The Effect Of Exercise-mediated Force-sensitive Factors YAP/TAZ On Bone In Mice

Research Background: Exercise can cause external mechanical on the bone,such as pulling,twisting and squeezing.The mechanical stimuli can be transmitted to the cells through the mechanical sensing receptors on the cell membrane and converted into biochemical signals for metabolic regulation.The Hippo pathway affect the modeling and remodeling of bone by regulating osteoblast differentiation in vitro,but the mechanism action on the Hippo-YAP/TAZ pathway in vivo is still unclear.As the force-sensitive factors,YAP/TAZ can affect the proliferation and differentiation process of bone marrow stem cells by sensing external mechanics stimulation.Recently,it has been found that under mechanical stimulation,the conformational changes of GPCRs can directly activate intracellular YAP/TAZ,causing osteoblast differentiation.Based on the background,this study intends to explore how the exercise regulates bone metabolism through external mechanical stimulation,especially through the ‘external mechanical-GPCRs-YAP/TAZ’ pathway.Besides,in order to provide a new perspective about exercise prevention and treatment osteoporosis by biomechanics.Research purposes: The aim of this study was to observe the changes in the mechanical sensitivity factor YAP/TAZ and to verify the effect on bone tissue.The mechanism of exercise-induced YAP/TAZ on bone tissue was explored by both positive and negative modes(exercise or tail suspension).Research methods: 50 male C57BL/6 mice of 4-week old were randomly divided into 5 groups.Including control group(C),down-jumping group(D),running group(R),swimming group(S)and tail suspension(TS).After the first week of adaptive growth,Group C had no treatment and naturally grew for 8 weeks.The frequency of group D was 6-7 times/min.Group R exercise on a treadmill in slope-9° and 0.8km/h.Group S was swimming at a temperature of 30±2°C,100cm×60cm×40cm water box.Group TS naturally grew in the first 5 weeks,tail suspension in the last 3 weeks.The first week of the experiment began with adaptive exercise training.Except the rest day,the training time was fixed.At the end of the 8 weeks training,took the lower limb bone of seven mice to measure the left femur and tibia morphological indexes.The right femur was used for micro-CT and RT-q PCR to detect m RNA expression levels of GPR19,YAP,TAZ and Runx2 et al.In addition,cultured bone stem cells of two mice from each group.Research results:(1)Compared with group C,at the end of the eight weeks exercise,the weekly body weight of the three exercise groups was significantly lower.The weight loss of group D was the most significant(P<0.01).(2)Compared with group C,the wet weight of the femur(P<0.01)and the tibia(P<0.05)in the group R was significantly increased.The length of femur and tibia increased significantly in group D(P<0.05)and group R(P<0.01).The femur width increased significantly in group D(P<0.05),group R(P<0.01)and group S(P<0.05)and the width of the tibia increased significantly in every exercise group(P<0.01).(3)Compared with group C,the values of BV/TV and Tb.Th in group R increased significantly(P<0.01),while the values of BV/TV and Tb.Th in group TS decreased significantly(P<0.05).The BV/TV parameter values of cancellous bone in group R increased significantly(P<0.05)and the value of Th.Sp decreased significantly(P<0.01).While the values of BV/TV decreased significantly(P<0.01)and Th.Sp increased significantly(P<0.05)in group TS.(4)Compared with group C,serum ALP activity in group D(P<0.05),group R(P<0.05)and group S(P<0.01)was significantly increased,while in group TS was decreased significantly(P<0.05).TRAP activity in group D(P<0.05)and group R(P<0.01)was significantly lower,while in group TS was increased significantly(P<0.001).(5)Compared with group C,group D,R and S had positive ALP staining in 7 days,and group R had the strongest positive reaction.Group D,R and S had 14 days of Von Kossa staining mineralized nodules significantly increased.The number of osteoclasts obtained by TRAP staining in group D,R and S decreased,while the number of osteoclasts in group TS was the highest.(6)Compared with group C,the expression of GPR19 m RNA in group TS was significantly increased(P<0.001).The expression of YAP1 m RNA in group D(P<0.05)and R(P<0.01)were significantly increased,while in group TS was decreased significantly(P<0.01).The expression of TAZ m RNA in group D,R and S was significantly increased(P<0.01).The level of Runx2 m RNA in group D and R was significantly increased(P<0.01),while in group S was significantly decreased(P<0.05).(7)Compared with group C,the expression of ALP m RNA in group D and R was significantly increased(P<0.01).The expression of OCN m RNA in group D(P<0.05)and R(P<0.01)was significantly increased.The expression of DC-STAMP m RNA in group D(P<0.05)and R(P<0.01)was significantly decreased.The expression of TRAP(P<0.01),NFATc1 and DC-STAMP m RNA(P<0.001)in TS group were significantly increased,while the expression of ALP and OCN m RNA were significantly decreased(P<0.05).Conclusions:(1)Exercise has a significant effect on the growth and development of bone,and the effect of jumping downhill and swimming is more prominent;long-term braking is not conducive to bone growth.(2)External mechanical stimulation caused by exercise can up-regulate the transcription levels of YAP and TAZ in the Hippo pathway of osteoblasts;increase the activity of ALP enzyme and decrease the activity of TRAP enzyme,thereby promoting the differentiation and function enhancement of osteoblasts and inhibiting osteoclasts.The function of the cells.(3)The YAP and TAZ transcription levels decreased after the loss of external mechanical stimulation,which reduced the transcriptional expression of Runx2,ALP and OCN,and increased the transcriptional activities of GPR19,TRAP,NFATc1 and DC-STAMP.