The IL-33/ST2 axis is protective against acute inflammation during the course of periodontitis.

Periodontitis, which is induced by repeated bacterial invasion and the ensuing immune reactions that follow, is the leading cause of tooth loss. Periodontal tissue is comprised of four different components, each with potential role in pathogenesis, however, most studies on immune responses focus on gingival tissue. Here, we present a modified ligature-induced periodontitis model in male mice to analyze the pathogenesis, which captures the complexity of periodontal tissue. We find that the inflammatory response in the peri-root tissues and the expression of IL-6 and RANKL by Thy-1.2- fibroblasts/stromal cells are prominent throughout the bone destruction phase, and present already at an early stage. The initiation phase is characterized by high levels of ST2 (encoded by Il1rl1) expression in the peri-root tissue, suggesting that the IL-33/ST2 axis is involved in the pathogenesis. Both Il1rl1- and Il33-deficient mice exhibit exacerbated bone loss in the acute phase of periodontitis, along with macrophage polarization towards a classically activated phenotype and increased neutrophil infiltration, indicating a protective role of the IL-33/ST2 axis in acute inflammation. Thus, our findings highlight the hidden role of the peri-root tissue and simultaneously advance our understanding of the etiology of periodontitis via implicating the IL-33/ST2 axis.

Osteoblast Lineage Cell-derived Sema3A Regulates Bone Homeostasis Independently of Androgens.

Semaphorin 3A (Sema3A) coordinates bone resorption and formation under the control of estrogen signaling. However, the contribution of osteoblast lineage cell-derived Sema3A to vertebral homeostasis has remained unclear. Moreover, it is unknown whether androgen signaling is involved in Sema3A expression in osteoblast lineage cells. In this study, we show that osteoblast lineage cell-derived Sema3A plays a key role in bone homeostasis independent of androgen signaling. Sema3a deletion with Sp7-Cre did not alter the trabecular bone mass in lumbar vertebrae, along with there being no significant difference in Sema3a mRNA expression. In contrast, osteoblast lineage cell-specific deletion of Sema3A with BGLAP-Cre led to decreased bone volume in both long bones and lumbar vertebrae. In addition, osteoblast lineage cell-derived Sema3A was not involved in orchidectomy-induced bone loss because androgen deficiency did not affect Sema3A protein expression. Thus, these results indicate that Sema3A derived from osteoblast lineage cells acts as an osteoprotective factor, even in vertebrae, and its expression is controlled in an androgen-independent manner.

The regulation of RANKL by mechanical force.

Receptor activator of nuclear factor-κB ligand (RANKL) is a key mediator of osteoclast differentiation and bone resorption. Osteoblast-lineage cells including osteoblasts and osteocytes express RANKL, which is regulated by several different factors, including hormones, cytokines, and mechanical forces. In vivo and in vitro analyses have demonstrated that various types of mechanosensing proteins on the cell membrane (i.e. mechanosensors) and intracellular mechanosignaling proteins play essential roles in the differentiation and functions of osteoblasts, osteoclasts, and osteocytes via soluble factors, such as sclerostin, Wnt ligands, and RANKL. This section provides an overview of the in vivo and in vitro evidence for the regulation of RANKL expression by mechanosensing and mechanotransduction.

Development of cyclic peptides with potent in vivo osteogenic activity through RaPID-based affinity maturation.

Osteoporosis is caused by a disequilibrium between bone resorption and bone formation. Therapeutics for osteoporosis can be divided into antiresorptives that suppress bone resorption and anabolics which increase bone formation. Currently, the only anabolic treatment options are parathyroid hormone mimetics or an anti-sclerostin monoclonal antibody. With the current global increases in demographics at risk for osteoporosis, development of therapeutics that elicit anabolic activity through alternative mechanisms is imperative. Blockade of the PlexinB1 and Semaphorin4D interaction on osteoblasts has been shown to be a promising mechanism to increase bone formation. Here we report the discovery of cyclic peptides by a novel RaPID (Random nonstandard Peptides Integrated Discovery) system-based affinity maturation methodology that generated the peptide PB1m6A9 which binds with high affinity to both human and mouse PlexinB1. The chemically dimerized peptide, PB1d6A9, showed potent inhibition of PlexinB1 signaling in mouse primary osteoblast cultures, resulting in significant enhancement of bone formation even compared to non-Semaphorin4D-treated controls. This high anabolic activity was also observed in vivo when the lipidated PB1d6A9 (PB1d6A9-Pal) was intravenously administered once weekly to ovariectomized mice, leading to complete rescue of bone loss. The potent osteogenic properties of this peptide shows great promise as an addition to the current anabolic treatment options for bone diseases such as osteoporosis.

