Estradiol Regulates ERK Phosphorylation Through ER? And Affects Cell Proliferation And Apoprosis

Objective: To explore the effect of Estradiol(E2)binding its receptor estrogen receptor ?(ER?)on the proliferation and apoptosis of C28I2.Methods: PCR amplified ESR2(recombinant human estrogen receptor ?)sequence and constructed adenovirus recombinant plasmid p Ad-ESR2,and packaged the recombinant adenovirus Ad-ESR2 in HEK293 cells.Treat human normal chondrocyte C28I2 with Ad-ESR2 and ESR2-si RNA,and set up control group(DMSO);E2 group(E2);Ad-ESR2group(Ad-ESR2);E2+Ad-GFP group(E2+Ad-GFP);E2+Ad-ESR2 group(E2+Ad-ESR2);E2+sicontrol group(sicontrol+E2);E2+ESR2-si1 group(ESR2-si1+E2);E2+ESR2-si3 group(ESR2-si3+E2).Western blotting was used to determine the expression of ER Stress and apoptosis-related proteins,q RT-PCR was used to detect the expression of proliferation-related marker genes,and the Ed U kit and flow cytometry were used to determine cell proliferation and apoptosis.ERK pathway inhibitor U0126 was used to set E2 group(E2);E2+U0126 group(E2+U0126);E2+Ad-ESR2 group(E2+Ad-ESR2);E2+Ad-ESR2+U0126group(E2+ Ad-ESR2+U0126).Through western blotting,it is explored that E2 in C28I2 cells affects ER stress and apoptosis through ER?regulating ERK signaling pathway.Results:(1)Successful construction of si RNA overexpressing adenovirus Ad-ESR2 and targeting ESR2;(2)Overexpression of Ad-ESR2 can promote IRE1? after E2 treatment [1.20±0.06 vs 0.95±0.05,P<0.05],PERK[1.29±0.04 vs 1.03±0.02,P<0.05] and the expression of XBP1s[1.17±0.03 vs 0.82±0.02,P<0.05],promote apoptosis Cleaved Caspase12[1.04±0.02 vs 0.87±0.04,P<0.05],inhibit proliferation related signs Gene PCNA[0.24±0.01 vs 0.59±0.07,P<0.05],Cyclin B1[0.22±0.01 vs 1.03±0.06,P<0.05],Cyclin D1[0.26±0.02 vs 0.63±0.02,P<0.05]expression,and ERK Phosphorylation activation decreased [0.83±0.03 vs1.95±0.12,P<0.05];(3)After transfection of ESR2-si RNA,the expression of endoplasmic reticulum stress-related protein and apoptosis-related protein decreased,which was related to cell proliferation.Related genes were up-regulated,and the intracellular p ERK/ERK ratio increased relatively.(4)After treatment with the specific ERK pathway inhibitor U0126,the expression of endoplasmic reticulum stress protein and apoptosis protein promoted by estrogen binding to ER? was antagonized.Conclusion: Estradiol targeting ER? regulates endoplasmic reticulum stress and apoptosis by activating ERK pathway phosphorylation,thereby inhibiting chondrocyte proliferation.