Programmed cell death receptor-1 suppresses osteogenic differentiation of rat bone marrow mesenchymal stem cells in a high-glucose microenvironment

Han Nianrong; Huang Yifei; Akram·Osman; Liu Yanlu; Hu Wei

doi:10.12307/2026.086

Abstract：BACKGROUND:Programmed cell death receptor-1(PD-1)and neural precursor cell expressed developmentally downregulated 4(NEDD4)are involved in the regulation of osteoblast differentiation,but the specific interaction between the two and the underlying regulatory mechanism still need to be further studied. OBJECTIVE:To investigate the mechanism of the effect of PD-1 regulation of NEDD4 on osteogenic differentiation of rat bone marrow mesenchymal stem cells in high-glucose environment. METHODS:(1)Immunoprecipitation-mass spectrometry was used to detect the interaction protein of PD-1.Co-immunoprecipitation was used to verify the interaction between PD-1 and NEDD4,and immunofluorescence was used to detect the localization of PD-1 and NEDD4.(2)Passage 3 rat bone marrow mesenchymal stem cells were randomly divided into normal glucose group(5.6 mmol/L),high glucose group(30 mmol/L),PD-1 knockdown empty group,PD-1 knockdown group,PD-1 overexpression empty group,and PD-1 overexpression group.Western blot assay was used to detect the protein expression of NEDD4.(3)Passage 3 rat bone marrow mesenchymal stem cells were randomly divided into normal glucose group(5.6 mmol/L),high glucose group(30 mmol/L),and NEDD4 knockdown group.qRT-PCR was used to measure the mRNA expression levels of NEDD4,zinc finger transcription factor Sp7(OSX)and Runt related transcription factor 2(Runx2)in each group.Alizarin Red S staining and alkaline phosphatase staining were used to evaluate their osteogenic differentiation ability.Western blot assay was used to detect the protein expression levels of Runx2,OSX,AKT,PI3K,p-PI3K,and p-AKT.(4)Subsequently,while PD-1 was overexpressed,NEDD4 knockdown treatment was performed to conduct a recovery experiment and observe changes in cell osteogenic differentiation. RESULTS AND CONCLUSION:(1)Immunoprecipitation-mass spectrometry,co-immunoprecipitation and immunofluorescence experiments showed that NEDD4 was the interactive protein of PD-1,and PD-1 and NEDD4 were co-localized.(2)The mRNA and protein expression levels of PD-1 in NEDD4 knockdown group were decreased in high glucose group(P<0.05).(3)NEDD4 knockdown group promoted osteogenic differentiation of rat bone marrow mesenchymal stem cells and activated PI3K/AKT pathway.(4)Osteoblast differentiation in the PD-1 overexpression+NEDD4 knockdown group was higher than that in the PD-1 overexpression+NEDD4 knockdown empty group,and the PI3K/AKT pathway was activated.It is concluded that PD-1 can regulate with NEDD4,affecting the activity of PI3K/AKT pathway and suppressing the osteogenic differentiation of bone marrow mesenchymal stem cells.