Dual Effects And Mechanisms Of Cholesterol In Osteogenic Differentiation Of Bone Marrow Stromal Cells

Part I The effect and mechanism of endogenous cholesterol in osteogenic differentiation of BMSCsObjective:To investigate the role of endogenous cholesterol in osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and to clarify the regulatory mechanism of Hh signalingpathway.Methods:the method of "in vitro cholesterol depletion experiments" was used to remove endogenous cholesterol from ST2 cells(a bone marrow stomal cell line)and primary BMSCs:ST2 cells and primary BMSCs were treated with starvation for 24 hours,a-MEM complete medium containing 1.5%cyclodextrin was used to remove endogenous cholesterol from the cells.After half an hour of incubation,?-MEM complete medium containing 40uM pravastatin sodium was used to block the biosynthesis of cholesterol.Intracellular cholesterol fluorescence staining kit was used to detect the fluorescence of endogenous cholesterol in ST2 cells by using fluorescent microscope at 4 times,10 times and 20 times respectively.At the same time,the changes of ST2 cell density before and after removal of endogenous cholesterol by 4,10 and 20 times magnification were observed by optical microscope.The supernatant of 5%Shh was added to the a-MEM complete culture medium for ST2 cells and primary BMSCs with removal of endogenous cholesterol,and 293T supernatant was used as control.After 3 days,the positive degree of ALP staining in ST2 cells and primary BMSCs was detected by ALP staining,the expression of Hh signal pathway target gene(Gli1,Ptchl)was detected by real-time quantitative PCR(qPCR)method.After 7 days,the expression of primary BMSCs'osteoblast-associated genes(Alpl,Sp7,Ibsp,Runx2 and Ocn)were detected by qPCR assay.In addition,2uM Purmo was added to the a-MEM complete culture medium for ST2 cells with removal of endogenous cholesterol,and dimethyl sulfoxide(Dimethyl sulfoxide,DMSO)was used as control.After 3 days,the positive degree and activity of ALP staining in ST2 cells were detected by ALPstaining and quantitative method.The expression levels of osteoblast-associated genes(Alpl)and Hh signal pathway target genes(Gli1,Ptchl)were detected by qPCR assay.Results:After the removal of endogenous cholesterol from ST2 cells,the relative total cell fluorescence decreased significantly by using fluorescence microscope with amplified by 4 times,10 times and 20 times.The removal rate of endogenous cholesterol in ST2 cells was 72%and the density of ST2 cells did not change significantly.The positive degree of ALP staining,the expression levels of Hh signal pathway target gene(Gli1,Ptch1)in primary BMSCs and ST2 cells in the depleted+Shh supernatant group was significantly lower than that in the control group.And the expression of primary BMSCs'osteoblast-associated genes(Alpl,Sp7,Ibsp,Runx2 and Ocn)were significantly lower than those in the control group.The positive degree and activity of ALP staining,the expression levels of osteoblast-associated gene(Alpl)were significantly decreased in ST2 cells in the depleted+Purmo group but the expression level of target gene(Gli1,Ptch1)in Hh signaling pathway did not change significantly.Conclusion:Endogenous cholesterol is essential for osteogenic differentiation of primary BMSCs through both Hh-dependent or-independent mechanisms.Therefore,physiological level of endogenous cholesterol can regulate the Hh signaling pathway and maintain osteogenic differentiation of the body,and it is not scientific to blindly reduce the endogenous cholesterol.This provides a theoretical basis and clinical guidance for the prevention and treatment of OP:maintaining physiological levels of endogenous cholesterol may prevent the occurrence of OP.Part II Inhibition and mechanism of exogenous cholesterol in osteogenic differentiation of BMSCs in basal osteogenic condition and Wnt inducerObjective:To investigate the effects of exogenous cholesterol in osteogenic differentiation of BMSCs in basal osteoblasts and Wnt