Abr, a Rho-regulating protein, modulates osteoclastogenesis by enhancing lamellipodia formation by interacting with poly(ADP-ribose) glycohydrolase.

BACKGROUND: Osteoclasts are multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage lineage. During osteoclast differentiation, Rho GTPases are involved in various processes, including cell migration, adhesion, and polarity. However, the role of Rho-regulatory molecules in the regulation of osteoclast differentiation remains unclear. In this study, among these genes, we focused on active breakpoint cluster region-related (Abr) protein that is a multifunctional regulator of Rho GTPases. METHODS AND RESULTS: We examined using knockdown and overexpression experiments in RANKL-stimulated RAW-D macrophages whether Abr regulates osteoclast differentiation and cell morphology. We observed an increase in Abr expression during osteoclast differentiation and identified expression of a variant of the Abr gene in osteoclasts. Knockdown of Abr suppressed osteoclast differentiation and resorption. Abr knockdown markedly inhibited the expression of osteoclast markers, such as Nfatc1, c-fos, Src, and Ctsk in osteoclasts. Conversely, overexpression of Abr enhanced the formation of multinucleated osteoclasts, bone resorption activity, and osteoclast marker gene expression. Moreover, Abr overexpression accelerated lamellipodia formation and induced the formation of well-developed actin in osteoclasts. Importantly, the Abr protein interacted with poly(ADP-ribose) glycohydrolase (PARG) and Rho GTPases, including RhoA, Rac1/2/3, and Cdc42 in osteoclasts. CONCLUSIONS: Taken together, these results indicate that Abr modulates osteoclastogenesis by enhancing lamellipodia formation via its interaction with PARG.

Rab44 Deficiency Induces Impaired Immune Responses to Nickel Allergy.

Rab44 was recently identified as an atypical Rab GTPase that possesses EF-hand and coiled-coil domains at the N-terminus, and a Rab-GTPase domain at the C-terminus. Rab44 is highly expressed in immune-related cells such as mast cells, macrophages, osteoclasts, and granulocyte-lineage cells in the bone marrow. Therefore, it is speculated that Rab44 is involved in the inflammation and differentiation of immune cells. However, little is known about the role of Rab44 in inflammation. In this study, we showed that Rab44 was upregulated during the early phase of differentiation of M1- and M2-type macrophages. Rab44-deficient mice exhibited impaired tumor necrosis factor alpha and interleukin-10 production after lipopolysaccharide (LPS) stimulation. The number of granulocytes in Rab44-deficient mice was lower, but the lymphocyte count in Rab44-deficient mice was significantly higher than that in wild-type mice after LPS stimulation. Moreover, Rab44-deficient macrophages showed impaired nickel-induced toxicity, and Rab44-deficient mice showed impaired nickel-induced hypersensitivity. Upon nickel hypersensitivity induction, Rab44-deficient mice showed different frequencies of immune cells in the blood and ears. Thus, it is likely that Rab44 is implicated in immune cell differentiation and inflammation, and Rab44 deficiency induces impaired immune responses to nickel allergies.

Rab34 plays a critical role as a bidirectional regulator of osteoclastogenesis.

Accumulating evidence suggests that Rab GTPases representing the largest branch of Ras superfamily have recently emerged as the core factors for the regulation of osteoclastogenesis through modulating vesicular transport amongst specific subcellular compartments. Among these, Rab34 GTPase has been identified to be important for the post-Golgi secretory pathway and for phagocytosis; nevertheless, its specific role in osteoclastogenesis has been completely obscure. Here, upon the in vitro model of osteoclast formation derived from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we reveal that Rab34 regulates osteoclastogenesis bidirectionally. More specifically, Rab34 serves as a negative regulator of osteoclast differentiation by promoting the lysosome-induced proteolysis of two osteoclastogenic surface receptors, c-fms and RANK, via the axis of early endosomes-late endosomes-lysosomes, leading to alleviate the transcriptional activity of two of the master regulator of osteoclast differentiation, c-fos and NFATc-1, eventually attenuating osteoclast differentiation and bone resorption. Besides, Rab34 plays a crucial role in modulating the secretory network of lysosome-related proteases including matrix metalloprotease 9 and Cathepsin K across the ruffled borders of osteoclasts, contributing to the regulation of bone resorption.

HSP90 drives the Rab11a-mediated vesicular transport of the cell surface receptors in osteoclasts.

