The Role of Unfolded Protein Response in Human Intervertebral Disc Degeneration: Perk and IRE1- α as Two Potential Therapeutic Targets

Abstract

Inflammation plays a key role in intervertebral disc degeneration (IDD). The association between inflammation and endoplasmic reticulum (ER) stress has been observed in many diseases. However, whether ER stress plays an important role in IDD remains unclear. Therefore, this study is aimed at investigating the expression of ER stress in IDD and at exploring the underlying mechanisms of IDD, ER stress, and inflammation. The expression of ER stress was activated in nucleus pulposus cells from patients who had IDD (D-NPCs) compared with patients without IDD (N-NPCs); and both the proliferation and synthesis capacity were decreased by inducer tunicamycin (Tm) and proinflammatory cytokines. Pretreatment of NPCs with 4-phenyl butyric acid (4-PBA) prevented the inflammatory cytokine-induced upregulation of unfolded protein response- (UPR-) related proteins and recovered cell synthetic ability. Furthermore, proinflammatory cytokine treatment significantly upregulated the expression of inositol-requiring protein 1 (IRE1-α) and protein kinase RNA-like ER kinase (PERK), but not activating transcription factor 6 (ATF6). Finally, knockdown of IRE1-α and PERK also restored the biological activity of NPCs. Our findings identified that IRE1-α and PERK might be the potential targets for IDD treatment, which may help illustrate the underlying mechanism of ER stress in IDD.

Figure 1

Activation of the UPR target genes in degenerated NPCs. (a) Transmission electron microscopy (TEM) images of endoplasmic reticulum (ER) in N-NPCs and D-NPCs. The morphology of ERs was showed with white arrows. The results showed more dilated and abundant ERs in D-NPCs. (b) The expressions of the UPR target genes (PERK, GRP78, and CHOP) in N-NPCs and D-NPCs were determined by real-time PCR. The results were normalized to GAPDH mRNA expression. (c) Western blot analysis showed the protein levels of PERK, GRP78, and CHOP in N-NPCs and D-NPCs. β-Actin was used as an internal control. The representative results were from three independent experiments. The error bars represent the SD from the mean values. ∗P < 0.05, ∗∗P < 0.01.

Figure 2

UPR activation leads to the decreased biological activity of NPCs. The expression of UPR genes in NPCs treated by Tm was analyzed by real-time PCR (a) and western blot (b). (c) Cell proliferation was evaluated by the CCK-8 assay at days 1 to 7 after TM treatment. (d) The expression of Agg, Col2, and SOX9 following the exposure of NPCs to Tm was confirmed by western blot. β-Actin was used as an internal control. The representative results were from three independent experiments. The error bars represent the SD from the mean values. ∗P < 0.05, ∗∗P < 0.01.

Figure 3

Inflammation triggers UPR and impairs biological activity of NPCs. The expression of UPR gene in NPCs treated by IL-1 and TNF-α synergistically without/with 4-PBA was analyzed by real-time PCR (a) and western blot (b). (c) Cell proliferation was evaluated by the CCK-8 assay at days 1 to 7 after IL-1 and TNF-α synergistically without/with 4-PBA treatment. (d) The expression of Agg, Col2, and SOX9 following the exposure of NPCs to IL-1 and TNF-α synergistically without/with 4-PBA was confirmed by western blot analysis. β-Actin was used as an internal control. The representative results were from three independent experiments. The error bars represent the SD from the mean values. ∗P < 0.05, ∗∗P < 0.01.

Figure 4

UPR activation regulated the synthesis of NPCs through the PERK and IRE1-α pathways. (a) The expression of PERK, ATF6, and IRE1-α proteins in degenerated and nondegenerated NP tissues was detected by IHC. Representative images are presented at the indicated magnifications. Scale bar, 50 μm. Compared with the controlled group, the presence of PERK and IRE1-α was significantly increased in degenerated tissues, whereas ATF6 was similar. (b) The expression of ATF6, IRE1-α, and PERK was downregulated after being transfected with PREK, ATF6, and IRE1-α siRNA as assayed by real-time PCR. (c) The expression of Agg, Col2, and SOX9 was significantly increased after knockdown of PERK and IRE1-α than ATF6 as assessed by western blot. β-Actin was used as an internal control. The representative results were from three independent experiments. The error bars represent the SD from the mean values.