E2F2 suppresses cell senescence and promotes odontogenic differentiation in dental pulp stem cells by stabilizing METTL3

Cell senescence has been widely demonstrated to limit the osteogenic and odontogenic potential of human dental pulp stem cells (DPSCs). This study aimed to elucidate the regulatory mechanism by which E2F transcription factor 2 (E2F2) influences senescence and osteogenic differentiation in young DPSCs (yDPSCs). yDPSCs and old DPSCs (oDPSCs) were isolated and characterized by flow cytometry. The endogenous expression levels of E2F2, methyltransferase-like 3 (METTL3), and senescence- or osteogenesis-related markers were determined by qRT-PCR and Western blot. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to assess cell senescence, while Alizarin Red S (ARS) and Alkaline Phosphatase (ALP) staining were used to evaluate the osteogenic differentiation capacity of yDPSCs. Dual-luciferase reporter and chromatin immunoprecipitation assays were conducted to confirm molecular interactions. Both yDPSCs and oDPSCs were positive for CD73 and CD105 and negative for HLA-DR. Compared with oDPSCs, yDPSCs exhibited stronger osteogenic differentiation potential and higher E2F2 expression. Knockdown of E2F2 increased the proportion of SA-β-gal-positive cells and upregulated the senescence markers p21 and p16, while it suppressed mineralization, ALP activity, and the expression of osteogenesis-associated markers (BMP2, OCN, OPN, RUNX2, Osterix) in yDPSCs. Mechanistically, METTL3 was identified as a transcriptional target of E2F2. Overexpression of METTL3 reversed the inhibitory effects of E2F2 knockdown on odontogenic/osteogenic differentiation and the promoting effects on cell senescence in yDPSCs. E2F2 plays a critical role in suppressing DPSC senescence and promoting their osteogenic differentiation.