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| FoxO1 Transcriptional Activation of Beclin-1 Expression Regulates Osteogenic Differentiation of Human Periodontal Stem Cells |
| HU Ya, ZHOU Jieyu, XU Huixia |
| Shenzhen People's Hospital, Guangdong Shenzhen 518020, China |
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Abstract Objective: To investigate the transcriptional activation effect of the transcription factor Forkhead box O1 (FoxO1) on the autophagy-related protein Beclin-1 in human dental periodontal stem cells, as well as the regulatory mechanisms of FoxO1 on autophagy, inflammation, and osteogenic differentiation in human periodontal ligament stem cells (PDLSCs). Methods: Human PDLSCs were divided into four groups: control group (human PDLSCs were cultured in Dulbecco's Modified Eagle Medium (DMEM) for 24hours, then replaced with fresh DMEM and cultured for one week), osteogenic induction group (human PDLSCs were cultured in DMEM for 24hours, then replaced with DMEM containing 100nmoL/L dexamethasone, 10mmoL/L beta-glycerophosphate, and 80 mg/L vitamin C and cultured for one week), empty vector group (EV; human PDLSCs were transfected with an empty vector plasmid for 24hours, then replaced with fresh DMEM and cultured for one week), and FoxO1 overexpression group (FoxO1-OE; human PDLSCs were transfected with a recombinant plasmid overexpressing FoxO1 for 24hours, then replaced with fresh DMEM and cultured for one week). The expression of FoxO1 and Beclin-1 in the cell lysate was analyzed using quantitative PCR (qPCR) and Western blot. Dual-luciferase assays were utilized to verify the binding of FoxO1 to the Beclin-1 promoter region. Enzyme-Linked Immunosorbent Assay (ELISA) was used to measure the levels of inflammatory cytokines (Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α)) in the culture supernatants of each group. Western blotting was employed to detect the expression of autophagy markers p62, Microtubule-associated protein 1 light chain 3-I (LC3-I), and Microtubule-associated protein 1 light chain 3-II (LC3-II). Alkaline Phosphatase (ALP) staining and Alizarin Red S staining were used to assess osteogenic differentiation and calcium nodule formation in each group. Immunofluorescence was utilized to detect the expression of the transcription factor Runx2. Results: Compared to the control group, the osteogenic induction group exhibited increased mRNA and protein levels of FoxO1 and Beclin-1 (P<0.05), and the same trend was observed when comparing the EV group to the FoxO1-OE group (P<0.05). The dual-luciferase assay demonstrated that FoxO1 significantly increased the luciferase activity of the Beclin-1 wild-type (WT) group (P<0.05), but had no effect on the Beclin-1 mutant (MUT) group (P > 0.05). Compared to the control group, the osteogenic induction group showed a decrease in p62 protein levels and an increase in the LC3-II/LC3-I ratio (P<0.05); similar differences were observed between the EV group and the FoxO1-OE group (P<0.05). Osteogenic induction did not significantly affect the levels of inflammatory cytokines (IL-1β, IL-6, TNF-α) (P>0.05), but FoxO1 overexpression significantly reduced these cytokines' levels (P<0.05). Both osteogenic induction and FoxO1 overexpression resulted in increased ALP and calcium nodule staining areas, accompanied by upregulated Runx2 expression (P<0.05). Conclusion: FoxO1 promotes autophagy through the activation of Beclin-1 expression in human PDLSCs, reduces inflammation, and thereby enhances osteogenic differentiation.
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2025, Vol. 31 
