KIAA1429-mediated M6A methylation inhibits osteoclast differentiation via stabilizing Lrp4 mRNA and protects against osteoporosis.

Introduction

Osteoporosis (OP) represents a prevalent skeletal metabolic disorder primarily defined by reduced bone mineral density and deteriorating bone microarchitecture. This condition is accompanied by diminished bone strength and heightened bone fragility, compromising skeletal integrity and increasing susceptibility to fractures [ 1 – 4 ]. OP and its accompanying fragility fractures increase disability and mortality rates among older persons, which results in significant healthcare costs [ 5 – 7 ]. OP occurs in all sexes and at any age but is more common in older male and postmenopausal female individuals [ 8 – 10 ]. Furthermore, the global increase in the aging population has led to an annual increase in new osteoporotic fracture cases [ 11 , 12 ]. In the US healthcare system, approximately $17 billion is spent annually to treat osteoporotic fractures, and this cost is projected to reach $50 billion by 2040 [ 13 ]. However, the cost of OP prevention or early OP treatment is relatively low compared with the cost of treating osteoporotic fractures [ 3 , 14 ]. Therefore, clarifying the pathological mechanisms underlying OP and exploring ideal diagnostic and therapeutic targets are significant steps to improve the quality of life of patients with OP and reducing their financial burden.

OP development is closely related to abnormal changes in intraosseous homeostasis, which is primarily maintained by osteoblasts and osteoclasts in a synchronized manner [ 15 – 17 ]. However, disturbance in the balance between bone formation and resorption primarily stems from an imbalance favoring osteoclast-mediated resorption over bone formation activity. This pathological shift is a major contributor to bone loss and the onset of OP [ 18 , 19 ].

As a common epigenetic modification in eukaryotes, N 6 -methyladenosine (m 6 A) methylation modification plays important functions in mRNA splicing, editing, stability, degradation, and polyadenylation [ 20 – 22 ]. The process of m 6 A methylation modification exists in a dynamic and reversible state, which is jointly regulated by methyltransferases (writers), demethylases (erasers), and methylated reading proteins (readers), and it participates in a variety of cellular biological processes, including cell differentiation, development, stress response, and disease genesis [ 21 , 23 ]. Recently, the role of m 6 A modification in OP has received widespread attention [ 24 , 25 ]. Methyltransferases, demethylases, and methylated reading proteins ameliorate OP by targeting multiple factors to regulate the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) [ 24 – 27 ]. As one of the important components of m 6 A methyltransferase, KIAA1429, also known as the virus-like m 6 A methyltransferase-associated protein (VIRMA), functions as the methyltransferase complex’s structural platform. It serves as a bridge connecting the catalytic core to the RNA substrate, thereby guiding m 6 A modification deposition at specific genomic locations [ 28 – 30 ]. KIAA1429 has been reported to act in an m 6 A-dependent manner and be involved in biological processes such as tumor migration, germ cell development, and metabolic disease genesis [ 30 – 32 ]. Although m 6 A methyltransferases play positive roles in bone biology, their underlying epigenetic mechanisms remain unclear [ 24 , 33 ]. Furthermore, compared with the research direction of osteoblasts, the osteoclast field of RNA methylation has been less studied, especially the significance of the bone biology of KIAA1429 and its molecular mechanism in osteoclast differentiation, which has not been elucidated.

In this study, we performed a comprehensive analysis using cellular models, animal models, and clinical bone sample-based study to confirm the potential clinical usefulness of KIAA1429 in the treatment of OP. In addition, we identified that KIAA1429-mediated m 6 A modifications regulated the expression of the low-density lipoprotein receptor-related protein 4 (LRP4), as well as the m 6 A methylation functional site of the Lrp4 gene, and demonstrated that LRP4 is a downstream target of KIAA1429 in the regulation of osteoclast differentiation. Notably, during the KIAA1429-mediated inhibition of osteoclast differentiation by LRP4, the methylated reading protein YT521-B homology-domain-containing protein 1 (YTHDC1) was involved in the m 6 A modification of LRP4, which upregulated LRP4 expression, thereby inhibiting osteoclast differentiation. Collectively, this study highlights the potential of KIAA1429 as a therapeutic target for OP and provides new insights into diagnostic and therapeutic strategies for OP.

Materials and methods

Public databases analysis

RNA-seq for targeted genes ( KIAA1429 ) were obtained from the Gene Expression Omnibus (GEO) dataset ( GSE225974 ), and heatmaps were drawn using R version 4.4.2.

Animals

This research complied with institutional ethical guidelines for laboratory animal welfare (Guide for the Care and Use of Laboratory Animals) and received approval from Shandong University Laboratory Animal Center. Female C57BL/6 mice (age: 3 months; n = 6 per cohort) were acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd. All subjects were maintained in identical specific pathogen-free (SPF) environmental conditions (temperature: 23 ± 2 °C; 12-h light/dark cycle), provided access to feed, and permitted unrestricted movement. Ovariectomy (OVX) surgery was performed regarding previous studies [ 27 ].

Micro-computed tomography (CT)

Mice were anesthetized and executed, and the area of the distal femur was collected and immersed in 75% ethanol at 4 °C for micro CT (PerkinElmen, USA) scanning and analysis. The parameters for scanning were set as follows: 90 kV, 88 μA, and 7 μm/pixel resolution. Quantification was performed using Analyze 12.0 (PerkinElmen, USA).

Bone histomorphometry and immunohistochemistry staining

Following micro-CT assessment, femoral samples underwent sequential processing: 24-h immersion in 4% paraformaldehyde fixation solution, succeeded by 21-day decalcification in 10% ethylenediaminetetraacetic acid (EDTA) at ambient conditions. Subsequently, longitudinal sections (3 μm thickness) were prepared after paraffin embedding of these specimens.

For hematoxylin and eosin (H&E) staining, paraffin sections were routinely deparaffinized to water by xylene and degraded ethanol. Staining with hematoxylin and eosin was performed sequentially according to the manufacturer’s protocol.

For Masson staining, paraffin sections were routinely deparaffinized to water by xylene and degraded ethanol. Staining was performed according to the manufacturer’s protocol.

For tartrate-resistant acid phosphatase (TRAP) staining, sections from each group were stained for TRAP according to the manufacturer’s protocol.

For immunohistochemistry staining, each section was incubated with anti-KIAA1249 antibody (no. 25712, 1:200, Proteintech, China), anti-LRP4 antibody (no. 56540-100UG, 1:200, Invitrogen, USA), anti-TNFAIP3 antibody (no. 5630S, 1: 200, Cell Signaling Technology, USA), and anti-CTSK antibody (no. 48353, 1:200, Santa Cruz, USA) at 4 °C overnight. The obtained sections were incubated with a secondary antibody for 1 h at room temperature.

Images were taken under the microscope (Olympus, Japan).

Single-cell sequencing and data analysis

Single-cell suspensions were generated from murine femoral bone tissue using established protocols [ 34 ]. Immediately after preparation, cell suspensions were processed according to the 10x Chromium 3′ v3 kit workflow (10x Genomics, USA). Library preparation preceded NovaSeq 6000 sequencing (LC-Bio Technologies, China). Raw sequencing data underwent quality control and disassembly via PISA software ( https://github.com/shiquan/PISA ), subsequently producing cell-by-gene count matrices. scRNA-seq data analysis used the R package Seurat, implementing comprehensive quality control measures that retained cells where nFeature_RNA > 200 and nCount_RNA between 200–30,000, with mitochondrial content percentage < 5%.

Technical variations across samples were addressed using canonical correlation analysis (CCA) for batch effect correction. Following LogNormalize normalization, dimensionality reduction via principal component analysis (PCA) determined feature space for Uniform Manifold Approximation and Projection (UMAP)-based cell clustering. Clusters were annotated through manual curation using established lineage-specific marker genes cross-referenced with the CellMarker database ( http://xteam.xbio.top/CellMarker/ ). This strategy enabled both marker gene identification per cluster and subsequent cell-type classification, with lineage-specific markers detailed in Supplementary Table S1.

Cell culture

RAW264.7 cells (Zhong Qiao Xin Zhou Biotechnology Co., Ltd., no. ZQ0098, Shanghai, China) were maintained under standard culture conditions; Dulbecco’s modified Eagle medium (DMEM) was supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic solution within a humidified 5% CO₂ incubator at 37 °C. Bone marrow-derived cells were obtained from murine long bones through medullary cavity flushing using DMEM. Subsequent overnight incubation of harvested cells in DMEM containing 10% FBS permitted separation of nonadherent populations for collection. Suspension cells were collected and cultured in DMEM containing 10% FBS and 40 ng/mL macrophage colony-stimulating factor (M-CSF) (no. 216-MCC, R&D Systems, USA) for 3 days to obtain bone marrow-derived macrophages (BMDM). The bone marrow mesenchymal stem cells (BMMSCs) were obtained from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (no. ZQ0465, Shanghai, China). BMMSCs were cultured in DMEM/F12 (1:1) medium (no. 11320033, Gibco, USA) containing 10% FBS and 1% antibiotics (no. 10378016, Gibco, USA).

Osteoclasts were induced to differentiate using 50 ng/mL RANKL (no. 462-TEC, R&D Systems, USA) to stimulate RAW264.7 cells for 7 days, and BMDM were treated with 40 ng/mL M-CSF and 50 ng/mL RANKL for 7 days. Cells without RANKL treatment were used as a negative control. Methods of bone marrow mesenchymal stem cell osteogenic differentiation induction were performed according to previous studies [ 27 ].

Tartrate-resistant acid phosphatase (TRAP) staining

Following osteoclast differentiation induction, fixed cells were incubated for 20 min in 4% formaldehyde at ambient temperature. TRAP staining was subsequently performed per manufacturer guidelines (Servicebio TRAP Detection Kit, no. G1050-50 T, Wuhan, China). Multinucleated osteoclasts (≥ 3 nuclei) exhibiting TRAP positivity were identified.

