The endoplasmic reticulum stress induced by tunicamycin affects the viability and autophagy activity of chondrocytes

Abstract Osteoarthritis (OA) is attributed to a reduction in chondrocytes within joint cartilage, and research has shown that endoplasmic reticulum (ER) stress and autophagy play important roles in the survival of chondrocytes. However, the relationship between ER stress and autophagy in chondrocytes remains unclear. In this study, we investigated the changes in apoptotic and autophagic activity in chondrocytes under ER stress. Following treatment with tunicamycin, the rate of apoptosis among chondrocytes increased. Western blot analysis showed the levels of unfolded protein response (UPR) related proteins increased, followed by elevated expression of light chain 3B‐II (LC3B‐II) and Beclin‐1. An ultrastructural investigation showed that a large number of pre‑autophagosomal structures or autophagosomes formed under tunicamycin treatment. However, the autophagy activity was significantly inhibited in chondrocytes after suppression of GRP78 by siRNA. The apoptosis ratio of chondrocytes pre‐treated with 3‐methyladenine was much higher than that of normal chondrocytes after exposure to tunicamycin. Our study revealed that the tunicamycin‐induced persistent UPR expression led to apoptosis of chondrocytes and activation of autophagy incorporation with GRP78. Blocking autophagy accelerated the apoptosis induced by ER stress, which confirmed the protective function of autophagy in the homeostasis of chondrocytes. These findings advance our understanding of chondrocyte apoptosis and provide potential molecular targets for preventing apoptotic death of chondrocytes.

degradation of the ECM, and thus, the normal function and viability of these cells are critical to the prevention of OA. 3 Therefore, the signaling events that determine the fate and function of chondrocytes, such as the unfolded protein response (UPR) and autophagy, are crucial to the maintenance of healthy cartilage.
The endoplasmic reticulum (ER) plays an important role in the synthesis and assembly of secretary protein. Cellular stress conditions, such as glucose deprivation, oxidative injury, aberrant Ca 2+ regulation, and the accumulation of unfolded protein, can disrupt proper ER function. To alleviate ER stress and restore normal ER function, the UPR is initiated. 4 First, with the assistance of protein kinase RNA-like ER kinase, some translational expression levels are inhibited including the pivotal factor X-box binding protein 1 (XBP1), which is involved in supporting the cell cycle. 5,6 Then, the UPR upregulates the expression of chaperone genes to facilitate the degradation of unfolded or misfolded proteins. If the ER stress persists, programmed cell execution is activated via activation of the transcription factor 4 (ATF4)/C/EBP homologous protein C/EBP (CHOP)/ growth arrest and DNA damage 34 (GADD34) pathway. 7 Previous studies have shown that ER stress in the chondrocytes in OA cartilage is related to cartilage degradation. 8 ER stress can induce the apoptosis of chondrocytes and upregulate the expression of matrix metalloproteinase (MMPs), which degrade the ECM. 9 Therefore, ER stress is one of the factors that contribute to the development of OA. The natural antibiotic tunicamycin (TM), which inhibits N-linked glycosylation, can disrupt protein maturation in the ER and has been proposed as an available tool for inducing and studying ER stress. 10 Autophagy is a cellular response to various stress conditions. During the process, damaged and dysfunctional organelles and molecules are recycled and digested into small molecules that can be used by the cell. Thus, autophagy is considered an essential protective process against cellular damage. 11 Increased autophagy in response to cellular stress also occurs in chondrocytes, according to evidence showing that autophagy disruption is related to apoptosis of chondrocytes and the development of OA. 12 Although ER stress and autophagy are functionally independent, these two systems are dynamically connected. They protect cells by relieving stress, but under some extreme conditions, they can both lead to apoptosis. 13 Furthermore, recent studies have claimed that ER stress can mediate autophagic processes. Using thin section electron microscopy, Bernales et al 14 discovered that the UPR in yeasts is accompanied by the formation of autophagosome-like structures.
Margariti et al 15 also found that XBP1 mRNA splicing triggers an autophagic response in endothelial cells (ECs) through the transcriptional regulation of Beclin-1. Moreover, the correlation between ER stress and mitophagy was reported in liver tissue and also shown to have a role in the chondrogenic and osteogenic differentiation of adipose stem cells in equine metabolic syndrome. 16 However, the relationship between the TM-induced ER stress and autophagy in articular chondrocytes remains elusive.
In the present study, we investigated the effect of the UPR induced by TM on the cellular viability of articular chondrocytes. By interfering with the UPR, we also evaluated the relationship between ER stress and autophagy in chondrocytes. Our observations suggest that the UPR induced by TM resulted in the activation of autophagy to protect chondrocytes from apoptosis, but persistent UPR induced the apoptosis of chondrocytes. Additionally, after the suppression of glucose-regulated protein (GRP) 78, the activation of autophagy induced by UPR was inhibited.

