Effects Of GLP-1 Receptor Agonist On The Osteogenic Differentiation Of Preosteoblast And The Underlying Mechanism

Glucagon-like peptide 1(GLP-1)is one of the incretin hormones that secreted from the ileal L cells lining the gut.It exerts many physiological functions including the potentiate of glucose-stimulated insulin secretion after a meal,promoting pancreatic beta cells proliferation and inhibiting their apoptosis,inhibition of glucagon secretion,restraining gastric emptying,increasing the feeling of satiety,and so on.GLP-1 could be rapidly degraded by dipeptidyl peptidase-Ⅳ(DPP-Ⅳ)in vivo,thus,researchers take efforts to developed a variety of GLP-1 analogues to strengthen their stability.Liraglutide have 97% homology with human GLP-1,with a half-life of about 12-14 h.Exendin-4 is a 39 amino acid polypeptide that shares 53% of its amino acid sequence with the N-terminal region of mammalian GLP-1,reached its peak blood drug concentration after 2 hours of injection.Liraglutide and Exendin-4 could not be degraded by DPP-IV,so both of them preserve multiple physiological functions,as well as overcome the shortcoming of the natural GLP-1.Liraglutide and Exendin-4 are now more and more widely accepted in clinical treatment.Beyond its well-known effects on glucose metabolism and the protection of pancreatic β cells,GLP-1 has been reported to have many extensive extrapancreatic effects,such as promoting bone formation and suppressing bone resorption.These results strongly suggest that GLP-1 and its receptor agonist may play a positive role in bone metabolism.Zhan et al.reported that liraglutide attenuates runt-related transcription factor 2(Runx2)expression in human vascular smooth muscle cells and inhibiting their osteoblastic differentiation.However,other articles reported the promoting effects of GLP-1 on osteogenic differentiation.Thus,the effect of GLP-1 and its analog on osteogenic differentiation is still controversial.TGF-β family members exert their cellular effects by binding to transmembrane receptors,when activated,the receptor kinase phosphorylates smads proteins,which move into the nucleus to stimulate the transcription of a set of target genes.Phosphorylated smad2/3 combined with smad4 and transfers to nucleus and regulates the transcription of osteogenic differentiation related genes.Since Wnt/β-catenin and PI3K/AKT signaling pathway could both regulate the process of osteogenic differentiation,we speculated that GLP-1 may regulate the expression of smads through Wnt/β-catenin and PI3K/AKT signaling pathway,and further affect the transcription and translation of targeted genes.Besides,Hedgehog is another important signaling pathway involved in the cell differentiation.Jie-Fen et al.also reported the promoting effect of strontium ranelate on rat bone mesenchymal stem cells osteoblast differentiation through Hedgehog/Gli1 signaling pathway.Therefore,this study aimed to investigate the effects of GLP-1 on the osteogenic differentiation and the underlying mechanism.The whole study mainly includes 4 parts:part 1,the promoting effects of liraglutide on the osteogenic differentiation of MC3T3-E1;part 2,the signaling pathway that may be involved in regulating the effects of liraglutide on the osteogenic differentiation of MC3T3-E1;part 3,the promoting effects of Exendin-4 on the osteogenic differentiation of MC3T3-E1;part 4,the signaling pathway that may be involved in the effects of Exendin-4 on the osteogenic differentiation of MC3T3-E1.From the perspectives of basic medicine,we demonstrated the important effects of GLP-1 analogue in the bone metabolism,and provided the theoretical basis for the clinical application of both liraglutide and exendin-4.Part 1 Liraglutide promotes the osteogenic differentiation of preosteoblastObjective: To determine the effects