The Effect And Related Mechanism Of TGR5 On Osteoblast Differentiation

BackgroundBone is a delicate dynamic organ regulated by osteoblasts and osteoclasts,which plays a critical role in regulating bone turnover.Osteoblasts are important for bone formation while osteoclasts are responsible for bone resorption.Osteoblasts are derived from mesenchymal stem cells and pre-osteoblasts through osteogenic differentiation.Dysfunction of osteogenic differentiation causes impairment of normal bone remodeling or bone turnover and is associated with several bone metabolic disorders,including osteoporosis.Decreased bone mineral density,collapse of bone homeostasis,and increased bone fragility are characterized as the major pathological features of osteoporosis.Osteoblast maturation is a complicated physiological process regulated in several steps including cell proliferation,differentiation,and mineralization.Successful osteogenic differentiation and mineralization of precursor cells into functional osteoblasts has been considered as a critical step in bone formation.Multiple intracellular signaling pathways have been reported to be involved in the process of osteoblast differentiation and mineralization.For example,activation of AMP-activated protein kinase(AMPK)could stimulate the phosphorylation of endothelial nitric oxide synthase(eNOS)and increase nitric oxide(NO)production in pre-osteoblast MC3T3-E1 cells,which promotes the differentiation and mineralization of osteoblastic cells,elevates osteoblast activity,and promotes bone formation.Activation of the AMPK/eNOS pathway leads to the expression of runt-related transcription factor 2(Runx-2),a central transcriptional factor involved in osteoblast maturation by regulating the expression of alkaline phosphatase(ALP),osteocalcin(OCN),osteonectin,osteopontin,and type I collagen.Stimulation of osteogenic differentiation has been considered as an important therapeutic strategy for the treatment of bone disorders,such as osteoporosis.Takeda G-protein coupled receptor 5(TGR5)is an important member of the G-protein-coupled receptor(GPCR)family.It was originally identified as a membrane receptor of bile acid.TGR5 has been reported to play a critical role in regulating bile acid and energy homeostasis,as well as glucose metabolism.TGR5 is present in diverse tissues and cells and possesses a variety of pharmacological properties by transducing extracellular signals through heterotrimeric G proteins.For example,it has been recently reported that TGR5 activation plays a pivotal role in inhibiting lipopolysaccharide(LPS)-induced acute gastric inflammation by suppressing activation of the I?B?/NF-?B signaling pathway.It has also been reported that TGR5 has therapeutic potential for kidney inflammation-related diseases by suppressing NF-?B and STAT3 signaling.TGR5 also displays its neuroprotective effects by mitigating neuroinflammation and improving outcomes in a azoxymethane(AOM)mouse model.However,little information regarding the effects of TGR5 in osteogenic differentiation has been reported before.ObjectiveThis topic proposed by in vivo and in vitro experiments to explore TGR5 effect on osteoblast differentiation and bone formation,and participate in the regulation of osteoblast differentiation of normal specific mechanism of action,not only research TGR5 in physiological function in the process of bone formation and for the provided new insight into the molecular mechanism of bone metabolism,as well as provides a new theoretical basis for clinical treatment of bone related diseases and targets.MethodsDysfunction of normal osteoblast differentiation is associated with bone loss related diseases such as osteoporosis.G-protein-coupled receptor TGR5 is considered to be an important regulator of bile acid and energy homeostasis.The effect of TGR5 on osteoblastic bone formation and matrix mineralization has been rarely reported.In this study,we first used rna-seq to detect the gene expression differences before and after the induction of-gp/AA preosteoblast cell line Mc3t3-el,and determined that the target gene TGR5 may be involved in the regulation of-gp/AA induced osteoblast differentiation and maturation,which can promote the differentiation of osteoblast precursor cells