Role And Mechanism Of Hedgehog Signaling In Regulating Osteogenic Differentiation Of BMSCs

Part one:Role and mechanism of Hedgehog signaling in regulating osteogenic differentiation of BMSCs in vitroObjective:To investigate the promoting effect of Hedgehog pathway activator Purmorphamine(Purmo)on the osteoblast differentiation of ST-2 cells and mouse bone marrow mesenchymal stem cells(BMSCs),and explore its molecular mechanism.Methods:ST-2 cell lines were recovered,mouse primary BMSCs were isolated and cultured in different conditions according to the purpose of the experiment.ST-2 cells and BMSCs were cultured in growth or osteogenic medium which contained DMSO as control,the experimental group further treated with 2?M Purmo.Alkaline phosphatase(ALP)staining was performed after 3 days of culture.The expression of Hedgehog pathway-related genes and osteogenic differentiation-related genes were detected by real-time quantitative PCR(qPCR)after 7 days of culture.Von Kossa staining was performed after 14 days of culture.In addition,cell clone crystal violet staining and ALP staining were performed in BMSCs group after 7 days of culture.In order to investigate the related molecular mechanism,we examined the expression of Wnt pathway-related genes after 7 days of osteogenic induction in BMSCs group by qPCR.In order to verify the relationship between Hedgehog pathway and Wnt pathway in the process of osteogenic-adipogenic differentiation of BMSCs,the BMSCs were cultured in osteogenic medium which contained 2?M Purmo,and then treated with or without the Wnt pathway inhibitor IWP2(20?M).ALP staining was performed after 3 days of culture,and qPCR was used to detect the expression of osteogenic related genes after 7 days of culture.Results 1:ALP staining showed that Alp was positively expressed in ST-2 cells after 3 days of Purmo treatment,and staining was negative in control group.Von Kossa staining showed that,the bone mineralized nodules were found in ST-2 cells cultured in osteogenic medium with Purmo after 14 days of culture,while no nodule was found in the control group.qPCR analysis showed that the expression levels of Hedgehog pathway target gene Ptch1,Gli1 and osteogenic differentiation-related gene Runx2,Osterix(Osx),Ibsp,Alpl in ST-2 cells treated with Purmo for 7 days were significantly higher than those in the control group(**p<0.01,***p<0.001).Results 2:Mouse BMSCs were successfully isolated and cultured,and had the potential to differentiate into osteoblasts and adipocytes.BMSCs were cultured in osteogenic inducing medium which contained DMSO,and treated with or without 2?M Purmo.After 7 days of culture,crystal violet staining showed no significant difference in cell clone number between the two groups(p>0.05).Colony ALP staining showed that the number of clones in the Purmo treatment group were significantly higher than that in the control group(*p<0.05).After 3 days of culture,ALP staining showed that the color of Purmo treatment group was significantly deeper than that of control group.Von Kossa staining showed that the color of Purmo treatment group was significantly deeper than that of control group after 14 days of treatment.We also found the expression levels of Hedgehog target gene Glil,Wnt target gene Wnt4,Wnt9a,WntlOb and osteogenic differentiation related gene Runx2,Osx,Ibsp,Alpl were significantly higher in Purmo treatment group than those of the control group(**p<0.05,**p<0.01,***p<0.001)after 7 days of culture by using qPCR.Results 3:We cultured the BMSCs in osteogenic inducing medium,and treated with 2?M Purmo or Purmo combined with Wnt pathway inhibitor IWP2(20?M).After 3 days of treatment,ALP staining showed that the Purmo-treated group was significantly darker than that of control group,and the Purmo-IWP2-treated group was significantly lighter than the Purmo-treated group.After 7 days of culture,the expression level of osteogenic differentiation related gene Runx2,Osx,Ibsp,Alpl,Osteocalcin(Ocn)in Purmo-treated group was significantly higher than control group,while the expression level of osteogenic differentiation related gene Osx,Ibsp,Alpl,Ocn in Purmo-IWP2-treated group was significantly lower than Purmo-treated group(*p<0.05),while there was no significant change in Runx2.Conclusion:Purmo can activate the Hedgehog pathway and promote osteogenic differentiation of ST-2 cells and BMSCs.Purmo can