Effect of PAD4 expression and transcriptional regulation on osteogenic function in osteoblasts exposed to high glucose

Objective:To explore the effect of peptidyl arginine deiminase 4 (PAD4) expression and transcriptional regulation on osteoblasts in high glucose environment and its related mechanism.Methods:hFOB1.19 osteoblasts were cultured in normal(2g/L)and high glucose(4g/L)medium and divided into normal group and high glucose group; sh-PAD4 and sh-NC were transfected into hFOB1.19 osteoblasts by lentivirus in high glucose environment and divided into sh-PAD4 group and sh-NC group.The expression of PAD4, H3cit, NFAT2, OCN and ALP were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and western blotting.The enrichment of PAD4, H3cit and H3K27ac at the transcription start site(TSS)of NFAT2, OCN and ALP was detected by chromatin immunoprecipitation PCR (ChIP-qPCR).Cell proliferation was determined by cell counting kit-8 (CCK-8) assay, and cell mineralization capacity was determined by alizarin red S staining.Results:Compared with the control group, the expression of PAD4, H3cit, NFAT2, OCN and ALP in the high glucose group increased, the enrichment levels of PAD4, H3cit and H3K27ac in NFAT2, OCN and ALP TSS in the high glucose group increased, and the cell proliferation level and mineralization ability increased(P< 0.05).Compared with the sh-NC group, the expression of PAD4, H3cit, NFAT2, OCN and ALP in the sh-PAD4 group decreased, the enrichment levels of PAD4, H3cit and H3K27ac in NFAT2, OCN and ALP TSS decreased, the cell proliferation level and mineralization ability decreased (P< 0.05).Conclusion:The expression of PAD4 and its mediated H3cit increase in the high glucose environment, and the latter is enriched in NFAT2, OCN and ALP TSS, which promotes the expression of osteogenic-related genes, proliferation and differentiation of osteoblasts.