inducers,and to clarify the regulatory mechanisms of Hh and Wnt signaling pathways in this process.Methods:5%Shh supernatant and 2uM Purmo were added to ST2 cells containing a-MEM complete culture medium respectively,293T supernatant and DMSO were used as control.After 3 days the positive degree of ALP staining in ST2 cells was detected by ALP staining.After 7 days the expression of Hh signal pathway target gene(Glil,Ptch1),osteoblast-associated genes(Alpl,Sp7 and Ibsp)were detected by qPCR method.After 14 days,the matrix mineralization ability of ST2 cells was detected by Von Kossa staining.Secondly,a-MEM complete culture medium containing different concentrations of exogenous cholesterol were added to ST2 cells to observe the activation of Hh signaling pathway and the maximum concentration of cholesterol not precipitated.20ug/ml exogenous cholesterol containing Cyclopamine(inhibitor of Smo protein)or Gant61(inhibitor of Gli1 transcription factor)were added to ST2 cells to detect the action sites of exogenous cholesterol activating Hh signaling pathway.20ug/ml exogenous cholesterol was added to ST2 cells containing a-MEM complete culture medium,ST2 cells and primary BMSCs containing basal osteogenic condition respectively.After 2 days,the expression level of osteoblast-associated genes(Alpl,Sp7,Ibsp,Runx2,Ocn and Coll)and Hh signaling pathway target gene(Gli1)were detected by qPCR method.After 7 days and 14 days,the positive degree of ALP staining in ST2 cells and primary BMSCs cells was detected by ALP staining,and the expression level of osteoblast-associated genes(Alpl,Sp7,Ibsp,Runx2 and Coll)and Hh signaling pathway target gene(Glil)in ST2 cells and primary BMSCs were detected by qPCR method.The expression of osteoblast-associated protein(Runx2,Osterix)in ST2 cells was detected by Western Blot assay.And 20ug/ml exogenous cholesterol was added to ST2 cells containing a-MEM complete culture medium and the proliferation of ST2 cells was detected at 6h,12h,24h,48h and 72h respectively.Finally,exogenous cholesterol was added to a-MEM complete culture medium containing 20%Wnt3a supernatant.After 3 days,the positive degree and activity of ALP staining in ST2 cells were detected by ALP staining and quantitative method.The expression level of osteoblast-associated genes(Alpl and Ibsp),Wnt signaling pathway target genes(Axin2 and Nkd2)and Hh signal pathway target genes(Gli1 and Ptchl)was detected by qPCR method.Results:The expression level of Hh target gene(Gli1,Ptchl)and osteoblast-associated genes(Alp1,Sp7 and Jbsp),the positive degree of ALP and Von Kossa staining in ST2 cells were significantly increased in Shh supernatant group and Purmo group.With the increase of cholesterol concentration,the expression level of target gene(Gli1)in Hh signaling pathway increased gradually,and the maximum concentration of non-precipitated is 20ug/ml.With the increase of Cyclopamine or Gant61 concentration,the expression level of Glil gene was decreased gradually.After treated with exogenous cholesterol,the positive degree of ALP staining,the expression level of osteoblast-associated genes(Alpl,Sp7,Ibsp,Runx2 and Coll)and osteoblast-associated protein(Runx2 and Osterix)in ST2 cells were significantly decreased.But,the target gene(Glil)of Hh signaling pathway was significantly increased;The positive degree of ALP staining and expression level of osteoblast-associated genes(Alp1,Sp7,Ibsp,Runx2 and Coll)decreased significantly in primary BMSCs,but the expression level of Glil and Ptchl in Hh signaling pathway did not change significantly.At the same time,the proliferation of ST2 cells was mild at 6h?12h?24h?48h and 72h respectively.After treated with Wnt3a supernatant and exogenous cholesterol simultaneously,the positive degree and activity of ALP staining and the expression level of osteoblast-associated genes(Alp1 and Ibsp)in ST2 cells were significantly decreased,while the expression level of Wnt signal pathway target genes(Axin2 and Nkd2)were significantly increased.At this time,the