activate the Wnt pathway and Hedgehog pathway and promote the osteogenic differentiation of BMSCs synergistically.Effect of Hedgehog pathway promoting osteogenic differentiation of BMSCs was inhibited after Wnt pathway was inhibited.Part two:Role and mechanism of Hedgehog signaling in regulating bone metabolism in VivoObjective:To investigate the role of Hedgehog pathway in bone metabolism in adult mouse and explore its molecular mechanism.Methods:The activation of Hedgehog pathway during bone growth in mice were tracked,the targeting cells were labeled and localized by genetic lineage tracing,fluorescent staining and LacZ staining.We crossed the Glil-CreERT2 mice with R26-mTmG mice,and the offspring Glil-CreERT2;R26-mTmG mice were treated with Tamoxifen injection at 4 weeks of age.The femur and tibia were harvested for frozen sections and immunofluorescence staining.GFP fluorescent protein was used to track Glil positive cells.Meanwhile,the tibias of 4 weeks-old Gli1+/-mice and their wild-type mice littermates were collected for LacZ staining to track the Glil positive cells.In addition,genetic lineage tracing and fluorescent staining were used to verify that the Hedgehog pathway regulates bone metabolism and which ligands are present(Shh/Ihh).Shh-CreERT2 mice were mated with R26-mTmG mice to obtain the Shh-CreERT2;R26-mTmG mice.The femur,tibia and skin of Shh-CreERT2;R26-mTmG mice were collected for frozen section after they were treated with tamoxifen at 4 weeks and 8 weeks.The GFP fluorescent protein was used to track Shh positive cells.At the same time,the Ihh positive cells were detected by paraffin section and immunohistochemistry in the tibia of 4 weeks-old wild-type mice.The role of membrane receptor Smo and transcription factor Gli in regulating bone metabolism in the Hedgehog pathway were further studied by using Smo osteoblast-specific knockout mice(Smoc/c;Osx-Cre).At the age of 8 weeks,the long bones of Smoc/c;Osx-Cre mice and their wide-type litternates were collected,Micro-CT scanning was performed to observe bone trabeculae,bone cortex and other related indexes,such as the relative volume of bone trabeculae(BV/TV),the number of trabecular bone(Tb.N),trabecular thickness(Tb.Th)and trabecular separation(Tb.Sp).Double calcein labelling was used to examine the double-standard spacing,and the bone mineralization deposition rate(MAR),Bone formation rate(BFR/BS)and mineralized surface ratio(MS/BS)were measured by using the Bioquant image analysis system.The changes of bone mass and the number of adipocytes in bone marrow were observed by HE staining.The expression of alkaline phosphatase in trabecular bone of mice was detected by ALP staining.The expression of Osteocalcin(Ocn)in bone tissue was detected by immunofluorescence TRAP staining and Bioquant image analysis system were used to detect the number of Trap positive osteoclasts in trabecular bone.The expression of Hedgehog pathway target gene and osteogenic differentiation related gene Runx2,Osx,Alpl,Ocn in bone tissue were examined by qPCR.We crossed Glil+/-mice to obtain the Gli1-/-mice and wild-type mice littermates.Gli2 c/c mice were mated with Osx-Cre mice to get the Gli2 osteoblast specific knockout mice(Gli2c/c;Osx-Cre mice).The long bones of the mice were collected at the age of 8 weeks,Micro-CT scanning was performed to observe bone trabeculae,bone cortex and calculate morphometric analysis of related indicators,such as the relative volume of bone trabeculae(BV/TV),the number of trabecular bone(Tb.N),trabecular thickness(Tb.Th)and trabecular separation(Tb.Sp).The changes of bone mass and the number of adipocytes in bone marrow were observed by HE staining.Results 1:Gli1-CreERT2;R26-mTmG mice were able to successfully obtain after Glil-Cre ERT2 mice were mated with R26-mTmG mice.Glil-CreERT2;R26-mTmG mice were treated with Tamoxifen at the age of 4 weeks,GFP-labeled Gli1-positive cells were found in mice tibia and femur tissues through immunofluorescence mice staining,which were mainly located in the growth plate and the region rich in mesenchymal stem precursor cells under the growth plate.Offspring of mice were obtained after Gli1+/-genotype mice were mated with wild-type mice.At the age of 4 weeks,the tibia of Glil+/-mice and wild-type control mice were collected for LacZ staining,and Gli1+/-mice were also found to be rich in Glil positive cells in