Piezo1 Mediated The Transdifferentiation Between Osteoblasts And Bone Marrow Adipocytes

Temporomandibular joint osteoarthritis（TMJ OA）is the severe manifestation of temporomandibular joint disorder.Abnormal subchondral bone remodeling is one of its main pathological changes.Aberrant mechanical loading plays an important role in the occurrence of OA.Recently,we developed the unilateral anterior crossbite（UAC）mouse model which simulated the occurrence and development of TMJ OA.UAC-induced TMJ OA is reversible.After the removal of abnormal occlusion stimulation,the osteoarthritic changes dismissed,which provides a useful animal model for studying subchondral bone remodeling in TMJ OA.Bone marrow adipose tissue（BMAT）is the important part of bone marrow,accounting for 10%of the total body fat.Moreover,BMAT gradually accumulates with aging and its volume can reach to 70%of the bone marrow volume at age of 25 in human.During various physiological or pathological changes,BMAT and bone mineral density show the trade-off relationship.The loss of trabecular bone occurs at the early stage of OA.It has been reported extensively that the balance of osteoblast and osteoclast activity was the key factor in bone homeostasis,whereas the role of bone marrow adipocytes（BMAds）,abundant in bone marrow,still remained unclear.BMAds are the main cellular components of BMAT,differentiated from bone marrow mesenchymal stem cells（BMSCs）,the same as osteoblasts.Terminally differentiated cells derived from the same progenitors have plasticity among each other,which means that they could transform into each other under certain conditions.Whether there is transdifferentiation between BMAds and osteoblasts in bone remodeling during OA and its function and mechanism deserve further investigation.The extracellular matrix stiffness significantly affects cell fate.Piezo1,the mechanosensitive ion channel,senses the extracellular matrix changes and further affect cell activities.Whether Piezo1 and its downstream signaling pathways are involved in the transdifferentiation between BMAds and osteoblasts remains to be studied.In the study,we conducted the following studies to explore the response of Lep R⁺cells,the transdifferitiation between BMAds and osteoblasts,the key roles of Piezo1 and its downstream molecular pathways during the process of TMJ condylar subchondral bone remodeling.Part 1:UAC led to adipogenic differentiation of Lep R⁺cells in the TMJ condylar subchondral bone of miceMethods:1.Histochemical staining and Micro-CT were utilized to detect the histological changes of TMJ condyle cartilage and subchondral bone in UAC and UAC removal（UAC-RE）models.Moreover,q RT-PCR was used to detect changes in the expression of osteogenic and adipogenic genes of TMJ condylar subchondral bone in these two mice models.2.Leptin receptor lineage tracing(Lep R^{Td T})mice was selected to specifically label Lep R⁺cells to track the differentiation commitments under UAC stimulation using fluorescent staining.Flow cytometry was performed to obtain Td T^{Lep R+}cells in vitro to study the factor influencing the differentiation choice of Td T^{Lep R+}cells.Results:1.Under UAC stimulation,the area of medullary cavity was increased,accompanied with decreased ratio of bone volume to tissue volume（BV/TV）and trabecular thickness（Tb.Th）while the trabecular separation（Tb.Sp）increased.The m RNA expression of the molecules related to adipogenesis in TMJ condylar subchondral bone of UAC mice increased,including peroxisome proliferator-activated receptor gamma（Pparg）,fatty acid binding protein 4（Fabp4）and adiponectin（Adipoq）.However,the m RNA expression of molecules related to osteogenesis reduced,including Osterix（OSX）,osteocalcin（OCN）and dentin matrix acidic phosphoprotein 1（DMP1）.These changes reversed significantly after the removal of UAC.2.UAC stimulation obviously decreased the number of Lep R⁺cells and the percentages of OSX⁺Td T⁺or OCN⁺Td T⁺to Td T⁺cells