O‐GlcNAcylation of YAP Enhances Nuclear Translocation to Regulate NRP1‐Mediated Osteogenic Differentiation in MC3T3‐E1 Cells

O‐GlcNAcylation of YAP Enhances Nuclear Translocation to Regulate NRP1‐Mediated Osteogenic Differentiation in MC3T3‐E1 Cells | 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 Research Article O‐GlcNAcylation of YAP Enhances Nuclear Translocation to Regulate NRP1‐Mediated Osteogenic Differentiation in MC3T3‐E1 Cells Xinyue Yang, Yao Weng, Anggun Dwi Andini, Xinyu Gan, Yuhan He, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8696147/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Our previous study demonstrated that O-GlcNAc transferase (OGT) promotes osteoblast differentiation in MC3T3‐E1 cells; however, the precise molecular mechanism remains unclear. In this study, we investigated whether OGT regulates osteoblast differentiation through Yes-associated protein (YAP) and neuropilin-1 (NRP1). Using MC3T3-E1 cells, we show that OGT directly O-GlcNAcylates YAP, promoting its nuclear translocation and transcriptional activation of osteogenic regulators RUNX2 and Osterix (OSX). Loss of OGT impaired YAP activity, NRP1 expression, and osteoblast differentiation. Treatment with lysophosphatidic acid (LPA), a YAP activator, restored YAP nuclear accumulation, re-established NRP1 expression, and rescued osteogenic marker expression in OGT-deficient cells. Functional studies further identified NRP1 as a downstream effector of the OGT–YAP axis required for osteogenesis.These findings establish a regulatory pathway in which OGT-mediated O-GlcNAcylation of YAP enhances NRP1-dependent osteogenic differentiation. Moreover, pharmacological activation of YAP by LPA compensates for OGT deficiency, highlighting the OGT–YAP–NRP1 axis as a potential therapeutic target for bone regeneration and osteoporosis. Neuropilin-1 (NRP1) Yes‐associated protein (YAP) O‐GlcNAc transferase (OGT) Lysophosphatidic acid (LPA) Osteoblast differentiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 | Introduction O-GlcNAc transferase (OGT) is an essential and evolutionarily conserved enzyme that catalyzes O-linked β-N-acetylglucosamine (O-GlcNAc) modification, a dynamic post-translational process regulating transcription, signal transduction, and cell differentiation ( 1 ). Notably, OGT expression is reduced in osteoporosis, and mice lacking OGT in bone cells exhibit severe bone mass loss ( 2 ). Our previous studies also demonstrated that O-GlcNAcylation promotes osteoblast differentiation by modulating mitochondria–cytoskeleton organization and calcium signaling in MC3T3-E1 cells( 3 , 4 ). O-GlcNAc is an essential, dynamic monosaccharide post-translational modification found on serine and threonine residues of thousands of nucleocytoplasmic proteins( 5 ). Changes in O-GlcNAcylation of proteins are involved in bone diseases such as osteoporosis( 6 ), periodontitis( 7 ), and osteoarthritis( 8 ). Importantly, O-GlcNAcylation not only modulates protein activity but can also regulate subcellular localization( 9 , 10 ), suggesting that OGT might influence osteogenesis through spatial control of key transcriptional regulators. Yes-associated protein (YAP) is a key transcriptional co-activator in the Hippo signaling pathway, which regulates cell proliferation, apoptosis, and organ size( 11 ). When the Hippo pathway is inactive, YAP translocates to the nuclear and promotes the expression of genes involved in growth and survival( 12 ). Previous studies have shown that OGT directly O-GlcNAcylates YAP at Ser109, disrupting its interaction with LATS1, preventing phosphorylation, and enhancing nuclear accumulation( 13 ). OGT-induced YAP O-GlcNAcylation has been implicated in tumor progression and vascular pathology( 14 , 15 ). However, whether OGT-mediated YAP O-GlcNAcylation is linked to its nuclear translocation during osteoblast differentiation and bone formation remains unclear. Notably, lysophosphatidic acid (LPA) is a potent activator of YAP ( 16 ). LPA, a natural bioactive phospholipid with pleiotropic effects on multiple tissues, acts primarily through G protein–coupled LPA receptors (LPAR1–6) to regulate pathways controlling proliferation, migration, survival, and differentiation ( 17 , 18 ). As an activator of the RhoA/ROCK pathway, LPA has been shown to rescue cerebellar function and related behavioral deficits caused by OGT deficiency and impaired O-GlcNAcylation ( 18 ). Nevertheless, whether LPA can restore the osteogenic differentiation capacity of OGT-deficient pre-osteoblastic cells remains to be elucidated. Previous studies indicate that LPA promotes osteogenesis in MC3T3-E1 cells via YAP activation. However, these studies mainly focused on mature osteoblast models, with limited validation in human cells or at the pre-osteoblastic stage( 19 , 20 ), and the downstream mechanisms of LPA–YAP signaling in osteoblasts remain largely unexplored. Neuropilin-1 (NRP1), a transmembrane glycoprotein and co-receptor for ligands such as VEGF and class 3 semaphorins( 21 ), plays critical roles in angiogenesis( 22 ), neuronal guidance( 23 ), immune regulation( 24 ), and tumor progression( 25 ). Importantly, NRP1 has emerged as a key molecule in osteogenesis, as we previously demonstrated that it interacts with Shroom3 to control osteo/odontogenesis in dental pulp stem cells( 26 ). NRP1 promotes osteogenic differentiation via VEGF–VEGFR2 signaling by upregulating alkaline phosphatase (ALP), osteocalcin (OCN), and osteoprotegerin (OPG) ( 27 ), and through SEMA3A-mediated activation of PI3K/Akt and Wnt/β-catenin pathways ( 28 ). Although LPA has been reported to enhance VEGF–VEGFR2 signaling and invasiveness in ovarian cancer cells ( 29 ), its effects on NRP1 expression and function in osteoblasts remain unclear. Moreover, in cancer cells, YAP/TEAD4 directly enhances NRP1 transcription ( 30 ), suggesting a potential mechanistic link between LPA-YAP activation and NRP1 expression in osteoblasts. Therefore, we aimed to investigate whether LPA-YAP activation drives NRP1 expression and contributes to osteogenic differentiation. In this study, we employed LPA as a YAP activator to restore osteogenic differentiation capacity and NRP1 expression in OGT-deficient MC3T3-E1 osteoblast-like cells. We investigated how OGT interacts with YAP to promote its O-GlcNAcylation and nuclear translocation during osteogenic differentiation. Nuclear YAP subsequently upregulates key osteogenic transcription factors, including RUNX2 and OSX. Furthermore, we demonstrated that NRP1 functions downstream of the OGT–YAP axis and contributes to osteoblast differentiation. In summary, our findings indicate that the LPA-enhanced OGT/YAP signaling axis, involving NRP1, plays a critical role in regulating osteogenic differentiation in MC3T3-E1 cells. 2 | Materials and Methods 2.1 | Cell Culture Pre-osteoblastic cell line MC3T3‐E1 and human embryonic kidney 293 (HEK293) cells were purchased from RIKEN BRC Cell Bank (Tsukuba, Japan) and maintained at 37°C incubator with 5% CO2. MC3T3‐E1 cells were cultured in minimum essential medium α (MEMα) (Gibco/Life Technologies Corporation, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco/Life Technologies Limited, Paisley, Scotland, UK), and 1% penicillin‐streptomycin mixed solution (Nacalai Tesque Inc., Kyoto, Japan). To induce differentiation, 50 mM ascorbic acid (Sigma‐Aldrich, St. Louis, MO, USA) and 2 mM β‐glycerophosphate (Nacalai Tesque Inc.) were added. The cells were cultured in a 60 mm dish (Thermo Fisher Scientific, Rochester, NY, USA) with 15 mm round glass coverslips (Matsunami, Kishiwada, Osaka, Japan) for immunostaining purposes. During Lysophosphatidic acid (LPA) treatment, LPA(Abcam,ab146430) was added to a final concentration of 5,10 and 20 µmol for 1 day, 3days, 6days. For HEK293 culture, MEM medium (Gibco/Life Technologies Corporation) was mixed with 10% fetal bovine serum, 1% penicillin‐streptomycin mixed solution, and 1% MEM non‐essential amino acids (Gibco/Life Technologies Corporation). 2.2 | Immunocytochemistry The samples were rinsed with phosphate-buffered saline (PBS) (Takara, Kusatsu, Shiga, Japan) and fixed with 4% paraformaldehyde‐PBS (Nacalai Tesque Inc.) for 15 min at room temperature. Next, the cells were permeabilized using 0.1% Triton X‐100 (Sigma‐Aldrich) for 2 min on the ice. For blocking step, 5% bovine serum albumin (BSA) (Nacalai Tesque Inc.) was used. Incubation overnight with primary antibody was conducted at 4°C. The next day, the samples were subjected to secondary antibody incubation at room temperature for 2 h, followed by using mounting medium with DAPI (ProLong™ Diamond Antifade Mountant with DAPI, Invitrogen™P36966, USA). The primary and secondary antibodies used can be found on Supporting Information S1: Data S1 . Cell imaging was performed using LSM 780 confocal laser scanning microscopy system (Zeiss, Oberkochen, Germany). The quantitative immunofluorescence analysis of YAP nuclear localization comprised three independent biological replicates at each time point (Days 0, 1, 3, and 6). For each biological replicate, three random fields of view were acquired. YAP nuclear and cytoplasmic distribution was quantified using FIJI/ImageJ (National Institutes of Health, Bethesda, MD, USA). DAPI staining was used to define nuclear boundaries, and the red channel was used to measure YAP fluorescence. Nuclear YAP intensity was obtained by applying DAPI-derived nuclear regions of interest (ROI) onto the YAP channel, while cytoplasmic YAP intensity was calculated as the total red fluorescence minus the nuclear YAP signal. The nuclear-to-cytoplasmic YAP ratio was calculated accordingly. All images were analyzed using a custom FIJI macro, including channel splitting, thresholding, ROI extraction, and IntDen measurement. The full ImageJ macro are provided in Supporting Information S2: Data S2. The raw dataset are provided in Supporting Information S3: Data S3 . 2.3 | Cellular Fractionation The procedure used has been previously documented( 31 ). In short, the cells were kept in buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulphonyl fluoride [PMSF]). After adding 10% Nonidet NP-40, the samples were vortexed and centrifuged at a high speed. The supernatant was then collected as the cytoplasmic sample and the pellet was resuspended in buffer C (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF). The nuclear pellet was sonicated and centrifuged to get the nuclear protein. 2.4 | Protein Collection Cells were washed with cold PBS, resuspended in lysate buffer (1 mM DTT, 1 mM PMSF, 1 µg/ml leupeptin, 2 µg/mL aprotinin, and 5 mM EGTA), and sonicated for 20 s. The cell lysates were then centrifuged at high speed. The supernatants were collected and the concentration was adjusted prior to western blot analysis. 2.5 | Coomassie Brilliant Blue (CBB) Staining Ten micrograms of protein were loaded to 10% SDS-PAGE gel. After electrophoresis, the gel was immersed in fixer solution (50% methanol, 10% acetic acid) for 30 min and stained for 20 min (0.6 g of Coomassie blue in 300 mL of 50% (v/v) methanol and 10% acetic acid). Destaining solution (10% methanol, 10% acetic acid) was then used until a contrast between protein band and gel was seen clearly. 2.6| Derive Ogt knockout cells line from MC3T3-E1 cells using CRISPR/Cas9 system For the Cas9 HDR experiments, the guide RNA (gRNA) for Cas9 was designed to target the 7th exon (Ensembl ID: ENSMUSE00000285748) of the major Ogt transcript (Ogt-201; CCDS: CCDS30318.1; Ensembl ID: ENSMUST00000044475.5). First, 3.6 µL of 100 µM Alt-R CRISPR-Cas9 target-specific crRNA (Mm.Cas9.OGT.1. AA; Integrated DNA Technologies, Coralville, IA, USA) and 3.7 µL of 100 µM Alt-R CRISPR-Cas9 tracrRNA (1072533; Integrated DNA Technologies, Coralville, IA, USA) were combined and heated at 95°C for 5 min. The crRNA:tracrRNA solution was then cooled at room temperature. The total 7.3 µL of the crRNA:tracrRNA solution was then combined with 4.8 µL of 62 µM Alt-R S.p. HiFi Cas9 Nuclease V3 (1081059; Integrated DNA Technologies, Coralville, IA, USA) and incubated at room temperature for 5 min to form the RNP complex. MC3T3-E1 cells resuspended in Opti-MEM. Next, 12.1 µL of the RNP complex, 270 µL Opti-MEM containing 2.4×10 6 MC3T3-E1 cells, 14.3 µL Opti-MEM, and 3.6 µLof Alt-R Cas9 Electroporation Enhancer (1075916; Integrated DNA Technologies, Coralville, IA, USA) were combined, and 100 µL of the combined media was transferred into an electroporation cuvette with electrodes of 2 mm gap size (EC-002; Nepa Gene, Chiba, Japan). Finally, 175 V of current (5 ms at 50 ms intervals, two pulses) for poring and 20 V of current (50 ms at 50 ms intervals, five pulses) for transfer were applied by using an electroporator (Super Electroporator NEPA21 Type II;Nepa Gene, Chiba, Japan). The resulting electroporated cells were transferred to a 6-well plate for cell grown and expanded. For isolating clone cells, the electroporated cell suspension was diluted and cultured in a 100 mm dish. When a single cell grew into a cell mass, each colony was isolated using a cloning ring. The knock-out efficiency was determined by measuring the protein expression of Ogt using Western blotting. 