TFEB protects nucleus pulposus cells against apoptosis and senescence via restoring autophagic flux

Osteoarthritis Cartilage. 2019 Feb;27(2):347-357. doi: 10.1016/j.joca.2018.10.011. Epub 2018 Nov 8.

Abstract

Objective: Excessive apoptosis and senescence of nucleus pulposus (NP) cells are major pathological changes in intervertebral disc degeneration (IVDD) development; previous studies demonstrated pharmacologically or genetically stimulation of autophagy may inhibit apoptosis and senescence in NP cells. Transcription factor EB (TFEB) is a master regulator of autophagic flux via initiating autophagy-related genes and lysosomal biogenesis. This study was performed to confirm whether TFEB was involved in IVDD development and its mechanism.

Methods: TFEB activity was detected in NP tissues in puncture-induced rat IVDD model by immunofluorescence as well as in tert-Butyl hydroperoxide (TBHP), the reactive oxygen species (ROS) donor to induce oxidative stress, treated NP cells by western blot. After TFEB overexpression in NP cells with lentivirus transfection, autophagic flux, apoptosis and senescence percentage were assessed. In in vivo study, the lentivirus-normal control (LV-NC) or lentivirus-TFEB (LV-TFEB) were injected into the center space of the NP tissue, after 4 or 8 weeks, Magnetic resonance imaging (MRI), X ray, Hematoxylin-Eosin (HE) and Safranin O staining were used to evaluate IVDD grades.

Results: The nuclear localization of TFEB declined in degenerated rat NP tissue as well as in TBHP treated NP cells. Applying lentivirus to transfect NP cells, TFEB overexpression restored the TBHP-induced autophagic flux blockage and protected NP cells against apoptosis and senescence; these protections of TFEB are diminished by chloroquine-medicated autophagy inhibition. Furthermore, TFEB overexpression ameliorates the puncture-induced IVDD development in rats.

Conclusions: Experimental IVDD inhibited the TFEB activity. TFEB overexpression suppressed TBHP-induced apoptosis and senescence via autophagic flux stimulation in NP cell and alleviates puncture-induced IVDD development in vivo.

Keywords: Apoptosis; Autophagy; Intervertebral disc degeneration; Senescence; TFEB.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Apoptosis / physiology*
  • Autophagy / drug effects
  • Autophagy / physiology
  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / genetics
  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / metabolism
  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / physiology*
  • Cells, Cultured
  • Cellular Senescence / drug effects
  • Cellular Senescence / physiology*
  • Chloroquine / pharmacology
  • Gene Expression Regulation / physiology
  • Genetic Therapy / methods
  • Intervertebral Disc Degeneration / metabolism
  • Intervertebral Disc Degeneration / pathology*
  • Intervertebral Disc Degeneration / therapy
  • Male
  • Nucleus Pulposus / drug effects
  • Nucleus Pulposus / metabolism
  • Nucleus Pulposus / pathology*
  • Oxidative Stress / drug effects
  • Oxidative Stress / physiology
  • Peroxides / pharmacology
  • Rats, Sprague-Dawley
  • Transfection
  • Up-Regulation / physiology

Substances

  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
  • Peroxides
  • TFEB protein, rat
  • Chloroquine
  • tert-butyl peroxide