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

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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.

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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 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).

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Authors and Affiliations

  1. Ferguson Laboratory for Orthopaedic and Spine Research, Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, University of Pittsburgh, 200 Lothrop Street, E1612 BSTWR, Pittsburgh, PA, 15213, USA

    Takashi Yurube, Hong Joo Moon, Robert A. Hartman, Nam V. Vo, James D. Kang & Gwendolyn A. Sowa

  2. Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan

    Takashi Yurube, Koji Takayama, Yohei Kawakami, Kotaro Nishida & Masahiro Kurosaka

  3. Damage Associated Molecular Pattern Molecule Laboratory, Department of Surgery, Hillman Cancer Center, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA, USA

    William J. Buchser & Michael T. Lotze

  4. Department of Neurosurgery, Guro Hospital, Korea University College of Medicine, Seoul, Korea

    Hong Joo Moon

  5. Department of Physical Medicine and Rehabilitation, University of Pittsburgh Medical Center, University of Pittsburgh, Pittsburgh, PA, USA

    Robert A. Hartman & Gwendolyn A. Sowa

  6. Department of Orthopaedic Surgery, Stem Cell Research Center, University of Pittsburgh Medical Center, University of Pittsburgh, Pittsburgh, PA, USA

    Koji Takayama & Yohei Kawakami

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  1. Takashi Yurube
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  2. William J. Buchser
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  3. Hong Joo Moon
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  9. Nam V. Vo
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  12. Gwendolyn A. Sowa
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Corresponding author

Correspondence to Gwendolyn A. Sowa.

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Conflict of interest

TY, WB, HM, RH, KT, YK, KN, MK, NV, ML, and GS have no conflicts of interest to declare. JDK has received research grants from Stryker and Synthes.

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Yurube, T., Buchser, W.J., Moon, H.J. et al. Serum and nutrient deprivation increase autophagic flux in intervertebral disc annulus fibrosus cells: an in vitro experimental study. Eur Spine J 28, 993–1004 (2019). https://doi.org/10.1007/s00586-019-05910-9

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  • DOI: https://doi.org/10.1007/s00586-019-05910-9

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