Liu Min, Xie Weiwei, Zheng Wei, Yin Danyang, Luo Rui, Guo Fengjin
Department of Cell Biology and Genetics, Core Facility of Development Biology, Basic Medical Science of Chongqing Medical University, Chongqing 400016, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2019 Feb 28;39(2):134-143. doi: 10.12122/j.issn.1673-4254.2019.09.02.
To investigate the effect of estradiol (E2)/estrogen receptor 1 (ESR1) on the proliferation of human chondrocytes and explore the molecular mechanism.
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.
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.
The targeted binding of E2 with ESR1 promotes the proliferation of human chondrocytes possibly by inhibiting the activation of ERK signaling pathway to promote cell autophagy and induce cell apoptosis.
研究雌二醇（E2）/雌激素受体1（ESR1）对人软骨细胞增殖的影响并探讨其分子机制。
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。运用蛋白质免疫印迹法（Western blotting）和实时定量聚合酶链反应（qPCR）检测人软骨细胞C28I2中ESR1蛋白和mRNA的表达。在不同腺病毒处理的细胞中，用蛋白质免疫印迹法检测E2对细胞自噬和凋亡相关蛋白表达以及细胞外信号调节激酶（ERK）信号通路磷酸化的影响。采用免疫荧光法观察细胞内自噬流，通过流式细胞术分析细胞凋亡率和细胞周期变化，并用qPCR检测增殖细胞核抗原（PCNA）、细胞周期蛋白B1（cyclin B1）和细胞周期蛋白D1（cyclin D1）mRNA的表达。使用蛋白质免疫印迹法和qPCR检测ERK特异性抑制剂对自噬和凋亡相关基因在蛋白和mRNA水平表达的抑制作用。
转染过表达ESR1的重组腺病毒并经E2处理C28I2细胞后，显著增强了自噬相关蛋白微管相关蛋白轻链3（LC3）、自噬相关蛋白7（ATG7）的表达，促进了LC3与溶酶体相关膜蛋白1（LAMP1）在细胞质中的共定位，增加了增殖相关标记基因PCNA、cyclin B1和cyclin D1的表达，并抑制了裂解的半胱天冬酶-3（cleaved caspase-3）、半胱天冬酶-12（caspase-12）和磷酸化ERK（pERK）的表达。对ESR1进行RNA干扰明显降低了C28I2细胞中自噬相关蛋白的表达水平，导致自噬流受抑制，凋亡相关蛋白和pERK表达增加，增殖标记基因表达下调。用ERK抑制剂阻断ERK激活明显抑制了E2/ESR1对自噬、增殖相关基因表达和细胞凋亡的作用。
E2与ESR1的靶向结合可能通过抑制ERK信号通路的激活来促进细胞自噬和诱导细胞凋亡，从而促进人软骨细胞的增殖。
