Lipocalin-2 expression in hypoxic murine osteocytes enhances RANKL-induced osteoclastogenesis

Lipocalin-2 expression in hypoxic murine osteocytes enhances RANKL-induced osteoclastogenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Lipocalin-2 expression in hypoxic murine osteocytes enhances RANKL-induced osteoclastogenesis Kohei Narita, Fumitoshi Ohori, Aseel Marahleh, Jinghan Ma, Jiayi Ren, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6679031/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Osteocytes regulate bone remodeling by interacting with osteoblasts and osteoclasts. Hypoxia influences osteocyte function and has been linked to increased osteoclastogenesis in pathological conditions such as orthodontic tooth movement (OTM) and bone metabolic diseases; however, the molecular mechanisms underlying these effects remain unclear. This study aimed to identify hypoxia-responsive genes in osteocytes and investigate their effects on osteoclastogenesis. Transcriptome analysis of murine long bone osteocyte-Y4 (MLO-Y4) osteocytes cultured under hypoxia (2% O₂) revealed that lipocalin-2 ( Lcn2 ) was the most significantly upregulated gene. Real-time RT-PCR confirmed increased Lcn2 expression and an elevated Rankl /osteoprotegerin ( Opg) ratio. Primary osteocytes were purified from DMP1-Topaz mice showed same hypoxic response. Functional analysis demonstrated that Lcn2 did not directly affect osteoclast precursors. However, it enhanced osteoclastogenesis via osteocytes in co-culture experiments. Western blot analysis demonstrated that LCN2 activated the MAPK signaling pathway in osteocytes. Furthermore, immunohistochemical analysis of hypoxic osteocytes on the compression side of OTM exhibited increased LCN2 expression. These findings suggest that LCN2 is upregulated in osteocytes under hypoxia and promotes osteoclastogenesis by increasing RANKL expression. This study provides new insights into the molecular mechanisms of bone resorption under hypoxic conditions and suggests Lcn2 as a potential therapeutic target for bone metabolic diseases. Biological sciences/Cell biology Biological sciences/Molecular biology osteocyte hypoxia lipocalin-2 osteoclast orthodontic tooth movement osteoclastogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Osteocytes are the primary cells embedded within the bone matrix and play a crucial role in maintaining bone homeostasis. 1 , 2 They extend dendritic processes, which allow interaction with osteoblasts and osteoclasts, thereby regulating bone remodeling. 3 , 4 When exposed to mechanical or inflammatory signals, osteocytes can alter the expression of key factors essential for osteoclast formation and bone resorption, such as RANKL, which is essential for osteoclast formation and bone resorption. 5 – 8 Hypoxia is a key factor in pathological conditions such as osteoporosis, rheumatoid arthritis, and cancer metastasis to bone, where it has been linked to the activation of osteoclasts and inflammation. 9 – 11 In orthodontic tooth movement (OTM), a hypoxic environment is created within the periodontal tissues on the compression side, 12 rendering exposure of osteocytes in this region to hypoxia. Recent studies suggest that hypoxia-induced osteocyte changes may increase bone resorption 13 , 14 ; however, the underlying molecular mechanisms remain unclear. The activation of hypoxia-inducible factor-1 alpha (HIF-1α) promotes the expression of several hypoxia-related genes, leading to alterations in cell metabolism and signaling pathways. 13 Activated HIF-1α in osteocytes has been shown to increase the expression of RANKL and promote osteoclast formation. 15 However, the broader transcriptional response of osteocytes to hypoxia and its impact on bone remodeling remain poorly understood. Lipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin (NGAL), is a secreted protein with diverse functions in inflammation, CKD, energy metabolism, iron homeostasis, and tumor progression. 16 – 18 Megalin and 24p3R, the known receptors of LCN2, are involved in intracellular signaling, inflammation, and cell survival. 19 , 20 These receptors have been identified in osteoblasts 21 ; however, their presence in osteocytes has not been confirmed. Recent studies suggest that LCN2 plays a role in bone metabolism and osteoclast formation, 21 , 22 as mechanical unloading has been shown to elevate LCN2 levels in osteoblasts. Moreover, osteoblast-derived LCN2 has been suggested to influence bone mass, energy metabolism, and appetite. 21 , 23 , 24 However, whether LCN2 is regulated in osteocytes under hypoxia and how it contributes to osteoclastogenesis and bone remodeling remains unknown. In this study, we performed transcriptome analysis to comprehensively analyze osteocyte gene expression changes under hypoxic conditions. We identified LCN2 as a significantly upregulated gene under hypoxic conditions and explored its potential function on osteoclastogenesis and bone resorption. Results Transcriptome analysis of MLO-Y4 cells under hypoxia and increased expression of LCN2 Principal component analysis (PCA) and heatmap analysis showed distinct gene expression patterns between the hypoxia-treated and control groups in MLO-Y4 osteocytes (Fig. 1 a). Among the DEGs, 1,638 genes were significantly upregulated, while 427 were significantly downregulated under hypoxia (Fig. 1 b). KEGG pathway analysis was performed to identify the biological pathways influenced by hypoxia. The analysis revealed significant changes in pathways related to inflammation, metabolism, and cell death. Particularly, inflammatory pathways such as TNF signaling (adj.P = 2.3 × 10⁻³), IL-17 signaling (adj.P = 1.1 × 10⁻²), and cytokine-cytokine receptor interaction (adj.P = 1.0 × 10⁻²) were significantly enriched. Additionally, metabolic pathways, including fatty acid metabolism (adj.P = 2.3 × 10⁻⁴), biosynthesis of cofactors (adj.P = 8.4 × 10⁻⁴) and apoptosis pathway (adj.P = 1.6 × 10⁻²) were activated under hypoxia (Fig. 1 c). A volcano plot analysis identified Lcn2 as the most upregulated gene in response to hypoxia (Fig. 1 d and Table 1 ). Table 1 Top 20 upregulated genes and bottom 20 downregulated expressed genes in hypoxia-stimulated osteocytes. Gene names log2(hypoxia/control) Gene names log2(hypoxia/control) Lcn2 5.657498247 Ifnb1 -5.03139778 Saa3 5.348509024 Hspb1 -4.794834403 Hp 5.091967973 Hsp25-ps1 -4.637273212 Cxcl5 4.56127558 Ifit3 -4.579191628 Cxcl2 4.325700288 Ifit3b -4.226851493 Ccl2 4.270203398 Hspa1b -4.214155473 Angptl4 3.99637083 Nr1d1 -4.032413927 Il6 3.991524473 Gm12902 -3.74775981 Il12b 3.750625547 Eno2 -3.702702262 Gpr84 3.572982729 Bhlhe40 -3.395421888 Csf3 3.565860988 Sik1 -3.274072262 Mmp3 3.505791142 Arl4d -3.258021924 Sema7a 3.455065655 Chaserr -3.206128235 Zc3h12a 3.435985302 Dnajb1 -3.161369148 Col3a1 3.308782705 Slc2a1 -3.125635175 Sdf2l1 3.263274845 Ero1a -3.095201693 Cxcl1 3.248553642 Ccl4 -3.006684166 Id3 3.184104711 Pet117 -2.994211064 Csf2 2.993232351 Ankrd37 -2.855582559 Gm6091 2.962308717 Gsta4 -2.763850474 GO analysis provided further insights into the cellular processes influenced by hypoxia. In the Biological Process category, pathways related to metabolic regulation were significantly enriched. For example, the ncRNA metabolic process (adj.P = 2.8 × 10⁻¹¹) and ncRNA processing (adj.P = 1.2 × 10⁻¹⁰) were highly significant. Additionally, pathways related to glycoprotein metabolic process (adj.P = 5.8 × 10⁻⁹) and protein glycosylation (adj.P = 4.7 × 10⁻⁸) were enriched (Fig. 1 e). In the Molecular Function category, genes involved in catalytic activity, acting on RNA (adj.P = 2.4 × 10 − 5 ), hexosyltransferase activity (adj.P = 5.2 × 10⁻ 4 ), and transferase activity were significantly affected (Fig. 1 f). In the Cellular Component category, the Golgi membrane (adj.P = 1.9 × 10⁻¹²), organelle inner membrane (adj.P = 2.3 × 10⁻¹⁰), and mitochondrial inner membrane (adj.P = 2.1 × 10⁻⁹) were significantly enriched (Fig. 1 g). Next, we performed real-time RT-PCR to measure the mRNA expression of Lcn2, Rankl , and Opg . The findings confirmed that Lcn2 and Rankl expression levels were significantly increased under hypoxia, while Opg expression remained unchanged, leading to a significantly elevated Rankl/Opg ratio in hypoxia-treated MLOY4 cells (Fig. 1 h–k). Increased expression of LCN2 and RANKL in primary osteocytes under hypoxia To isolate primary osteocytes, fluorescence-activated cell sorting was performed using DMP1-Topaz mice, which express a green fluorescent protein (GFP) under the control of the Dmp1 promoter (Fig. 2 a). The isolated cells displayed a characteristic stellate morphology of osteocytes (Fig. 2 b). Real-time RT-PCR analysis also showed that primary osteocytes cultured under hypoxia significantly increased Lcn2 and Rankl mRNA expression. In contrast, Opg mRNA expression remained unchanged, significantly increasing Rankl/Opg under hypoxia (Fig. 2 c-f). LCN2 induces RANKL expression in MLO-Y4 cells Immunofluorescence staining was performed to investigate whether MLO-Y4 cells express receptors for LCN2. The results confirmed the presence of megalin and 24p3R, both known LCN2 receptors, in MLO-Y4 cells (Fig. 3 a). Next, we examined the effect of LCN2 on RANKL and OPG expression. MLO-Y4 cells were treated with rmLCN2, and gene expression levels were assessed using real-time RT-PCR. After 1 day of rmLCN2 treatment, no significant changes were observed in Rankl or Opg expression, and the Rankl / Opg ratio remained unchanged (Fig. 3 b-d). However, after 3 days of rmLCN2 treatment, RANKL expression was significantly upregulated (Fig. 3 e), while OPG expression remained unchanged (Fig. 3 f). Consequently, the Rankl / Opg ratio was significantly increased (Fig. 3 g), indicating that prolonged exposure to LCN2 enhances RANKL expression in MLO-Y4 cells. LCN2 enhances osteoclastogenesis via osteocytes To investigate whether LCN2 directly influences osteoclast differentiation, osteoclast precursors were cultured under four conditions: M-CSF alone, M-CSF + RANKL, M-CSF + RANKL + LCN2, and M-CSF + LCN2. TRAP staining demonstrated that osteoclast formation occurred only in the M-CSF + RANKL and M-CSF + RANKL + LCN2 groups, indicating that LCN2 alone does not directly induce osteoclastogenesis (Fig. 4 a, b). To assess the indirect effects of LCN2 on osteoclast differentiation via osteocytes, osteoclast precursors were co-cultured with MLO-Y4 cells in the presence of vitamin D3 and prostaglandin E2. TRAP staining revealed a significant increase in TRAP-positive multinucleated osteoclasts in the LCN2-treated group compared to the control, suggesting that LCN2 enhances osteoclastogenesis through its effects on osteocytes (Fig. 4 c, d). LCN2 activates ERK1/2, p38, and JNK MAPKs signaling in MLO-Y4 cells To elucidate the signaling pathways involved in LCN2-mediated effects, we examined the activation of MAPK pathways in MLO-Y4 cells treated with rmLCN2 (Fig. 5 a). Full-length blots are provided in Supplementary Fig. S1 . Western blot analysis demonstrated that ERK1/2 phosphorylation (p-ERK1/2) significantly increased at 15 min post-treatment before declining (Fig. 5 b, c). JNK phosphorylation (p-JNK) peaked at 5 min and rapidly decreased (Fig. 5 d, e). p38 phosphorylation (p-p38) increased at 15 min and subsequently declined (Fig. 5 f, g). Expression of HIF-1α and LCN2 in osteocytes during OTM Immunohistochemical analysis showed that HIF-1α and LCN2 expression increased significantly in osteocytes at the compression side during OTM (Fig. 6 a). Expression levels peaked on day 2, with HIF-1α slightly decreasing on day 6, while LCN2 remained elevated (Fig. 6 b, c). Discussion Osteocytes play a central role in maintaining bone homeostasis by regulating osteoblast and osteoclast activity. 1 , 2 The expression of RANKL by osteocytes is crucial for osteoclast differentiation. 5 , 6 Recent studies suggest that hypoxia can promote osteoclastogenesis both directly—by acting on osteoclast precursors—and indirectly—by influencing osteocytes and osteoblasts. 23 – 28 However, the mechanisms by which hypoxic osteocytes contribute to osteoclast formation and bone remodeling remain incompletely understood. In diseases, such as osteoporosis, rheumatoid arthritis, bone metastasis, and OTM, hypoxia has been implicated in the activation of osteoclasts and inflammation. 