USP18 attenuates endoplasmic reticulum stress via the PERK-eIF2α-ATF4 axis to reduce apoptosis in hepatocellular carcinoma cells

Abstract

Ubiquitin-specific peptidase 18 (USP18) is a specific interferon-stimulated gene 15 demodifying enzyme that plays an important role in apoptosis. In this study, we investigated the role of USP18 in apoptosis in hepatocellular carcinoma cells, especially its ability to regulate apoptosis through endoplasmic reticulum (ER) stress. We found that protein levels of Bcl-2-associated protein x and cytochrome c were down-regulated by USP18, which suppressed the classical mitochondrial-mediated apoptosis pathway. USP18 also inhibited apoptosis through the unfolded protein response (UPR) pathway by inhibiting the phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and the expression of CCAAT/enhance-binding protein homologous protein, which is a downstream marker molecule of ER stress. The UPR triggered by ER stress eventually led to the cleavage of downstream effecter proteases, including caspase-3, leading to apoptosis. Furthermore, USP18 combined with a PERK agonist regulated apoptosis through the PERK-eukaryotic initiation factor-2α-activating transcription factor 4 axis of the UPR. Our results show that USP18 participates in the regulation of hepatocellular carcinoma cell apoptosis through different pathways, especially the ER stress pathway, and that it plays a complex role in cell stress responses and apoptosis regulation.

Keywords: Apoptosis; Endoplasmic reticulum stress; Hepatocellular carcinoma; Protein kinase RNA-like Endoplasmic reticulum kinase; Ubiquitin-specific peptidase 18.

Fig. 1

USP18 regulated the proliferation of HCC cells. (a) Correlation between expression level of USP18 and overall survival of HCC patients from UALCAN, KM-plotter and HPA databases, n = number of samples. (b) Expression levels of USP18 protein and mRNA in HCC cell lines were significantly higher than that in normal liver cells. (c, d) Huh7 and SMMC7721 cells were transfected with siRNAs or overexpression plasmids, respectively. Protein expression of USP18 was detected by western blot. (e, f) Overexpression or knockdown of USP18 regulated cell proliferation of Huh7 and SMMC7721 cells, as shown in cell viability assays using the Cell Counting Kit-8. Data are presented as the mean ± SD. Statistical analyses were performed with one-way ANOVA. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns = not significant.

Fig. 2

USP18 regulated apoptosis of HCC cells. (a, b) Flow cytometry analysis showed that expression change of USP18 affected apoptosis of Huh7 and SMMC7721 cells. (c, d) Western blotting results showed that regulation of USP18 expression levels affected apoptosis-related proteins in the classical mitochondrial pathway in Huh7 and SMMC7721 cells. Data are presented as the mean ± SD. Statistical analyses were performed with one-way ANOVA. ^*P < 0.05, ^***P < 0.001.

Fig. 3

USP18 inhibited apoptosis via ER stress. Huh7 cells were treated with 5 mM ER stress inhibitor 4-PBA or 3 µM ER stress agonist TM, respectively. The final concentration of DMSO were 0.05% and 0.03% in vehicle control groups, respectively. (a) Flow cytometry analysis showed that apoptosis in Huh7 cells was regulated by expression change of USP18, ER stress agonist, or ER stress inhibitor. (b) Western blotting analysis showing levels of apoptosis-related protein cleaved-caspase-3 and ER stress-related proteins CHOP and GRP78 in Huh7 cells. (c) The fluorescence intensity of CHOP was detected by immunofluorescence assay. USP18 in red, CHOP in green, DAPI nuclear staining in blue. The representative images were acquired under × 200 magnification. Data are presented as the mean ± SD. Statistical analyses were performed with one-way ANOVA. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 4

USP18 regulates ER stress and PERK-eIF2α-ATF4 signaling in Huh7 cells. (a) Levels of ER stress-related proteins in Huh7 cells were detected by western blotting. Cells were treated with 3 µM ER stress agonist TM. (b). Western blotting analysis of UPR-related proteins p-PERK, ATF4, p-eIF2α, and CHOP in Huh7 cells. Cells were treated with 2.5 µM PERK agonist CCT020312. The final concentration of DMSO is 0.025% in vehicle control group. Data are presented as the mean ± SD.

Fig. 5

A proposed working model of apoptosis regulated by USP18 through the PERK-eIF2α-ATF4 signaling pathway. USP18 inhibits the expression of CHOP, a marker gene of ER stress, by regulating the PERK-eIF2α-ATF signaling pathway, which relieves the inhibition of downstream anti-apoptotic protein Bcl-2, thereby inhibiting cell apoptosis. USP18 can also directly affect apoptosis by regulating the classical mitochondrial-mediated apoptosis pathway. Solid arrows indicate promotion, and dashed arrows indicate inhibitory effects.