while the percentages of BODIPY⁺Td T⁺or Adipo Q⁺Td T⁺to Td T⁺cells were significantly increased.In addition,the expression of Leptin in the subchondral bone of TMJ significantly reduced.Exogenous supplementation of Leptin increased BV/TV and Tb.Th while Tb.Sp reduced in UAC mice.The m RNA expression of Pparg,Fabp4 and Adipoq were significantly decreased,while that of Osx、Ocn and Dmp1 increased.Moreover,Leptin supplementation increased the percentages of OSX⁺Td T⁺or OCN⁺Td T⁺to Td T⁺cells and decreased the percentages of BODIPY⁺Td T⁺or Adipo Q⁺Td T⁺to Td T⁺cells.3.Td T^{Lep R+}cells were selected by flow cytometry.Lower level of Leptin promoted adipogenesis,presented with more Oil Red O-positive staining and higher m RNA expression of Pparg,CCAAT/enhancer binding proteinα（Cebpa）,Fabp4 and Adipoq.Moreover,higher level of Leptin promoted osteogenesis,presented with more alkaline phosphatase（ALP）-positive staining and higher m RNA expression of runt-related transcription factor 2（Runx2）,Alp,Ocn and Dmp1.Conclusion:1.UAC led to reversible changes of increased adipogenic and weakened osteogenic differentiation in TMJ condylar subchondral bone.2.The differentiation commitments of Td T^{Lep R+}cells were changed under mechanical loading and biological environment in the TMJ condylar subchondral bone,inclined to adipogenesis under UAC stimulation with lower Leptin.Supplementation of Leptin promoted the osteogenic differentiation of Td T^{Lep R+}cells,which also reversed TMJ condylar subchondral bone loss caused by UAC.Part 2:Piezo1-mediated the transdifferentiation of DMP1⁺osteoblasts into adipocytes in TMJ condylar subchondral boneMethods:1.DMP1-Cre^ERT2crossed with R26^{td Tomato}mice was used to specifically label mature osteoblasts to track the differentiation commitments of DMP1⁺cells under UAC.2.The MC3T3-E1 cell line was cultured on soft（elastic modulus:0.12±0.004 MPa）and stiff（elastic modulus:28.85±2.58 MPa）polydimethylsiloxane（PDMS）substrates respectively.Fluorescence staining,q RT-PCR and western blot were performed to detect the differentiation changes of osteoblasts and the molecular mechanism.3.Osteoblast-specific knockout of Piezo1 were obtained by crossing DMP1-Cre^ERT2 mice with Piezo1-flox mice.Histochemical staining,Micro-CT and fluorescent staining were used to explore the responses of TMJ condylar subchondral bone after specific knockout of Piezo1 in DMP1⁺osteoblasts to UAC stimulation.Results:1.UAC significantly increased the percentages of DMP1^{Td T+}BODIPY⁺and DMP1^{Td T+}Adipo Q⁺cells to Td T⁺cells.2.Compared with MC3T3-E1 cultured on the hard substrate,cells on soft substrate presented lower expression of Piezo1,accompanied with shrunken cytoskeleton.In the osteogenic differentiation-inducing environment,the cells cultured on the soft substrate formed fewer mineralized nodules with lower m RNA expression of Ocn and Dmp1,as well as lower AKT and ERK phosphorylation.Under the adipogenic differentiation-inducing environment,there were more lipid droplets with higher expression of Pparg,Fabp4 and Adipoq,accompanied with lower AKT and ERK phosphorylation.3.In the TMJ condylar subchondral bone of DMP1^{Cre ER};Piezo1^-/-mice,compared to that of DMP1^{Cre ER};Piezo1^fl/fl mice,marrow cavity area increased with decreased BV/TV,Tb.Th and increased Tb.Sp.However,the number of OSX and OCN positive cells decreased while the number of BODIPY、PPARγand FABP4 positive cells presented with no significant increase.4.UAC stimulation induced obvious TMJ condylar subchondral bone loss with increased number of BODIPY,PPARγand FABP4 positive cells in DMP1^{Cre ER};Piezo1^fl/fl mice.However,there was no significant difference in BV/TV,Tb.Th,Tb.N and Tb.Sp with the decreased number of BODIPY,FABP4 and PPARγpositive cells in DMP1^{Cre ER};Piezo1^-/-mice stimulated with UAC.5.BV/TV,Tb.Th,Tb.N and Tb.Sp of TMJ subchondral bone were no longer significantly changed in DMP1^{Cre ER};Piezo1^-/-mice under UAC