The key royal jelly component 10-hydroxy-2-decenoic acid protects against bone loss by inhibiting NF-κB signaling downstream of FFAR4.

The supplementation of royal jelly (RJ) is known to provide a variety of health benefits, including anti-inflammatory and anti-obesity effects. RJ treatment also reportedly protects against bone loss, but no single factor in RJ has yet been identified as an anti-osteoporosis agent. Here we fractionated RJ and identified 10-hydroxy-2-decenoic acid (10H2DA) as a key component involved in inhibiting osteoclastogenesis based on mass spectrometric analysis. We further demonstrated free fatty acid receptor 4 (FFAR4) as directly interacting with 10H2DA; binding of 10H2DA to FFAR4 on osteoclasts inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced activation of NF-κB signaling, thereby attenuating the induction of nuclear factor of activated T cells (NFAT) c1, a key transcription factor for osteoclastogenesis. Oral administration of 10H2DA attenuated bone resorption in ovariectomized mice. These results suggest a potential therapeutic approach of targeting osteoclast differentiation by the supplementation of RJ, and specifically 10H2DA, in cases of pathological bone loss such as occur in postmenopausal osteoporosis.

RANKL biology: bone metabolism, the immune system, and beyond.

Receptor activator of NF-κB (RANK) ligand (RANKL) induces the differentiation of monocyte/macrophage-lineage cells into the bone-resorbing cells called osteoclasts. Because abnormalities in RANKL, its signaling receptor RANK, or decoy receptor osteoprotegerin (OPG) lead to bone diseases such as osteopetrosis, the RANKL/RANK/OPG system is essential for bone resorption. RANKL was first discovered as a T cell-derived activator of dendritic cells (DCs) and has many functions in the immune system, including organogenesis, cellular development. The essentiality of RANKL in the bone and the immune systems lies at the root of the field of "osteoimmunology." Furthermore, this cytokine functions beyond the domains of bone metabolism and the immune system, e.g., mammary gland and hair follicle formation, body temperature regulation, muscle metabolism, and tumor development. In this review, we will summarize the current understanding of the functions of the RANKL/RANK/OPG system in biological processes.

Mechanotransduction via the Piezo1-Akt pathway underlies Sost suppression in osteocytes.

Osteocytes function as critical regulators of bone homeostasis by coordinating the functions of osteoblasts and osteoclasts, and are constantly exposed to mechanical force. However, the molecular mechanism underlying the mechanical signal transduction in osteocytes is not well understood. Here, we found that Yoda1, a selective Piezo1 agonist, increased intracellular calcium mobilization and dose-dependently decreased the expression of Sost (encoding Sclerostin) in the osteocytic cell line IDG-SW3. We also demonstrated that mechanical stretch of IDG-SW3 suppressed Sost expression, a result which was abrogated by treatment with the Piezo1 inhibitor GsMTx4, and the deficiency of Piezo1. Furthermore, the suppression of Sost expression was abolished by treatment with an Akt inhibitor. Taken together, these results indicate that the activation of the Piezo1-Akt pathway in osteocytes is required for mechanical stretch-induced downregulation of Sost expression.

Suppression of hematopoietic cell kinase ameliorates the bone destruction associated with inflammation.

