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Multiple small GTPases play crucial roles in bone homeostasis by regulating the differentiation and function of bone cells, including osteoclasts and osteoblasts. Here, we investigated whether developmentally regulated GTP-binding protein 2 (Drg2), a subfamily of the GTPase superfamily, could affect bone mass by regulating osteoclast and osteoblast differentiation. Downregulation of Drg2 using siRNA in bone marrow-derived macrophages inhibited osteoclast differentiation and function and Rac1 activation in vitro. Comparatively, Drg2 downregulation in calvarial-derived osteoprogenitor cells enhanced osteoblast differentiation and function in vitro. Rac1 activation was also suppressed by Drg2 downregulation in osteoprogenitor cells. Both osteoclast and osteoblast differentiation regulated by Drg2 downregulation were restored by suppressing Rac1 activity. Drg2-deficient mice showed increased bone mass due to a dramatic reduction in osteoclast numbers without significantly affecting the number of osteoblasts. Furthermore, Drg2 downregulation strongly inhibited RANKL-induced bone loss in vivo. In summary, Drg2 contributes to bone homeostasis by regulating the differentiation and function of osteoclasts and osteoblasts through Rac1 activation. In particular, the effect of Drg2 on osteoclasts is strong enough to regulate bone mass in vivo; therefore, Drg2 has significant potential for use as a therapeutic target in bone loss-related diseases., Competing Interests: Competing interests: The authors declare no competing interests. Ethical approval: All animal experiments were approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee (CNU IACUC-H-2021-62) and performed according to the approved guidelines., (© 2025. The Author(s).)

Several CC subfamily chemokines have been reported to regulate bone metabolism by affecting osteoblast or osteoclast differentiation. However, the role of monocyte chemotactic protein 3 (MCP-3), a CC chemokine, in bone remodeling is not well understood. Here, we show that MCP-3 regulates bone remodeling by promoting osteoblast differentiation and inhibiting osteoclast differentiation. In a Ccr3-dependent manner, MCP-3 promoted osteoblast differentiation by stimulating p38 phosphorylation and suppressed osteoclast differentiation by upregulating interferon beta. MCP-3 increased bone morphogenetic protein 2-induced ectopic bone formation, and mice with MCP-3-overexpressing osteoblast precursor cells presented increased bone mass. Moreover, MCP-3 exhibited therapeutic effects by abrogating receptor activator of nuclear factor kappa-B ligand-induced bone loss. Therefore, MCP-3 has therapeutic potential for diseases involving bone loss due to its positive role in osteoblast differentiation and negative role in osteoclast differentiation., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Stanniocalcin 1 (STC1) is a calcium- and phosphate-regulating hormone that is expressed in all tissues, including bone tissues, and is involved in calcium and phosphate homeostasis. Previously, STC1 expression was found to be increased by 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] administration in renal proximal tubular cells. In this study, we investigated whether STC1 directly regulates osteoblast differentiation or reciprocally controls the effects of 1,25(OH) 2 D 3 on osteoblasts to contribute to bone homeostasis. We found that STC1 inhibited osteoblast differentiation in vitro and bone morphogenetic protein 2 (BMP2)-induced ectopic bone formation in vivo. Moreover, 1,25(OH) 2 D 3 increased STC1 expression through direct binding to the Stc1 promoter of the vitamin D receptor (VDR). STC1 activated the 1,25(OH) 2 D 3 -VDR signaling pathway through the upregulation of VDR expression mediated by the inhibition of Akt phosphorylation in osteoblasts. STC1 further increased the effects of 1,25(OH) 2 D 3 on receptor activator of nuclear factor-κB ligand (RANKL) secretion and inhibited osteoblast differentiation by exhibiting a positive correlation with 1,25(OH) 2 D 3 . The long-bone phenotype of transgenic mice overexpressing STC1 specifically in osteoblasts was not significantly different from that of wild-type mice. However, compared with that in the wild-type mice, 1,25(OH) 2 D 3 administration significantly decreased bone mass in the STC1 transgenic mice. Collectively, these results suggest that STC1 negatively regulates osteoblast differentiation and bone formation; however, the inhibitory effect of STC1 on osteoblasts is transient and can be reversed under normal conditions. Nevertheless, the synergistic effect of STC1 and 1,25(OH) 2 D 3 through 1,25(OH) 2 D 3 administration may reduce bone mass by inhibiting osteoblast differentiation., (© 2024. The Author(s).)

