Abstract: Objective To explore the mechanism by which inositol-requiring enzyme-1α (IRE1α) regulates autophagy function of chondrocytes through calcium homeostasis endoplasmic reticulum protein (CHERP). Methods 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, IRE1α and p-IRE1α 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 IRE1α were detected using immunofluorescence assay. Results 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 (P<0.05). Rapamycin obviously up-regulated LC3 II/LC3 I ratio (P<0.001) in C28/I2 cells, but this effect was significantly attenuated by co-treatment with 4μ8c (P<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 (P<0.01), ATG5 (P<0.001) and ATG7 (P<0.001), lowered or even lost expressions of IRE1α and p-IRE1α proteins (P<0.01), and increased expression of CHERP (P<0.05) and intracellular calcium ion content (P<0.001). Bafilomycin A1 treatment obviously increased LC3 II/LC3 I ratio in the chondrocytes from both wild-type and ERN1 knockout mice (P<0.01 or 0.05), but the increment was more obvious in the wild-type chondrocytes (P<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 (P<0.05). In C28/I2 cells, overexpression of CHERP obviously decreased the fluorescence intensity of LC3, and overexpression of IRE1α enhanced the fluorescence intensity and partially rescued the fluorescence reduction of LC3 caused by CHERP. Conclusion IRE1α deficiency impairs autophagy in chondrocytes by upregulating CHERP and increasing intracellular calcium ion content.

Key words: IRE1α; autophagic flux; calcium ions; calcium homeostasis endoplasmic reticulum protein; chondrocytes