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. 2021 Mar 20;41(3):336–343. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2021.03.04

雌二醇通过ERβ调控ERK磷酸化影响细胞增殖和凋亡

Estradiol activates ERK phosphorylation by binding to ERβ to inhibit proliferation and promote apoptosis of human chondrocytes

Mengying ZHANG 1, Yuyou YANG 1, Min LIU 1, Li LIANG 1, Rui LUO 1, Danyang YIN 1, Fengjin GUO 1,*
PMCID: PMC8075796  PMID: 33849823

Abstract

Objective

To explore the effect of estradiol (E2) binding to its receptor ERβ on the proliferation and apoptosis of C28I2 cells.

Methods

We cloned the sequence of ESR2 into a recombinant adenovirus plasmid (pAd-ESR2) and packaged the plasmid in HEK293 cells. Normal human chondrocyte C28I2 cells were transfected with Ad-ESR2 or small interfering RNA targeting ESR2-siRNA (ESR2-siRNA), and the effects of treatment with DMSO or E2 on the expression of the proteins associated with endoplasmic reticulum (ER) stress and cell apoptosis were determined using Western blotting. qRT-PCR was used to detect the expressions of proliferation-related marker genes, and an EdU kit and flow cytometry were used to assess cell proliferation and apoptosis. We also tested the effects of U0126 (an ERK pathway inhibitor) and E2, alone or in combination, on ER stress, apoptosis and the ERK signaling pathway in C28I2 cells infected with Ad-ESR2 using Western blotting.

Results

Overexpression of Ad-ESR2 in C28I2 cells significantly promoted the expressions of IRE1α, PERK, XBP1s, and cleaved caspase-12, inhibited proliferation related marker genes PCNA, cyclin B1, cyclin D1, and decreased the level of ERK phosphorylation following E2 treatment (all P < 0.05). Interference of ESR2 caused significant reduction in the expressions of ER stress-related proteins and apoptosis-related proteins, up-regulated the genes related to cell proliferation, and increased intracellular pERK/ERK ratio in C28I2 cells. The effect of E2 binding to ERβ, which promoted the expressions of ER stress associated proteins and apoptosis related proteins, was obviously antagonized by treatment of the cells with U0126.

Conclusion

The binding of E2 to ERβ promotes ER stress and apoptosis in human chondrocytes by activating ERK pathway phosphorylation inhibit cell proliferation.

Keywords: estradiol, estrogen receptor β, endoplasmic reticulum stress, apoptosis, proliferation, ERK signaling pathway

软骨损伤大多由创伤引起，软骨以及软骨下骨的局部缺损随着年龄的增长侵袭范围逐渐增大，软骨功能也随之逐渐退化，最终发展为关节炎[1]。软骨组织由于其本身具有的无血管、无神经、无淋巴等特性，决定其损伤后的自身修复能力非常有限，研究显示由于软骨损伤导致的骨关节炎，严重影响了全球数百万患者的生存质量[2]。雌激素参与细胞的生长、发育和分化等多种生理过程，现已知类固醇激素17β-雌二醇（E2）是最有效的生理性雌激素。由重组人雌激素受体β（ESR2）基因编码的人雌激素受体β（ERβ）是雌激素的核受体，与雌激素反应元件结合以调节靶基因的转录，ESR2基因位于ERK基因的下游[3]。雌激素在骨生长与骨重建中都扮演了重要的角色[4-6]。有研究报道骨关节炎患者，其软骨细胞线粒体功能逐渐下降的同时，伴随着软骨细胞凋亡增加和自噬减少[7]。

研究显示雌二醇在骨发育中扮演重要角色[4-6]，内质网应激调控多种细胞凋亡[8-10]与增殖[11-12]，但ERβ是否以及如何通过内质网应激在软骨细胞及骨代谢中发挥作用尚不清楚，本研究旨在初步探讨雌二醇靶向其受体ERβ在软骨细胞C28I2中对增殖、凋亡的效应与机制。

