Role And Mechanism Of GSK3?-mediated Apoptosis In Glucocorticoid Induced Osteonecrosis Of The Femoral Head

Glucocorticoids(GCs)are often used as drugs to treat some inflammatory and autoimmune diseases such as systemic lupus erythematosus and nephrotic syndrome.However,part of patients receiving long-term GCs therapy will develop osteonecrosis of the femoral head(ONFH),presenting with pain in hip,limitation of motion,trouble in walking.Steroid induced avascular necrosis of the femoral head(SANFH)can hardly be reversed by drugs and finally part of patients need a total hip arthroplasty(THA).Although the pathogenic mechanism of SANFH has been well studied,it can not be explained by a single theory up to now.To date,many theories tried to explain the pathogenic mechanism of SANFH.The theory of apoptosis have been well studied by researchers.Researchers pointed out that GCs induce apoptosis of osteocytes and osteoblasts directly,but induce ischemia indirectly.Other researchers pointed out that cell apoptosis other than cell death is associated with SANFH.Thus,GCs-induced apoptosis of osteocytes and osteoblasts is the important pathogenic mechanism of SANFH.Glycogen synthase kinase 3?(GSK3?)is a key regulator of multiple pivotal cell processes including glucose metabolism,cell cycle,signal transduction,gene transcription,gene translation,cell proliferation,cell survival and cell death.GSK3? can not only regulate cell differentation through the Wnt/?-catenin pathway,but also mediate apoptosis of various cell types including osteoblasts.GSK3?-mediated apoptosis might be associated with p53,Bax and ERK.It might be also associated with cyclophilin D(Cy P-D),adeninenueleotidetranslocase(ANT)and mitochondrial permeability transition pore(m PTP).GSK3? has also been reported to mediate apoptosis of osteoblasts.However,it is unknown whether GSK3?-mediated apoptosis plays role in SANFH and how GSK3?-mediated apoptosis of osteoblasts.In the present study,we established the model of rat SANFH to know whether GSK3?-mediated apoptosis plays role in SANFH.Also,a dexamethasone(Dex)-induced apoptosis of osteoblasts model was also established to investigate the mechanism of GSK3?-mediated apoptosis of osteoblasts.Using GSK3? shRNA and gene chip techniques,we screened the genes and pathways associated with GSK3?-mediated osteoblast apoptosis.And then,we study the effects of Piezo-type mechanosensitive ion channel component 2(Piezo2)on Dex-induced apoptosis of osteoblasts and the expression of OPG,RANKL of osteoblasts,communication betweenosteoblasts and osteoclasts.Part I GSK3?-mediated osteoblast apoptosis is involved in glucocorticoid induced osteonecrosis of the femoral head in rats Objective: To study the role and mechanism of GSK3? in glucocorticoid induced osteonecrosis of the femoral head in rats.Methods: An rat model of glucocorticoid induced osteonecrosis of the femoral head(ONFH)was established,and Li Cl(a GSK3? inhibitor)was used to feed rats.H&E staining and TUNEL staining were used to evaluate percentage of empty lacunae and apoptotic cells respectively.Bone loss were evaluated by micro-CT(bone volume per tissue volume(BV/TV),trabecular number(Tb.N),trabecular thickness(Tb.Th)and trabecular separation(Tb.Sp)).Western Blot was used to examine the protein expression of total GSK3?,p-Ser-9 GSK3?,Bcl-2 and Bax from femoral head.Results: ONFH group showed a high percentage of empty lacunae and apoptotic cells,whilst Li Cl treatment improved the situation.ONFH group showed bone loss,evidenced by decreased BV/TV,decreased Tb.N,decreased Tb.Th and increased Tb.Sp,whilst Li Cl treatment increased the bone mass.ONFH group showed decreased p-Ser-9GSK3?,decreased Bcl-2 and increased Bax,whilst Li Cl treatment reversed these effects.Conclusions: GSK3?