TY - JOUR
T1 - Serum and nutrient deprivation increase autophagic flux in intervertebral disc annulus fibrosus cells
T2 - an in vitro experimental study
AU - Yurube, Takashi
AU - Buchser, William J.
AU - Moon, Hong Joo
AU - Hartman, Robert A.
AU - Takayama, Koji
AU - Kawakami, Yohei
AU - Nishida, Kotaro
AU - Kurosaka, Masahiro
AU - Vo, Nam V.
AU - Kang, James D.
AU - Lotze, Michael T.
AU - Sowa, Gwendolyn A.
N1 - Funding Information:
Acknowledgements The authors thank Drs. Thomas P. Lozito (Department of Orthopaedic Surgery, Center for Cellular and Molecular Engineering, University of Pittsburgh, Pittsburgh, PA), Mayumi Morizane (Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, PA), Masahiro Shuda (Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA), and Tetsuya Watan-abe (Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh Asthma Institute, University of Pittsburgh, Pittsburgh, PA) for their expertise. We also thank Mr. Kevin Ngo and Ms. Qing Dong (Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA) for their technical assistance. This work was supported in part by The Albert B. Ferguson, Jr., M.D. Orthopaedic Fund of The Pittsburgh Foundation, NIH AG044376, and The Uehara Memorial Foundation (Grant Nos. AG044376, 2012400067).
Funding Information:
The authors thank Drs. Thomas P. Lozito (Department of Orthopaedic Surgery, Center for Cellular and Molecular Engineering, University of Pittsburgh, Pittsburgh, PA), Mayumi Morizane (Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, PA), Masahiro Shuda (Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA), and Tetsuya Watanabe (Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh Asthma Institute, University of Pittsburgh, Pittsburgh, PA) for their expertise. We also thank Mr. Kevin Ngo and Ms. Qing Dong (Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA) for their technical assistance. This work was supported in part by The Albert B. Ferguson, Jr., M.D. Orthopaedic Fund of The Pittsburgh Foundation, NIH AG044376, and The Uehara Memorial Foundation (Grant Nos. AG044376, 2012400067).
Publisher Copyright:
© 2019, Springer-Verlag GmbH Germany, part of Springer Nature.
PY - 2019/5/1
Y1 - 2019/5/1
N2 - 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.[Figure not available: see fulltext.].
AB - 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.[Figure not available: see fulltext.].
KW - Annulus fibrosus (AF) cells
KW - Apoptosis
KW - Autophagy
KW - Intervertebral disc
KW - Senescence
KW - Serum and nutrient deprivation
UR - http://www.scopus.com/inward/record.url?scp=85062771006&partnerID=8YFLogxK
U2 - 10.1007/s00586-019-05910-9
DO - 10.1007/s00586-019-05910-9
M3 - Article
C2 - 30847707
AN - SCOPUS:85062771006
SN - 0940-6719
VL - 28
SP - 993
EP - 1004
JO - European Spine Journal
JF - European Spine Journal
IS - 5
ER -