Rab11a, which ubiquitously localizes to early and recycling endosomes, is required for regulating the vesicular transport of cellular cargos. Interestingly, our previous study revealed that Rab11a served as a negative regulator of osteoclastogenesis by facilitating the lysosomal proteolysis of (1) colony-stimulating factor-1 (c-fms) receptor and (2) receptor activator of nuclear factor-κB (RANK) receptor, thereby resulting in inhibition of osteoclast (OC) differentiation, maturation, and bone-resorbing activity. However, the molecular mechanisms of how Rab11a negatively affected osteoclastogenesis were largely unknown. Heat shock protein (HSP90), including two isoforms HSP90α and HSP90ß, necessitates the stability, maturation, and activity of a broad range of its clients, and is essentially required for a vast array of signal transduction pathways in nonstressful conditions. Furthermore, cumulative evidence suggests that HSP90 is a vital element of the vesicular transport network. Indeed, our recent study revealed that HSP90, a novel effector protein of Rab11b, modulated Rab11b-mediated osteoclastogenesis. In this study, we also found that Rab11a interacted with both HSP90α and HSP90ß in OCs. Upon blockade of HSP90 ATPase activity by a specific inhibitor(17-allylamino-demethoxygeldanamycin), we showed that (1) the ATPase domain of HSP90 was a prerequisite for the interaction between HSP90 and Rab11a, and (2) the interaction of HSP90 to Rab11a sufficiently maintained the inhibitory effects of Rab11a on osteoclastogenesis. Altogether, our findings undoubtedly indicate a novel role of HSP90 in regulating Rab11a-mediated osteoclastogenesis.

Coronin1C Is a GDP-Specific Rab44 Effector That Controls Osteoclast Formation by Regulating Cell Motility in Macrophages.

Osteoclasts are multinucleated bone-resorbing cells that are formed by the fusion of macrophages. Recently, we identified Rab44, a large Rab GTPase, as an upregulated gene during osteoclast differentiation that negatively regulates osteoclast differentiation. However, the molecular mechanisms by which Rab44 negatively regulates osteoclast differentiation remain unknown. Here, we found that the GDP form of Rab44 interacted with the actin-binding protein, Coronin1C, in murine macrophages. Immunoprecipitation experiments revealed that the interaction of Rab44 and Coronin1C occurred in wild-type and a dominant-negative (DN) mutant of Rab44, but not in a constitutively active (CA) mutant of Rab44. Consistent with these findings, the expression of the CA mutant inhibited osteoclast differentiation, whereas that of the DN mutant enhanced this differentiation. Using a phase-contrast microscope, Coronin1C-knockdown osteoclasts apparently impaired multinuclear formation. Moreover, Coronin1C knockdown impaired the migration and chemotaxis of RAW-D macrophages. An in vivo experimental system demonstrated that Coronin1C knockdown suppresses osteoclastogenesis. Therefore, the decreased cell formation and fusion of Coronin1C-depleted osteoclasts might be due to the decreased migration of Coronin1C-knockdown macrophages. These results indicate that Coronin1C is a GDP-specific Rab44 effector that controls osteoclast formation by regulating cell motility in macrophages.

Dimethyl fumarate prevents osteoclastogenesis by decreasing NFATc1 expression, inhibiting of erk and p38 MAPK phosphorylation, and suppressing of HMGB1 release.

Osteoclasts are multinucleated bone-resorbing cells derived from monocyte/macrophage progenitor cells. Excessive formation and resorbing activities of osteoclasts are involved in the bone-destructive pathologies of rheumatoid arthritis and osteoporosis. Recently, it has been found that nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor for anti-oxidative stress genes, functions in osteoclastogenesis. Dimethyl fumarate (DMF) is a potent activator of Nrf2 and has been shown to inhibit osteoclastogenesis. Here, we investigated the mechanisms of this inhibition by examining the activation of several signalling pathways during the differentiation of bone marrow-derived macrophages into osteoclasts. DMF inhibited the differentiation of osteoclasts in a dose-dependent manner and suppressed the bone-resorbing activity of osteoclasts. DMF treatment decreased the expression of nuclear factor of activated T-cells cytoplasmic-1, and significantly decreased phosphorylation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in osteoclasts. We also found that DMF inhibited the extracellular release of high mobility group box 1, associated with an up-regulation of heme oxygenase-1, likely mediated through Nrf2 activation. Our results indicate that DMF inhibits osteoclast differentiation through multiple pathways.

