Study On The Role And Mechanism Of Focal Adhesion Proteins Pinch1/2 In The Regulation Of Bone Homeostasis

Osteoporosis is a systemic skeletal disease characterized by the decrease of bone mass and the microarchitectural deterioration of bone tissue,leading to an increment of bone fragility and fractures risk.Nowadays,it causes huge financial burden and consumes a lot of medical resources with ageing of the population.The body maintains bone mass homeostasis through continuous bone remodeling,in which osteoclasts break down the old bone matrix while osteoblasts complete bone reconstruction.Osteocytes are the most abundant cells in bone tissues which derived from bone marrow mesenchymal stem cells,and they make a great contribution to bone homeostasis maintainence in physiological and disease states.Osteocytes can secrete and express many key factors,such as RANKL,OPG,and Sclerostin,etc.controlling the formation and function of osteoclasts and osteoblasts.Osteocytes are long-lived cells and some will die by apoptosis,however,the key factors controlling their function and survival are still unknown until now.Mammalian cells have two functional PINCH proteins,PINCH1 and PINCH2,which play significant roles in cell and extracellular matrix adhesion,cell migration,proliferation,and survival.The role of PINCH1 and PINCH2 in regulating bone homeostasis is unclarified.This thesis aims to study role regulation of PINCH on regulation of bone homeostasis to obtain new clues to the prevention and treatment strategies for osteoporosis.To exam the role of PINCH in regulating bone homeostasis,mouse model with osteocyte Pinch deletion is analyzed.Micro-CT examination of bone mass showed that osteocyte Pinch deletion lead to significant bone loss especially bone mineral density.Bone mass is controlled by osteoblast-mediated bone formation and osteoclast-mediated bone resorption.In our study,we found that bone formation was significantly reduced by the loss of Pinch in osteocyte measured by calcein double labelling,but bone resorption was not affected detected by TRAP staining.Although the Pinch deletion in osteocytes lead to an up-regulation of Rankl expression,Opg also has a proportional up-regulation.Furthermore,single knockout of Pinch1 or global knockout of Pinch2 didn't affect bone mass,indicating that Pinch1 and Pinch2 had functional compensation in regulating bone remodeling.Bone loss in osteocytic-Pinch deletion mice was due to changes in bone formation.The molecular mechanism of Pinch regulating bone homeostasis was further explored in the thesis.We found that Pinch1 could directly bind to Igf1 r to partially affect Sclerostin expression.This result was also verified by CRISPR-Cas9 technology to knockout Pinch1 in MLO-Y4 cells in vitro.The high expression of Sclerostin in osteocytes affected the differentiation of bone marrow mesenchymal stem cells,as well as inhibited the Wnt/?-Catenin signaling pathway in primary osteoblasts,which played an important role in bone formation.Meanwhile,the deletion of osteocyte Pinch changed the microenvironment of the bone marrow cavity,resulting in enhanced adipogenic differentiation and weakened osteogenic differentiation of mesenchymal stem cells,and this could be partially explained by the decreased expression of Yap1 / Taz in bone marrow mesenchymal stem cells.As for the osteocytes,Pinch deletion resulted in increased apoptosis without affecting autophagy caused by the low expression of integrin.Osteocytes are mechanical sensing devices for long bones.To investigate whether Pinch is involved in the mechanical signal transduction of osteocytes,two mouse models,of hindlimb unloading and ulnar loading model,were utilized in the experiments this study.In the hindlimb unloading model,mice with osteocyte Pinch deletion exhibited more severe bone loss.However,in the ulnar loading model,the thickness of the bone matrix could be increased to cortical bone in control mice,and this increase was lost in mice with osteocyte Pinch deletion.Thus the response of osteocytes to mechanical signal was decreased by osteocytic Pinch deletion.In summary,this thesis describes the key role of Pinch protein in regulating bone homeostasis and bone mechanical transduction.Our results indicates that modulate osteocyte Pinch expression could be a potentially strategy for prevention and treatment of human metabolic bone disease.