Objectives: To investigate the role of non-receptor tyrosine kinases (NRTKs) in inflammation-induced osteoclastogenesis.Methods: Microarray analyses of global mRNA expression during receptor activator of NF-κB ligand (RANKL) and RANKL plus tumor necrosis factor (TNF)-α-induced osteoclast differentiation were performed. The inhibitory effect on TNF-α-induced osteoclast differentiation of A-419259, a potent inhibitor of hematopoietic cell kinase (Hck), was examined. The in vivo therapeutic effect of A-419259 treatment on lipopolysaccharide (LPS)-induced inflammatory bone destruction was evaluated.Results: We confirmed that Hck expression was selectively increased among the NRTKs during the osteoclast differentiation induced by RANKL and TNF-α, but not by RANKL alone. RANKL and TNF-α-induced osteoclast differentiation and they were dose-dependently inhibited by A-419259 treatment through inhibition of the expression of key regulators of osteoclastogenesis, including Prdm1 and Nfatc1. Notably, LPS-induced inflammatory bone loss in murine calvarial bones was ameliorated by the administration of A-419259.Conclusions: Our results demonstrate that the administration of A-419259 is effective for the inhibition of osteoclast differentiation induced by TNF-α in the presence of RANKL. Therefore, an inhibitor of Hck may be useful as a potent anti-osteoclastogenic agent for the treatment of inflammatory bone destruction.

[The bone and Wnt signaling : overview.]

The Wnt signaling pathway is known to play an important role in various biological processes including embryonic development and tissues homeostasis. Following the identification of the mutations in LRP5, encoding for the Wnt co-receptor low density lipoprotein receptor-related protein 5, associated with bone disorders in human, numerous studies have demonstrated the importance of Wnt signaling in bone cells. The Wnt signaling pathway is one of the key regulators of bone metabolism, hence the treatment using a monoclonal antibody against sclerostin, a bone-specific endogenous Wnt inhibitor, could improve bone mass and decrease fracture risk.

Autoregulation of Osteocyte Sema3A Orchestrates Estrogen Action and Counteracts Bone Aging.

Osteocyte survival is key to bone homeostasis and is perturbed in menopause and aging. However, it remains unknown how osteocyte-mediated maintenance of the skeleton is regulated by the osteoprotective factor semaphorin 3A (Sema3A), a secreted protein that is known to reduce bone resorption and enhance bone formation. Here, we show that estrogen induces osteocyte expression of Sema3A, which acts on its receptor on osteocytes to promote their survival and maintain bone homeostasis. Postnatal global and conditional deletion of Sema3a in osteoblastic cells resulted in a severe osteoporotic phenotype marked by fewer osteocytes. This phenotype was recapitulated by osteocyte-specific deficiency of either Sema3A or its receptor component neuropilin-1 (Nrp1). A stimulator of soluble guanylate cyclase-cGMP signaling mimicked Sema3A action and ameliorated bone loss after ovariectomy. We further show that serum levels of SEMA3A decreased with age or after menopause in humans. Thus, we provide a mechanistic insight into the estrogen action and a promising therapeutic approach to protect against bone-related aging.

Osteocyte regulation of orthodontic force-mediated tooth movement via RANKL expression.

Orthodontic tooth movement is achieved by the remodeling of the alveolar bone surrounding roots of teeth. Upon the application of orthodontic force, osteoclastic bone resorption occurs on the compression side of alveolar bone, towards which the teeth are driven. However, the molecular basis for the regulatory mechanisms underlying alveolar bone remodeling has not been sufficiently elucidated. Osteoclastogenesis is regulated by receptor activator of nuclear factor-κB ligand (RANKL), which is postulated to be expressed by the cells surrounding the tooth roots. Here, we show that osteocytes are the critical source of RANKL in alveolar bone remodeling during orthodontic tooth movement. Using a newly established method for the isolation of periodontal tissue component cells from alveolar bone, we found that osteocytes expressed a much higher amount of RANKL than other cells did in periodontal tissue. The critical role of osteocyte-derived RANKL was confirmed by the reduction of orthodontic tooth movement in mice specifically lacking RANKL in osteocytes. Thus, we provide in vivo evidence for the key role of osteocyte-derived RANKL in alveolar bone remodeling, establishing a molecular basis for orthodontic force-mediated bone resorption.

