Sertraline induces endoplasmic reticulum stress in hepatic cells

Abstract

Sertraline is used for the treatment of depression, and is also used for the treatment of panic, obsessive-compulsive, and post-traumatic stress disorders. Previously, we have demonstrated that sertraline caused hepatic cytotoxicity, with mitochondrial dysfunction and apoptosis being underlying mechanisms. In this study, we used microarray and other biochemical and molecular analyses to identify endoplasmic reticulum (ER) stress as a novel molecular mechanism. HepG2 cells were exposed to sertraline and subjected to whole genome gene expression microarray analysis. Pathway analysis revealed that ER stress is among the significantly affected biological changes. We confirmed the increased expression of ER stress makers by real-time PCR and Western blots. The expression of typical ER stress markers such as PERK, IRE1α, and CHOP was significantly increased. To study better ER stress-mediated drug-induced liver toxicity; we established in vitro systems for monitoring ER stress quantitatively and efficiently, using Gaussia luciferase (Gluc) and secreted alkaline phosphatase (SEAP) as ER stress reporters. These in vitro systems were validated using well-known ER stress inducers. In these two reporter assays, sertraline inhibited the secretion of Gluc and SEAP. Moreover, we demonstrated that sertraline-induced apoptosis was coupled to ER stress and that the apoptotic effect was attenuated by 4-phenylbutyrate, a potent ER stress inhibitor. In addition, we showed that the MAP4K4-JNK signaling pathway contributed to the process of sertraline-induced ER stress. In summary, we demonstrated that ER stress is a mechanism of sertraline-induced liver toxicity.

Keywords: Apoptosis; Drug-induced liver toxicity; Endoplasmic reticulum stress; MAPK pathway; Reporter gene assay; Sertraline.

Fig. 1

(A) Hierarchical Cluster Analysis (HCA) of gene expression profiles in control and sertraline-treated groups. The values of the log 2 intensities of genes were hierarchically clustered using Ward’s distance metric. The intensity of the entire gene set was used; no specific cut off was applied for the analysis. Each column represents the results from an individual hybridization. Each row represents the log 2 intensity value for one particular gene. Samples are labeled according to the convention of Dose (µM)_Technical replicate number. (B) Numbers of differentially expressed genes in HepG2 cells in response to treatment with 12.5, 25, and 50 µM sertraline. A gene was identified as differentially expressed if the fold-change was greater than 1.5 (up- or down-regulated) and the P-value was less than 0.05 in comparison to the control group. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)

Fig. 2

qPCR confirmation of microarray data. Genes involved in ER stress response were determined by qPCR. HepG2 cells were treated for 6 (A) and 2 h (B) with increasing concentrations (3.13, 6.25, 12.5, 25, and 50 µM) of sertraline, with DMSO as the vehicle control. Values were means ± SD of three separate experiments. *P <0.05, **P <0.01, and ***P <0.001 as compared with DMSO controls.

Fig. 3

Effects of sertraline in HepG2 cells on the expression of proteins involved in the ER stress response and on splicing of XBP mRNA. (A) Total cellular proteins were extracted at 2 and 6 h after sertraline treatment. The level of ER stress related proteins including phospho-PERK, PERK, phospho-eIF2α, eIF2α, CHOP, ATF-4, and PDI was determined by Western blotting. GAPDH was used as a loading control. Data are typical of three experiments. (B and C) Relative protein level to DMSO control by densitometric analysis. (D) HepG2 cells were treated with the indicated concentration of sertraline for 6 h. The total RNA was isolated and semi-quantitative RT-PCR analysis was performed to detect both spliced (263 bp) and unspliced (289 bp) XBP mRNA. GAPDH was also amplified and used as an internal control. A representative DNA gel from three independent experiments is shown.

Fig. 4

Effects of brefeldin A and thapsigargin on the secretion of Gluc and SEAP proteins in HepG2 cells. As ER stress inducers, brefeldin A and thapsigargin were tested for their inhibitory effects on the molecules involved in ER stress in HepG2 cells and Gluc and SEAP secretion in the Gluc and SEAP reporter cells. (A and B) HepG2 cells were treated with various concentrations of brefeldin A and thapsigargin for 6, 16, and 24 h. The main ER stress response related proteins (PERK, CHOP, ATF4, and PDI) were determined by Western blotting. (C and D) After treatment with increasing concentrations of brefeldin A and thapsigargin for 24 h, the activities of Gluc and SEAP were determined as described in Section 2; the results shown are mean ± SD of four separate experiments. ^###P < 0.001 represents relative SEAP activity was significantly different from the control; ***P <0.001 represents relative Gluc activity was significantly different form the control. Relative Gluc or SEAP activity was calculated based on % of each (Gluc or SEAP activity in medium per Fluc activity) over its vehicle control.

Fig. 5

Effects of sertraline on the secretion of Gluc and SEAP proteins in HepG2 cells. (A) (HepG2-Gluc-Fluc) or (B) (HepG2-SEAP-Fluc) cells were treated with various concentrations of sertraline for 2 and 6 h. The activities of Gluc (A) or SEAP (B) was measured *P <0.05, **P <0.01, and ***P < 0.001 as compared with DMSO controls.

Fig. 6

Effects of ER stress response in the induction of sertraline induced apoptosis. (A) Total cellular proteins were extracted at 2 and 6 h after sertraline treatment. The levels of ER stress related caspase protein pro-caspase 4 was determined by Western blotting. (B) HepG2 cells were pretreated with 1 mM 4-PBA for 2 h prior to 25 µM sertraline treatment for additional 2 h. The expression of CHOP was assessed by Western blotting. GAPDH was used as a loading control. (C) After treatment with 1 mM 4-PBA for 2 h, HepG2 cells were treated with various concentrations of sertraline for additional 2 h. Apoptosis was determined by caspase 3/7 activity. The bar graph shows the mean ± SD of four separate experiments. **P <0.005 versus treatment of sertraline alone.

Fig. 7

Effect of silencing MAP4K4 on sertraline-induced ER stress. (A) Two HepG2 cell lines with silenced gene expression of MAP4K4 (sh1-MAP4K4 and sh2-MAP4K4) or a scramble control (SC) were treated with sertraline at 25 µM for 6 h. Total cellular proteins were extracted. The expression levels of MAP4K4 and ER stress related proteins (PERK, p-PERK, CHOP, PDI, and pro-caspase-4) were detected by Western blotting. GAPDH was used as a loading control B) After treatment with 10 µM SP600125 (JNK inhibitor) for 2 h (HepG2-Fluc-Gluc) cells were treated with indicated concentrations of sertraline for 6 h. The relative Gluc activity was calculated; the results shown are mean ± SD of four separate experiments. *P <0.05, **P <0.01 versus treatment with sertraline alone.

Fig. 8

A scheme for proposed mechanism on sertraline-induced liver toxicity.