Nat10-mediated N4-acetylcytidine modification enhances Nfatc1 translation to exacerbate osteoclastogenesis in postmenopausal osteoporosis

Significance

Understanding the role of the novel RNA modification N4-acetylcytidine (ac4C) in osteoclast differentiation is essential for improving postmenopausal osteoporosis (PMOP) therapy. Nat10-mediated ac4C promotes bone loss of PMOP by increasing osteoclastogenesis. Mechanistically, Nat10-mediated ac4C enhances the translation efficiency (TE) of Nfatc1, thereby inducing Nfatc1 expression and consequent osteoclast maturation. Nat10 can be an epigenetic therapeutic target to prevent PMOP bone loss.

Abstract

Increased differentiation or activity of osteoclasts is the key pathogenic factor of postmenopausal osteoporosis (PMOP). N4‐acetylcytidine (ac4C) modification, catalyzed by Nat10, is a novel posttranscriptional mRNA modification related to many diseases. However, its impact on regulating osteoclast activation in PMOP remains uncertain. Here, we initially observed that Nat10-mediated ac4C positively correlates with osteoclast differentiation of monocytes and low bone mass in PMOP. The specific knockout of Nat10 in monocytes and remodelin, a Nat10 inhibitor, alleviates ovariectomized (OVX)-induced bone loss by downregulating osteoclast differentiation. Mechanistically, epitranscriptomic analyses reveal that the nuclear factor of activated T cells cytoplasmic 1 (Nfatc1) is the key downstream target of ac4C modification during osteoclast differentiation. Subsequently, translatomic results demonstrate that Nat10-mediated ac4C enhances the translation efficiency (TE) of Nfatc1, thereby inducing Nfatc1 expression and consequent osteoclast maturation. Cumulatively, these findings reveal the promotive role of Nat10 in osteoclast differentiation and PMOP from a novel field of RNA modifications and suggest that Nat10 can be a target of epigenetic therapy for preventing bone loss in PMOP.

Postmenopausal osteoporosis (PMOP) is a metabolic bone disease characterized by decreased bone mass, deterioration of bone microarchitecture, and increased fracture risk, primarily due to a decline in estrogen levels following menopause in women. Previous studies have shown that the prevalence of osteoporosis in postmenopausal women is significantly higher than that in men (1). Fragility fractures caused by PMOP are extremely common. Notably, hip fractures occur in approximately one-third of women after reaching 65 y (2). Osteoclast activation is the key pathogenic factor in PMOP (3). However, currently available bone resorption inhibitors, such as bisphosphonates and denosumab, fail to satisfy the long-term needs for bone health and may result in severe side effects (4, 5). Therefore, exploring the mechanism of osteoclast differentiation from a novel perspective is necessary to develop new anti-bone resorptive strategies for PMOP.

The latest research suggests that epigenetic rather than genetic factors largely determine PMOP (6). Abnormal epigenetic modifications, such as DNA methylation and histone modifications, are crucial for the progression of PMOP (7, 8). However, the reversibility of epigenetic inheritance implies that reversing abnormal modifications may bring new opportunities for treating PMOP (9, 10). Although epigenetic therapy targets for PMOP have been reported, no ideal efficacy has been achieved, necessitating further exploration and discovery.

As the cutting-edge in RNA epigenetics, mRNA modification plays a crucial role in the posttranscriptional regulation of gene expression in eukaryotes (11). As recently recognized, mRNA acetylation modification (12). N4-acetylcytidine (ac4C) regulates various diseases, including inflammation, metabolic diseases, and cancers (13–15). N-acetyltransferase 10 (Nat10) is the sole known ac4C writer in mammals, catalyzing the acetylation of cytidine residues in mRNA. Emerging evidence has suggested that Nat10 may be associated with the development of osteogenic differentiation of mesenchymal stem cells and inflammatory bone loss (16–19). Whether Nat10-mediated ac4C participates in the progression of PMOP by regulating osteoclast differentiation remains an area of investigation.

In this study, we found that Nat10-mediated ac4C modification facilitates bone loss in ovariectomized (OVX) mice and PMOP females by regulating osteoclast differentiation. High-throughput acRIP-seq (acetylated RNA immunoprecipitation sequencing) and acChem-seq (NaBH4-based ac4C chemical sequencing) revealed that ac4C modification of the nuclear factor of activated T cells cytoplasmic 1 (Nfatc1) plays a crucial role in the regulation of osteoclast differentiation. Furthermore, Ribo-seq (Ribosome profiling sequencing) and RNA-seq demonstrated that Nat10-mediated ac4C improves the translation efficiency (TE) of Nfatc1, ultimately increasing Nfatc1 expression and consequent osteoclast maturation, thereby promoting OVX-induced bone loss. The findings above highlight the potential for targeting Nat10 as a promising epigenetic therapeutic strategy against bone resorption in PMOP.

1. Results

1.1. NAT10 and ac4C Modification Increased in PMOP.

To explore the importance of RNA acetylation in PMOP, we first detected the abundance of ac4C in femur tissues from PMOP patients and normal individuals using a dot blot assay. The results showed that the ac4C abundance was significantly greater in femur mRNA from patients with PMOP compared to the controls (Fig. 1 A and B). Furthermore, we observed elevated mRNA and protein levels of NAT10—the only known ac4C writer that catalyzes RNA acetylation—in femur tissues of PMOP patients (Fig. 1 C–E). In addition to human samples, the levels of mRNA ac4C and Nat10 increased in the femurs of OVX mice (Fig. 1 F–J), a typical mouse model of PMOP (20). In contrast to our findings, Yang et al. reported downregulation of ac4C and Nat10 expression in femoral tissues of PMOP patients and OVX mice (17). The contradictory results may stem from differences in the inclusion and exclusion criteria for clinical samples. Since Nat10 has been proved to play a critical role in regulating developmental disorders (14, 21–23), the inclusion of patients with developmental dysplasia of the hip as a control group by Yang et al. may led to different results. To further validate the levels of ac4C and Nat10 in osteoporosis, we established a senile osteoporosis mouse model and observed similar results to those found in our OVX mice (SI Appendix, Fig. S1).