[Semaphorin and osteoporosis.]

Although Semaphorins were originally identified as neuronal axon guidance molecules, recent research has revealed the functions of Semaphorins in many organs, tissues and cells. Among Semaphorin family members, Semaphorin 3A(Sema3A)and Sema4D are highly expressed in bone cells and play critical roles in the regulation of bone homeostasis. Other semaphorins and their receptors are also shown to be involved in bone metabolism. In contrast, the function of Semaphorins expressed in lung is not well understood althogh many of Semaphorins are highly expressed in lung among various tissues examined. As growing evidence reveals that the link between chronic obstructive pulmonary disease(COPD)and osteoporosis, the crosstalk between bone and lung through Semaphorin signaling should be investigated.

Global epigenomic analysis indicates protocadherin-7 activates osteoclastogenesis by promoting cell-cell fusion.

Gene expression is dependent not only on genomic sequences, but also epigenetic control, in which the regulation of chromatin by histone modification plays a crucial role. Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) are related to transcriptionally activated and silenced sequences, respectively. Osteoclasts, the multinucleated cells that resorb bone, are generated by the fusion of precursor cells of monocyte/macrophage lineage. To elucidate the molecular and epigenetic regulation of osteoclast differentiation, we performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis for H3K4me3 and H3K27me3 in combination with RNA sequencing. We focused on the histone modification change from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(-) and identified the protocadherin-7 gene (Pcdh7) to be among the genes epigenetically regulated during osteoclastogenesis. Pcdh7 was induced by RANKL stimulation in an NFAT-dependent manner. The knockdown of Pcdh7 inhibited RANKL-induced osteoclast differentiation due to the impairment of cell-cell fusion, accompanied by a decreased expression of the fusion-related genes Dcstamp, Ocstamp and Atp6v0d2. This study demonstrates that Pcdh7 plays a key role in osteoclastogenesis by promoting cell-cell fusion.

Successful natural interferon-ß plus ribavirin therapy in a chronic hepatitis C patient after discontinuation of interferon-α treatment due to arrhythmia and interstitial pneumonia.

A 71-year-old female patient with hepatitis C virus genotype 1 had previously discontinued interferon (IFN)-α plus ribavirin therapy, pegylated IFN-α (pegIFN-α) monotherapy, and natural IFN-α monotherapy because of arrhythmia, interstitial pneumonia, and severe neurovegetative symptoms. She subsequently completed 72 weeks of natural IFN-ß plus ribavirin therapy without remarkable adverse effects and achieved a sustained viral response, suggesting differences in the pharmacological properties and biological effects of IFN-α and IFN-ß. Thus, natural IFN-ß plus ribavirin therapy may be a treatment option for patients with poor tolerance to IFN-α or pegIFN-α treatments.

[Bone and Stem Cells. Molecular mechanisms of the differentiation and activation of osteoclasts derived from hematopoietic cells].

Mononuclear myeloid lineage cells, which are attracted to bone surfaces by chemokines and other factors, differentiate into multinucleated bone resorbing osteoclasts by cell fusion. Receptor activator of nuclear factor-κB ligand (RANKL) , which is expressed in mesenchymal cells, including osteocytes and hypertrophic chondrocytes, is essential for osteoclast differentiation and function. Osteoclasts have the capacity to resorb bone and impaired osteoclast differentiation and/or function leads to osteopetrosis, a rare disease in which mineralized bone cannot be removed. In contrast, excessive osteoclastogenesis causes diseases such as osteoporosis. Recent findings suggest that osteoclasts can also function as positive and negative regulators of osteoblastic bone formation. Thus, understanding of the molecular mechanisms that regulate osteoclastogenesis is important to develop therapeutic approaches to prevent bone diseases. This paper reviews recent findings of the molecular mechanisms regulating osteoclast differentiation and function.

[The regulatory mechanisms of bone metabolism by semaphorin].

