The Molecular Mechanism Of Gαi1/3 Mediated BMP2 Signal Transduction And Osteogenesis

Research background and objective Bone morphogenetic protein 2(BMP2)plays an important role in bone development,bone homeostasis,and bone regeneration.Although recombinant human bone morphogenetic protein(rhBMP2)has been used in clinical fracture treatment and spinal fusion,it may cause a series of side effects such as heterotopic ossification,osteolysis caused by abnormal activation of osteoclasts,and bone cysts.How to enhance the function of endogenous BMP2 is very important.The previous research of our group found that the G protein inhibitory a subunit 1/3(Gail/3),which is only responsible for GPCR signal transduction,can mediate a variety of tyrosine kinase receptor signal transduction(VEGFR,TrkB,IL4R,etc.).BMP2 mainly activates multiple scaffold proteins through receptor serine/threonine kinase(RS/TK),activates Erk-MAPK,PI3K-Akt and Smads signals,thereby promoting the survival,regeneration and bone formation of osteoblast-related cells.However,how BMP2 activates downstream signaling pathways and whether there is a key protein mediation still needs further research.In this paper,we first clarify the role of Gαil and Gai3(Gail/3)in bone metabolism;then explore the role and molecular mechanism of Gail/3 protein in BMP2 downstream signal transduction;finally explore the role of Gail/3 protein in BMP2 mediated The key role in osteogenesis and bone regeneration.Methods Part One:Collected 20 patients with fragmented bone tissue during fracture surgery in the Department of Orthopedics,Second Affiliated Hospital of Soochow University.According to the bone density,β-CTx and PNIP test results,the patients were divided into normal group and osteoporosis group.The expression level of Gail and Gai3 mRNAin bone tissue were detected by polymerase chain reaction(PCR);the expression level of Gail and Gai3 protein in bone tissue were detected by Western blot.Breed wild-type(WT)and Gail/3 knock-out(DKO)mice,and stain the bones of the three days after birth through the doublestaining experiment of bones to analyze the bone development of WT and DKO mice.MicroCT was performed on the femurs of 6W and OW mice to analyze the bone mass in WT and DKO mice.At the same time,the mouse femurs were stained with H&E and Masson to observe the number of trabecular bones.The load capacity of femur of DKO mice was tested by three-point stress test.Finally,isolate the primary mononuclear macrophages(Bone marrow-derived macrophages,BMDMs)derived from the bone marrow of WT and DKO mice,induce osteoclast formation,process TRAP staining,and observe the effect of Gail/3 on osteoclast differentiation.Part Two:Using CRISPR/Cas9 and other molecular biological methods to regulate the expression of Gail/3 in mouse embryo fibrogenesis cells(MEFs),osteogenic precursor cell line(MC3T3-E1)and primary mouse bone marrow mesenchymal stem cells(BMSCs).Above cells were treated with BMP2(20ng/mL),the expression of proteins related to ErkMAPK,PI3K-Akt and Smads signaling pathways were detect by Western blot.At the same time,the expression of adaptor protein Gab1 in the above cells was detected.Knock out the adaptor protein Gab1(Gab1-KO)in MEFs,treat WT-Gab1 and Gab1-KO for different time with BMP2(20ng/mL),and detect the activation of Erk-MAPK,PI3K-Akt and Smads signaling pathways in cells by Western blot.The Western blot bands were quantitatively analyzed by ImageJ software.Part Three:Establish Gail/3 knockdown/overexpression MC3T3-E1 or BMSCs and negative control cells by lentivirus transfection,adenovirus transfection and other technologies.BMP2(20ng/mL)treated Gail/3 knockdown MC3T3-E1 cells for different times.qPCR detects the expression levels of Runx2,OPN,Sp7,ALP genes in the cells.BMP2(20ng/mL)treated MC3T3-E1/BMSCs cells with Gail/3 knocked down at different times,and Western blot was used to detect the expression of Runx2 and Sp7 in the above cells.BMP2 induced osteogenic differentiation of the above-mentioned MC3T3-E1 and BMSCs.After 21 days,the formation of calcium salt nodules was detected by Alizarin Red,and the secretion of alkaline phosphatase(ALP)was detected after 14 days.Construct WT,DKO mouse femoral fracture models,Micro-CT scan,H&E,Masson staining to detect the bone regeneration at the fracture;immunohistochemistry to detect the expression of Runx2,Sp7,OPN in the new bone tissue.Results Part One:The expression of biochemical indicators related to bone formation(PINP)and osteoclast-related biochemical indicators(β-CTx)in the blood of osteoporosis patients increased;the mRNA and protein expression levels of Gai 1 and Gαi3 in osteoporotic patients Elevated.Gail/3 knockout(DKO)mice have delayed skeletal development,cranial hypoplasia,and short clavicle;the bone mass and bone mass of adult and old DKO mice are lower than those of WT mice of the same age.Knockdown of Gail/3 has no effect on osteoclast differentiation of BMDMs.Part Two:In MEFs/MC3T3-E1/BMSCs,Gail/3 knockout or silencing can significantly inhibit BMP2-induced activation of Erk-MAPK,PI3K-Akt and Smads signals.Knockout of Gail or Gai3 partially inhibited BMP2-induced signal pathway activation.Knockout of Gαi2 has no effect on the signal activation induced by BMP2.Exogenous introduction of Gail or Gαi3 into Gail/3 double-knockout MEFs can partially restore the effect of BMP2-induced signal transduction;conversely,overexpression of Gαi1/3 promotes BMP2-induced Erk-MAPK,PI3K-Akt and Smads activation.The deletion of Gail/3 affects the phosphorylation of Gab1 induced by BMP2;knocking out Gab1 inhibits downstream signal transduction induced by BMP2.Part Three:Knockdown of Gail/3 in MC3T3-E1/BMSCs inhibits the expression of Runx2,Sp7 and other osteogenesis-related genes.After knocking down Gail/3,the ability of BMP2 promoting calcium nodule formation and ALP secretion was weakened.Overexpression of Gail/3 can increase the effect of BMP2 on the osteogenic ability of MC3T3-E1.In vivo experiments found that the bone regeneration of DKO mice was weaker than that of WT mice after fracture,and the expression of Runx2,OPN,and Sp7 in the new bone tissue decreased.Conclusions (1)Gαi1/3 is highly expressed in the bone tissue of patients with osteoporosis.The loss of Gail/3 will affect bone formation and bone quality,and Gail/3 may be involved in osteogenesis.(2)Gαi1/3 as a key protein mediates BMP2 to activate downstream Akt-mTOR,Smads,Erk-MAPK signaling pathways;Gαil/3 protein is essential for the activation of Gabl,Gail/3 can activate Akt-mTOR through Gab1,Smads,Erk-MAPK signal.(3)Gail/3 regulates the osteogenic effects of BMP2 in promoting osteogenesis-related cells,including calcium salt formation and the expression of osteogenic related proteins;Gail/3 deletion inhibits the promoting effect of BMP2 on fracture healing.