Alizarin Red S (ARS) and alkaline phosphatase (ALP) detection

After designated osteogenic induction periods, cell cultures underwent fixation in 4% paraformaldehyde (20 min) followed by two PBS washes. ARS staining employed Solarbio reagent (no. G1450) for 30 min under ambient conditions. Concurrently, ALP activity was visualized via BCIP/NBT chromogenic reaction kit (no. C3206, Beyotime, China). Stained samples were quantified using ImageJ software (NIH, Bethesda, MD, USA).

Quantitative real-time polymerase chain reaction (PCR)

Cellular and murine femoral distal cancellous bone RNA was purified via TRIzol extraction (no. 15596026, Invitrogen, USA). Complementary DNA synthesis and quantitative PCR followed established methodology [ 27 ]. Transcript levels underwent normalization via comparative CT method using Gapdh as endogenous control. Primer sequences are presented in Supplementary Table S2.

RNA m 6 A quantification

Total RNA purified from cultured cells and distal femur trabecular bone using TRIzol (no. 15596026, Invitrogen, USA) served as input material. Relative N 6 -methyladenosine modification levels were determined spectrophotometrically at 450 nm following the EpiQuik m 6 A RNA Methylation Quantification Kit protocol (no. P-9005–48, EpiGentek, USA).

Enzyme-linked immunosorbent assay (ELISA)

Mice were anesthetized and executed, serum was collected, and CTX-1 and PINP were detected by commercial ELISA kits according to the instruction manual. All of the ELISA kits were obtained from Elabscience Biotechnology Co., Ltd. (Wuhan, China).

Patient tissue samples

Postmenopausal female patients (age > 50 years) who underwent hip replacement surgery at Qilu Hospital of Shandong University (Qingdao) were selected. All cases were strictly excluded from secondary osteoporosis (e.g., metabolic bone disease, bone metastasis of malignant tumors, multiple myeloma, history of bone radiotherapy, history of long-term glucocorticoid use, malabsorption syndrome), combined severe liver and kidney diseases [estimated glomerular filtration rate (eGFR) < 30 mL/min, Child–Pugh class C cirrhosis], active autoimmune diseases, and history of anti-osteoporosis drug treatment. Finally, 20 patients were included. According to the criteria of the World Health Organization, the patients were divided into the OP group [femoral neck or vertebral body bone mineral density (BMD) T value ≤ −2.5] and the non-OP group (femoral neck and vertebral body BMD T value > −2.5) according to the BMD T value of patients [ 35 , 36 ]. All human bone tissues used in the experiment no longer served a purpose during surgery, and the collection of bone specimens did not have any impact on the treatment. The cancellous bone in the center of the non-load-bearing area of the femoral head was collected (drilled vertically with an 8 mm diameter bone drill to a depth of 10 mm), and the samples were frozen in liquid nitrogen and then transferred to the laboratory for standardized processing. The study was approved by the Medical Ethics Committee of Qilu Hospital of Shandong University (Qingdao, China), and all patients signed an informed consent form before surgery.

Lentivirus-mediated overexpression and knockout

CRISPR/Cas9-engineered lentiviral vectors targeting Kiaa1429 , Lrp4 , and Tnfaip3 for knockout studies, alongside a Kiaa1429 overexpression construct, were procured from Shanghai Genechem Co., Ltd. For transduction, RAW264.7 cells at approximately 50% confluence were exposed to lentiviral particles in the presence of polybrene (8 µg/mL final concentration). Following a 12-h incubation period, the viral supernatant was aspirated, and cultures were refreshed with standard growth medium. Puromycin selection commenced 72 h post transduction to establish stable pools. Effective target gene knockout or overexpression was subsequently confirmed through quantitative reverse transcription-PCR (qRT-PCR) and western blot analysis prior to further experimentation.

Western blotting (WB)

Total protein was extracted from cells using RIPA lysis buffer (no. ST506, Beyotime, China) containing phenylmethylsulfonyl fluoride (PMSF). PAGE Gel Fast Preparation Kit was used for WB assay (no. PG112, Epizyme Biotech, China). The specific procedure referred to a previous report [ 27 ]; in brief, equal amounts of protein samples were separated on polyacrylamide gels and transferred to nitrocellulose membranes, which were closed for 1 h using 5% skimmed milk powder, and the membranes obtained were incubated with anti-KIAA1429 antibody (no. 25712, 1:1000, Proteintech, China), anti-LRP4 antibody (no. 56540-100UG, 1:1000, Invitrogen, USA), anti-TNFAIP3 antibody (no. 5630S, 1:1000, Cell Signaling Technology, USA), anti-NFATC1 antibody (no. 7294, 1:1000, Santa Cruz Biotechnology, USA), anti-CTSK antibody (no. 48353, 1:1000, Santa Cruz Biotechnology, USA), anti-C-FOS antibody (no. ab190289, 1:1000, Abcam, UK), anti-YTHDC1 antibody (no. 14392–1-AP, Proteintech, China), anti-p65 antibody (no. A19653, 1:1000, ABclonal, China), anti-p-p65 antibody (no. 3033 T, 1:1000, Cell Signaling Technology, USA), anti-IκBα antibody (no. 4812, 1:1000, Cell Signaling Technology, USA), anti-p-IκBα antibody (no. 2859, 1:1000, Cell Signaling Technology, USA), anti-p-IKKα/β antibody (no. 2687 T, 1:1000, Cell Signaling Technology, USA), anti-IKKα antibody (no. sc-7606, 1:1000, Santa Cruz Biotechnology, USA), and anti-Lamin B antibody (no. AB0054, 1:1000, Abways, China) at 4 °C overnight. Following secondary antibody incubation, immunocomplexes were detected using a Tanon 5200 chemiluminescent imaging system (Tanon Science & Technology Co., China).

Actin tracker

Actin cytoskeletal structures were visualized using TRITC-conjugated phalloidin (no. CA1610, Solarbio, China). Cells grown on coverslips at 50% confluence underwent fixation in 4% paraformaldehyde (20 min) followed by two phosphate-buffered saline (PBS) washes. Permeabilization was performed with 0.1% Triton X-100 (5 min). Subsequently, 200 µL of prepared TRITC-phalloidin working solution was applied to coverslips and incubated protected from light (30 min, room temperature). Nuclear counterstaining employed 200 µL DAPI solution (100 nM, 30 s). Samples were examined using an Olympus BX53 fluorescence microscope.

Adeno-associated virus (AAV) injection

An AAV for KIAA1429 overexpression and empty vector-carrying AAV were constructed by Genechem Co., Ltd. (Shanghai, China). The vector information was AAV9-CMV- Kiaa1429 . Intramedullary femoral injection of the viruses was utilized, and the specific steps were referred to previous studies [ 27 , 37 ]. An internal pressure syringe was inserted through the defect, and 10 μL of Kiaa1429 AAV (1 × 10 11 vg/mL) was injected directly into the bone marrow cavity of the femur by penetrating the periosteum and bone cortex. After repositioning the quadriceps–patellar complex, the medial arthrotomy was closed using 5–0 Vicryl sutures. Employing an analogous surgical approach, 10 μL of AAV-empty vector (1 × 10 11 vg/mL) was delivered via intrasynovial injection to control subjects.

MeRIP-seq and analysis

Total RNA was isolated from both transfected and wild-type RAW264.7 cells ( n = 3 biological replicates per group) using TRIzol reagent (Invitrogen no. 15596026, USA). Transcriptome profiling and m⁶A-specific RNA immunoprecipitation sequencing (MeRIP-seq) were concurrently conducted with three technical replicates for each biological sample. Polyadenylated mRNA purification employed the Arraystar Seq-Star™ isolation kit (Aksomics, China), followed by m⁶A-methylated RNA enrichment with anti-m⁶A antibody (Abcam, no. ab286164, UK). Strand-specific sequencing libraries from both m⁶A-enriched fractions and input mRNA controls were prepared using KAPA’s mRNA-SEQ kit, followed by paired-end sequencing (Illumina HiSeq 4000, LC-Bio Technologies, China).

Raw sequencing reads were aligned to the Mus musculus reference genome (GRCm39) using HISAT2 version 2.1.0 with default parameters. m⁶A peaks were identified through comparative analysis between immunoprecipitated and input samples using exomePeak2 (version 3.16) with a negative binomial model, requiring |fold change (FC)| > 1 and false discovery rate (FDR)-adjusted p < 0.05 (Benjamini–Hochberg correction). Peak distributions were visualized in the Integrative Genomics Viewer (Broad Institute, USA). Differential gene expression analysis was conducted in DESeq2 (version 1.38.3) using the Wald test, applying thresholds of |FC| > 1 and FDR-adjusted p < 0.05. Biological interpretation of differentially expressed genes and m⁶A-modified transcripts was performed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using clusterProfiler (version 4.8.1). Enrichment significance was assessed via the hypergeometric test with Benjamini–Hochberg multiple testing correction, requiring FDR < 0.05.

Dual-luciferase reporter assay

A dual luciferase reporter gene assay was used to validate that KIAA1429 directly targets Lrp4 mRNA. Luciferase vectors were constructed to contain either the Lrp4 wild-type (WT) 3′-untranslated region (UTR) or the Lrp4 mutant (MUT) 3′-UTR sequence. RAW264.7 was then co-transfected with the luciferase vector using Lipofectamine 3000 Transfection Agent (Invitrogen, USA). Subsequently, 24 h after transfection, it was analyzed using the Luciferase Reporter Detection System (Promega, USA).

Genotyping

The Ctsk -CreERT2 transgenic line was acquired from Cyagen (USA). CRISPR–Cas9 technology was used by the same provider to generate Lrp4 flox/+ mice. Intercrossing of Lrp4 flox/+ heterozygotes produced homozygous Lrp4 flox/flox animals. These were subsequently bred with Ctsk -CreERT2 mice to obtain Ctsk -CreERT2; Lrp4 flox/flox dual-transgenic progeny. Experimental groups consisted of 12-week-old female mice carrying both genotypes. To induce conditional knockout, animals received intraperitoneal tamoxifen injections (MCE, no. HY-13757A, USA; 1 mg daily for five consecutive days), generating Lrp4 conditional knockout ( Lrp4 -cKO) mice.

