title: Expression of silent mating type information regulator 2 homolog 1 and its role in human intervertebral disc cell homeostasis.
abstract: BACKGROUND
  Intervertebral disc tissue homeostasis is modulated by a variety of molecules. Silent mating type information regulator 2 homolog 1 (SIRT1) plays a key role in various physiological processes. The aim of the present study was to verify the expression of SIRT1 and determine SIRT1 function in human intervertebral disc cell homeostasis.
  
  
  METHODS
  Human nucleus pulposus (NP) cells were obtained from 24 surgical patients (mean age: 39.4 years) and monolayer-cultured. SIRT1 expression was investigated using RT-PCR analysis and immunohistochemical staining. Quantitative real-time RT-PCR was performed to detect mRNA expression of SIRT1 and other genes: aggrecan, collagen type 2 and Sox9. The effect of SIRT1 on the extracellular matrix metabolism of NP cells was examined using recombinant human SIRT1 protein and a protein delivery reagent. Cell number and proliferation activity were measured following SIRT1 treatment. To reveal the deacetylation potential of transfected recombinant human SIRT1, western blotting for acetylated p53 was utilized. R-phycoerythrin was used for the negative control.
  
  
  RESULTS
  SIRT1 expression was confirmed at both mRNA and protein levels in almost all NP cells. Real-time RT-PCR analysis showed SIRT1 mRNA expression significantly increased with donor age (P <0.05, ρ = 0.492). Pfirrmann grade 3 discs showed significantly higher SIRT1 mRNA expression than other grades. SIRT1 treatment significantly reduced aggrecan, Sox9 and collagen type 2 mRNA expression in a dose-dependent manner in all disease classes and disc degeneration grades. Proliferation activity was decreased by SIRT1 treatment in lumbar spinal stenosis and lumbar disc herniation, Pfirrmann grade 3 and grade 4 discs. In contrast, it was significantly upregulated in idiopathic scoliosis, Pfirrmann grade 2 discs. The negative control protein did not affect extracellular matrix metabolism or proliferation activity.
  
  
  CONCLUSIONS
  We demonstrate for the first time that SIRT1 is expressed by human NP cells. SIRT1 expression was significantly elevated in an early degeneration stage. SIRT1 affected both extracellular matrix metabolism and proliferation activity; the effect of SIRT1 was altered according to disease class and disc degeneration grade. SIRT1 appears to play a key role in homeostasis during the human intervertebral disc degeneration process.
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Japan 650-0017.
authors.forename: Zhongying
authors.initials: Z
authors.lastname: Zhang
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Kakutani
authors.forename: Koichiro
authors.initials: K
authors.lastname: Maeno
authors.forename: Toru
authors.initials: T
authors.lastname: Takada
authors.forename: Takashi
authors.initials: T
authors.lastname: Yurube
authors.forename: Minoru
authors.initials: M
authors.lastname: Doita
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kurosaka
authors.forename: Kotaro
authors.initials: K
authors.lastname: Nishida
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D053674:Aggrecans / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006706:Homeostasis / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000378:metabolism* / Q000473:pathology; D007405:Intervertebral Disc Displacement / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D012600:Scoliosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D056564:Sirtuin 1 / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D013130:Spinal Stenosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D014162:Transfection; D055815:Young Adult
chemical_list: D053674:Aggrecans; D024043:Collagen Type II; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; C447939:SIRT1 protein, human; D056564:Sirtuin 1