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

Takashi Yurube, William J. Buchser, Hong Joo Moon, Robert A. Hartman, Koji Takayama, Yohei Kawakami, Kotaro Nishida, Masahiro Kurosaka, Nam V. Vo, James D. Kang, Michael T. Lotze, Gwendolyn A. Sowa

Research output: Contribution to journalArticle

Abstract

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.].

Original languageEnglish
JournalEuropean Spine Journal
DOIs
Publication statusPublished - 2019 Jan 1

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Intervertebral Disc
Food
Autophagy
Serum
HMGB1 Protein
Galactosidases
Cyclin-Dependent Kinase Inhibitor p16
Apoptosis
Oxygen
Intervertebral Disc Degeneration
Cell Aging
In Situ Nick-End Labeling
Annulus Fibrosus
In Vitro Techniques
Caspase 3
Cultured Cells
Cell Death
Cell Count
Western Blotting
Cell Proliferation

Keywords

  • Annulus fibrosus (AF) cells
  • Apoptosis
  • Autophagy
  • Intervertebral disc
  • Senescence
  • Serum and nutrient deprivation

ASJC Scopus subject areas

  • Surgery
  • Orthopedics and Sports Medicine

Cite this

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

In: European Spine Journal, 01.01.2019.

Research output: Contribution to journalArticle

Yurube, T, Buchser, WJ, Moon, HJ, Hartman, RA, Takayama, K, Kawakami, Y, Nishida, K, Kurosaka, M, Vo, NV, Kang, JD, Lotze, MT & Sowa, GA 2019, 'Serum and nutrient deprivation increase autophagic flux in intervertebral disc annulus fibrosus cells: an in vitro experimental study', European Spine Journal. https://doi.org/10.1007/s00586-019-05910-9
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. / Serum and nutrient deprivation increase autophagic flux in intervertebral disc annulus fibrosus cells : an in vitro experimental study. In: European Spine Journal. 2019.
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abstract = "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.].",
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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.

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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

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