Glil expression level of target gene in Hh signaling pathway was significantly increased,but Ptchl did not change significantly.Conclusion:Exogenous cholesterol may activate the Hh signaling pathway by acting on the level of Smo protein or its upstream regulatory factor in the Hh signal transduction pathway.Although exogenous cholesterol could not activate the Wnt signaling pathway,it could increase the activity of the activated Wnt signaling pathway.At the same time,exogenous cholesterol inhibits osteogenic differentiation through both Hh-independent and Wnt-independent mechanisms.This provides a theoretical basis and clinical guidance for the prevention and treatment of OP:excessive intake of exogenous cholesterol may increase the incidence of OP.Part HI Regulation and mechanism of osteogenic differentiation of BMSCs by exogenous cholesterol in Hh inducer and hydroxycholesterol in growth mediumObjective:To investigate the effect of exogenous cholesterol in osteogenic differentiation of BMSCs in Hh inducer and its molecular mechanism.The effects of hydroxycholesterol in osteogenic differentiation in other BMSCs cell lines and the regulation mechanism of Hh signaling pathway in this process were preliminarily studied.Methods:2uM Purmo and 5%Shh supernatants were added to a-MEM complete culture medium in ST2 cells to activate Hh signaling pathway.On this basis,the expression level of Hh signal pathway target gene(Gli1)in ST2 cells was detected by qPCR at 15 h after ST2 cells were treated with exogenous cholesterol.The positive degree and activity of ALP staining in ST2 cells were detected by ALP staining and quantitative method 3 days later.The expression levels of osteoblast-associated genes(Alp1,Sp7 and Ibsp)and target genes of Hh signaling pathway(Glil and Ptch1)in ST2 cells were detected by qPCR assay at 3 days,7 days and 14 days respectively.After 14 days,the matrix mineralization ability of ST2 cells was detected by Von Kossa staining.Secondly,lOuM 22(S)-hydroxycholesterol and 25-hydroxycholesterol were added to ?-MEM complete culture medium in ST2 cells respectively.The expression level of Hh signal pathway target gene(Gli1)was detected by qPCR assay at 15 hours and 7 days respectively.Finally,different concentrations(5uM and 10uM)of 22(S)-hydroxycholesterol and 25-hydroxycholesterol were added to ?-MEM complete culture medium in ST2 cells respectively.The positive degree of ALP staining in ST2 cells were detected by ALP staining 3 days later.Results:After ST2 cells were treated with Purmo or Shh supernatants respectively,the positive degree of ALP and Von Kossa staining,the activity of ALP and the expression level of osteoblast-associated genes(Alpl,Sp7 and Ibsp)and target genes of Hh signaling pathway(Gli1 and Ptchl)were significantly increased.On this basis,the positive degree of ALP and Von Kossa staining,the activity of ALP and the expression level of osteoblast-associated genes(Alpl,Sp7 and Ibsp)and target genes of Hh signaling pathway(Glil and Ptchl)were significantly decreased after ST2 cells were treated with exogenous cholesterol.After ST2 cells were treated with 22(S)-hydroxycholesterol and 25-hydroxycholesterol respectively,the target gene(Gli1)of Hh signaling pathway in ST2 cells was increased significantly.After the treatment with different concentrations of 22(S)-hydroxycholesterol and 25-hydroxycholesterol,the positive degree of ALP staining in ST2 cells was increased.Conclusion:Exogenous cholesterol can reduce the activity of activated Hh signaling pathway and inhibit the osteogenic differentiation of ST2 cells.Exogenous cholesterol and its oxides(hydroxycholesterol)can inhibit or promote osteogenic differentiation through a Hh-dependent molecular mechanisms.Hydroxycholesterol can preliminarily promote osteogenic differentiation in various BMSCs cell lines,which provides a theoretical basis and clinical guidance for the prevention and treatment of OP:hydroxycholesterol is expected to become a bone formation accelerator in the treatment of OP.