1. 材料和方法

1.1. 材料

人正常软骨细胞C28I2、人胚胎肾细胞293，BJ5183菌株及pAd-Track-CMV腺病毒包装体系均来自于本实验室。吉凯基因ESR2质粒（133C89F），2×Taq PCR酶、SYBR qPCR MasterMix酶、逆转录试剂盒（Vazyme）；HindⅢ、BglⅡ、EcoRⅠ、EcoRⅤ内切酶、LIC连接酶（Takara）、Pac1酶（BioLabs）；Pme1酶（Thermo Scientific）；PEI转染试剂（Sigma-Aldrich）；DMEM细胞培养基、胎牛血清（BI）；DNA胶回收试剂盒（Omega）；RNA提取试剂盒（BioTeke）；RIPA强裂解液、EdU-488细胞增殖检测试剂盒（Beyotime）；蛋白MARKER（BioRad）；脱脂奶粉（伊利）；抗Caspase3抗体、抗IRE1α抗体、抗PERK抗体、抗ERK1/2抗体、抗p-ERK1/2抗体（CST）；抗ESR2抗体（Proteintech）、抗Caspase12抗体（NOVUS）；抗XBP1抗体（Affinity）；抗GAPDH抗体（碧云天）；U0126（MedChemExpress）；羊抗兔二抗（BOSTER）；羊抗鼠二抗（Absin）；ECL化学发光液（Affinity）；培养皿（NEST）。

1.2. 方法

1.2.1. Ad-ESR2重组腺病毒包装、鉴定与感染复数测定

从GeneBank获得ESR2编码序列，设计引物（含Hind Ⅲ、Bgl Ⅱ酶切位点），ESR2: Forward primer: （5'-GTCAGATCCGCTAGAGATCTCTGCAGGTCGACTCTAGA-3'），Revise primer: （5'-GATATCTTATCTAGAAGCTTTCCTTGTAGTCCATACCC-3'），以吉凯基因ESR2质粒（133C89F）为模板PCR扩增得到目的片段后，成功获得重组腺病毒质粒pAd-ESR2，酶切及测序鉴定正确。培养HEK-293细胞于对数生长期时接种于6孔板，待细胞达到70%~80%融合，将PacⅠ酶切并纯化后的Ad-ESR2质粒转染HEK-293细胞。待8~ 10 d后，大部分细胞出现细胞病变效应，收集细胞和上清并离心后反复冻融3次以上，收集病毒原液感染HEK293细胞，进行扩增，储存第三代腺病毒于-80 ℃冰箱备用。正确计算Ad-ESR2腺病毒滴度[13]后，感染C28I2细胞48 h，根据GFP阳性细胞荧光强度确定Ad-ESR2的最佳感染复数（MOI），检测Ad-ESR2的表达效率。

1.2.2. ESR2-siRNA的转染

将人正常软骨细胞C28I2于六孔板中以1×106/孔的密度铺盘，培养12 h待贴壁细胞密度约60%时更换为1 mL的无血清DMEM，根据PEI转染试剂说明准备转染稀释液：每个转染孔设置两组1.5 mL Ep管，其中一组Ep管用500 µL/管无血清DMEM以稀释5 µL PEI，另一组Ep管用500 µL无血清DMEM以稀释5 µL siRNA（转染浓度为5×10-8 mol/L），用枪头轻轻混匀后将Ep管室温孵育10 min，将PEI稀释液与siRNA稀释液合并吹打混匀室温孵育20 min后加入六孔板，继续培养6 h后更换为完全培养基。ESR2-siRNA由擎科生物设计并合成，项目订单TSX20200703-023-0011。ESR2-siRNA1: Forward primer: （5'-CCAGUUAUCACAUCUGUAUTT-3'），Revise primer: （5'-AUACAGAUGUGAUAACUGGTT-3'）；ESR2-siRNA2: Forward primer: （5'-GCAUUCAAGGACAUAAUGATT-3'），Revise pri-mer: （5'-UCAUUAUGUCCUUGAAUGCTT-3'）；ESR2-siRNA3: Forward primer: （5'-CAAAUCAGUGUACAAUCGATT-3')，Revise primer: （5'-UCGAUUGUACACUGAUUUGTT-3'）；si-control: Forward primer: （5'-UUCUCCGAACGUGUCACGUTT-3'），Revise primer: （5'-ACGUGACACGUUCGGAGAATT-3'）。

