Abstract
The intervertebral disc is the largest avascular organ. Autophagy is an important cell survival mechanism by self-digestion and recycling damaged components under stress, primarily nutrient deprivation. Resident cells would utilize autophagy to cope with the harsh disc environment. Our objective was to elucidate the roles of human disc cellular autophagy. In human disc cells, serum deprivation and pro-inflammatory interleukin-1β (IL-1β) stimulation increased autophagy marker microtubule-associated protein 1 light chain 3 (LC3)-II and decreased autophagy substrate p62/sequestosome 1 (p62/SQSTM1), indicating enhanced autophagy. Then, RNA interference (RNAi) of autophagy-related gene 5 (ATG5), essential for autophagy, showed decreases in ATG5 protein (26.8%–27.4%, p < 0.0001), which suppressed early-stage autophagy with decreased LC3-II and increased p62/SQSTM1. Cell viability was maintained by ATG5 RNAi in serum-supplemented media (95.5%, p = 0.28) but reduced in serum-free media (80.4%, p = 0.0013) with IL-1β (69.9%, p = 0.0008). Moreover, ATG5 RNAi accelerated IL-1β-induced changes in apoptosis and senescence. Meanwhile, ATG5 RNAi unaffected IL-1β-induced catabolic matrix metalloproteinase release, down-regulated anabolic gene expression, and mitogen-activated protein kinase pathway activation. Lysosomotropic chloroquine supplementation presented late-stage autophagy inhibition with apoptosis and senescence induction, while catabolic enzyme production was modest. Disc-tissue analysis detected age-related changes in ATG5, LC3-II, and p62/SQSTM1. In summary, autophagy protects against human disc cellular apoptosis and senescence rather than extracellular matrix catabolism.
Highlights
Up to 85% of people experience back pain during their lives [1]
No significant differences in Cell Counting Kit-8 (CCK-8) dehydrogenase activity were detected between the control and autophagy-related gene 5 (ATG5) RNA interference (RNAi) groups in 10% fetal bovine serum (FBS)-supplemented Dulbecco’s modified Eagle’s medium (DMEM)—non-targeting
No significant differences in Cell Counting Kit-8 (CCK-8) dehydrogenase activity were detected between the control and ATG5 RNAi groups in 10% FBS-supplemented DMEM—non-targeting small interfering RNA (siRNA), 100.0%; ATG5 siRNA, 95.5 ± 5.6% (p = 0.28)
Summary
Up to 85% of people experience back pain during their lives [1]. Back pain causes disability that increases medical expenses and affects the workforce [1]. RNAi-mediated ATG5 knockdown, excluding off-target effects from consistent findings of different siRNA sequences, inhibited human disc cellular autophagy without mTOR-signaling modulation. We measured released catabolic MMP-3 and MMP-13 and anti-catabolic TIMP-1 and TIMP-2 protein amounts, all were unchanged by ATG5 RNAi. We further monitored MAPK-signaling pathways, major MMP production, and activation cascades [7,43]; ERK1/2, p38, and JNK phosphorylation levels were all insensitive to ATG5 RNAi. human disc-cell matrix metabolism would not involve ATG5-dependent autophagy. The present findings were consistent regardless of these issues
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