WTAP increases BMP2 expression to promote osteoblast differentiation and inhibit osteoblast senescence via m6A methylation of Sp1.

Abstract

Pro-differentiation and anti-senescence treatment may be potential strategies for senile osteoporosis therapy. However, the regulatory mechanism underlying osteoblast differentiation and senescence in senile osteoporosis remain to be clarified. In the present study, the preosteoblast cell line MC3T3-E1 was used to induce osteoblast differentiation. The H₂O₂ was applied to induce senescence. H₂O₂ treatment significantly inhibited the expression of Wilms tumor 1-associating protein (WTAP), runtrelated transcription factor 2 (Runx2), Osterix and specific protein 1 (Sp1), inhibited the alkaline phosphatase (ALP) activity, upregulated the senescence-associated β-galactosidase (SA-β-Gal), and increased the mRNA levels of p16 and p21. WTAP overexpression significantly reversed the effect of H₂O₂, during the osteoblast differentiation of MC3T3-E1 cells. The RIP-qRT-PCR and MeRIP-qRT-PCR assays confirmed that N6-methyladenosine (m⁶A) modification of Sp1 mRNA was significantly decreased by H₂O₂ treatment, but was increased by WTAP overexpression. The m⁶A modification of Sp1 mRNA significantly increased the stability of Sp1 mRNA. The ChIP-qRT-PCR assay and luciferase reporter gene assay showed that Sp1 could bind to the promoter of BMP2. BMP2 knockdown reversed the effect of Sp1 on osteoblast differentiation and senescence. In conclusion, WTAP increased BMP2 expression to promote osteoblast differentiation and inhibit osteoblast senescence via increasing m⁶A methylation of Sp1 mRNA. This study sheds new light on our understanding of mechanisms underlying osteoblast differentiation and senescence, and provides potential strategies for senile osteoporosis therapy.