Oral diseases exhibit a significant association with metabolic syndrome, including dyslipidemia. However, direct evidence supporting this relationship is lacking, and the involvement of cholesterol metabolism in the pathogenesis of periodontitis (PD) has yet to be determined. In this study, we showed that high cholesterol caused periodontal inflammation in mice. Cholesterol homeostasis in human gingival fibroblasts was disrupted by enhanced uptake through C-X-C motif chemokine ligand 16 (CXCL16), upregulation of cholesterol hydroxylase (CH25H), and the production of 25-hydroxycholesterol (an oxysterol metabolite of CH25H). Retinoid-related orphan receptor α (RORα) mediated the transcriptional upregulation of inflammatory mediators; consequently, PD pathogenesis mechanisms, including alveolar bone loss, were stimulated. Our collective data provided direct evidence that hyperlipidemia is a risk factor for PD and supported that inhibition of the CXCL16-CH25H-RORα axis is a potential treatment mechanism for PD as a systemic disorder manifestation., (© 2023. The Author(s).)

STAT5 is a transcription factor that is activated by various cytokines, hormones, and growth factors. Activated STAT5 is then translocated to the nucleus and regulates the transcription of target genes, affecting several biological processes. Several studies have investigated the role of STAT5 in adipogenesis, but unfortunately, its role in adipogenesis remains controversial. In the present study, we generated adipocyte-specific Stat5 conditional knockout (cKO) (Stat5 fl/fl ;Apn-cre) mice to investigate the role of STAT5 in the adipogenesis of bone marrow mesenchymal stem cells (BMSCs). BMSC adipogenesis was significantly inhibited upon overexpression of constitutively active STAT5A, while it was enhanced in the absence of Stat5 in vitro. In vivo adipose staining and histological analyses revealed increased adipose volume in the bone marrow of Stat5 cKO mice. ATF3 is the target of STAT5 during STAT5-mediated inhibition of adipogenesis, and its transcription is regulated by the binding of STAT5 to the Atf3 promoter. ATF3 overexpression was sufficient to suppress the enhanced adipogenesis of Stat5-deficient adipocytes, and Atf3 silencing abolished the STAT5-mediated inhibition of adipogenesis. Stat5 cKO mice exhibited reduced bone volume due to an increase in the osteoclast number, and coculture of bone marrow-derived macrophages with Stat5 cKO adipocytes resulted in enhanced osteoclastogenesis, suggesting that an increase in the adipocyte number may contribute to bone loss. In summary, this study shows that STAT5 is a negative regulator of BMSC adipogenesis and contributes to bone homeostasis via direct and indirect regulation of osteoclast differentiation; therefore, it may be a leading target for the treatment of both obesity and bone loss-related diseases.

Aging is associated with cellular senescence followed by bone loss leading to bone fragility in humans. However, the regulators associated with cellular senescence in aged bones need to be identified. Hypoxia-inducible factor (HIF)-2α regulates bone remodeling via the differentiation of osteoblasts and osteoclasts. Here, we report that HIF-2α expression was highly upregulated in aged bones. HIF-2α depletion in male mice reversed age-induced bone loss, as evidenced by an increase in the number of osteoblasts and a decrease in the number of osteoclasts. In an in vitro model of doxorubicin-mediated senescence, the expression of Hif-2α and p21, a senescence marker gene, was enhanced, and osteoblastic differentiation of primary mouse calvarial preosteoblast cells was inhibited. Inhibition of senescence-induced upregulation of HIF-2α expression during matrix maturation, but not during the proliferation stage of osteoblast differentiation, reversed the age-related decrease in Runx2 and Ocn expression. However, HIF-2α knockdown did not affect p21 expression or senescence progression, indicating that HIF-2α expression upregulation in senescent osteoblasts may be a result of aging rather than a cause of cellular senescence. Osteoclasts are known to induce a senescent phenotype during in vitro osteoclastogenesis. Consistent with increased HIF-2α expression, the expression of p16 and p21 was upregulated during osteoclastogenesis of bone marrow macrophages. ChIP following overexpression or knockdown of HIF-2α using adenovirus revealed that p16 and p21 are direct targets of HIF-2α in osteoclasts. Osteoblast-specific (Hif-2α fl/fl ;Col1a1-Cre) or osteoclast-specific (Hif-2α fl/fl ;Ctsk-Cre) conditional knockout of HIF-2α in male mice reversed age-related bone loss. Collectively, our results suggest that HIF-2α acts as a senescence-related intrinsic factor in age-related dysfunction of bone homeostasis.