Fig. 1.

NAT10 and ac4C modification increased in the PMOP. (A–C) The levels of ac4C (A and B) and NAT10 mRNA (C) in femurs from the control and PMOP humans (n = 15 per group). Values are normalized to the control group. (D–E) Representative immunofluorescence staining showed the NAT10 expression in femurs from the control and PMOP humans. Mean fluorescence intensity (MFI) values are normalized to the control group (n = 15 per group). White dotted line: trabecular bone surface, B: bone, BM: bone marrow. (Scale bar, 50 μm.) (F–H) The levels of ac4C (F and G) and Nat10 mRNA (H) in femurs from the sham and OVX mice. Values are normalized to the sham group (n = 15 per group). (I and J) Representative immunofluorescence staining showed the Nat10 expression in femurs from the sham and OVX mice. MFI values are normalized to the control group (n = 15 per group). White dotted line: trabecular bone surface, B: bone, BM: bone marrow. (Scale bar, 50 μm.) (K) Enzyme-linked immunosorbent assay (ELISA) showed Nat10 protein levels in BMDMs extracted from control and PMOP patients (n = 15 per group). (L) Linear correlation between NAT10 protein levels in BMDMs and T-value of BMD (n = 30, P < 0.01, Pearson’s correlation coefficient test). (M) Linear correlation between NAT10 protein levels in BMDMs and β-CTX concentration in plasma (n = 30, P < 0.01, Pearson’s correlation coefficient test). The data are shown as the means ± SDs; *P < 0.05; **P < 0.01; ***P < 0.001.

Moreover, as precursor cells of osteoclasts, enzyme-linked immunosorbent assay (ELISA) results showed that NAT10 protein levels in the bone marrow–derived monocytes (BMDMs) of PMOP females were higher than those in the control samples (Fig. 1K). Interestingly, we noticed that NAT10 protein levels in BMDMs were negatively correlated with the T-value of BMD and positively correlated with the bone turnover maker of β-CTX, which is related to osteoclast bone resorption (Fig. 1 L and M). These data suggest that NAT10 and ac4C modification might be closely related to PMOP progression by regulating osteoclastogenesis.

1.2. Nat10 Promotes Osteoclastogenesis In Vitro.

We first clarified the relationship between estrogen and Nat10 expression in osteoclastogenesis. As the primary bone-resorbing cell, the osteoclast lineage is derived from monocyte/macrophage lineage precursors after receptor activator of nuclear factor κB ligand (RANKL) activation. We observed that estrogen did not alter Nat10 RNA and protein levels in BMDMs (SI Appendix, Fig. S2 A–C). Correspondingly, estrogen did not affect Nat10 expression during the early, middle, or late stages of RANKL-induced osteoclast differentiation, consistent with previous results (SI Appendix, Fig. S2 D–F) (24). In vivo, estrogen deficiency increases the RANKL/OPG ratio in bone marrow serum (SI Appendix, Fig. S2G), which is critical for osteoclast activation (25). Nat10 expression in BMDMs from OVX mice was elevated along with the increased RANKL/OPG ratio (SI Appendix, Fig. S2H). We expectedly found that the expression of Nat10 and Ctsk increased gradually in a dose-dependent manner regulated by RANKL (SI Appendix, Fig. S2 I–K). These results suggest that although Nat10 is not directly affected by estrogen during osteoclast differentiation, the upregulation of Nat10 expression in osteoclasts is driven by increased RANKL levels resulting from estrogen deficiency in vivo. Subsequently, analysis of published RNA profiles (Fig. 2 A and B) showed that Nat10 was more highly expressed in osteoclasts than monocytes from both humans and mice. We also observed gradually increased Nat10 protein levels during osteoclast differentiation in vitro (Fig. 2 C–E). Concurrently, mRNA ac4C levels increased as osteoclast differentiation progressed (Fig. 2F). The above results indicate that Nat10 and ac4C are associated with osteoclast differentiation in vitro.

Fig. 2.

Nat10 promotes osteoclastogenesis in vitro. (A and B) Nat10 mRNA expression of monocytes and osteoclasts from human (GSE246769 dataset) and mouse (GSE247553 dataset) data. (C and D) Immunofluorescence staining showed elevated Nat10 expression (red) in RANKL-induced BMDMs. (Scale bar, 100 μm.) MFI values are normalized to 0 d group (n = 3 per group). (E and F) The Nat 10 protein (E) and mRNA ac4C (F) levels in RANKL-induced BMDMs at 0, 3, and 5 d. Values are normalized to 0 d group (n = 3 per group). (G and H) The Nat10 protein (G) and mRNA ac4C (H) levels in BMDMs from Nat10^ΔLysM mice and control mice. Values are normalized to the control group (n = 3 per group). (I) TRAP staining of RANKL-induced BMDMs from Nat10^ΔLysM and control mice at indicated days. (Scale bar, 100 μm.) Quantification analysis of the TRAP⁺ osteoclast (Oc) (>3 nuclei) per field and the mean area per Oc at 5 d after RANKL induction (n = 6 per group). (J) Calcr and Ctsk mRNA levels of BMDMs from Nat10^ΔLysM and control mice at 5 d after RANKL induction (n = 3 per group). (K) Western blotting and quantification analysis of Ctsk at 0, 3, and 5 d after RANKL induction. Values are normalized to the control group (n = 3 per group). (L) Scanning electron microscope displayed the bone slice pits absorbed by RANKL-induced BMDMs from control and Nat10^ΔLysM mice at 7 d. (Scale bar, 100 μm.) Quantification analysis of bone resorption pit area (n = 3 per group). The data are shown as the means ± SDs; *P < 0.05; **P < 0.01; ***P < 0.001.