and under the growth plate,while the LacZ staining of wild-type mice was negative.Shh-CreERT2;R26-mTmG genotype mice were injected with Tamoxifen at 4 weeks and 8 weeks of age,and then the femur,tibia and skin were collected for frozen sections and immunofluorescence examination.GFP fluorescence protein was used to track the Shh positive cells.GFP fluorescence expression was found in the skin tissue,suggesting the existence of Shh positive cells,but there was no GFP fluorescence protein in the bone tissue.The tibial tissues of wild-type mice were collected at 4 weeks of age for paraffin section and Ihh immunohistochemistry,and Ihh positive cells were found to be expressed.Results 2:After genotype identification and grouping,Micro-CT scanning was performed in Smoc/c;Osx-Cre mice and the control mice at the age of 8 weeks.Compared with control mice,Smo conditioned knockout mice had significantly reduced trabecular volume,increased trabecular septum,and a thinner thickness of the cortical bone at the metaphyseal region of the upper tibia.Related parameters of bone tissue morphology quantitative analysis software analysis found that the Smo conditional knockout,compared with the control mice,the tibial BV/TV,Tb.N and Tb.Th decreased significantly,while the Tb.Sp increased significantly,the difference was statistically significant differences(*p<0.05).Double calcein labelling showed that the double-labeled spacing was significantly smaller in Smoc/c;Osx-Cre mice compared with the control mice at 8 weeks of age.The measurement of bone mineralization deposition rate(MAR),bone formation rate(BFRS/BS),mineralization surface proportion(MS/BS)reflect the dynamic parameters of bone formation were lower in Smoc/c;Osx-Cre mice compared with the control mice(*p<0.05).HE staining showed that the bone mass of Smo conditional knockout mice was significantly reduced compared with the control mice,while the number of adipocytes in bone marrow was significantly increased(*p<0.05).ALP staining showed that Alp activity was significantly lower in trabecular bone of Smo conditional knockout mice than that of control mice at 8 weeks of age.The expression of Osteocalcin(Ocn)in the trabecular bone of Smo conditional knockout mice was significantly lower than that of the control group.Trap staining and quantitative analysis of osteoclasts using Bioquant image analysis system showed no significant difference in the number of Trap positive osteoclasts on trabecular bone in Smo conditioned knockout mice compared with the control mice(*p>0.05).qPCR results of bone tissue showed that the expression levels of Smo and osteogenic differentiation-related genes,such as Osx,Alpl and Ocn were significantly lower than those in the control mice at 8-week old age.(*p<0.05),while Runx2 showed no significant changes.Results 3:After genotype identification and grouping,long bones were collected at 8 weeks of age.Micro-CT scan analysis results showed that compared with the control mice,the tibial BV/TV,Tb.N and Tb.Th were significantly decreased in Gli1 knockout mice(*p<0.05),and the Tb.Sp showed no significant change.HE staining showed that the bone mass of Gli1 knockout mice was less than control mice,while the adipocytes in bone marrow increased significantly.After we obtained the Gli2c/c;Osx-Cre mice and their control littermates,long bones were collected at 8 weeks of age.Micro-CT scanning analysis showed that compared with the control mice,the tibial BV/TV and Tb.N were decreased significantly in Gli2 conditional knockout mice,the difference is statistically significant differences(*p<0.05).There was no significant change in Tb.Th,and the Tb.Sp was increased,but the difference was no statistically significant(p>0.05).HE staining showed that Gli2 conditional knockout mice had significantly less bone mass and increased number of adipocytes in bone marrow compared with control mice.Conclusion:The Hedgehog pathway is involved in the regulation of osteoblast differentiation in adult mice and plays a role in the form of Ihh ligand.Smo conditional knockout in the Hedgehog pathway significantly inhibits the osteogenesis in vivo,but has no significant effect on the activity of osteoclasts,resulting in slowed down bone growth rate and decreased bone mass.After both downstream transcription factors Gli1 and Gli2 were knocked out,it could partially inhibit in vivo osteogenesis.