2.7 | Western Blot Analysis Ten micrograms of protein were separated using 10% SDS- PAGE and blotted into PVDF membranes (Merck, Darmstadt, Germany). After blocking, the membranes were exposed to the primary antibody overnight at 4°C. Secondary antibody incubation was performed next day in room temperature. The bands were visualized through chemiluminescent after adding HRP substrate (WBLUF0100; Millipore, Burlington, MA, USA). Band density was analyzed by using ImageJ (National Institutes of Health, Bethesda, MD, USA) and normalization by internal control was performed to quantify the amount of protein. The ratio between nuclear and cytoplasmic YAP was calculated by comparing the densitometric analysis of the nuclear and cytoplasmic YAP bands, normalized by western blot results. For quantification of O-GlcNAcylated protein, the intensity of the immunoprecipitated (IP) O‐GlcNAcylated protein band was divided by the intensity of the corresponding total protein band in the input lane. This normalization accounts for variations in sample loading and transfer efficiency. Normalized values were obtained for MC3T3‐E1 osteoblast differentiation at days 0, 3, and 6. Fold changes were calculated by dividing the normalized values at days 3 and 6 by the corresponding value at day 0. List of antibodies used can be found on Supporting Information S1: Data S1 . 2.8 | Alizarin Red Staining Cells were incubated in 95% ethanol for 10 min after fixation, followed by staining with 10 mg/mL alizarin red solution (Wako Pure Chemical Industries, Kanagawa, Japan). After the color developed, the reaction was stopped using distilled water. The cell pictures were then taken by using Leica DMi1 phase contrast microscope (Leica, Wetzlar, Germany). 2.9 | Alkaline Phosphatase (ALP) Staining The samples were dipped into the staining solution (Napthol AS-BI phosphate (Sigma), N‐N′ dimethylformamide (Wako Pure Chemical Industries), 0.2 M Tris‐HCl buffer pH 8.3–8.5, Fast blue RR salt (Sigma) for 10 min and the reaction was stopped by distilled water. For nuclear staining, 1% methyl green stain solution pH 4.0 (Muto Pure Chemicals, Co. Ltd., Tokyo, Japan) was used. Cell pictures were taken immediately after the procedure was finished. 2.10 | Immunoprecipitation (IP) for O-GlcNAcylated Proteins The cells on the different differentiation days were incubated with 200 µM Ac4GAlNAz (CLK-1086‐5; Jena Bioscience, Jena, Germany) overnight. On the following day, cells were resuspended in lysis buffer (2 mM Tris pH 7.5, 15 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% Triton X‐100, 0.25 mM sodium pyrophosphate, 0.1 mM β‐ glycerophosphate, 0.1 mM Na3VO4, 1 µg/mL leupeptin) and sonicated. The samples were centrifuged at high speed for 15 min. The supernatant was collected and divided into two tubes: input and IP sample. The IP sample was incubated with 200 µM DBCO‐PEG4‐ Biotin (760749; Sigma) for 1 h at 4°C with rotation. The IP process was performed using T1 beads from the Dynabeads Streptavidin Trial Kit (Invitrogen) according to the manufacturer's protocol. Briefly, the T1 beads were washed three times with PBS. During the washing step, after the PBS was added, the tube was vortexed for 30 s. The beads and supernatant were separated using a magnetic stand for 1 min. After that, the beads were incubated with the protein sample that had been incubated with DBCO‐PEG4‐Biotin for 30 min at room temperature. The beads were washed with 0.1% BSA five times after incubation. Finally, the beads were resuspended in elution buffer (10 mM EDTA pH 8.2% and 95% formamide) and incubated at 65°C for 2 min. The supernatant separated from the beads was then analyzed by western blot analysis. 2.11 | Immunoprecipitation MC3T3-E1 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors, sonicated, and centrifuged at 15,000 rpm for 15 min at 4°C. The supernatant was incubated with 3 µL of anti-NRP1, anti-YAP, or anti-OGT antibody, or species-matched IgG control, for 2 h at 4°C with rotation. Pre-equilibrated protein A/G PLUS-Agarose beads (Santa Cruz, sc-2003) were added and incubated overnight at 4°C. Beads were washed four times with RIPA buffer, and bound proteins were eluted with 1× SDS sample buffer by boiling for 5 min, followed by SDS-PAGE and immunoblotting. 2.12 | Cell Transfection For NRP1 overexpression, the mouse NRP1 coding sequence (Sp-3flag-NM\_008737) was cloned into the multiple cloning site (MCS) of the GV218 lentiviral vector (GeneChem Corporation, Shanghai, China), resulting in the PL-NRP1 (KL60938-1) construct. The GV218 vector (11.1 kb) contains a CMV promoter for high-level expression in mammalian cells, a ubiquitin promoter driving enhanced green fluorescent protein (EGFP) expression, and an ampicillin resistance gene for bacterial selection. The vector also includes pBR322 ori, 5′ long terminal repeat (5′LTR), and BamHI/AgeI cloning sites. For NRP1 knockdown, a short hairpin RNA (shRNA) targeting mouse NRP1 (NM\_003873.7) was designed and inserted into the GV493 lentiviral backbone (GeneChem Corporation, Shanghai, China) under the control of the U6 promoter, generating PL-NRP1-RNAi (PSC109402-1). The GV493 vector (10,880 bp) contains CBh promoter-driven copGFP for transduction efficiency monitoring, a puromycin resistance cassette for selection, and ampicillin resistance for bacterial propagation. The vector backbone includes 5′LTR and 3′LTR sequences and EcoRI/AgeI restriction sites for cloning. Lentiviral particles were produced in HEK293T cells and used to transduce target cells, followed by antibiotic selection to generate stable cell lines. During the cell transfection, opti-MEM (Gibco/Invitrogen Corporation, Paisley, Scotland, UK) was used together with PEI max (Polysciences Inc., Warrington, PA, USA). 2.13| Protein–Protein Docking Protein structures of YAP (UniProt ID: P46938), OGT1 (UniProt ID: Q8CGY8), and NRP1 (UniProt ID: P97333) were obtained from the AlphaFold Protein Structure Database ( https://alphafold.ebi.ac.uk/ ). Protein–protein docking was performed using AlphaFold3 ( https://alphafoldserver.com ), and the top-ranked model according to the ranking score was selected as the optimal docking conformation. The binding free energy of the selected complex was evaluated using the HawkDock server ( http://cadd.zju.edu.cn/hawkdock/ ), which also ranked amino acid residues based on their contribution to binding free energy. Residues with high binding contribution were visualized using PyMOL 2.4 (Schrödinger, LLC) to provide an intuitive representation of the interaction interfaces. 2.14 | Statistical Analysis The statistical analysis was conducted by using GraphPad Prism software version 8.0 (GraphPad Software, San Diego, CA, USA). Mean values accompanied by standard deviations (SD) were derived from a minimum of three independent replicates. The data were analyzed by one-way analysis of variance (ANOVA), two‐way ANOVA, or t‐test according to the type of the data, and the p < 0.05 was considered as significant (*). 3 | Results 3.1 | YAP translocates to the nucleus during osteoblast differentiation. We first evaluated YAP expression and distribution during the osteogenic differentiation of MC3T3-E1 cells. Immunofluorescence staining revealed that during early osteoblast differentiation, OGT gradually translocated from the nucleus to the cytoplasm (Fig. 1 A), consistent with our previous observation [32]. In contrast, YAP progressively accumulated in the nucleus, with a marked increase in nuclear fluorescence signal over time(Fig. 1 A). By day 3/6, the nuclear-to-cytoplasmic ratio of YAP was clearly higher compared with undifferentiated cells (Fig. 1 B), indicating enhanced nuclear localization as differentiation progressed. Western blot analysis demonstrated a gradual increase in total YAP protein expression from day 0 to day 6 (Figs. 1 C, D). Differentiation marker Osterix (OSX) increased until Day 12 during osteoblast differentiation in MC3T3-E1 cells ( Supplementary Data Fig. 1 A). Subcellular fractionation further confirmed these findings: in cytoplasmic fractions, YAP levels decreased over the course of differentiation, whereas nuclear fractions showed a notable increase in YAP abundance from day 3 onwards (Fig. 1 E, F). The purity of subcellular fractions was validated using α-tubulin (cytoplasmic marker) and LSD1 (nuclear marker). Using CRISPR/Cas9 technology, we generated OGT knockout MC3T3-E1 cells. The detailed editing and single-clone selection procedures are described in the Methods section and our previous publication( 3 ). Western blot analysis confirmed the knockout efficiency of the established OGT KO clones (Fig. 1 G), and the clone with the most efficient depletion, OGT KO #2, was selected for subsequent experiments. Loss of OGT resulted in a marked reduction in YAP protein expression (Fig. 1 H,I). These results indicate that osteoblast differentiation is accompanied by a redistribution of YAP from the cytoplasm to the nucleus, a process that may be correlated with OGT . 3.2 | OGT interacts with YAP, promotes its O-GlcNAcylation, and facilitates nuclear localization during osteoblast differentiation Next, we investigated the relationship between YAP and OGT during early osteoblast differentiation. In contrast to control MC3T3-E1 cells, OGT knockdown resulted in a marked retention of YAP in the cytoplasm at all examined stages, as shown by the absence of overlapping nuclear DAPI and YAP fluorescence signals (Fig. 2 A), suggesting that OGT is required for YAP nuclear translocation. To further investigate whether OGT regulates YAP through O‐GlcNAcylation, we established an O‐GlcNAc protein immunoprecipitation (IP) assay (Fig. 2 B). MC3T3-E1 cells were differentiated for 0, 3, or 6 days and total protein extracts were prepared. Protein loading was normalized using CBB staining (Supplementary Data Fig. 1 F ) , allowing comparison of overall O-GlcNAcylation between time points. CBB-normalized analyses showed that the global O-GlcNAcylation of cellular proteins was incerasing at day 3 and day6. Consistent with this overall trend, Western blot analysis of IP fractions demonstrated that O‐GlcNAcylated YAP levels were markedly elevated at day 3 and day 6 (Fig. 2 C,D), indicating a dynamic regulation of YAP O‐GlcNAcylation during differentiation. Given that OGT catalyzes the transfer of O-GlcNAc to substrate proteins, we examined whether it directly interacts with YAP. Reciprocal co‐immunoprecipitation experiments confirmed a physical association between OGT and YAP both in undifferentiated cells (day 0) and in cells at day 3 of osteoblast differentiation (Fig. 2 E, F). This consistent interaction across stages supports a model in which OGT physically binds YAP to promote its O‐GlcNAcylation, thereby enabling nuclear localization and transcriptional activity during osteogenesis. 3.3 | LPA activates YAP and rescues the impaired osteogenic differentiation of OGT-knockout MC3T3-E1 cells To further elucidate the role of O-GlcNAcylated YAP in osteoblast differentiation, OGT-knockout MC3T3‐E1 cells were treated with LPA, a known activator of YAP, and their osteogenic potential was evaluated. Immunofluorescence analysis showed increase in YAP nuclear fluorescence following treatment with LPA (5, 10, and 20 µmol) compared to the untreated control group (Supplementary Fig. 1G) . Immunofluorescence staining revealed that LPA markedly promoted the nuclear re-localization of YAP in OGT-deficient cells, as evidenced by the pronounced nuclear red fluorescence signal(Fig. 3 A). Western blot analysis demonstrated that LPA treatment increased the protein expression of the osteogenic transcription factors RUNX2 and OSX in OGT-knockout cells at day 3 of osteoblast differentiation in a dose-dependent manner (Fig. 3 B). Quantification showed that, compared with untreated knockout cells, LPA significantly upregulated RUNX2 expression at both 10 µmol and 20 µmol (p < 0.05), and similarly enhanced OSX expression at 10 µmol (p < 0.05) (Fig. 3 C, D). Functionally, ALP staining revealed that OGT-knockout MC3T3‐E1 cells exhibited reduced osteogenic differentiation compared with the control cells, as indicated by weak red staining. Strikingly, LPA treatment restored ALP activity in knockout cells in a dose-dependent manner, with 5 µmol and 10 µmol producing robust ALP staining comparable to control group levels (Fig. 3 E), indicating functional recovery of osteoblast differentiation. Finally, we aimed to investigate whether LPA can promote YAP O-GlcNAcylation. We examined the effect of LPA on YAP O-GlcNAcylation in wild-type MC3T3-E1 cells (Fig. 3 F). Our analysis demonstrated that LPA treatment increased both the O-GlcNAcylation level and total protein expression of YAP (Fig. 3 F, G; Supplementary Fig. 1B,C ), suggesting that LPA may facilitate YAP nuclear translocation by enhancing its O-GlcNAcylation and protein erxpression. Collectively, these findings indicate that LPA-mediated YAP activation not only restores its nuclear localization, protein expression, and O-GlcNAcylation, but also rescues the impaired osteogenic differentiation capacity caused by OGT loss, underscoring the critical role of O-GlcNAcylation–YAP signaling in bone formation. 