Genomic DNA isolation involved collection of 1 mm tail biopsies. Tissue samples were processed using the M5 Animal Tissue Direct PCR Kit (Mei5bio, no. MF280, China) following manufacturer specifications, with subsequent DNA amplification. Electrophoresis separation was performed on PCR products using 2% agarose gels prepared in 1 × Tris/acetic acid/EDTA (TAE) buffer containing GelRed (5 μL per 100 mL). Imaging and documentation employed the Bio-Rad GelDoc Go System (USA). Primer sequences are presented in Supplementary Table S3.

siRNA and plasmid transfection

Mouse Ythdc1 -siRNA and negative control (si-NC) were obtained from GenePharma Co., Ltd. (Shanghai, China). siRNA was diluted with diethyl pyrocarbonate (DEPC) water. For single wells of 6-well plates, 100 pmol siRNA and 5 μL of Lipofectamine 3000 Transfection Reagent (no. L3000075, Invitrogen, USA) were added to 250 μL of Opti-MEM I (no. 11058021, Invitrogen, USA) and thoroughly mixed, and left for 5 min at room temperature. For transfection, the medium was replaced with 1.5 mL of Opti-MEM I. After 4 h, the medium was replaced with fresh medium. Subsequent experiments were performed 48 h after transfection. Supplementary Table S4 presents the primers that were used.

The process of overexpression (OE) Lrp4 plasmid construction was referred to in the previous study [ 38 ], and the process of plasmid transfection was the same as siRNA transfection. The amount of plasmid was 4 μg, and the amount of Lipofectamine 3000 transfection reagent was 10 μL.

RNA immunoprecipitation (RIP) assay

RNA immunoprecipitation (RIP) assays were conducted per manufacturer’s instructions (Geneseed Kit, no. P0101240302, China). Concisely, transfected RAW264.7 cells were harvested and lysed in RIP-specific buffer. Magnetic beads were separately conjugated to either anti-YTHDC1 antibody (Proteintech, no. 14392–1-AP, China), anti-m⁶A antibody (Abcam, no. ab286164, UK), or control IgG. Following overnight incubation of lysates with antibody-bound beads at 4 °C, precipitated RNA complexes were isolated using TRIzol reagent (Invitrogen no. 15596026, USA). RNA enrichment levels were subsequently quantified via qRT-PCR.

Co-immunoprecipitation (Co-IP)

For Co-IP, total cellular proteins were extracted using lysis buffer, and then, the extracts were incubated with anti-LRP4 antibody (no. 56540-100UG, 1:1000, Invitrogen, USA), anti-TNFAIP3 antibody (no. 5630S, 1:1000, Cell Signaling Technology, USA), or IgG at 4 °C overnight. Then, the mixture was incubated with Protein A&G beads (no. B23201 , Selleck, Germany) at 4 °C overnight. Precipitates were washed four times with lysis buffer and analyzed by western blotting with designated antibodies.

Immunofluorescence

For immunofluorescence, after antigen extraction, each section was incubated with an anti-LRP4 antibody (no. 56540-100UG, 1:200, Invitrogen, USA) and anti-TNFAIP3 antibody (no. 5630S, 1:200, Cell Signaling Technology, USA) at 4 °C overnight. Obtained sections were incubated with secondary antibody for 1 h at room temperature. Nuclei were detected using DAPI. Images were taken under confocal microscopy (Olympus, Japan).

Nuclear translocation

To detect NF-κB-p65 subunit location, Tnfaip3 knockout RAW264.7 cells were treated with or without Lrp4 overexpression. The cytoplasmic and nuclear protein fractions were extracted with Triton X-100 and sodium dodecyl sulfate (SDS) lysis buffer separately. The following were detected by western blotting: p65, Lamin B, and GAPDH.

Statistical analysis

Statistical evaluations were implemented using GraphPad Prism (version 9.0.0; GraphPad, USA). Between-group comparisons used two-tailed unpaired Student’s t -tests. Multiple group comparisons employed one-way analysis of variance (ANOVA). Data are presented throughout as mean values ± standard deviation (SD). Statistical significance was defined at p < 0.05. Unless otherwise specified, sample sizes comprised ≥ 3 biological replicates per experimental condition, with all investigations independently replicated thrice for verification.

Results

KIAA1429 expression was decreased in osteoporotic bone tissue and correlated with osteoclast differentiation

To investigate the potential role of m 6 A methylation in osteoclast differentiation, we performed bioinformatics analysis using the public dataset GSE225974 in the GEO database. The heatmap indicated significant differences in m 6 A-related genes (Fig. 1 A). RNA-seq results indicated that KIAA1429 expression was downregulated in osteoclasts from patients with osteoporosis, and its expression was correlated negatively with that of Cathepsin K ( CTSK ) and Matrix Metallopeptidase 9 ( MMP9 ) (Supplementary Fig. S1A, B). Previous studies have proved the mechanisms by which Wt1-associated protein (WTAP), Methyltransferase 3 (METTL3), and Methyltransferase 14 (METTL14) regulate osteoclast differentiation [ 24 , 27 , 39 ]. RNA-seq analysis concurred with these results. The osteoclasts from patients with osteoporosis showed low expression of the m 6 A writer genes such as WTAP , METTL3 , and METTL14 (Supplementary Fig. S1C). However, the role of KIAA1429 in osteoclast differentiation was not elucidated. Fig. 1 KIAA1429 and m 6 A modification were decreased in osteoporotic bone tissues and were closely associated with osteoclast differentiation. A Heatmap of m 6 A regulator expression in the peripheral blood mononuclear cell (PBMC) and osteoclast (OC) from the GEO database ( GSE225974 ), with high and low expression levels shown in red and blue, respectively. B Schematic workflow of the scRNA-seq. C A UMAP analysis results displaying 18 cell clusters. D Annotation of 18 cell clusters to eight cell types. E Volcano plots of different cellular subset in sham and OVX mice in scRNA-seq. F UMAP expression distribution of specific marker genes for osteoclasts and Kiaa1429 . G Violin plot demonstrating the difference in Kiaa1429 expression in osteoclasts between sham and OVX mice. H Representative images of tartrate-resistant acid phosphatase (TRAP) staining of RAW264.7 cells on days 0, 3, and 5 during the osteoclast differentiation. I mRNA levels of osteoclast-related genes ( Ctsk , c-Fos , and Mmp-9 ) in RAW264.7 on days 0, 3, and 5 during the osteoclast differentiation were assayed by qRT-PCR analysis. J mRNA level of Kiaa1429 in RAW264.7 on days 0, 3, and 5 during the osteoclast differentiation was assayed by qRT-PCR analysis. K The m 6 A content from RAW264.7 was measured by colorimetric quantification assay on days 0, 3, and 5 during the osteoclast differentiation. L Representative images of TRAP staining in primary bone marrow-derived macrophages (BMDM) on days 0, 3, and 5 during the osteoclast differentiation. M mRNA levels of osteoclast-related genes in BMDM on days 0, 3, and 5 during the osteoclast differentiation were assayed by qRT-PCR analysis. N mRNA level of Kiaa1429 in BMDM on days 0, 3, and 5 during the osteoclast differentiation was assayed by qRT-PCR analysis. O The m 6 A content from BMDM was measured by colorimetric quantification assay on days 0, 3, and 5 during the osteoclast differentiation. P mRNA level of Kiaa1429 in bone tissues of sham and OVX mice was assayed by qRT-PCR analysis. Q The m 6 A content in bone tissues of sham and OVX mice was measured by colorimetric quantification assay. R Representative images of immunohistochemistry staining for KIAA1429 in sham and OVX mice. S The serum concentration of CTX-1 in sham and OVX mice. T Co-expression analysis of Kiaa1429 mRNA expression with CTX-1 in sham and OVX mice. U mRNA level of KIAA1429 in bone tissues of postmenopausal patients with osteoporosis and patients without osteoporosis was assayed by qRT-PCR analysis. V The m 6 A content in bone tissues of patients with osteoporosis and patients without osteoporosis was measured by colorimetric quantification assay. W The serum concentration of CTX-1 in postmenopausal patients with osteoporosis and patients without osteoporosis. X Co-expression analysis of the KIAA1429 mRNA with CTX-1 in postmenopausal patients with osteoporosis and patients without osteoporosis. Y Co-expression analysis of the KIAA1429 mRNA with L1–L4 BMD T-scores or hip BMD T-scores in postmenopausal patients with osteoporosis and patients without osteoporosis. The values are mean ± SD of at least three independent experiments; n.s. not significant; n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

We successfully established OVX mice for in vivo experiments. Micro-computed tomography (micro CT) analysis showed a decrease in trabecular bone volume/total volume (Tb. BV/TV), trabecular thickness (Tb. Th), trabeculae number (Tb. No.), and bone mineral density (BMD), and an increase in trabecular separation (Tb. Sp) (Supplementary Fig. S1D). Hematoxylin and eosin (H&E) and Masson staining indicated trabecular bone loss in OVX mice (Supplementary Fig. S1E–G). TRAP staining demonstrated a substantial upregulation of bone resorption in OVX mice (Supplementary Fig. S1H).

Bone specimens from sham and OVX mice were selected for single-cell RNA sequencing analysis (Fig. 1 B). Computational processing utilized the Seurat R package, where quality-controlled and normalized data revealed cellular RNA distributions (Supplementary Fig. S1I). A strong correlation ( r = 0.90) between nCount_RNA and nFeature_RNA indicated adequate post-filtering cellular integrity (Supplementary Fig. S1J). From 4370 filtered cells, we identified genes exhibiting substantial expression variation across osteoporotic bone tissue. The FindVariableFeatures function isolated the top 3000 variably expressed transcripts for subsequent analysis, confirming numerous dynamic genes in OVX specimens (Supplementary Fig. S1K).

