Study On Bone Mechanosensitivity In Osteocyte-specific NFATc3 Gene Knockout Mouse

As a load-bearing organ of the body,bone is stress sensitive.In order to respond to the changes of external biomechanical loading,bone can initiate targeted bone remodeling to dynamically adjust its own structure,so as to achieve the optimal state.Mechanical loading is considered to be one of the main driving factors of bone mechanical adaptation.Only physiological moderate stress stimulation can effectively promote the net increase in bone mass,as well as maintain bone microstructure,matrix mineralized level and load-bearing capacity.As the most abundant cells in bone,osteocytes(OCY)are not only the core stresssensitive receptors,but also the regulators of bone metabolic process.OCY lives in an interstitial fluid environment within the bone lacunar-canalicular system(LCS),the pressure gradient generated by external stress stimulation can drive fluid flow and produce a "magnified" fluid shear stress(FSS)on the surface of OCY.The previous study of our research group has found that when OCY senses FSS stimulation,it will produce a unique multi-spike calcium oscillatory response in the cytoplasm and the calcium oscillations will be decoded and interpreted by the downstream decoder.Transcription factor NFAT has been proved as a decoder sensitive to low-frequency calcium oscillations,which regulates downstream genes by dephosphorylation and translocation into the nucleus.Numerous studies have shown that NFAT can regulate the activities and functions of OB and OC,and recent results combining WB and immunofluorescence experiments of our group have revealed that the NFATc3 isoform acts as the core decoder of calcium oscillations in OCY.However,there are no relevant studies systematically describe the role of NFATc3 in skeletal mechanical signal transduction.In summary,this study aimed to utilize an OCY-specific NFATc3 knockout mouse model and investigated the changes of bone phenotype and metabolism in normal and NFATc3 knockout mice under physiological loading condition and simulated microgravity through in vivo and in situ experiments.Then,in vitro FSS stimulation was applied to the extracted primary OCY in two groups,and the effects of NFATc3 gene on osteocytic calcium signaling response,activity and cytokine expressions were explored.This study provides a theoretical and experimental basis for systematically elucidating the important scientific question regarding how the skeleton decodes and transduces external stress stimuli,enriches the understanding of bone mechanical adaptation mechanisms and mechanical signal transduction under various mechanical environments,and also provides an important reference for skeletal diseases with OCY as target cells,which is expected to yield high economic and social benefits.The research is mainly divided into the following three parts:Part Ⅰ: Study on the mechanical sensitivity of bone in NFATc3 knockout mice under normal stress environment Backgrounds: Mechanical stimulation is an important regulator of bone metabolism.Moderate stress stimulation higher than the minimum threshold can promote bone mass and bone quality through a dose-dependent bone formation response.NFATc3 isoform has been screened as the core decoder in OCY,and it is of great scientific significance to clarify the role of NFATc3 in bone mechanical adaptation and homeostasis regulation and to explore its potential mechanisms under normal stress environment.Methods: Firstly,we purchased osteocyte-specific NFATc3 gene knockout mice,which were established by Cre-LoxP site-specific recombinase system.Mice tail identification and NFATc3 immunohistochemical staining were used to verify the genotype and knockout efficiency.Secondly,two-week physiological axial compressive loading was applied to the right tibiae of normal(WT group)and gene knockout(NFATc3-cKO group)mice using a rodent long bone mechanical loading device,with the left tibiae as the control.Then,the bone phenotypes and bone remodeling-related parameters of the tibiae in two groups were analyzed by using Micro-CT scanning,immunofluorescence double labeling,toluidine blue staining,Trap staining,H&E staining and immunohistochemical staining.Finally,the tibiae of mice in two groups were extracted,and real-time calcium response of in situ OCY under dynamic loading was collected and analyzed.Results: The genotypes of the purchased knockout mice were all flox-homozygous containing Cre enzyme,and the NFATc3 positive rate of OCY in the tibiae of NFATc3-cKO mice decreased significantly,which proved the reliability of using these gene knockout mice in the follow-up experimental researches.The results of in vivo experiments showed that physiological axial cyclic loading could significantly improve bone mass and bone microstructure of tibial trabeculae,increase the thickness of cortical bone in the middle shaft of tibiae,enhance the number of OB and bone formation rate,inhibit OC formation,and reduce the number of atypical OCY in WT group.In addition,the expression of Sclerostin and RANKL in the loaded tibiae of WT group was significantly down-regulated,while the expression of OPG was significantly up-regulated,which promoted the bone anabolism of OB and inhibited the bone resorption function of OC.However,physiological compressive loading failed to exert statistically significant changes in bone phenotype,bone metabolism,OCY bioactivity and regulatory function of tibiae in NFATc3-cKO mice.Interestingly,obvious calcium oscillatory spikes of in situ OCY could both be observed in two groups when stimulated with compressive loading,and there were no statistical differences between the related calcium