3.4 | NRP1 plays an important role in osteoblast differentiation Recent studies indicated that NRP1 is involved in bone formation[26–28]. To investigate NRP1’s function in MC3T3-E1 cells’ osteoblast differentiation, first we examined protein expression levels of NRP1 during differentiation of the preosteoblastic cell line MC3T3-E1. NRP1 expression is increasing during the osteoblast differentiation day3 to day12 (Fig. 4 A,B). Immunofluorescence staining further revealed dynamic changes in subcellular localization of NRP1 and OGT (Fig. 4 C). At day 6, extensive co-localization was observed, suggesting that NRP1 may interact with OGT during osteogenic differentiation. To more precisely examine the role of NRP1 in osteoblast differentiation, we examined the effect of NRP1 overexpression and knockdown on the expression of osteogenic transcription factors. NRP1 overexpression was achieved by lentiviral transduction of a GV218 vector carrying the full-length mouse NRP1 coding sequence, while NRP1 knockdown was performed using a GV493 vector expressing an NRP1-targeting shRNA, The detailed procedures are described in the Methods section. Western blot results show that NRP1 was successfully overexpressed(Fig. 4 D) and knock down(Fig. 4 H). We performed single-cell cloning on ovNRP1 cells and shNRP1 cells, obtaining several clones that expressed very high levels of NRP1 protein and ALP activity in ovNRP1 cells(Fig. 4 D,E) and very low levels of NRP1 protein and ALP activity in shNRP1 cells(Fig. 4 H,J). Functional assays demonstrated that NRP1 overexpression in NRP1-OE cells significantly promoted osteoblast differentiation, as evidenced by increased ALP staining on day 7 and intensified Alizarin Red S staining on day 20 (Fig. 4 E). NRP1 knockdown in NRP1-sh cells significantly impaired osteogenic capacity, as indicated by decreased ALP and mineral deposition (Fig. 4 J). Collectively, these findings indicate that NRP1 positively regulates osteoblast differentiation. Furthermore, we examined the effect of NRP1 on YAP protein expression. Overexpression of NRP1 in MC3T3-E1 cells reduced YAP protein levels (Fig. 4 F, G), whereas knockdown of NRP1 increased YAP expression (Fig. 4 H, I). These results suggest that NRP1 functions as a negative upstream regulator of YAP protein stability or synthesis in MC3T3-E1 cells. 3.5 | NRP1 Acts Downstream of OGT-Mediated YAP Nuclear Translocation to Promote Osteogenesis To investigate the relationship between NRP1 and OGT-Mediated YAP Nuclear Translocation during the osteoblast differentiation, we examined the mRNA and protein expression level of NRP1 in OGT knock out MC3T3-E1 cells. In OGT-ko cells, expression of NRP1 was decreased at mRNA levels (Fig. 5 A) and protein levels (Fig. 5 D,E). Immunostaining result on OGT-ko cells also showed that NRP1 expression decreased (Fig. 5 B). We investigated whether OGT physically interacts with NRP1. Co-immunoprecipitation analysis revealed no evidence of a direct interaction between the two proteins in MC3T3‐E1 cells (Supplementary Fig. 1D) . Consistently, O-GlcNAcylation Western blotting did not detect O-GlcNAc–modified NRP1 in MC3T3-E1 cells at day 0, 3, or 6 of osteogenic differentiation (Supplementary Fig. 1E) . Next, we used LPA to stimulate YAP and examined NRP1 protein levels in OGT-knockout cells at day 3 of osteoblast differentiation. NRP1 expression was decreased in OGT-KO cells but was restored following treatment with 10µmol LPA (Fig. 5 G,H), suggesting that NRP1 may act downstream of OGT-mediated YAP nuclear translocation to promote osteogenesis. Consistently, immunofluorescence staining showed that NRP1 fluorescence signals were also restored by LPA treatment in NRP1-knockdown MC3T3-E1 cells ( Fig. 5 C ) . Immunofluorescence staining further demonstrated enhanced YAP fluorescence signals in NRP1-deficient MC3T3-E1 cells ( Fig. 5 F), consistent with our previous western blot results(Fig. 4 H,I). To further explore the regulatory relationship between NRP1 and YAP, we employed the STRING database to predict interactions among NRP1, YAP, and OGT. Strikingly, this analysis indicated potential direct interactions among all three proteins (Fig. 6 A). Subsequent co-immunoprecipitation confirmed specific bindings between YAP and NRP1, as well as YAP and OGT (Fig. 6 B). To show the details, we used the predicted molecular docking model to illustrate the interaction among NRP1, YAP, and OGT. The structural representation demonstrates that OGT (blue) and YAP (pink) bind to NRP1 (brown), forming a potential ternary complex. Detailed views of the interaction interface indicate specific amino acid residues involved in the binding, including ARG317, ASP531, MET535, and LYS526 from NRP1, as well as residues such as ASN165, PHE54, and ARG74 from YAP. The magnified inset highlights the hydrogen bonding and hydrophobic interactions contributing to the stability of the complex, suggesting a direct structural basis for the functional cooperation of NRP1, YAP, and OGT(Fig. 6 C). 4 | Discussion Previous studies have demonstrated that O-GlcNAcylation is a crucial posttranslational modification regulating the stability and subcellular localization of transcription factors ( 5 ). For instance, O-GlcNAcylation of FOXA1 at multiple residues enhances its stability and chromatin assembly( 32 ). In line with these findings, we show that OGT-mediated O-GlcNAcylation of YAP promotes its nuclear accumulation and transcriptional activation of NRP1, thereby facilitating osteogenesis. Interestingly, we previously observed that during early osteoblast differentiation, OGT relocalizes from the nucleus to the cytoplasm( 33 ). We speculate that this cytoplasmic translocation enables OGT to interact with YAP and promote its O-GlcNAcylation, thereby facilitating YAP nuclear translocation and subsequently initiating and enhancing osteogenic differentiation. However, further investigations are required to substantiate this hypothesis. Although YAP is a well-established coactivator of the Hippo pathway in bone formation and mechanotransduction ( 34 ), its regulation by O-GlcNAcylation has remained largely unexplored. Our findings define the OGT–YAP–NRP1 axis and extend the biological scope of O-GlcNAcylation to bone metabolism. GlcNAcylation has been reported to stabilize YAP and enhance its transcriptional activity in pathological contexts, including endometrial cancer ( 15 ) and myofibroblastic activation( 35 ), primarily by preventing phosphorylation-dependent degradation. Here, we uncover a previously unrecognized physiological role of YAP O-GlcNAcylation in promoting osteogenic differentiation. This highlights its context-dependent function and suggests therapeutic potential for targeting this axis in bone-related disorders. Pharmacological modulation of YAP O-GlcNAcylation is already under investigation in oncology( 35 , 36 ). However, our results suggest that systemic inhibition of YAP O-GlcNAcylation could compromise skeletal integrity, leading to osteoporosis or impaired bone healing. Interestingly, we did not detect a functional interaction between NRP1 and OGT (Supplementary Fig. 1D) , nor O-GlcNAc modification of NRP1 in our study( Supplementary Fig. 1E) . Previous reports on NRP1 glycosylation are limited, mainly emphasizing the GAG modification at S612, which regulates tumor migration( 37 ), adipogenesis( 38 ), and angiogenesis( 39 ). More recently, LC-MS based glycoproteomic analysis by Tuhin Das et al. identified four O-linked glycosylation sites (S612, S637, T638, S641) on NRP1( 40 ). Notably, all four sites are located on its extracellular domain, and OGT-mediated O-GlcNAcylation of extracellular regions of membrane proteins is exceedingly rare. Therefore, it is plausible that in MC3T3-E1 cells, OGT regulates NRP1 indirectly rather than through direct O-GlcNAc modification, which is consistent with our observations. In this study, we demonstrate that LPA promotes YAP nuclear translocation by enhancing its O-GlcNAcylation. Previous studies have suggested that LPA can activate YAP through the Hippo pathway by phosphorylating LATS or through the RhoA/ROCK pathway, inhibiting YAP phosphorylation, and thus promoting its nuclear translocation in MC3T3-E1 cells( 41 ). We propose that LPA may utilize these alternative pathways, such as Hippo or RhoA/ROCK, to promote YAP nuclear localization in OGT-deficient cells. Future work should focus on elucidating the underlying mechanisms. LPA, a potent osteogenic factor that enhances bone regeneration via the Wnt/β-catenin pathway( 42 ), is also broadly implicated in cancer and inflammation( 43 , 44 ). Although the LPA receptor antagonist Ki16425 shows antitumor efficacy and alleviates tumor-associated organ damage, it also blocks LPS-induced Ca 2+ responses and fails to prevent bone loss in ovariectomized mice( 45 ), suggesting that systemic suppression of LPA signaling may impair osteogenesis. Consistent with this, our study identifies a novel LPA–OGT–YAP–NRP1 axis, which validates the osteogenic capacity of LPA in osteoblast-like cells and further establishes a mechanistic link between LPA and NRP1. Given that NRP1 plays an essential role in vascular development and interacts with VEGF/VEGFR and Sema3A/plexinA1( 46 ), and that previous studies have demonstrated LPA-mediated angiogenic regulation through PI3K–Akt signaling in endothelial cells( 47 ), and NF-κB–dependent pathways in chondrocytes ( 48 ), it is plausible that LPA–NRP1 signaling serves as a molecular hub integrating osteogenic and angiogenic programs. These findings highlight potential skeletal side effects of LPA-targeted therapies and suggest that future studies should explore the dual role of LPA in coupling osteogenesis and angiogenesis via NRP1 signaling. NRP1 functions as an integrator of diverse signaling pathways to regulate cell differentiation( 38 ). In bone, NRP1 and NRP2 cooperate with VEGF isoforms to fine-tune angiogenesis( 49 ). Meanwhile, activation of YAP/TAZ in VEGF- or BMP2-transfected MSCs enhances both osteogenesis and angiogenesis( 50 ). Our results suggest that NRP1 upregulation may participate in this process. Interestingly, we also found that NRP1 forms a negative feedback loop with YAP(Fig. 4 F,G,H,I), as NRP1 knockdown in HDMECs abolishes LATS1 activation and increases YAP expression( 51 ), consistent with our observations. This feedback may function as a safeguard to balance osteogenic and angiogenic signaling and prevent aberrant YAP activation. Collectively, our findings establish NRP1 as a regulator of osteogenesis, and point to NRP1–YAP crosstalk as a promising therapeutic target in regenerative medicine. This study primarily relied on in vitro MC3T3-E1 models; thus, in vivo validation of the OGT–YAP–NRP1 axis is necessary. The specific O-GlcNAcylation sites on YAP and their regulatory mechanisms remain to be defined, and the downstream signaling pathways of NRP1 in osteogenesis warrant further exploration. Future studies should employ site-specific mutant mouse models and pharmacological approaches to confirm the physiological relevance of this pathway in bone formation and repair. In addition, the long-term skeletal effects of modulating LPA/YAP O-GlcNAcylation require careful evaluation. Development of tissue- or pathway-specific inhibitors may optimize antitumor efficacy while minimizing adverse skeletal outcomes. Ultimately, precision medicine strategies that integrate cancer therapy with bone-protective approaches could maximize therapeutic benefit without compromising skeletal health. 