9 – 12 Therefore, understanding the adaptive responses of osteocytes to hypoxia holds clinical significance. In this study, transcriptome analysis of hypoxia-exposed MLO-Y4 osteocytes revealed significant changes in expression of several genes, with Lcn2 showing a particular upregulation. LCN2 is widely known as a factor involved in inflammation, iron metabolism, and energy homeostasis. 16 – 22 Previous studies have shown that hypoxia induces Lcn2 expression in astrocytes and cardiomyocytes. 29 , 30 These findings are consistent with the findings that osteocytes also upregulate Lcn2 under hypoxic conditions. This suggests a broader role for Lcn2 in cellular adaptation to hypoxia. Pathway analysis indicated the activation of inflammatory pathways, including the TNF and IL-17 signaling pathways and cytokine-cytokine receptor interaction. Osteocytes contribute to the production of cytokines and regulate osteoclastogenesis under inflammatory conditions. 31 – 33 Consistent with these findings, our data showed that hypoxic stimulation upregulated inflammation-related cytokine genes, including Il6 , Il12B , and Ccl2 , and increased RANKL expression. Enhanced TNF-α signaling in osteocytes has been shown to promote RANKL expression and enhance bone resorption. 34 This suggests that hypoxia-activated inflammatory signaling in osteocytes may induce osteoclast formation. In low-oxygen environments, ATP production via oxidative phosphorylation is reduced, leading to a shift toward glycolysis and fatty acid oxidation. Hypoxia has been reported to induce oxidative stress in adipose tissue and enhance lipolysis. 35 Furthermore, intensified lipid metabolism may affect intracellular energy supply and bone remodeling. 36 , 37 These findings suggest a mechanism by which osteocytes adapt to hypoxic stress. Hypoxia is also a well-established inducer of apoptosis. KEGG pathway analysis revealed activation of apoptosis-related pathways under hypoxic conditions, aligning with previous findings that apoptotic osteocytes promote osteoclast differentiation by HIF-1α signaling. 28 Among the genes upregulated under hypoxia, Lcn2 exhibited the most significant upregulation. Lcn2 regulates inflammatory pathways and lipid metabolism 38 – 40 and may also play a role in apoptosis. 41 The concurrent upregulation of inflammatory and metabolic pathways observed in KEGG analysis suggests that LCN2 may regulate multiple processes. While the precise functions of LCN2 in osteocytes remain to be fully elucidated, our findings support its involvement in hypoxia-induced osteocyte adaptations. We further employed Gene Ontology (GO) analysis to investigate the effects of hypoxia on osteocyte function. In the Biological Process category, significant alterations were observed in RNA metabolic pathways, including ncRNA metabolic processes and ncRNA processing. This suggests that hypoxia induces significant changes in osteocyte RNA regulation as part of adaptive responses. Recent studies indicate that HIF-1α interacts with ncRNAs to regulate metabolic adaptation and stress responses. 42 Our identification of altered ncRNA-related gene expression under hypoxia suggests a potential role for ncRNAs in osteocyte adaptation. In the Molecular Function category, significant changes were observed in catalytic activity, acting on RNA, hexosyltransferase activity, and transferase. The alterations in catalytic activity, targeting RNA suggest modifications in enzyme activity that may influence mRNA stability and translation. Changes in hexosyltransferase activity indicate potential glycosylation modifications, which could affect cell signaling and the function of membrane proteins in osteocytes. Furthermore, significant changes were detected in intracellular membrane structures such as the Golgi apparatus, organellar membranes, and mitochondrial membranes. Previous studies in tumor cells have shown that hypoxic stress induces Golgi adaptation, affecting protein secretion and modifications. 43 A similar mechanism in osteocytes may enhance LCN2 secretion. Furthermore, osteocytes may adjust mitochondrial function under hypoxic conditions as part of their metabolic response, potentially increasing ATP production through enhanced electron transport. 44 A related mechanism has been reported in osteoclasts, where hypoxia-induced ATP production promotes short-term bone resorption. 45 Our findings suggest that osteocytes undergo similar mitochondrial adaptations, which may influence energy metabolism and bone remodeling. We validated our transcriptome findings using real-time RT-PCR, confirming a significant upregulation of LCN2 and RANKL mRNA levels in response to hypoxia, while OPG expression remained unchanged. This resulted in an increased RANKL/OPG ratio. Similarly, in primary osteocytes, hypoxic treatment led to a substantial elevation in both LCN2 and RANKL, further increasing the RANKL/OPG ratio. These findings suggest that osteocytes upregulate LCN2 expression under hypoxic conditions and enhance RANKL production, thereby modulating the RANKL/OPG ratio. We found that MLO-Y4 cells express both megalin and 24P3R proteins, suggesting that LCN2 may function in osteocytes through receptor-mediated signaling. When LCN2 was added to MLO-Y4 cells, RANKL expression remained unchanged at day 1 but significantly increased after 3 days of stimulation, leading to a higher RANKL/OPG ratio. These findings indicate that LCN2 does not immediately induce RANKL expression in osteocytes but exerts its effects over time. To determine whether LCN2 directly influences osteoclast formation, we treated osteoclast precursor cultures with LCN2 and assessed TRAP staining. No direct stimulation of osteoclastogenesis was observed, aligning with Rucci et al., 21 who reported that LCN2 indirectly regulates osteoclastogenesis via osteoblasts rather than acting directly on osteoclast precursors. However, Kim et al. showed that LCN2 directly suppresses osteoclast differentiation. 45 These differences are likely due to variations in the origin of the recombinant protein. Rucci et al. used mammalian cell-derived recombinant LCN2, which likely retains proper post-translational modifications. In contrast, Kim et al. used Escherichia coli -expressed recombinant LCN2, which may lack these modifications. The absence of these modifications may alter receptor interactions and signaling, potentially explaining the differences in observed effects. In co-culture experiments with MLO-Y4 cells and osteoclast precursors, the addition of LCN2 significantly increased osteoclast formation. However, when LCN2 was added to osteoclast precursors alone, no increase in osteoclastogenesis was observed, suggesting that LCN2 acts indirectly through osteocytes. This effect is likely mediated by the upregulation of RANKL expression in osteocytes. Our findings align with those of Rucci et al., 21 who demonstrated that osteoblast-derived LCN2 enhances RANKL expression and promotes osteoclastogenesis. To investigate the signaling mechanisms involved, we examined MAPK (ERK1/2, p38, and JNK) activation following LCN2 stimulation and found that LCN2 activated these pathways. This is consistent with prior research showing that LCN2 regulates neuroinflammation in microglia via p38 MAPK signaling in the central nervous system. 46 A similar mechanism may operate in osteocytes, further supporting LCN2-mediated MAPK activation. Additionally, previous studies have shown that MAPK signaling, particularly ERK and p38 MAPK, regulates RANKL expression in osteocyte, osteoblastic cells and bone marrow stromal cells under inflammatory conditions. 34 , 47 – 49 Thus, MAPK activation may also contribute to RANKL regulation in osteocytes. Our findings suggest that LCN2 expression in osteocytes is upregulated under hypoxic conditions. LCN2 secreted by osteocytes may act through paracrine and/or autocrine signaling, increasing RANKL expression via MAPK activation and subsequently promoting osteoclastogenesis. Finally, in an OTM model, immunohistochemical analysis revealed significantly increased HIF-1α and LCN2 expression in osteocytes on the compression side. Since orthodontic force is thought to induce hypoxia in the compressed periodontal ligament, 9 osteocytes in the compression zone are likely subjected to hypoxic stress. By day 2 of OTM, we observed a significant increase in HIF-1α-positive osteocytes, confirming their hypoxic state. Furthermore, the number of LCN2-positive osteocytes significantly increased on days 2 and 6, suggesting that hypoxia-induced LCN2 expression in osteocytes contributes to osteoclast formation on the compression side during OTM. This study has several limitations. First, the precise mechanisms by which LCN2 signaling, particularly via 24p3R and megalin, regulates RANKL expression remain unclear. While our data confirm that osteocyte-derived LCN2 increases RANKL, we have not directly established whether this occurs specifically through MAPK signaling. The potential involvement of other pathways, such as NF-κB or STAT3, is not fully characterized. Future studies should incorporate MAPK inhibition and gene silencing to determine whether MAPKs are essential mediators of LCN2-induced RANKL expression. Furthermore, knockdown or knockout experiments targeting LCN2 and its receptors (24p3R and Megalin) will be necessary to define their precise role in osteoclastogenesis. Secondly, the in vivo role of LCN2 in bone remodeling requires further investigation. This study primarily focused on in vitro experiments using MLO-Y4 cells, leaving its impact on bone resorption and remodeling at the in vivo level unresolved. Specifically, to clarify the role of LCN2 in OTM, future research should employ OTM models using LCN2 knockout mice to assess how LCN2 deficiency affects hypoxia-induced bone remodeling. In conclusion, our study demonstrates that osteocyte-derived LCN2, upregulated under hypoxia, may promote RANKL expression, thereby enhancing osteoclast formation (Fig. 7 ). These findings provide new insights into the molecular mechanisms underlying tooth movement in orthodontic therapy and bone metabolic disorders such as osteoporosis and rheumatoid arthritis, highlighting LCN2 as a potential therapeutic target. Materials and methods Mice and reagents This study was approved by the Institutional Animal Care and Use Committee of the Tohoku University Environmental & Safety Committee (Approval Number: 2018DnA-028-06). All procedures were conducted in accordance with the Regulations for Animal Experiments and Related Activities at Tohoku University. We also complied with ARRIVE guidelines. All mice were housed in specific pathogen-free conditions under a 12-hour light/dark cycle with ad libitum access to feed (Labo MR Stock, Nosan Corporation, Kanagawa, Japan). Eight-week-old C57BL/6J male mice (wild-type; WT) were purchased from CLEA Japan (Tokyo, Japan). C57BL/6-Tg (Dmp1-Topaz) 1lkal/J mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed in specific pathogen-free conditions under a 12-h light/dark cycle and provided ad libitum access to food (Labo MR Stock, Nosan Corporation, Kanagawa, Japan). MLO-Y4 cells were purchased from AddexBio Technologies (San Diego, CA, USA). Recombinant mouse Lcn-2 (rmLCN2) for the in vitro experiments was purchased from R&D Systems (Minneapolis, MN, USA). Recombinant mouse macrophage colony-stimulating factor (M-CSF) was obtained from the M-CSF expressing CMG14-12 cell line. Recombinant mouse RANKL was purchased from PeproTech (Rocky Hill, NJ, USA). The following polyclonal antibodies were used: SLC22A17 (Abonova, Taipei, Taiwan), megalin (Bioss, Woburn, MA, USA), HIF1-α (GeneTex, Irvine, CA, USA), and LCN2 (Aviva Systems Biology, San Diego, CA, USA). IgG polyclonal isotype control was purchased from Abcam (Cambridge, UK). Cell culture and hypoxic culture MLO-Y4 cells (2.2 × 10 6 cells/dish) were cultured in 10 cm culture dishes (Corning Costar, USA) in α-minimum essential medium (α-MEM) (Fujifilm, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) at 37°C under 5% CO₂ for 24 h. For hypoxic treatment, cells were incubated in a BIONIX hypoxic culture kit (Sugiyama-Gen, Tokyo, Japan), where the oxygen concentration was adjusted to 2% O₂ using the gas control reagent. The culture system was then sealed in a gas-barrier pouch and incubated at 37°C with 5% CO₂ for 24 h. Transcriptome analysis and bioinformatics analysis Total RNA was extracted from hypoxia-treated MLO-Y4 cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and treated with RNase-Free DNase Set (Qiagen) to remove residual genomic DNA. RNA quality was assessed using the RNA Integrity Number (RIN), with all samples exhibiting RIN values of ≥ 8, indicating high-quality RNA. Poly (A)-tailed mRNA was selectively captured for library preparation while preserving strand information. Library quality was verified, confirming the absence of primer dimers and a single peak distribution with a median fragment size of approximately 300 bp, ensuring high-quality libraries. The final library concentrations ranged from 46 to 138 nM, providing sufficient material for sequencing. Unique dual indices (10 bp) were used to minimize misassignment due to index hopping. Sequencing was performed using NovaSeq 6000, and raw output files (.cbcl format) were converted to fastq format. Quality control analysis using FastQC confirmed that each sample yielded ≥ 25 million reads. The DRAGEN RNA pipeline was used to map the fastq data to the mouse reference genome, followed by quantifying transcripts per million (TPM) values at both gene and transcript levels. Differential expression analysis (n = 3 per group) was conducted using the DRAGEN Differential Expression tool. Differentially expressed genes (DEGs) were identified based on TPM values, and TPM values were used to generate an input file for iDEP. Clustering, enrichment, and pathway analyses were performed using the iDEP tool. Preparation of primary osteocytes We isolated osteocytes using a previously described method. 