Endoplasmic reticulum (ER) stress can initiate autophagy via unfolded protein response (UPR). As a key downstream gene of UPR, DDIT3/CHOP is expressed in chondrocytes. However, the regulation mechanism of DDIT3/CHOP on autophagy in chondrocytes remains unclear. In this study, the expression levels of autophagic markers Beclin1 and LC3B were found to decrease while p62 increase in the tibial growth plate and costal primary chondrocytes from DDIT3/CHOP KO mice. In vitro, overexpressing DDIT3/CHOP induced autophagy in ATDC5 chondrocytes, displaying an elevated immunofluorescence signal of LC3B and elevated numbers of autophagosomes and autolysosomes. Analysis of the gain- and loss-of-function indicated that the protein level of Beclin1 and the ratio of LC3BII/I increased in DDIT3/CHOP overexpression cells, whereas decreased in DDIT3/CHOP knockdown cells. The decreased level of p62 and additional accumulation of LC3BII caused by chloroquine (CQ) further indicated that DDIT3/CHOP enhanced autophagic flux. Mechanistically, we found that DDIT3/CHOP binds directly to the promoter of SIRT1 to promote its expression by CHIP, qRT-PCR, and Western blot analysis. In addition, SIRT1 enhanced autophagic activity in ATDC5 cells, and inhibition or activation of SIRT1 partially reversed the effect of overexpressing or downregulating DDIT3/CHOP on autophagy. Furthermore, AKT signaling was found to be responsible for DDIT3/CHOP-regulated autophagy in ATDC5 cells. SIRT1 knockdown reversed the effect of DDIT3/CHOP overexpression on AKT signaling. In conclusion, our data clarifies that DDIT3/CHOP promotes autophagy in ATDC5 chondrocytes through the SIRT1-AKT pathway. These results were also confirmed in the primary chondrocytes.

内质网 (ER) 应激可通过未折叠蛋白反应 (UPR) 启动自噬。作为 UPR 的关键下游基因，DDIT3/CHOP 在软骨细胞中表达。然而，DDIT3/CHOP对软骨细胞自噬的调控机制尚不清楚。在这项研究中，发现自噬标志物 Beclin1 和 LC3B 的表达水平降低，而 p62 在来自 DDIT3/CHOP KO 小鼠的胫骨生长板和肋骨原代软骨细胞中增加。在体外，过表达 DDIT3/CHOP 诱导 ATDC5 软骨细胞自噬，显示 LC3B 免疫荧光信号升高以及自噬体和自噬溶酶体数量增加。功能获得和功能丧失的分析表明，DDIT3/CHOP 过表达细胞中 Beclin1 的蛋白质水平和 LC3BII/I 的比率增加，而在 DDIT3/CHOP 敲低细胞中降低。氯喹 (CQ) 引起的 p62 水平降低和 LC3BII 的额外积累进一步表明 DDIT3/CHOP 增强了自噬通量。在机制上，我们发现 DDIT3/CHOP 直接与 SIRT1 的启动子结合以促进其通过 CHIP、qRT-PCR 和蛋白质印迹分析的表达。此外，SIRT1 增强了 ATDC5 细胞的自噬活性，抑制或激活 SIRT1 可部分逆转过表达或下调 DDIT3/CHOP 对自噬的影响。此外，还发现 AKT 信号是 ATDC5 细胞中 DDIT3/CHOP 调节的自噬的原因。SIRT1 敲低逆转了 DDIT3/CHOP 过表达对 AKT 信号的影响。总之，我们的数据阐明了 DDIT3/CHOP 通过 SIRT1-AKT 途径促进 ATDC5 软骨细胞的自噬。