To clarify the role of Nat10 in osteoclast metabolism, we established monocyte-specific Nat10 knockout mice (Nat10^ΔLysM) by crossing mice bearing loxP sites flanking exon 4 of Nat10 (Nat10^flox/flox) with LysM-Cre mice (SI Appendix, Fig. S3A). Nat10 protein expression and mRNA ac4C abundance were effectively abolished in BMDMs of Nat10^ΔLysM mice (Fig. 2 G and H), while there was no significant difference in the number of splenic mononuclear cells (SI Appendix, Fig. S3B). Furthermore, we found that BMDMs in Nat10^ΔLysM mice were unable to form multinucleated osteoclasts during RANKL-induced osteoclast differentiation (Fig. 2I) and exhibited significantly reduced levels of Ctsk and Calcr (Fig. 2 J and K). Regarding bone resorption function, BMDMs in Nat10^ΔLysM mice showed little to no evidence of resorption pits on bovine bone slices (Fig. 2L).

Similarly, Nat10 knockout RAW264.7 cells lost the capability for osteoclast differentiation and bone resorption, with significant downregulated ac4C abundance (SI Appendix, Fig. S4). Conversely, overexpression of Nat10 induced ac4C mRNA abundance and enhanced the osteoclast differentiation and bone resorption capability of osteoclasts on bovine bone slices (SI Appendix, Fig. S5). These findings suggest that Nat10 exerts a positive effect on RANKL-induced osteoclast differentiation.

1.3. Nat10 Deletion in Monocytes Alleviated OVX-Induced Bone Loss.

To further determine the role of Nat10 on bone mass in vivo, we first investigated the bone homeostasis in Nat10 conditional knockout mice and their control littermates (Nat10^flox/flox). Nat10^ΔLysM mice exhibited higher BMD, bone volume fraction (BV/TV), and trabecular number (Tb. N), as well as lower trabecular separation (Tb. Sp) compared with the control mice (SI Appendix, Fig. S6 A–F). H&E staining showed that the Nat10 deletion effectively increased the number and thickness of trabecular bones in Nat10^ΔLysM mice (SI Appendix, Fig. S6G). However, Nat10 deletion did not significantly alter the number of OCN⁺ osteoblasts on the trabecular bone surface between control and Nat10^ΔLysM mice (SI Appendix, Fig. S6 H and I). Predictably, Nat10 deletion in monocytes significantly downregulated the number of TRAP⁺ osteoclasts on the trabecular bone surface in Nat10^ΔLysM mice compared to the control (SI Appendix, Fig. S6 J and K). Moreover, Nat10 conditional knockout mice exhibited high bone mass at the early stage and maintained a steady state thereafter. These results suggest that Nat10 may regulate the growth and development of monocytes or their differentiation into osteoclasts, and Nat10 knockout in monocytes may not significantly impact osteogenic activity.

Furthermore, OVX or sham operation was performed in 10-week-old female Nat10^ΔLysM mice or control mice. Eight weeks postoperation, both the bone formation marker PINP (N-terminal collagen type I extension propeptide) and the bone resorption marker CTX-I (C-terminal cross-linking telopeptide of type I collagen) were significantly elevated in serum of control–OVX mice. Nat10 knockout markedly reduced CTX-I levels, particularly following OVX. However, there was no significant difference in PINP levels between the corresponding group of Nat10^ΔLysM mice and control mice (Fig. 3 A and B). Micro-CT displayed representative images of the distal femurs from the different mice groups at 0- and 8-week postoperation, respectively (Fig. 3 C and D). At basal condition (0 wk), Nat10^ΔLysM mice exhibited higher BMD, BV/TV, and Tb. N, as well as lower Tb. Sp compared with the control group (Fig. 3E). Four weeks after the operation, the control–OVX mice exhibited obvious bone loss, characterized by deteriorated BMD and trabecular parameters. OVX-induced bone loss was also observed in Nat10^ΔLysM-OVX mice; however, their BMD was higher than that of the control–sham mice with comparable trabecular parameters (SI Appendix, Fig. S7 A and B). Compared to 4 wk postoperation, control–OVX mice exhibited more severe bone loss at 8 wk postoperation. However, the Nat10-deficient mice demonstrated a stronger resistance to OVX-induced bone loss (Fig. 3F).

Fig. 3.

Nat10 deletion in monocytes alleviated OVX-induced bone loss. (A and B) Serum PINP (N-terminal collagen type I extension propeptide) and CTX-I (C-terminal cross-linking telopeptide of type I collagen) in Nat10^ΔLysM and control mice at 8 wk postoperation (Sham and OVX) (n = 5 per group). (C and D) Representative micro-CT images of the distal femurs from Nat10^ΔLysM and control mice at 0 and 8 wk postoperation (Sham and OVX), respectively. (E and F) Quantification analysis of bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular separation (Tb. Sp) (n = 5 per group). (G and H) H&E staining (G) and TRAP staining (H) of distal femurs from Nat10^ΔLysM and control mice at 8 wk postoperation (Sham and OVX). TRAP⁺ osteoclasts on the bone surface were marked by black arrows. (Scale bar, 200 μm.) Enlarged images. (Scale bar, 50 μm.) (I) The number of TRAP⁺ osteoclasts per bone surface (n = 5 per group). Oc: Osteoclasts; BS: Bone Surface. (J) Immunofluorescence staining showed the Nat10⁺/Ctsk⁺ osteoclasts (white arrows) on the trabecular bone surface (white dotted line) in Nat10^ΔLysM and control mice at 8 wk postoperation (Sham and OVX). (Scale bar, 50 μm.) (K) The number of Nat10⁺/Ctsk⁺ osteoclasts per bone surface (n = 5 per group). Oc: Osteoclasts; BS: Bone Surface. The data are shown as the means ± SDs; *P < 0.05; **P < 0.01; ***P < 0.001.