Principal component analysis (PCA) reduced dimensionality prior to UMAP visualization, yielding 40 significant principal components (Supplementary Fig. S1L). Uniform Manifold Approximation and Projection (UMAP) resolved 18 transcriptionally distinct clusters (Fig. 1 C), with sample-specific distributions shown in Supplementary Fig. S1M. Cluster identities were annotated using CellMarker database references. The top three differentially expressed markers per cluster are shown in Supplementary Fig. S1N, with annotated gene expression patterns in Supplementary Fig. S1O. These clusters comprised eight cellular populations: monocytes, B cells, macrophages, osteoblasts, bone marrow mesenchymal stem cells (BMMSCs), osteoclasts, erythroid cells, and neutrophils (Fig. 1 D).

Differential expression analysis demonstrated significantly reduced Kiaa1429 expression specifically within osteoclast subpopulations (Fig. 1 E). Canonical osteoclast markers ( Tcf7 , Acp5 , and Nfatc1 ) confirmed cluster identity, with Kiaa1429 co-expression observed in these cells (Fig. 1 F). Notably, Kiaa1429 levels were substantially diminished in osteoclasts from OVX mice (Fig. 1 G).

RAW264.7 cells underwent osteoclastogenic induction. Tartrate-resistant acid phosphatase (TRAP) staining confirmed successful differentiation by days 3 and 5 (Fig. 1 H). Transcript levels of osteoclast-related genes ( Ctsk , c-Fos , and Mmp9 ) increased during differentiation, while Kiaa1429 , Wtap , Mettl3 , and Mettl14 mRNA levels decreased (Fig. 1 I–J and P). Concomitant reduction in total RNA m⁶A methylation was observed at both time points (Fig. 1 K). These expression patterns and m⁶A changes were recapitulated in primary bone marrow-derived macrophages (BMDMs) (Fig. 1 L–O; Supplementary Fig. S1Q).

Furthermore, we verified the expression of Kiaa1429 by in vivo experiments. The mRNA level of Kiaa1429 and the m 6 A level in total RNA were significantly low in the bone tissues of OVX mice (Fig. 1 P, Q ). In addition, immunohistochemistry staining demonstrated a significant reduction in KIAA1429 protein expression in the bone tissue of OVX mice (Fig. 1 R). As shown in Fig. 1 S–T, the C-terminal telopeptide of type I collagen (CTX-1) level increased in OVX mice serum, and the Kiaa1429 mRNA level in the bone tissues of the mice correlated negatively with serum CTX-1 content.

On the basis of bone mineral density (BMD) measured by dual-energy X-ray absorptiometry (DXA), we collected 20 bone tissue samples and serum from patients with and without osteoporosis, respectively, and then summarized the patients’ baseline information. The non-OP group had a mean age of 61.1 ± 9.00 years, and the OP group had a mean age of 65.3 ± 8.63 years. There was no significant difference between groups ( p = 0.301), indicating comparable age distributions. Both groups had a similar mean height: 163 ± 6.27 cm (non-OP) and 163 ± 4.03 cm (OP) ( P = 0.933). Mean weight was 59.3 ± 9.67 kg (non-OP) and 62.6 ± 8.13 kg (OP), with no significant difference ( p = 0.420). The body mass index was 21.6 ± 2.65 kg/m 2 (non-OP) and 22.5 ± 3.55 kg/m 2 (OP), and the difference was not statistically significant ( p = 0.504). The non-OP group had a relatively normal L1–L4 BMD T-score (−0.55 ± 0.24), while the OP group exhibited severe osteoporosis (−3.68 ± 0.77). The difference was highly significant ( p < 0.001). Similarly, the non-OP group had a hip BMD T-score of −0.85 ± 0.26, whereas the OP group had a much lower T-score (−3.67 ± 0.73), with a highly significant difference ( p < 0.001) (Supplementary Table S5). The KIAA1429 mRNA expression and the m 6 A level in total RNA were significantly reduced in the bone tissues of patients with osteoporosis (Fig. 1 U, V). In addition, CTX-1 levels increased in the serum of patients with osteoporosis, and the KIAA1429 mRNA level in the bone tissues of patients with osteoporosis correlated negatively with serum CTX-1 content (Fig. 1 W, X). Meanwhile, the results demonstrated significant positive correlations between KIAA1429 expression and both L1–L4 BMD T-scores ( p < 0.0001, r = 0.6207) and hip BMD T-scores ( p < 0.0001, r = 0.5746) (Fig. 1 Y). In summary, KIAA1429 expression decreased during osteoclast differentiation and in osteoporotic bone tissue.

KIAA1429 regulated osteoclast differentiation

We efficiently established Kiaa1429 knockout RAW264.7 cells by employing the CRISPR–Cas9 technique, and then we selected knockout (KO) KIAA1429-2 RAW264.7 cells for subsequent functional experiments (Fig. 2 A–C). To address whether KIAA1429 knockout affects cell proliferation and viability in RAW264.7 cells, we performed CCK-8 assays under standardized conditions (0, 24, 48, and 72 h post seeding). The results revealed no statistically differences in cell viability between the KIAA1429 knockout group and vector group at any time point (Supplementary Fig. S2A). The relative m 6 A content decreased after Kiaa1429 knockout (Fig. 2 D). As shown in Supplementary Fig. S2B, Kiaa1429 knockout enhanced the mRNA levels of osteoclast-related genes, including c-Fos , Ctsk , Mmp9 , and Nuclear factor of activated T cell cytoplasmic 1 ( Nfatc1 ). The protein levels of NFATC1, CTSK, and C-FOS indicated the same trend (Fig. 2 E). TRAP and fibrous actin (F-actin) staining demonstrated the promotional effect of Kiaa1429 knockout on osteoclast differentiation (Fig. 2 F, G). Fig. 2 KIAA1429 knockout promoted osteoclast differentiation and KIAA1429 overexpression inhibited osteoclast differentiation in vitro. A Workflow of generating Kiaa1429 knockout RAW264.7 cells using CRISPR/Cas9 technology and subsequent osteoclast differentiation related-tests. B , C KIAA1429 expression in vector and Kiaa1429 knockout RAW264.7 cells, measured by qRT-PCR and western blotting. D The m 6 A content was measured in Kiaa1429 knockout cells. E Immunoblotting of osteoclast-related proteins (NFATC1, CTSK, and C-FOS) in control and Kiaa1429 knockout cells with or without RANKL treatment. F , G Representative images of TRAP staining and F-actin staining in Kiaa1429 knockout cells. H Workflow of osteoclast differentiation related-tests in control and Kiaa1429 -overexpressed cell. I , J KIAA1429 expression in Kiaa1429 -overexpressed RAW264.7 cells, measured by qRT-PCR and western blotting. K The m 6 A content in Kiaa1429 -overexpressed cells was measured by colorimetric quantification assay. L Immunoblotting of osteoclast-related proteins (NFATC1, CTSK, and C-FOS) in control and Kiaa1429 -overexpressed cells with or without RANKL treatment. ( M , N ) Representative images of TRAP staining and F-actin staining in Kiaa1429 -overexpressed cells. The values are mean ± SD of at least three independent experiments; n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

In addition, we performed a series of tests using Kiaa1429 -overexpressed cells to determine the role of KIAA1429 in osteoclast differentiation (Fig. 2 H). qRT-PCR and western blotting results confirmed the significant elevation of KIAA1429 expression after Kiaa1429 overexpression (Fig. 2I , J). The relative m 6 A content increased after Kiaa1429 overexpression owing to its methylase function (Fig. 2 K). As shown in Supplementary Fig. S2C, Kiaa1429 overexpression suppressed the mRNA expression of osteoclast-related genes. The levels of osteoclast differentiation-related proteins revealed the same trends (Fig. 2 L). TRAP and F-actin staining demonstrated the inhibitory effect of Kiaa1429 overexpression on osteoclast differentiation (Fig. 2 M, N). Thus, the preliminary results indicated that KIAA1429 inhibited osteoclast differentiation.

We investigated the role of KIAA1429 in osteogenic differentiation by inducing BMMSC differentiation toward osteoblasts. The results of Alizarin Red S (ARS) and alkaline phosphatase (ALP) staining confirmed the successful induction of BMMSC osteogenic differentiation (Supplementary Fig. S3A). Then, we detected the changes of mRNA expression on different days, which showed that the mRNA expressions of osteogenic differentiation-related genes, including Runt-related transcription factor 2 ( Runx2 ), Collagen type I ( Col1) , and Osteocalcin ( Ocn ), increased in proportion to the duration of osteogenic induction, whereas Kiaa1429 expression showed no apparent difference (Supplementary Fig. S3B). Therefore, we focused on the biological functions of KIAA1429 during osteoclast differentiation in this study. In addition, we knocked out Kiaa1429 in BMMSCs to further validate the effect of KIAA1429 on osteogenic differentiation. We efficiently established Kiaa1429 knockout BMMSCs by employing the CRISPR–Cas9 technique and selected KO KIAA1429-2 BMMSCs cells for subsequent functional experiments (Supplementary Fig. S3C, D). ARS and ALP staining proved that KIAA1429 knockout did not alter the osteogenic capacity of BMMSCs during osteogenic differentiation (Supplementary Fig. S3E, F). Then, the mRNA expressions of osteogenic differentiation-related genes, including Runx2 , Col1 , and Ocn , were also not altered by KIAA1429 knockout during osteogenic differentiation (Supplementary Fig. S3G). The above results confirmed that KIAA1429 did not change the fate of osteogenic differentiation in BMMSCs.

