SLAT negatively regulates RANKL-induced osteoclast differentiation

RANKL induces the formation of osteoclasts, which are responsible for bone resorption. Herein, we investigated the role of SWAP-70-like adapter of T cells (SLAT) in RANKL-induced osteoclastogenesis. Expression levels of SLAT were reduced during RANKL-induced osteoclastogenesis. Overexpression of SLAT in BMMs inhibited TRAP-positive multinuclear osteoclast formation and attenuated the expression of NFATc1, which is an important modulator in osteoclastogenesis. Furthermore, silencing of SLAT by RNA interference enhanced osteoclast formation as well as NFATc1 expression. In addition, SLAT was involved in RANKL-induced JNK activation in osteoclasts. Taken together, our data suggest that SLAT acts as a negative modulator of RANKL-induced osteoclastogenesis.     

miR-31 controls osteoclast formation and bone resorption by targeting RhoA

Introduction

Increased activity of osteoclasts is responsible for bone loss and joint destruction in rheumatoid arthritis. For osteoclast development and bone resorption activity, cytoskeletal organization must be properly regulated. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that suppress expression of their target genes. This study was conducted to identify crucial miRNAs to control osteoclasts.

Methods

miRNA expression in the bone marrow-derived macrophages (BMM) with or without receptor activator of nuclear factor κB ligand (RANKL) stimulation was analyzed by miRNA array. To examine the role of specific miRNAs in osteoclast formation, bone resorption activity and actin ring formation, the BMM were retrovirally transduced with miRNA antagomirs. To confirm whether the suppressive effects on osteoclastogenesis by miR-31 inhibition were mediated by targeting RhoA, osteoclast formation was analyzed in the presence of the RhoA inhibitor, exoenzyme C3.

Results

miR-31 was identified as one of the highly upregulated miRNAs during osteoclast development under RANKL stimulation. Inhibition of miR-31 by specific antagomirs suppressed the RANKL-induced formation of osteoclasts and bone resorption. Phalloidin staining of osteoclasts revealed that actin ring formation at the cell periphery was severely impaired by miR-31 inhibition, and clusters of small ringed podosomes were observed instead. In these osteoclasts, expression of RhoA, one of the miR-31 target genes, was upregulated by miR-31 inhibition in spite of the impaired osteoclastogenesis. Treatment with the RhoA inhibitor, exoenzyme C3, rescued the osteoclastogenesis impaired by miR-31 inhibition.

Conclusions

miR-31 controls cytoskeleton organization in osteoclasts for optimal bone resorption activity by regulating the expression of RhoA.     

The transmembrane adaptor protein, linker for activation of T cells (LAT), regulates RANKL-induced osteoclast differentiation

RANKL induces the formation of osteoclasts, which are responsible for bone resorption. Herein we investigate the role of the transmembrane adaptor proteins in RANKL-induced osteoclastogenesis. LAT positively regulates osteoclast differentiation and is up-regulated by RANKL via c-Fos and NFATc1, whereas LAB and LIME act as negative modulators of osteoclastogenesis. In addition, silencing of LAT by RNA interference or overexpression of a LAT dominant negative in bone marrow-derived macrophage cells attenuates RANKL-induced osteoclast formation. Furthermore, LAT is involved in RANKL-induced PLC(γ) activation and NFATc1 induction. Thus, our data suggest that LAT acts as a positive regulator of RANKL-induced osteoclastogenesis.     

RANKL stimulates inducible nitric-oxide synthase expression and nitric oxide production in developing osteoclasts. An autocrine negative feedback mechanism triggered by RANKL-induced interferon-beta via NF-kappaB that restrains osteoclastogenesis and bone resorption

Nitric oxide (NO) is a multifunctional signaling molecule and a key vasculoprotective and potential osteoprotective factor. NO regulates normal bone remodeling and pathological bone loss in part through affecting the recruitment, formation, and activity of bone-resorbing osteoclasts. Using murine RAW 264.7 and primary bone marrow cells or osteoclasts formed from them by receptor activator of NF-kappaB ligand (RANKL) differentiation, we found that inducible nitric-oxide synthase (iNOS) expression and NO generation were stimulated by interferon (IFN)-gamma or lipopolysaccharide, but not by interleukin-1 or tumor necrosis factor-alpha. Surprisingly, iNOS expression and NO release were also triggered by RANKL. This response was time- and dose-dependent, required NF-kappaB activation and new protein synthesis, and was specifically blocked by the RANKL decoy receptor osteoprotegerin. Preventing RANKL-induced NO (via iNOS-selective inhibition or use of marrow cells from iNOS-/- mice) increased osteoclast formation and bone pit resorption, indicating that such NO normally restrains RANKL-mediated osteoclastogenesis. Additional studies suggested that RANKL-induced NO inhibition of osteoclast formation does not occur via NO activation of a cGMP pathway. Because IFN-beta is also a RANKL-induced autocrine negative feedback inhibitor that limits osteoclastogenesis, we investigated whether IFN-beta is involved in this novel RANKL/iNOS/NO autoregulatory pathway. IFN-beta was induced by RANKL and stimulated iNOS expression and NO release, and a neutralizing antibody to IFN-beta inhibited iNOS/NO elevation in response to RANKL, thereby enhancing osteoclast formation. Thus, RANKL-induced IFN-beta triggers iNOS/NO as an important negative feedback signal during osteoclastogenesis. Specifically targeting this novel autoregulatory pathway may provide new therapeutic approaches to combat various osteolytic bone diseases.     