1.2.3. QPCR检测E2联合Ad-ESR2处理后C28I2细胞PCNA、CyclinB1和CyclinD1 mRNA表达

人正常软骨细胞C28I2细胞融合度达到60%时，加入Ad-ESR2和ESR2-siRNA处理24 h后加入E2（10-8 mol/L）[14]，24 h后提取细胞总RNA。六孔板每孔加入1 mL裂解液RL使贴壁细胞在冰上充分裂解2 min后用枪头吹打混匀收集液体于1.5 mL无酶EP管，4 ℃最大转速离心10 min，上清液转移至一新的无酶Ep管并加入0.2 mL三氯甲烷充分振荡15 s室温下孵育3 min，4 ℃、15 000 r/min离心10 min。最上层水相转移至新的无酶Ep管并加入等体积的70%乙醇吹打混匀，共同加入吸附柱中4 ℃最大转速离心1 min，经反复漂洗、去蛋白等步骤，将得到RNA融入30 μL无RNase水中，定量后一部分-80 ℃保存，一部分逆转录为cDNA进行QPCR。相对表达量计算：2-ΔΔCt [ΔΔCt=（Ct，目标-Ct，GAPDH）干预组-（Ct，目标-Ct，GAPDH）对照组]；内参：GAPDH。QPCR反应条件及PCNA，CyclinB，CyclinD1，GAPDH引物见已发表论文[14-15]。

1.2.4. 细胞蛋白提取以及Western blot检测

人正常软骨细胞C28I2细胞融合度达到60%时加入Ad-ESR2和ESR2-siRNA处理24 h后加入E2（10-8 mol/L）[14]和U0126（10 μmol/L，24 h后用PBS洗3次，用细胞裂解液提取细胞总蛋白质进行免疫印迹检测，每组蛋白质样品经10% SDS-PAGE分离后，采用250 mA恒流电转至PVDF膜上，5% BSA在摇床上室温封闭1 h后，一抗（1∶ 1000）4 ℃孵育，摇床过夜；次日，洗膜（1×TBST）3次后，HRP标记的二抗（1∶8000）室温孵育2 h；洗膜（1× TBST）3次后使用ECL发光液，机器曝光。

1.2.5. 流式细胞术（FCM）检测C28I2细胞凋亡和周期的变化

经分组处理48 h的人正常软骨细胞C28I2后收集于1.5 mL离心管中，用PBS洗2遍后轻轻吹散制成细胞悬液，凋亡组细胞立即送检，周期组细胞使用75%预冷乙醇固定后送至重庆医科大学生命科学院302流式细胞室检测细胞凋亡率和细胞周期的变化。

1.2.6. 免疫荧光实验

将C28I2细胞进行分组处理48 h后，加入EdU继续孵育2 h，室温下分别用4% PFA固定15 min和0.3% Triton X-100透化15 min。加入Click反应液避光孵育30 min，1×PBS洗3次，5 min/次，加入Hoechest室温避光孵育10 min，1×PBS洗3次，5 min/次，拍摄照片。细胞图像通过显微镜用×10摄取。使用ImageJ专业图像分析软件对单个细胞累积吸光度值进行量化。

1.3. 统计学分析

各组实验结果均采用Graphpad prism6软件进行统计分析，数据以均数±标准差进行描述，采用单因素方差分析，以P < 0.05为差异具有统计学意义。所有实验都是独立重复3次。

