Cyclic stretch-induced apoptosis in rat annulus fibrosus cells is mediated in part by endoplasmic reticulum stress through nitric oxide production

Abstract

Various mechanical stresses in vivo induce disc cell apoptosis and intervertebral disc (IVD) degeneration, but the underlying molecular mechanism is not fully known. The aim of this study was to investigate the role of endoplasmic reticulum stress in cyclic stretch-induced apoptosis of rat annulus fibrosus (AF) cells. Flexercell Tension Plus system was used to apply cyclic stretch to rat annulus fibrosus cells at a frequency of 0.5 Hz with 20% elongation for 12, 24, 36, or 48 h. Apoptosis was detected by flow cytometry, and nuclei morphologic changes were visualized by Hoechst 33258 staining and caspase-8, 9 activity assays. The expression of the markers of endoplasmic reticulum stress including CHOP, GRP78, and caspase-12 were determined by RT-PCR and Western blot. Mitochondrial membrane potential change was observed by JC-1 staining in situ. In addition, the levels of the nitric oxide (NO) were determined with the Griess reaction and fluorescence staining. The results indicated that cyclic stretch at a frequency of 0.5 Hz with 20% elongationinduced apoptosis in rat AF cells. Prolonged exposure of the unphysiologically cyclic stretch to AF cells caused NO overproduction, up-regulation of endoplasmic reticulum stress markers including CHOP, GRP78, and caspase-12, depolarization of mitochondria and activation of caspase-9. However, cyclic stretch at this level had no effect on caspase-8 activity. In addition, specific inhibitor of caspase-12 (Z-ATAD-FMK) and caspase-9 (Z-LEHD-FMK) partly suppressed cyclic stretch-induced AF cell apoptosis and the anti-apoptotic effects of the caspase inhibitors were additive. Our data suggest that endoplasmic reticulum stress, likely mediated by NO, contributes to the AF cell apoptosis induced by cyclic stretch in addition to the mitochondrial pathway. These findings could be helpful to understand the mechanism of disc cell apoptosis, the root cause of IVD degeneration.

Upregulated expression of PERK in spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament

“…The system induced the transcription of messenger ribonucleic acids of several growth factors, including BMP-2, BMP-4, prostaglandin I 2 synthase, and osteoblast-specific transcription factor in the cells from OPLL patients [9,19,24,30]. At the same time, several studies have suggested that ER stress response can be activated by mechanical stress in some cellular pathological processes, such as apoptosis in rat annulus fibrosus cells or human connective tissue cells [32]. Thus, based on above biological information, we hypothesized that PERK-mediated ER stress might be downstream mechanism in development of OPLL induced by some primary genetic or mechanical factors.…”

Sestrin-Mediated Inhibition of Stress-Induced Intervertebral Disc Degradation Through the Enhancement of Autophagy

“…ER stress comprises stimuli that regulate a wide range of cellular responses including apoptosis, proliferation, inflammation, and differentiation in mammalian cells [35]. Many studies have shown that activation of the ER stress-induced apoptosis pathway contributes to various degenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and osteoarthritis [12, 13, 36]. 2-DG is well known to induce ER stress by inhibiting glycolysis and N-glycosylation of proteins [35, 37].…”

Cyclic stretch enhances apoptosis in human lumbar ligamentum flavum cells via the induction of reactive oxygen species generation

“…21 Recently, mechanical forces have been shown to induce apoptosis in several cell types. [22][23][24][25] Apoptosis is a cellular suicidal program crucial to the development and maintenance of multicellular organisms. 8 To our knowledge, the investigation described in the current study is the first to report the apoptosis in LF cells after mechanical stretch in vitro.…”

“…These observations are consistent with previous reports. [22][23][24][25] Cyclic stretch can induce ROS generation and ROS are well-known to enhance apoptosis. 10,11 In this study, we found that cyclic stretch enhanced ROS production in LF cells in a time-dependent manner.…”