Autophagy in rat annulus fibrosus cells: evidence and possible implications

Chao Shen,Li-Yang Dai,Jun Yan,Lei-Sheng Jiang

doi:10.1186/ar3443

Abstract

IntroductionProgrammed cell death of intervertebral disc (IVD) cells plays an important role in IVD degeneration, but the role of autophagy, a closely related cell death event, in IVD cells has not been documented. The current study was designed to investigate the effect of interleukin (IL)-1β on the occurrence of autophagy of rat annulus fibrosus (AF) cells and the interrelationship between autophagy and apoptosis.MethodsRat AF cells were isolated and exposed, in tissue cultures with or without serum, to IL-1β in different concentrations for 24 hours. Ultrastructural analysis, flow cytometry and lysosomal activity assessment were performed after the in vitro treatment to determine the presence and levels of autophagy. The mRNA expression of autophagy-related proteins (Beclin-1, Bcl-2 and microtubule associated protein 1 light chain 3 (LC3)) were evaluated using real-time PCR. 3-methyladenine (3-MA), a PI3K inhibitor, was used to determine the interaction between autophagy and apoptosis via the suppression of autophagy.ResultsAutophagy was detected in rat AF cells under serum starvation condition by transmission electron microscopy. PCR and flow cytometry results showed that IL-1β enhanced the autophagy-induction effect of serum deprivation in a dose-dependent manner. However, IL-1β alone failed to induce autophagy in AF cells cultured without serum starvation. When autophagy was suppressed by 3-MA, the apoptosis incidence was increased. Serum supplement also partly reversed the autophagy incidence without affecting the apoptosis incidence in the same cells.ConclusionsIL-1β up-regulates serum deprivation-induced autophagy of AF cells in a dose-dependent manner. Autophagy may represent a protective mechanism against apoptosis in AF cells and IVD degeneration.

Highlights

Programmed cell death of intervertebral disc (IVD) cells plays an important role in IVD degeneration, but the role of autophagy, a closely related cell death event, in IVD cells has not been documented
Serum-starvation-induced autophagy in isolated rat annulus fibrosus (AF) cells To verify whether autophagy occurs in rat AF cells, we used electronic microscopy to visualize autophagy vesicles in the cytoplasm
The results showed that autophagy incidence was gradually increased over time but IL-1b did not induce autophagy when the AF cells were cultured with 10% fetal bovine serum (FBS)

Summary

Introduction

Programmed cell death of intervertebral disc (IVD) cells plays an important role in IVD degeneration, but the role of autophagy, a closely related cell death event, in IVD cells has not been documented. Intervertebral disc (IVD) degeneration, associated with aging, is the common cause of neck or back pain in adults and often leads to reduction in quality of life [1]. Studies have suggested that IVD degeneration is a cell-mediated pathogenic process [4,5,6]: the disc cells, known as nucleus pulposus (NP) and annulus. The programmed cell death is believed to play an essential role in tissue homeostasis as well as the pathogenesis of IVD degeneration [8,9,10]. The evidence from clinical and animal model studies has suggested that loss of disc cellularity is associated with apoptosis during the process of IVD degeneration [11,12,13]. Treatment targeting programmed cell death interception will be a potential direction for retarding or preventing IVD degeneration. Significant progress has been made in understanding apoptosis that is involved in IVD degeneration, the underlying mechanisms are not well understood

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