KIAA1429 overexpression inhibited bone resorption in OVX mice

To determine whether KIAA1429 directly regulates m 6 A-mediated methylation during bone resorption, we constructed an adeno-associated virus (AAV) delivery system to overexpress Kiaa1429 by injection into the femoral bone marrow cavity through the periosteum and bone cortex (Fig. 3 A). Micro-CT analysis showed significantly higher bone mass (higher Tb. BV/TV) and upregulated trabecular bone structure (higher Tb. No., Tb. Th, and BMD; lower Tb. Sp) in the Kiaa1429 -overexpressed group compared with that in the control group (Fig. 3 B, C). H&E and Masson staining demonstrated that the trabecular bone area was significantly increased in the Kiaa1429 overexpression group (Fig. 3 D, E). To further test the effect of Kiaa1429 overexpression on bone resorption in vivo, we performed TRAP staining and immunohistochemistry staining, which showed that bone resorption was significantly reduced in the Kiaa1429 overexpression group (Fig. 3 F, G ). Taken together, these results suggested that Kiaa1429 overexpression could inhibit bone resorption, thereby restoring bone loss in vivo. Fig. 3 Kiaa1429 overexpression inhibited bone resorption in vivo. A Flowchart of the AAV injection experiment into the bone marrow cavity. B Representative micro-CT images of trabecular bone from the femoral metaphysis. C Micro-CT measurements of Tb. BV/TV; Tb. Th; Tb. No.; Tb. Sp; and BMD in the femurs after treatment with overexpressed negative control (AAV-NC) and overexpressed Kiaa1429 adeno-associated virus (AAV-KIAA1429). D Representative images of H&E staining in AAV-NC and AAV-KIAA1429 groups. E Representative images of Masson staining in AAV-NC and AAV-KIAA1429 groups. F Representative images of TRAP staining in AAV-NC and AAV-KIAA1429 groups. G Representative images of immunohistochemistry staining for CTSK in AAV-NC and AAV-KIAA1429 groups. The values are mean ± SD of at least three independent experiments; n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

To further explore the effectiveness of this delivery system in vivo, we obtained BMDM from the femurs of mice (Supplementary Fig. S4A). KIAA1429 expression was significantly upregulated in the BMDM from the Kiaa1429 overexpression group, and the m 6 A abundance in total RNA was also increased (Supplementary Fig. S4B–D). The transcript levels of osteoclast-related genes in the Kiaa1429 overexpression group were downregulated, and the protein levels revealed the same trend (Supplementary Fig. S4E, F). TRAP staining and F-actin staining demonstrated decreased differentiation ability of osteoclasts in the Kiaa1429 -overexpressed group (Supplementary Fig. S4G, H). Collectively, we successfully constructed an AAV delivery system that overexpressed Kiaa1429 and observed that KIAA1429 suppressed BMDM differentiation into osteoclasts.

LRP4 was identified as a downstream target of KIAA1429-mediated m 6 A modification

To dissect the mechanism underlying KIAA1429 in osteoclast differentiation, we performed RNA-seq and MeRIP-seq on Kiaa1429 -overexpressed RAW264.7 cells. Consistent with the finding of previous studies [ 30 ], MeRIP-seq results showed that the “GGAC” sequence motif was highly enriched in m 6 A immunoprecipitated RNA (Fig. 4 A). In addition, pie charts and metagenetic analysis of m 6 A peak distribution indicated that prevalence of m 6 A peaks in the coding sequences (CDSs) and 3′ untranslated regions (UTRs) of mRNAs, with highest enrichment around the 3′-UTR (Fig. 4 B, C ). Next, we analyzed the differentially expressed genes (DEGs) using MeRIP-seq and RNA-seq, in which the number of differentially expressed genes found in RNA-seq was 2526, and in MeRIP-seq, it was 1532, finding remarkable associations between KIAA1429 expressions and 239 genes (Fig. 4 D, E). Subsequently, we performed gene ontology (GO) enrichment analysis for the 239 candidate genes and found significant differences in the pathway “positive regulation of peptidyl-tyrosine phosphorylation” (Fig. 4 F). We screened the key genes by validating the transcript levels of the related genes in the pathway. We found that the Lrp4 mRNA level was significantly high in Kiaa1429 -overexpressed cells and showed the most pronounced difference (Fig. 4 G). Meanwhile, the RAW264.7 cells and BMDM that were induced to differentiate toward osteoclasts showed notably reduced Lrp4 mRNA expression in osteoclasts (Fig. 4 H). Immunohistochemical staining results showed pronounced reduction in LRP4 protein expression in the femoral tissues of OVX mice compared with that of the sham group (Fig. 4I ). Fig. 4 LRP4 was identified as a downstream target of KIAA1429-mediated m 6 A modification in osteoclast differentiation. A HOMER with MeRIP-seq identified the top consensus motif of m 6 A peaks in RAW264.7 with or without Kiaa1429 overexpression. B , C The percentage of total m 6 A peak distribution in the specified regions in RAW264.7 cells with OE NC and KIAA1429 overexpression. D Volcano plot of RNA-seq and MeRIP-seq in OE NC and Kiaa1429 -overexpressed cells. E Venn diagram exhibiting overlap among differentially expressed genes (DEGs) on the basis of MeRIP-seq and RNA-seq. F Gene ontology (GO) enrichment analysis revealed high enrichment of the positive regulation of peptidyl-tyrosine phosphorylation pathway on the basis of RNA-seq and MeRIP-seq. G mRNA expression of DEGs upon Kiaa1429 overexpression, measured by qRT-PCR. H mRNA level of Lrp4 in RAW264.7 and BMDM during osteoclast differentiation, measured by qRT-PCR. I Representative images of immunohistochemistry staining for LRP4 in sham and OVX mice and quantitative analysis. J MeRIP-qPCR demonstrated the direct binding between m 6 A and Lrp4 mRNA. K Visualization of m 6 A peak in Lrp4 transcripts based on MeRIP-seq. L Construction of a luciferase reporter containing wild-type (WT) Lrp4 3′-UTR and mutation (Mut) Lrp4 3′-UTR at the m 6 A consensus sequence. M Luciferase activity in Kiaa1429 knockout or Kiaa1429 -overexpressed RAW264.7 cells. The values are mean ± SD of at least three independent experiments; n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

MeRIP-qPCR analysis against potential m 6 A site-specific primers showed that Kiaa1429 overexpression increased the m 6 A modification of Lrp4 mRNA while Kiaa1429 knockout downregulated the m 6 A modification of Lrp4 mRNA (Fig. 4 J). MeRIP-seq results showed that Kiaa1429 overexpression upregulated the m 6 A peak of the 3′-UTR segment of the Lrp4 gene (Fig. 4 K). Hence, we hypothesized that KIAA1429 directly regulates the m 6 A modification in 3′-UTR of Lrp4 mRNA, thereby promoting its expression. Subsequently, we constructed luciferase reporter genes containing wild-type or mutant 3′-UTRs by replacing the adenosine (A) in the mutant reporter gene with thymine (T) to eliminate the m 6 A modification (Fig. 4 L). Upon Kiaa1429 overexpression, the luciferase reporter gene containing the wild-type 3′-UTR exhibited a pronounced increase in luciferase activity, whereas mutation of the m 6 A site eliminated this enhancement (Fig. 4 M). These findings supported the hypothesis that LRP4 may be a direct downstream target of KIAA1429, and that the 3′-UTR of Lrp4 was the site of KIAA1429-mediated m 6 A targeting.

KIAA1429-mediated LRP4 inhibited osteoclast differentiation in vitro

To further characterize the effects of LRP4 on osteoclast differentiation, we performed a series of restoration assays. First, we transfected Kiaa1429 knockout cells with Lrp4 plasmid. qRT-PCR and western blotting results showed that the LRP4 expression increased notably after transfection (Fig. 5 A, B). Then, functional assays were performed to assess the role of LRP4 in Kiaa1429 -mediated osteoclast differentiation. LRP4 upregulation suppressed the promotion of osteoclastic factors by Kiaa1429 knockout (Fig. 5 C, D). TRAP staining and F-actin staining demonstrated that LRP4 upregulation reversed the increased positivity of osteoclasts induced by KIAA142 knockout (Fig. 5 E, F). Fig. 5 LRP4 overexpression rescued the osteoclast differentiation induced by Kiaa1429 knockout, and LRP4 knockout reversed the osteoclast differentiation induced by Kiaa1429 overexpression. A , B LRP4 expression in Lrp4 -overexpressed RAW264.7 cells, assayed by qRT-PCR and western blotting. C mRNA expressions of Ctsk , C-fos , Nfatc1 , and Mmp9 in Kiaa1429 knockout cells with or without Lrp4 overexpression after RANKL treatment. D Immunoblotting of osteoclast-related proteins (NFATC1, CTSK, and C-FOS) in Kiaa1429 knockout cells with or without Lrp4 overexpression after RANKL treatment. The vector group was used as the control group for KO-KIAA1429, and NC group was used as the control group for OE-LRP4. ( E , F ) TRAP staining and F-actin in Kiaa1429 knockout cells with or without Lrp4 overexpression after RANKL treatment. G , H qRT-PCR and western blotting confirmation of Lrp4 knockout in RAW264.7 cells. I , J The effect of Lrp4 knockout on osteoclast differentiation was measured by qRT-PCR and western blotting after Kiaa1429 overexpression. K , L TRAP staining and F-actin in Lrp4 knockout cells with or without Kiaa1429 overexpression after RANKL treatment. The values are mean ± SD of at least three independent experiments; n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

To further confirm the role of LRP4 in KIAA1429-mediated osteoclast differentiation, we generated Lrp4 knockout RAW264.7 cells and selected KO LRP4-2 cells for subsequent functional assays (Fig. 5 G, H). Similarly, we transfected Kiaa1429 -overexpressed cells with KO LRP4-2 and found that Lrp4 knockout increased osteoclast differentiation and reinstated the inhibitory effect of osteoclastic factors at the protein and mRNA levels in the Kiaa1429 overexpression group (Fig. 5I , J). TRAP staining and F-actin staining demonstrated that Lrp4 knockout reversed the decreased positivity of osteoclasts caused by Kiaa1429 overexpression (Fig. 5 K–L). These findings suggested that LRP4 altered KIAA1429-mediated osteoclast differentiation, and LRP4 was a downstream target of KIAA1429 in regulating osteoclast differentiation.

