Early estrogen-induced gene 1, a novel RANK signaling component,is essential for osteoclastogenesis

The receptor activator of NF-κB (RANK) and immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptors are essential factors involved in regulating osteoclast formation and bone remodeling. Here, we identify early estrogen-induced gene 1 (EEIG1) as a novel RANK ligand (RANKL)-inducible protein that physically interacts with RANK and further associates with Gab2, PLCγ2 and Tec/Btk kinases upon RANKL stimulation. EEIG1 positively regulates RANKL-induced osteoclast formation, likely due to its ability to facilitate RANKL-stimulated PLCγ2 phosphorylation and NFATc1 induction. In addition, an inhibitory peptide designed to block RANK-EEIG1 interaction inhibited RANKL-induced bone destruction by reducing osteoclast formation. Together, our results identify EEIG1 as a novel RANK signaling component controlling RANK-mediated osteoclast formation, and suggest that targeting EEIG1 might represent a new therapeutic strategy for the treatment of pathological bone resorption.     

Inactivation of glycogen synthase kinase-3β is required for osteoclast differentiation

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase originally identified as a regulator of glycogen deposition. Although the role of GSK-3β in osteoblasts is well characterized as a negative regulator of β-catenin, its effect on osteoclast formation remains largely unidentified. Here, we show that the GSK-3β inactivation upon receptor activator of NF-κB ligand (RANKL) stimulation is crucial for osteoclast differentiation. Regulation of GSK-3β activity in bone marrow macrophages by retroviral expression of the constitutively active GSK-3β (GSK3β-S9A) mutant inhibits RANKL-induced osteoclastogenesis, whereas expression of the catalytically inactive GSK-3β (GSK3β-K85R) or small interfering RNA (siRNA)-mediated GSK-3β silencing enhances osteoclast formation. Pharmacological inhibition of GSK-3β further confirmed the negative role of GSK-3β in osteoclast formation. We also show that overexpression of the GSK3β-S9A mutant in bone marrow macrophages inhibits RANKL-mediated NFATc1 induction and Ca(2+) oscillations. Remarkably, transgenic mice expressing the GSK3β-S9A mutant show an osteopetrotic phenotype due to impaired osteoclast differentiation. Further, osteoclast precursor cells from the transgenic mice show defects in expression and nuclear localization of NFATc1. These findings demonstrate a novel role for GSK-3β in the regulation of bone remodeling through modulation of NFATc1 in RANKL signaling.     

TLR2-dependent modulation of osteoclastogenesis by Porphyromonas gingivalis through differential induction of NFATc1 and NF-kappaB

Osteolytic diseases, including rheumatoid arthritis, osteomyelitis, and periodontitis, are usually associated with bacterial infections. However, the precise mechanisms by which bacteria induce bone loss still remain unclear. Evidence exists that Toll-like receptor (TLR) signaling regulates both inflammation and bone metabolism and that the receptor activator of NF-κB ligand (RANKL) and its receptor RANK are the key regulators for bone remodeling and for the activation of osteoclasts. Here, we investigate the direct effects of the periodontal pathogen Porphyromonas gingivalis on osteoclast differentiation and show that P. gingivalis differentially modulates RANKL-induced osteoclast formation contingent on the state of differentiation of osteoclast precursors. In addition, although an optimal induction of cytokines by P. gingivalis is dependent on TLR2 and TLR4, as well as myeloid differentiation factor 88 and Toll/IL-1R domain-containing adaptor-inducing IFN-β, P. gingivalis utilizes TLR2/ myeloid differentiation factor 88 in modulating osteoclast differentiation. P. gingivalis modulates RANKL-induced osteoclast formation by differential induction of NFATc1 and c-Fos. More importantly, RANKL-mediated lineage commitment also has an impact on P. gingivalis-induced cytokine production. RANKL inhibits P. gingivalis-induced cytokine production by down-regulation of TLR/NF-κB and up-regulation of NFATc1. Our findings reveal novel aspects of the interactions between TLR and RANK signaling and provide a new model for understanding the mechanism underlying the pathogenesis of bacteria-mediated bone loss.     

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.     

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.     

MCP-1-induced human osteoclast-like cells are tartrate-resistant acid phosphatase, NFATc1, and calcitonin receptor-positive but require receptor activator of NFkappaB ligand for bone resorption

MCP-1 (monocyte chemotactic protein-1) is a CC chemokine that is induced by receptor activator of NFkappaB ligand (RANKL) in human osteoclasts. In the absence of RANKL, treatment of human peripheral blood mononuclear cells with macrophage colony-stimulating factor and MCP-1 resulted in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells that are positive for calcitonin receptor (CTR) and a number of other osteoclast markers, including nuclear factor of activated t cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). Although NFATc1 was strongly induced by MCP-1 and was observed in the nucleus, MCP-1 did not permit the formation of bone-resorbing osteoclasts, although these cells had the typical TRAP(+)/CTR(+) multinuclear phenotype of osteoclasts. Despite a similar appearance to osteoclasts, RANKL treatment was required in order for TRAP(+)/CTR(+) multinuclear cells to develop bone resorption activity. The lack of bone resorption was correlated with a deficiency in expression of certain genes related to bone resorption, such as cathepsin K and MMP9. Furthermore, calcitonin blocked the MCP-1-induced formation of TRAP(+)/CTR(+) multinuclear cells as well as blocking osteoclast bone resorption activity, indicating that calcitonin acts at two stages of osteoclast differentiation. Ablation of NFATc1 in mature osteoclasts did not prevent bone resorption activity, suggesting NFATc1 is involved in cell fusion events and not bone resorption. We propose that the MCP-1-induced TRAP(+)/CTR(+) multinuclear cells represent an arrested stage in osteoclast differentiation, after NFATc1 induction and cellular fusion but prior to the development of bone resorption activity.     

RANKL-induced schlafen2 is a positive regulator of osteoclastogenesis

Osteoclasts are hematopoietic lineage derived-multinucleated cells that resorb bone. Their activity in balance with that of osteoblast is essential for bone homeostasis. Receptor activator of NF-κB ligand (RANKL) is known as an essential cytokine for the osteoclastogenesis, and c-Jun signaling in cooperation with NFAT family is crucial for RANKL-regulated osteoclastogenesis. We show here that schlafen2 (Slfn2), a member of a new family of growth regulatory genes involved in thymocyte development, is critical for osteoclastogenesis. RANKL selectively induces Slfn2 expression in osteoclast precursors via Rac1 signaling pathway. Targeted inhibition of Slfn2 by small interfering RNAs (siRNAs) markedly inhibits the formation of osteoclasts by diminishing the activation of c-Jun and the expression of c-Jun and NFATc1. In contrast, the overexpression of Slfn2 markedly increased phosphorylation and transactivation of c-Jun by RANKL. Together, these results indicate that Slfn2 has an essential role in osteoclastogenesis, functioning upstream of c-Jun and NFATc1.