The Role Of USP22 In High-phosphate Induced Calcification Of Human Aortic Smooth Muscle Cells

Objective: Vascular calcification is a very common type of vascular pathophenotype,which is closely associated with senescence,atherosclerotic heart disease,diabetes mellitus,and chronic kidney disease.There is evidence that vascular calcification present in ? 90% of men and ? 67% of women over the age of 70 years.For women,cardiovascular disease is more likely to occur after menopause than before menopause,and the increase in this trend is in line with the increase of postmenopausal population,suggesting that the cardiovascular system of premenopausal women may benefit from higher levels of endogenous estrogen.Experiments have shown that smooth muscle cells isolated from the aorta of normal female pigs have higher levels of osteoprotegerin than ovariectomized pigs and are resistant to mineralization compared with ovariectomized pigs.USP22,a ubiquitin specific Peptidase,is highly conserved and widely expressed in human tissues.Recent studies have shown that USP22 inhibits SOX2 transcription and promotes differentiation of mouse embryonic stem cells,which is an important process in vascular calcification.At the same time,USP22 can enhance androgen receptor-mediated transcriptional activity.Therefore,this study aimed to explore the relationship between USP22 and estrogen/estrogen receptor(estrogen receptor,ER),and whether USP22 could play a role in the process of vascular calcification through this mechanism.Methods: Human aortic smooth muscle cells are induced by high phosphorus to establish the cell model of vascular calcification,and the model was evaluated by calcium colorimetric assay kit.The changes of USP22 protein level during calcification were observed by Western blotting.Effect of USP22 on the transcriptional activity of ER was detected by dual-luciferase assay.The interaction between USP22 and ER was detected by co-immunoprecipitation.After overexpression/silencing of USP22 in human aortic smooth muscle cells,calcium colorimetric assay kit was used to detect calcification.Results: The model of vascular calcification based on human aortic smooth muscle cells was successfully established.With Western blotting,the expression of RUNX2 and USP22 increased in calcification model.Dual-luciferase assay showed that USP22 inhibited the transcriptional activity of ER;Co-ip confirmed that USP22 could combine with ER;Cell calcification increased and RUNX2 expression increased when USP22 was overexpressed,and when USP22 was silenced Cell calcification inhibited.Conclusion: high phosphorus can induce calcification of human aortic smooth muscle cells.The expression of USP22 increased when human aortic smooth muscle cells calcified,suggesting that it promoted the process of calcification.With dual-luciferase assay and co-ip results,USP22 may inhibit the transcriptional activity of ER by directly binding to ER.However,the effect of overexpression/silencing of USP22 on calcification showed that it promoted the calcification through ER.