5 | Conclusions In summary, our study elucidates a novel molecular mechanism by which LPA promotes osteogenic differentiation through the OGT-mediated O-GlcNAcylation of YAP. LPA stimulation enhances YAP O-GlcNAcylation, facilitating its nuclear translocation and subsequent transcriptional activation of the downstream target NRP1. This signaling axis amplifies osteogenic gene expression and promotes bone formation. Our findings reveal a previously unrecognized link between metabolic signaling and transcriptional regulation in osteoblast differentiation, highlighting the OGT–YAP–NRP1 pathway as a potential therapeutic target for metabolic bone diseases. Abbreviations OGT O–GlcNAc transferase YAP Yes–associated protein 1 NRP1 Neuropilin–1 LPA Lysophosphatidic acid CHIP Chromatin immunoprecipitation NC Negative control Declarations Conflicts of Interest The authors declare no conflicts of interest. Funding: This study was supported by the National Natural Science Foundation of China (81900991), National Natural Science Foundation of LiaoNing province (2025-MS-228), and a Grant-in‐Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (23K18431, 22H03511, 21K19644, H.O.; 22H06790, M.I.; 21K17211, Y.F.). Author Contribution Author ContributionsConceptualization: Xinyue Yang, Hirohiko Okamura,Yaqiong Yu. Methodology: Xinyue Yang, Anggun Dwi Andini , Xinyu Gan ,Yao Weng, Mika Ikegame, Hirohiko Okamura. Investigation: Yao Weng, Yilin Zheng, Xinyu Gan, Yuhan He.Validation: Yao Weng, Anggun Dwi Andini , Xinyu Gan , Yuhan He.Formal analysis: Hirohiko Okamura. Data curation: Mika Ikegame, Hirohiko Okamura. Visualization: Xinyue Yang. Writing;Xinyue Yang, Anggun Dwi Andini ,Yao Weng, Mika Ikegame, Hirohiko Okamura. Resources: Xinyue Yang, Mika Ikegame.Project administration: Yaqiong Yu ,Hirohiko Okamura. Funding acquisition: Yaqiong Yu ,Mika Ikegame, Hirohiko Okamura. Supervision: Yaqiong Yu ,Mika Ikegame, Hirohiko Okamura. Acknowledgments This study was supported by the National Natural Science Foundation of China (81900991), the China Postdoctoral Science Foundation (2019M651174), and a Grant-in‐Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (23K18431, 22H03511, 21K19644, H.O.; 22H06790, M.I.; 21K17211, Y.F.). Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. References Levine ZG, Walker S. The Biochemistry of O-GlcNAc Transferase: Which Functions Make It Essential in Mammalian Cells? Annu Rev Biochem. 2016;85:631–57. Du Y, Gao X, Chen J, Chen X, Liu H, He W, et al. OGT mediated HDAC5 O-GlcNAcylation promotes osteogenesis by regulating the homeostasis of epigenetic modifications and proteolysis. J Orthop Transl. 2025;50:14–29. 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Biomater Sci. 2019;7(11):4588–602. Collins JM, Lang A, Parisi C, Moharrer Y, Nijsure MP, Thomas Kim JH, et al. YAP and TAZ couple osteoblast precursor mobilization to angiogenesis and mechanoregulation in murine bone development. Dev Cell. 2024;59(2):211–227.e5. Li M, Wang P, Li J, Zhou F, Huang S, Qi S, et al. NRP1 transduces mechanical stress inhibition via LATS1/YAP in hypertrophic scars. Cell Death Discov. 2023;9(1):341. Additional Declarations No competing interests reported. Supplementary Files GraphicalHighlights.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8696147","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":592778332,"identity":"16d572f1-f6b0-4300-b93c-903083ef013a","order_by":0,"name":"Xinyue Yang","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinyue","middleName":"","lastName":"Yang","suffix":""},{"id":592778334,"identity":"50a38c51-4209-46b2-a47c-149d2fcbf83f","order_by":1,"name":"Yao Weng","email":"","orcid":"","institution":"Okayama University","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Weng","suffix":""},{"id":592778335,"identity":"77957158-72eb-43d6-891f-7d0dd8fb7ed4","order_by":2,"name":"Anggun Dwi Andini","email":"","orcid":"","institution":"Okayama University","correspondingAuthor":false,"prefix":"","firstName":"Anggun","middleName":"Dwi","lastName":"Andini","suffix":""},{"id":592778337,"identity":"11d0a69b-9a4e-40a7-bcf8-2d3372fb32e8","order_by":3,"name":"Xinyu Gan","email":"","orcid":"","institution":"Okayama University","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Gan","suffix":""},{"id":592778339,"identity":"dbb5dc52-b949-487f-92ca-9740ad8a4e6d","order_by":4,"name":"Yuhan He","email":"","orcid":"","institution":"Okayama University","correspondingAuthor":false,"prefix":"","firstName":"Yuhan","middleName":"","lastName":"He","suffix":""},{"id":592778340,"identity":"d17be833-d237-4429-b441-dfd73c3f2bf2","order_by":5,"name":"Yilin Zheng","email":"","orcid":"","institution":"Okayama University","correspondingAuthor":false,"prefix":"","firstName":"Yilin","middleName":"","lastName":"Zheng","suffix":""},{"id":592778341,"identity":"f87fb27f-fdd6-476c-92bf-528e37a58f43","order_by":6,"name":"Ikegame Mika","email":"","orcid":"","institution":"Okayama University","correspondingAuthor":false,"prefix":"","firstName":"Ikegame","middleName":"","lastName":"Mika","suffix":""},{"id":592778342,"identity":"32227da6-7a87-4bc1-99cc-546e3438c3e1","order_by":7,"name":"Okamura Hirohiko","email":"","orcid":"","institution":"Okayama University","correspondingAuthor":false,"prefix":"","firstName":"Okamura","middleName":"","lastName":"Hirohiko","suffix":""},{"id":592778344,"identity":"b036a181-5887-4f60-b84e-ca19553a8f97","order_by":8,"name":"Yaqiong Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYNACAyBmZj74+E+FhBw/8VrY2ZINeM5YGEs2EG0TP4+ZBG9bReIGQloMbuQYfnhTYJMn78xjbCA5T4JxAwPzw0c38GsxlpxjkFZseJit8IHhNglmcwY2Y+Mc/FoMpHkMDidubGbebJC4TYLNsoGHTZqAFuPfEC0MZhIH50jwGBwgrMUMbMt8ZhYzycYGCQmCWiTPPCuzBPolcQMzW7IxwzEJA8lmAn7hO568+cabPzaJ8/sPH3zMUFNX38/e/PAxPi0KBzgMGHhALjwAE2LGoxwE5BvYH4C1yDcQUDkKRsEoGAUjFwAAn6NLd+RWTvwAAAAASUVORK5CYII=","orcid":"","institution":"China Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yaqiong","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2026-01-26 03:38:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8696147/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8696147/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102964188,"identity":"f4a2b2f7-2b5f-4671-a71e-c46e02e8a759","added_by":"auto","created_at":"2026-02-19 04:21:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":124214,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYAP translocates to the nucleus during osteoblast differentiation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunofluorescence staining of YAP and OGT in MC3T3‐E1 cells at day 0, day 1, day 3 and day 6 of osteoblast differentiation.(B) Quantification of the nuclear-to-cytoplasmic ratio of YAP during differentiation, analyzed using ImageJ. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test.(C) Western blot analysis of total YAP expression during differentiation; GAPDH was used as a loading control.(D) Quantification of YAP expression levels shown in (C). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test.(E) Western blot analysis of nuclear and cytoplasmic fractions using antibodies against YAP, LSD1 (nuclear marker), and α-tubulin (cytoplasmic marker) in MC3T3‐E1 cells at day 0, day 3, day 6 of osteoblast differentiation.(F) Quantification of YAP distribution shown in (E). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. (G) Western blot analysis of OGT expression in control and OGT-Knockout cells. GAPDH was used as a loading control. (H) Western blot analysis of YAP expression in control and OGT-Knockout cells. GAPDH was used as a loading control.(I) Quantification of YAP expression levels shown in (H). All data are presented as mean ± SD; n = 3; *p \u0026lt; 0.05; **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-8696147/v1/8f4f2d65457dfe9456292859.png"},{"id":102919860,"identity":"cb73f7b2-6fa8-43a0-91d4-a015d5faeb3b","added_by":"auto","created_at":"2026-02-18 12:12:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":130163,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOGT regulates YAP nuclear localization through O-GlcNAcylation during osteoblastogenesis. \u003c/strong\u003e(A) Immunofluorescence staining of YAP and OGT in control and OGT-knockout MC3T3‐E1 cells at day 0, day 3 and day 6 of osteoblast differentiation. (B) Schematic diagram of the procedure for O‐GlcNAcylated protein immunoprecipitation. (C) Western blot analysis of YAP in MC3T3‐E1 cells input and O‐GlcNAc-IP samples at day 0, day 3 and day 6 of osteoblast differentiation. (D) Quantification of O-GlcNAcylated YAP shown in (D), normalized to total input level. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. (E) Reciprocal co‐immunoprecipitation (Co‐IP) of OGT and YAP in MC3T3‐E1 cells at day 0 of osteoblast differentiation. (F) Reciprocal co‐immunoprecipitation (Co‐IP) of OGT and YAP in MC3T3‐E1 cells at day 3 of osteoblast differentiation. All data are presented as mean ± SD; n = 3; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-8696147/v1/c385e400c999f90e56ae8721.png"},{"id":102964428,"identity":"fd862cf2-b86c-44e0-8db2-574cb321be1b","added_by":"auto","created_at":"2026-02-19 04:22:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":121336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYAP Nuclear Re-Localization Restores Osteogenic Differentiation in OGT-KO MC3T3-E1 Cells \u003c/strong\u003e(A)Immunostaining results of YAP in control and OGT-KO MC3T3-E1 cells adding 10μM LPA during osteoblast differentiation day0/day3/day6. (B)Western blot analysis of RUNX2,OSX protein in control and OGT-KO MC3T3-E1 cells in the presence of LPA(0/5/10/20 μM) at day 3 of osteoblast differentiation.(C)Quantification of RUNX2 expression in control and OGT-KO MC3T3-E1 cells in the presence of LPA(0/5/10/20 μM) at day 3 of osteoblast differentiation, normalized by β-actin. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. (D)Quantification of OSX expression in control and OGT-KO MC3T3-E1 cells in the presence of LPA(0/5/10/20 μM) at day 3 of osteoblast differentiation, normalized by β-actin. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. (E)ALP activity in OGT-KO MC3T3-E1 cells in the presence of LPA (5/10/20 μM) on day7, visualized by azo dye method. (F) Western blot analysis of YAP in input and O‐GlcNAc-IP samples from MC3T3‐E1 cells at day 3 of differentiation and treated by 10μM LPA.(G) Quantification of O-GlcNAcylated YAP shown in (F), normalized to total input level. All data are presented as mean ± SD; n = 3; *p \u0026lt; 0.05; **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-8696147/v1/79022b67c99723fc50e2ea49.png"},{"id":102963813,"identity":"e9b536e5-2f02-4240-9fc3-8a029d6dcf4b","added_by":"auto","created_at":"2026-02-19 04:20:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":126532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNRP1 affects osteoblast differentiation of MC3T3‐E1 cells.\u003c/strong\u003e(A) Western blot analysis of NRP1 protein expression during osteoblast differentiation from day 0 to day 12. GAPDH was used as a loading control. (B) Quantification of NRP1 protein expression levels shown in (A). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. (C) Immunofluorescence staining of NRP1 and OGT in MC3T3‐E1 cells at day 0, day 3, and day 6 of osteoblast differentiation.(D) Western blot analysis of NRP1 expression in control and NRP1-overexpressing cells. β-actin was used as a loading control.(E) ALP activity and calcium deposition in NRP1-overexpressing clones (1, 2, and 3) on day 7 and day 20, visualized by the azo dye method and Alizarin Red S staining, respectively.(F) Western blot analysis of NRP1 and YAP expression in control and NRP1-overexpressing cells. GAPDH was used as a loading control.\u003c/p\u003e\n\u003cp\u003e(G) Quantification of YAP protein expression levels shown in (F). (H)Western blot analysis of NRP1 and YAP expression in control and NRP1-knockdown cells. β-actin was used as a loading control.(I) Quantification of YAP protein expression levels shown in (H). (J) ALP activity and calcium deposition in NRP1-knockdown clones (1, 2, and 3) on day 7 and day 20, visualized by the azo dye method and Alizarin Red S staining, respectively. All data are presented as mean ± SD; n = 3; *p \u0026lt; 0.05; **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-8696147/v1/60f241d7a3c9a38b2713e624.png"},{"id":102964386,"identity":"4379ad18-510f-4206-8973-d8d3ba7eb330","added_by":"auto","created_at":"2026-02-19 04:22:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":136477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNRP1 is regulated by OGT-mediated nuclear translocation of YAP during osteoblast differentiation of MC3T3‐E1 cells. \u003c/strong\u003e(A) Gene expression levels of OGT and NRP1 in OGT-knockout MC3T3‐E1 cells measured by real-time PCR. (B) Immunofluorescence staining of NRP1 and OGT in control, OGT-knockout MC3T3‐E1 cells. (C) Immunofluorescence staining of NRP1 and OGT in control, NRP1-knockdown MC3T3‐E1 cells and 10 μM LPA-treated NRP1-knockdown MC3T3‐E1 cells. (D)Western blot analysis of NRP1 and OGT protein levels in control, OGT-knockout MC3T3‐E1 cells. β-actin was used as a loading control. (E) Quantification of NRP1 protein expression levels shown in (D). (F) Immunofluorescence staining of YAP and OGT in control, NRP1-knockdown MC3T3‐E1 cells and 10 μM LPA-treated NRP1-knockdown MC3T3‐E1 cells.(G) Western blot analysis of NRP1 protein expression in OGT-knockout MC3T3‐E1 cells treated with different concentrations of LPA (0, 5 and 10 μM) at day 3 of differentiation. β-actin was used as a loading control.(H) Quantification of NRP1 expression levels shown in (G), normalized to β-actin. