Li X, Yin D, Fan M, Yang Y, Liang L, Feng N, Li X, Guo F
Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, China.
Department of Pathology, Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing 100011, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2022 Jun 20;42(6):785-793. doi: 10.12122/j.issn.1673-4254.2022.06.01.
To explore the mechanism by which inositol-requiring enzyme-1 (IRE1) regulates autophagy function of chondrocytes through calcium homeostasis endoplasmic reticulum protein (CHERP).
Cultured human chondrocytes (C28/I2 cells) were treated with tunicamycin, 4μ8c, rapamycin, or both 4μ8c and rapamycin, and the expressions of endoplasmic reticulum (ER) stress- and autophagy-related proteins were detected with Western blotting. Primary chondrocytes from ERN1 knockout (ERN1 CKO) mice and wild-type mice were examined for ATG5 and ATG7 mRNA expressions, IRE1 and p-IRE1 protein expressions, and intracellular calcium ion content using qPCR, Western blotting and flow cytometry. The effect of bafilomycin A1 treatment on LC3 Ⅱ/LC3 Ⅰ ratio in the isolated chondrocytes was assessed with Western blotting. Changes in autophagic flux of the chondrocytes in response to rapamycin treatment were detected using autophagy dual fluorescent virus. The changes in autophagy level in C28/I2 cells overexpressing CHERP and IRE1 were detected using immunofluorescence assay.
Tunicamycin treatment significantly up-regulated ER stress-related proteins and LC3 Ⅱ/LC3 Ⅰ ratio and down-regulated the expression of p62 in C28/I2 cells ( < 0.05). Rapamycin obviously up-regulated LC3 Ⅱ/LC3 Ⅰ ratio ( < 0.001) in C28/I2 cells, but this effect was significantly attenuated by co-treatment with 4μ8c ( < 0.05). Compared with the cells from the wild-type mice, the primary chondrocytes from ERN1 knockout mice showed significantly down-regulated mRNA levels of ERN1 ( < 0.01), ATG5 ( < 0.001) and ATG7 ( < 0.001), lowered or even lost expressions of IRE1 and p-IRE1 proteins (P < 0.01), and increased expression of CHERP ( < 0.05) and intracellular calcium ion content ( < 0.001). Bafilomycin A1 treatment obviously increased LC3 Ⅱ/ LC3 Ⅰ ratio in the chondrocytes from both wild-type and ERN1 knockout mice ( < 0.01 or 0.05), but the increment was more obvious in the wild-type chondrocytes ( < 0.05). Treatment with autophagy dual-fluorescence virus resulted in a significantly greater fluorescence intensity of LC3-GFP in rapamycin-treated ERN1 CKO chondrocytes than in wild-type chondrocytes ( < 0.05). In C28/I2 cells, overexpression of CHERP obviously decreased the fluorescence intensity of LC3, and overexpression of IRE1 enhanced the fluorescence intensity and partially rescued the fluorescence reduction of LC3 caused by CHERP.
IRE1 deficiency impairs autophagy in chondrocytes by upregulating CHERP and increasing intracellular calcium ion content.
探讨肌醇需求酶1（IRE1）通过内质网钙稳态蛋白（CHERP）调节软骨细胞自噬功能的机制。
用衣霉素、4μ8c、雷帕霉素或4μ8c与雷帕霉素联合处理培养的人软骨细胞（C28/I2细胞），采用蛋白质免疫印迹法检测内质网（ER）应激和自噬相关蛋白的表达。用实时定量聚合酶链反应（qPCR）、蛋白质免疫印迹法和流式细胞术检测内质网激酶1基因敲除（ERN1 CKO）小鼠和野生型小鼠原代软骨细胞中自噬相关基因5（ATG5）和自噬相关基因7（ATG7）的mRNA表达、IRE1和磷酸化IRE1（p-IRE1）蛋白表达以及细胞内钙离子含量。用蛋白质免疫印迹法评估巴佛洛霉素A1处理对分离软骨细胞中微管相关蛋白1轻链3Ⅱ（LC3Ⅱ）/微管相关蛋白1轻链3Ⅰ（LC3Ⅰ）比值的影响。用自噬双荧光病毒检测雷帕霉素处理后软骨细胞自噬通量的变化。用免疫荧光法检测过表达CHERP和IRE1的C28/I2细胞自噬水平的变化。
衣霉素处理显著上调C28/I2细胞中ER应激相关蛋白和LC3Ⅱ/LC3Ⅰ比值，并下调p62表达（P<0.05）。雷帕霉素明显上调C28/I2细胞中LC3Ⅱ/LC3Ⅰ比值（P<0.千分之一），但4μ8c联合处理可显著减弱该作用（P<0.05）。与野生型小鼠细胞相比，ERN1基因敲除小鼠原代软骨细胞中ERN1的mRNA水平显著下调（P<0.01），ATG5（P<0.千分之一）和ATG7（P<0.千分之一）的mRNA水平也显著下调，IRE1和p-IRE1蛋白表达降低甚至缺失（P<0.01），CHERP表达增加（P<0.05），细胞内钙离子含量增加（P<0.千分之一）。巴佛洛霉素A1处理明显增加野生型和ERN1基因敲除小鼠软骨细胞中LC3Ⅱ/LC3Ⅰ比值（P<0.01或P<0.05），但野生型软骨细胞中增加更明显（P<0.05）。自噬双荧光病毒处理后，雷帕霉素处理的ERN1 CKO软骨细胞中LC3-绿色荧光蛋白（GFP）的荧光强度明显高于野生型软骨细胞（P<0.05）。在C28/I2细胞中，过表达CHERP明显降低LC3的荧光强度，过表达IRE1增强荧光强度，并部分挽救CHERP引起的LC3荧光降低。
IRE1缺乏通过上调CHERP和增加细胞内钙离子含量损害软骨细胞自噬。
