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

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.

Nucleus pulposus cells regulate macrophages in degenerated intervertebral discs via the integrated stress response-mediated CCL2/7-CCR2 signaling pathway

“…The ISR, which is an important stress response, maintains homeostasis by downregulating global protein translation and upregulating the translation of stress-related proteins such as ATF4 13 . Our study showed that the proportion of p-eIF2α-positive cells was increased in degenerated disc tissues, which was consistent with a previous study 57 , proving that the ISR was activated in IDD. Although the ISR protects against adverse stimulation, its benefits to cells and tissues vary with duration and degree 13 , 58 .…”

Chondrocyte-like cells in nucleus pulposus and articular chondrocytes have similar transcriptomic profiles and are paracrine-regulated by hedgehog from notochordal cells and subchondral bone

“…The importance of hedgehog pathways in NP and AC homeostasis has been reported. Modulation of hedgehog pathways causes osteoarthritis ( Lin et al, 2009 ) and IVD degeneration in mice ( Bao et al, 2021 ). Gli1 has been found to be expressed in NP ( Li et al, 2019 ).…”

Endoplasmic reticulum stress associates with the development of intervertebral disc degeneration

“…Inhibition of ATF4 expression can alleviate TNF-α-induced ROS production and apoptosis in NP cells ( 135 ). In addition, starvation-mediated TUNEL-positive frequency and apoptotic protein expression in AF cells can be significantly inhibited by PERK inhibitors ( 51 ).…”