Essential Role Of Cofilin In Regulating NF-?B Nuclear Translocation On Osteoblast Osteogenesis Under Mechanical Strain

Mechanical stimulation plays an essential role in bone metabolism.The skeleton has the ability to perfectly adapt to external forces of the operating environment,by altering its morphology and metabolism in order to meet different needs.The maintenance of bone mass relies on mechanical stimulation,and at the same time the bone mass adapts to mechanical stimulation.Proper strain can stimulate the growth and reconstruction of bone tissue,promote new bone formation in many clinical treatments,such as distraction osteogenesis,fracture repair,orthodontic tooth movement and so forth.Repeating and increased loading,such as regular exercise,can also induce new bone formation,whereas lack of mechanical stimulation such as prolonged bed rest,neuromuscular palsy,or space flight,will cause the loss of bone mass.Over the past few decades,osteoblasts have become widely recognized as mechanical-sensing cells and effector cells within the bone.Osteoblasts coordinate the remodeling process by converting external mechanical forces into biochemical reactions,a process known as Mechanical transduction process.Exploring the biomechanical signal transduction mechanism of osteoblasts is of great significance for clarifying the mechanism of bone remodeling and finding new targets for regulating bone growth.NF-?B is one of the most important transcription factors involved in bone metabolism,regulating inflammation,affecting innate and adaptive immune responses^1,2.In bone formation,NF-?B signaling is directly involved in osteoclast differentiation and activation ^3,4.Chronic NF-?B activation affects mesenchymal stem cell?MSC?differentiation,weakens osteoblast-mediated bone formation ⁵.Many studies suggest that NF-?B is involved in intracellular processes under mechanical stimulation.So,what is the role of NF-?B in osteoblasts under mechanotransduction?In our previous work,we performed differential proteomic analysis of osteoblasts before and after mechanical loading,in which the expression of Cofilin was significantly increased after loading,and it was Clarified the positive effect of Cofilin on osteoblastic osteogenic differentiation in mechanical stress stimulation⁹.The nuclear translocation of RelA/p65?NF-?B subunit?requires a dynamic alteration of the actin cytoskeleton.Interfering with the actin cytoskeleton changes inhibits RelA/p65 nuclear aggregation¹⁰.Therefore,actin cytoskeletal remodeling may affect NF-?B activity and participate in a series of reactions.Cofilin1 is an actin-binding protein that plays an important role in actin filament dynamics and recombination by stimulating actin filament cleavage and de-polymerization¹¹.In combination with our previous experiments,Cofilin is also a mechanosensitive protein,which works as an important factor affecting the regulation of cytoskeletal proteins.Combined with the literature review,we speculate that Cofilin plays a key role in the regulation of NF-?B in osteoblasts.In view of the above considerations,we designed two parts of the study:Part 1.Effects of NF-?B on Osteoblast Osteogenesis under Mechanical Strain ObjectiveTo explore the effect of NF-?B on Osteoblast Osteogenesis under 12%static mechanical strain.MethodsFirst,the lentiviral transfection method was used to establish the models of RelA?the major subunit of NF-?B?over-expression and RelA interference in osteoblasts MG63.Subsequently,Flexcell mechanics loading system was used to load 12%of the static strain on MG63 cells with different RelA expression.This study set 1h,4h,8h,12h different force duration,and set non-force control group.After that,all the cells were collected all the cells,enzyme kinetic assay was used to detect the expression of alkaline phosphatase in different groups of cells.Alizarin red staining was used to detect the formation of calcium nodules.Elisa detected the secretion of osteocalcin and type I collagen.The expression of Runx-2,Osterix and collagen I were detected by real-time quantitative RT-PCR.ResultsIn MG63 group with RelA overexpression,the osteoblast-related proteins and gene expression were different in different loading groups after 12%SMS loading.With the extension of time,the expressions of osteoblast-associated ALP,osteocalcin and type I collagen showed a slight increase,then decreased.Compared with MG63 cells,the three osteoblast-associated proteins in RelA overexpression group tended to increase weakly and required longer stretch stimulation,and the expression of calcium nodules was also significantly lower than that in MG63 group.The gene expression of Runx-2,Osterix and type I collagen was detected by RT-PCR,and the results showed similar trend with the experimental results of protein expression.However,in RelA interference group,the expression of osteogenesis-related proteins was significantly higher than that in normal cells,and the expression of osteogenesis-related genes was also much higher than that in normal cells.At the same time,only a relatively short duration of force was required.Osteogenesis-related protein and gene expression were significantly increased after 1h;ConclusionUnder 12%static strain stimulation,the osteoblast's osteogenic properties changed significantly.The 12%static strain at different time had different effect on the osteoblastic performance.NF-?B plays an important negative regulatory role on the osteogenic properties of osteoblasts under mechanical stimulation.This provides a theoretical basis for understanding the mechanism of action within osteoblasts stimulated by mechanical mechanics and also improves the overall understanding of the role of NF-?B in osteoblasts under mechanical strain.Part 1.Mechanism of Cofilin in osteoblasts for regulating NF-?B under mechanical strain.ObjectiveTo investigate the relationship between Cofilin expression and NF-?B activity in osteoblasts under mechanical strain and confirm that mechanical strain can promote osteogenesis by up-regulating Cofilin expression and then inhibiting NF-?B activity.MethodsThe lentiviral transfection methods were used to upregulate and interfere with the expression of Cofilin in osteoblasts MG63 respectively.The cells were load ed with12%static strain,and set 15min,30min,45min,60min different force duration,while non-force control group was set.After that,we collected all the cells.The expression of IkB?,p-IkB?,IKK and p-p65 and the expression of p-p65 in nuclear proteins were detected by Western Blotting.The dual-luciferase reporter gene was used to verify the ability of NF-?B to bind to promoter DNA and to verify the regulatory effect of Cofilin on NF-?B.ResultsCompared with MG63 group with Cofilin overexpression and Normal MG63 group,NF-?B inhibitory protein I?B?decreased faster and more obvious in Cofilin interference group.P-p65 also quickly gathered in the cells and as a result of nuclear protein WB,p-p65 clearly showed an intranuclear aggregation shift.This indicated that the activation of NF-?B in Cofilin-interference group occurred rapidly and remained active under different time-consuming application of force.The result of dual luciferase report also confirmed that NF-?B in Cofilin interference group had strong binding ability with promoter DNA,and it could increase the activity of NF-?B only by a short time of mechanical stimulation.ConclusionUnder 12%static tension,the activity of NF-?B in osteoblasts changed significantly.Appropriate mechanical stimuli could activate NF-?B effectively,and the highest activity of NF-?B was found in osteoblasts at 30 min,and the binding ability to promoter DNA was the strongest,too.Under mechanical strain,Cofilin played an important role in NF-?B performance.The mechanical strain could inhibit the activity of NF-?B by up-regulating the expression of Cofilin and further promote the osteogenic differentiation of osteoblasts,which confirmed the existence of Cofilin/NF-?B in osteoblasts.