Dot blot assay showed that the OVX-induced increases in femoral ac4C mRNA levels can be partially attenuated in Nat10^ΔLysM mice (SI Appendix, Fig. S7C). Histological H&E staining indicated that the Nat10 deletion in monocytes effectively increased the number and thickness of trabecular bones in OVX mice (Fig. 3G). Moreover, TRAP staining showed that the number of TRAP⁺ osteoclasts on the trabecular bone surface in the control–OVX group was significantly higher than that of the control–sham group, consistent with the characteristics of high bone turnover caused by OVX (26). However, Nat10-deficient mice effectively resisted OVX-induced osteoclast activation on the trabecular bone surface compared to the control mice (Fig. 3 H-I). Further evidence of immunofluorescence assays showed that OVX caused a sharp elevation in the number of Nat10⁺/Ctsk⁺ osteoclasts on the trabecular bone surface in the control mice. At the same time, the Nat10 deletion effectively resisted OVX-induced Nat10⁺/Ctsk⁺ osteoclast proliferation (Fig. 3 J and K). In summary, it is conceivable that the Nat10 deletion in monocytes can alleviate OVX-induced bone loss by inhibiting the formation of osteoclasts on the trabecular bone surface in vivo.

1.4. Remodelin Prevents OVX-Induced Bone Loss by Inhibiting Osteoclastogenesis.

To explore the potential role of Nat10 as a therapeutic target for PMOP, we attempted to inhibit Nat10 enzyme activity by using remodelin, a specific inhibitor of Nat10. We found that remodelin effectively reduced ac4C mRNA levels in BMDMs at a concentration of 8 μM without inhibitory effects on cell viability (SI Appendix, Fig. S8 A and B). Remodelin treatment impaired osteoclastogenesis in a dose-dependent manner, with a remarkable reduction of the number and the size of mature osteoclast (multinucleated (>3 nuclei) and highly TRAP-expressing cells) (Fig. 4A and SI Appendix, Fig. S8C). Meanwhile, significantly reduced mRNA and protein levels of osteoclast marker Ctsk in the remodelin-treated group were detected (SI Appendix, Fig. S8 D and E). Bone resorption assays revealed that the bone resorption area decreased with increasing concentrations of the remodelin (Fig. 4B and SI Appendix, Fig. S8F). Therefore, inhibiting Nat10 using remodelin prevents osteoclastogenesis in vitro.

Fig. 4.

Remodelin prevents OVX-induced bone loss from inhibiting osteoclastogenesis. (A) TRAP staining of RANKL-induced BMDMs at 5 d incubated with different concentrations of remodelin. (Scale bar, 200 μm.) (B) Scanning electron microscopy displayed the bone slice pits absorbed by RANKL-induced BMDMs at 7 d incubated with different concentrations of remodelin. (Scale bar, 100 μm.) (C) Representative micro-CT images of distal femurs from indicated mice groups at 4 wk postoperation. (D) Quantification analysis of BMD, bone volume/tissue volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular separation (Tb. Sp) (n = 5 per group). (E and F) H&E staining (E) and TRAP staining (F) of distal femurs from indicated mice groups at 4 wk postoperation. TRAP+ osteoclasts on the bone surface were marked by black arrows. (Scale bar, 200 μm.) Enlarged images. (Scale bar, 50 μm.) (G) Immunofluorescence staining showed the Nat10⁺/Ctsk⁺ osteoclasts (white arrows) on the trabecular bone surface (white dotted line) from indicated mice groups at 4 wk postoperation. (Scale bar, 50 μm.) The data are shown as the means ± SDs; *P < 0.05; **P < 0.01; ***P < 0.001.

Furthermore, OVX mice and sham mice were treated with remodelin (5 mg/kg) via intraperitoneal injection every other day for 4 wk. In vivo, remodelin effectively resisted the OVX-induced high bone turnover state, significantly reducing serum CTX-I and PINP levels, which was also observed in the sham group (SI Appendix, Fig. S8G). Furthermore, micro-CT monitoring showed that remodelin effectively resisted OVX-induced bone loss at 2 and 4 wk, as evidenced by improvements in BMD and trabecular parameters, whereas these values showed no significant changes in sham mice treated with remodelin (Fig. 4 C and D and SI Appendix, Fig. S8 H and I). Histological results were consistent with those of micro-CT. H&E staining showed remodelin significantly increased the number and thickness of trabecular bones in OVX mice, but not in the sham mice (Fig. 4E). Additionally, we observed that remodelin significantly downregulated the number of OCN⁺ osteoblasts on the bone surface in both sham and OVX mice (SI Appendix, Fig. S8 J and K). However, Yang et al. reported that remodelin inhibited osteogenic differentiation and exacerbated bone loss in OVX mice (17), which is opposite to our results. This discrepancy may be attributed to differences in the administration route, dosage, and duration of remodelin treatment. Yang et al. administered a high dose of remodelin (100 mg/kg) via oral gavage every other day for two months following OVX operation, whereas we utilized a lower dose (5 mg/kg) delivered via intraperitoneal injection every other day for one month. Our previous data indicated that high doses of remodelin will impair cell proliferation (SI Appendix, Fig. S7A). Additionally, a recent study using a low-dose intraperitoneal injection protocol, similar to ours, successfully reversed inflammatory bone loss (27).