34 Osteocytes were isolated from the calvariae of 5–6-day-old DMP1-Topaz mice. Calvariae were dissected under aseptic conditions and sequentially digested at 37°C with agitation using a 0.2% (w/v) collagenase solution (Fujifilm) for 20 min, followed by a 5 mM ethylenediaminetetraacetic acid (EDTA) solution for 15 min. The digestion process consisted of the following sequential steps: collagenase (fraction 1), EDTA (fraction 2), collagenase (fraction 3), collagenase (fraction 4), and EDTA (fraction 5). All fractions, except fraction 1, were cultured overnight in α-MEM supplemented with 10% FBS and 1% P/S under 5% CO₂ at 37℃. Adherent cells were harvested using trypsin–EDTA (Thermo Fisher/Sigma-Aldrich, Japan) and filtered through a 40 µm nylon cell strainer (Falcon, USA). Topaz-positive osteocytes were subsequently isolated by fluorescence-activated cell sorting (FACS; FACSAria II, BD Biosciences, Franklin Lakes, NJ, USA). The isolated Topaz-positive cells were used as primary osteocytes, which were cultured in α-MEM in a 24-well plate at a density of 3.0 × 10 4 cells/well. The plates were cultured for 24 h under normal (20% O 2 ) or hypoxia (2% O 2 ) conditions using the BIONIX hypoxic culture kit. LCN2 protein treatment MLO-Y4 cells (1.0 × 10 5 cells/well) were cultured in a 12-well plate (Corning Costar) in α-MEM with 10% FBS and 1% P/S at 37°C under 5% CO₂ for 24 h. rmLCN2 was dissolved in sterile PBS and added to the culture medium at a final concentration of 100 ng/mL. Cells were incubated with rmLCN2 for 1 or 3 days before further analysis. Real-time RT-PCR analysis Total RNA was extracted from MLO-Y4 osteocytic cells using the RNeasy Mini Kit. cDNA was synthesized using SuperScript IV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Real-time RT-PCR was performed using TB Green Premix Ex Taq II (Takara, Shiga, Japan) on a Thermal Cycle Dice Real-Time System TP800 (Takara). The cycling conditions were initial denaturation at 95°C for 30 s, followed by 50 cycles of 95°C for 5 s and 60°C for 30 s. The following gene-specific primers were used: Gapdh : 5′-GGTGGAGCCAAAAGGGTCA-3′ (forward), 5′-GGGGGCTAAGCAGTTGGT-3′ (reverse), Actb : 5′-GAAATCGTGCGTGACATCAAA-3′ (forward), 5′-TGTAGTTTCATGGATGCCACAG-3′ (reverse), Rankl : 5′-CCTGAGGCCAGCCATTT-3′ (forward), 5′-CTTGGCCCAGCCTCGAT-3′ (reverse), Opg : 5′-ATCAGAGCCTCATCACCTT-3′ (forward), 5′-CTTAGGTCCAACTACAGAGGAAC-3′ (reverse), Lcn2 : 5′-CCAGTTCGCCATGGTATTTT-3′ (forward), 5′-CACACTCACCACCCATTCAG-3′ (reverse). The expression levels of the target genes were analyzed using the 2 −ΔΔCt method, with Gapdh as the reference gene for LCN2-treated cells and Actb as the reference gene for hypoxia-treated cells. Immunofluorescence staining MLO-Y4 cells (1 × 10³ cells/well) were seeded in 96-well plates and cultured overnight at 37°C under 5% CO₂ in α-MEM with 10% FBS and 1% P/S. Cells were then washed three times with PBS and fixed using 4% formaldehyde solution in PBS for 15 min at room temperature. After washing with PBS, cells were permeabilized with 0.5% Triton X-100 (v/v) in PBS for 15 min. After another PBS wash, cells were blocked using 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature. For immunofluorescence staining, cells were incubated overnight at 4°C with the following primary antibodies, diluted in 3% BSA in PBS: SLC22A17, megalin polyclonal antibody (1:400 dilution), (1:400 dilution), IgG polyclonal isotype control. The next day, cells were washed with PBS and incubated for 1 h at room temperature in the dark with Alexa Fluor-conjugated secondary antibodies (1:100 dilution in 3% BSA in PBS). Finally, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min before visualizing fluorescent signals under a fluorescence microscope (Olympus IX71, Tokyo, Japan). Preparation of osteoclast precursors C57BL/6J mice were sacrificed, and the femora and tibiae were immediately dissected. The epiphyses were removed, and bone marrow cells were flushed out with α-MEM. The cell suspension was filtered through a 40 µm nylon cell strainer and cultured in α-MEM supplemented with 10% FBS, 1% P/S, and M-CSF (100 ng/mL) for 3 days. Adherent bone marrow macrophages (BMMs) were collected using trypsin-EDTA (Sigma-Aldrich) and used as osteoclast precursors. Osteoclast differentiation Osteoclast precursors were seeded in 96-well plates at 5.0 × 10⁴ cells/well and cultured under the following conditions: M-CSF (100 ng/mL) alone, M-CSF (100 ng/mL) + RANKL (100 ng/mL), M-CSF (100 ng/mL) + RANKL (100 ng/mL) + rmLCN2 (100 ng/mL), M-CSF (100 ng/mL) + rmLCN2 (100 ng/mL). The medium was changed every 2 days. On day 4, cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and stained with tartrate-resistant acid phosphatase (TRAP) staining consisting of acetate buffer (pH 5.0), naphthol AS-MX phosphate, fast red violet LB salt, and 50 mM sodium tartrate. Cells were considered osteoclasts if they were TRAP-positive and had two or more nuclei. Co-culture of MLO-Y4 and osteoclast precursors MLO-Y4 osteocytic cells (1.0 × 10³ cells/well) were seeded in 96-well plates and cultured for 6 h in α-MEM supplemented with 10% FBS, 1% P/S at 37°C under 5% CO₂. Afterward, osteoclast precursors (2.0 × 10⁴ cells/well) were added to the wells simultaneously with 10⁻⁸ M 1,25-dihydroxyvitamin D₃ (Sigma-Aldrich) and 10⁻⁶ M prostaglandin E₂ (PGE₂; Sigma-Aldrich). One group was additionally treated with and without rmLCN2(100 ng/mL). The medium was changed every 2 days. On day 7, cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and TRAP staining. Cells were considered osteoclasts if they were TRAP-positive and had two or more nuclei. Western blot analysis MLO-Y4 cells were cultured in α-MEM supplemented with 10% FBS, 1% P/S at 37°C under 5% CO₂ overnight. Cells were then serum-starved in 2% FBS for 4 h, followed by 1% FBS for 2 h. After starvation, cells were treated with rmLCN2 (100 ng/mL) for 0, 5, 15, 30, or 60 min. Control wells (0 min) received no LCN2. Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Millipore, Burlington, MA, USA) supplemented with 1% protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min on ice and insoluble material was removed by centrifugation. Protein samples were mixed 3:1 with Laemmli sample buffer (Bio-Rad, CA, USA) containing β-mercaptoethanol (Bio-Rad) and denatured at 95°C for 5 min. Equal amounts of protein were separated using Mini-PROTEAN TGX Precast Gels (Bio-Rad) and transferred onto 0.2 µm PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in BlockAce (DS Pharma Biomedical, Osaka, Japan) at room temperature for 1–2 h, followed by overnight incubation at 4°C with the following primary antibodies: Phospho-p38 MAPK (Thr180/Tyr182) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA, 1:3000 dilution). p38 MAPK rabbit monoclonal antibody (Cell Signaling Technology, 1:3000 dilution). Phospho-ERK1/2 (Thr202/Tyr204) rabbit monoclonal antibody (Cell Signaling Technology, 1:3000 dilution). ERK1/2 MAPK rabbit monoclonal antibody (Cell Signaling Technology, 1:3000 dilution). Phospho-SAPK/JNK (Thr183/Tyr185) rabbit polyclonal antibody (Cell Signaling Technology, 1:3000 dilution). SAPK/JNK MAPK rabbit polyclonal antibody (Cell Signaling Technology, 1:3000 dilution). β-actin mouse monoclonal antibody (Sigma-Aldrich, 1:5000 dilution). After washing with tris-buffered saline with Tween-20 (TBS-T), membranes were incubated with HRP-conjugated anti-rabbit IgG (Cell Signaling Technology, 1:5000 dilution) or anti-mouse IgG (Cytiva, Tokyo, Japan, 1:10,000 dilution) for 1 h at room temperature. Signals were detected using an enhanced chemiluminescence (ECL) system (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, IL, USA). Band intensity was quantified using ImageJ software (NIH, USA). Experimental tooth movement OTM was performed as previously described. 50 – 53 Male WT mice (8–12-week-old) (n = 4) were anesthetized with an intraperitoneal injection of a mixture of medetomidine, midazolam, and butorphanol prior to appliance placement. A nickel-titanium (Ni-Ti) closed-coil spring (TOMY SEIKO Co. Ltd., Tokyo, Japan) was attached between the upper incisor and left first molar. The appliance was secured using a stainless-steel wire (0.01 mm diameter), which was fixed to a hole drilled in the upper anterior alveolar bone and tied to the first molar. The first molar was moved mesially with a force of 10 g. Each group contained four mice, and OTM was carried out for 0, 2, and 6 days. Histological preparation and immunohistochemistry Calvariae and maxillae were harvested and fixed overnight in 4% paraformaldehyde at 4°C. Samples were then decalcified in 14% EDTA at 4°C for 3 days (calvariae) or 1 month (maxillae). Following dehydration, the specimens were embedded in paraffin and sectioned: Coronal sections (5 µm thick) for calvariae and horizontal sections (4 µm thick) for maxillae. Maxillae sections were obtained approximately 150 µm from the root branch of the upper-left first molar. For immunohistochemistry, paraffin sections were deparaffinized, rehydrated, and treated with 3% hydrogen peroxide (H₂O₂) for 15 min to block endogenous peroxidase activity. Sections were then blocked with 5% skim milk for 30 min at 37°C and incubated overnight at 4°C with HIF-1α (1:100 dilution), LCN2 (1:50 dilution) polyclonal antibodies and IgG polyclonal isotype control. After washing, sections were processed using the VECTASTAIN Elite ABC Kit (PK-6105, Vector Laboratories Inc., Burlingame, CA, USA) and developed with 3,3′-diaminobenzidine (DAB, Vector Laboratories). Hematoxylin was used for counterstaining. The percentage of HIF-1α- and LCN2-positive osteocytes was quantified within a region parallel to the long axis of the root, starting from the periodontal ligament. Statistical analysis Statistical analyses were performed using JMP Pro 17 software (JMP Statistical Discovery, Cary, NC, USA). For comparisons between the two groups, Student’s t-test was used. For multiple group comparisons, ANOVA followed by the Tukey–Kramer post hoc test was applied. Statistical significance was set at p < 0.05. Declarations Competing interests The authors declare no competing interests. Ethics approval All animal experiments were performed in accordance with the ARRIVE guidelines and were approved by the Regulations for Animal Experiments and Related Activities at Tohoku University (2018DnA-028-06). Funding This work was supported by JST SPRING, Grant Number JPMJSP2114. JSPS KAKENHI Grant Numbers JP21K10178 and JP22K17244 from the Japan Society for the Promotion of Science. Author Contribution K.N., H.K., and F.O. contributed to designing this study. K.N., H.K., F.O., J.R., A.M., J.M., A.L., Z.F., and K.M. performed the experiments. K.N., H.K., and F.O. analyzed the data and confirmed the results. K.N., H.K., and F.O. drafted the manuscript. H.K. supervised the project. All authors approved the final version. Acknowledgement A part of this study was supported by a support system for young researchers who use research equipment, instruments, and devices at Tohoku University. We thank the Biomedical Research Core of Tohoku University Graduate School of Medicine for supporting fluorescence-activated cell sorting (FACS). Data Availability The RNA-seq datasets generated and analyzed during this study are available in the NCBI Gene Expression Omnibus (GEO) under accession number GSE298138. All other data supporting the findings of this study are included in the article and its Supplementary Information files. References Bonewald, L. F. The amazing osteocyte. 