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. All data are presented as mean ± SD; n = 3; *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-8696147/v1/ebb8d724a52d5c5a96db60b2.png"},{"id":102964134,"identity":"5e1825a6-c8b3-47c7-adb6-fb4698c8602d","added_by":"auto","created_at":"2026-02-19 04:21:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":87153,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction of OGT,NRP1 and YAP proteins in MC-3T3 E1 cells \u003c/strong\u003e(A)PPI analysis between the OGT,NRP1 and YAP protein using the STRING database. (B)Reciprocal co-immunoprecipitation(Co-IP) of NRP1 and YAP in MC-3T3 E1 cells differentited day0. (C) Molecular docking analysis. Hydrogen bonds are shown in yellow. YAP (green), OGT1 (blue-purple), NRP1 (flesh color).\u003c/p\u003e","description":"","filename":"Picture6.png","url":"https://assets-eu.researchsquare.com/files/rs-8696147/v1/f1a79ed88b9598a8a52a0da8.png"},{"id":102964460,"identity":"6795acb1-d9b3-4401-b1fc-5f05a3e83cd5","added_by":"auto","created_at":"2026-02-19 04:22:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":35680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract：O‐GlcNAcylation of YAP Enhances Nuclear Translocation to Regulate NRP1‐Mediated Osteogenic Differentiation in MC3T3‐E1 Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study reveals that OGT-mediated O-GlcNAcylation promotes YAP nuclear translocation, activating osteogenic genes (RUNX2, OSX) and upregulating NRP1—a factor essential for osteoblast differentiation. Pharmacological YAP activation by LPA rescues the impairments in NRP1 expression and osteogenesis caused by OGT deficiency. These findings establish the OGT–YAP–NRP1 axis as a pivotal mechanism and therapeutic target for bone regeneration.\u003c/p\u003e","description":"","filename":"Picture7.png","url":"https://assets-eu.researchsquare.com/files/rs-8696147/v1/02916ae3e8fe99a08f2e9393.png"},{"id":105563864,"identity":"6f128ef0-1f78-4dc9-ba67-2437b5bba71f","added_by":"auto","created_at":"2026-03-27 12:48:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2062113,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8696147/v1/6e375247-faee-4636-9c18-99528e58afaa.pdf"},{"id":102964202,"identity":"03beb88a-590f-4176-8b7b-efa4c7dee12e","added_by":"auto","created_at":"2026-02-19 04:21:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19245,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalHighlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-8696147/v1/18a93ec917a3e3455d31566d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"O‐GlcNAcylation of YAP Enhances Nuclear Translocation to Regulate NRP1‐Mediated Osteogenic Differentiation in MC3T3‐E1 Cells","fulltext":[{"header":"1 | Introduction","content":"\u003cp\u003eO-GlcNAc transferase (OGT) is an essential and evolutionarily conserved enzyme that catalyzes O-linked β-N-acetylglucosamine (O-GlcNAc) modification, a dynamic post-translational process regulating transcription, signal transduction, and cell differentiation (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Notably, OGT expression is reduced in osteoporosis, and mice lacking OGT in bone cells exhibit severe bone mass loss (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Our previous studies also demonstrated that O-GlcNAcylation promotes osteoblast differentiation by modulating mitochondria\u0026ndash;cytoskeleton organization and calcium signaling in MC3T3-E1 cells(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). O-GlcNAc is an essential, dynamic monosaccharide post-translational modification found on serine and threonine residues of thousands of nucleocytoplasmic proteins(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Changes in O-GlcNAcylation of proteins are involved in bone diseases such as osteoporosis(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), periodontitis(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), and osteoarthritis(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Importantly, O-GlcNAcylation not only modulates protein activity but can also regulate subcellular localization(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), suggesting that OGT might influence osteogenesis through spatial control of key transcriptional regulators.\u003c/p\u003e \u003cp\u003eYes-associated protein (YAP) is a key transcriptional co-activator in the Hippo signaling pathway, which regulates cell proliferation, apoptosis, and organ size(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). When the Hippo pathway is inactive, YAP translocates to the nuclear and promotes the expression of genes involved in growth and survival(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Previous studies have shown that OGT directly O-GlcNAcylates YAP at Ser109, disrupting its interaction with LATS1, preventing phosphorylation, and enhancing nuclear accumulation(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). OGT-induced YAP O-GlcNAcylation has been implicated in tumor progression and vascular pathology(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). However, whether OGT-mediated YAP O-GlcNAcylation is linked to its nuclear translocation during osteoblast differentiation and bone formation remains unclear.\u003c/p\u003e \u003cp\u003eNotably, lysophosphatidic acid (LPA) is a potent activator of YAP (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). LPA, a natural bioactive phospholipid with pleiotropic effects on multiple tissues, acts primarily through G protein\u0026ndash;coupled LPA receptors (LPAR1\u0026ndash;6) to regulate pathways controlling proliferation, migration, survival, and differentiation (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). As an activator of the RhoA/ROCK pathway, LPA has been shown to rescue cerebellar function and related behavioral deficits caused by OGT deficiency and impaired O-GlcNAcylation (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Nevertheless, whether LPA can restore the osteogenic differentiation capacity of OGT-deficient pre-osteoblastic cells remains to be elucidated. Previous studies indicate that LPA promotes osteogenesis in MC3T3-E1 cells via YAP activation. However, these studies mainly focused on mature osteoblast models, with limited validation in human cells or at the pre-osteoblastic stage(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), and the downstream mechanisms of LPA\u0026ndash;YAP signaling in osteoblasts remain largely unexplored.\u003c/p\u003e \u003cp\u003eNeuropilin-1 (NRP1), a transmembrane glycoprotein and co-receptor for ligands such as VEGF and class 3 semaphorins(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), plays critical roles in angiogenesis(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), neuronal guidance(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), immune regulation(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), and tumor progression(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Importantly, NRP1 has emerged as a key molecule in osteogenesis, as we previously demonstrated that it interacts with Shroom3 to control osteo/odontogenesis in dental pulp stem cells(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). NRP1 promotes osteogenic differentiation via VEGF\u0026ndash;VEGFR2 signaling by upregulating alkaline phosphatase (ALP), osteocalcin (OCN), and osteoprotegerin (OPG) (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), and through SEMA3A-mediated activation of PI3K/Akt and Wnt/β-catenin pathways (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Although LPA has been reported to enhance VEGF\u0026ndash;VEGFR2 signaling and invasiveness in ovarian cancer cells (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), its effects on NRP1 expression and function in osteoblasts remain unclear. Moreover, in cancer cells, YAP/TEAD4 directly enhances NRP1 transcription (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), suggesting a potential mechanistic link between LPA-YAP activation and NRP1 expression in osteoblasts. Therefore, we aimed to investigate whether LPA-YAP activation drives NRP1 expression and contributes to osteogenic differentiation.\u003c/p\u003e \u003cp\u003eIn this study, we employed LPA as a YAP activator to restore osteogenic differentiation capacity and NRP1 expression in OGT-deficient MC3T3-E1 osteoblast-like cells. We investigated how OGT interacts with YAP to promote its O-GlcNAcylation and nuclear translocation during osteogenic differentiation. Nuclear YAP subsequently upregulates key osteogenic transcription factors, including RUNX2 and OSX. Furthermore, we demonstrated that NRP1 functions downstream of the OGT\u0026ndash;YAP axis and contributes to osteoblast differentiation. In summary, our findings indicate that the LPA-enhanced OGT/YAP signaling axis, involving NRP1, plays a critical role in regulating osteogenic differentiation in MC3T3-E1 cells.\u003c/p\u003e"},{"header":"2 | Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 | Cell Culture\u003c/h2\u003e \u003cp\u003ePre-osteoblastic cell line MC3T3‐E1 and human embryonic kidney 293 (HEK293) cells were purchased from RIKEN BRC Cell Bank (Tsukuba, Japan) and maintained at 37\u0026deg;C incubator with 5% CO2. MC3T3‐E1 cells were cultured in minimum essential medium α (MEMα) (Gibco/Life Technologies Corporation, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco/Life Technologies Limited, Paisley, Scotland, UK), and 1% penicillin‐streptomycin mixed solution (Nacalai Tesque Inc., Kyoto, Japan). To induce differentiation, 50 mM ascorbic acid (Sigma‐Aldrich, St. Louis, MO, USA) and 2 mM β‐glycerophosphate (Nacalai Tesque Inc.) were added. The cells were cultured in a 60 mm dish (Thermo Fisher Scientific, Rochester, NY, USA) with 15 mm round glass coverslips (Matsunami, Kishiwada, Osaka, Japan) for immunostaining purposes. During Lysophosphatidic acid (LPA) treatment, LPA(Abcam,ab146430) was added to a final concentration of 5,10 and 20 \u0026micro;mol for 1 day, 3days, 6days. For HEK293 culture, MEM medium (Gibco/Life Technologies Corporation) was mixed with 10% fetal bovine serum, 1% penicillin‐streptomycin mixed solution, and 1% MEM non‐essential amino acids (Gibco/Life Technologies Corporation).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 | Immunocytochemistry\u003c/h2\u003e \u003cp\u003eThe samples were rinsed with phosphate-buffered saline (PBS) (Takara, Kusatsu, Shiga, Japan) and fixed with 4% paraformaldehyde‐PBS (Nacalai Tesque Inc.) for 15 min at room temperature. Next, the cells were permeabilized using 0.1% Triton X‐100 (Sigma‐Aldrich) for 2 min on the ice. For blocking step, 5% bovine serum albumin (BSA) (Nacalai Tesque Inc.) was used.\u003c/p\u003e \u003cp\u003eIncubation overnight with primary antibody was conducted at 4\u0026deg;C. The next day, the samples were subjected to secondary antibody incubation at room temperature for 2 h, followed by using mounting medium with DAPI (ProLong\u0026trade; Diamond Antifade Mountant with DAPI, Invitrogen\u0026trade;P36966, USA). The primary and secondary antibodies used can be found on \u003cb\u003eSupporting Information S1: Data S1\u003c/b\u003e. Cell imaging was performed using LSM 780 confocal laser scanning microscopy system (Zeiss, Oberkochen, Germany).\u003c/p\u003e \u003cp\u003eThe quantitative immunofluorescence analysis of YAP nuclear localization comprised three independent biological replicates at each time point (Days 0, 1, 3, and 6). For each biological replicate, three random fields of view were acquired. YAP nuclear and cytoplasmic distribution was quantified using FIJI/ImageJ (National Institutes of Health, Bethesda, MD, USA). DAPI staining was used to define nuclear boundaries, and the red channel was used to measure YAP fluorescence. Nuclear YAP intensity was obtained by applying DAPI-derived nuclear regions of interest (ROI) onto the YAP channel, while cytoplasmic YAP intensity was calculated as the total red fluorescence minus the nuclear YAP signal. The nuclear-to-cytoplasmic YAP ratio was calculated accordingly. All images were analyzed using a custom FIJI macro, including channel splitting, thresholding, ROI extraction, and IntDen measurement. The full ImageJ macro are provided in \u003cb\u003eSupporting Information S2: Data S2.