LRP6/filamentous-actin Signaling Mediates Osteogenic Commitment In Force-Induced Periodontal Ligament Stem Cells

Background and objectiveAlveolar bone can experience a remodeling process in response to mechanical stress from orthodontic loads to establish new homeostasis,which is mainly influenced by mechanoadaptive events from the periodontium.Periodontal ligament stem cells(PDLSCs),the main functional osteoprogenitors in the periodontal ligament(PDL),are involved in adaptive activities by mechanosensing and signaling.Specifically,PDLSCs perceive biophysical stimuli and convert them into the osteogenic niche,which directly acts on cellular self-renewal and differentiation and consequently on adjacent alveolar bone turnover.To date,mechanotransduction mechanisms whereby PDLSCs translate mechanical stress into biochemical signals and thereby trigger osteogenic programs necessary for alveolar bone remodeling are being deciphered.In the past years,our insights into force-instigated intracellular signaling cascades that regulate osteogenic activities in PDLSCs have gained momentum.However,knowledge on mechanosensors that link extrinsic stress to intrinsic biochemical response is limited.Low-density lipoprotein receptor-related protein 6(LRP6)is a WNT transmembrane receptor that plays a range of important functions in cell mechanosensing by activating the Wnt/β-Catenin signaling.Recently,LRP6 has been qualified as a key monitor for tooth development,oligodontia,and oral squamous cell carcinoma.However,it remains to be clarified whether LRP6 can act as a key mechanosensor of PDLSCs to sense mechanical stimuli and subsequently regulates PDLSCs osteogenic commitment.This study aimed to illuminate key sensors mechanosignaling that regulate osteogenic activities in mechanically induced PDLSCs during orthodontic tooth movement.To provide the biological basis for engineering mechanotargeting drugs that target the control of alveolar bone remodeling and attenuate relapse of orthodontic treatment.Materials and methods(1)The effect of mechanical force loading on the LRP6 expression in PDLSCs and periodontal ligament tissues.Human PDLSCs were derived and cultured by the explant culture methods.The model of PDLSCs was successfully established.Crystal violet staining was performed to evaluate the colony-forming ability of PDLSCs.Flow cytometry was adopted to determine and analyze the expression of the mesenchymal marker of PDLSCs.Alizarin Red Staining and Oil Red O staining were used to assess the multidirectional differentiation of PDLSCs.The Flexcell FX-5000TM Cell Stretching Bioreactor and tooth movement model were first established to determine the expression profile of LRP6.Western blotting(WB),quantitative real-time PCR(qRT-PCR),and cellular immunofluorescence were used to determine the LRP6 expression of PDLSCs.Tissue immunofluorescence was adopted to determine the LRP6 expression of periodontal ligament tissues.Hematoxylin-eosin staining(H&E staining)and micro-Computed Tomography(micro-CT)were adopted to the built of tooth movement model.(2)The role of LRP6 on force-induced osteogenic commitment of PDLSCs.The loss-of-function assay was used to investigate the role of LRP6 on force-regulated osteogenic commitment in PDLSCs.Alkaline phosphatase(ALP)staining,ALP activity assay,WB,and qRT-PCR were used to estimate the ability of osteogenic differentiation of PDLSCs.Immunofluorescence,WB,and qRT-PCR were used to estimate the ability of proliferation of PDLSCs.(3)Potential mechanisms underlying the role of LRP6 on mechanically induced osteogenic commitment of PDLSCs.Crystalline violet staining was used to visualize cell morphological change.Phalloidin staining,WB,and qRT-PCR were adopted to affirm filamentous actin(F-actin)alteration.YAP nucleoplasmic localization was assessed by immunofluorescence and WB.YAP transcriptional response was evaluated by qRT-PCR.Cytochalasin D(0.2 μg/ml)was used to determine the effects of F-actin on osteogenic commitment and YAP switch behavior in mechanically induced PDLSCs.In here,ALP staining,ALP activity assay,WB,and qRT-PCR were used to estimate the ability of osteogenic differentiation of PDLSCs.Immunofluorescence,WB,and qRT-PCR were used to estimate the ability of proliferation of PDLSCs.Results(1)LRP6 was robustly activated in mechanically induced PDLSCs and PDL tissues.(2)LRP6 loss impeded the ability of osteogenic differentiation and proliferation in force-dependent PDLSCs.(3)LRP6 knockout caused cell morphological aberration of mechanically induced PDLSCs.Intracellular F-actin showed significant elongation,disrupted arrangement,and broken continuity,as well as F-actin depolymerization in mechanically PDLSCs with LRP6 deficiency.Moreover,mechanical stimuli significantly facilitate F-actin polymerization,and disrupted F-actin polymerization impeded the ability of proliferation and osteogenic differentiation of mechanically induced PDLSCs.(4)In force-induced PDLSCs,LRP6 loss caused YAP nucleoplasmic relocation,YAP phosphorylation,and YAP inactivation.Similarly,F-actin depolymerization also caused YAP nucleoplasmic relocation,YAP phosphorylation,and YAP inactivation.Besides,LRP6 deficiency limited the activation of Wnt/β-Catenin signaling in mechanically induced PDLSCs.ConclusionWe identified that LRP6 in PDLSCs acted as the mechanosensor regulating mechanical stress-inducible osteogenic commitment via the F-actin/YAP cascade.We thus proposed a novel mechanism for LRP6 beyond the Wnt/β-Catenin signaling to regulate osteogenic activities.Furthermore,exploiting mechanotherapeutics that specifically target LRP6 may provide a promising avenue for anchorage maintenance and relapse reduction during orthodontic tooth movement.