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(a) Principal Component Analysis (PCA): Each point represents a sample (red: hypoxia, blue: control). (b) Heatmap: Rows represent genes, columns represent samples, and colors indicate normalized expression values (red: high expression, green: low expression). (c) KEGG pathway analysis: The top 30 statistically significant pathways are shown. The vertical axis represents KEGG pathways, while the horizontal axis represents statistical significance (Adjusted P-Value). Dot size indicates the number of associated genes, and color represents the direction of expression change (red: upregulated, blue: downregulated). (d) Volcano Plot: Each dot represents a gene. The horizontal axis represents the log\u003csub\u003e2\u003c/sub\u003e fold change, and the vertical axis represents statistical significance (−log\u003csub\u003e10\u003c/sub\u003e p-value). Red indicates significantly upregulated genes, while blue indicates significantly downregulated genes. (e-g) Gene Ontology (GO) Analysis: The top 30 statistically significant pathways are displayed. (e) Biological Process, (f) Molecular Function, and (g) Cellular Component. Dot size indicates the number of associated genes, and color represents the direction of expression change (red: upregulated, blue: downregulated). (h–k) Expression Changes of Specific Genes: Gene expression levels in MLO-Y4 cells under hypoxic conditions were measured using real-time RT-PCR and presented as box plots. Data are shown as mean ± standard deviation (SD). Statistical significance was set at *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6679031/v1/7e332d94e139b793a6173728.png"},{"id":83771871,"identity":"f4fcbcf5-1376-43e3-9fb2-442c20777586","added_by":"auto","created_at":"2025-06-02 12:39:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":247695,"visible":true,"origin":"","legend":"\u003cp\u003eReal-time RT-PCR analysis of primary osteocytes under hypoxia. (a) Number of Topaz-positive cells isolated using fluorescence-activated cell sorting (FACS): The number of Topaz-positive cells isolated using FACS is shown. (b) Morphology of isolated Topaz-positive cells: The morphology of isolated Topaz-positive cells is shown. Scale bar: 100 µm. (c–f) Gene expression analysis using real-time RT-PCR. Expression levels of (c) \u003cem\u003eLcn2\u003c/em\u003e, (d) \u003cem\u003eRankl\u003c/em\u003e, (e) \u003cem\u003eOpg\u003c/em\u003e, and (f) \u003cem\u003eRankl/Opg\u003c/em\u003e ratio in primary osteocytes under hypoxia and control conditions. Data are presented as mean ± SD. Statistical significance was set at *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6679031/v1/e495b01aa7d84de57bc47e6b.png"},{"id":83771874,"identity":"662d9fa4-dd98-42ea-8951-f18847e1ef38","added_by":"auto","created_at":"2025-06-02 12:39:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":496164,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of LCN2 receptors and real-time RT-PCR analysis of LCN2 treatment in MLO-Y4 cells. (a) Immunofluorescence staining of LCN2 receptors in MLO-Y4 cells. Expression of LCN2 receptors, Megalin and 24p3R, was analyzed using immunofluorescence staining. Nuclei were stained with DAPI. Scale bar: 100 µm. (b–d) Gene expression analysis after 1 day of LCN2 treatment. MLO-Y4 cells were cultured with rmLCN2 protein for 1 day, and gene expression levels of (b) \u003cem\u003eRankl\u003c/em\u003e, (c) \u003cem\u003eOpg\u003c/em\u003e, and (d) \u003cem\u003eRankl/Opg \u003c/em\u003eratio were analyzed using real-time RT-PCR. (e–g) Gene expression analysis after 3 days of LCN2 treatment. MLO-Y4 cells were cultured with rmLCN2 protein for 3 days, and gene expression levels of (e) \u003cem\u003eRankl\u003c/em\u003e, (f) \u003cem\u003eOpg\u003c/em\u003e, and (g) \u003cem\u003eRankl/Opg\u003c/em\u003e ratio were analyzed using real-time RT-PCR. Data are presented as mean ± SD. Statistical significance was set at *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6679031/v1/0b6e16684dee77fc90288d1a.png"},{"id":83771876,"identity":"37df7d08-fecc-42de-9243-34813f8f3f8a","added_by":"auto","created_at":"2025-06-02 12:39:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":842922,"visible":true,"origin":"","legend":"\u003cp\u003eLCN2 enhanced osteoclastogenesis via MLO-Y4 cells. (a) Microscopic images of osteoclasts. Osteoclast precursors were cultured under the following conditions: macrophage colony-stimulating factor (M-CSF), M-CSF (100 ng/mL) + receptor activator of nuclear factor-κB ligand (RANKL) (100 ng/mL), M-CSF + RANKL + rmLCN2 (100 ng/mL), or M-CSF + rmLCN2. Representative TRAP-stained images of osteoclasts under each condition are shown. Scale bar = 100 µm. (b) Quantification of TRAP-positive osteoclasts: The number of TRAP-positive cells with three or more nuclei was quantified. Data are presented as mean ± standard deviation (SD). (c) Osteoclast formation in the MLO-Y4 co-culture system: Osteoclast precursors were co-cultured with MLO-Y4 cells in the presence of vitamin D3 and prostaglandin E2, with or without rmLCN2. TRAP staining was used to visualize osteoclast formation. Scale bar = 100 µm.\u003c/p\u003e\n\u003cp\u003e(d) Quantification of TRAP-positive osteoclasts in the co-culture system. The number of TRAP-positive osteoclasts was quantified in control and rmLCN2-treated groups. Data are presented as mean ± SD. Statistical significance was determined using the Tukey–Kramer test (n = 4), with *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 considered significant.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6679031/v1/5682db55e6e43de7deb650d0.png"},{"id":83771872,"identity":"4f76b1b1-706b-481b-a551-ff768960da2a","added_by":"auto","created_at":"2025-06-02 12:39:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":374817,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of LCN2 on ERK1/2, p38, and JNK MAPK phosphorylation in MLO-Y4 cells. MLO-Y4 cells were incubated with 100 ng/mL of rmLCN2 for 0, 5, 15, 30, and 60 min; 0 indicates the condition without rmLCN2 treatment. (a) Western blot analysis: Cells were lysed and analyzed by western blotting using antibodies against phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK, JNK, and β-actin. (b-g) Quantification of protein phosphorylation levels. Band densities were measured using ImageJ software. (b) p-ERK1/2, (d) p-JNK, and (f) p-p38 phosphorylation levels were normalized to β-actin. (c), (e), and (g) phosphorylation levels were normalized to their respective total protein levels. Data are expressed as mean ± SD (n = 3). Statistical significance was determined using Tukey-Kramer test, with *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6679031/v1/fcbd325e9c9e0d7bc574f92b.png"},{"id":83771966,"identity":"3ebb1973-10d1-4775-ba96-3ae78fc50bab","added_by":"auto","created_at":"2025-06-02 12:47:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":624026,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of HIF-1α and LCN2 expression in osteocytes on the compression side during orthodontic tooth movement (OTM). (a) Immunohistochemical analysis of HIF-1α and LCN2 expression. The expression of HIF-1α and LCN2 in osteocytes was examined during OTM. The percentage of HIF-1α- and LCN2-positive osteocytes was evaluated within a 400 × 200 µm region on the mesial periodontal ligament compression side, approximately 150 µm from the distobuccal root branch of the upper left first molar. After initiating OTM, observations were conducted on days 0, 2, and 6. Arrowheads indicate HIF-1α- or LCN2-positive osteocytes. Black arrows indicate the direction of orthodontic force. M: mesial side; D: distal side; a: alveolar bone; p: periodontal ligament; r: root. Black scale bar = 50 µm. White scale bar = 25 µm. (b) Quantification of HIF-1α- and LCN2-Positive Osteocytes during OTM: The percentage of HIF-1α- and LCN2-positive osteocytes was quantified at each time point. Data are presented as mean ± SD. Statistical significance was determined using the Tukey–Kramer test (n = 4), with *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 considered significant.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6679031/v1/e793d067f5c14a851f64314b.png"},{"id":83771877,"identity":"57a676a9-47c5-4120-8ffd-9170bc5353e5","added_by":"auto","created_at":"2025-06-02 12:39:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":221818,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of how hypoxic stress induces LCN2 in osteocytes and stimulates bone resorption. Under hypoxia conditions, osteocytes increase the expression of LCN2, which, through autocrine and paracrine signaling, enhances RANKL expression. Increased RANKL then drives the differentiation of osteoclast precursor cells into osteoclasts, thereby promoting bone resorption.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6679031/v1/3c0ccb526eccbde7c823a8fa.png"},{"id":100069467,"identity":"0ef5c408-ba17-4e02-aed2-29c152384ab4","added_by":"auto","created_at":"2026-01-12 16:14:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4185254,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6679031/v1/13a554d2-c0f1-49a4-88b3-450835afddb4.pdf"},{"id":83771967,"identity":"6646cadf-94a3-47f7-95b6-194d5219f409","added_by":"auto","created_at":"2025-06-02 12:47:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":329199,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.docx","url":"https://assets-eu.researchsquare.com/files/rs-6679031/v1/ed5b89627c32a18ac2802c89.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lipocalin-2 expression in hypoxic murine osteocytes enhances RANKL-induced osteoclastogenesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteocytes are the primary cells embedded within the bone matrix and play a crucial role in maintaining bone homeostasis.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e They extend dendritic processes, which allow interaction with osteoblasts and osteoclasts, thereby regulating bone remodeling.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e When exposed to mechanical or inflammatory signals, osteocytes can alter the expression of key factors essential for osteoclast formation and bone resorption, such as RANKL, which is essential for osteoclast formation and bone resorption.\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHypoxia is a key factor in pathological conditions such as osteoporosis, rheumatoid arthritis, and cancer metastasis to bone, where it has been linked to the activation of osteoclasts and inflammation.\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e In orthodontic tooth movement (OTM), a hypoxic environment is created within the periodontal tissues on the compression side,\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e rendering exposure of osteocytes in this region to hypoxia. Recent studies suggest that hypoxia-induced osteocyte changes may increase bone resorption\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e; however, the underlying molecular mechanisms remain unclear. The activation of hypoxia-inducible factor-1 alpha (HIF-1α) promotes the expression of several hypoxia-related genes, leading to alterations in cell metabolism and signaling pathways.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Activated HIF-1α in osteocytes has been shown to increase the expression of RANKL and promote osteoclast formation.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e However, the broader transcriptional response of osteocytes to hypoxia and its impact on bone remodeling remain poorly understood.\u003c/p\u003e \u003cp\u003eLipocalin-2 (LCN2), also known as neutrophil gelatinase-associated lipocalin (NGAL), is a secreted protein with diverse functions in inflammation, CKD, energy metabolism, iron homeostasis, and tumor progression.\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Megalin and 24p3R, the known receptors of LCN2, are involved in intracellular signaling, inflammation, and cell survival.