\u003c/b\u003e The raw dataset are provided in \u003cb\u003eSupporting Information S3: Data S3\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 | Cellular Fractionation\u003c/h2\u003e \u003cp\u003eThe procedure used has been previously documented(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In short, the cells were kept in buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulphonyl fluoride [PMSF]). After adding 10% Nonidet NP-40, the samples were vortexed and centrifuged at a high speed. The supernatant was then collected as the cytoplasmic sample and the pellet was resuspended in buffer C (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF). The nuclear pellet was sonicated and centrifuged to get the nuclear protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 | Protein Collection\u003c/h2\u003e \u003cp\u003eCells were washed with cold PBS, resuspended in lysate buffer (1 mM DTT, 1 mM PMSF, 1 \u0026micro;g/ml leupeptin, 2 \u0026micro;g/mL aprotinin, and 5 mM EGTA), and sonicated for 20 s. The cell lysates were then centrifuged at high speed. The supernatants were collected and the concentration was adjusted prior to western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 | Coomassie Brilliant Blue (CBB) Staining\u003c/h2\u003e \u003cp\u003eTen micrograms of protein were loaded to 10% SDS-PAGE gel. After electrophoresis, the gel was immersed in fixer solution (50% methanol, 10% acetic acid) for 30 min and stained for 20 min (0.6 g of Coomassie blue in 300 mL of 50% (v/v) methanol and 10% acetic acid). Destaining solution (10% methanol, 10% acetic acid) was then used until a contrast between protein band and gel was seen clearly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6| Derive Ogt knockout cells line from MC3T3-E1 cells using CRISPR/Cas9 system\u003c/h2\u003e \u003cp\u003eFor the Cas9 HDR experiments, the guide RNA (gRNA) for Cas9 was designed to target the 7th exon (Ensembl ID: ENSMUSE00000285748) of the major Ogt transcript (Ogt-201; CCDS: CCDS30318.1; Ensembl ID: ENSMUST00000044475.5). First, 3.6 \u0026micro;L of 100 \u0026micro;M Alt-R CRISPR-Cas9 target-specific crRNA (Mm.Cas9.OGT.1. AA; Integrated DNA Technologies, Coralville, IA, USA) and 3.7 \u0026micro;L of 100 \u0026micro;M Alt-R CRISPR-Cas9 tracrRNA (1072533; Integrated DNA Technologies, Coralville, IA, USA) were combined and heated at 95\u0026deg;C for 5 min. The crRNA:tracrRNA solution was then cooled at room temperature. The total 7.3 \u0026micro;L of the crRNA:tracrRNA solution was then combined with 4.8 \u0026micro;L of 62 \u0026micro;M Alt-R S.p. HiFi Cas9 Nuclease V3 (1081059; Integrated DNA Technologies, Coralville, IA, USA) and incubated at room temperature for 5 min to form the RNP complex. MC3T3-E1 cells resuspended in Opti-MEM. Next, 12.1 \u0026micro;L of the RNP complex, 270 \u0026micro;L Opti-MEM containing 2.4\u0026times;10\u003csup\u003e6\u003c/sup\u003e MC3T3-E1 cells, 14.3 \u0026micro;L Opti-MEM, and 3.6 \u0026micro;Lof Alt-R Cas9 Electroporation Enhancer (1075916; Integrated DNA Technologies, Coralville, IA, USA) were combined, and 100 \u0026micro;L of the combined media was transferred into an electroporation cuvette with electrodes of 2 mm gap size (EC-002; Nepa Gene, Chiba, Japan). Finally, 175 V of current (5 ms at 50 ms intervals, two pulses) for poring and 20 V of current (50 ms at 50 ms intervals, five pulses) for transfer were applied by using an electroporator (Super Electroporator NEPA21 Type II;Nepa Gene, Chiba, Japan). The resulting electroporated cells were transferred to a 6-well plate for cell grown and expanded. For isolating clone cells, the electroporated cell suspension was diluted and cultured in a 100 mm dish. When a single cell grew into a cell mass, each colony was isolated using a cloning ring. The knock-out efficiency was determined by measuring the protein expression of Ogt using Western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 | Western Blot Analysis\u003c/h2\u003e \u003cp\u003eTen micrograms of protein were separated using 10% SDS- PAGE and blotted into PVDF membranes (Merck, Darmstadt, Germany). After blocking, the membranes were exposed to the primary antibody overnight at 4\u0026deg;C. Secondary antibody incubation was performed next day in room temperature. The bands were visualized through chemiluminescent after adding HRP substrate (WBLUF0100; Millipore, Burlington, MA, USA). Band density was analyzed by using ImageJ (National Institutes of Health, Bethesda, MD, USA) and normalization by internal control was performed to quantify the amount of protein.\u003c/p\u003e \u003cp\u003eThe ratio between nuclear and cytoplasmic YAP was calculated by comparing the densitometric analysis of the nuclear and cytoplasmic YAP bands, normalized by western blot results. For quantification of O-GlcNAcylated protein, the intensity of the immunoprecipitated (IP) O‐GlcNAcylated protein band was divided by the intensity of the corresponding total protein band in the input lane. This normalization accounts for variations in sample loading and transfer efficiency. Normalized values were obtained for MC3T3‐E1 osteoblast differentiation at days 0, 3, and 6. Fold changes were calculated by dividing the normalized values at days 3 and 6 by the corresponding value at day 0. List of antibodies used can be found on \u003cb\u003eSupporting Information S1: Data S1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 | Alizarin Red Staining\u003c/h2\u003e \u003cp\u003eCells were incubated in 95% ethanol for 10 min after fixation, followed by staining with 10 mg/mL alizarin red solution (Wako Pure Chemical Industries, Kanagawa, Japan). After the color developed, the reaction was stopped using distilled water. The cell pictures were then taken by using Leica DMi1 phase contrast microscope (Leica, Wetzlar, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 | Alkaline Phosphatase (ALP) Staining\u003c/h2\u003e \u003cp\u003eThe samples were dipped into the staining solution (Napthol AS-BI phosphate (Sigma), N‐N\u0026prime; dimethylformamide (Wako Pure Chemical Industries), 0.2 M Tris‐HCl buffer pH 8.3\u0026ndash;8.5,\u003c/p\u003e \u003cp\u003eFast blue RR salt (Sigma) for 10 min and the reaction was stopped by distilled water. For nuclear staining, 1% methyl green stain solution pH 4.0 (Muto Pure Chemicals, Co. Ltd., Tokyo, Japan) was used. Cell pictures were taken immediately after the procedure was finished.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 | Immunoprecipitation (IP) for O-GlcNAcylated Proteins\u003c/h2\u003e \u003cp\u003eThe cells on the different differentiation days were incubated with 200 \u0026micro;M Ac4GAlNAz (CLK-1086‐5; Jena Bioscience, Jena, Germany) overnight. On the following day, cells were resuspended in lysis buffer (2 mM Tris pH 7.5, 15 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% Triton X‐100, 0.25 mM sodium pyrophosphate, 0.1 mM β‐ glycerophosphate, 0.1 mM Na3VO4, 1 \u0026micro;g/mL leupeptin) and sonicated. The samples were centrifuged at high speed for 15 min. The supernatant was collected and divided into two tubes: input and IP sample. The IP sample was incubated with 200 \u0026micro;M DBCO‐PEG4‐ Biotin (760749; Sigma) for 1 h at 4\u0026deg;C with rotation. The IP process was performed using T1 beads from the Dynabeads Streptavidin Trial Kit (Invitrogen) according to the manufacturer's protocol. Briefly, the T1 beads were washed three times with PBS. During the washing step, after the PBS was added, the tube was vortexed for 30 s. The beads and supernatant were separated using a magnetic stand for 1 min. After that, the beads were incubated with the protein sample that had been incubated with DBCO‐PEG4‐Biotin for 30 min at room temperature. The beads were washed with 0.1% BSA five times after incubation. Finally, the beads were resuspended in elution buffer (10 mM EDTA pH 8.2% and 95% formamide) and incubated at 65\u0026deg;C for 2 min. The supernatant separated from the beads was then analyzed by western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 | Immunoprecipitation\u003c/h2\u003e \u003cp\u003eMC3T3-E1 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors, sonicated, and centrifuged at 15,000 rpm for 15 min at 4\u0026deg;C. The supernatant was incubated with 3 \u0026micro;L of anti-NRP1, anti-YAP, or anti-OGT antibody, or species-matched IgG control, for 2 h at 4\u0026deg;C with rotation. Pre-equilibrated protein A/G PLUS-Agarose beads (Santa Cruz, sc-2003) were added and incubated overnight at 4\u0026deg;C. Beads were washed four times with RIPA buffer, and bound proteins were eluted with 1\u0026times; SDS sample buffer by boiling for 5 min, followed by SDS-PAGE and immunoblotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 | Cell Transfection\u003c/h2\u003e \u003cp\u003eFor NRP1 overexpression, the mouse NRP1 coding sequence (Sp-3flag-NM\\_008737) was cloned into the multiple cloning site (MCS) of the GV218 lentiviral vector (GeneChem Corporation, Shanghai, China), resulting in the PL-NRP1 (KL60938-1) construct. The GV218 vector (11.1 kb) contains a CMV promoter for high-level expression in mammalian cells, a ubiquitin promoter driving enhanced green fluorescent protein (EGFP) expression, and an ampicillin resistance gene for bacterial selection. The vector also includes pBR322 ori, 5\u0026prime; long terminal repeat (5\u0026prime;LTR), and BamHI/AgeI cloning sites.\u003c/p\u003e \u003cp\u003eFor NRP1 knockdown, a short hairpin RNA (shRNA) targeting mouse NRP1 (NM\\_003873.7) was designed and inserted into the GV493 lentiviral backbone (GeneChem Corporation, Shanghai, China) under the control of the U6 promoter, generating PL-NRP1-RNAi (PSC109402-1). The GV493 vector (10,880 bp) contains CBh promoter-driven copGFP for transduction efficiency monitoring, a puromycin resistance cassette for selection, and ampicillin resistance for bacterial propagation. The vector backbone includes 5\u0026prime;LTR and 3\u0026prime;LTR sequences and EcoRI/AgeI restriction sites for cloning. Lentiviral particles were produced in HEK293T cells and used to transduce target cells, followed by antibiotic selection to generate stable cell lines.\u003c/p\u003e \u003cp\u003eDuring the cell transfection, opti-MEM (Gibco/Invitrogen Corporation, Paisley, Scotland, UK) was used together with PEI max (Polysciences Inc., Warrington, PA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13| Protein\u0026ndash;Protein Docking\u003c/h2\u003e \u003cp\u003eProtein structures of YAP (UniProt ID: P46938), OGT1 (UniProt ID: Q8CGY8), and NRP1 (UniProt ID: P97333) were obtained from the AlphaFold Protein Structure Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/\u003c/span\u003e\u003cspan address=\"https://alphafold.ebi.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein\u0026ndash;protein docking was performed using AlphaFold3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafoldserver.com\u003c/span\u003e\u003cspan address=\"https://alphafoldserver.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the top-ranked model according to the ranking score was selected as the optimal docking conformation. The binding free energy of the selected complex was evaluated using the HawkDock server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cadd.zju.edu.cn/hawkdock/\u003c/span\u003e\u003cspan address=\"http://cadd.zju.edu.cn/hawkdock/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which also ranked amino acid residues based on their contribution to binding free energy. Residues with high binding contribution were visualized using PyMOL 2.4 (Schr\u0026ouml;dinger, LLC) to provide an intuitive representation of the interaction interfaces.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 | Statistical Analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was conducted by using GraphPad Prism software version 8.0 (GraphPad Software, San Diego, CA, USA). Mean values accompanied by standard deviations (SD) were derived from a minimum of three independent replicates. The data were analyzed by one-way analysis of variance (ANOVA), two‐way ANOVA, or t‐test according to the type of the data, and the p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as significant (*).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 | Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 | YAP translocates to the nucleus during osteoblast differentiation.