Furthermore, we focused on the effects of remodelin on osteoclasts at the trabecular bone surface. Remodelin treatment markedly reduced the quantity of TRAP⁺ osteoclasts on the trabecular bone surface in both sham and OVX mice (Fig. 4F and SI Appendix, Fig. S8L). Using immunofluorescence, we next confirmed that remodelin significantly reduced the number of Nat10⁺/Ctsk⁺ osteoclasts on the trabecular bone surface in sham and OVX mice (Fig. 4G and SI Appendix, Fig. S8M). These results suggest that although remodelin inhibits both osteoclastic and osteoblastic metabolism by downregulating Nat10 activity, it ultimately effectively resists OVX-induced bone loss. Therefore, the Nat10 gene can be a potential target for the treatment of PMOP.

1.5. Nfatc1 Is the Key Target of Nat10 for ac4C Modification.

As previously reported, Nat10 was identified as the “writer” of ac4C modification in eukaryotes (12). Nevertheless, the exact targets regulated by Nat10-mediated ac4C during osteoclastogenesis remain to be clarified. We employed acRIP-seq and acChem-seq (28) on Nat10^flox/flox (control) and Nat10^ΔLysM BMDMs to identify ac4C‐modified transcripts in osteoclasts and reveal the molecular mechanism by which ac4C modification promotes osteoclastogenesis.

Using acRIP‐seq analysis, we determined that the ac4C motif “CXXCXXCXX” (29) was highly enriched in control BMDMs during osteoclast differentiation (Fig. 5A). The coding sequence (CDS) region of mRNA was the primary location for ac4C modification (Fig. 5B), consistent with previous reports (12). The level of ac4C modification in the CDS and 5’UTR regions in Nat10^ΔLysM BMDMs was lower than that of control cells (Fig. 5C). Through differential analysis of the ac4C peaks between the control and Nat10^ΔLysM BMDMs, we identified 1,440 down-regulated and 940 up-regulated ac4C peaks (Fig. 5D). Gene ontology (GO) enrichment analysis was performed to analyze the down-regulated differentially expressed genes (DEGs), which were significantly enriched in the osteoclast differentiation pathway (Fig. 5E). The above results suggest that Nat10 regulates ac4C modification of osteoclast differentiation-associated genes.

Fig. 5.

Nfatc1 is the key target of Nat10 for ac4C modification. (A) Sequence motifs enriched within ac4C peaks in RANKL-induced control BMDMs at 3 d. (B) Pie charts showed the percentage distribution of ac4C peaks in control BMDMs. (C) Peak density of the ac4C peaks across the mRNA transcripts in Nat10^ΔLysM versus control BMDMs. (D) Volcano plot analysis of differential ac4C modification genes. (E) Gene ontology (GO) analysis of the ac4C downregulated genes from acRIP‐seq data in Nat10^ΔLysM and control BMDMs. (F) Venn diagram showed the gene set within the GO term “osteoclast differentiation” that exhibited significant downregulation of ac4C abundance and deletion of C > T mismatches. (G) The integrative genomics viewer (IGV) diagram displayed the read distributions (acRIP-seq) and acChem-seq-derived ac4C site across target transcript of Nfatc1. Black boxes showed reproducible and significantly downregulated ac4C peaks in Nat10^ΔLysM BMDMs. The acChem-seq-derived ac4C site was marked with red bars below the ac4C peaks. The corresponding nucleotide and amino acid sequences were revealed in the red box, with the red “C” indicating the ac4C modification site. (H) acRIP-qPCR analysis showed ac4C levels of Nfatc1 in control and Nat10^ΔLysM BMDMs (n = 3 per group). (I) Nat10 RIP-qPCR demonstrated the interaction between Nfatc1 mRNA and Nat10 (n = 3 per group). The data are shown as the means ± SDs; *P < 0.05; **P < 0.01; ***P < 0.001.

To identify the precise sites of ac4C modification, we introduced acChem-seq to rigorously identify ac4C target genes. The acChem-seq allows for precisely identifying ac4C modification sites with C > T mismatches at single-based resolution during osteoclast differentiation (SI Appendix, Fig. S9A) (28). The results showed a more than 50% reduction of ac4C modification in the Nat10^ΔLysM BMDMs compared to the control (SI Appendix, Fig. S9B), confirming the pivotal role of Nat10 in catalyzing ac4C modifications of BMDMs during osteoclast differentiation. We next identified a gene set within the GO term “osteoclast differentiation” that exhibited significant downregulation of ac4C abundance and deletion of C > T mismatches: Nfatc1, Tob2, Ocstamp, Ireb2, Tnfrsf11a, CD109, Tgfb1, Tcirg1, Sbno2, and Nf1 (Fig. 5F). Integrative Genomics View (IGV) software was used to visualize the distribution of ac4C peaks and chemical sequencing-derived ac4C sites across the target transcripts of these ten genes. Notably, Nfatc1 has been identified as a key transcription factor involved in osteoclast differentiation, with the RANKL/RANK (Tnfrsf11a) signaling pathway serving as its upstream activator (30). Interestingly, only the Nfatc1 transcript exhibited significant and reduplicative ac4C peaks near its chemical sequencing-derived ac4C site (Fig. 5G and SI Appendix, Fig. S10). To further validate the ac4C abundance of Nfatc1 and Tnfrsf11a, acRIP‐qPCR assays revealed a significant reduction in the ac4C modification of Nfatc1 in Nat10^ΔLysM BMDMs (Fig. 5H), but not in Tnfrsf11a (SI Appendix, Fig. S9J). The results above suggest that the ac4C acetylation of Nfatc1 catalyzed by Nat10 plays a critical role in osteoclastogenesis. Consistently, Nat10 RIP-qPCR indicated that the Nat10 protein directly binds to the Nfatc1 mRNA in BMDMs (Fig. 5I) but does not bind to Tnfrsf11a (SI Appendix, Fig. S9K). These results indicate that Nfatc1 mRNA is the key target of Nat10‐mediated ac4C modification during osteoclastogenesis.

