Iris Koreana NAKAI Inhibits Osteoclast Formation via p38-Mediated Nuclear Factor of Activated T Cells 1 Signaling Pathway

Background@#Iris Koreana NAKAI (IKN) is a flowering perennial plant that belongs to the Iridaceae family. In this study, we aimed to demonstrate the effects of IKN on osteoclast differentiation in vitro and in vivo. We also sought to verify the molecular mechanisms underlying its anti-osteoclastogenic effects. @*Methods@#Osteoclasts were formed by culturing mouse bone marrow macrophage (BMM) cells with macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). Bone resorption assays were performed on dentin slices. mRNA expression levels were analyzed by quantitative polymerase chain reaction. Western blotting was performed to detect protein expression or activation. Lipopolysaccharide (LPS)-induced osteoclast formation was performed using a mouse calvarial model. @*Results@#In BMM cultures, an ethanol extract of the root part of IKN suppressed RANKL-induced osteoclast formation and bone resorptive activity. In contrast, an ethanol extract of the aerial parts of IKN had a minor effect on RANKL-induced osteoclast formation. Mechanistically, the root part of IKN suppressed RANKL-induced p38 mitogen-activated protein kinase (MAPK) activation, effectively abrogating the induction of c-Fos and nuclear factor of activated T cells 1 (NFATc1) expression. IKN administration decreased LPS-induced osteoclast formation in a calvarial osteolysis model in vivo. @*Conclusions@#Our study suggested that the ethanol extract of the root part of IKN suppressed osteoclast differentiation and function partly by downregulating the p38 MAPK/c-Fos/NFATc1 signaling pathways. Thus, the root part

The Role of Toll-Like Receptors in Osteoclastogenesis

Bone homeostasis is maintained by a balance in the levels of osteoclast and osteoblast activity. Osteoclasts are bone-resorbing cells and have been shown to act as key players in various osteolytic diseases. Osteoclasts differentiate from monocyte/macrophage lineage cells in the presence of receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. Osteoblasts support osteoclastogenesis by producing several osteoclast differentiation factors. Toll-like receptors (TLRs) are members of the pattern recognition receptor family that are involved in recognizing pathogen-associated molecular patterns and damage-associated molecular patterns in response to pathogen infection. TLRs regulate osteoclastogenesis and bone resorption through either the myeloid differentiation primary response 88 or the Toll/interleukin-1 receptor domaincontaining adapter-inducing interferon-β signaling pathways. Since osteoclasts play a central role in the progression of osteolytic diseases, extensive research focusing on TLR downstream signaling in these cells should be conducted to advance the development of effective TLR modulators. In this review, we summarize the currently available information on the role of TLRs in osteoclast differentiation and osteolytic diseases.

The Role of Toll-Like Receptors in Osteoclastogenesis

Bone homeostasis is maintained by a balance in the levels of osteoclast and osteoblast activity. Osteoclasts are bone-resorbing cells and have been shown to act as key players in various osteolytic diseases. Osteoclasts differentiate from monocyte/macrophage lineage cells in the presence of receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. Osteoblasts support osteoclastogenesis by producing several osteoclast differentiation factors. Toll-like receptors (TLRs) are members of the pattern recognition receptor family that are involved in recognizing pathogen-associated molecular patterns and damage-associated molecular patterns in response to pathogen infection. TLRs regulate osteoclastogenesis and bone resorption through either the myeloid differentiation primary response 88 or the Toll/interleukin-1 receptor domaincontaining adapter-inducing interferon-β signaling pathways. Since osteoclasts play a central role in the progression of osteolytic diseases, extensive research focusing on TLR downstream signaling in these cells should be conducted to advance the development of effective TLR modulators. In this review, we summarize the currently available information on the role of TLRs in osteoclast differentiation and osteolytic diseases.

