title: Autophagy in the Degenerating Human Intervertebral Disc: In Vivo Molecular and Morphological Evidence, and Induction of Autophagy in Cultured Annulus Cells Exposed to Proinflammatory Cytokines-Implications for Disc Degeneration.
abstract: METHODS
  Autophagy-related gene expression and ultrastructural features of autophagy were studied in human discs.
  
  
  OBJECTIVE
  To obtain molecular/morphological data on autophagy in human disc degeneration and cultured human annulus cells exposed to proinflammatory cytokines.
  
  
  BACKGROUND
  Autophagy is an important process by which cytoplasm and organelles are degraded; this adaptive response to sublethal stresses (such as nutrient deprivation present in disc degeneration) supplies needed metabolites. Little is known about autophagic processes during disc degeneration.
  
  
  METHODS
  Human disc specimens were obtained after institutional review board approval. Annulus mRNA was analyzed to determine autophagy-related gene expression levels. Immunolocalization and ultrastructural studies for p62, ATG3, ATG4B, ATG4C, ATG7, L3A, ULK-2, and beclin were conducted. In vitro experiments used IL-1β- or TNF-α-treated human annulus cells to test for autophagy-related gene expression.
  
  
  RESULTS
  More degenerated versus healthier discs showed significantly greater upregulation of well-recognized autophagy-related genes (P ≤ 0.028): beclin 1 (upregulated 1.6-fold); ATG8 (LC3) (upregulated 2.0-fold); ATG12 (upregulated 4.0-fold); presenilin 1 (upregulated 1.6-fold); cathepsin B (upregulated 4.5-fold). p62 was localized, and ultrastructure showed autophagic vacuolization and autophagosomes with complex, redundant whorls of membrane-derived material. In vitro, proinflammatory cytokines significantly upregulated autophagy-related genes (P ≤ 0.04): DRAM1 (6.24-fold); p62 (4.98-fold); PIM-2 oncogene, a positive regulator of autophagy (3-fold); WIPI49 (linked to starvation-induced autophagy) (upregulated 2.3-fold).
  
  
  CONCLUSIONS
  Data provide initial molecular and morphological evidence for the presence of autophagy in the degenerating human annulus. In vivo gene analyses showed greater autophagy-related gene expression in more degenerated than healthier discs. In vitro data suggested a mechanism implicating a role of TNF-α and IL-1β in disc autophagy. Findings suggest the importance of future work to investigate the relationship of autophagy to apoptosis, cell death, cell senescence, and mitochondrial dysfunction in the aging and degenerating disc.
  
  
  METHODS
  N/A.
authors.affiliation: From the Department of Orthopaedic Surgery, Carolinas HealthCare System, Charlotte, NC.
authors.forename: Helen E
authors.initials: HE
authors.lastname: Gruber
authors.forename: Gretchen L
authors.initials: GL
authors.lastname: Hoelscher
authors.forename: Jane A
authors.initials: JA
authors.lastname: Ingram
authors.forename: Synthia
authors.initials: S
authors.lastname: Bethea
authors.forename: Edward N
authors.initials: EN
authors.lastname: Hanley
mesh_terms: D000328:Adult; D000368:Aged; D051017:Apoptosis Regulatory Proteins / Q000032:analysis / Q000235:genetics; D001343:Autophagy / Q000187:drug effects / Q000235:genetics*; D000071193:Autophagy-Related Protein 7; D000071183:Autophagy-Related Proteins; D000071186:Beclin-1; D002352:Carrier Proteins / Q000235:genetics; D002401:Cathepsin B / Q000235:genetics; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000235:genetics; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007223:Infant; D053583:Interleukin-1beta / Q000494:pharmacology; D007403:Intervertebral Disc / Q000737:chemistry / Q000648:ultrastructure*; D055959:Intervertebral Disc Degeneration / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D008159:Lumbar Vertebrae; D008297:Male; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000235:genetics; D008875:Middle Aged; D053764:Presenilin-1 / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D016601:RNA-Binding Proteins / Q000032:analysis / Q000235:genetics; D012447:Sacrum; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D044764:Ubiquitin-Activating Enzymes / Q000032:analysis / Q000235:genetics; D044763:Ubiquitin-Conjugating Enzymes / Q000032:analysis / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects; D055815:Young Adult
chemical_list: D051017:Apoptosis Regulatory Proteins; D000071183:Autophagy-Related Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D002352:Carrier Proteins; C512709:DRAM1 protein, human; D053583:Interleukin-1beta; C477012:MAP1LC3A protein, human; C502070:MAP1LC3B protein, human; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; C118585:P62 protein, human; C490587:PIM2 protein, human; D053764:Presenilin-1; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014409:Tumor Necrosis Factor-alpha; C522812:WIPI1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D017346:Protein Serine-Threonine Kinases; C541703:Ulk2 protein, human; C474025:ATG4A protein, human; C507524:ATG4B protein, human; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C509349:ATG7 protein, human; D000071193:Autophagy-Related Protein 7; D044764:Ubiquitin-Activating Enzymes; C455621:ATG3 protein, human