NAT10 Promotes Osteogenic Differentiation of Mesenchymal Stem Cells by Mediating N4-Acetylcytidine Modification of Gremlin 1

Zhenbiao Zhu; Xiaowei Xing; Shisi Huang; Yuanyuan Tu

doi:10.1155/2021/8833527

NAT10 Promotes Osteogenic Differentiation of Mesenchymal Stem Cells by Mediating N4-Acetylcytidine Modification of Gremlin 1

Stem Cells Int. 2021 Apr 12:2021:8833527. doi: 10.1155/2021/8833527. eCollection 2021.

Authors

Zhenbiao Zhu¹, Xiaowei Xing¹, Shisi Huang², Yuanyuan Tu¹

Affiliations

¹ Department of Orthopaedics, Central South University Xiangya School of Medicine Affiliated Haikou Hospital, Haikou, Hainan, China.
² Department of Oncology, Central South University Xiangya School of Medicine Affiliated Haikou Hospital, Haikou, Hainan, China.

Abstract

Objective: To investigate the function of NAT10 in mesenchymal stem cell (MSC) osteogenic differentiation and study the mechanism by which NAT10 affects MSC osteogenesis by mediating Gremlin 1 N4-acetylcytidine (ac⁴C) modification.

Methods: Osteogenic differentiation of MSCs was induced, and the osteogenic ability was evaluated with alizarin red S (ARS) and alkaline phosphatase (ALP) assays. The NAT10 expression level during MSC osteogenesis was measured by western blot (WB). MSCs were transfected with lentiviruses to inhibit (Sh-NAT10) or overexpress NAT10 (Over-NAT10), and the osteogenic differentiation ability was assessed by ARS, ALP, and osteogenic gene marker assays. β-Catenin, Akt, and Smad signaling pathway component activation levels were assessed, and the expression levels of key Smad signaling pathway molecules were determined by PCR and WB. The Gremlin 1 mRNA ac⁴C levels were analyzed using RIP-PCR, and the Gremlin 1 mRNA degradation rate was determined. Sh-Gremlin 1 was transfected to further investigate the role of NAT10 and Gremlin 1 in MSC osteogenesis.

Results: During MSC osteogenesis, NAT10 expression, ARS staining, and the ALP level gradually increased. Decreasing NAT10 expression inhibited, and increasing NAT10 expression promoted MSC osteogenic differentiation. NAT10 affected the BMP/Smad rather than the Akt and β-Catenin signaling pathway activation by regulating Gremlin 1 expression. The Gremlin 1 mRNA ac⁴C level was positively regulated by NAT10, which accelerated Gremlin 1 degradation. Sh-Gremlin 1 abolished the promotive effect of NAT10 on MSC osteogenic differentiation.

Conclusion: NAT10 positively regulated MSC osteogenic differentiation through accelerating the Gremlin 1 mRNA degradation by increasing its ac⁴C level. These results may provide new mechanistic insight into MSC osteogenesis and bone metabolism in vivo.