2. 结果

2.1. Ad-ESR2重组腺病毒和ESR2-siRNA的构建和鉴定

以HEK293真核细胞cDNA为模板，PCR获得ESR2目的基因1593 bp（图 1A）。EcoRⅠ和EcoRⅤ酶切鉴定重组穿梭质粒pAdTrack-ESR2，得到约6000 bp和4600 bp片段（图 1B）。共同电转腺病毒穿梭质粒pAdTrack-ESR2和腺病毒骨架质粒pAd-Easy-1得到pAdESR2重组腺病毒质粒，酶切及测序鉴定重组腺病毒质粒构建成功（图 1C、E）；重组腺病毒Ad- ESR2在HEK293细胞中包装成功（图 1D），Ad-ESR2滴度为6.5× 1010 PFU/mL；Ad-ESR2腺病毒的最佳感染复数MOI= 240（图 1F）。C28I2细胞经Ad- ESR2感染48 h后，Western blot鉴定ERβ的蛋白表达量与对照组相比明显上调（P < 0.05，图 1G、H）。ESR2-siRNA转染C28I2细胞48 h后，相较于对照组si-control，细胞中ERβ的蛋白水平明显降低（P < 0.05）；量化分析显示ESR2- si1，ESR2-si2，ESR2-si3的敲降率分别为56%，54%，72%，因此，选用ESR2-si1和ESR2-si3为后续实验siRNA序列（图 1I、J）。

1.

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Ad-ESR2重组腺病毒和ESR2-siRNA的构建和鉴定

Construction and identification of Ad-ESR2 recombinant adenovirus and ESR2-siRNA. A: ESR2 PCR result. M: 2000 DNA Ladder; 1: ESR2 cDNA. B: The result of restriction enzyme digestion of pAd-Track-ESR2. M: DL 10000; 1: pAd-Track-CMV without digestion; 2: pAd-Track-CMV digested by EcoRI and EcoRV; 3: pAd-Track-ESR2 digested by EcoRI and EcoRV. C: The result of Pac1 digestion of pAd-ESR2. M: DL 20000; 1: pAd-Easy without digestion; 2: pAd-ESR2 digested by Pac1. D: Packaging of adenovirus pAdESR2 in HEK293. E: ESR2 sequencing result graph. F: Determination of MOI value of Ad-ESR2 in C28I2. G: Protein level of ERβ in the Ad-ESR2-infected C28I2 cells. H: Quantification analysis of the protein level of ERβ in the Ad-ESR2-infected C28I2 cells. *P < 0.05 vs AdGFP. I: Protein level of ERβ in the ESR2-siRNA-transfected C28I2 cells. J: Quantification analysis of the protein level of ERβ in the ESR2-siRNA-transfected C28I2 cells. *P < 0.05 vs si-control.

2.2. E2靶向ERβ对C28I2细胞增殖的影响

C28I2细胞分组处理48 h后，Real-time PCR结果显示（图 2A~C），与对照组E2+Ad-GFP相比，E2+AdESR2实验组周期相关基因PCNA、CyclinB1、CyclinD1均显著降低（P < 0.05）；与对照组E2+sicontrol相比，E2+ ESR2-si1实验组的PCNA、CyclinB1、CyclinD1 mRNA水平均升高（P < 0.05）；实验组E2+ESR2-si3的PCNA、CyclinB1的mRNA水平增加（P < 0.05）。使用EdU-488细胞增殖检测试剂盒，根据EdU定位S期细胞与所有细胞数Hoechest的占比，与对照组E2组（98%）相比，E2+ Ad-ESR2组S期细胞（24%）减少（P < 0.05，图 2D、E）。用流式细胞仪分析细胞周期的变化，结果显示与对照组E2+sicontrol相比，实验组E2+ESR2-si1和E2+ESR2- si3的DNA合成期（S）明显增加（P < 0.05，图 2F、G）。

2.

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E2结合ERβ对C28I2细胞增殖的作用

Effects of E2 and ERβ on the proliferation of C28I2 cells. A-C: qPCR analysis of mRNA level of cell cycle related marker genes (PCNA, cyclin B1, and cyclin D1) in chondrocytes treated by Ad-ESR2 and ESR2-siRNA with E2 (*P < 0.05 vs E2+Ad-GFP, #P < 0.05 vs E2+sicontrol). D: EdU assay of cell cycle in Ad-ESR2-infected C28I2 cells with E2 (48 h after treatment, original magnification: ×10). E: Quantification analysis of relative fluorescence intensity level of EdU, *P < 0.05 vs E2. F: Effect of ESR2-siRNA on cell cycle in C28I2 cells with E2 by FCM. G: Quantification analysis of the FCM.

