E2 Targeting ESR1 Regulates Chondrocyte Proliferation Via ERK Signaling Pathway

Objective:To investigate the effect of E2/ESR1?estrogen receptor 1,ER??on proliferation of C28I2 chondrocyte and explore the molecular mec hanism.This study lays a foundation for further exploring the biologi cal function of ESR1 gene and provides a certain experimental basis and theoretical basis for the study of cartilage-related diseases.Methods:The Ad-Easy adenovirus vector system was used to construct and package the over-expression adenovirus Ad-ESR1.Western blot and QPCR were used to detect the expression of the mRNA and protein in C28I2 cells.Different treatment groups of adenovirus were set under the induction of E2,western blot was used to detect the expression of autophagy,apoptosis-related protein and phosphorylation of ERK signaling pathway in C28I2 cells.IHC was used to observe the intracellular autophagic flow.FCM was used to detect the apoptosis rate and cell cycle;QPCR detects the expression of PCNA?CyclinB1 and CyclinD1.The adenovirus treated mouse metatarsus in vitro for 5 days,observed the bone length and mineralization by Alcian blue-alizarin red staining.The cells were treated with specific inhibitor of ERK named U0126,and the inhibitory effects about autophagy,apoptosis-related protein on this pathway were detected by western blot.QPCR was used to detect the expression of proliferation-related marker genes.Results:The recombinant adenovirus Ad-ESR1 was successfully constructed and packaged?Titer=2×10⁸ PFU/mL?.High-concentration E2(10^-5mol/L?10^-6mol/L)inhibited autophagy and increased the expression of apoptotic proteins in C28I2 cells,while low-concentration E2(10^-9mol/L)up-regulated the expression of LC3 and ATG7 and decreased the expression of apoptosis-related proteins.Overexpression of ESR1 further promoted the expression of LC3 and ATG7 after treatment of low-concentration E2,promoted the colocalization of LC3 and LAMP1 in the cytoplasm and the expression of the proliferation-related marker genes PCNA,Cyclin B1 and Cyclin D1,supressed the expression of cleaved caspase3?cleaved caspase12 and pERK.Ad-ESR1 increased the bone length and mineralization of mouse metatarsus.After inhibiting the expression of ESR1 by siRNA,combined with E2 treatment of C28I2 cells,ESR1-siRNA reduced the effect of E2 on autophagy,and on the other hand reduced the inhibition of E2 on apoptosis,and the cell proliferation was down-regulated.The intracellular pERK is relatively increased.After the activation of the ERK signaling pathway was blocked by specific inhibitors,the biological effects of E2 targeting ESR1 were inhibited.Conclusions:Different concentrations of E2 have different biological effects on chondrocytes.The targeted binding of E2 to ESR1 can promote the proliferation of chondrocytes by promoting autophagy and inhibiting apoptosis.Besides,ESR1 also regulates endochondral bone formation.The biological role of E2 targeting ESR1 to regulate proliferation is associated with the ERK signaling pathway.