receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclast formation and its bone resorbing activity. Mechanistically, DHA treatment markedly abolished phosphorylation of IκBα, and slightly affected a p38 MAPK dependent pathway. Moreover, DHA treatment induced down-regulation of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator for osteoclast differentiation and its target proteins, such as Src and cathepsin K. These results indicate that DHA represses RANKL-induced osteoclastogenesis of bone marrow macrophages through reduced NFATc1 expression and impaired phosphorylation of IκBα.

Rutaecarpine attenuates osteoclastogenesis by impairing macrophage colony stimulating factor and receptor activator of nuclear factor κ-B ligand-stimulated signalling pathways.

Rutaecarpine is a major alkaloid isolated from Evodia rutaecarpa. Here, we investigated the effects of rutaecarpine on osteoclast differentiation induced by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κ-B ligand (RANKL) in bone marrow-derived macrophages (BMMs). Treatment with rutaecarpine significantly inhibited osteoclastogenesis and prevented bone resorption of BMM-derived osteoclasts. Mechanistically, rutaecarpine decreased the protein level of nuclear factor of activated T cells cytoplasmic-1 (NFATc1) and the phosphorylation of other signalling pathways during the osteoclast differentiation. Thus, rutaecarpine may be useful as a therapeutic agent for the treatment of bone diseases.

The Rho-specific guanine nucleotide exchange factor Plekhg5 modulates cell polarity, adhesion, migration, and podosome organization in macrophages and osteoclasts.

Osteoclasts are multinucleated bone-resorbing cells that are formed by fusion of monocyte/macrophage lineage. Osteoclasts and macrophages generate podosomes that are actin-based dynamic organelles implicated in cell adhesion, spreading, migration, and degradation. However, the detailed mechanisms of podosome organization remain unknown. Here, we identified the Rho-specific guanine-nucleotide exchange factor (Rho-GEF) Plekhg5 as an up-regulated gene during differentiation of osteoclasts from macrophages. Knockdown of Plekhg5 with small interfering RNA in both macrophages and osteoclasts induced larger cell formation with impaired cell polarity and resulted in an elongated and flattened shape. In macrophages, Plekhg5 depletion enhanced random migration, but impaired directional migration, adhesion, and matrix degradation. Plekhg5 in osteoclasts affected random migration, podosome organization, and bone resorption. Plekhg5 depletion affected signaling and localization of several Rho downstream effectors. In fact, end-binding protein 1 (EB1), cofilin and vinculin were abnormally localized in Plekhg5-depleted cells, and mDia1 and LIM kinase (LIMK)1 were upregulated in Plekhg5-depleted cells compared with control cells. However, overexpression of Plekhg5 in macrophages induced an increase in its mRNA level, but failed to increase the protein level, indicating that overexpressed Plekhg5 was degraded in macrophages but not HEK293T cells. Thus, Plekhg5 affects cell polarity, migration, adhesion, degradation, and podosome organization in macrophages and osteoclasts.

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 vitrovia 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 1ß 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.

The dental resin monomers HEMA and TEGDMA have inhibitory effects on osteoclast differentiation with low cytotoxicity.

The dental resin monomers 2-hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) are released from the resin matrix due to unpolymerized monomers; once released, they influence various biological functions and the viability of cells in the oral environment. Although HEMA and TEGDMA have various effects on cells, including inflammation, inhibition of cell proliferation or differentiation, and apoptosis, the effects of these monomers on osteoclasts remain unknown. In this study, we investigated the effects of HEMA and TEGDMA on osteoclast differentiation of bone marrow-derived macrophages or murine monocytic cell line RAW-D. Both HEMA and TEGDMA inhibited osteoclast formation and their bone-resorbing activity at non-cytotoxic concentrations. Moreover, HEMA and TEGDMA decreased the expression of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator of osteoclast differentiation, and of osteoclast markers that are transcriptionally regulated by NFATc1, including Src and cathepsin K. Regarding their effects on signaling pathways involved in osteoclast differentiation, HEMA impaired the phosphorylation of extracellular signal-regulated kinase and Jun N-terminal kinase, whereas TEGDMA attenuated the phosphorylation of Akt and Jun N-terminal kinase. Thus, HEMA and TEGDMA inhibit osteoclast differentiation through different signaling pathways. This is the first report on the effects of the monomers HEMA and TEGDMA on osteoclasts. Copyright © 2017 John Wiley & Sons, Ltd.