\u003c/h2\u003e \u003cp\u003eWe first evaluated YAP expression and distribution during the osteogenic differentiation of MC3T3-E1 cells. Immunofluorescence staining revealed that during early osteoblast differentiation, OGT gradually translocated from the nucleus to the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), consistent with our previous observation [32]. In contrast, YAP progressively accumulated in the nucleus, with a marked increase in nuclear fluorescence signal over time(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). By day 3/6, the nuclear-to-cytoplasmic ratio of YAP was clearly higher compared with undifferentiated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), indicating enhanced nuclear localization as differentiation progressed. Western blot analysis demonstrated a gradual increase in total YAP protein expression from day 0 to day 6 (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). Differentiation marker Osterix (OSX) increased until Day 12 during osteoblast differentiation in MC3T3-E1 cells (\u003cb\u003eSupplementary Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Subcellular fractionation further confirmed these findings: in cytoplasmic fractions, YAP levels decreased over the course of differentiation, whereas nuclear fractions showed a notable increase in YAP abundance from day 3 onwards (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F). The purity of subcellular fractions was validated using α-tubulin (cytoplasmic marker) and LSD1 (nuclear marker). Using CRISPR/Cas9 technology, we generated OGT knockout MC3T3-E1 cells. The detailed editing and single-clone selection procedures are described in the Methods section and our previous publication(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Western blot analysis confirmed the knockout efficiency of the established OGT KO clones (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), and the clone with the most efficient depletion, OGT KO #2, was selected for subsequent experiments. Loss of OGT resulted in a marked reduction in YAP protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH,I). These results indicate that osteoblast differentiation is accompanied by a redistribution of YAP from the cytoplasm to the nucleus, a process that may be correlated with OGT .\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 | OGT interacts with YAP, promotes its O-GlcNAcylation, and facilitates nuclear localization during osteoblast differentiation\u003c/h2\u003e \u003cp\u003eNext, we investigated the relationship between YAP and OGT during early osteoblast differentiation. In contrast to control MC3T3-E1 cells, OGT knockdown resulted in a marked retention of YAP in the cytoplasm at all examined stages, as shown by the absence of overlapping nuclear DAPI and YAP fluorescence signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting that OGT is required for YAP nuclear translocation. To further investigate whether OGT regulates YAP through O‐GlcNAcylation, we established an O‐GlcNAc protein immunoprecipitation (IP) assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). MC3T3-E1 cells were differentiated for 0, 3, or 6 days and total protein extracts were prepared. Protein loading was normalized using CBB staining \u003cb\u003e(Supplementary Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e, allowing comparison of overall O-GlcNAcylation between time points. CBB-normalized analyses showed that the global O-GlcNAcylation of cellular proteins was incerasing at day 3 and day6. Consistent with this overall trend, Western blot analysis of IP fractions demonstrated that O‐GlcNAcylated YAP levels were markedly elevated at day 3 and day 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC,D), indicating a dynamic regulation of YAP O‐GlcNAcylation during differentiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven that OGT catalyzes the transfer of O-GlcNAc to substrate proteins, we examined whether it directly interacts with YAP. Reciprocal co‐immunoprecipitation experiments confirmed a physical association between OGT and YAP both in undifferentiated cells (day 0) and in cells at day 3 of osteoblast differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). This consistent interaction across stages supports a model in which OGT physically binds YAP to promote its O‐GlcNAcylation, thereby enabling nuclear localization and transcriptional activity during osteogenesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 | LPA activates YAP and rescues the impaired osteogenic differentiation of OGT-knockout MC3T3-E1 cells\u003c/h2\u003e \u003cp\u003eTo further elucidate the role of O-GlcNAcylated YAP in osteoblast differentiation, OGT-knockout MC3T3‐E1 cells were treated with LPA, a known activator of YAP, and their osteogenic potential was evaluated. Immunofluorescence analysis showed increase in YAP nuclear fluorescence following treatment with LPA (5, 10, and 20 \u0026micro;mol) compared to the untreated control group \u003cb\u003e(Supplementary Fig.\u0026nbsp;1G)\u003c/b\u003e. Immunofluorescence staining revealed that LPA markedly promoted the nuclear re-localization of YAP in OGT-deficient cells, as evidenced by the pronounced nuclear red fluorescence signal(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Western blot analysis demonstrated that LPA treatment increased the protein expression of the osteogenic transcription factors RUNX2 and OSX in OGT-knockout cells at day 3 of osteoblast differentiation in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Quantification showed that, compared with untreated knockout cells, LPA significantly upregulated RUNX2 expression at both 10 \u0026micro;mol and 20 \u0026micro;mol (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and similarly enhanced OSX expression at 10 \u0026micro;mol (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). Functionally, ALP staining revealed that OGT-knockout MC3T3‐E1 cells exhibited reduced osteogenic differentiation compared with the control cells, as indicated by weak red staining. Strikingly, LPA treatment restored ALP activity in knockout cells in a dose-dependent manner, with 5 \u0026micro;mol and 10 \u0026micro;mol producing robust ALP staining comparable to control group levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), indicating functional recovery of osteoblast differentiation. Finally, we aimed to investigate whether LPA can promote YAP O-GlcNAcylation. We examined the effect of LPA on YAP O-GlcNAcylation in wild-type MC3T3-E1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Our analysis demonstrated that LPA treatment increased both the O-GlcNAcylation level and total protein expression of YAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G; \u003cb\u003eSupplementary Fig.\u0026nbsp;1B,C\u003c/b\u003e), suggesting that LPA may facilitate YAP nuclear translocation by enhancing its O-GlcNAcylation and protein erxpression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollectively, these findings indicate that LPA-mediated YAP activation not only restores its nuclear localization, protein expression, and O-GlcNAcylation, but also rescues the impaired osteogenic differentiation capacity caused by OGT loss, underscoring the critical role of O-GlcNAcylation\u0026ndash;YAP signaling in bone formation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 | NRP1 plays an important role in osteoblast differentiation\u003c/h2\u003e \u003cp\u003eRecent studies indicated that NRP1 is involved in bone formation[26\u0026ndash;28]. To investigate NRP1\u0026rsquo;s function in MC3T3-E1 cells\u0026rsquo; osteoblast differentiation, first we examined protein expression levels of NRP1 during differentiation of the preosteoblastic cell line MC3T3-E1. NRP1 expression is increasing during the osteoblast differentiation day3 to day12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B). Immunofluorescence staining further revealed dynamic changes in subcellular localization of NRP1 and OGT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). At day 6, extensive co-localization was observed, suggesting that NRP1 may interact with OGT during osteogenic differentiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo more precisely examine the role of NRP1 in osteoblast differentiation, we examined the effect of NRP1 overexpression and knockdown on the expression of osteogenic transcription factors. NRP1 overexpression was achieved by lentiviral transduction of a GV218 vector carrying the full-length mouse NRP1 coding sequence, while NRP1 knockdown was performed using a GV493 vector expressing an NRP1-targeting shRNA, The detailed procedures are described in the Methods section. Western blot results show that NRP1 was successfully overexpressed(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and knock down(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). We performed single-cell cloning on ovNRP1 cells and shNRP1 cells, obtaining several clones that expressed very high levels of NRP1 protein and ALP activity in ovNRP1 cells(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD,E) and very low levels of NRP1 protein and ALP activity in shNRP1 cells(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH,J). Functional assays demonstrated that NRP1 overexpression in NRP1-OE cells significantly promoted osteoblast differentiation, as evidenced by increased ALP staining on day 7 and intensified Alizarin Red S staining on day 20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). NRP1 knockdown in NRP1-sh cells significantly impaired osteogenic capacity, as indicated by decreased ALP and mineral deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Collectively, these findings indicate that NRP1 positively regulates osteoblast differentiation. Furthermore, we examined the effect of NRP1 on YAP protein expression. Overexpression of NRP1 in MC3T3-E1 cells reduced YAP protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G), whereas knockdown of NRP1 increased YAP expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, I). These results suggest that NRP1 functions as a negative upstream regulator of YAP protein stability or synthesis in MC3T3-E1 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 | NRP1 Acts Downstream of OGT-Mediated YAP Nuclear Translocation to Promote Osteogenesis\u003c/h2\u003e \u003cp\u003eTo investigate the relationship between NRP1 and OGT-Mediated YAP Nuclear Translocation during the osteoblast differentiation, we examined the mRNA and protein expression level of NRP1 in OGT knock out MC3T3-E1 cells. In OGT-ko cells, expression of NRP1 was decreased at mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD,E). Immunostaining result on OGT-ko cells also showed that NRP1 expression decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We investigated whether OGT physically interacts with NRP1. Co-immunoprecipitation analysis revealed no evidence of a direct interaction between the two proteins in MC3T3‐E1 cells \u003cb\u003e(Supplementary Fig.\u0026nbsp;1D)\u003c/b\u003e. Consistently, O-GlcNAcylation Western blotting did not detect O-GlcNAc\u0026ndash;modified NRP1 in MC3T3-E1 cells at day 0, 3, or 6 of osteogenic differentiation \u003cb\u003e(Supplementary Fig.\u0026nbsp;1E)\u003c/b\u003e. Next, we used LPA to stimulate YAP and examined NRP1 protein levels in OGT-knockout cells at day 3 of osteoblast differentiation. NRP1 expression was decreased in OGT-KO cells but was restored following treatment with 10\u0026micro;mol LPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG,H), suggesting that NRP1 may act downstream of OGT-mediated YAP nuclear translocation to promote osteogenesis. Consistently, immunofluorescence staining showed that NRP1 fluorescence signals were also restored by LPA treatment in NRP1-knockdown MC3T3-E1 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Immunofluorescence staining further demonstrated enhanced YAP fluorescence signals in NRP1-deficient MC3T3-E1 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), consistent with our previous western blot results(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH,I). To further explore the regulatory relationship between NRP1 and YAP, we employed the STRING database to predict interactions among NRP1, YAP, and OGT. Strikingly, this analysis indicated potential direct interactions among all three proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Subsequent co-immunoprecipitation confirmed specific bindings between YAP and NRP1, as well as YAP and OGT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo show the details, we used the predicted molecular docking model to illustrate the interaction among NRP1, YAP, and OGT. The structural representation demonstrates that OGT (blue) and YAP (pink) bind to NRP1 (brown), forming a potential ternary complex. Detailed views of the interaction interface indicate specific amino acid residues involved in the binding, including ARG317, ASP531, MET535, and LYS526 from NRP1, as well as residues such as ASN165, PHE54, and ARG74 from YAP. The magnified inset highlights the hydrogen bonding and hydrophobic interactions contributing to the stability of the complex, suggesting a direct structural basis for the functional cooperation of NRP1, YAP, and OGT(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e"},{"header":"4 | Discussion","content":"\u003cp\u003ePrevious studies have demonstrated that O-GlcNAcylation is a crucial posttranslational modification regulating the stability and subcellular localization of transcription factors (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). For instance, O-GlcNAcylation of FOXA1 at multiple residues enhances its stability and chromatin assembly(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In line with these findings, we show that OGT-mediated O-GlcNAcylation of YAP promotes its nuclear accumulation and transcriptional activation of NRP1, thereby facilitating osteogenesis. Interestingly, we previously observed that during early osteoblast differentiation, OGT relocalizes from the nucleus to the cytoplasm(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). We speculate that this cytoplasmic translocation enables OGT to interact with YAP and promote its O-GlcNAcylation, thereby facilitating YAP nuclear translocation and subsequently initiating and enhancing osteogenic differentiation. However, further investigations are required to substantiate this hypothesis. Although YAP is a well-established coactivator of the Hippo pathway in bone formation and mechanotransduction (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), its regulation by O-GlcNAcylation has remained largely unexplored. Our findings define the OGT\u0026ndash;YAP\u0026ndash;NRP1 axis and extend the biological scope of O-GlcNAcylation to bone metabolism.