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

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.

Keywords: N4‐acetylcytidine; Nat10; Nfatc1; osteoporosis.

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.

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.

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.

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.

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.

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.