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

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.

NAT10: An RNA cytidine transferase regulates fatty acid metabolism in cancer cells

“…[1][2][3] Studies related to NAT10 mediated ac4C modification have shown its biological impact in many cellular events such as ribosome biogenesis, translational efficiency, bone remodelling, cell proliferation and epithelial to mesenchymal transition (EMT). 1,[4][5][6][7][8][9][10] N4-cytidine acetylation at position 1 773 (ac4C1773) of 18S rRNA is shown to be essential for 18S rRNA related RNA processing and ribosome biogenesis in saccharomyces cerevisiae. 4 Another study in human reported acetylation at position 1 842 (ac4C1842) evidently showing the impact of ac4C modification in 18S rRNA biogenesis.…”

“…Gremlin 1 is an antigonist of bone morphogenic protein 2 (BMP2) and BMP4 which play essential role in the bone development. 8 NAT10 promotes cell proliferation by acetylation CEP170 mRNA transcript in multiple myeloma. The stability of acetylated CEP170 transcript is associated with cell proliferation and chromosomal instability (CIN).…”

“…RNA cytidine acetylation is an important epitranscriptomic event catalyzed by N ‐acetyltransferase 10 (NAT10) at the N4 position referred to N4‐acetylcytidine (ac4C) modification 1–3 . Studies related to NAT10 mediated ac4C modification have shown its biological impact in many cellular events such as ribosome biogenesis, translational efficiency, bone remodelling, cell proliferation and epithelial to mesenchymal transition (EMT) 1,4–10 …”

“…Similarly, NAT10 regulates osteogenic differentiation of mesenchymal stem cells (MSCs) via Gremlin 1 ac4C mediated decay. Gremlin 1 is an antigonist of bone morphogenic protein 2 (BMP2) and BMP4 which play essential role in the bone development 8 …”

MSC based gene delivery methods and strategies improve the therapeutic efficacy of neurological diseases

“…Numerous viral vectors are widely used to modify the gene expression profile of MSCs, including retroviruses [ 9 , 10 ], lentiviruses [ 11 , 12 ], adenoviruses, adeno-associated viruses (AAVs) [ 13 , 14 ], baculoviruses [ 15 , 16 ]. Gene delivery using viral vectors exploits the natural capacity of viruses to infect MSCs and insert viral genes into the host genome, including any genes of interest (transgenes) ligated to the viral genome or replacing viral gene(s) ( Fig.…”