1.6. Nfatc1 Translation Efficiency Is Enhanced by Nat10-Mediated ac4C.

Studies have reported that Nat10 could enhance TE and influence ac4C, especially when the ac4C modification sites are primarily located in the CDS region (29). TE directly affects the expression levels of nascent proteins, implying that a reduced ac4C level will decrease the expression of nascent proteins during osteoclast differentiation. However, the surface sensing of translation (SUnSET) assay was performed to validate that the elimination of ac4C modifications in Nat10^ΔLysM BMDMs moderately lowered the expression of nascent proteins during osteoclastogenesis (Fig. 6A), indicating that Nat10 is not essential for global mRNA translation in BMDMs. Therefore, we used Ribo-seq and RNA-seq to reveal the role of ac4C modification in the regulation of TE between the control and Nat10^ΔLysM BMDMs 3 d after RANKL induction. We first performed the volcano map analysis of the genes with differences in the translation level and transcriptional level (FDR < 0.05, |log₂ Fold Change| > 1) (SI Appendix, Fig. S11 A and B). The results suggested that Nat10 depletion resulted in TE fold reduction in 674 genes, and 573 genes showed upregulation. Plotting TE against RNA‐seq data using the formula previously reported(31) showed 122 genes to differ at the transcript level and 3,526 genes to differ only at translational efficiency (Fig. 6B). A combined analysis with transcriptome and genes of ac4C (+) or ac4C (−) in control and Nat10^ΔLysM cells was performed. The results showed that the transcript level of ac4C (+) mRNAs in control BMDMs was not significantly different from that of ac4C (−) mRNAs in Nat10^ΔLysM BMDMs (SI Appendix, Fig. S11C). However, when compared with control cells, the overall TE of genes in Nat10^ΔLysM BMDMs was slightly reduced (|log₂ Fold Change| > 1) (Fig. 6C).

Fig. 6.

Nfatc1 translation efficiency (TE) is enhanced by Nat10-mediated ac4C. (A) Nascent protein levels of control and Nat10^ΔLysM BMDMs 3 d after RANKL induction. (B) Difference direction of TE and transcription level comparison and classification between control and Nat10^ΔLysM BMDMs at 3 d after RANKL induction. (C) Overall TE of genes in control and Nat10^ΔLysM BMDMs. (D–G) Representative graphs showing the overlay of Nfatc1 transcripts from Ribo-Seq (ribosome-protected fragment, RPF) and RNA-Seq and their corresponding average individual normalized reads for RPF and RNA and the ratio of RPF/RNA (ribosome occupancy) in control and Nat10^ΔLysM BMDMs at 3 d after RANKL induction. (H) The fold decrease in TE of the top 5 genes among 10 potential downstream genes identified by the intersection of ac4C omics data with the GO term “osteoclast differentiation”. (I) Sucrose gradient analysis in control and Nat10^ΔLysM BMDMs (n = 3 per group). (J) qRT-PCR analysis of changes in Nfatc1 binding to Nat10 between overexpressing (OE) and mutant Nfatc1 (n = 3 per group). (K) qRT‐PCR analysis of changes in ac4C‐modified Nfatc1 levels between overexpressing and mutant Nfatc1 (n = 3 per group). (L–N) Nfatc1 mRNA and protein levels in HEK-293 T cells with overexpressing (OE) and mutant Nfatc1. Values are normalized to the OE group (n = 3 per group). The data are shown as the means ± SDs; *P < 0.05; **P < 0.01; ***P < 0.001.

We next analyzed the TE of the 10 potential downstream genes identified by the intersection of ac4C omics data with the GO term of osteoclast differentiation. Ribosome occupancy results demonstrated decreasing trend of TE in five genes (Nfatc1, Cd109, Ocstamp, Nf1, and Ireb2) (Fig. 6 D–G and SI Appendix, Fig. S12); the Nfatc1 gene showed the greatest reduction (Fig. 6H). Consistently, sucrose gradient analysis showed that Nfatc1 mRNA was significantly reduced in the polysome fractions in Nat10^ΔLysM BMDMs, with a concomitant increase in lighter 80S fractions compared to the control BMDMs. This verified that ac4C modification enhances the TE of Nfatc1 mRNA during osteoclastogenesis (Fig. 6I).

Due to the potential ac4C site of Nfatc1 mRNA identified by the combined analysis of acRIP-seq and acChem-seq, we further confirmed the regulatory effects of ac4C on the transcription and expression levels of Nfatc1. Nfatc1 point mutation vectors were constructed by converting cytosine residues within the ac4C motifs to either adenine (C-A mutation) or guanine (C-G mutation) (SI Appendix, Fig. S13A). HEK293T cells were transfected with equal amounts of Nfatc1-overexpressing (OE) or mutant plasmids. The mutations significantly inhibited the interaction between Nat10 and Nfatc1 mRNA (Fig. 6J), resulting in a marked decrease in the ac4C levels of Nfatc1 (Fig. 6K). However, the mutations significantly reduced the Nfatc1 protein levels without affecting their mRNA levels in HEK293T cells (Fig. 6 L–N). Furthermore, we observed that mutations significantly reduced osteoclast differentiation capacity compared to the OE group (SI Appendix, Fig. S13 B–D). Similarly, the bone resorption ability of mutations was impaired (SI Appendix, Fig. S13 E and F).

These results suggest that Nat10‐mediated ac4C modification regulates the TE of Nfatc1 rather than mRNA transcription during osteoclastogenesis.