2.3. E2靶向ERβ对C28I2细胞ER stress及介导凋亡的影响

C28I2细胞分组处理48 h后，收集各组细胞蛋白进行Western blot，结果显示E2 +Ad- ESR2处理后，Cleaved Caspase12、PERK、IRE1α、XBP1s的表达相较于E2+Ad-GFP对照组明显增加（P < 0.05，图 3A、B）；转染ESR2-siRNA后，Cleaved Caspase12、PERK、IRE1α、XBP1s的表达与对照组E2+sicontrol相比均减少（P < 0.05，图 3C、D）。流式细胞术分析显示，与E2+Ad-GFP对照组的凋亡率25.81%相比，E2+Ad-ESR2实验组的凋亡率显著升高（88.41%）（P < 0.05，图 3E、F）。E2 + ESR2-siRNA实验组与E2+sicontrol对照组的凋亡率相比无显著差异。

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E2结合ERβ对C28I2细胞ER stress及介导凋亡的影响

Effects of E2 and ERβ on ER stress and apoptosis of C28I2 cells. A: E2 combines with Ad-ESR2 to affect protein levels of ER stress and apoptosis related protein levels detected by Western blotting. B: Quantification analysis of the A, *P < 0.05 vs E2+Ad-GFP, C: E2 combines with ESR2- siRNA to affect protein levels of ER stress and apoptosis related protein levels detected by Western blotting, D: Quantification analysis of the C, #P < 0.05 vs E2+sicontrol. E: FCM detects the results of Ad-ESR2 and ESR2-siRNA on C28I2 cell apoptosis under E2 treatment. F: Quantification analysis of the apoptotic rate, *P < 0.05 vs E2+Ad-GFP.

2.4. C28I2细胞中E2靶向ERβ通过ERK信号通路影响ER stress及介导凋亡

C28I2细胞分组处理48 h后，收集各组细胞蛋白进行Western blot，结果显示，与对照组E2+Ad-GFP相比，实验组E2+Ad-ESR2中pERK/ERK的相对表达水平明显下降（P < 0.05，图 4A、B），而在转染ESR2-siRNA后，pERK/ERK的相对表达水平增加（P < 0.05，图 4C、D）。使用ERK通路抑制剂U0126（10 µmol/L）阻断ERK信号通路后，能够显著抑制E2+Ad-ESR2诱导的内质网应激相关蛋白PERK、IRE1α、XBP1s和凋亡相关蛋白cleaved caspase3、cleaved caspase12的表达（P < 0.05，图 4E~G）。

4.

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E2结合ERβ影响ER stress及凋亡与ERK的磷酸化

E2 combined with ERβ affects ER stress, apoptosis and ERK phosphorylation. A, B: Effect of E2 combined with AdESR2 on protein levels of pERK and ERK (*P < 0.05 vs E2+Ad-GFP). C, D: Effect of E2 combined with ESR2-siRNA on protein levels of pERK and ERK (#P < 0.05 vs E2+sicontrol). E-G: Effect of U0126 on ER stress and apoptosis in cells treated with E2+AdESR2 detected by Western blotting (#P < 0.05 vs E2; *P < 0.05 vs E2+Ad-ESR2).