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e These receptors have been identified in osteoblasts\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e; however, their presence in osteocytes has not been confirmed. Recent studies suggest that LCN2 plays a role in bone metabolism and osteoclast formation,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e as mechanical unloading has been shown to elevate LCN2 levels in osteoblasts. Moreover, osteoblast-derived LCN2 has been suggested to influence bone mass, energy metabolism, and appetite.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e However, whether LCN2 is regulated in osteocytes under hypoxia and how it contributes to osteoclastogenesis and bone remodeling remains unknown.\u003c/p\u003e \u003cp\u003eIn this study, we performed transcriptome analysis to comprehensively analyze osteocyte gene expression changes under hypoxic conditions. We identified LCN2 as a significantly upregulated gene under hypoxic conditions and explored its potential function on osteoclastogenesis and bone resorption.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome analysis of MLO-Y4 cells under hypoxia and increased expression of LCN2\u003c/h2\u003e \u003cp\u003ePrincipal component analysis (PCA) and heatmap analysis showed distinct gene expression patterns between the hypoxia-treated and control groups in MLO-Y4 osteocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Among the DEGs, 1,638 genes were significantly upregulated, while 427 were significantly downregulated under hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eKEGG pathway analysis was performed to identify the biological pathways influenced by hypoxia. The analysis revealed significant changes in pathways related to inflammation, metabolism, and cell death. Particularly, inflammatory pathways such as TNF signaling (adj.P\u0026thinsp;=\u0026thinsp;2.3 \u0026times; 10⁻\u0026sup3;), IL-17 signaling (adj.P\u0026thinsp;=\u0026thinsp;1.1 \u0026times; 10⁻\u0026sup2;), and cytokine-cytokine receptor interaction (adj.P\u0026thinsp;=\u0026thinsp;1.0 \u0026times; 10⁻\u0026sup2;) were significantly enriched. Additionally, metabolic pathways, including fatty acid metabolism (adj.P\u0026thinsp;=\u0026thinsp;2.3 \u0026times; 10⁻⁴), biosynthesis of cofactors (adj.P\u0026thinsp;=\u0026thinsp;8.4 \u0026times; 10⁻⁴) and apoptosis pathway (adj.P\u0026thinsp;=\u0026thinsp;1.6 \u0026times; 10⁻\u0026sup2;) were activated under hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). A volcano plot analysis identified \u003cem\u003eLcn2\u003c/em\u003e as the most upregulated gene in response to hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTop 20 upregulated genes and bottom 20 downregulated expressed genes in hypoxia-stimulated osteocytes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene names\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003elog2(hypoxia/control)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGene names\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003elog2(hypoxia/control)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLcn2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.657498247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIfnb1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-5.03139778\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSaa3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.348509024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHspb1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-4.794834403\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.091967973\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHsp25-ps1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-4.637273212\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCxcl5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.56127558\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIfit3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-4.579191628\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCxcl2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.325700288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIfit3b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-4.226851493\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCcl2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.270203398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHspa1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-4.214155473\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAngptl4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.99637083\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNr1d1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-4.032413927\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIl6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.991524473\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGm12902\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-3.74775981\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIl12b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.750625547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEno2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-3.702702262\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGpr84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.572982729\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBhlhe40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-3.395421888\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsf3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.565860988\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSik1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-3.274072262\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMmp3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.505791142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eArl4d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-3.258021924\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSema7a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.455065655\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChaserr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-3.206128235\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZc3h12a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.435985302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDnajb1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-3.161369148\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCol3a1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.308782705\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlc2a1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-3.125635175\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSdf2l1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.263274845\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEro1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-3.095201693\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCxcl1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.248553642\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCcl4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-3.006684166\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eId3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.184104711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePet117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-2.994211064\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsf2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.993232351\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnkrd37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-2.855582559\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGm6091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.962308717\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGsta4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-2.763850474\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGO analysis provided further insights into the cellular processes influenced by hypoxia. In the Biological Process category, pathways related to metabolic regulation were significantly enriched. For example, the ncRNA metabolic process (adj.P\u0026thinsp;=\u0026thinsp;2.8 \u0026times; 10⁻\u0026sup1;\u0026sup1;) and ncRNA processing (adj.P\u0026thinsp;=\u0026thinsp;1.2 \u0026times; 10⁻\u0026sup1;⁰) were highly significant. Additionally, pathways related to glycoprotein metabolic process (adj.P\u0026thinsp;=\u0026thinsp;5.8 \u0026times; 10⁻⁹) and protein glycosylation (adj.P\u0026thinsp;=\u0026thinsp;4.7 \u0026times; 10⁻⁸) were enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). In the Molecular Function category, genes involved in catalytic activity, acting on RNA (adj.P\u0026thinsp;=\u0026thinsp;2.4 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e), hexosyltransferase activity (adj.P\u0026thinsp;=\u0026thinsp;5.2 \u0026times; 10⁻\u003csup\u003e4\u003c/sup\u003e), and transferase activity were significantly affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). In the Cellular Component category, the Golgi membrane (adj.P\u0026thinsp;=\u0026thinsp;1.9 \u0026times; 10⁻\u0026sup1;\u0026sup2;), organelle inner membrane (adj.P\u0026thinsp;=\u0026thinsp;2.3 \u0026times; 10⁻\u0026sup1;⁰), and mitochondrial inner membrane (adj.P\u0026thinsp;=\u0026thinsp;2.1 \u0026times; 10⁻⁹) were significantly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eNext, we performed real-time RT-PCR to measure the mRNA expression of \u003cem\u003eLcn2, Rankl\u003c/em\u003e, and \u003cem\u003eOpg\u003c/em\u003e. The findings confirmed that \u003cem\u003eLcn2\u003c/em\u003e and \u003cem\u003eRankl\u003c/em\u003e expression levels were significantly increased under hypoxia, while \u003cem\u003eOpg\u003c/em\u003e expression remained unchanged, leading to a significantly elevated \u003cem\u003eRankl/Opg\u003c/em\u003e ratio in hypoxia-treated MLOY4 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh\u0026ndash;k).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIncreased expression of LCN2 and RANKL in primary osteocytes under hypoxia\u003c/h3\u003e\n\u003cp\u003eTo isolate primary osteocytes, fluorescence-activated cell sorting was performed using DMP1-Topaz mice, which express a green fluorescent protein (GFP) under the control of the \u003cem\u003eDmp1\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The isolated cells displayed a characteristic stellate morphology of osteocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eReal-time RT-PCR analysis also showed that primary osteocytes cultured under hypoxia significantly increased \u003cem\u003eLcn2\u003c/em\u003e and \u003cem\u003eRankl\u003c/em\u003e mRNA expression. In contrast, \u003cem\u003eOpg\u003c/em\u003e mRNA expression remained unchanged, significantly increasing \u003cem\u003eRankl/Opg\u003c/em\u003e under hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-f).\u003c/p\u003e\n\u003ch3\u003eLCN2 induces RANKL expression in MLO-Y4 cells\u003c/h3\u003e\n\u003cp\u003eImmunofluorescence staining was performed to investigate whether MLO-Y4 cells express receptors for LCN2. The results confirmed the presence of megalin and 24p3R, both known LCN2 receptors, in MLO-Y4 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Next, we examined the effect of LCN2 on RANKL and OPG expression. MLO-Y4 cells were treated with rmLCN2, and gene expression levels were assessed using real-time RT-PCR. After 1 day of rmLCN2 treatment, no significant changes were observed in \u003cem\u003eRankl\u003c/em\u003e or \u003cem\u003eOpg\u003c/em\u003e expression, and the \u003cem\u003eRankl\u003c/em\u003e/\u003cem\u003eOpg\u003c/em\u003e ratio remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-d). However, after 3 days of rmLCN2 treatment, RANKL expression was significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), while OPG expression remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Consequently, the \u003cem\u003eRankl\u003c/em\u003e/\u003cem\u003eOpg\u003c/em\u003e ratio was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg), indicating that prolonged exposure to LCN2 enhances RANKL expression in MLO-Y4 cells.