1.7. Nfatc1 Overexpression Reverses the Inhibitory Effect of Nat10 Deletion on Osteoclast Differentiation and Bone Resorption.

To further validate the role of Nfatc1 in the Nat10-mediated promotion of osteoclast differentiation, we conducted Nfatc1 overexpression experiments on Nat10-knockout RAW 264.7 cells (SI Appendix, Fig. S14 A and B). The results of TRAP staining showed that Nfatc1 overexpression partially restored the capability of osteoclast differentiation in Nat10-knockout RAW264.7 cells (SI Appendix, Fig. S14 C and D). Further bone resorption assays demonstrated that Nfatc1 overexpression rescued the bone resorption ability of Nat10-knockout RAW264.7 cells during osteoclastogenesis (SI Appendix, Fig. S14 E and F).

Finally, we constructed an Nfatc1-overexpressing model in Nat10^ΔLysM mice. The immunofluorescence staining confirmed the specificity of Nfatc1 overexpression in bone marrow macrophages (SI Appendix, Fig. S14G). Nfatc1 protein levels in BMDMs from the Nfatc1 overexpressing group (AAV-Nfatc1) were significantly higher than those in the Nat10^flox/flox, Nat10^ΔLysM, and AAV-vector groups (SI Appendix, Fig. S14 H and I). Micro-CT results showed that BMD and trabecular parameters of femurs in the Nfatc1-overexpressing group were significantly lower than those in the vector and Nat10^ΔLysM groups (SI Appendix, Fig. S14 J and K). These results indicate that Nfatc1 overexpression can reverse the inhibitory effect of the Nat10 deletion on bone resorption.

Consistently, H & E staining exhibited fewer and thinner trabeculae bones in the overexpressing group compared to the vector and Nat10^ΔLysM groups (SI Appendix, Fig. S14L). TRAP staining further indicated that the number of TRAP-positive osteoclasts on the trabecular bone surface was partially rescued in the overexpressing group (SI Appendix, Fig. S14 M and N). Thus, the forced overexpression of Nfatc1 can reverse the inhibitory effects of Nat10 deletion on osteoclast differentiation and bone resorption. In summary, these data underscore the critical role of Nfatc1 in Nat10-mediated osteoclast differentiation and bone resorption.

2. Discussion

As one of the more peculiar chemical modifications, ac4C acetylation has been confirmed to exist in eukaryotic and prokaryotic tRNAs, rRNAs, and mRNAs. Early studies on ac4C acetylation primarily focused on tRNA and rRNA, highlighting its critical role in regulating protein translation accuracy and organismal thermotolerance (32). Few studies have investigated ac4C modifications in mRNAs compared to tRNAs and rRNAs. With the advent of cutting-edge techniques for identifying the epitranscriptome of ac4C, Arango et al. discovered ac4C modification of mRNA in HeLa cells, and its biological significance has been gradually recognized (12). ac4C modification in mRNA regulates protein translation, RNA stability, and other biological processes (33), which has been demonstrated to be associated with the progression of various tumor diseases (34). However, few studies have focused on the effect of ac4C modification in regulating nontumor diseases, such as inflammation, cardiac remodeling, and neurodegenerative diseases (15, 35, 36). Here, we found that ac4C modification positively promotes osteoclast differentiation and bone loss of PMOP. Using a multiomics screening strategy combining acRIP-seq, acChem-seq, Ribo-seq, and RNA-seq data to reveal the mechanisms of Nat10-mediated ac4C in osteoclastogenesis, we found that Nfatc1 is the key downstream target of ac4C modification, a promising enhancement of the TE of Nfatc1 mRNA.

As a member of the general control nonrepressible 5 (GCN5)-related N-acetyltransferase (GNAT) family, Nat10 is currently the only known acetyltransferase for catalyzing RNA ac4C in eukaryotes (37). The GNAT family proteins are correlated with the development of bone diseases. For example, NAT1 promoted osteoclastogenesis in luminal breast cancer and accelerated bone metastasis of cancer cells (38). NAT2 polymorphisms have been demonstrated to be associated with the progression and metastasis of osteosarcoma (39). A few in vitro studies have found that Nat10 plays a crucial role in the osteogenic differentiation of mesenchymal stem cells (16, 40). However, the primary characteristic of the pathological condition of PMOP is osteoclast activation, and the underlying functions and mechanisms of Nat10 in osteoclast differentiation remain unclear. Herein, we found that Nat10 was markedly increased in the femurs of PMOP humans and OVX mice and osteoclast differentiation of monocytes. Nat10 positively regulates osteoclast differentiation, and the expression of Nat10 mediating osteoclastogenesis is regulated by RANKL in a dose-dependent manner. RANKL is an essential cytokine for osteoclast differentiation, activation, and survival, and in vivo estrogen deficiency is a key factor contributing to elevated RANKL levels (25, 41). Based on our findings, we suppose that Nat10 activation is mediated by RANKL-induced downstream signaling pathways, rather than direct regulation by estrogen.

Additionally, Nat10 deletion in monocytes negatively impacts osteoclastogenesis at the transcriptional, expression, and functional levels, ultimately alleviating OVX-induced bone loss. Recent studies described that NAT10 in peripheral blood mononuclear cells was negatively correlated with ankylosing spondylitis characterized by enhanced osteogenesis (42), and that Nat10 inhibition could alleviate inflammatory alveolar bone loss (18), indirectly supporting our findings. In vivo experiments demonstrated that the Nat10-specific inhibitor remodelin significantly reduced both osteoblast and osteoclast numbers on the bone surface, but effectively preventing OVX-induced bone loss. OVX mimics the high bone turnover state of PMOP, characterized by osteoclastic activity surpassing osteoblastic activity, and the inhibitory effect of remodelin on osteoclasts may play a predominant role at this stage.