Inhibitory effect of luteolin on osteoclast differentiation and function

Osteoclasts are multinucleated cells that play a crucial role in bone resorption, and are formed by the fusion of mononuclear osteoclasts derived from osteoclast precursors of the macrophage lineage. Compounds that specifically target functional osteoclasts would be ideal candidates for anti-resorptive agents for clinical applications. In the present study, we investigated the effects of luteolin, a flavonoid, on the regulation of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, functions and signaling pathway. Addition of luteolin to a coculture system of mouse bone marrow cells and ST2 cells in the presence of 10⁻⁸ M 1α,25(OH)₂D₃ caused significant inhibition of osteoclastogenesis. Luteolin had no effects on the 1α,25(OH)₂D₃-induced expressions of RANKL, osteoprotegerin and macrophage colony-stimulating factor mRNAs. Next, we examined the direct effects of luteolin on osteoclast precursors using bone marrow macrophages and RAW264.7 cells. Luteolin completely inhibited RANKL-induced osteoclast formation. Moreover, luteolin inhibited the bone resorption by mature osteoclasts accompanied by the disruption of their actin rings, and these effects were reversely induced by the disruption of the actin rings in mature osteoclasts. Finally, we found that luteolin inhibited RANKL-induced osteoclastogenesis through the suppression of ATF2, downstream of p38 MAPK and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) expression, respectively. Taken together, the present results indicate that naturally occurring luteolin has inhibitory activities toward both osteoclast differentiation and functions through inhibition of RANKL-induced signaling pathway as well as actin ring disruption, respectively.     

Antioxidants, like coenzyme Q10, selenite, and curcumin, inhibited osteoclast differentiation by suppressing reactive oxygen species generation

Coenzyme Q10 (CoQ10), selenium, and curcumin are known to be powerful antioxidants. Osteoclasts are capable of resorbing mineralized bone and excessive bone resorption by osteoclasts causes bone loss-related diseases. During osteoclast differentiation, the reactive oxygen species (ROS) acts as a secondary messenger on signal pathways. In this study, we investigated whether antioxidants can inhibit RANKL-induced osteoclastogenesis through suppression of ROS generation and compared the relative inhibitory activities of CoQ10, sodium selenite, and curcumin on osteoclast differentiation. We found that antioxidants markedly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in both bone marrow-derived monocytes (BMMs) and RAW 264.7 cells. Antioxidants scavenged intracellular ROS generation within osteoclast precursors during RANKL-stimulated osteoclastogenesis. These also acted to significantly suppress the gene expression of NFATc1, TRAP, and osteoclast-associated immunoglobulin-like receptor (OSCAR), which are genetic markers of osteoclast differentiation in a dose-dependent manner. These antioxidants also suppressed ROS-induced IκBα signaling pathways for osteoclastogenesis. Specially, curcumin displayed the highest inhibitory effect on osteoclast differentiation when concentrations were held constant. Together, CoQ10, selenite, and curcumin act as inhibitors of RANKL-induced NFATc1 which is a downstream event of NF-κB signal pathway through suppression of ROS generation, thereby suggesting their potential usefulness for the treatment of bone disease associated with excessive bone resorption.     

Inhibition of receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation and bone resorption by Gryllus bimaculatus extract: An in vitro study

Excessive bone-resorbing osteoclast activity during bone remodeling is a major feature of bone diseases, such as osteoporosis. Therefore, the inhibition of osteoclast formation and bone resorption can be an effective therapeutic target for various bone diseases. Gryllus biomaculatus (GB) has recently been approved as an alternative food source because of its high nutritional value and environmental sustainability. Traditionally, GB has been known to have various pharmacological properties, including antipyretic and blood pressure-lowering activity, and it has recently been reported to have various biological activities, including protective effects against inflammation, oxidative stress, insulin resistance, and alcohol-induced liver injury. However, the effect of GB on osteoclast differentiation and bone metabolism has not yet been demonstrated. In this study, we confirmed the inhibitory effect of GB extract (GBE) on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. To determine the effect of GBE on RANKL-induced osteoclast differentiation and function, we performed TRAP and F-actin staining, as well as a bone-resorbing assay. The intracellular mechanisms of GBE responsible for the regulation of osteoclastogenesis were revealed by Western blot analysis and quantitative real-time polymerase chain reaction. We investigated the relationship between GBE and expression of osteoclast-specific molecules to further elucidate the underlying mechanisms. It was found that GBE significantly suppressed osteoclastogenesis by decreasing the phosphorylation of Akt, p38, JNK, and ERK, as well as Btk-PLCγ2 signaling, in pathways involved in early osteoclastogenesis as well as through the subsequent suppression of c-Fos, NFATc1, and osteoclastogenesis-specific marker genes. Additionally, GBE inhibited the formation of F-actin ring-positive osteoclasts and bone resorption activity of mature osteoclasts. Our findings suggest that GBE is a potential functional food and therapeutic candidate for bone diseases involving osteoclasts.     