\u003c/p\u003e\n\u003ch3\u003eLCN2 enhances osteoclastogenesis via osteocytes\u003c/h3\u003e\n\u003cp\u003eTo investigate whether LCN2 directly influences osteoclast differentiation, osteoclast precursors were cultured under four conditions: M-CSF alone, M-CSF\u0026thinsp;+\u0026thinsp;RANKL, M-CSF\u0026thinsp;+\u0026thinsp;RANKL\u0026thinsp;+\u0026thinsp;LCN2, and M-CSF\u0026thinsp;+\u0026thinsp;LCN2. TRAP staining demonstrated that osteoclast formation occurred only in the M-CSF\u0026thinsp;+\u0026thinsp;RANKL and M-CSF\u0026thinsp;+\u0026thinsp;RANKL\u0026thinsp;+\u0026thinsp;LCN2 groups, indicating that LCN2 alone does not directly induce osteoclastogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003eTo assess the indirect effects of LCN2 on osteoclast differentiation via osteocytes, osteoclast precursors were co-cultured with MLO-Y4 cells in the presence of vitamin D3 and prostaglandin E2. TRAP staining revealed a significant increase in TRAP-positive multinucleated osteoclasts in the LCN2-treated group compared to the control, suggesting that LCN2 enhances osteoclastogenesis through its effects on osteocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d).\u003c/p\u003e\n\u003ch3\u003eLCN2 activates ERK1/2, p38, and JNK MAPKs signaling in MLO-Y4 cells\u003c/h3\u003e\n\u003cp\u003eTo elucidate the signaling pathways involved in LCN2-mediated effects, we examined the activation of MAPK pathways in MLO-Y4 cells treated with rmLCN2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Full-length blots are provided in Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Western blot analysis demonstrated that ERK1/2 phosphorylation (p-ERK1/2) significantly increased at 15 min post-treatment before declining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c). JNK phosphorylation (p-JNK) peaked at 5 min and rapidly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, e). p38 phosphorylation (p-p38) increased at 15 min and subsequently declined (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, g).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExpression of HIF-1α and LCN2 in osteocytes during OTM\u003c/h2\u003e \u003cp\u003eImmunohistochemical analysis showed that HIF-1α and LCN2 expression increased significantly in osteocytes at the compression side during OTM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Expression levels peaked on day 2, with HIF-1α slightly decreasing on day 6, while LCN2 remained elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOsteocytes play a central role in maintaining bone homeostasis by regulating osteoblast and osteoclast activity.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e The expression of RANKL by osteocytes is crucial for osteoclast differentiation.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Recent studies suggest that hypoxia can promote osteoclastogenesis both directly\u0026mdash;by acting on osteoclast precursors\u0026mdash;and indirectly\u0026mdash;by influencing osteocytes and osteoblasts.\u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25 CR26 CR27\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e However, the mechanisms by which hypoxic osteocytes contribute to osteoclast formation and bone remodeling remain incompletely understood. In diseases, such as osteoporosis, rheumatoid arthritis, bone metastasis, and OTM, hypoxia has been implicated in the activation of osteoclasts and inflammation.\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Therefore, understanding the adaptive responses of osteocytes to hypoxia holds clinical significance.\u003c/p\u003e \u003cp\u003eIn this study, transcriptome analysis of hypoxia-exposed MLO-Y4 osteocytes revealed significant changes in expression of several genes, with \u003cem\u003eLcn2\u003c/em\u003e showing a particular upregulation. LCN2 is widely known as a factor involved in inflammation, iron metabolism, and energy homeostasis.\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20 CR21\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Previous studies have shown that hypoxia induces \u003cem\u003eLcn2\u003c/em\u003e expression in astrocytes and cardiomyocytes.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e These findings are consistent with the findings that osteocytes also upregulate \u003cem\u003eLcn2\u003c/em\u003e under hypoxic conditions. This suggests a broader role for \u003cem\u003eLcn2\u003c/em\u003e in cellular adaptation to hypoxia.\u003c/p\u003e \u003cp\u003ePathway analysis indicated the activation of inflammatory pathways, including the TNF and IL-17 signaling pathways and cytokine-cytokine receptor interaction. Osteocytes contribute to the production of cytokines and regulate osteoclastogenesis under inflammatory conditions.\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Consistent with these findings, our data showed that hypoxic stimulation upregulated inflammation-related cytokine genes, including \u003cem\u003eIl6\u003c/em\u003e, \u003cem\u003eIl12B\u003c/em\u003e, and \u003cem\u003eCcl2\u003c/em\u003e, and increased RANKL expression. Enhanced TNF-α signaling in osteocytes has been shown to promote RANKL expression and enhance bone resorption.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e This suggests that hypoxia-activated inflammatory signaling in osteocytes may induce osteoclast formation. In low-oxygen environments, ATP production via oxidative phosphorylation is reduced, leading to a shift toward glycolysis and fatty acid oxidation. Hypoxia has been reported to induce oxidative stress in adipose tissue and enhance lipolysis.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e Furthermore, intensified lipid metabolism may affect intracellular energy supply and bone remodeling.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e These findings suggest a mechanism by which osteocytes adapt to hypoxic stress. Hypoxia is also a well-established inducer of apoptosis. KEGG pathway analysis revealed activation of apoptosis-related pathways under hypoxic conditions, aligning with previous findings that apoptotic osteocytes promote osteoclast differentiation by HIF-1α signaling.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Among the genes upregulated under hypoxia, \u003cem\u003eLcn2\u003c/em\u003e exhibited the most significant upregulation. \u003cem\u003eLcn2\u003c/em\u003e regulates inflammatory pathways and lipid metabolism\u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and may also play a role in apoptosis.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e The concurrent upregulation of inflammatory and metabolic pathways observed in KEGG analysis suggests that LCN2 may regulate multiple processes. While the precise functions of LCN2 in osteocytes remain to be fully elucidated, our findings support its involvement in hypoxia-induced osteocyte adaptations.\u003c/p\u003e \u003cp\u003eWe further employed Gene Ontology (GO) analysis to investigate the effects of hypoxia on osteocyte function. In the Biological Process category, significant alterations were observed in RNA metabolic pathways, including ncRNA metabolic processes and ncRNA processing. This suggests that hypoxia induces significant changes in osteocyte RNA regulation as part of adaptive responses. Recent studies indicate that HIF-1α interacts with ncRNAs to regulate metabolic adaptation and stress responses.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e Our identification of altered ncRNA-related gene expression under hypoxia suggests a potential role for ncRNAs in osteocyte adaptation. In the Molecular Function category, significant changes were observed in catalytic activity, acting on RNA, hexosyltransferase activity, and transferase. The alterations in catalytic activity, targeting RNA suggest modifications in enzyme activity that may influence mRNA stability and translation. Changes in hexosyltransferase activity indicate potential glycosylation modifications, which could affect cell signaling and the function of membrane proteins in osteocytes. Furthermore, significant changes were detected in intracellular membrane structures such as the Golgi apparatus, organellar membranes, and mitochondrial membranes. Previous studies in tumor cells have shown that hypoxic stress induces Golgi adaptation, affecting protein secretion and modifications.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e A similar mechanism in osteocytes may enhance LCN2 secretion. Furthermore, osteocytes may adjust mitochondrial function under hypoxic conditions as part of their metabolic response, potentially increasing ATP production through enhanced electron transport.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e A related mechanism has been reported in osteoclasts, where hypoxia-induced ATP production promotes short-term bone resorption.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e Our findings suggest that osteocytes undergo similar mitochondrial adaptations, which may influence energy metabolism and bone remodeling.\u003c/p\u003e \u003cp\u003eWe validated our transcriptome findings using real-time RT-PCR, confirming a significant upregulation of LCN2 and RANKL mRNA levels in response to hypoxia, while OPG expression remained unchanged. This resulted in an increased RANKL/OPG ratio. Similarly, in primary osteocytes, hypoxic treatment led to a substantial elevation in both LCN2 and RANKL, further increasing the RANKL/OPG ratio. These findings suggest that osteocytes upregulate LCN2 expression under hypoxic conditions and enhance RANKL production, thereby modulating the RANKL/OPG ratio.\u003c/p\u003e \u003cp\u003eWe found that MLO-Y4 cells express both megalin and 24P3R proteins, suggesting that LCN2 may function in osteocytes through receptor-mediated signaling. When LCN2 was added to MLO-Y4 cells, RANKL expression remained unchanged at day 1 but significantly increased after 3 days of stimulation, leading to a higher RANKL/OPG ratio. These findings indicate that LCN2 does not immediately induce RANKL expression in osteocytes but exerts its effects over time.\u003c/p\u003e \u003cp\u003eTo determine whether LCN2 directly influences osteoclast formation, we treated osteoclast precursor cultures with LCN2 and assessed TRAP staining. No direct stimulation of osteoclastogenesis was observed, aligning with Rucci et al.,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e who reported that LCN2 indirectly regulates osteoclastogenesis via osteoblasts rather than acting directly on osteoclast precursors. However, Kim et al. showed that LCN2 directly suppresses osteoclast differentiation.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e These differences are likely due to variations in the origin of the recombinant protein. Rucci et al. used mammalian cell-derived recombinant LCN2, which likely retains proper post-translational modifications. In contrast, Kim et al. used \u003cem\u003eEscherichia coli\u003c/em\u003e-expressed recombinant LCN2, which may lack these modifications. The absence of these modifications may alter receptor interactions and signaling, potentially explaining the differences in observed effects.\u003c/p\u003e \u003cp\u003eIn co-culture experiments with MLO-Y4 cells and osteoclast precursors, the addition of LCN2 significantly increased osteoclast formation. However, when LCN2 was added to osteoclast precursors alone, no increase in osteoclastogenesis was observed, suggesting that LCN2 acts indirectly through osteocytes. This effect is likely mediated by the upregulation of RANKL expression in osteocytes. Our findings align with those of Rucci et al.,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e who demonstrated that osteoblast-derived LCN2 enhances RANKL expression and promotes osteoclastogenesis.\u003c/p\u003e \u003cp\u003eTo investigate the signaling mechanisms involved, we examined MAPK (ERK1/2, p38, and JNK) activation following LCN2 stimulation and found that LCN2 activated these pathways. This is consistent with prior research showing that LCN2 regulates neuroinflammation in microglia via p38 MAPK signaling in the central nervous system.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e A similar mechanism may operate in osteocytes, further supporting LCN2-mediated MAPK activation. Additionally, previous studies have shown that MAPK signaling, particularly ERK and p38 MAPK, regulates RANKL expression in osteocyte, osteoblastic cells and bone marrow stromal cells under inflammatory conditions.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e Thus, MAPK activation may also contribute to RANKL regulation in osteocytes.\u003c/p\u003e \u003cp\u003eOur findings suggest that LCN2 expression in osteocytes is upregulated under hypoxic conditions. LCN2 secreted by osteocytes may act through paracrine and/or autocrine signaling, increasing RANKL expression via MAPK activation and subsequently promoting osteoclastogenesis.