Osteoclastogenesis relies on a complex signaling cascade activated by macrophage colony-stimulating factor (M-CSF) and RANKL. Core transcription factors, such as c-Fos, PU.1, Mitf, and Nfatc1, are crucial signaling hubs that extensively regulate osteoclastogenesis. The epigenetic modifications of core transcription factors play crucial roles in osteoclastogenesis, including histone and nucleic acid modifications (8, 43). Previous studies have found that Nat10-mediated ac4C typically regulates the pathological states of diseases by modifying key transcription factors. Examples include the major transcription factor RUNX2 in osteogenic differentiation of mesenchymal stem cells (17), FOXP1 associated with poor prognosis in cancers (13), and the core pluripotency regulator OCT4 in embryonic stem cells (14). Our omics results from acRIP-seq and acChem-seq suggest that the Nat10 knockout leads to the most notable downregulation of ac4C modification in the core transcription factor Nfatc1 during osteoclast differentiation. During the later stage of RANKL-induced osteoclastogenesis, Nfatc1 is a key transcription factor that promotes the fusion of osteoclast precursor cells and facilitates bone resorption functions.

Previous studies also highlighted the crucial role of histone methylation of Nfatc1 in osteoclast maturation from a metabolic-epigenetic perspective (8). Additionally, zoledronic acid, a traditional anti-bone resorptive drug, has been proven to inhibit the resorptive function of osteoclasts by modifying Nfatc1 through m6A methylation (43). These findings highlight the important role of epigenetic modifications of Nfatc1 in osteoclastogenesis, supporting the fact that Nfatc1 is a key downstream target in the regulation of osteoclastogenesis by ac4C modification.

From the early synthesis in the nucleus to later degradation in cytoplasm, mRNA modifications play many roles, but their underlying mechanisms remain unclear (44). Previous studies have proposed that ac4C acetylation promoted osteogenic differentiation of mesenchymal stem cells by increasing the stability of RUNX2, Gremlin 1, and VEGFA mRNA (16, 17, 40), accelerated the malignant progression of cervical cancer by enhancing the TE of FOXP1 (13), and promoted mesenchymal-to-epithelial transition by increasing the stability and TE of NR2F1 mRNA (21). Daniel Arango et al. summarized the location-dependent regulation of mRNA function by ac4C modification: ac4C widely distributed in the CDS region may influence the TE of mRNA, whereas ac4C enriched in the 5’UTR and the 3’UTR region may respectively affect the upstream translation initiation rate and the mRNA stability (29). According to our results, the ac4C modification of Nfatc1 is located at the CDS region, suggesting that ac4C may regulate the TE of Nfatc1. This hypothesis was confirmed by translatomics and polysome profiling, which showed that Nat10 deletion led to the most significant decrease in the TE of Nfatc1 among genes involved in GO term “osteoclast differentiation” with downregulated ac4C modification.

In summary, this study offers compelling in vitro and in vivo evidence illustrating the promotive effect of Nat10-mediated ac4C in osteoclast differentiation and PMOP progression. As an essential epitranscriptomic regulator, Nat10 enhances the TE of Nfatc1 through ac4C modification, thereby promoting osteoclast differentiation and OVX-induced bone loss (SI Appendix, Fig. S15). These findings introduce theoretical insights into the understanding of PMOP from the emerging field of RNA modifications, facilitating the development of promising epigenetic therapy strategies for PMOP.

2.1. Limitations of the Study.

The ac4C acetylation provides a novel perspective for understanding osteoclast activation in PMOP from an epigenetic standpoint. However, the development of PMOP involves complex interactions between osteoclasts and osteoblasts. Therefore, focusing on the regulatory role of epigenetics in the crosstalk between osteoclasts and osteoblasts can provide a more comprehensive understanding of the epigenetic mechanisms underlying PMOP. Our study clarifies the downstream mechanisms by which Nat10-mediated ac4C promotes osteoclastogenesis. As a nuclear protein, Nat10 may be subject to regulation by upstream signals in the cytoplasm, and the specific mechanisms still need to be explored.

3. Materials and Methods

3.1. Human Subject Ethics Statement.

Femoral trabecular bone samples were obtained from female patients undergoing total hip arthroplasty (THA) at Nanfang Hospital, Southern Medical University. Inclusion Criteria: 1) Control group: female patients aged over 50 y, with menopause duration greater than 1 y, hip BMD T-score > −2.5, no history of hip or vertebral fragility fractures, and undergoing THA due to acute trauma leading to complex comminuted fractures, severe displacement, or high risk of femoral head necrosis due to compromised blood supply. PMOP group: female patients aged over 50 y, with menopause duration greater than 1 y, hip BMD T-score ≤ −2.5 or a history of hip fragility fracture, and undergoing THA. Exclusion Criteria: Patients with a history of spine or hip surgeries, secondary osteoporosis (e.g., using corticosteroid, diabetes, hyperthyroidism), secondary hip arthritis (e.g., septic arthritis, rheumatoid arthritis, gouty arthritis), or prior standardized antiosteoporotic treatment were excluded. All participants provided written informed consent for the use of their clinical data and surgical specimens. The protocol for this study was approved by the Clinical Research Ethics Committee of Nanfang Hospital, Southern Medical University (Approval No. NFEC-202312-K52-01). Detailed information is listed in SI Appendix, Table S1.

Details of the experiments, e.g., cell culture, OVX model establishment, generation and characterization of conditional knockout mice, micro-CT analysis, molecular cloning and transfection, histological experiments, acRIP-seq, ribo-seq, and statistical analysis, are described in SI Appendix, Materials and Methods.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

Matrix data have been deposited in Gene Expression Omnibus (GSE279364) (45).