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Introduction: Hypertension is a major risk factor for cardiovascular disease (CVD) and is associated with increased bone loss due to excessive activity of the local renin-angiotensin system (RAS). Angiotensinogen/Angiotensin (ANG) II/Angiotensin II type 1 receptor (AT1R) axis is considered as the core axis regulating RAS activity. Azilsartan is an FDA-approved selective AT1R antagonist that is used to treat hypertension. This study aimed to determine whether azilsartan affects formation of osteoclast, resorption of bone, and the expression of cytokines linked with osteoclastogenesis during lipopolysaccharide (LPS)-triggered inflammation in vivo ., Methods: In vivo , following a 5-day supracalvarial injection of LPS or tumor necrosis factor-alpha (TNF-α) with or without azilsartan, the proportion of bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, which are identified as osteoclasts on mice calvariae were counted. The mRNA expression levels of TRAP, cathepsin K, receptor activator of NF-κB ligand (RANKL), and TNF-α were also evaluated. In vitro , the effect of azilsartan (0, 0.01, 0.1, 1, and 10 μM) on RANKL and TNF-α-triggered osteoclastogenesis were investigated. Also, whether azilsartan restrains LPS-triggered TNF-α mRNA and protein expression in macrophages and RANKL expression in osteoblasts were assessed. Furthermore, western blotting for analysis of mitogen-activated protein kinases (MAPKs) signaling was conducted., Results: Azilsartan-treated calvariae exhibited significantly lower bone resorption and osteoclastogenesis than those treated with LPS alone. In vivo , LPS with azilsartan administration resulted in lower levels of receptor activator of RANKL and TNF-α mRNA expression than LPS administration alone. Nevertheless, azilsartan did not show inhibitory effect on RANKL- and TNF-α-triggered osteoclastogenesis in vitro . Compared to macrophages treated with LPS, TNF-α mRNA and protein levels were lower in macrophages treated by LPS with azilsartan. In contrast, RANKL mRNA and protein expression levels in osteoblasts were the same in cells co-treated with azilsartan and LPS and those exposed to LPS only. Furthermore, azilsartan suppressed LPS-triggered MAPKs signaling pathway in macrophages. After 5-day supracalvarial injection, there is no difference between TNF-α injection group and TNF-α with azilsartan injection group., Conclusion: These findings imply that azilsartan prevents LPS-triggered TNF-α production in macrophages, which in turn prevents LPS-Triggered osteoclast formation and bone resorption in vivo ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fan, Kitaura, Ren, Ohori, Noguchi, Marahleh, Ma, Kanou, Miura, Narita, Lin and Mizoguchi.)

The skeleton is an organ of dual functionality; on the one hand, it provides protection and structural competence. On the other hand, it participates extensively in coordinating homeostasis globally given that it is a mineral and hormonal reservoir. Bone is the only tissue in the body that goes through strategically consistent bouts of bone resorption to ensure its integrity and organismal survival in a temporally and spatially coordinated process, known as bone remodeling. Bone remodeling is directly enacted by three skeletal cell types, osteoclasts, osteoblasts, and osteocytes; these cells represent the acting force in a basic multicellular unit and ensure bone health maintenance. The osteocyte is an excellent mechanosensory cell and has been positioned as the choreographer of bone remodeling. It is, therefore, not surprising that a holistic grasp of the osteocyte entity in the bone is warranted. This review discusses osteocytogenesis and associated molecular and morphological changes and describes the osteocytic lacunocanalicular network (LCN) and its organization. We highlight new knowledge obtained from transcriptomic analyses of osteocytes and discuss the regulatory role of osteocytes in promoting osteoclastogenesis with an emphasis on the case of osteoclastogenesis in anosteocytic bones. We arrive at the conclusion that osteocytes exhibit several redundant means through which osteoclast formation can be initiated. However, whether osteocytes are true "orchestrators of bone remodeling" cannot be verified from the animal models used to study osteocyte biology in vivo . Results from studying osteocyte biology using current animal models should come with the caveat that these models are not osteocyte-specific, and conclusions from these studies should be interpreted cautiously., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Marahleh, Kitaura, Ohori, Noguchi and Mizoguchi.)