Fexaramine Inhibits Receptor Activator of Nuclear Factor-κB Ligand-induced Osteoclast Formation via Nuclear Factor of Activated T Cells Signaling Pathways

BACKGROUND: Osteoclasts are bone resorbing cells and are responsible for bone erosion in diseases as diverse as osteoporosis, periodontitis, and rheumatoid arthritis. Fexaramine has been developed as an agonist for the farnesoid X receptor (FXR). This study investigated the effects of fexaramine on receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclast formation and signaling pathways. METHODS: Osteoclasts were formed by culturing mouse bone marrow-derived macrophages (BMMs) with macrophage colony-stimulating factor (M-CSF) and RANKL. Bone resorption assays were performed using dentine slices. The mRNA expression level was analyzed by real-time polymerase chain reaction. Western blotting assays were conducted to detect the expression or activation level of proteins. Lipopolysaccharide-induced osteoclast formation was performed using a mouse calvarial model. RESULTS: Fexaramine inhibited RANKL-induced osteoclast formation, without cytotoxicity. Furthermore, fexaramine diminished the RANKL-stimulated bone resorption. Mechanistically, fexaramine blocked the RANKL-triggered p38, extracellular signal-regulated kinase, and glycogen synthase kinase 3β phosphorylation, resulting in suppressed expression of c-Fos and NF of activated T cells (NFATc1). Consistent with the in vitro anti-osteoclastogenic effect, fexaramine suppressed lipopolysaccharide-induced osteoclast formation in the calvarial model. CONCLUSIONS: The present data suggest that fexaramine has an inhibitory effect on osteoclast differentiation and function, via downregulation of NFATc1 signaling pathways. Thus, fexaramine could be useful for the treatment of bone diseases associated with excessive bone resorption.

Attenuation of RANKL-induced Osteoclast Formation via p38-mediated NFATc1 Signaling Pathways by Extract of Euphorbia Lathyris L

BACKGROUND: Osteoclasts are the only cell type capable of breaking down bone matrix, and its excessive activation is responsible for the development of bone-destructive diseases. Euphorbia lathyris L. (ELL) is an herbal plant that belongs to the Euphorbiaceae family. This study investigated the effects of the methanol extract of the aerial part of ELL on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast formation and signaling pathways. METHODS: Osteoclasts were formed by co-culturing mouse bone marrow with osteoblasts or by culturing mouse bone marrow-derived macrophages (BMMs) with macrophage colony-stimulating factor (M-CSF) and RANKL. Bone resorption assays were performed using dentine slices. The expression level of mRNA was analyzed by real-time polymerase chain reaction (PCR) or reverse transcription (RT)-PCR. Western blotting assays were performed to detect the expression or activation level of proteins. RESULTS: ELL inhibited RANKL-induced osteoclast formation without cytotoxicity. Furthermore, the RANKL-stimulated bone resorption was diminished by ELL. Mechanistically, ELL blocked the RANKL-triggered p38 mitogen-activated protein kinase (MAPK) phosphorylation, which resulted in the suppression of the expression of c-Fos and nuclear factor of activated T cells (NFATc1). In osteoblasts, ELL had little effect on the mRNA expression of RANKL and osteoprotegerin (OPG). CONCLUSIONS: The present data suggest that ELL has an inhibitory effect on osteoclast differentiation and function via downregulation of the p38/c-Fos/NFATc1 signaling pathways. Thus, ELL could be useful for the treatment of bone diseases associated with excessive bone resorption.