\u003c/p\u003e \u003cp\u003eFinally, in an OTM model, immunohistochemical analysis revealed significantly increased HIF-1α and LCN2 expression in osteocytes on the compression side. Since orthodontic force is thought to induce hypoxia in the compressed periodontal ligament,\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e osteocytes in the compression zone are likely subjected to hypoxic stress. By day 2 of OTM, we observed a significant increase in HIF-1α-positive osteocytes, confirming their hypoxic state. Furthermore, the number of LCN2-positive osteocytes significantly increased on days 2 and 6, suggesting that hypoxia-induced LCN2 expression in osteocytes contributes to osteoclast formation on the compression side during OTM.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, the precise mechanisms by which LCN2 signaling, particularly via 24p3R and megalin, regulates RANKL expression remain unclear. While our data confirm that osteocyte-derived LCN2 increases RANKL, we have not directly established whether this occurs specifically through MAPK signaling. The potential involvement of other pathways, such as NF-κB or STAT3, is not fully characterized. Future studies should incorporate MAPK inhibition and gene silencing to determine whether MAPKs are essential mediators of LCN2-induced RANKL expression. Furthermore, knockdown or knockout experiments targeting LCN2 and its receptors (24p3R and Megalin) will be necessary to define their precise role in osteoclastogenesis. Secondly, the in vivo role of LCN2 in bone remodeling requires further investigation. This study primarily focused on in vitro experiments using MLO-Y4 cells, leaving its impact on bone resorption and remodeling at the in vivo level unresolved. Specifically, to clarify the role of LCN2 in OTM, future research should employ OTM models using LCN2 knockout mice to assess how LCN2 deficiency affects hypoxia-induced bone remodeling.\u003c/p\u003e \u003cp\u003eIn conclusion, our study demonstrates that osteocyte-derived LCN2, upregulated under hypoxia, may promote RANKL expression, thereby enhancing osteoclast formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These findings provide new insights into the molecular mechanisms underlying tooth movement in orthodontic therapy and bone metabolic disorders such as osteoporosis and rheumatoid arthritis, highlighting LCN2 as a potential therapeutic target.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMice and reagents\u003c/h2\u003e \u003cp\u003eThis study was approved by the Institutional Animal Care and Use Committee of the Tohoku University Environmental \u0026amp; Safety Committee (Approval Number: 2018DnA-028-06). All procedures were conducted in accordance with the Regulations for Animal Experiments and Related Activities at Tohoku University. We also complied with ARRIVE guidelines. All mice were housed in specific pathogen-free conditions under a 12-hour light/dark cycle with \u003cem\u003ead libitum\u003c/em\u003e access to feed (Labo MR Stock, Nosan Corporation, Kanagawa, Japan).\u003c/p\u003e \u003cp\u003eEight-week-old C57BL/6J male mice (wild-type; WT) were purchased from CLEA Japan (Tokyo, Japan). C57BL/6-Tg (Dmp1-Topaz) 1lkal/J mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed in specific pathogen-free conditions under a 12-h light/dark cycle and provided ad libitum access to food (Labo MR Stock, Nosan Corporation, Kanagawa, Japan). MLO-Y4 cells were purchased from AddexBio Technologies (San Diego, CA, USA). Recombinant mouse Lcn-2 (rmLCN2) for the in vitro experiments was purchased from R\u0026amp;D Systems (Minneapolis, MN, USA). Recombinant mouse macrophage colony-stimulating factor (M-CSF) was obtained from the M-CSF expressing CMG14-12 cell line. Recombinant mouse RANKL was purchased from PeproTech (Rocky Hill, NJ, USA). The following polyclonal antibodies were used: SLC22A17 (Abonova, Taipei, Taiwan), megalin (Bioss, Woburn, MA, USA), HIF1-α (GeneTex, Irvine, CA, USA), and LCN2 (Aviva Systems Biology, San Diego, CA, USA). IgG polyclonal isotype control was purchased from Abcam (Cambridge, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and hypoxic culture\u003c/h2\u003e \u003cp\u003eMLO-Y4 cells (2.2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/dish) were cultured in 10 cm culture dishes (Corning Costar, USA) in α-minimum essential medium (α-MEM) (Fujifilm, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) at 37\u0026deg;C under 5% CO₂ for 24 h. For hypoxic treatment, cells were incubated in a BIONIX hypoxic culture kit (Sugiyama-Gen, Tokyo, Japan), where the oxygen concentration was adjusted to 2% O₂ using the gas control reagent. The culture system was then sealed in a gas-barrier pouch and incubated at 37\u0026deg;C with 5% CO₂ for 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome analysis and bioinformatics analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from hypoxia-treated MLO-Y4 cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and treated with RNase-Free DNase Set (Qiagen) to remove residual genomic DNA. RNA quality was assessed using the RNA Integrity Number (RIN), with all samples exhibiting RIN values of \u0026ge;\u0026thinsp;8, indicating high-quality RNA.\u003c/p\u003e \u003cp\u003ePoly (A)-tailed mRNA was selectively captured for library preparation while preserving strand information. Library quality was verified, confirming the absence of primer dimers and a single peak distribution with a median fragment size of approximately 300 bp, ensuring high-quality libraries. The final library concentrations ranged from 46 to 138 nM, providing sufficient material for sequencing. Unique dual indices (10 bp) were used to minimize misassignment due to index hopping. Sequencing was performed using NovaSeq 6000, and raw output files (.cbcl format) were converted to fastq format. Quality control analysis using FastQC confirmed that each sample yielded\u0026thinsp;\u0026ge;\u0026thinsp;25\u0026nbsp;million reads. The DRAGEN RNA pipeline was used to map the fastq data to the mouse reference genome, followed by quantifying transcripts per million (TPM) values at both gene and transcript levels. Differential expression analysis (n\u0026thinsp;=\u0026thinsp;3 per group) was conducted using the DRAGEN Differential Expression tool. Differentially expressed genes (DEGs) were identified based on TPM values, and TPM values were used to generate an input file for iDEP. Clustering, enrichment, and pathway analyses were performed using the iDEP tool.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of primary osteocytes\u003c/h2\u003e \u003cp\u003eWe isolated osteocytes using a previously described method.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Osteocytes were isolated from the calvariae of 5\u0026ndash;6-day-old DMP1-Topaz mice. Calvariae were dissected under aseptic conditions and sequentially digested at 37\u0026deg;C with agitation using a 0.2% (w/v) collagenase solution (Fujifilm) for 20 min, followed by a 5 mM ethylenediaminetetraacetic acid (EDTA) solution for 15 min. The digestion process consisted of the following sequential steps: collagenase (fraction 1), EDTA (fraction 2), collagenase (fraction 3), collagenase (fraction 4), and EDTA (fraction 5). All fractions, except fraction 1, were cultured overnight in α-MEM supplemented with 10% FBS and 1% P/S under 5% CO₂ at 37℃. Adherent cells were harvested using trypsin\u0026ndash;EDTA (Thermo Fisher/Sigma-Aldrich, Japan) and filtered through a 40 \u0026micro;m nylon cell strainer (Falcon, USA). Topaz-positive osteocytes were subsequently isolated by fluorescence-activated cell sorting (FACS; FACSAria II, BD Biosciences, Franklin Lakes, NJ, USA). The isolated Topaz-positive cells were used as primary osteocytes, which were cultured in α-MEM in a 24-well plate at a density of 3.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well. The plates were cultured for 24 h under normal (20% O\u003csub\u003e2\u003c/sub\u003e) or hypoxia (2% O\u003csub\u003e2\u003c/sub\u003e) conditions using the BIONIX hypoxic culture kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLCN2 protein treatment\u003c/h2\u003e \u003cp\u003eMLO-Y4 cells (1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were cultured in a 12-well plate (Corning Costar) in α-MEM with 10% FBS and 1% P/S at 37\u0026deg;C under 5% CO₂ for 24 h. rmLCN2 was dissolved in sterile PBS and added to the culture medium at a final concentration of 100 ng/mL. Cells were incubated with rmLCN2 for 1 or 3 days before further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eReal-time RT-PCR analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from MLO-Y4 osteocytic cells using the RNeasy Mini Kit. cDNA was synthesized using SuperScript IV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s protocol. Real-time RT-PCR was performed using TB Green Premix Ex Taq II (Takara, Shiga, Japan) on a Thermal Cycle Dice Real-Time System TP800 (Takara). The cycling conditions were initial denaturation at 95\u0026deg;C for 30 s, followed by 50 cycles of 95\u0026deg;C for 5 s and 60\u0026deg;C for 30 s. The following gene-specific primers were used: \u003cem\u003eGapdh\u003c/em\u003e: 5\u0026prime;-GGTGGAGCCAAAAGGGTCA-3\u0026prime; (forward), 5\u0026prime;-GGGGGCTAAGCAGTTGGT-3\u0026prime; (reverse), \u003cem\u003eActb\u003c/em\u003e: 5\u0026prime;-GAAATCGTGCGTGACATCAAA-3\u0026prime; (forward), 5\u0026prime;-TGTAGTTTCATGGATGCCACAG-3\u0026prime; (reverse), \u003cem\u003eRankl\u003c/em\u003e: 5\u0026prime;-CCTGAGGCCAGCCATTT-3\u0026prime; (forward), 5\u0026prime;-CTTGGCCCAGCCTCGAT-3\u0026prime; (reverse), \u003cem\u003eOpg\u003c/em\u003e: 5\u0026prime;-ATCAGAGCCTCATCACCTT-3\u0026prime; (forward), 5\u0026prime;-CTTAGGTCCAACTACAGAGGAAC-3\u0026prime; (reverse), \u003cem\u003eLcn2\u003c/em\u003e: 5\u0026prime;-CCAGTTCGCCATGGTATTTT-3\u0026prime; (forward), 5\u0026prime;-CACACTCACCACCCATTCAG-3\u0026prime; (reverse). The expression levels of the target genes were analyzed using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method, with \u003cem\u003eGapdh\u003c/em\u003e as the reference gene for LCN2-treated cells and \u003cem\u003eActb\u003c/em\u003e as the reference gene for hypoxia-treated cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eMLO-Y4 cells (1 \u0026times; 10\u0026sup3; cells/well) were seeded in 96-well plates and cultured overnight at 37\u0026deg;C under 5% CO₂ in α-MEM with 10% FBS and 1% P/S. Cells were then washed three times with PBS and fixed using 4% formaldehyde solution in PBS for 15 min at room temperature. After washing with PBS, cells were permeabilized with 0.5% Triton X-100 (v/v) in PBS for 15 min. After another PBS wash, cells were blocked using 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature. For immunofluorescence staining, cells were incubated overnight at 4\u0026deg;C with the following primary antibodies, diluted in 3% BSA in PBS: SLC22A17, megalin polyclonal antibody (1:400 dilution), (1:400 dilution), IgG polyclonal isotype control. The next day, cells were washed with PBS and incubated for 1 h at room temperature in the dark with Alexa Fluor-conjugated secondary antibodies (1:100 dilution in 3% BSA in PBS). Finally, cells were stained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) for 5 min before visualizing fluorescent signals under a fluorescence microscope (Olympus IX71, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of osteoclast precursors\u003c/h2\u003e \u003cp\u003eC57BL/6J mice were sacrificed, and the femora and tibiae were immediately dissected. The epiphyses were removed, and bone marrow cells were flushed out with α-MEM. The cell suspension was filtered through a 40 \u0026micro;m nylon cell strainer and cultured in α-MEM supplemented with 10% FBS, 1% P/S, and M-CSF (100 ng/mL) for 3 days. Adherent bone marrow macrophages (BMMs) were collected using trypsin-EDTA (Sigma-Aldrich) and used as osteoclast precursors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eOsteoclast differentiation\u003c/h2\u003e \u003cp\u003eOsteoclast precursors were seeded in 96-well plates at 5.0 \u0026times; 10⁴ cells/well and cultured under the following conditions: M-CSF (100 ng/mL) alone, M-CSF (100 ng/mL)\u0026thinsp;+\u0026thinsp;RANKL (100 ng/mL), M-CSF (100 ng/mL)\u0026thinsp;+\u0026thinsp;RANKL (100 ng/mL)\u0026thinsp;+\u0026thinsp;rmLCN2 (100 ng/mL), M-CSF (100 ng/mL)\u0026thinsp;+\u0026thinsp;rmLCN2 (100 ng/mL). The medium was changed every 2 days. On day 4, cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and stained with tartrate-resistant acid phosphatase (TRAP) staining consisting of acetate buffer (pH 5.0), naphthol AS-MX phosphate, fast red violet LB salt, and 50 mM sodium tartrate. Cells were considered osteoclasts if they were TRAP-positive and had two or more nuclei.