-mediated osteoblast apoptosis is involved in glucocorticoid induced osteonecrosis of the femoral head in rats.Part II GSK3? mediates osteoblast apoptosis induced by dexamethasone via the mitochondrial pathway Objective: To investigate the mechanism of GSK3? in Dex-induced osteoblast apoptosis.Methods: Primary osteoblasts from rats were treated by Dex and MTT assay and FITC-Annexin V/PI assay were done to evaluated levels of cell viability and cell apoptosis respectively.Then GSK3?-siRNA was added to osteoblasts and GSK3?mRNA and protein expression were examined by q RT-PCR and Western Blot.Apoptosis was evaluated by TUNEL assay and FITC-Annexin V/PI assay after treatment with Dex and GSK3?-siRNA.Mitochondrial membrane potential was also evaluated.Protein levels of caspase-9,caspase-3,PARP and cytochrome C were also evaluated by Western Blot.Results: Dex inhibited proliferation and promoted apoptosis of primary rat osteoblastsin a dose-dependent manner.GSK3?-siRNA transfection decreased expression of GSK3? mRNA.Dex treatment decreased the expression of p-Ser-9 GSK3?.GSK3?-siRNA transfection decreased protein expression of both GSK3? and p-Ser-9GSK3?.Dex treatment increased percentage of TUNEL-positive cells and decreased mitochondrial membrane potential,whilst GSK3?-siRNA treatment reversed them.Dex treatment increased the levels of cleaved caspase-9,cleaved caspase-3,PARP and promotes the release of cytochrome C from mitochondria to cytoplasm,whilst GSK3?-siRNA treatment reversed them.Conclusions: GSK3? can mediate Dex-induced osteoblast apoptosis via the mitochondrial pathway.Part III Effect of GSK3?-shRNA on gene expression profiling of dexamethasonetreated osteoblasts Objective: To study the mechanism of GSK3? in Dex-induced apoptosis of MC3T3-E1 osteoblasts by using gene chip techniques.Methods: The stably transfected MC3T3-E1 cells expressing GSK3?-shRNA were obtained.Then cells were treated by Dex and divided into 3 groups: Control group,Dex groups and Dex + GSK3?-shRNA group.Next,apoptosis was evaluated by flow cytometer,and a DNA microarray was performed and gene expressions was analyzed.Some differentially expressed genes were further validated by q RT-PCR.Results: The stably transfected cells were obtained.Dex increased the apoptosis of osteoblasts while GSK3?-shRNA treatment decreased apoptosis.Results from gene chip analysis showed Piezo2,Hoxb8,Kif18 a,Dlk1,Tnfsf14,Casq2,Bcl2l14 genes and apoptotic pathway,MAPK pathway,TGF? pathway,Wnt pathway might be involved in the mechanism of GSK3? in the process of Dex-induced osteoblast apoptosis.Conclusions: GSK3?-shRNA treatment alters genes expression profiling of Dex-treated osteoblasts and diverse signaling pathways.Furthermore,Piezo2,Hoxb8,Kif18 a,Dlk1,Tnfsf14,Casq2 and Bcl2l14 genes might be associated with the mechanism of GSK3?-mediated Dex-induced osteoblast apoptosis.Part IV Roles of Piezo-type mechanosensitive ion channel component 2 in Dex-induced osteoblast apoptosis and the effect of it on OPG and RANKL expressions of osteoblasts Objective: To study the roles of Piezo-type mechanosensitive ion channel component 2in Dex-induced osteoblast apoptosis and the effect of it on OPG and RANKLexpressions of osteoblasts.Methods: Western Blot was used to evaluated the expression of Piezo2 in MC3T3-E1 osteoblasts treated by Dex and GSK3?-shRNA.Then OPG and RANKL protein levels were examined after knockdown of MC3T3-E1 cells by Piezo2-siRNA.Next,apoptotic rate of MC3T3-E1 cells was evaluated by TUNEL assay and FITC-Annexin V/PI assay after treatment with Piezo2-siRNA and Dex.Results: The protein level of Piezo2 was decreased by Dex treatment but increased by GSK3?-shRNA treatment.Piezo2-siRNA treatment increased protein expression of OPG and decreased the level of RANKL.Piezo2-siRNA decreased the apoptotic rate of both untreated MC3T3-E1 osteoblasts and Dex-treated MC3T3-E1 osteoblasts.Conclusions: Piezo2 alters expressions of OPG and RANKL in MC3T3-E1 osteoblasts and plays role in Dex-induced osteoblast apoptosis.