Regulation Of PTH1R Nuclear Transport On Bone Metabolism And Its Mechanism

Objective:1.Explore the effect of PTH intervention on PTH1R nuclear transport2.Clarify the effect of PTH1R on bone metabolism after entering the nucleus3.Explore the possible mechanism of PTH1R in promoting bone formation.Methods:1.Observation of PTH1R protein expression level and intracellular distribution changes in MC3T3-E1 cell under PTH intervention by immunofluorescence technique;2.Mass spectrometry,immunofluorescence colocalization and Chip-seq technology were used to analyze the proteins and genes interacting with PTH1R in the nucleus of MC3T3-E1 cell,and to explore and verify its mechanism;3.Multi-omics verification of changes in mouse bone formation and its mechanism analysis after PTH1R nuclear localization sequence knockout.Results:1.The results of immunofluorescence experiments showed that the nuclear transport efficiency of PTH1R was improved under the intervention of PTH,and the results of mass spectrometry indicated that PTH1R interacted with TRAP150 after entering the nucleus;2.The nuclear localization of PTH1R and TR under immunofluorescence is similar.The KEGG pathway enrichment analysis results show that the most enriched pathway is Wnt signaling pathway,and the enrichment score is 3.218639;3.The results of qRT-PCR experiments showed that the transcription levels of osteogenic markers Runx2,BMP2,ALP and OSX were down-regulated after blocking PTH1R into the nucleus,The results of skeletal staining of offspring and Micro-CT bone scans of mouse femurs showed that after blocking PTH1R nuclear transport,the skeletal development of mice was delayed,the cortical bone was significantly thinner,the separation degree of trabecular bone was significantly increased;4.The results of RNA-seq analysis showed that the expression of Ptx3,Bmp4 and other genes was significantly down-regulated after blocking PTH1R into the nucleus;the bone formation-related signal pathways(TGF-β signaling pathway,ECM receptor interaction,Wnt signaling pathway,etc.)were significantly down-regulated;bone resorption-related signal Pathways(PPAR signaling pathway,GAG degradation,etc.)were significantly up-regulated.Conclusion:1.PTH1R interacts with TRAP150 in the atypical pathway through the stimulation of PTH into the nucleus,and after its entry into the nucleus,it may act on the Wnt signaling pathway through TRAP150 bridging and synergize with TR to regulate bone metabolism;2.Bone formation in mice is inhibited after blocking PTH1R nuclear transport,and PTH1R nuclear transport is important for regulating bone formation in mice;3.The internal mechanism of this effect may be that PTH1R promotes the interaction of TGF-β signaling pathway,Wnt signaling pathway and extracellular matrix receptor interaction after entering the nucleus,while inhibiting PPAR signaling pathway and glycosaminoglycan degradation.