Peroxiredoxin II (Prx II), an antioxidant enzyme in the Prx family, reduces oxidative stress by decreasing the intracellular ROS levels. Osteoblast differentiation is promoted by bone morphogenetic protein 2 (BMP2), which upregulates the expression of osteoblast differentiation marker genes, through Smad1/5/9 phosphorylation. We found that Prx II expression was increased by a high dose of lipopolysaccharide (LPS) but was not increased by a low dose of LPS. Prx II itself caused a decrease in the osteogenic gene expression, alkaline phosphatase (ALP) activity, and Smad1/5/9 phosphorylation induced by BMP2. In addition, BMP2-induced osteogenic gene expression and ALP activity were higher in Prx II knockout (KO) cells than they were in wild-type (WT) cells. These inhibitory effects were mediated by protein phosphatase 2A Cα (PP2A Cα), which was increased and is known to induce the dephosphorylation of Smad1/5/9. The overexpression of Prx II increased the expression of PP2A Cα, and PP2A Cα was not expressed in Prx II KO cells. Moreover, PP2A Cα reduced the level of BMP2-induced osteogenic gene expression and Smad1/5/9 phosphorylation. LPS inhibited BMP2-induced Smad1/5/9 phosphorylation and the suppressed phosphorylation was restored by the PP2A inhibitor okadaic acid (OA). Bone phenotype analyses using microcomputed tomography (μCT) revealed that the Prx II KO mice had higher levels of bone mass than the levels of the WT mice. We hypothesize that Prx II has a negative role in osteoblast differentiation through the PP2A-dependent dephosphorylation of Smad1/5/9.

Osteoarthritis-the most common form of age-related degenerative whole-joint disease 1 -is primarily characterized by cartilage destruction, as well as by synovial inflammation, osteophyte formation and subchondral bone remodelling 2,3 . However, the molecular mechanisms that underlie the pathogenesis of osteoarthritis are largely unknown. Although osteoarthritis is currently considered to be associated with metabolic disorders, direct evidence for this is lacking, and the role of cholesterol metabolism in the pathogenesis of osteoarthritis has not been fully investigated 4-6 . Various types of cholesterol hydroxylases contribute to cholesterol metabolism in extrahepatic tissues by converting cellular cholesterol to circulating oxysterols, which regulate diverse biological processes 7,8 . Here we show that the CH25H-CYP7B1-RORα axis of cholesterol metabolism in chondrocytes is a crucial catabolic regulator of the pathogenesis of osteoarthritis. Osteoarthritic chondrocytes had increased levels of cholesterol because of enhanced uptake, upregulation of cholesterol hydroxylases (CH25H and CYP7B1) and increased production of oxysterol metabolites. Adenoviral overexpression of CH25H or CYP7B1 in mouse joint tissues caused experimental osteoarthritis, whereas knockout or knockdown of these hydroxylases abrogated the pathogenesis of osteoarthritis. Moreover, retinoic acid-related orphan receptor alpha (RORα) was found to mediate the induction of osteoarthritis by alterations in cholesterol metabolism. These results indicate that osteoarthritis is a disease associated with metabolic disorders and suggest that targeting the CH25H-CYP7B1-RORα axis of cholesterol metabolism may provide a therapeutic avenue for treating osteoarthritis.

Supplementation of mesenchymal stem cells (MSCs) at sites of bone resorption is required for bone homeostasis because of the non-proliferation and short lifespan properties of the osteoblasts. Calcium ions (Ca 2+ ) are released from the bone surfaces during osteoclast-mediated bone resorption. However, how elevated extracellular Ca 2+ concentrations would alter MSCs behavior in the proximal sites of bone resorption is largely unknown. In this study, we investigated the effect of extracellular Ca 2+ on MSCs phenotype depending on Ca 2+ concentrations. We found that the elevated extracellular Ca 2+ promoted cell proliferation and matrix mineralization of MSCs. In addition, MSCs induced the expression and secretion of osteopontin (OPN), which enhanced MSCs migration under the elevated extracellular Ca 2+ conditions. We developed in vitro osteoclast-mediated bone resorption conditions using mouse calvaria bone slices and demonstrated Ca 2+ is released from bone resorption surfaces. We also showed that the MSCs phenotype, including cell proliferation and migration, changed when the cells were treated with a bone resorption-conditioned medium. These findings suggest that the dynamic changes in Ca 2+ concentrations in the microenvironments of bone remodeling surfaces modulate MSCs phenotype and thereby contribute to bone regeneration.