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.

The Transcription Factor EB (TFEB) Regulates Osteoblast Differentiation Through ATF4/CHOP-Dependent Pathway.

Osteoblasts are bone-forming cells that produce large amounts of collagen type I and various bone matrix proteins. Although osteoblast differentiation is highly regulated by various factors, it remains unknown whether lysosomes are directly involved in osteoblast differentiation. Here, we demonstrate the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, modulates osteoblast differentiation. The expression levels of TFEB as well as those of endosomal/lysosomal proteins were up-regulated during osteoblast differentiation using mouse osteoblastic MC3T3-E1 cells. By gene knockdown (KD) experiments with small interfering RNA (siRNA), TFEB depletion caused markedly reduced osteoblast differentiation as compared with the control cells. Conversely, overexpression (OE) of TFEB resulted in strikingly enhanced osteoblastogenesis compared to the control cells. By analysis of down-stream effector molecules, TFEB KD was found to cause marked up-regulation of activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP), both of which are essential factors for osteoblastogenesis. In contrast, TFEB OE promoted osteoblast differentiation through reduced expression of ATF4 and CHOP without differentiation agents. Given the importance of ATF4 and CHOP in osteoblastogenesis, it is clear that the TFEB-regulated signaling pathway for osteoblast differentiation is involved in ATF4/CHOP-dependent signaling pathway.

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.

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.

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.

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.

Fisetin inhibits osteoclastogenesis through prevention of RANKL-induced ROS production by Nrf2-mediated up-regulation of phase II antioxidant enzymes.

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 RANKL-mediated ROS production by Nrf2-mediated up-regulation of phase II antioxidant enzymes.

Structural and phylogenetic comparison of napsin genes: the duplication, loss of function and human-specific pseudogenization of napsin B.

Aspartic proteinases form a widely distributed protein superfamily, including cathepsin D, cathepsin E, pepsins, renin, BACE and napsin. Human napsin genes are located on human chromosome 19q13, which comprises napsin A and napsin B. Napsin B has been annotated as a pseudogene because it lacks an in-frame stop codon; its nascent chains are cotranslationally degraded. Until recently, there have been no studies concerning the molecular evolution of the napsin protein family in the human genome. In the present study, we investigated the evolution and gene organization of the napsin protein family. Napsin B orthologs are primarily distributed in primates, while napsin A orthologs are the only napsin genes in other species. The corresponding regions of napsin B in the available sequences from primate species contain an in-frame stop codon at a position equivalent to that of human napsin A. In addition, a rare single-nucleotide polymorphism (SNP) that creates a proper stop codon in human napsin B was identified using HapMap populations. Recombinant protein expression and three-dimensional comparative modeling revealed that napsin B exhibits residual activity toward synthetic aspartic protease substrates compared with napsin A, presumably through a napsin B-specific Arg287 residue. Thus, napsin B was duplicated from napsin A during the early stages of primate evolution, and the subsequent loss of napsin B function during primate evolution reflected ongoing human-specific napsin B pseudogenization.

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.

The coffee diterpene kahweol prevents osteoclastogenesis via impairment of NFATc1 expression and blocking of Erk phosphorylation.

Osteoclasts (OCLs) are multinucleated bone resorbing cells whose differentiation is regulated by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating 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.

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 (H(2)O(2)) 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.