of liraglutide on the proliferation and osteogenic differentiation of MC3T3-E1 and the dose-effect relationship.Methods:1 MC3T3-E1 cells cultured in osteogenic medium were treated with 0 mol/L,10-9mol/L,10-8mol/L and 10-7mol/L liraglutide for 24,48 and 72 hours.We then observed the effects of liraglutide at different concentration on the proliferation of MC3T3-E1 cells by CKK-8 kit as well as flow cytometry.2 MC3T3-E1 cells cultured in ordinary medium were set to blank control group;cells cultured in osteogenic medium were set to osteogenic control group;0 mol/L,10-9mol/L,10-8mol/L and 10-7mol/L liraglutide were added into osteogenic medium to set osteogenic intervention group.We then determined the GLP-1 receptor(GLP-1R)distribution by immunofluorescent staining after 48 hours of administration.After 3,7,14 and 21 days of intervention,we detected alkaline phosphatase(ALP)activities and calcium depositions of MC3T3-E1 cells,and further detected GLP-1R,Runx2 and osteocalcin(OCN)mRNA and protein levels by real-time RT-PCR and western blot analysis.Rusults:1 Liraglutide has no significant influence on the cell viability and cell cycle phase of MC3T3-E1 cells.2 GLP-1R exists in the cytoplasm and nucleus of MC3T3-E1 cells.Liraglutide could dose dependently increase the expression of GLP-1R;the concentration of liraglutide which led to the maximum effects were 10-7 mol/L at day 14.3 Liraglutide could dose dependently increase the ALP activities and calcium depositions of MC3T3-E1 cells.4 Both real-time RT-PCR and western blot analysis results suggested liraglutide could dose dependently increase the expression of Runx2 and OCN.The expression of Runx2 reached its peak value at day 7,and the expression of OCN reached its peak value at day 21.Part 2 Both Wnt/β-catenin and PI3K-AKT signaling pathway were involved in the promoting effects of liraglutide on preosteoblast osteogenic differentiationObjective: To determine whether smad2/3 participate in the promoting effects of liraglutide on the osteogenic differentiation of MC3T3-E1 cells.And determine the preponderant pathway which mediated the effects of liraglutide on smad 2/3 mRNA and protein expression.Methods:1 MC3T3-E1 cells cultured in ordinary medium were set to blank control group;cells cultured in osteogenic medium were set to osteogenic control group;0 mol/L,10-9mol/L,10-8mol/L and 10-7mol/L liraglutide were added into osteogenic medium to set osteogenic intervention group.We then detected smad2 and smad3 mRNA levels by real-time RT-PCR and p-smad2,smad2,p-smad3,smad3 protein levels and western blot analysis.2 We used small interfering RNA(SiRNA)to inhibit the smad 2/3 mRNA expression,and observed whether Smad2/3-SiRNA offset the liraglutide induced increase of osteogenic differentiation.MC3T3-E1 cells cultured in osteogenic medium were treated by normal saline(control group),empty vector transfection group,empty vector+liraglutide,Smad2-SiRNA(smad2 gene silencing group),Smad2-SiRNA+liraglutide,Smad3-SiRNA(smad3 gene silencing group)and Smad3-SiRNA+liraglutide respectively for 3,7 or 21 days.We detected smad2,smad3 and Runx2 mRNA levels by real-time RT-PCR as well as p-smad2,smad2,p-smad3,smad3 and Runx2 protein levels by western blot analysis.We also detected ALP activities and calcium depositions of MC3T3-E1 cells to evaluate the differentiation degrees.3 We used DKK-1(Wnt/β-catenin inhibitor)and LY294002(PI3K-AKT inhibitor)to determine the preponderant pathway which mediated the effects of liraglutide on smad 2/3 expression.Results:1 Liraglutide could dose dependently increase the expression of smad 2/3 mRNA levels.2 Liraglutide could dose dependently increase p-smad2,smad2,p-smad2/smad2,p-smad3,smad3 and p-smad3/smad3 relative protein levels.3 SiSmad 2/3 transfection offset the effects of liraglutide on osteogenic