into osteoblasts.Then,we used the TGR5 agonist GPBARA and siRNA technology to prove that the activity of TGR5 can regulate the expression of Mc3t3-el osteoblastic differentiation related genes(ALP,OCN,Osx),osteoblastic differentiation transcription factor Runx2,and affect cell ALP activity and matrix calcification.After that,we further studied the mechanism and found that TGR5 promoted the differentiation of Mc3t3-el into osteoblasts by activating AMPK signaling pathway.Finally,we demonstrated in vivo that TGR5 affects bone development and osteoblastic differentiation in mice by affecting the AMPK signaling pathway in transgenic mice with TGR5 deletion.Results(1)Differences in gene expression before and after induction of Mc3t3-el in ?-GP/AA osteoblast cell lines? glycerol phosphate/ascorbic acid(beta glycerophosphate/ascorbic acid,? GP/AA)can induce the differentiation of osteoblast and formation.We have established osteoblast precursors(OBPs)to osteoblast with preosteoblast cell line Mc3t3-el,which can induce osteoblast differentiation.We treated Mc3t3-e124h,4h8 and 72h with 4 mM-gp and 25 g/mlAA,respectively,and then collected different groups of RNA and sent them to the company for second-generation sequencing.RNA-seq and biological reaction path Analysis(Ingenuity Pathway Analysis,IPA)found that beta GP/AA has 1488 gene expression induced by 24 h,48 h to induce has 1417 genes expression,induction of 72 h has 337 genes expression,including TGR5 is the most obvious expression in three groups.Therefore,we speculate that TGR5 may be involved in regulating the differentiation and maturation of osteoblasts induced by-gp/AA,and can promote the differentiation of osteoblast precursor cells into osteoblasts.(2)?-GP/AA induces and promotes the expression of TGR5 in Mc3t3-e1TGR5 is expressed in different types of cells and tissues.However,it is unknown whether TGR5 is expressed in MC3T3-E1 cells.Therefore,we assessed whether TGR5 is expressed in MC3T3-E1 cells by RT-PCR and western blot analysis.Murine MIN6 cells were used as a positive control for expression of TGR5.As expected,TGR5 could be detected in MC3T3-E1 cells at both the gene levels and the protein levels,as determined by RT-PCR and western blot analysis,respectively.Next,we found that the expression of TGR5 in MC3T3-E1 cells was increased in a time-dependent manner in response to osteogenic medium(OM)treatment from day 3 to day 14.These results implicate that TGR5 might serve as an important factor during the osteoblastic differentiation process of MC3T3-E1 cells.(3)TGR5 agonist can promote the differentiation of Mc3t3-e1 cells into osteoblastsTo clarify the possible involvement of TGR5 in osteoblastic differentiation of MC3T3-E1 cells,the specific TGR5 agonist GPBARA(3?M)was used to treat MC3T3-E1 cells in OM.ALP activity,alizarin red S staining,and expressions of osteoblastic differentiation marker genes were measured to index osteoblast differentiation.As shown in Fig.2A,the presence of GPBARA(3?M)markedly elevated ALP activity compared to group treated with OM alone.Correspondingly,alizarin red S staining results indicated that the addition of GPBARA(3?M)promoted matrix mineralization of MC3T3-E1 cells on day 14.Notably,real-time PCR results indicate that activation of TGR5 using GPBARA(3?M)enhanced the expression of osteogenic marker genes,including ALP,OCN,Osx,and Col-I.(4)TGR5 promotes the expression of Mc3t3-e1 osteoblast-specific transcription factor Runx2Marker gene expression of osteoblastic differentiation is mainly regulated by the transcriptional factor Runx-2.As expected,real-time PCR and western blot analysis revealed that GPBARA(3?M)treatment significantly increased the expression of Runx-2 at both the gene and protein levels.(5)Knockdown of TGR5 inhibits the differentiation of Mc3t3-el into osteoblastsIn order to further verify the biological function of TGR5 in osteoblastic differentiation,the expression of TGR5 was knocked out by transfection with TGR,siRNA.Western blot showed that TGR5 knockout was successful.Importantly,the silencing portion of TGR5 blocked the expression of ALP activity,matrix mineralization,and osteoblast differentiation marker genes,with statistically significant differences(p<0.05).These results suggest that TGR5 may play an important role in osteogenic differentiation.