KIAA1429-mediated LRP4-regulated osteoclast differentiation and bone resorption in vivo

The above in vivo experiments confirmed that Kiaa1429 overexpression inhibited bone resorption and rescued bone loss in OVX mice. Considering that Lrp4 was a downstream target gene of KIAA1429-mediated m 6 A methylation modification, we constructed osteoclast-specific Lrp4 -deficient mice to further validate the regulation of KIAA1429 in LRP4 during osteoclast differentiation (Fig. 6 A). We obtained conditional Lrp4 knockout mice ( Ctsk -CreERT2; Lrp4 fl/fl ) by breeding Lrp4 fl/fl mice with Ctsk -CreERT2 mice, and PCR was implemented to confirm that conditional Lrp4 knockout mice could specifically delete Lrp4 from osteoclasts in the bone marrow cavity (Supplementary Fig. S5A–E). Fig. 6 Kiaa1429 overexpression could not rescue bone loss by LRP4 deficiency in vivo. A Flowchart of the AAV injection experiment into the bone marrow cavity in Ctsk -CreERT2; Lrp4 fl/fl OVX mice and Lrp4 fl/fl OVX mice. B Representative micro-CT images of trabecular bone from the femoral metaphysis in Ctsk -CreERT2; Lrp4 fl/fl OVX mice and those of other groups. C Micro-CT measurements of Tb. BV/TV; Tb. Th; Tb. No.; Tb. Sp; and BMD in the femurs of Ctsk -CreERT2; Lrp4 fl/fl OVX mice after treatment with AAV-NC or AAV-KIAA1429. D Representative images of H&E staining and quantitative analysis in Ctsk -CreERT2; Lrp4 fl/fl OVX mice and Lrp4 fl/fl OVX mice after treatment with AAV-NC or AAV-KIAA1429. E Representative images of Masson staining and quantitative analysis in Ctsk -CreERT2; Lrp4 fl/fl OVX mice and Lrp4 fl/fl OVX mice after treatment with AAV-NC or AAV-KIAA1429. F Representative images of TRAP staining and quantitative analysis in Ctsk -CreERT2; Lrp4 fl/fl OVX mice and Lrp4 fl/fl OVX mice after treatment with AAV-NC or AAV-KIAA1429. G Representative images of immunohistochemistry staining for CTSK and quantitative analysis in Ctsk -CreERT2; Lrp4 fl/fl OVX mice, and Lrp4 fl/fl OVX mice after treatment with AAV-NC or AAV-KIAA1429. The values are mean ± SD of at least three independent experiments; n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Next, the adeno-associated virus (AAV) delivery system of overexpressing Kiaa1429 was used to treat Ctsk -CreERT2; Lrp4 fl/fl OVX mice. Micro-CT analysis demonstrated significantly lower trabecular bone volume (lower Tb. BV/TV) and impaired trabecular structure (lower Tb. Th, Tb. No., and BMD, and higher Tb. Sp) in the Ctsk -CreERT2; Lrp4 fl/fl OVX mice compared with Lrp4 fl/fl OVX mice, whereas Kiaa1429 overexpression could not reverse the enhancement of bone resorption in the Ctsk -CreERT2; Lrp4 fl/fl OVX mice (Fig. 6 B, C). H&E and Masson staining also revealed that Kiaa1429 overexpression could not restore the loss of trabecular bone area in the Ctsk -CreERT2; Lrp4 fl/fl OVX mice group (Fig. 6 D, E). TRAP staining and immunohistochemical staining were performed to examine the changes in bone resorption capacity. TRAP staining showed much higher osteoclast positivity in femoral sections of Ctsk -CreERT2; Lrp4 fl/fl OVX mice in those of Lrp4 fl/fl OVX mice, whereas Kiaa1429 overexpression failed to reverse the enhancement of bone resorption (Fig. 6 F). Immunohistochemical staining results demonstrated increased positivity for the osteoclast differentiation-associated protein CTSK in the Ctsk -CreERT2; Lrp4 fl/fl OVX mice; Kiaa1429 overexpression did not reverse this phenomenon (Fig. 6 G). Briefly, these findings indicated that Lrp4 knockout in osteoclasts attenuated the inhibitory effect of Kiaa1429 overexpression on bone resorption in OVX mice, further suggesting that Lrp4 was a target gene of KIAA1429 for the regulation of osteoclast differentiation.

KIAA1429 improved Lrp4 mRNA stability through a YTHDC1 dependent mechanism

m 6 A readers are thought to be critical for the regulation of gene expression [ 27 ]. To determine the mechanism underlying the regulation of Lrp4 mRNA expression by KIAA1429-mediated m 6 A, we analyzed the interactions between Lrp4 and m 6 A-binding proteins, including YTH structural domain family (YTHDF) proteins, IGF2BP and HNRNP proteins. RNA-seq results indicated that Ythdc1 expression was significantly increased in Kiaa1429 -overexpressed cells (Fig. 7 A). In addition, qRT-PCR assay results also demonstrated the most significant increase in Ythdc1 expression in the Kiaa1429 overexpression group (Fig. 7 B). RNA immunoprecipitation (RIP) analysis using YTHDC1 antibody demonstrated that YTHDC1 bound to Lrp4 mRNA, and that Kiaa1429 overexpression resulted in increased binding, while the amount of binding was decreased by Kiaa1429 deletion (Fig. 7 C). We transfected the cells with Ythdc1 siRNA to attenuate its intracellular expression. Owing to its highest knockdown efficiency, siYTHDC1-2 was used for subsequent functional experiments (Fig. 7 D, E). To further verify whether KIAA1429-mediated LRP4 is dependent on reader YTHDC1 to regulate osteoclast differentiation, we performed a series of restoration assays. We transfected Kiaa1429 -overexpressed cells with si-YTHDC1 and found that Ythdc1 knockdown reversed the promotion effect of Kiaa1429 overexpression on LRP4 at the mRNA levels (Fig. 7 F–H). In addition, Ythdc1 knockdown reduced the inhibitory effect of KIAA1429 overexpression on osteoclastic differentiation (Fig. 7 I–K). Overall, these results suggested that KIAA1429-mediated m 6 A modification increased Lrp4 mRNA stability during osteoclast differentiation and enhanced its expression in a YTHDC1-dependent manner (Fig. 7 L). Fig. 7 KIAA1429 improved Lrp4 mRNA stability through a YTHDC1-mediated methylation recognition. A Heatmap of m 6 A readers expression in RNA-seq, with high and low expression levels shown in red and blue, respectively. B qRT-PCR was used to determine the alterations in mRNA expressions of m 6 A readers upon Kiaa1429 overexpression. C Validation of direct binding between YTHDC1 and Lrp4 mRNA was performed by RIP-qPCR. D , E qRT-PCR and western blotting confirmation of Ythdc1 knockdown RAW264.7 cells. ( F , G , H ) LRP4 expression was measured by qRT-PCR and western blotting in Kiaa1429 overexpression cells after treatment with si-YTHDC1. (I) mRNA expressions of Ctsk , C-fos , Nfatc1 and Mmp9 in Kiaa1429 overexpression cells after treatment with si- YTHDC J , K Representative images of TRAP and F-actin staining and quantitative analysis in Kiaa1429 overexpression cells after treatment with si- YTHDC1 . L Flow chart of the increased stability of Lrp4 mRNA in a YTHDC1-dependent manner. The values are mean ± SD of at least three independent experiments; n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

KIAA1429-mediated LRP4-regulated TNFAIP3 in osteoclasts

Previous studies have shown that the Wnt signaling pathway has become one of the most critical signaling axes in bone biology, and it plays an important regulatory role, especially in osteogenic differentiation [ 40 , 41 ]. In addition, at the neuromuscular junction (NMJ), the regulation of the Wnt signaling pathway by LRP4 depends on ligand activation, which induces conformational changes in LRP4 and affects the transduction of the Wnt signaling pathway [ 42 , 43 ]. In this study, we explored the effect of KIAA1429-mediated LRP4 on the Wnt signaling pathway in precursor cells. We detected the mRNA expression of key components of the Wnt signaling pathway ( Gsk-3β , β-Catenin , and Axin2 ) in precursor cells via qRT-PCR, and the results showed that KIAA1429-mediated LRP4 did not cause changes in the Wnt signaling pathway (Supplementary Fig. S6A).

Next, we analyzed the data from RNA-seq and MeRIP-seq to gain mechanistic insight into how KIAA1429-mediated LRP4 functioned in osteoclast differentiation. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated remarkable enrichment of DEGs in the TNFα signaling pathway (Fig. 8 A), which was known to be involved in the regulation of inflammatory responses. Furthermore, increasing evidence suggested that it also played a key role in the regulation of osteoclast differentiation [ 44 ]. We performed qRT-PCR to detect the genes enrichment in the TNFα pathway, showing that the mRNA level of tumor necrosis factor alpha-induced protein 3 ( Tnfaip3 ) was significantly upregulated in Kiaa1429 overexpression cells (Fig. 8 B). Similarly, a notable upregulation in TNFAIP3 protein levels was observed in Kiaa1429 -overexpressed cells (Fig. 8 C). Fig. 8 KIAA1429-mediated LRP4-regulated TNFAIP3 in osteoclasts. A Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis of signaling pathway based on RNA-seq and MeRIP-seq. B qRT-PCR was used to determine the alterations in mRNA expression of DEGs upon Kiaa1429 overexpression. C Western blotting was performed for TNFAIP3 in Kiaa1429 -overexpressed RAW264.7 cells. D , E qRT-PCR and western blotting were performed for TNFAIP3 in Lrp4 -overexpressed cells. F Representative images of immunohistochemistry staining for TNFAIP3 and quantitative analysis in Ctsk -CreERT2; Lrp4 fl/fl and Lrp4 fl/fl OVX mice. G , H TNFAIP3 was measured by qRT-PCR and western blotting in RAW264.7 and BMDM during osteoclast differentiation. I , J The effect of Lrp4 knockout on TNFAIP3 expression was measured by qRT-PCR and western blotting after Kiaa1429 overexpression. K , L , M The effect of Lrp4 overexpression on TNFAIP3 expression was measured by qRT-PCR and western blotting after Kiaa1429 knockout. N Co-immunoprecipitation of TNFAIP3 with endogenous LRP4 in RAW264.7 and BMDM. O Co-localization of LRP4 and TNFAIP3 after treatment with or without RANKL in RAW264.7 cells was exhibited in confocal immunofluorescent images. The values are mean ± SD of at least three independent experiments; n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

