title: FoxO transcription factors support oxidative stress resistance in human chondrocytes.
abstract: OBJECTIVE
  A major signaling pathway that regulates cellular aging is the insulin/insulin-like growth factor 1 (IGF-1)/phosphatidylinositol 3-kinase (PI3K)/Akt/FoxO transcription factor axis. We previously observed that FoxO transcription factors are dysregulated in aged and OA cartilage. The objective of this study was to investigate the impact of down-regulated FoxO transcription factors on chondrocytes.
  
  
  METHODS
  Small interfering RNAs (siRNAs) targeting FOXO1 (siFOXO1) and FOXO3 (siFOXO3) were transfected into human articular chondrocytes. Cell viability following treatment with the oxidant tert-butyl-hydroperoxide (tBHP) was measured by MTT assay. Caspase 3/7 activation and apoptotic cells were examined. Gene and protein expression of antioxidant proteins and autophagy-related proteins and changes in inflammatory mediators following treatment with interleukin-1β were assessed. Cells transfected with FOXO plasmids were also analyzed.
  
  
  RESULTS
  Cell viability was significantly reduced by siFOXO after treatment with tBHP. Apoptosis accompanied by caspase activation was significantly increased in siFOXO-transfected chondrocytes. Knockdown of FOXO1 and FOXO1+3 resulted in significant reductions in levels of glutathione peroxidase 1 (GPX-1), catalase, light chain 3 (LC3), Beclin1, and sirtuin 1 (SIRT-1) proteins following treatment with tBHP. In contrast, the constitutive active form of FOXO3 increased cell viability while inducing GPX-1, Beclin1, and LC3 in response to tBHP. Expression and production of ADAMTS-4 and chemerin were significantly increased in siFOXO-transfected chondrocytes.
  
  
  CONCLUSIONS
  Reduced expression of FoxO transcription factors in chondrocytes increased susceptibility to cell death induced by oxidative stress. This was associated with reduced levels of antioxidant proteins and autophagy-related proteins. Our data provide evidence for a key role of FoxO transcription factors as regulators of chondrocyte oxidative stress resistance and tissue homeostasis.
authors.affiliation: The Scripps Research Institute, La Jolla, California.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Akasaki
authors.forename: Oscar
authors.initials: O
authors.lastname: Alvarez-Garcia
authors.forename: Masahiko
authors.initials: M
authors.lastname: Saito
authors.forename: Beatriz
authors.initials: B
authors.lastname: Caramés
authors.forename: Yukihide
authors.initials: Y
authors.lastname: Iwamoto
authors.forename: Martin K
authors.initials: MK
authors.lastname: Lotz
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D001343:Autophagy; D002358:Cartilage, Articular / Q000166:cytology; D053148:Caspase 3 / Q000187:drug effects / Q000378:metabolism; D053453:Caspase 9 / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000235:genetics* / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D008875:Middle Aged; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics*; D034741:RNA, Small Interfering; D055815:Young Adult; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
chemical_list: C085126:FOXO1 protein, human; C499517:FOXO3 protein, human; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D053583:Interleukin-1beta; D016877:Oxidants; D034741:RNA, Small Interfering; D020122:tert-Butylhydroperoxide; D053148:Caspase 3; D053453:Caspase 9