Preliminary Experimental Research On The Effects And Mechanisms Of NFAT On Mechanical Signal Transduction In Osteocyte

Skeletal disease is closely related to mechanical loading.The mechanical loading in physiological level is capable of maintaining and promoting bone mass.However,a lack of external mechanical loading on skeleton will induce a rapid loss of bone mass.The accumulation of bone micro damage can be induced when bone is subjected to fatigue overloading,which is very common in stress fracture.Therefore,it is of tremendous significance to deeply understand the mechanism of mechanical adaptation and how bone responds and transduces external stress stimulation.There are three kinds of cells in bone,namely osteoblast（OB）,osteoclast（OC）and osteocyte（OCY）.OCY was originally identified as a terminal differentiated inert cell which is deeply buried in bone mineralized matrix without any biological function.However,our team has discovered a unique multi-peak calcium oscillation in OCY under fluid shear stress（FSS）but a single calcium peak in OB for the first time in the world,and the sensitivity of OCY to stress was much higher than that of OB,which revealed how bone responded to external stress stimulation by calcium oscillation of OCY.However,it is still unknown how OCY decodes and transduces this multi-peak calcium oscillation.The current study firstly developed a set of general FSS loading system through the hardware design and computer programming,which established a scientific and effective experimental platform for cellular research of fluid mechanics.Secondly,we investigated and analyzed the effects of FSS on OCY in cellular morphology,biological activity and expression of related cytokines with this platform.The results indicated that FSS stimulation in physiological level could induce morphological variation and significantly inhibited apoptosis in OCY and regulatory effects on osteogenesis and bone resorption by activating Wnt//β-catenin signaling pathway and regulating RANKL/OPG.And FSS stimulation exerted a significant time-depended manner in the expression of bone-metabolism-related signal molecules secreted by OCY.Thirdly,we constructed a cellular model with NFAT signal blocked in molecular level and found that the positive effects of FSS in physiological level on biological activities of OCY had declined.Meanwhile,the Wnt/β-catenin pathway was significantly inhibited and the inhibitory effects of RANKL/OPG on bone resorption was also decreased after the suppression of NFAT signal.This research enriches the understanding of the role of calcium-oscillating-mediated NFAT signaling pathway in the mechanical signal transduction of OCY and also provides an important experimental basis for scientific community to systematically understand how bone responds to and transduces external stress stimulation.The whole research is divided into the following three parts.Part I:Development of fluid shear stress loading system based on LabVIEW.Backgrounds.At present,the cybernation part in the existing fluid shear stress（FSS）loading systems abroad can only regulate the magnitude of FSS with a minor amplitude and high expenses.The similar domestic devices exhibited non-conformant standards and large limitations,which cannot ensure the comparability of flow distribution.Thus,the current study developed a universal FSS loading system based on LabVIEW.Methods.The development of the hardware part of the FSS loading system was used by a peristaltic pump and a self-designed multichannel fluid chamber.The FSS mode controlling program based on LabVIEW was designed to control the FSS loading device via a computer with RS485 interfacing technology and Modbus protocols.Then the systematic standardization and FEM simulative analysis were conducted.Results.The current study developed a universal FSS loading system,which could provide three magnitude-adjustable loading modes by hardware and software parts.The results of systematic standardization of the relationship between shear force and rotary speed showed a high-positive linear correlation,indicating that the required FSS could be directly attained by computer setting and/or hardware adjusting.The FEM analysis demonstrated that the magnitude of FSS underneath of fluid chamber was homogeneous.Conclusion.The FSS loading system can control the fluid flow circularly and continuously via hardware and software and convey precise and stable flow rate.The development of the system has established a scientific and effective experimental platform for the studies of cellular hydromechanics,which is of vital importance of revealing FSS-induced cellular signaling transduction mechanisms to improve the treatment of related diseases and