3. 讨论

自研究证明了ER的存在以来，ERβ从大鼠前列腺和卵巢成功克隆，随后被证明其具有与ERα不同的配体结合特异性和对雌激素激动剂的信号响应[16-19]。ERβ在卵巢、肺、男性生殖器官、前列腺、结肠、肾脏等多种器官和系统中均有表达。ERβ相对于ERα以更高的亲和力结合雌二醇和B环不饱和雌激素[20]。研究发现，在女性供体的人类牙周膜细胞PDLC中用雌激素处理24 h后，ESR2/ESR1的比率增加了1倍，表明雌激素受体以性别特异性模式表达[21]。在成骨细胞中，ERα激活会增加ERβ的表达，而ERβ可以抑制ERα表达，并且在缺乏ERβ的小鼠骨骼中ERα升高[22-24]。ERα和ERβ信号转导通路联系密切、互相影响[25-26]。通过选择性抑制ERβ会增加大鼠成骨细胞的增殖，而选择性抑制ERα可以减少增殖。因此，ERα和ERβ对基底成骨细胞的增殖具有相反的作用，这种情况在其他各种细胞模型中都得到了很好的证实[27-30]。本实验通过构建Ad-ESR2腺病毒和敲低ESR2 siRNA，探究其影响人正常软骨细胞C28I2增殖与凋亡的机制。

ESR2参与调控多种细胞的增殖与凋亡，ERβ被认为具有抗增殖和促凋亡作用，因此被描述为一种肿瘤抑制因子，研究表明，卵巢癌和乳腺癌中ERβ的过表达下调细胞周期蛋白D1，从而体现抗细胞增殖的作用[31]。经过量雌二醇处理后ERα促进了的细胞增殖，而ERβ抑制了细胞的基础增殖[32]。在COLO205结肠癌细胞中有研究发现，ERβ诱导DNA的断裂从而导致细胞凋亡；在LoVo结肠癌细胞中ERβ通过增加的p53信号传导，从而增加细胞凋亡并减少细胞增殖[33-34]。研究证明，ERβ的过表达通过减少Cyclin E和增加p21，调节细胞周期，减少HCT8人结肠癌细胞的增殖[35]。在结肠癌细胞HT29和SW480中有研究发现，ERβ通过调节细胞周期的G1期基因使细胞增殖被抑制[36]。本实验结果发现，E2与Ad-ESR2共同处理C28I2细胞后，DNA合成期细胞显著降低，细胞增殖显著抑制；同时ERK磷酸化水平明显抑制，流式细胞术及QPCR结果均表明C28I2细胞凋亡增加；采用U0126特异性阻断ERK磷酸化激活，显示E2靶向ERβ激活的内质网应激及促进的凋亡均被抑制，提示E2靶向结合ERβ调控细胞增殖、内质网应激与凋亡等生物学功能依赖于ERK信号途径。

ERK通路的磷酸化激活与细胞增殖密切相关，ERβ和ERα在多种不同类型细胞中通过激活ERK信号引发各种生物学效应[37-38]。有研究报道黄豆苷元通过EsR依赖性MEK/ERK和PI3K/Akt途径在MG-63细胞中增强细胞增殖[39]。有研究发现BPA诱导的成骨细胞在用不同浓度植物雌激素治疗后，其细胞活力，增殖和分化均增加[40]。而本研究发现软骨细胞中雌二醇E2通过靶向结合ERβ，通过激活ERK磷酸化激活，促进内质网应激及介导凋亡，进而抑制软骨细胞的增殖。内质网应激是指当内质网受到外界的刺激后，激活内质网上的三条信号通路（即IRE1，PERK和ATF6），从而重建内质网稳态；但当应激时间过长或应激反应超过了细胞负荷，便导致细胞凋亡。后续我们将进一步深入探讨E2靶向ERβ发挥的生物学效应调控内质网应激及凋亡参与骨、软骨生长的具体机制。

综上所述，本实验通过成功构建Ad-ESR2腺病毒和ESR2 siRNA，证明E2靶向结合ERβ通过ERK信号通路调控内质网应激及介导的凋亡，进而抑制细胞增殖，实现E2结合ERβ对软骨细胞增殖多靶点、多方位的调控。为进一步研究E2与ERα、ERβ在骨发育及骨相关疾病中的作用奠定实验基础。

Biography

张梦颖，硕士，E-mail: 479160704@qq.com

Funding Statement

重庆市人力资源和社会保障局（重庆市留学人员创业创新支持计划，渝人社办2018-389）

Contributor Information

张 梦颖 (Mengying ZHANG), Email: 479160704@qq.com.

郭 风劲 (Fengjin GUO), Email: guo.fengjin@cqmu.edu.cn.

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