To determine whether KIAA1429 acted as a regulator to TNFAIP3 by modulating LRP4, we performed a series of restoration assays. The results showed a marked increase in Tnfaip3 mRNA and protein levels in Lrp4 overexpression group (Fig. 8 D, E). Similarly, protein positivity of TNFAIP3 was significantly decreased in bone tissues of Ctsk -CreERT2; Lrp4 fl/fl OVX mice compared with Lrp4 fl/fl OVX mice (Fig. 8 F). To investigate the expression of TNFAIP3 during osteoclast differentiation, we induced osteoclast differentiation in both RAW264.7 and BMDM cells then detected the expression of TNFAIP3, and the results revealed an obvious decrease in TNFAIP3 mRNA and protein expression during osteoclast differentiation (Fig. 8 G, H). Therefore, we knocked out Lrp4 in Kiaa1429 -overexpressed cells to further validate the relationship between KIAA1429, LRP4, and TNFAIP3. Elevated protein and mRNA levels of Tnfaip3 after Kiaa1429 overexpression were reversed by Lrp4 knockout (Fig. 8I , J). Meanwhile, we overexpressed Lrp4 in Kiaa1429 knockout cells and found that Lrp4 overexpression rescued TNFAIP3 protein and mRNA levels suppressed by Kiaa1429 knockout (Fig. 8 K–M). The physical interaction between endogenous LRP4 and TNFAIP3 was determined by co-immunoprecipitation (co-IP), and the results revealed that TNFAIP3 could be detected in immunoprecipitation complexes specifically bound to LRP4 antibody in RAW264.7 and BMDM (Fig. 8 N). In addition, the result of confocal imaging demonstrated a great downregulation in the binding capacity of LRP4 to TNFAIP3 after treatment with RANKL in RAW264.7 cells (Fig. 8 O). The above results suggested that KIAA1429-mediated LRP4 is involved in the regulation of osteoclast differentiation through interaction with TNFAIP3.

KIAA1429/LRP4-mediated TNFAIP3-inhibited osteoclast differentiation through inhibition of NF-κB pathway activity

To further determine the effect of TNFAIP3 on osteoclast differentiation, we efficiently deleted the Tnfaip3 gene using the CRISPR–Cas9 genome editing strategy and selected KO TNFAIP3-2 RAW264.7 cells to perform a series of restoration assays (Fig. 9 A, B). We overexpressed Lrp4 in Tnfaip3 knockout cells and found that the protein and mRNA levels of osteoclast differentiation-related genes were downregulated upon Lrp4 overexpression, but these levels were reversed by Tnfaip3 knockout (Fig. 9 C, D). Furthermore, TRAP and F-actin staining revealed that Tnfaip3 knockout rescued the decreased positivity of osteoclasts caused by Lrp4 overexpression (Fig. 9 E, F). These findings demonstrated that TNFAIP3 was a downstream target of LRP4 in the regulation of osteoclast differentiation. Fig. 9 KIAA1429/LRP4-mediated TNFAIP3 inhibited osteoclast differentiation by activating the NF-κB pathway. A , B TNFAIP3 expression was measured by qRT-PCR and western blotting in Tnfaip3 knockout RAW264.7 cells. C mRNA expressions of Ctsk , C-fos , Nfatc1 , and Mmp9 in Tnfaip3 knockout cells with or without Lrp4 overexpression after RANKL treatment. D Immunoblotting of osteoclast-related proteins (NFATC1, CTSK, and C-FOS) in Tnfaip3 knockout cells with or without Lrp4 overexpression after RANKL treatment. E , F TRAP staining and fibrous actin (F-actin) staining in Tnfaip3 knockout cells with or without Lrp4 overexpression after RANKL treatment. G , H The activation of the NF-κB pathway (Phospho-p65, p65, Phospho-lkBα, lkBa, Phospho-IKKα/β, and IKKα) was measured by western blotting in Tnfaip3 knockout RAW264.7 cells with or without Lrp4 overexpression. I Expression of p65 in cytoplasmic or nuclear extracts was detected using western blotting in Tnfaip3 knockout RAW264.7 cells with or without Lrp4 overexpression. GAPDH and Lamin B were shown as cytoplasmic and nuclear internal controls, respectively. The values are mean ± SD of at least three independent experiments; n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Previous studies have proved that the NF-κB signaling pathway significantly promoted osteoclast differentiation [ 45 ]. TNFAIP3, which encodes A20, is a cytoplasmic ubiquitin ligase and a master negative regulator of the NF-κB signaling pathway, which inhibits NF-κB pathway activity [ 46 ]. However, the regulatory role of TNFAIP3 in osteoclast differentiation is unclear. Similarly, our previous study showed that activation of LRP4 inhibited NF-κB pathway activity [ 38 ]. To explore the relationship between LRP4 regulation of TNFAIP3 and NF-κB pathway, we overexpressed Lrp4 in Tnfaip3 knockout cells and detected NF-κB pathway activity. The results indicated that Tnfaip3 deletion reversed the inhibition of phospho-p65, phospho-IκBα, and phospho-IKKα/β caused by Lrp4 overexpression (Fig. 9 G, H). As shown in Fig. 9I , Tnfaip3 knockout reversed the reduction in the translocation of ground p65 from the cytoplasm to the nucleus, which was induced by Lrp4 overexpression. The above results suggested that KIAA1429-mediated m 6 A methylation inhibited osteoclast differentiation by regulating LRP4, thereby affecting TNFAIP3 recruitment, which results in the inhibition of NF-κB pathway activation.

Discussion

The maintenance of bone homeostasis includes osteoclast-dominated bone resorption and osteoblast-dominated osteogenesis, which are in dynamic balance to maintain a constant bone mass and normal biological function of the skeleton [ 16 , 47 ]. Osteoclasts are formed by the differentiation of monocytes and macrophage-like precursor cells of bone marrow hematopoietic stem cells [ 48 – 50 ]. Hyperdifferentiation of precursor cells to osteoclasts disrupts the equilibrium of bone metabolism, induces bone destruction, and leads to the development of OP [ 51 , 52 ]. Postmenopausal OP is a bone disease caused by decreasing estrogen levels, in which enhanced osteoclast activity and increased bone resorption are key factors [ 19 , 53 ]. Thus, osteoclast hyperfunction remains a critical element in the development of OP. The current study reports for the first time the regulatory mechanism of KIAA1429-mediated m 6 A modification on osteoclast differentiation and postmenopausal OP.

Our study carries significance from several perspectives: Firstly, KIAA1429 expression was found to be downregulated in bone tissue and osteoclasts in postmenopausal OP. Secondly, KIAA1429-mediated m 6 A modification was found to affect osteoclast differentiation by regulating LRP4 expression. Thirdly, KIAA1429-mediated enhancement of LRP4 was found to be regulated in a YTHDC1-dependent manner. Finally, KIAA1429-mediated LRP4 was identified to affect osteoclast differentiation by regulating the TNFAIP3/NF-κB signaling axis, thereby ameliorating OP. These findings suggested that the molecular pathway involved in KIAA1429-mediated m 6 A modification in osteoclasts may contribute to developing potential therapeutic strategies for OP.

The m 6 A modification is one of the most common epigenetic modifications on eukaryotic mRNAs, which reinforces a crucial role in the regulation of gene expression and is vital for skeletal development and metabolism [ 33 , 54 ]. Recent studies have shown that m 6 A methyltransferases influenced the development of OP by participating in the regulation of various osteoblasts [ 24 , 25 ]. In 2023, we demonstrated that methyltransferase WTAP-mediated modification of m 6 A methylation affects BMMSC differentiation and thus improves OP [ 27 ]. However, KIAA1429, an essential component of methyltransferases, has not yet been intensively studied in the development of OP; thus, our current study focused on KIAA1429-mediated m 6 A methylation on postmenopausal osteoporosis and osteoclast differentiation. The establishment of a murine model of postmenopausal OP revealed that m 6 A modification levels were significantly reduced in the bone tissue of OVX mice. These findings are also consistent with the significantly reduced mRNA and protein levels of KIAA1429 in OVX bone tissue. In addition, KIAA1429’s gain of function prevented estrogen deficiency-induced postmenopausal OP and osteoclastic hyperfunction, establishing KIAA1429 indispensable in defining the differentiation fate of osteoclasts to ensure bone health. Thus, our study supported a regulatory role for m 6 A and KIAA1429 in osteoclast differentiation.

Furthermore, while investigating the dynamic expression changes of KIAA1429 during osteoclastic differentiation, we also evaluated the expression levels of other m 6 A methyltransferases. The m 6 A methyltransferase is mainly composed of METTL3, METTL14, WTAP, and KIAA1429 [ 55 ]. METTL3 is the catalytic subunit of mRNA modified by N 6 -methyladenosine (m 6 A), which provides methyl groups by binding S -adenosine methionine (SAM) and modifies the internal RNA with m 6 A [ 56 ]. METTL14 forms a heterodimer with METTL3 and enhances substrate recognition through its RNA-binding domains (such as the RGG motif) [ 57 ]. As the largest scaffold component of the m⁶A methyltransferase complex, KIAA1429 guides the complex to target regions by recognizing specific RNA sequences [ 58 , 59 ]. Each component is interdependent within the cell and performs its own function. The absence or functional impairment of any one component may significantly reduce the overall m 6 A level. In addition, our study revealed that during osteoclast differentiation, both the expression level of KIAA1429 and the abundance of m 6 A showed a decreasing trend. Meanwhile, the expression levels of other m 6 A methyltransferases (such as METTL3, METTL14, and WTAP) also decreased, with no compensatory increase observed. Therefore, given the unique role of KIAA1429 in the m 6 A regulatory mechanism, the changes in the expression of KIAA1429 during osteoclast differentiation may not be compensated by other m 6 A regulators.

