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

European spine journal

article

Takashi Yurube¹  William J. Buchser³  Hong Joo Moon¹  Robert A. Hartman¹  Koji Takayama²  Yohei Kawakami²  Kotaro Nishida²  Masahiro Kurosaka²  Nam V. Vo¹  James D. Kang¹  Michael T. Lotze³  Gwendolyn A. Sowa¹ 

[1] Ferguson Laboratory for Orthopaedic and Spine Research, Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, University of Pittsburgh;Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine;Damage Associated Molecular Pattern Molecule Laboratory, Department of Surgery, Hillman Cancer Center, University of Pittsburgh Cancer Institute, University of Pittsburgh;Department of Neurosurgery, Guro Hospital, Korea University College of Medicine;Department of Physical Medicine and Rehabilitation, University of Pittsburgh Medical Center, University of Pittsburgh;Department of Orthopaedic Surgery, Stem Cell Research Center, University of Pittsburgh Medical Center, University of Pittsburgh

关键词: Intervertebral disc;    Annulus fbrosus (AF) cells;    Serum and nutrient deprivation;    Autophagy;    Apoptosis;    Senescence;   

DOI  :  10.1007/s00586-019-05910-9

来源: Springer

【 摘 要 】

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. 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. 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. 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. These slides can be retrieved under Electronic Supplementary Material.

【 授权许可】

Unknown