Bone is continuously remodeled by osteoclastic bone resorption and osteoblastic bone formation to maintain the structural integrity and mineral homeostasis. This process is called "bone remodeling" . These bone cells are regulated by mechanical stimulation and systemic (hormonal) factors in addition to autocrine, paracrine factors and cell-cell interactions. Recently, we reported that two semaphorin molecules Sema4D and Sema3A have a crucial role in the regulation of bone remodeling. Sema4D derived from osteoclasts inhibits osteoblast differentiation not to hamper osteoclastic bone resorption. Sema3A derived from osteoblast lineage cells inhibits osteoclast differentiation and promotes osteoblast differentiation synchronously to increase bone mass. These studies provide a scientific basis for future therapeutic approaches to bone diseases.

New insights into osteoclastogenic signaling mechanisms.

Bone is continuously renewed through a dynamic balance between bone resorption and formation. This process is the fundamental basis for the maintenance of normal bone mass and architecture. Osteoclasts play a crucial role in both physiological and pathological bone resorption, and receptor activator of nuclear factor-κB ligand (RANKL) is the key cytokine that induces osteoclastogenesis. Here we summarize the recent advances in the understanding of osteoclastogenic signaling by focusing on the investigation of RANKL signaling and RANKL-expressing cells in the context of osteoimmunology. The context afforded by osteoimmunology will provide a scientific basis for future therapeutic approaches to diseases related to the skeletal and immune systems.

Osteoprotection by semaphorin 3A.

The bony skeleton is maintained by local factors that regulate bone-forming osteoblasts and bone-resorbing osteoclasts, in addition to hormonal activity. Osteoprotegerin protects bone by inhibiting osteoclastic bone resorption, but no factor has yet been identified as a local determinant of bone mass that regulates both osteoclasts and osteoblasts. Here we show that semaphorin 3A (Sema3A) exerts an osteoprotective effect by both suppressing osteoclastic bone resorption and increasing osteoblastic bone formation. The binding of Sema3A to neuropilin-1 (Nrp1) inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation by inhibiting the immunoreceptor tyrosine-based activation motif (ITAM) and RhoA signalling pathways. In addition, Sema3A and Nrp1 binding stimulated osteoblast and inhibited adipocyte differentiation through the canonical Wnt/ß-catenin signalling pathway. The osteopenic phenotype in Sema3a−/− mice was recapitulated by mice in which the Sema3A-binding site of Nrp1 had been genetically disrupted. Intravenous Sema3A administration in mice increased bone volume and expedited bone regeneration. Thus, Sema3A is a promising new therapeutic agent in bone and joint diseases.

[Bone and calcium update; bone research update. Osteoclastogenesis and osteoimmunology].

Bone is constantly renewed by the balanced action of osteoblastic bone formation and osteoclastic bone resorption. This restructuring process called "bone remodeling" is important not only for maintaining bone mass and strength, but also for mineral homeostasis. Excessive osteoclast activity leads to pathological bone resorption, as seen in a variety of local or generalized osteopenic conditions such as rheumatoid arthritis, cancer bone metastasis and osteoporosis. The immune and skeletal systems share various molecules including cytokines, signaling molecules, transcription factors and membrane receptors. The scope of osteoimmunology has been extended to encompass a wide range of molecular and cellular interactions, the elucidation of which will provide a scientific basis for future therapeutic approaches to diseases related to the immune and skeletal systems.

Evidence for osteocyte regulation of bone homeostasis through RANKL expression.

Osteocytes embedded in bone have been postulated to orchestrate bone homeostasis by regulating both bone-forming osteoblasts and bone-resorbing osteoclasts. We find here that purified osteocytes express a much higher amount of receptor activator of nuclear factor-κB ligand (RANKL) and have a greater capacity to support osteoclastogenesis in vitro than osteoblasts and bone marrow stromal cells. Furthermore, the severe osteopetrotic phenotype that we observe in mice lacking RANKL specifically in osteocytes indicates that osteocytes are the major source of RANKL in bone remodeling in vivo.