LRP4, or low-density lipoprotein receptor-related protein 4, is a member of the low-density lipoprotein receptor family. LRP4 binds neuronal aggregation proteins at neuromuscular junctions and forms complexes with muscle-specific kinases to form neuromuscular synapses [ 38 , 60 ]. In recent years, an increasing number of studies have reported the involvement of LRP4 in metabolic diseases of bone, such as osteoarthritis, OP, and disc degeneration [ 38 , 61 , 62 ]. Our previous studies have shown that digoxin acted on LRP4 to protect cartilage, thereby ameliorating osteoarthritis [ 38 ]. LRP4, a target of digoxin, was able to prevent disc degeneration by modulating inflammation, promoting extracellular matrix (ECM) anabolism, and reducing apoptosis [ 61 ]. However, osteoclasts are one of the major cells involved in bone metabolism, and the mechanism by which LRP4 affects osteoclast function has not been further investigated. Our results revealed that the m 6 A methyltransferase KIAA1429 was able to modify LRP4, thereby altering the fate of osteoclast differentiation. The m 6 A methyltransferase KIAA1429 normally catalyzes m 6 A modifications near the 3′-UTR and termination codon of target mRNAs [ 63 , 64 ]. Our MeRIP-seq and RNA-seq results indicated that higher levels of m 6 A abundance were observed when KIAA1429 was overexpressed and LRP4 was a key downstream target of KIAA1429 in osteoclasts. Visualization of the m 6 A peak of the Lrp4 transcript by MeRIP-seq showed that m 6 A modification during Kiaa1429 overexpression was mainly concentrated in the 3′-UTR region of Lrp4 . In addition, RIP assay results demonstrated that after overexpression of KIAA1429, the expression level of Lrp4 mRNA bound by YTHDC1 protein was elevated. YTHDC1 belongs to a family of m 6 A readers that modulate the fate of m 6 A-modified transcripts at different stages of the mRNA life cycle, from gene transcription to translation [ 65 ]. YTHDC1 has an evolutionarily conserved 100–150-amino-acid YTH (YT521-homology) domain, which contains critical tryptophan residues (Trp-377 and Trp-428) that form an aromatic cage and binding pocket for m 6 A within the DRA * CH motifs (D = A/G/U, R = A/G, A * is the methylated adenosine and H = A/C/U) [ 66 – 68 ]. On the basis of previous studies and the experimental results of our study, the YTH domain of the YTHDC1 protein may enhance the stability of Lrp4 mRNA by binding to the m 6 A in the 3′-UTR region of Lrp4 mRNA. We identified an epigenetically mediated mechanism whereby KIAA1429-mediated m 6 A modification promotes LRP4 expression in a YTHDC1-dependent manner.

In vivo experiments showed that Kiaa1429 overexpression did not rescue bone mass and inhibit osteoclast differentiation in osteoclast-specific Lrp4 knockout OVX mice, which further suggested that Lrp4 was a downstream target gene of KIAA1429 in osteoclasts. Our findings provide a novel model of KIAA1429-mediated regulation in osteoclastic differentiation.

Tumor necrosis factor α-induced protein 3 (TNFAIP3), also known as A20, was previously suggested to be involved in the development of a variety of autoimmune or inflammation-associated diseases by inhibiting NF-κB function mainly through deubiquitination [ 69 , 70 ]. The DUB domain of TNFAIP3 removes K63-linked ubiquitin chains on RIP1, TRAF6, and NEMO, leading to depolymerization of the IKK complex and downregulation of the inflammatory response [ 71 , 72 ]. Studies have reported that TNFAIP3 exerted anti-osteoclastogenic effects by inhibiting TRAF6-dependent autophagy in human periodontal ligament cells [ 73 ]. TNFAIP3 regulated the osteogenic/chondrogenic transdifferentiation and vascular calcification of vascular smooth muscle cells by inhibiting the activity of NF-κB p65 [ 74 ]. These findings are consistent with our current report on the inhibition of the NF-κB signaling pathway by TNFAIP3 in osteoclast differentiation. LRP4, as a transmembrane protein, was shown to be involved in the regulation of the NF-κB signaling pathway, but the interplay between both has not been demonstrated. Our study found that KIAA1429-mediated LRP4 could protect against OP by suppressing osteoclastic differentiation through recruiting TNFAIP3 to inhibit the NF-κB signaling pathway, not only enhancing our understanding of the mechanism of the LRP4-mediated NF-κB signaling pathway but also elucidating the unique mechanism of KIAA1429/LRP4-mediated regulation of the NF-κB pathway in osteoclasts by TNFAIP3. As shown in Fig. 8 N and 8 O, co-immunoprecipitation confirms a physical interaction between LRP4 and TNFAIP3 in precursor cells, while immunofluorescence staining indicates loss of their co-localization during osteoclast differentiation. We propose that LRP4 may function as a regulator that stabilizes TNFAIP3 in precursor cells, thereby modulating NF-κB signaling. The loss of LRP4 during osteoclast differentiation may reduce TNFAIP3 stability or its inhibitory effect on NF-κB, leading to increased activation of this pathway and enhanced osteoclastogenesis. However, our study has not yet explored the structural domains of LRP4 protein interacting with TNFAIP proteins. In future studies, we will further clarify the key structural domains of the LRP4–TNFAIP3 interaction, reveal the precise molecular mechanism of the LRP4–TNFAIP3 interaction, and provide a theoretical basis for the development of regulatory strategies targeting this interaction.

Several limitations of this study warrant consideration. First, while we employed OVX mice and mouse osteoclasts as experimental models for postmenopausal osteoporosis. It is important to note that inherent species differences and the complexity of human disease pathophysiology may limit direct translation of these findings to clinical settings. Second, although our analysis of bone tissue samples from 20 patients (10 with osteoporosis and 10 without osteoporosis) provided preliminary evidence supporting the association between KIAA1429 and osteoporosis severity, the relatively small clinical sample size necessitates cautious interpretation of these results. In the future, we will conduct further research by increasing the number of clinical samples and through preclinical animal models and human clinical trials.

Conclusions

Our study reveals for the first time that YTHDC1 binds to KIAA1429-mediated m 6 A modification of Lrp4 mRNA and increases LRP4 expression, identifying a previously unknown mechanism, while NF-κB pathway inactivation in osteoclast differentiation is regulated by LRP4-recruited TNFAIP3. Our findings provide new insights into the critical regulatory role of m 6 A in OP (Fig. 10 ). Fig. 10 KIAA1429-mediated m 6 A methylation inhibits osteoclast differentiation and protects against osteoporosis. KIAA1429 mediates m 6 A modification of Lrp4 mRNA, and then recruits YTHDC1 to increase Lrp4 stability and expression. Enhancement of LRP4 recruits TNFAIP3, leading to inactivation of NF-κB signaling, which in turn inhibits osteoclast differentiation

Supplementary Information

Additional file 1. Additional file 2.

Abbreviations

Adenosine

Adeno-associated virus

Alkaline phosphatase

One-way analysis of variance

Alizarin Red S

Bone mineral density

Bone marrow-derived macrophages

Bone marrow mesenchymal stem cells

Canonical correlation analysis

Coding sequences

Co-immunoprecipitation

Collagen type I

Cathepsin K

Ethylenediaminetetraacetic acid

Demethylases

Fibrous actin

Fetal bovine serum

Gene ontology

Hematoxylin and eosin

Kyoto Encyclopedia of Genes and Genomes

Virus-like m6A methyltransferase-associated protein

Low-density lipoprotein receptor-related protein 4

N 6 -methyladenosine

M⁶A-specific RNA immunoprecipitation sequencing

Methyltransferase 14

Methyltransferase 3

Micro-computed tomography

Matrix metallopeptidase 9

Mutant

Nuclear factor of activated T cell cytoplasmic 1

Osteocalcin

Osteoporosis

Ovariectomy

Phosphate-buffered saline

Principal component analysis

Methylated reading proteins

RNA immunoprecipitation

Runt-related transcription factor 2

Standard deviation

Specific pathogen-free

Thymine

Trabecular bone volume/total volume

Trabeculae number

Trabecular separation

Trabecular thickness

Tumor necrosis factor alpha-induced protein 3

Tartrate-resistant acid phosphatase

Untranslated region

Methyltransferases

Wild-type

Wt1-associated protein

YT521-B homology-domain-containing protein 1

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jincheng Liu and Qingyang Fu contributed to this work equally.

Acknowledgements

The authors gratefully acknowledge the Key Laboratory of Qingdao in Medicine and Engineering for consultation and instrument availability that supported this work.

Author contributions

JL, QF, KW, and HS contributed to the conceptualization, methodology, and original draft writing. JL, QF, ML, JC, and MX carried out the data analysis and review, editing, and writing of the manuscript. XZ, SL, LL, XW, and WX participated in the formal analysis and the use of software. JL, QF, KW, and HS contributed to the visualization and the investigation. JL, QF, KW, and HS contributed to the resources and validation. KW and HS contributed to supervision. KW and HS were responsible for the project administration and funding acquisition. All authors read and approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (NSFC82302751), the Young Taishan Scholars tsqn (no. 202312390), Shandong Excellent Young Scientists Fund Program (Overseas) (2024HWYQ-016), Shandong Provincial Natural Science Foundation (no. ZR2023MH050), Qingdao Natural Science Foundation (no. 23-2-1-179-zyyd-jch), and Qingdao Key Clinical Specialty of Orthopedics Center (QDZDZK2022095).

Data availability

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

Declarations

Ethics approval and consent to participate

Human subjects research was approved by the Medical Ethics Committee of Qilu Hospital (Qingdao) of Shandong University (approval no. KYLL-2024036, date: 28 October 2024) and performed in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all subjects involved in the study. All animal studies were approved by the Animal Care and Ethics Committee of Shandong University (approval no. 24015, date: 24 April 2024). The Animal Care and Ethics Committee of Shandong University adheres to the principles and guidelines set forth by the International Council for Laboratory Animal Science (ICLAS), ensuring that our animal research meets international ethical standards.

Consent for publication

Not applicable.

Competing Interests

The authors declare no competing interests.

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