stimulation compared to those without UAC stimulation.The number of OSX,OCN,BODIPY,PPARγand FABP4 positive cells also no longer changed significantly.Conclusion:1.DMP1⁺osteoblasts could transdifferentiate into BMAds in the soft matrix environment in bone marrow under UAC stimulation.2.Piezo1 plays the mediating role in the transdifferentiation of DMP1⁺osteoblasts to BMAds in softer matrix stiffness.The knockout of Piezo1 in DMP1⁺osteoblasts dismissed this transdifferentiation related to matrix stiffness.Part3:Piezo1 mediated the transdifferentiation of Adipo Q⁺adipocytes into osteoblasts during osteogenesis of TMJ condylar subchondral boneMethods:1.Adipo Q-Cre^ERT2crossed with R26^{td Tomato}mice was used to specifically label bone marrow adipocytes to track differentiation commitments of Adipo Q⁺cells under UAC-RE.2.The 3T3-L1 cell line was cultured on soft（elastic modulus:0.12±0.004 MPa）and stiff（elastic modulus:28.85±2.58 MPa）polydimethylsiloxane（PDMS）substrates.Fluorescence staining,q RT-PCR and western blot were performed to detect the differentiation changes of adipocytes and the corresponding mechanism.3.Adipocyte-specific knockout of Piezo1 were obtained by crossing Adipo Q-Cre^ERT2 mice with Piezo1-flox mice.Histochemical staining,Micro-CT and fluorescent staining were used to explore the responses of TMJ condylar subchondral bone after specific knockout of Piezo1 in Adipo Q⁺cells to UAC-RE and corresponding molecular mechanism.Results:1.UAC-RE decreased the number of Adipo Q^{Td T+}cells and significantly increased the percentage of Adipo Q^{Td T+}OSX⁺and Adipo Q^{Td T+}OCN⁺cells to Td T⁺cells in subchondral bone marrow of TMJ when compared to that of UAC mice.2.Compared with 3T3-L1 cultured on soft substrates,the expression of Piezo1 cultured on hard substrates was significantly increased.Under osteogenic differentiation induction,cells cultured on hard substrates formed more mineralized nodules,with higher m RNA levels of Ocn and Dmp1,as well as p-AKT and p-ERK.Cells cultured on the rigid substrate form less lipid droplets under adipogenic differentiation induction,expressing less Pparg,Fabp4and Adipoq while AKT phosphorylation and ERK phosphorylation were higher.3.In the TMJ condylar subchondral bone of Adipo Q^{Cre ER};Piezo1^-/-mice,compared to that of Adipo Q^{Cre ER};Piezo1^fl/fl mice,marrow cavity area enlarged with decreased BV/TV,Tb.Th and increased Tb.Sp.Moreover,the number of FABP4 and PPARγpositive cells decreased.4.UAC-RE reversed the loss of subchondral bone in Adipo Q^{Cre ER};Piezo1^fl/fl mice and significantly decreased the number of PPARγand FABP4 positive cells with increased OSX and OCN positive cells.However,after stimulation by UAC-RE,the TMJ subchondral bone morphological differences were no longer significant in Adipo Q^{Cre ER};Piezo1^-/-mice.Conclusion:1.Adipo Q⁺adipocytes could transdifferentiate into osteoblasts when the surface area of trabecular bone increased in the process of osteogenesis resulted from UAC-RE.2.Piezo1 mediates the transdifferentiation of Adipo Q⁺BMAds into osteoblasts in stiffer matrix.The knockout of Piezo1 in Adipo Q⁺BMAds dismissed this transdifferentiation related to matrix stiffness.In conclusion,the study explored the changes of Lep R⁺cells commitments,transdifferentiation between osteoblasts and adipocytes and also the mediated role of Piezo1in the process of TMJ subchondral bone remodeling through animal models,linage tracing,in vitro assays and tissue-specific Piezo1 knockout mice.The following conclusions were obtained.1.Under UAC stimulation,Lep R⁺cells obtained the reversibly enhanced adipogenic differentiation and weakened osteogenic differentiation in condylar subchondral bone of TMJ.2.In the TMJ condylar subchondral bone,DMP1⁺osteoblasts and Adipo Q⁺adipocytes can transdifferentiate into each other.3.Piezo1 mediated the transdifferentiation between DMP1⁺osteoblasts and Adipo Q⁺adipocytes associated with changes in matrix stiffness.