[Targeted binding of estradiol with ESR1 promotes proliferation of human chondrocytes in vitro by inhibiting activation of ERK signaling pathway].
[Targeted binding of estradiol with ESR1 promotes proliferation of human chondrocytes in vitro by inhibiting activation of ERK signaling pathway].
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DOI：
10.12122/j.issn.1673-4254.2019.09.02
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发表时间：
2019-02-28
影响因子：
--
通讯作者：
Guo, Fengjin
中科院分区：
文献类型：
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作者：
Liu, Min;Xie, Weiwei;Guo, Fengjin
目的：探讨雌二醇（E2）/雌激素受体1（ESR1）对人软骨细胞体外增殖的影响并探讨其分子机制。方法：采用Ad-Easy腺病毒包装系统构建并包装过表达ESR1的腺病毒Ad-ESR1。采用Western blotting和qPCR检测人软骨细胞C28I2细胞中ESR1蛋白和mRNA的表达。在不同腺病毒处理的细胞中，采用Western blotting检测E2对细胞自噬、凋亡相关蛋白表达及ERK信号通路磷酸化的影响。免疫荧光法观察细胞内自噬流，流式细胞仪分析细胞凋亡率及细胞周期变化，qPCR检测PCNA、cyclin B1、cyclin D1 mRNA的表达。采用Western blotting和qPCR检测ERK特异性抑制剂在蛋白和mRNA水平上对自噬和凋亡相关基因表达的抑制作用。结果：转染过表达ESR1的重组腺病毒并E2处理C28I2细胞后，自噬相关蛋白LC3、ATG7的表达显着增强，促进LC3和LAMP1在细胞质中的共定位，增加了自噬相关蛋白LC3、ATG7的表达。增殖相关标记基因 PCNA、cyclin B1 和 cyclin D1，并抑制 cleaved caspase-3、caspase-12 和 pERK 的表达。 ESR1的RNA干扰明显降低C28I2细胞中自噬相关蛋白的表达水平，导致自噬流受到抑制，凋亡相关蛋白和pERK表达增加，并下调增殖标志基因的表达。 ERK抑制剂阻断ERK激活，可明显抑制E2/ESR1对自噬、增殖相关基因表达和细胞凋亡的影响。结论：E2与ESR1靶向结合可促进人软骨细胞体外增殖，可能是通过抑制ERK信号通路的激活，促进细胞自噬、诱导细胞凋亡。
OBJECTIVE: To investigate the effect of estradiol (E2)/estrogen receptor 1 (ESR1) on the proliferation of human chondrocytes in vitro and explore the molecular mechanism.METHODS: The Ad-Easy adenovirus packaging system was used to construct and package the ESR1-overexpressing adenovirus Ad-ESR1. Western blotting and qPCR were used to detect the expression of ESR1 protein and mRNA in human chondrocyte C28I2 cells. In the cells treated with different adenoviruses, the effects of E2 were tested on the expressions of proteins related with cell autophagy and apoptosis and the phosphorylation of ERK signaling pathway using Western blotting. Immunofluorescence assay was used to observe the intracellular autophagic flow, flow cytometry was performed to analyze the cell apoptosis rate and the cell cycle changes, and qPCR was used to detect the expressions of PCNA, cyclin B1 and cyclin D1 mRNAs. The inhibitory effect of the specific inhibitor of ERK on the expressions of autophagy- and apoptosis-related genes at both the protein and mRNA levels were detected using Western blotting and qPCR.RESULTS: Transfection with the recombinant adenovirus overexpressing ESR1 and E2 treatment of C28I2 cells significantly enhanced the expressions of autophagy-related proteins LC3, ATG7, promoted the colocalization of LC3 and LAMP1 in the cytoplasm, increased the expressions of the proliferation-related marker genes PCNA, cyclin B1 and cyclin D1, and supressed the expressions of cleaved caspase-3, caspase-12 and pERK. RNA interference of ESR1 obviously lowered the expression levels of autophagy-related proteins in C28I2 cells, causing also suppression of the autophagic flow, increments of the expressions of apoptosis-related proteins and pERK, and down-regulated the expressions of the proliferation marker genes. Blocking ERK activation with the ERK inhibitor obviously inhibited the effects of E2/ESR1 on autophagy, proliferationrelated gene expressions and cell apoptosis.CONCLUSIONS: The targeted binding of E2 with ESR1 promotes the proliferation of human chondrocytes in vitro possibly by inhibiting the activation of ERK signaling pathway to promote cell autophagy and induce cell apoptosis.