Chitooligosaccharide inhibits RANKL-induced osteoclastogenesis and ligation-induced periodontitis by suppressing MAPK/ c-fos/NFATC1 signaling

Considering the high rate of osteoclast-related diseases worldwide, research targeting osteoclast formation/function is crucial. In vitro, we demonstrated that chitooligosaccharide (CS) dramatically inhibited osteoclastogenesis as well as osteoclast function dose-dependently. CS suppressed osteoclast-specific genes expression during osteoclastogenesis. Furthermore, we found that CS attenuated receptor activator of nuclear factor kappa B ligand (RANKL)-mediated mitogen-activated protein kinase (MAPK) pathway involving p38, erk1/2, and jnk, leading to the reduced expression of c-fos and nuclear factor of activated T cells c1 (NFATc1) during osteoclast differentiation. In vivo, we found CS protected rats from periodontitis-induced alveolar bone loss by micro-computerized tomography and histological analysis. Overall, CS inhibited RANKL-induced osteoclastogenesis and ligature-induced rat periodontitis model, probably by suppressing the MAPK/c-fos/NFATc1 signaling pathway. Therefore, CS may be a safe and promising treatment for osteoclast-related diseases.     

Lyn inhibits osteoclast differentiation by interfering with PLCγ1-mediated Ca signaling

Osteoclasts differentiate from macrophage-lineage cells to become specialized for bone resorption function. By a proteomics approach, we found that Lyn was down-regulated by the osteoclast differentiation factor, receptor activator of NF-κB ligand (RANKL). The forced reduction of Lyn caused a striking increase in the RANKL-induced PLCγ1, Ca²⁺, and NFATc1 responses during differentiation. These data suggest that Lyn plays a negative role in osteoclastogenesis by interfering with the PLCγ1-mediated Ca²⁺ signaling that leads to NFATc1 activation. Consistent with the in vitro results, in vivo injection of Lyn specific siRNA into mice calvariae provoked a fulminant bone resorption. Our study provides the first evidence of the involvement of Lyn in the negative regulation of osteoclastogenesis by RANKL.     

Asiaticoside,a component of Centella asiatica attenuates RANKL-induced osteoclastogenesis via NFATc1 and NF-κB signaling pathways

Identification of natural compounds that inhibit osteoclastogenesis will facilitate the development of antiresorptive treatment of osteolytic bone diseases. Asiaticoside is a triterpenoid derivative isolated from Centella asiatica, which exhibits varying biological effects like angiogenesis, anti-inflammation, wound healing, and osteogenic differentiation. However, its role in osteoclastogenesis remains unknown. Here, we show that Asiaticoside can suppress RANKL-induced osteoclast formation and bone resorption in a dose-dependent manner. Asiaticoside attenuated the expression of osteoclast marker genes including Ctsk, Atp6v0d2, Nfatc1, Acp5, and Dc-stamp. Furthermore, Asiaticoside inhibited RANKL-mediated NF-κB and NFATc1 activities, and RANKL-induced calcium oscillation. Collectively, this study demonstrates that Asiaticoside inhibited osteoclast formation and function through attenuating RANKL-induced key signaling pathways, which may indicate that Asiaticoside is a potential antiresorptive agent against osteoclast-related osteolytic bone diseases.     

MHC class II transactivator negatively regulates RANKL-mediated osteoclast differentiation by downregulating NFATc1 and OSCAR

Nuclear factor of activated T cells (NFAT) c1 plays a key role in receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation and function via induction of osteoclast-specific target genes including osteoclast-associated receptor (OSCAR), cathepsin K, and tartrate-resistant acid phosphatase. To elucidate which downstream target genes are regulated by NFATc1 during osteoclastogenesis, we used microarray analyses to examine gene expression profiles in the context of bone marrow-derived macrophages overexpressing a constitutively active form of NFATc1. Herein, we demonstrate that MHC class II transactivator (CIITA) is up-regulated downstream of NFATc1. Overexpression of CIITA in osteoclast precursors attenuates RANKL-induced osteoclast formation through down-regulation of NFATc1 and OSCAR. Epigenetic overexpression of CIITA regulates NFATc1 and OSCAR by competing with c-Fos and NFATc1 for CBP/p300 binding sites. Furthermore, silencing of CIITA by RNA interference in osteoclast precursors enhances osteoclast formation as well as NFATc1 and OSCAR expression. Taken together, our data reveal that CIITA can act as a modulator of RANKL-induced osteoclastogenesis.