The Functional Analysis of MLKs-JNK Pathways in Osteoclast Formation

OBJECTIVES: Osteoclasts (OCs) are bone-resorbing multinucleated cells derived from hematopoietic progenitors of the monocyte-macrophage lineage. OC precursors, such as bone marrow-derived macrophages (BMMs), are formed in the presence of macrophage colony-stimulating factor (M-CSF) and differentiate into OCs in response to M-CSF and receptor activator of nuclear factor kappaB ligand (RANKL). In this study, we investigated the role of mixed lineage kinases (MLKs)-c-Jun amino-terminal kinase (JNK) pathways in OC formation. METHODS: We performed an OC formation assay and reverse transcription polymerase chain reaction (RT-PCR) analysis. RESULTS: We first explored the role of JNK on osteoclst formation using mouse bone marrow (BM) culture system. We found that OC formation was impaired when the JNK inhibitor was added either in early or late stage, suggesting the requirement for JNK activation during OC formation. MLKs are serine/threonine kinases that regulate signaling by the JNK. Since the JNK activity is specifically required for osteoclastogenesis, we examined the messenger ribonucleic acid (mRNA) levels of MLKs in BMs, BMMs and OCs by RT-PCR. Among MLKs, the level of MLK3 mRNA expression is highest in BMs, BMMs and OCs. Moreover, we found that the mRNA expression of MLK2 and MLK3 is increased with the differentiation of BMs to BMMs, and is sustained in OCs. Finally we investigated the role of MLK3 in OC differentiation using gene knock-down techniques. The silencing of MLK3 in BMMs partly attenuated RANKL-induced OC differentiation. CONCLUSIONS: These data suggest that JNK and MLK3 may positively regulate OC formation.

The Functional Analysis of MLKs-JNK Pathways in Osteoclast Formation

OBJECTIVES: Osteoclasts (OCs) are bone-resorbing multinucleated cells derived from hematopoietic progenitors of the monocyte-macrophage lineage. OC precursors, such as bone marrow-derived macrophages (BMMs), are formed in the presence of macrophage colony-stimulating factor (M-CSF) and differentiate into OCs in response to M-CSF and receptor activator of nuclear factor kappaB ligand (RANKL). In this study, we investigated the role of mixed lineage kinases (MLKs)-c-Jun amino-terminal kinase (JNK) pathways in OC formation. METHODS: We performed an OC formation assay and reverse transcription polymerase chain reaction (RT-PCR) analysis. RESULTS: We first explored the role of JNK on osteoclst formation using mouse bone marrow (BM) culture system. We found that OC formation was impaired when the JNK inhibitor was added either in early or late stage, suggesting the requirement for JNK activation during OC formation. MLKs are serine/threonine kinases that regulate signaling by the JNK. Since the JNK activity is specifically required for osteoclastogenesis, we examined the messenger ribonucleic acid (mRNA) levels of MLKs in BMs, BMMs and OCs by RT-PCR. Among MLKs, the level of MLK3 mRNA expression is highest in BMs, BMMs and OCs. Moreover, we found that the mRNA expression of MLK2 and MLK3 is increased with the differentiation of BMs to BMMs, and is sustained in OCs. Finally we investigated the role of MLK3 in OC differentiation using gene knock-down techniques. The silencing of MLK3 in BMMs partly attenuated RANKL-induced OC differentiation. CONCLUSIONS: These data suggest that JNK and MLK3 may positively regulate OC formation.

Rolipram, a Phosphodiesterase 4 Inhibitor, Stimulates Inducible cAMP Early Repressor Expression in Osteoblasts

Phosphodiesterase (PDE) 4 inhibitors have been shown to induce the cAMP-mediated signaling pathway by inhibiting cAMP hydrolysis. This study investigated the effect of a PDE4 inhibitor on the expression of the inducible cAMP early repressor (ICER), which is an endogenous inhibitor of CRE- mediated transcription, in osteoblastic cells. RT-PCR analysis revealed that rolipram, a PDE4 inhibitor, stimulates the ICER mRNA in a dose dependent manner. The induction of ICER mRNA expression by rolipram was suppressed by the inhibitors of protein kinase A (PKA) and p38 MAPK, suggesting the involvement of PKA and p38 MAPK activation in ICER expression by rolipram. It was previously shown that rolipram induced the expression of TNF-related activation-induced cytokine (TRANCE, also known as RANKL, ODF, or OPGL) in osteoblasts. This paper provides evidences that a transcriptional repressor like ICER might modulate TRANCE mRNA expression by rolipram in osteoblasts.