Parathyroid Hormone Promotes Human Umbilical Vein Endothelial Cell Migration And Proliferation Through Orai1-mediated Calcium Signaling

Background:Secondary hyperparathyroidism（SHPT）is an independent risk factor for cardiovascular events（CVD）and death,which seriously affects the survival rate and quality of life of patients.Parathyroid hormone（PTH）is the main endocrine regulator of extracellular calcium and phosphorus levels.Stored-operated calcium entry（SOCE）regulates Ca²⁺entry in non excitatory cells,and Orai1 is the main component of SOCE.SOCE plays a key role in the occurrence and development of CVD.SHPT related endothelial dysfunction may be related to calcium homeostasis disorder,and the specific mechanism is still unclear.Objective:The purpose of this study was to study the effect and related mechanism of PTH on human umbilical vein endothelial cells（HUVECs）,and the effects of storage operated Ca²⁺entry（SOCE）and NFAT pathway on endothelial dysfunction.Fully understand the pathogenesis of SHPT related cardiovascular diseases and find new prevention and treatment targets.Methods:（1）Western blot and CCK8 cell proliferation toxicity test were used to detect the effect of PTH on the expression level and proliferation of SOCE-related proteins in HUVECs.（2）Calcium imaging and CCK8 cell proliferation toxicity test were used to detect the role of Orai1 protein in mediating soce mediated calcium influx and the proliferation activity of PTH treated HUVECs,respectively.（3）RNA sequencing（RNA seq）was used to detect the differentially expressed genes between PTH treated and non PTH treated HUVECs.Some of these genes were confirmed by Western blot and quantitative RT-PCR.（4）Western blot and immunofluorescence were used to detect the effect of PTH on NFAT nuclear translocation of HUVECs after treatment with BTP2,Cs A,W7 and Orai1 si RNA transfection.（5）The effects of BTP2,Cs A,W7 and Orai1 si RNA transfection on the expression of COL1A1 in PTH treated HUVECs were detected by Western blot.（6）Cell migration assay and CCK8 cell proliferation toxicity assay were used to detect whether COL1A1 regulates cell migration and proliferation in PTH treated HUVECs.Results:（1）Compared with the control group,after HUVECs were treated with PTH（100 p M）for 24 h,the protein expression levels of Orai1 and STIM1 increased significantly,By contrast,Orai2 and Orai3 protein expression levels were not affected by any tested concentration of PTH,and the proliferation ability of HUVECs increased.（2）PTH increased the Ca²⁺influx and proliferation of HUVECs by regulating Orai1.（3）Transcriptome sequencing found 1655 differentially expressed genes（823up-regulated and 832 down regulated）in PTH treated HUVECs,Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysed enhanced expression levels of adhesion signal and two key genes COL1A1 and NFATc1.It was verified by Western blot and quantitative RT-PCR.（4）Orai1 is involved in the regulation of nuclear translocation of NFATC1 induced by PTH.（5）PTH regulates COL1A1 expression by enhancing NFATC1 nuclear translocation.（6）PTH can regulate cell migration and proliferation of HUVECs by enhancing COL1A1 expression.Conclusions:This study is the first,to our knowledge,to demonstrate that PTH increased Orai1protein expression and Orai1-mediated SOCE activity in HUVECs,leading to the nuclear translocation of NFATC1 to subsequently enhance COL1A1 expression and COL1A1-mediated migration and proliferation of HUVECs.Our results suggest that Orai1 and the downstream calmodulin/calcineurin/NFATC1/COL1A1 signaling pathway may play an important role in PTH-induced endothelial dysfunction and may be key potential therapeutic targets for preventing or treating SHPT-induced CVD.