(6)TGR5 promotes osteoblast differentiation by activating AMPK signaling pathwayENOS phosphorylation and amp-activated protein kinase(AMPK)production of NO play a key role in osteoblast differentiation.Therefore,we examined whether GPBARA(3 M)could affect the Mc3t3-elenos/NO signaling pathway.The results showed that GPBARA(3 M)could significantly promote the phosphorylation of Mc3t3-elenos in a time-dependent manner.Daf-fm DA staining was used to detect the production of Mc3t3-e1no.The results showed that GPBARA treatment increased the production time of Mc3t3-e1no from 1h to 6h.In addition,GPBARA(3 M)was found to promote the phosphorylation of Mc3t3-elampk and ACC in a time-dependent manner.Therefore,we speculated that TGR5 promotes the differentiation of Mc3t3-el into osteoblasts by activating AMPK signaling pathway.(7)AMPK inhibitors inhibit Mc3t3-el differentiation into osteoblastsTo further confirm the role of AMPK in the regulation of GPBARA induced osteogenic differentiation,we treated Mc3t3-el with a specific AMPK inhibitor compound C.Mc3t3-el cells were cultured in conditioned osteogenic medium for 14 days.It was found that the presence of complex C inhibited GPBARA induced ALP activity,matrix mineralization and osteoblast differentiation marker gene expression.Meanwhile,we also found that GPBARA induced runx-2 expression could be inhibited by blocking AMPK activation with compound C.These results suggest that the role of TGR5 in osteoblastic differentiation is mediated by AMPK.(8)TGR5 is essential for osteoblast differentiation during bone formationIn order to prove the effect of TGR5 on bone development and osteoblastic differentiation of mice in vivo,we constructed transgenic mice with TGR5 deletion and analyzed the effect of TGR5 expression on bone formation by in situ hybridization.The results showed that:in E14.5,compared with the wild-type TGR5 mice,the area of ECM mineralization in tgr5-deficient mice was significantly smaller,and the differences were statistically significant(p<0.05).Von Kossa staining was used to detect the calcium content in bone and the mineralization degree of ECM.Compared with wild-type TGR5 mice,the bone calcium content of tgr5-deficient mice was lower,and the degree of mineralization of ECM was lower,and the differences were statistically significant(p<0.05).Alcian blue staining showed that most of the bone ECM of tgr5-deficient mice were chondrocytes,and the differences were statistically significant(p<0.05).In addition,we also found that compared with the wild-type TGR5 mice,the expressions of OCN and col-i,the related genes of osteoblastic differentiation,and the expression of Runx2,the transcription factor of osteoblastic differentiation,were significantly reduced in the bone of tgr5-deficient mice,with statistically significant differences(p<0.05).Therefore,in the process of bone formation and development,TGR5 can affect the deposition of calcium in bone and regulate the expression of genes related to the differentiation of osteoblasts.(9)TGR5 affects bone development by regulating the AMPK signaling pathwayIn front of the main through the activation of AMPK has proved TGR5 signaling pathways promote the differentiation of osteoblast,our way through the tail vein injection of mice to lack TGR5 give AMPK agonist GSK621(2 mg/kg)injection,found that lack of AMPK agonist can promote TGR5 mice bone development,will promote the osteoblast differentiation related gene OCN,Col-i and the expression of transcription factor Runx2.Therefore,in the process of bone formation and development,TGR5 can affect bone development through AMPK signaling pathway and regulate the expression of genes related to osteoblast differentiation.ConclusionsIn conclusion,TGR5 can affect the expression of osteoblast-related genes through the regulation of AMPK signaling pathway,thereby affecting the differentiation of osteoblasts and the development of bones.This study not only elucidated the physiological function of TGR5 in the bone formation process,but also provided new insights into the molecular mechanism of bone metabolism and new theoretical basis and targets for clinical treatment of bone-related diseases.