Efferent fibers innervate gustatory and mechanosensitive afferent fibers in frog fungiform papillae.

A possibility of efferent innervation of gustatory and mechanosensitive afferent fiber endings was studied in frog fungiform papillae with a suction electrode. The amplitude of antidromic impulses in a papillary afferent fiber induced by antidromically stimulating an afferent fiber of glossopharyngeal nerve (GPN) with low voltage pulses was inhibited for 40 s after the parasympathetic efferent fibers of GPN were stimulated orthodromically with high voltage pulses at 30 Hz for 10 s. This implies that electrical positivity of the outer surface of papillary afferent membrane was reduced by the efferent fiber-induced excitatory postsynaptic potential. The inhibition of afferent responses in the papillae was blocked by substance P receptor blocker, L-703,606, indicating that substance P is probably released from the efferent fiber terminals. Slow negative synaptic potential, which corresponded to a slow depolarizing synaptic potential, was extracellularly induced in papillary afferent terminals for 45 s by stimulating the parasympathetic efferent fibers of GPN with high voltage pulses at 30 Hz for 10 s. This synaptic potential was also blocked by L-703,606. These data indicate that papillary afferent fiber endings are innervated by parasympathetic efferent fibers.

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.

The receptor potential of frog taste cells in response to cold and warm stimuli.

Temperature sensitivity of frog taste cells was studied. The taste cell designated Type thermosensitive (TS) I cell was depolarized by warm stimulus at 30 degrees C and hyperpolarized by cold stimulus at 10 degrees C. The taste cell designated Type TS II cell was depolarized by the cold stimulus and hyperpolarized by the warm stimulus. Menthol solution at 20 degrees C, which selectively activates transient receptor potential (TRP) M8 channels sensitive to cold stimuli, depolarized Type TS II cells but not Types TS I cells. Thermal stimuli-induced receptor potentials were all blocked by a nonselective cation channel blocker flufenamic acid. The results indicate that Type TS I cells have warm sensor channels alone, Type TS II cells have cold sensor channels alone and both the channels are a nonselective cation channel. The candidate of cold sensor channel in Type TS II cells is a TRPM8 channel and that of warm sensor channel in Type TS I cells is likely to be a TRPM4-like channel from the published data. In a subset of taste cells, Types TS III and TS IV cells were found. The former was depolarized by both cold and warm stimuli, but the latter was hyperpolarized by both stimuli. Types TS III and TS IV cells might have both TRPM4-like and TRPM8 channels. It is supposed that depolarizations induced by both cold and warm stimuli were dominant in Type TS III cells and hyperpolarizations induced by both the thermal stimuli were dominant in Type TS IV cells.

Effect of gap junction blocker beta-glycyrrhetinic acid on taste disk cells in frog.

A gap junction blocker, 18beta-glycyrrhetinic acid (beta-GA), increased the membrane resistance of Ia, Ib and II/III cells of frog taste disk by 50, 160, and 300 M Omega, respectively, by blocking the gap junction channels and hemichannels. The amplitudes of gustatory depolarizing potentials in the disk cells for 4 basic taste stimuli were reduced to 40-60% after intravenous injection of beta-GA at 1.0 mg/kg. beta-GA of 1.0 mg/kg did not affect the resting potentials and the reversal potentials for tastant-induced depolarizing potentials in any taste disk cells. The percentage of cells responding to each of 4 basic taste stimuli and varying numbers of 4 taste qualities did not differ between control and beta-GA-treated taste disk cells. This implies that gustatory depolarizing response profiles for 4 basic taste stimuli were very similar in control and beta-GA-treated taste disk cells. It is concluded that beta-GA at 1.0 mg/kg reduced the amplitude of gustatory depolarizing potentials in taste disk cells by strongly blocking depolarizing currents flowing through the gap junction channels and hemichannels, but probably weakly affected the gustatory transduction mechanisms for 4 taste stimuli.