differentiation of MC3T3-E1 cells.4 Both Wnt/β-catenin and PI3K-AKT signaling pathways are involved in the promoting effects of liraglutide on smad 2/3 related proteins.Part 3 Exendin-4 promotes the osteogenic differentiation of preosteoblastObjective: To determine the effects of Exendin-4 on the osteogenic differentiation of MC3T3-E1 and the dose-effect relationship.Methods:1 MC3T3-E1 cells cultured in ordinary medium was set to blank control group;cells cultured in osteogenic medium was set to osteogenic control group;0 mol/L,10-9mol/L,10-8mol/L and 10-7mol/L Exendin-4 were added into osteogenic medium to set osteogenic intervention group.After 3,7,14 and 21 days of intervention,we detected GLP-1R mRNA and protein levels by real-time RT-PCR and western blot analysis.2 ALP activities and calcium depositions were also detected to evaluate the degrees of differentiation,and we further detected Runx2 and OCN mRNA and protein levels by real-time RT-PCR and western blot analysis.Results:1 Both real-time RT-PCR and western blot analysis results suggested Exendin-4 could dose dependently increase the GLP-1R mRNA and protein levels.2 Exendin-4 could dose dependently increase the ALP activities and calcium depositions of MC3T3-E1 cells.3 Exendin-4 could dose dependently increase the Runx2 and OCN mRNA and protein levels of MC3T3-E1 cells.Part 4 Exendin-4 promotes the osteogenic differentiation of preosteoblast via Hedgehog/Gli1 signaling pathwayObjective: To determine whether Hedgehog/Gli1 signaling pathway was participated in the promoting effect of exendin-4 during the osteogenic differentiation progress of MC3T3-E1 cells.Methods:1 MC3T3-E1 cells cultured in ordinary medium was set to blank control group;cells cultured in osteogenic medium was set to osteogenic control group;0 mol/L,10-9mol/L,10-8mol/L and 10-7mol/L Exendin-4 were added into osteogenic medium to set osteogenic intervention group.After 3,7,14 and 21 days of intervention,we detected Hedgehog and Gli1 mRNA and protein levels by real-time RT-PCR and western blot analysis.2 We used small interfering RNA(SiRNA)to inhibit the Gli1 expression,and observed whether SiGli1 offset the Exendin-4 induced increase of osteogenic differentiation.MC3T3-E1 cells cultured in osteogenic medium were treated by normal saline(control group),empty vector transfection group,empty vector+Exendin-4,Gli1-RNA(Gli1 gene silencing group),Gli1-RNA+Exendin-4 respectively for 3,7 or 21 days.We detected Gli1 and Runx2 mRNA and protein levels by real-time RT-PCR and western blot analysis.We also detected ALP activities and calcium depositions of MC3T3-E1 cells to evaluate their differentiation degrees.3 We used Cyclopamine(Hedgehog/Gli1 inhibitor)to determine whether Hedgehog/Gli1 signaling pathway mediated the effects of Exendin-4 on Gli1 expression.Results:1 Both real-time RT-PCR and western blot analysis results suggested Exendin-4 could dose dependently increase the Hedgehog and Gli1 mRNA and protein levels.2 SiGli1 transfection offset the effects of Exendin-4 on osteogenic differentiation of MC3T3-E1 cells.3 Hedgehog/Gli1 signaling pathway was involved in the promoting effects of Exendin-4 on Gli1.Conclusion:1 Liraglutide has no significant influence on proliferation of MC3T3-E1 cells.2 GLP-1R exists in the cytoplasm and nucleus of MC3T3-E1 cells,and liraglutide could dose dependently increase the expression of GLP-1R.3 Liraglutide could dose dependently increase the expression of smad 2/3 mRNA and their relative protein levels through Wnt/β-catenin and PI3K-AKT signaling pathway,promoting the osteogenic differentiation of MC3T3-E1 cells.4 Exendin-4 could dose dependently increase the expression of GLP-1R.5 Exendin-4 could dose dependently promote the osteogenic differentiation of MC3T3-E1 cells through Hedgehog/Gli1 signaling pathway.