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.

Key words: bone marrow mesenchymal stem cell, programmed cell death receptor 1 (PD-1), neural precursor cell expressed developmentally downregulated 4 (NEDD4), PI3K, AKT, signaling pathway, high-glucose microenvironment