| Chondrocyte isolation and culture
Rat articular cartilage was obtained from the femoral and tibial con-

| Cell viability assay
The chondrocytes were seeded and cultured in 96-well plates. After culturing for 24 h, the viability of the chondrocytes was measured every day using a CCK-8 cell count kit (KeyGen Biotech. Co., Ltd., Nanjing, China). When the chondrocytes were in the logarithmic growth phase (day 4), the cells were divided into two groups: the control (CON) group, which received no treatment, and the TM group, which was treated with 0.5 μg/ml TM (Abcam, Cambridge, MA, USA). This concentration has been widely applied in previous studies to induce ER stress. 20 The differences in the viability between the two groups were compared after treatment for 24, 48, and 72 h. At each time point, 10 μl of CCK-8 kit solution was added to each well, and the plates were incubated at 37°C for 1 h before cell viability was measured using a microplate reader (#M581789; Westingarea, Shanghai, China).

| Apoptosis assay
The chondrocytes were seeded and cultured in 6-well plates for 4 days before treatment with TM (0.5 μg/ml). Following treatment for 0, 6, 12, and 24 h, the apoptosis ratio of the cells was quantified using an Annexin V-FITC flow cytometric kit (KeyGen Biotech. Co., Ltd., Nanjing, China) according to the manufacturer's instructions.
Briefly, the cells were harvested and washed in PBS three times.

| Western blot analysis
Following treatment with TM (0.5 μg/ml), the cells were harvested at the designated time points, and Western blot analysis was performed as previously described. 23 In brief, the cells were prepared in lysate buffer containing protease and phosphatase inhibitors.  Then, following 24 h treatment with TM, the expression levels of two proteins related to autophagy (LC3B and Beclin-1) were analyzed by western blotting, and autophagosome formation was observed via TEM as described above.

| Statistical analyses
The data are reported as mean ± standard error of the mean (SEM) values. One-way analysis of variance (ANOVA) was used to analyze values among multiple groups, and Student's t test was used to compare values between two groups. All data were analyzed using

| ER stress induced by TM decreased the viability of chondrocytes and increased the rate of apoptosis
The viability of chondrocytes increased obviously during the first 4 days in culture. However, after exposure to TM (0.5 μg/ml) for 72 h, the viability of the chondrocytes was significantly less than that in the CON group (*P < .05 or **P < .01; Figure 1). To further determine the cytotoxicity of TM in chondrocytes, we accessed the rate of apoptosis using Annexin V-FITC staining and flow cytometry.
As shown in Figure 2, following 12-24 h of TM treatment, the apoptosis rate of chondrocytes increased rapidly ( Figure 2). These results indicated that the ER stress induced by TM decreased chondrocyte viability and increased the rate of apoptosis in a time-and dose-dependent manner.

| The ER expanded during induction of the UPR, which was accompanied by autophagosome formation
To determine the ultrastructural changes in chondrocytes treated with TM, we observed the cellular morphology by TEM. As shown in Figure 3, massive expansion of the ER occurred following treatment with TM (0.5 μg/ml), especially after 12-24 h of treatment.
Unexpectedly, during the induction of the UPR, large amounts of autophagosomes with a high electron density accumulated in the cells. As shown in Figure 3, the number of autophagosomes increased after 12 h, and some undegraded substrates were contained within the autophagosomes, which may indicate the formation of mature autophagosomes and an increase in autophagy activity. 24