The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors.

Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.

Rab44, a novel large Rab GTPase, negatively regulates osteoclast differentiation by modulating intracellular calcium levels followed by NFATc1 activation.

Rab44 is an atypical Rab GTPase that contains some additional domains such as the EF-hand and coiled-coil domains as well as Rab-GTPase domain. Although Rab44 genes have been found in mammalian genomes, no studies concerning Rab44 have been reported yet. Here, we identified Rab44 as an upregulated protein during osteoclast differentiation. Knockdown of Rab44 by small interfering RNA promotes RANKL-induced osteoclast differentiation of the murine monocytic cell line, RAW-D or of bone marrow-derived macrophages (BMMs). In contrast, overexpression of Rab44 prevents osteoclast differentiation. Rab44 was localized in the Golgi complex and lysosomes, and Rab44 overexpression caused an enlargement of early endosomes. A series of deletion mutant studies of Rab44 showed that the coiled-coil domain and lipidation sites of Rab44 is important for regulation of osteoclast differentiation. Mechanistically, Rab44 affects nuclear factor of activated T-cells c1 (NFATc1) signaling in RANKL-stimulated macrophages. Moreover, Rab44 depletion caused an elevation in intracellular Ca2+ transients upon RANKL stimulation, and particularly regulated lysosomal Ca2+ influx. Taken together, these results suggest that Rab44 negatively regulates osteoclast differentiation by modulating intracellular Ca2+ levels followed by NFATc1 activation.

Actin binding LIM 1 (abLIM1) negatively controls osteoclastogenesis by regulating cell migration and fusion.

Actin binding LIM 1 (abLIM1) is a cytoskeletal actin-binding protein that has been implicated in interactions between actin filaments and cytoplasmic targets. Previous biochemical and cytochemical studies have shown that abLIM1 interacts and co-localizes with F-actin in the retina and muscle. However, whether abLIM1 regulates osteoclast differentiation has not yet been elucidated. In this study, we examined the role of abLIM1 in osteoclast differentiation and function. We found that abLIM1 expression was upregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation, and that a novel transcript of abLIM1 was exclusively expressed in osteoclasts. Overexpression of abLIM1 in the murine monocytic cell line, RAW-D suppressed osteoclast differentiation and decreased expression of several osteoclast-marker genes. By contrast, small interfering RNA-induced knockdown of abLIM1 enhanced the formation of multinucleated osteoclasts and markedly increased the expression of the osteoclast-marker genes. Mechanistically, abLIM1 regulated the localization of tubulin, migration, and fusion in osteoclasts. Thus, these results indicate that abLIM1 negatively controls osteoclast differentiation by regulating cell migration and fusion mediated via actin formation.

The intranuclear PEX domain of MMP involves proliferation, migration, and metastasis of aggressive adenocarcinoma cells.

Members of matrix metalloproteinase (MMP) family promote cancer cell migration, invasion, and metastasis through alteration of the tumor milieu, intracellular signaling pathways, and transcription. We examined gene expression signatures of colon adenocarcinoma cell lines with different metastatic potentials and found that rapidly metastatic cells powerfully expressed genes encoding MMP3 and MMP9. The non-proteolytic PEX isoform and proteolytic isoforms of MMPs were significantly expressed in the metastatic cells in vitro. Knockdown of MMP3 attenuated cancer cell migration and invasion in vitro and lung metastasis in vivo. Profound nuclear localization of MMP3/PEX was found in tumor-stroma marginal area. In contrast, MMP9 was localized in central area of subcutaneous tumors. Overexpression of the PEX isoform of MMP3 promoted proliferation and migration of the rapidly metastatic cells in vitro. Taken together, the non-proteolytic PEX isoform of MMPs locating in cell nuclei involves proliferation, migration, and subsequent metastasis of aggressive adenocarcinoma cells.

HSP-enriched properties of extracellular vesicles involve survival of metastatic oral cancer cells.