extensive application in histological engineering.Part II:Research on regulatory effects of FSS stimulation on morphology,activity and expression of related gene and protein of osteocytes.Backgrounds.Substantial studies have focused only on the effects of interstitial fluid shear stress（FSS）on instantaneous signaling response of osteocytes such as calcium signaling and many other intracellular messenger molecules of osteocytes.However,the effects of interstitial FSS on cellular function and bone metabolism-modulating cytokine expression of osteocytes have not yet been fully identified,and most previous studies only investigated the expression of these cytokines in the gene level with inconsistent and conflicting results and a lack of systematic quantification in the protein level.More importantly,it is still unknown whether the gene and protein expression of these bone-metabolism-modulating-related cytokines exhibits a time-dependent manner in response to interstitial FSS stimulation.Methods.Self-designed loading systems for cellular shear stress was used to exerted physiological fluid stimulation on osteocyte-like MLO-Y4 cell line.Cellular morphology,cytoskeletal microstructure,biological activity,and gene and protein expression of related cytokines（i.e.,RANKL,OPG,Wnt3a,β-catenin,SOST and DKK1）was systematically investigated.Results.Many dendritic pseudopodia around the cellular periphery after FSS stimulation were observed in the SEM and cytoskeleton staining images,with the orientation of most filopodia of the MLO-Y4 cells aligned with the direction of the fluid flow.Increased cellular volume and fusiform-like cell outline with elongated and dense microfilament was also observed after FSS stimulation.The total apoptosis rates were significantly decreased in the FSS-stimulated cells.What’s more,the expression of Wnt3a,β-catenin and OPG was prominently up-regulated while RANKL,SOST and DKK1 was significantly down-regulated after FSS stimulation with significantly different RANKL/OPG ratio in different stimuli time points.Conclusion.The cytoskeleton recombination,the filopodia formation and the inhabited of osteocytes apoptosis can be induced by physiological FSS stimulation.The Wnt/β-catenin signaling pathway can be activated,and the inhibiting effects of SOST and DKK1 on the signaling pathway can also be suppressed by FSS stimulation.Also,the bioactivity of osteoclasts and bone resorption can be regulated by inhibiting RANKL/OPG.Moreover,the external FSS stimulation exerts the potential time-dependent activation effects of these cytokines in osteocytes.This study offers proof-of-principle evidence for the experimental scheme of signal transduction mechanism of osteocytes and also provides a scientific and reasonable gist for the research of hydromechanical experimental design.Part III:Effects and mechanism of NFAT signaling pathway on cellular mechanical signal transduction of osteocytes.Backgrounds.NFAT is a significant factor in cytoplasm with decoding properties on calcium signaling of low frequency.The influential effects of NFAT on regulating genetic transcription and expression of osteoblasts and osteoclasts were already verified.However,the similar effects of NFAT on osteocytes,especially on stress-induced calcium signal transduction of osteocytes and bone-metabolism-modulating-related cytokines secreted by osteocytes have not been reported yet.Methods.NFAT signaling pathway was interrupted by Cyclosporine A and physiological FSS stimulation was applied to MLO-Y4 cells.Cellular morphology,bioactivity and gene and protein expression of related cytokines secreted by osteocytes was investigated.Subtypes of NFAT factors in osteocyte were quantified in gene and protein level using MLO-Y4 cells and tibia and cranium of matured mice,laying a foundation of the construction of lentiviral vector based on hyper-expressed NFAT subtypes and the study on the effects of FSS stimulation on biological characteristics of osteocyte using gene silencing technology.Results.The facilitated effects of FSS on the bioactivity of osteocytes and the Wnt/β-catenin signaling pathway were inhibited after the suppression of NFAT signaling pathway.The inhibiting effects of FSS-induced low-expressed RANKL/OPG on the differentiation of osteoclast and bone resorption were also declined.NFATc1 and NFATc3were found out hyper-expressed in osteocyte by PCR and immunofluorescence technology.Conclusion.NFAT has a decoding effect on FSS-induced osteocytes calcium oscillation.Calcium signaling affects intracellular gene transcription and expression via NFAT signaling pathway.The cytoskeleton-Ca²⁺-NFAT in bone cells is a critical signaling pathway of responding and transducing external mechanical stress.