Periodontal disease is one of the most prevalent chronic disorders worldwide. It is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss. Here, we focused on the role of adipokines, which are locally expressed by periodontal tissues, in the regulation of catabolic gene expression leading to periodontal inflammation. The expression of the nicotinamide phosphoribosyltransferase (NAMPT) adipokine was dramatically increased in inflamed human and mouse gingival tissues. NAMPT expression was also increased in lipopolysaccharide- and proinflammatory cytokine-stimulated primary cultured human gingival fibroblasts (GF). Adenovirus-mediated NAMPT (Ad-Nampt) overexpression upregulated the expression and activity of COX-2, MMP1 and MMP3 in human GF. The upregulation of IL-1β- or Ad-Nampt-induced catabolic factors was significantly abrogated by the intracellular NAMPT (iNAMPT) inhibitor, FK866 or by the sirtuin (SIRT) inhibitor, nicotinamide (NIC). Recombinant NAMPT protein or extracellular NAMPT (eNAMPT) inhibition using a blocking antibody did not alter NAMPT target gene expression levels. Moreover, intragingival Ad-Nampt injection mediated periodontitis-like phenotypes including alveolar bone loss in mice. SIRT2, a part of the SIRT family, was positively associated with NAMPT actions in human GF. Furthermore, in vivo inhibition of the NAMPT-NAD + -SIRT axis by NIC injection in mice ameliorated the periodontal inflammation and alveolar bone erosion caused by intragingival injection of Ad-Nampt. Our findings indicate that NAMPT is highly upregulated in human GF, while its enzymatic activity acts as a crucial mediator of periodontal inflammation and alveolar bone destruction via regulation of COX-2, MMP1, and MMP3 levels.

Plant homeodomain finger protein 20 (PHF20), a methyl lysine effector protein, is a component MOF-NSL lysine acetyltranferase complex. Global deletion of PHF20 has shown spinal bone defects and reduced skeletal formation. However, the molecular basis of PHF20 involved in skeletal development has not been elucidated yet. The objective of this study was to determine the role of PHF20 in osteoblast differentiation and mineralization. Expression of PHF20 was gradually increased during osteoblast differentiation. Overexpression of PHF20 enhanced ALP activity and mineralized nodule formation as well as the expression of osteogenic markers including Runx2. In contrast, inhibition of PHF20 expression reduced osteoblast differentiation and mineralization. Mechanistically, PHF20 increased the promoter activity of osteogenic genes including Og2, Alp, and Bsp through direct association with Runx2. Moreover, PHF20 increased the enrichment of H3K4me3 on the promoter of Runx2 followed by increased Runx2 promoter activity. Interestingly, Bix-01294, a histone methylation inhibitor, decreased mineralized nodule formation through decreasing the levels of H3K4me3 and Runx2. Overexpression of PHF20 restored the Bix-01294 effects. Taken together, these results indicate that methyl lysine-binding protein PHF20 might be a novel regulator of osteoblast differentiation.

Rheumatoid arthritis (RA) and osteoarthritis (OA), two common types of arthritis, affect the joints mainly by targeting the synovium and cartilage. Increasing evidence indicates that a significant network connects synovitis and cartilage destruction during the progression of arthritis. We recently demonstrated that hypoxia-inducible factor (HIF)-2α causes RA and OA by regulating the expression of catabolic factors in fibroblast-like synoviocytes (FLS) or chondrocytes. To address the reciprocal influences of HIF-2α on FLS and chondrocytes, we applied an in vitro co-culture system using a transwell apparatus. When co-cultured with HIF-2α-overexpressing chondrocytes, FLS exhibited increased expression of matrix metalloproteinases and inflammatory mediators, similar to the effects induced by tumor-necrosis factor (TNF)-α treatment of FLS. Moreover, chondrocytes co-cultured with HIF-2α-overexpressing FLS exhibited upregulation of Mmp3 and Mmp13, which is similar to the effects induced by interleukin (IL)-6 treatment of chondrocytes. We confirmed these differential HIF-2α-induced effects via distinct secretory mediators using Il6-knockout cells and a TNF-α-blocking antibody. The FLS-co-culture-induced gene expression changes in chondrocytes were significantly abrogated by IL-6 deficiency, whereas TNF-α neutralization blocked the alterations in gene expression associated with co-culture of FLS with chondrocytes. Our results further suggested that the observed changes might reflect the HIF-2α-induced upregulation of specific receptors for TNF-α (in FLS) and IL-6 (in chondrocytes). This study broadens our understanding of the possible regulatory mechanisms underlying the crosstalk between the synovium and cartilage in the presence of HIF-2α, and may suggest potential new anti-arthritis therapies.