Cancer cells often secrete extracellular vesicles (EVs) that carry heat shock proteins (HSPs) with roles in tumor progression. Oral squamous cell carcinoma (OSCC) belongs to head and neck cancers (HNC) whose lymph-node-metastases often lead to poor prognosis. We have examined the EV proteome of OSCC cells and found abundant secretion of HSP90-enriched EVs in lymph-node-metastatic OSCC cells. Double knockdown of HSP90α and HSP90ß, using small interfering RNA significantly reduced the survival of the metastatic OSCC cells, although single knockdown of each HSP90 was ineffective. Elevated expression of these HSP90 family members was found to correlate with poor prognosis of HNC cases. Thus, elevated HSP90 levels in secreted vesicles are potential prognostic biomarkers and therapeutic targets in metastatic OSCC.

Anti-EGFR antibody cetuximab is secreted by oral squamous cell carcinoma and alters EGF-driven mesenchymal transition.

Genetic amplification, overexpression, and increased signaling from the epidermal growth factor receptor (EGFR) are often found in oral squamous cell carcinoma (OSCC) and thus EGFR is frequently targeted molecularly by the therapeutic antibody cetuximab. We assessed effects of cetuximab in control of EGF-driven malignant traits of OSCC cells. EGF stimulation promoted progression level of mesenchymal traits in OSCC cells, which were attenuated by cetuximab but incompletely. We pursued a potential mechanism underlying such incomplete attenuation of OSCC malignant traits. Cetuximab promoted secretion of EGFR-EVs by OSCC cells and failed to inhibit EGF-driven secretion of EGFR-EVs. Cetuximab was also found to be robustly secreted with the EGFR-EVs by the OSCC cells. Thus, EGF promotes the level of mesenchymal traits of OSCC cells and secretion of EGFR-EVs, which involve cetuximab resistance.

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 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.

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 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.

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.

Punicalagin attenuates osteoclast differentiation by impairing NFATc1 expression and blocking Akt- and JNK-dependent pathways.

Punicalagin is a bioactive polyphenol that is classified as an ellagitannin. Although punicalagin has been shown to have various pharmacological effects, such as anti-oxidative, anti-inflammatory, and anti-tumor effects, no studies have reported the effects of punicalagin on osteoclasts (OCLs). In this study, we investigated the effects of punicalagin on OCL differentiation by receptor activator of nuclear factor kappa-B ligand in the murine monocytic RAW-D cell line and bone marrow-derived macrophages (BMMs). Treatment with punicalagin significantly inhibited OCL formation from RAW-D cells and BMMs and prevented bone resorption of BMM-derived OCLs. Moreover, punicalagin impaired multinucleation and actin-ring formation in OCLs, and decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a master regulator of OCL differentiation, and concomitantly reduced the expression levels of Src and cathepsin K, which are transcriptionally regulated by NFATc1. The effects of punicalagin on intracellular signaling during the OCL differentiation of BMMs indicated that punicalagin-treated OCLs displayed markedly reduced phosphorylation of Jun N-terminal kinase and Akt, and partially impaired phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and inhibitor of nuclear factor kappa-B alpha compared with untreated OCLs. Thus, punicalagin may affect bone metabolism by inhibiting OCL differentiation.

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.

Castalagin Exerts Inhibitory Effects on Osteoclastogenesis Through Blocking a Broad Range of Signaling Pathways with Low Cytotoxicity.

Castalagin is a rare plant polyphenol that is classified as a hydrolyzable tannin. Although it has antioxidant, antitumorigenic, and leishmanicidal effects, the utility of castalagin against bone diseases remain to be elucidated. Here, we investigated the effects of castalagin on the differentiation of osteoclasts (OCLs), multinucleated bone-resorbing cells. After stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL), the formation of OCLs from bone marrow-derived macrophages was significantly inhibited by castalagin even at 1 µM. However, castalagin displayed little cytotoxicity at a higher concentration of 50 µM. The effects of castalagin on intracellular signaling during OCL differentiation showed that castalagin suppresses RANKL-stimulated phosphorylation of major signaling pathways including protein kinase B (Akt), extracellular signal-regulated kinase, Jun N-terminal kinase, p38 mitogen-activated protein kinases, and inhibitor of nuclear factor kappa B alpha. Moreover, following castalagin treatment, the protein levels of nuclear factor of activated T-cells, cytoplasmic 1, a master regulator for OCL differentiation, and NF-κB were decreased. Thus, castalagin exerts inhibitory effects on osteoclastogenesis through blockage of a broad range of signaling pathways, but has low cytotoxicity.