Serum and nutrient deprivation increase autophagic flux in intervertebral disc annulus fibrosus cells: an in vitro experimental study

• Published in: European spine journal Vol. 28; no. 5; pp. 993 - 1004
• Main Authors: Yurube, Takashi, Buchser, William J., Moon, Hong Joo, Hartman, Robert A., Takayama, Koji, Kawakami, Yohei, Nishida, Kotaro, Kurosaka, Masahiro, Vo, Nam V., Kang, James D., Lotze, Michael T., Sowa, Gwendolyn A.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.05.2019; Springer Nature B.V
• Subjects: Animals; Annulus Fibrosus - cytology; Annulus Fibrosus - metabolism; Apoptosis; Apoptosis - physiology; Autophagy; Autophagy - physiology; Caspase; Caspase-3; Cell death; Cell fate; Cell proliferation; Cells, Cultured; Cellular Senescence; Cellular stress response; Culture Media; Cyclin-dependent kinase inhibitors; Cytometry; Degeneration; HMGB1 protein; INK4 protein; Intervertebral Disc - cytology; Intervertebral Disc - metabolism; Intervertebral discs; Medicine; Medicine & Public Health; Metabolic flux; Metabolism; Neurosurgery; Nucleus pulposus; Nutrient concentrations; Nutrient deficiency; Nutrients; Original Article; Oxygen; Oxygen - metabolism; Phagocytosis; Rabbits; Senescence; Surgical Orthopedics; Western blotting; β-Galactosidase; Senescence; Annulus fibrosus (AF) cells; Serum and nutrient deprivation; Autophagy; Intervertebral disc; Apoptosis
• ISSN: 0940-6719; 1432-0932
• DOI: 10.1007/s00586-019-05910-9

Purpose The loss of nutrient supply is a suspected contributor of intervertebral disc degeneration. However, the extent to which low nutrition affects disc annulus fibrosus (AF) cells is unknown as nutrient deprivation has mainly been investigated in disc nucleus pulposus cells. Hence, an experimental study was designed to clarify the effects of limited nutrients on disc AF cell fate, including autophagy, the process by which cells recycle their own damaged components. Methods Rabbit disc AF cells were cultured in different media with varying serum concentrations under 5% oxygen. Cellular responses to changes in serum and nutrient concentrations were determined by measuring proliferation and metabolic activity. Autophagic flux in AF cells was longitudinally monitored using imaging cytometry and Western blotting for LC3, HMGB1, and p62/SQSTM1. Apoptosis (TUNEL staining and cleaved caspase-3 immunodetection) and cellular senescence (senescence-associated β-galactosidase assay and p16/INK4A immunodetection) were measured. Results Markers of apoptosis and senescence increased, while cell proliferation and metabolic activity decreased under the withdrawal of serum and of nutrients other than oxygen, confirming cellular stress. Time-dependent increases in autophagy markers, including LC3 puncta number per cell, LC3-II expression, and cytoplasmic HMGB1, were observed under conditions of reduced nutrition, while an autophagy substrate, p62/SQSTM1, decreased over time. Collectively, these findings suggest increased autophagic flux in disc AF cells under serum and nutrient deprivation. Conclusion Disc AF cells exhibit distinct responses to serum and nutrient deprivation. Cellular responses include cell death and quiescence in addition to reduced proliferation and metabolic activity, as well as activation of autophagy under conditions of nutritional stress. Graphical abstract These slides can be retrieved under Electronic Supplementary Material.