A Coordinated Mechanism By Canonical Wnt And Hippo Signaling Pathway Regulates Osteogenesis Of Human Periodontal Ligament Stem Cells

Objectives:The purpose of this study was to investigate the effect of canonical Wnt signaling pathway on osteogenic differentiation of human periodontal stem cells and the expression of Hippo signaling pathway during this process.To investigate the molecular mechanism of canonical Wnt signaling pathway which mediates Hippo signaling pathway regulating the osteogenic differentiation of PDLSCs,to reveal the cross-regulation of two signaling pathways in the osteogenic differentiation of stem cells,and to provide theoretical basis for seed cell optimization in oral tissue regeneration engineering.Methods:Primary human periodontal stem cells were isolated and passaged.Flow cytometry was performed to identify the expression levels of surface markers on stem cells.By osteogenesis and lipogenesis induction,the multipotent differentiation ability of stem cells was detected.PDLSCs were respectively treated with recombinant proteins Wnta3a and dkk-1 to activate and block the canonical Wnt signaling pathway by q-PCR and Western blot upon the expressions of ?-catenin?Wnt3a?LRP5?DVL?Axin and GSK3?,and to detect the changes in the osteogenic capacity of PDLSCs and the components such as TAZ?LATS1 and Mobl in the Hippo signaling pathway.The transcriptional family TCF/LEF activity of canonical Wnt signaling pathway and the transcriptional family TEAD activity of hippo signaling pathway were detected by luciferase reporting system.Lentivirus transfection and rescue experiments were conducted,Wnta3a+shNC,Wnta3a+shTAZ,DKK-1+oeNC,DKK-1+oeTAZ four groups were designed to verify whether TAZ was a downstream target of the canonical Wnt pathway,and Co-IP was used to verify whether ?-catenin formed a protein complex with LATS1.Results:The activation of Wnt/?-catenin signaling pathway promoted bone formation and entailed osteogenic lineage differentiation of periodontal ligament stem cells(hPDLSCs),and exerted its osteogenic effect by inhibition of Hippo signaling pathway.Analysis of luciferase reporter assay and rescue experiment revealed TAZ,the transcriptional co-activators in the Hippo pathway,is key target of Wnt/?-catenin signaling pathway.TAZ expression counteracted the DKK-1-induced reduction in proliferation and osteogenesis.Moreover,TAZ knockdown offset the Wnt3a-induced augmentation of osteogenesis.Moreover,we found that ?-catenin interacted directly with LATS1 and formed a complex by co-immunoprecipitation,and that inhibition of ?-catenin or LATS1 by their dominant-negative forms dramatically suppressed the formation of this complex.Conclusions:These findings conjointly indicate Wnt/?-catenin signaling positively modulates osteogenic differentiation of hPDLSCs via the inhibition of Hippo signaling.Interaction between ?-catenin and LATS1 contributes to enhancing the cooperative effect of ?-catenin and TAZ on osteoblast differentiation.