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eGlcNAcylation has been reported to stabilize YAP and enhance its transcriptional activity in pathological contexts, including endometrial cancer (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and myofibroblastic activation(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), primarily by preventing phosphorylation-dependent degradation. Here, we uncover a previously unrecognized physiological role of YAP O-GlcNAcylation in promoting osteogenic differentiation. This highlights its context-dependent function and suggests therapeutic potential for targeting this axis in bone-related disorders. Pharmacological modulation of YAP O-GlcNAcylation is already under investigation in oncology(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). However, our results suggest that systemic inhibition of YAP O-GlcNAcylation could compromise skeletal integrity, leading to osteoporosis or impaired bone healing. Interestingly, we did not detect a functional interaction between NRP1 and OGT\u003cb\u003e(Supplementary Fig.\u0026nbsp;1D)\u003c/b\u003e, nor O-GlcNAc modification of NRP1 in our study(\u003cb\u003eSupplementary Fig.\u0026nbsp;1E)\u003c/b\u003e. Previous reports on NRP1 glycosylation are limited, mainly emphasizing the GAG modification at S612, which regulates tumor migration(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), adipogenesis(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), and angiogenesis(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). More recently, LC-MS based glycoproteomic analysis by Tuhin Das et al. identified four O-linked glycosylation sites (S612, S637, T638, S641) on NRP1(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Notably, all four sites are located on its extracellular domain, and OGT-mediated O-GlcNAcylation of extracellular regions of membrane proteins is exceedingly rare. Therefore, it is plausible that in MC3T3-E1 cells, OGT regulates NRP1 indirectly rather than through direct O-GlcNAc modification, which is consistent with our observations.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eIn this study, we demonstrate that LPA promotes YAP nuclear translocation by enhancing its O-GlcNAcylation. Previous studies have suggested that LPA can activate YAP through the Hippo pathway by phosphorylating LATS or through the RhoA/ROCK pathway, inhibiting YAP phosphorylation, and thus promoting its nuclear translocation in MC3T3-E1 cells(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). We propose that LPA may utilize these alternative pathways, such as Hippo or RhoA/ROCK, to promote YAP nuclear localization in OGT-deficient cells. Future work should focus on elucidating the underlying mechanisms.\u003c/p\u003e \u003cp\u003eLPA, a potent osteogenic factor that enhances bone regeneration via the Wnt/β-catenin pathway(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), is also broadly implicated in cancer and inflammation(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Although the LPA receptor antagonist Ki16425 shows antitumor efficacy and alleviates tumor-associated organ damage, it also blocks LPS-induced Ca\u003csup\u003e2+\u003c/sup\u003e responses and fails to prevent bone loss in ovariectomized mice(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), suggesting that systemic suppression of LPA signaling may impair osteogenesis. Consistent with this, our study identifies a novel LPA\u0026ndash;OGT\u0026ndash;YAP\u0026ndash;NRP1 axis, which validates the osteogenic capacity of LPA in osteoblast-like cells and further establishes a mechanistic link between LPA and NRP1. Given that NRP1 plays an essential role in vascular development and interacts with VEGF/VEGFR and Sema3A/plexinA1(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e), and that previous studies have demonstrated LPA-mediated angiogenic regulation through PI3K\u0026ndash;Akt signaling in endothelial cells(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), and NF-κB\u0026ndash;dependent pathways in chondrocytes (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), it is plausible that LPA\u0026ndash;NRP1 signaling serves as a molecular hub integrating osteogenic and angiogenic programs. These findings highlight potential skeletal side effects of LPA-targeted therapies and suggest that future studies should explore the dual role of LPA in coupling osteogenesis and angiogenesis via NRP1 signaling.\u003c/p\u003e \u003cp\u003eNRP1 functions as an integrator of diverse signaling pathways to regulate cell differentiation(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In bone, NRP1 and NRP2 cooperate with VEGF isoforms to fine-tune angiogenesis(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Meanwhile, activation of YAP/TAZ in VEGF- or BMP2-transfected MSCs enhances both osteogenesis and angiogenesis(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Our results suggest that NRP1 upregulation may participate in this process. Interestingly, we also found that NRP1 forms a negative feedback loop with YAP(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF,G,H,I), as NRP1 knockdown in HDMECs abolishes LATS1 activation and increases YAP expression(\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), consistent with our observations. This feedback may function as a safeguard to balance osteogenic and angiogenic signaling and prevent aberrant YAP activation. Collectively, our findings establish NRP1 as a regulator of osteogenesis, and point to NRP1\u0026ndash;YAP crosstalk as a promising therapeutic target in regenerative medicine.\u003c/p\u003e \u003cp\u003eThis study primarily relied on in vitro MC3T3-E1 models; thus, in vivo validation of the OGT\u0026ndash;YAP\u0026ndash;NRP1 axis is necessary. The specific O-GlcNAcylation sites on YAP and their regulatory mechanisms remain to be defined, and the downstream signaling pathways of NRP1 in osteogenesis warrant further exploration. Future studies should employ site-specific mutant mouse models and pharmacological approaches to confirm the physiological relevance of this pathway in bone formation and repair. In addition, the long-term skeletal effects of modulating LPA/YAP O-GlcNAcylation require careful evaluation. Development of tissue- or pathway-specific inhibitors may optimize antitumor efficacy while minimizing adverse skeletal outcomes. Ultimately, precision medicine strategies that integrate cancer therapy with bone-protective approaches could maximize therapeutic benefit without compromising skeletal health.\u003c/p\u003e"},{"header":"5 | Conclusions","content":"\u003cp\u003eIn summary, our study elucidates a novel molecular mechanism by which LPA promotes osteogenic differentiation through the OGT-mediated O-GlcNAcylation of YAP. LPA stimulation enhances YAP O-GlcNAcylation, facilitating its nuclear translocation and subsequent transcriptional activation of the downstream target NRP1. This signaling axis amplifies osteogenic gene expression and promotes bone formation. Our findings reveal a previously unrecognized link between metabolic signaling and transcriptional regulation in osteoblast differentiation, highlighting the OGT\u0026ndash;YAP\u0026ndash;NRP1 pathway as a potential therapeutic target for metabolic bone diseases.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOGT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eO\u0026ndash;GlcNAc transferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eYAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eYes\u0026ndash;associated protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNRP1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeuropilin\u0026ndash;1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLysophosphatidic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCHIP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChromatin immunoprecipitation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNegative control\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study was supported by the National Natural Science Foundation of China (81900991), National Natural Science Foundation of LiaoNing province (2025-MS-228), and a Grant-in‐Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (23K18431, 22H03511, 21K19644, H.O.; 22H06790, M.I.; 21K17211, Y.F.).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor ContributionsConceptualization: Xinyue Yang, Hirohiko Okamura,Yaqiong Yu. Methodology: Xinyue Yang, Anggun Dwi Andini , Xinyu Gan ,Yao Weng, Mika Ikegame, Hirohiko Okamura. Investigation: Yao Weng, Yilin Zheng, Xinyu Gan, Yuhan He.Validation: Yao Weng, Anggun Dwi Andini , Xinyu Gan , Yuhan He.Formal analysis: Hirohiko Okamura. Data curation: Mika Ikegame, Hirohiko Okamura. Visualization: Xinyue Yang. Writing;Xinyue Yang, Anggun Dwi Andini ,Yao Weng, Mika Ikegame, Hirohiko Okamura. Resources: Xinyue Yang, Mika Ikegame.Project administration: Yaqiong Yu ,Hirohiko Okamura. Funding acquisition: Yaqiong Yu ,Mika Ikegame, Hirohiko Okamura. Supervision: Yaqiong Yu ,Mika Ikegame, Hirohiko Okamura.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis study was supported by the National Natural Science Foundation of China (81900991), the China Postdoctoral Science Foundation (2019M651174), and a Grant-in‐Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (23K18431, 22H03511, 21K19644, H.O.; 22H06790, M.I.; 21K17211, Y.F.).\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLevine ZG, Walker S. The Biochemistry of O-GlcNAc Transferase: Which Functions Make It Essential in Mammalian Cells? 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Cell Death Discov. 2023;9(1):341.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Neuropilin-1 (NRP1), Yes‐associated protein (YAP), O‐GlcNAc transferase (OGT), Lysophosphatidic acid (LPA), Osteoblast differentiation","lastPublishedDoi":"10.21203/rs.3.rs-8696147/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8696147/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOur previous study demonstrated that O-GlcNAc transferase (OGT) promotes osteoblast differentiation in MC3T3‐E1 cells; however, the precise molecular mechanism remains unclear. In this study, we investigated whether OGT regulates osteoblast differentiation through Yes-associated protein (YAP) and neuropilin-1 (NRP1). Using MC3T3-E1 cells, we show that OGT directly O-GlcNAcylates YAP, promoting its nuclear translocation and transcriptional activation of osteogenic regulators RUNX2 and Osterix (OSX). Loss of OGT impaired YAP activity, NRP1 expression, and osteoblast differentiation. Treatment with lysophosphatidic acid (LPA), a YAP activator, restored YAP nuclear accumulation, re-established NRP1 expression, and rescued osteogenic marker expression in OGT-deficient cells. Functional studies further identified NRP1 as a downstream effector of the OGT\u0026ndash;YAP axis required for osteogenesis.These findings establish a regulatory pathway in which OGT-mediated O-GlcNAcylation of YAP enhances NRP1-dependent osteogenic differentiation. Moreover, pharmacological activation of YAP by LPA compensates for OGT deficiency, highlighting the OGT\u0026ndash;YAP\u0026ndash;NRP1 axis as a potential therapeutic target for bone regeneration and osteoporosis.\u003c/p\u003e","manuscriptTitle":"O‐GlcNAcylation of YAP Enhances Nuclear Translocation to Regulate NRP1‐Mediated Osteogenic Differentiation in MC3T3‐E1 Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-18 12:12:32","doi":"10.21203/rs.3.rs-8696147/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fae22b7f-b18e-45bf-9e4d-55ee00f38820","owner":[],"postedDate":"February 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T01:55:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-18 12:12:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8696147","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8696147","identity":"rs-8696147","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}