title: The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells.
abstract: BACKGROUND
  The goals of this study were to examine the oxemic regulation of Wnt signaling to explore whether Wnt signaling accelerates the age-related degeneration of nucleus pulposus cells, and if so, to define the mechanism underlying this effect. We investigated the expression of Klotho, a newly identified antiaging gene, and whether its regulation is attributable to the suppression of Wnt signaling.
  
  
  METHODS
  Rat nucleus pulposus cells were cultured under normoxic (21% O2) or hypoxic (2% O2) conditions, and the expression and promoter activity of Wnt signaling and Klotho were evaluated. The effect of Klotho protein was examined with transfection experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, senescence-associated β-galactosidase staining, and cell-cycle analysis. To determine the methylation status of the Klotho promoter region, bisulfite genomic sequencing analysis was performed. Its relation with the activation of Wnt signaling was assessed. We also examined whether the expression of Klotho could block the effects of pathological Wnt expression in nucleus pulposus cells.
  
  
  RESULTS
  Nucleus pulposus cells exhibited increased β-catenin mRNA and protein under the hypoxic condition. Klotho protein was expressed in vivo, and protein and messenger RNA expression decreased under the hypoxic condition. Klotho treatment decreased cell proliferation and induced the quiescence of nucleus pulposus cells. In addition, Klotho treatment inhibited expression of β-catenin gene and protein compared with untreated control cells.
  
  
  CONCLUSIONS
  These data indicate that Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells. These results suggest that the expression of Klotho is regulated by the balance between upregulation and downregulation of Wnt signaling.
authors.affiliation: Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan. a.hiyama@tokai-u.jp
authors.forename: Akihiko
authors.initials: A
authors.lastname: Hiyama
authors.forename: Fumiyuki
authors.initials: F
authors.lastname: Arai
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sakai
authors.forename: Katsuya
authors.initials: K
authors.lastname: Yokoyama
authors.forename: Joji
authors.initials: J
authors.lastname: Mochida
mesh_terms: D000375:Aging; D000818:Animals; D015153:Blotting, Western; D015687:Cell Hypoxia; D002478:Cells, Cultured; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005966:Glucuronidase / Q000378:metabolism*; D007403:Intervertebral Disc / Q000378:metabolism*; D055959:Intervertebral Disc Degeneration / Q000378:metabolism; D000090265:Klotho Proteins; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D060449:Wnt Signaling Pathway / Q000502:physiology*
chemical_list: D005966:Glucuronidase; D000090265:Klotho Proteins