The Hippo-YAP/β-catenin signaling axis coordinates odontogenic differentiation in dental pulp stem cells: Implications for dentin-pulp regeneration

Abstract

Objective: This study investigated the interplay between Hippo-YAP and β-catenin signaling in regulating odontogenic differentiation of human dental pulp stem cells (DPSCs) and explored its potential implications for dentin-pulp regeneration.

Methods: Using lentivirus-mediated YAP overexpression/silencing, β-catenin siRNA knockdown, and pharmacological Wnt inhibition (via WIF-1), we assessed DPSC proliferation, migration, mineralization, and molecular markers (via qRT-PCR, immunofluorescence). In vivo validation employed subcutaneous transplantation of DPSC-seeded scaffolds in immunocompromised mice.

Results: YAP activation enhanced DPSC proliferation (1.44-fold), migration (1.39-fold), invasion (1.54-fold), and differentiation, as evidenced by elevated ALP activity (1.46-fold) and mineralization (1.36-fold). We observed transcriptional upregulation of odontogenic markers (RUNX2, DSPP, DMP1, OCN, ALP) and Wnt pathway components (β-catenin, Cyclin D1, c-Myc). Immunofluorescence revealed coordinated YAP and β-catenin expression patterns during differentiation. β-catenin silencing or Wnt inhibition abolished YAP-mediated functional enhancements and simultaneously suppressed YAP expression, partially confirming bidirectional regulation. In vivo, YAP-overexpressing DPSCs exhibited 1.27- to 1.62-fold induction of dentin-specific markers and β-catenin, whereas YAP silencing impaired these markers expression.

Conclusions: Our findings demonstrate that coordinated YAP and β-catenin signaling drives DPSC odontogenesis, with potential implications for dentin regeneration. Although reciprocal regulation is evident, direct molecular interactions require further validation.

Fig 1. Spatiotemporal activation dynamics of YAP and β-catenin during DPSC odontogenic differentiation.

(A) Immunofluorescence shows progressive increases in YAP and β-catenin protein levels in DPSCs over 14 days of odontogenic induction. Nuclei were stained with DAPI (blue). Scale bar: 25 µm. (B) Spatial immunohistochemical mapping in hydrogen peroxide-bleached murine incisors (mimicking inflammatory pulp conditions) demonstrates enriched YAP/β-catenin co-expression in odontoblast layers adjacent to predentin. Scale bar: 100 µm in100 × ; 25 µm in 400 × . (C) qRT-PCR quantification confirms progressive upregulation of YAP, β-catenin, and Wnt targets (Cyclin D1, c-Myc) during differentiation. Data: mean ± SD; *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 2. YAP overexpression potentiates DPSCs’ regenerative capacity.

(A, E) Scratch wound healing assay shows a 1.39-fold accelerated closure in YAP-overexpressing DPSCs (OE-YAP) vs. controls at 48 h. (B, F) Transwell invasion assay demonstrates a 1.54-fold increased invasion capacity in OE-YAP cells. (C, I) Alkaline phosphatase (ALP) staining and activity quantification reveal a 1.46-fold elevation in OE-YAP cells at day 14. (D, J) Alizarin Red staining (red nodules) and mineralization quantification show a 1.36-fold higher mineralization in OE-YAP cells. (G) qRT-PCR confirms that OE-YAP upregulates odontogenic markers (RUNX2, DSPP, DMP1, OCN, and ALP). (H) CCK-8 proliferation assay shows a 1.44-fold increase in OE-YAP cells at 72 h. Data: mean ± SD; *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bar: 100 µm.

Fig 3. YAP knockdown suppresses DPSCs’ regenerative properties.

(A–J) Functional assays demonstrate that YAP silencing (sh-YAP) significantly attenuates key regenerative capacities compared to controls: (i) wound healing (A, E); (ii) Transwell invasion (B, F); (iii) ALP activity (C, I); (iv) mineralization (D, J); (v) proliferation (H). (G) qRT-PCR confirms that the sh-YAP group downregulates odontogenic markers. Data: mean ± SD; *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bar: 100 µm.

Fig 4. YAP enhances β-Catenin and Wnt-related gene expression in odontogenic DPSCs.

(A) Lentiviral-mediated YAP overexpression (OE-YAP) and knockdown (sh-YAP) efficiency validated by immunofluorescence. (B, D) qRT-PCR shows OE-YAP upregulates Wnt pathway components (β-catenin, c-Myc, and Cyclin D1), while sh-YAP suppresses them. (C) Immunofluorescence reveals OE-YAP increases cytoplasmic β-catenin (red), whereas sh-YAP diminishes it. Data: mean ± SD; *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bar: 25 µm.

Fig 5. β-catenin ablation reverts YAP-driven functional enhancement.

(A, C) The scratch wound closure assay: β-catenin siRNA reduces OE-YAP migration by 42.87% at 48 h (representative images in A, quantification in C). (B, D) The Transwell assay: β-catenin knockdown decreases OE-YAP invasion by 56.12% (representative images in B, quantification in D). (E) CCK-8 analysis shows that β-catenin siRNA reduces OE-YAP proliferative capacity by 54.97%. Data: mean ± SD; *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. control. Scale bar: 100 µm.

Fig 6. β-catenin ablation suppresses YAP expression and odontogenic capacity.

(A) Immunofluorescence demonstrates that β-catenin knockdown not only abolishes OE-YAP-induced β-catenin upregulation but also reduces YAP expression, indicating β-catenin-dependent stabilization of YAP. Scale bar: 25 µm. (C) qRT-PCR validates β-catenin siRNA knockdown efficacy. (B, E) ALP activity is decreased by 25.48% in β-catenin-silenced OE-YAP cells compared to controls. Scale bar: 100 µm. (D, F) Mineralization capacity is reduced by 41.52% following β-catenin ablation. Scale bar: 100 µm. (G) β-catenin siRNA downregulates YAP-induced markers and reciprocally suppresses YAP mRNA expression. Data: mean ± SD; *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 7. Pharmacological Wnt inhibition mimics β-catenin silencing in suppressing regenerative potential.

(A-D) Wnt inhibitory factor-1 (WIF-1) treatment phenocopies β-catenin siRNA effects, including wound closure reduction (AB), invasion capacity decrease (CE), and proliferation decline (D). Data: mean ± SD; *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bar: 100 µm.

Fig 8. Pharmacological Wnt inhibition mimics β-catenin silencing in suppressing molecular expression and odontogenic differentiation.

(A) Immunofluorescence demonstrates diminished expression of both YAP and β-catenin in WIF-1-treated OE-YAP cells. Scale bar: 25 µm (B) qRT-PCR confirms WIF-1-mediated downregulation of β-catenin and its downstream targets c-Myc and Cyclin D1 in OE-YAP cells. (CD) WIF-1 treatment abolishes YAP-mediated enhancement of ALP activity and mineralization capacity. Scale bar: 100 µm. Data: mean ± SD; *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 9. In vivo validation of YAP-mediated dentin regeneration.

(A) Immunohistochemistry of implanted DPSCs grafts shows OE-YAP upregulates dentinogenic markers (DSPP, DMP1, RUNX2, OCN, and ALP) and β-catenin compared to controls. Scale bar: 100 µm (100×); 25 µm (400×). (B) Quantification confirms 1.36- to 1.62-fold induction of odontogenic markers and 1.27-fold β-catenin increase in OE-YAP grafts. (C) Schematic illustration of the tooth slice/scaffold design for ectopic transplantation. Data: mean ± SD; *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.