Docosahexaenoic acid (DHA) is an omega-3 fatty acid that has a range of positive impacts on human health, including anti-inflammatory effects and inhibition of osteoclast formation via G-protein-coupled receptor 120 (GPR120). Orthodontic force was reported to induce tumor necrosis factor-α (TNF-α) expression, which activates osteoclast differentiation during orthodontic tooth movement (OTM). The aim of this study was to investigate the influence of DHA on TNF-α-induced osteoclast formation and OTM in vivo . We examined osteoclast formation and bone resorption within the calvaria of both wild-type (WT) and GPR120-deficient (GPR120-KO) mice injected with phosphate-buffered saline (PBS), TNF-α, TNF-α and DHA, or DHA. DHA inhibited TNF-α-induced osteoclast formation and bone resorption in WT mice but had no effect in GPR120-KO mice. OTM experiments were performed in mouse strains with or without regular injection of DHA, and the effects of DHA on osteoclast formation in the alveolar bones during OTM were examined. DHA also suppressed OTM in WT but not GPR120-KO mice. Our data showed that DHA suppresses TNF-α-induced osteoclastogenesis and bone resorption via GPR120. TNF-α has considerable significance in OTM, and therefore, DHA may also inhibit TNF-α-induced osteoclast formation and bone resorption in OTM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ma, Kitaura, Ogawa, Ohori, Noguchi, Marahleh, Nara, Pramusita, Kinjo, Kanou, Kishikawa, Ichimura and Mizoguchi.)

Osteoimmunology peeks into the interaction of bone and the immune system, which has largely proved to be a multiplex reaction. Osteocytes have been shown to regulate bone resorption through the expression of RANKL in physiologic and pathologic conditions. TNF-α, a product of the immune system, is an important cytokine regulating bone resorption in inflammatory conditions either directly or by increasing RANKL and M-CSF expressions by osteoblasts and stromal cells. The effect of TNF-α on a wide range of cell types has been documented; however, the direct effect of TNF-α on osteocytes has not been established yet. In this study, primary osteocytes were isolated by cell sorting from neonatal calvaria of Dmp1-Topaz mice, which express the green fluorescent protein under the influence of dentin matrix protein 1 promoter. The results show that osteocytes have a significantly higher RANKL mRNA expression when cultured with TNF-α. A co-culture system of osteocytes and TNF receptors I and II deficient osteoclast precursors treated with TNF-α show a significant increase in TRAP-positive cells while cultures without TNF-α failed to show TRAP-positive cells. Additionally, in vivo experiments of TNF-α injected to mouse calvaria show an increase in TRAP-positive cell number in the suture mesenchyme and an increase in the percentage of RANKL-positive osteocytes compared to PBS-injected calvaria. Osteocytes cultured with TNF-α show up-regulation of MAPKs phosphorylation measured by western blot, and adding MAPKs inhibitors to osteocytes cultured with TNF-α significantly decreases RANKL mRNA expression compared to osteocytes cultured with TNF-α alone. We also found that TNF-α activates the NF-κB pathway in osteocytes measured as a function of p65 subunit nuclear translocation. TNF-α directly affects osteocyte RANKL expression and increases osteoclastogenesis; our results demonstrate that osteocytes guard an important role in inflammatory bone resorption mediated by TNF-α., (Copyright © 2019 Marahleh, Kitaura, Ohori, Kishikawa, Ogawa, Shen, Qi, Noguchi, Nara and Mizoguchi.)

Docosahexaenoic acid (DHA) is an n-3 fatty acid that is an important structural component of the cell membrane. DHA exerts potent anti-inflammatory effects through G protein-coupled receptor 120 (GPR120), which is a functional receptor for n-3 fatty acids. DHA also regulates osteoclast formation and function. However, no studies have investigated the effect of DHA on inflammation-induced osteoclast formation in vivo . In the present study, we investigated whether DHA influences osteoclast formation, bone resorption and the expression of osteoclast-associated cytokines during lipopolysaccharide (LPS)-induced inflammation in vivo , and then we elucidated the underlying mechanisms by using in vitro experiments. In vitro experiments revealed both receptor activator of NF-kB ligand (RANKL)- and tumor necrosis factor-α (TNF-α)-induced osteoclast formation was inhibited by DHA. Supracalvarial administration of LPS with or without DHA was carried out for 5 days and then the number of osteoclasts, ratio of bone resorption pits and the level of type I collagen C-terminal cross-linked telopeptide were measured. All measurements were significantly lower in LPS+DHA-co-administered mice than LPS-administered mice. However, this DHA-induced inhibition was not observed in LPS-, DHA-, and selective GPR120 antagonist AH7614-co-administered mice. Furthermore, the expression of RANKL and TNF-α mRNAs was lower in the LPS+DHA-co-administered group than in the LPS-administered group in vivo . TNF-α mRNA levels were decreased in macrophages co-treated with LPS+DHA compared with cells treated with LPS in vitro . In contrast, RANKL mRNA expression levels from osteoblasts co-treated with DHA and LPS in vitro were equal to that in cells treated with LPS alone. Finally, the inhibitory effects of DHA on osteoclast formation in vitro were not observed by using osteoclast precursors from GPR120-deficient mice, and inhibition of LPS-induced osteoclast formation and bone resorption by DHA in vivo was not observed in GPR120-deficient mice. These results suggest that DHA inhibits LPS-induced osteoclast formation and bone resorption in vivo via GPR120 by inhibiting LPS-induced TNF-α production in macrophages along with direct inhibition of osteoclast formation.