signaling parameters.Conclusion: Specific knockout of NFATc3 gene does not affect the ability of OCY to encode calcium oscillations upstream,but the positive effects of physiological mechanical loading are counteracted downstream due to a significant decrease in the amount of NFATc3 dephosphorylating and translocating into the nucleus,indicating a significant decline in stress sensitivity and mechanical response ability in NFATc3-cKO mice.This part of the experiment can enrich the knowledge of the mechanism of NFATc3 transcription factor in decoding and transducing mechanical signaling under normal stress environment.Part Ⅱ: Study on the mechanical sensitivity of bone in NFATc3 knockout mice under stress-deficient environment Backgrounds: Stress-deficient environments(e.g.,prolonged bed rest,limb paralysis,space flight)can trigger uncoupling of normal bone remodeling process and enhance bone resorption,which leads to rapid bone loss and progressive bone quality deterioration.The previous part investigated the role of NFATc3 in transducing normal mechanical stimuli,while the transduction effect of NFATc3 gene under simulated microgravity remains to be systematically described.This part of the study can provide new perspectives for further explaining the bone loss mechanisms in microgravity environment.Methods: Mice in the WT and NFATc3-cKO groups were hindlimb suspended with tail suspension cages for 4 weeks to simulate low gravity environment.Then,bilateral tibiae were taken out to systematically analyze the changes of tibial phenotype and metabolism in two groups under insufficient loading condition.The relevant evaluation indices were same as the first part of the study.Results: Compared with the mice raised under normal gravity,after 4 weeks of hindlimb suspension,the bone mass of proximal tibial trabeculae in WT group was significantly decreased,the microarchitecture was obviously destroyed,the bone mineralized deposition was significantly inhibited,the number of OB on the surface of trabeculae was significantly decreased,the number of OC in proximal tibiae and the empty lacunae of OCY were significantly increased.The results of immunohistochemical staining revealed that the expression of Sclerostin and the ratio of RANKL/OPG were both significantly up-regulated,suggesting that bone anabolism was significantly inhibited and the bone resorption activity was significantly increased.The specific knockout of NFATc3 gene aggravated the detrimental effects of simulated microgravity on bone mass and bone microstructure,and could further inhibit the increase of OB number and promote OC formation.By further reducing the survival rate of OCY,its ability to regulate OB and OC suffered greater damage.Conclusion: Specific knockout of NFATc3 gene can exacerbate the extent of bone loss under weightlessness,and bone homeostasis and mechanical sensitivity are more severely compromised.This part of the experiment further proves the important role of NFATc3 transcription factor in transducing bone mechanical signals.Part Ⅲ: Effects of NFATc3 on mechanical signal transduction and biological activity of in vitro primary OCY under normal stress environment Backgrounds: FSS generated by interstitial fluid flow is an intrinsic driving force of mechanical signal transduction in bone and has a positive regulatory effect on bone formation.Based on the first part of the experiment,it is important to further investigate how NFATc3 regulates OCY bioactivity and bone remodeling in vitro and transduces the biomechanical signals under physiological level of FSS stimulation.Methods: After extracting the primary OCY from the long bones of two groups of mice,the physiological FSS with 2 Pa was applied to the primary OCY for 3 hours by using the self-made in vitro FSS loading system.Then,the primary OCY in each group was collected,and the viability and expression levels of important cytokines were analyzed by CCK-8,qRT-PCR and WB assays.In addition,the primary OCY was incubated with calcium probe,and dynamic calcium signaling response of OCY induced by in vitro FSS was collected.Results: Compared with the cells statically cultured for 3 hours in the same environment,physiological FSS could significantly increase the cell viability of primary OCY in Ctrl group,but this up-regulation effect could not be observed in NFATc3-cKO group.The results of qRT-PCR assay showed that,at the gene expression level,as the primary OCY in Ctrl group,in vitro physiological FSS stimulation could also significantly alter the expression levels of bone remodeling-related factors in the primary OCY of NFATc3-cKO group,indicating that NFATc3 knockout did not counteract the positive effects of FSS loading.The results of WB assay revealed that,at the protein expression level,the downregulation effect of FSS stimulation on the expression of some negative cytokines(RANKL,Dkk1,Sclerostin)regulating bone remodeling in primary OCY was significantly inhibited after NFATc3-cKO,which offset the positive effects of FSS stimulation on promoting OB activity and inhibiting OC differentiation.As in the first part of the experiment,both groups of in vitro primary OCY could exhibit obvious calcium oscillatory response after receiving FSS stimulation.Conclusion: Conditional knockout of NFATc3 gene can counteract the promoting effect of FSS stimulation on OCY viability and inhibit OCY from exerting regulatory functions on targeted factors during the translation phase,which is consistent with the results of in vivo animal experiments in the first part.This part of the experiment provides a new perspective for us to understand the biological response and transduction process of OCY induced by FSS stimulation.