| TM induced the expression of proteins related to the UPR (GRP78, ATF4, CHOP, and GADD34) and autophagy (LC3B and Beclin-1)
Western blot analysis showed that the protein levels of UPR-related regulators, including GRP78, ATF4, CHOP, and GADD34, were low in the normal chondrocytes ( Figure 4). Intriguingly, the expression levels of these four proteins in chondrocytes were all significantly increased after TM treatment for 24 h (*P < .05 or **P < .01). At the same time, the expression level of Beclin-1, which is related to the nucleation of autophagic vesicles, was significantly increased after TM treatment for 12-24 h (*P < .05). Meanwhile, a rapid increase in LC3B-II expression was seen after 12-24 h of TM treatment ( Figure 5). The Western blot results indicated that TM induced the UPR as well as an increase in the autophagy activity in chondrocytes in vitro.

| Overactivation of the UPR induced by TM attenuated the autophagy activity in chondrocytes after deprivation of GRP78
In this study, we examined the effect of the UPR on autophagy

| Autophagy is induced during ER stress to protect against cell death
After 24 h of treatment with TM, the apoptosis rate among chondrocytes that had been pre-treated with 3-MA was much higher than that among normal chondrocytes (**P < .01; Figure 9). This result confirmed the protective effect of autophagy in chondrocytes exposed to TM.

| D ISCUSS I ON
The ER is a vast membranous network that serves as the major assembly and folding site for almost all secreted and integral membrane proteins. Furthermore, the ER is involved in maintaining intracellular Ca 2+ equilibration as well as cellular signal reception, F I G U R E 1 Proliferation of chondrocytes in CON and TM groups. TM was added to chondrocytes of the TM group on day 4 in culture. After different durations of TM treatment, the viability index of the chondrocytes in each group was assessed using a CCK-8 assay. *P < .05; **P < .01 transmission, and responses. 25 Many studies have shown that ER stress induces cell function changes and apoptosis that contribute to a variety of degenerative diseases, such as diabetes, 26 congestive cardiomyopathy, 27 Parkinson's disease, 28  In another experiment, autophagic vesicles and the expression levels of autophagy-related 5 (ATG-5) and LC3 decreased with increasing age, followed by a reduction in the density of chondrocytes in cartilage and an increase in the apoptosis rate among chondrocytes. 33 Rapamycin, an inducer of autophagy, was shown to reduce the severity of experimental OA when administered via intraperitoneal injection. 34 Therefore, toward the discovery of a new approach to the early prevention of OA, it is meaningful to study the relationship between ER stress and autophagy in chondrocytes.  39 However, the intracellular pathways connecting ER stress and autophagy are very complicated and remain unclear. It has been reported that the eukaryotic initiation factor 2 alpha (eIF2α)-ATF4 pathway has a novel regulatory role in autophagy gene transcription in response to stress in fibroblasts. 40 Kouroku et al 41 revealed that PERK/eIF2α phosphorylation is involved in polyglutamine-induced LC3 conversion and that autophagy activation protects cells from ER stress-mediated cell death by degrading polyglutamine aggregates. The IRE1α-JNK1 pathway is also a major regulator of autophagy activation. Prior research revealed that JNK1 phosphorylates BCL2 to disrupt the interaction of Beclin-1 and BCL2, which induces autophagy activation in tumor cells. 42 In addition, CHOP overexpression during ER stress causes down-regulation of BCL2 in tumor cells. 43 In conclusion, the present study demonstrated that treatment of chondrocytes with TM to induce ER stress resulted in activation of the UPR, and persistent UPR induced apoptosis among the chondrocytes. Our experiments also confirmed that the UPR can activate autophagy through the GRP78 pathway to protect chondrocytes from apoptosis.

ACK N OWLED G M ENTS
Hao Wu, Zhichao Meng, and Yang Jiao are co-first authors. The present study was supported by the National Natural Science Foundation of Beijing (No. 7144252).

F I G U R E 9
Pre-treatment with 3-MA led to increased apoptosis among TM-treated chondrocytes. After 24 h of treatment with TM, the apoptosis rate among chondrocytes pre-treated with 3-MA was much higher than that among normal chondrocytes. **P < .01