Different calcium ion concentrations affect epithelial mesenchymal transformation of human peritoneal mesothelial cells via endoplasmic reticulum stress
Objective To study the effects of different calcium ion concentrations on epithelial mesenchymal transformation(EMT)of human peritoneal mesothelial cell(HPMC)via endoplasmic reticulum stress(ERS).Methods HPMC cell line HMrSV5 was cultured in vitro and treated in groups.The cells in the control group,high calcium group 1,and high calcium group 2 were treated with medium containing calcium ion concentrations of 1.25,1.75,and 2.25 mmol/L,respectively.The solvent control group was treated with medium containing 1.25 mmol/L physiological calcium ion concentration and 0.1%dimethyl sulfoxide(DMSO),the high calcium+solvent group was treated with medium containing 2.25 mmol/L calcium ion concentration and 0.1%DMSO,the high calcium+4-phenylbutyric acid(4-PBA)group was treated with medium containing 2.25 mmol/L calcium ion concentration and 1 mmol/L ERS inhibitor 4-PBA,and each group was treated for 48 hours.Morphological changes of cells in each group were observed under light microscope.The expressions of epithelial cell phenotype marker zonula occluden-1(ZO-1)and mesenchymal cell phenotype marker α-smooth muscle actin(α-SMA)in the cells were observed by immunofluorescence staining.The expressions of EMT marker genes E-cadherin,ZO-1,α-SMA and Vimentin were detected by fluorescence quantitative polymerase chain reaction(PCR).The expressions of ERS marker proteins phosphorylated protein kinase R-like endoplasmic reticulum kinase(p-PERK),phosphorylated eukaryotic initiation factor 2α(p-eIF2α),transcription activating factor 4(ATF4)and C/EBP homologous protein(CHOP)were detected by Western blotting.Results Compared with the control group,the morphology of HMrSV5 cells became slender and fibrotic,the fluorescence intensity of ZO-1 increased,and the fluorescence intensity of α-SMA decreased in high calcium 1 and high calcium 2 groups,indicating that the cells transformed from epithelial cells to mesenchyme cells.The mRNA expressions of E-cadherin and ZO-1 were significantly decreased,while the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK,p-eIF2α,ATF4 and CHOP were significantly increased,moreover,the expressions of the above marker genes or proteins in the high calcium 2 group was more obvious than those in the high calcium 1 group[E-cadherin mRNA(2-ΔΔCt):0.53±0.05 vs.0.75±0.09,ZO-1 mRNA(2-ΔΔCt):0.42±0.06 vs.0.69±0.06,α-SMA mRNA(2-ΔΔCt):1.81±0.16 vs.1.32±0.14,Vimentin mRNA(2-ΔΔCt):2.05±0.22 vs.1.48±0.16,p-PERK protein(p-PERK/β-actin):0.81±0.09 vs.0.59±0.06,p-eIF2α protein(p-eIF2α/β-actin):0.87±0.10 vs.0.50±0.06,ATF4 protein(ATF4/β-actin):0.93±0.10 vs.0.72±0.06,CHOP protein(CHOP/β-actin):0.79±0.09 vs.0.46±0.04,all P<0.05].Compared with the solvent control group,the morphological changes of cells,the expressions of EMT marker genes and ERS marker proteins after high calcium ion concentration of 2.25 mmol/L were consistent with those in the high calcium 2 group than control group.Compared with the high calcium+solvent group,the cell morphology recovered the characteristics of polygonal and pebble-like epithelial cells in the high calcium+4-PBA group,the fluorescence intensity of ZO-1 increased,the fluorescence intensity of α-SMA decreased,and the mRNA expressions of E-cadherin and ZO-1 in the cells were significantly increased[E-cadherin mRNA(2-ΔΔCt):0.86±0.09 vs.0.57±0.04,ZO-1 mRNA(2-ΔΔCt):0.81±0.06 vs.0.48±0.05,both P<0.05],the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK,p-eIF2α,ATF4 and CHOP were significantly decreased[α-SMA mRNA(2-ΔΔCt):1.21±0.13 vs.1.77±0.15,Vimentin mRNA(2-ΔΔCt):1.30±0.14 vs.1.94±0.20,p-PERK protein(p-PERK/β-actin):0.38±0.04 vs.0.92±0.11,p-eIF2α protein(p-eIF2α/β-actin):0.34±0.05 vs.1.05±0.13,ATF4 protein(ATF4/β-actin):0.57±0.06 vs.0.97±0.11,CHOP protein(CHOP/β-actin):0.51±0.04 vs.0.90±0.12,all P<0.05].Conclusion High calcium ion concentrations of 1.75 mmol/L and 2.25 mmol/L promote EMT of HPMC via activating ERS.
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