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCo-culture of MLO-Y4 and osteoclast precursors\u003c/h2\u003e \u003cp\u003eMLO-Y4 osteocytic cells (1.0 \u0026times; 10\u0026sup3; cells/well) were seeded in 96-well plates and cultured for 6 h in α-MEM supplemented with 10% FBS, 1% P/S at 37\u0026deg;C under 5% CO₂. Afterward, osteoclast precursors (2.0 \u0026times; 10⁴ cells/well) were added to the wells simultaneously with 10⁻⁸ M 1,25-dihydroxyvitamin D₃ (Sigma-Aldrich) and 10⁻⁶ M prostaglandin E₂ (PGE₂; Sigma-Aldrich). One group was additionally treated with and without rmLCN2(100 ng/mL). The medium was changed every 2 days. On day 7, cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and TRAP staining. Cells were considered osteoclasts if they were TRAP-positive and had two or more nuclei.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eMLO-Y4 cells were cultured in α-MEM supplemented with 10% FBS, 1% P/S at 37\u0026deg;C under 5% CO₂ overnight. Cells were then serum-starved in 2% FBS for 4 h, followed by 1% FBS for 2 h. After starvation, cells were treated with rmLCN2 (100 ng/mL) for 0, 5, 15, 30, or 60 min. Control wells (0 min) received no LCN2. Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Millipore, Burlington, MA, USA) supplemented with 1% protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min on ice and insoluble material was removed by centrifugation. Protein samples were mixed 3:1 with Laemmli sample buffer (Bio-Rad, CA, USA) containing β-mercaptoethanol (Bio-Rad) and denatured at 95\u0026deg;C for 5 min. Equal amounts of protein were separated using Mini-PROTEAN TGX Precast Gels (Bio-Rad) and transferred onto 0.2 \u0026micro;m PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in BlockAce (DS Pharma Biomedical, Osaka, Japan) at room temperature for 1\u0026ndash;2 h, followed by overnight incubation at 4\u0026deg;C with the following primary antibodies: Phospho-p38 MAPK (Thr180/Tyr182) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA, 1:3000 dilution). p38 MAPK rabbit monoclonal antibody (Cell Signaling Technology, 1:3000 dilution). Phospho-ERK1/2 (Thr202/Tyr204) rabbit monoclonal antibody (Cell Signaling Technology, 1:3000 dilution). ERK1/2 MAPK rabbit monoclonal antibody (Cell Signaling Technology, 1:3000 dilution). Phospho-SAPK/JNK (Thr183/Tyr185) rabbit polyclonal antibody (Cell Signaling Technology, 1:3000 dilution). SAPK/JNK MAPK rabbit polyclonal antibody (Cell Signaling Technology, 1:3000 dilution). β-actin mouse monoclonal antibody (Sigma-Aldrich, 1:5000 dilution). After washing with tris-buffered saline with Tween-20 (TBS-T), membranes were incubated with HRP-conjugated anti-rabbit IgG (Cell Signaling Technology, 1:5000 dilution) or anti-mouse IgG (Cytiva, Tokyo, Japan, 1:10,000 dilution) for 1 h at room temperature. Signals were detected using an enhanced chemiluminescence (ECL) system (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, IL, USA). Band intensity was quantified using ImageJ software (NIH, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eExperimental tooth movement\u003c/h2\u003e \u003cp\u003eOTM was performed as previously described.\u003csup\u003e\u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e Male WT mice (8\u0026ndash;12-week-old) (n\u0026thinsp;=\u0026thinsp;4) were anesthetized with an intraperitoneal injection of a mixture of medetomidine, midazolam, and butorphanol prior to appliance placement. A nickel-titanium (Ni-Ti) closed-coil spring (TOMY SEIKO Co. Ltd., Tokyo, Japan) was attached between the upper incisor and left first molar. The appliance was secured using a stainless-steel wire (0.01 mm diameter), which was fixed to a hole drilled in the upper anterior alveolar bone and tied to the first molar. The first molar was moved mesially with a force of 10 g. Each group contained four mice, and OTM was carried out for 0, 2, and 6 days.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eHistological preparation and immunohistochemistry\u003c/h2\u003e \u003cp\u003eCalvariae and maxillae were harvested and fixed overnight in 4% paraformaldehyde at 4\u0026deg;C. Samples were then decalcified in 14% EDTA at 4\u0026deg;C for 3 days (calvariae) or 1 month (maxillae). Following dehydration, the specimens were embedded in paraffin and sectioned: Coronal sections (5 \u0026micro;m thick) for calvariae and horizontal sections (4 \u0026micro;m thick) for maxillae. Maxillae sections were obtained approximately 150 \u0026micro;m from the root branch of the upper-left first molar. For immunohistochemistry, paraffin sections were deparaffinized, rehydrated, and treated with 3% hydrogen peroxide (H₂O₂) for 15 min to block endogenous peroxidase activity. Sections were then blocked with 5% skim milk for 30 min at 37\u0026deg;C and incubated overnight at 4\u0026deg;C with HIF-1α (1:100 dilution), LCN2 (1:50 dilution) polyclonal antibodies and IgG polyclonal isotype control. After washing, sections were processed using the VECTASTAIN Elite ABC Kit (PK-6105, Vector Laboratories Inc., Burlingame, CA, USA) and developed with 3,3\u0026prime;-diaminobenzidine (DAB, Vector Laboratories). Hematoxylin was used for counterstaining. The percentage of HIF-1α- and LCN2-positive osteocytes was quantified within a region parallel to the long axis of the root, starting from the periodontal ligament.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using JMP Pro 17 software (JMP Statistical Discovery, Cary, NC, USA). For comparisons between the two groups, Student\u0026rsquo;s t-test was used. For multiple group comparisons, ANOVA followed by the Tukey\u0026ndash;Kramer post hoc test was applied. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eEthics approval\u003c/h2\u003e\n\u003cp\u003eAll animal experiments were performed in accordance with the ARRIVE guidelines and were approved by the Regulations for Animal Experiments and Related Activities at Tohoku University (2018DnA-028-06).\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by JST SPRING, Grant Number JPMJSP2114. JSPS KAKENHI Grant Numbers JP21K10178 and JP22K17244 from the Japan Society for the Promotion of Science.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eK.N., H.K., and F.O. contributed to designing this study. K.N., H.K., F.O., J.R., A.M., J.M., A.L., Z.F., and K.M. performed the experiments. K.N., H.K., and F.O. analyzed the data and confirmed the results. K.N., H.K., and F.O. drafted the manuscript. H.K. supervised the project. All authors approved the final version.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eA part of this study was supported by a support system for young researchers who use research equipment, instruments, and devices at Tohoku University. We thank the Biomedical Research Core of Tohoku University Graduate School of Medicine for supporting fluorescence-activated cell sorting (FACS).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe RNA-seq datasets generated and analyzed during this study are available in the NCBI Gene Expression Omnibus (GEO) under accession number GSE298138. All other data supporting the findings of this study are included in the article and its Supplementary Information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBonewald, L. F. The amazing osteocyte. \u003cem\u003eJ. Bone Min. Res.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e (2), 229\u0026ndash;238. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jbmr.320\u003c/span\u003e\u003cspan address=\"10.1002/jbmr.320\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarahleh, A., Kitaura, H., Ohori, F., Noguchi, T. \u0026amp; Mizoguchi, I. The osteocyte and its osteoclastogenic potential. \u003cem\u003eFront. Endocrinol. 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Exacerbating orthodontic tooth movement in mice with salt-sensitive hypertension. \u003cem\u003eJ. Dent. Sci.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e (2), 764\u0026ndash;769. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jds.2024.10.020\u003c/span\u003e\u003cspan address=\"10.1016/j.jds.2024.10.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"osteocyte, hypoxia, lipocalin-2, osteoclast, orthodontic tooth movement, osteoclastogenesis","lastPublishedDoi":"10.21203/rs.3.rs-6679031/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6679031/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteocytes regulate bone remodeling by interacting with osteoblasts and osteoclasts. Hypoxia influences osteocyte function and has been linked to increased osteoclastogenesis in pathological conditions such as orthodontic tooth movement (OTM) and bone metabolic diseases; however, the molecular mechanisms underlying these effects remain unclear. This study aimed to identify hypoxia-responsive genes in osteocytes and investigate their effects on osteoclastogenesis. Transcriptome analysis of murine long bone osteocyte-Y4 (MLO-Y4) osteocytes cultured under hypoxia (2% O₂) revealed that lipocalin-2 (\u003cem\u003eLcn2\u003c/em\u003e) was the most significantly upregulated gene. Real-time RT-PCR confirmed increased \u003cem\u003eLcn2\u003c/em\u003e expression and an elevated \u003cem\u003eRankl\u003c/em\u003e/osteoprotegerin (\u003cem\u003eOpg)\u003c/em\u003e ratio. Primary osteocytes were purified from DMP1-Topaz mice showed same hypoxic response. Functional analysis demonstrated that \u003cem\u003eLcn2\u003c/em\u003e did not directly affect osteoclast precursors. However, it enhanced osteoclastogenesis via osteocytes in co-culture experiments. Western blot analysis demonstrated that LCN2 activated the MAPK signaling pathway in osteocytes. Furthermore, immunohistochemical analysis of hypoxic osteocytes on the compression side of OTM exhibited increased LCN2 expression. These findings suggest that LCN2 is upregulated in osteocytes under hypoxia and promotes osteoclastogenesis by increasing RANKL expression. This study provides new insights into the molecular mechanisms of bone resorption under hypoxic conditions and suggests \u003cem\u003eLcn2\u003c/em\u003e as a potential therapeutic target for bone metabolic diseases.\u003c/p\u003e","manuscriptTitle":"Lipocalin-2 expression in hypoxic murine osteocytes enhances RANKL-induced osteoclastogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-02 12:39:28","doi":"10.21203/rs.3.rs-6679031/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-10T05:29:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-09T14:33:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-09T08:34:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-02T08:14:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187473453821911891085413669528849769708","date":"2025-06-01T04:42:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32823870143926115523304029393592107337","date":"2025-05-31T01:20:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254742634591111614318017195854866083157","date":"2025-05-30T08:44:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-30T06:32:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-30T06:26:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-29T09:26:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-29T04:31:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-16T08:40:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2ef65cbf-a328-4ee9-8eb9-0646c4e49984","owner":[],"postedDate":"June 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49337875,"name":"Biological sciences/Cell biology"},{"id":49337876,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-01-12T16:07:37+00:00","versionOfRecord":{"articleIdentity":"rs-6679031","link":"https://doi.org/10.1038/s41598-025-34575-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-06 15:57:58","publishedOnDateReadable":"January 6th, 2026"},"versionCreatedAt":"2025-06-02 12:39:28","video":"","vorDoi":"10.1038/s41598-025-34575-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-34575-2","workflowStages":[]},"version":"v1","identity":"rs-6679031","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6679031","identity":"rs-6679031","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}