Pharmacological Disruption of Phosphorylated Eukaryotic Initiation Factor-2α/Activating Transcription Factor 4/Indian Hedgehog Protects Intervertebral Disc Degeneration via Reducing the Reactive Oxygen Species and Apoptosis of Nucleus Pulposus Cells.

Xiaotao Wu,Zhanyang Qian,Xin Hong,Junping Bao,Lei Liu,Hui Che

doi:10.3389/fcell.2021.675486

Abstract

Excessive reactive oxygen species (ROS) and apoptosis in nucleus pulposus (NP) cells accelerate the process of intervertebral disc degeneration (IDD). Here, we integrated pathological samples and in vitro and in vivo framework to investigate the impact of phosphorylation of eukaryotic initiation factor-2α (eIF2α)/activating transcription factor 4 (ATF4)/Indian hedgehog (Ihh) signaling in the IDD. From the specimen analysis of the IDD patients, we found phosphorylated eIF2α (p-eIF2α), ATF4 and Ihh protein levels were positively related while the NP tissue went degenerative. In vitro, tumor necrosis factor (TNF)-α caused the NP cell degeneration and induced a cascade of upregulation of p-eIF2α, ATF4, and Ihh. Interestingly, ATF4 could enhance Ihh expression through binding its promoter region, and silencing of ATF4 decreased Ihh and protected the NP cells from degeneration. Moreover, ISRIB inhibited the p-eIF2α, which resulted in a suppression of ATF4/Ihh, and alleviated the TNF-α-induced ROS production and apoptosis of NP cells. On the contrary, further activating p-eIF2α aggravated the NP cell degeneration, with amplification of ATF4/Ihh and a higher level of ROS and apoptosis. Additionally, applying cyclopamine (CPE) to suppress Ihh was efficient to prevent NP cell apoptosis but did not decrease the ROS level. In an instability-induced IDD model in mice, ISRIB suppressed p-eIF2α/ATF4/Ihh and prevented IDD via protecting the anti-oxidative enzymes and decreased the NP cell apoptosis. CPE prevented NP cell apoptosis but did not affect anti-oxidative enzyme expression. Taken together, p-eIF2α/ATF4/Ihh signaling involves the ROS level and apoptosis in NP cells, the pharmacological disruption of which may provide promising methods in preventing IDD.

Highlights

Intervertebral disc degeneration (IDD) is a potential cause of chronic degenerative disc disease and pathophysiological disorders, including discogenic back pain, disc herniation, spinal stenosis, and spinal instability (Dowdell et al, 2017)
nucleus pulposus (NP) tissues of G1 indicate a nearly healthy condition without visible intervertebral disc degeneration (IDD), those of G3 indicate mild IDD, and those of G5 indicate severe IDD
The results of western blot (WB) analysis indicated that the total eukaryotic initiation factor-2α (eIF2α) was stable and phosphorylated eIF2α (p-eIF2α) protein gradually accumulated as the NP tissues degenerated from G1 to G5, which was accompanied by an upregulation of activating transcription factor 4 (ATF4) and Indian hedgehog (Ihh) expression (P < 0.05; Figures 1C,D)

Summary

Introduction

Intervertebral disc degeneration (IDD) is a potential cause of chronic degenerative disc disease and pathophysiological disorders, including discogenic back pain, disc herniation, spinal stenosis, and spinal instability (Dowdell et al, 2017). It is clear that it is the result of multiple factors, containing biomechanical factors, molecular biology factors, and genetic metabolism factors. These inducements lead to changes in the microenvironment of the intervertebral disc, resulting in the NP cells gradually losing their original phenotype and functions (Roberts et al, 2006). The damage to the amount or quality of the NP cells causes the transformation of collagen type and the decrease in aggrecan and water content, which infers that excessive NP cell apoptosis may be the origin of the IDD (Ding et al, 2013). Our understanding of the ROS-sensitive signaling network in NP cells is limited

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