title: Loss of chondrogenic potential in dedifferentiated chondrocytes correlates with deficient Shc-Erk interaction and apoptosis.
abstract: OBJECTIVE
  If dedifferentiated chondrocytes could be induced to redifferentiate in vitro, then we might thereby be furnished with a population of phenotypically stable cells for autologous implantation in reconstructive surgery. We therefore investigated the redifferentiation capabilities of chondrocytes which, having migrated from alginate beads to form a monolayer, were subsequently passaged. We also characterized the molecular traits of irreversibly dedifferentiated cells.
  
  
  METHODS
  Human chondrocytes that had migrated from alginate beads to form a monolayer (passage 1) were passaged seven times (passages 2-8). Cells from each passage were then recultivated in alginate beads. We assessed the synthesis of type-II collagen, cartilage-specific proteoglycans, adhesion molecules (integrins), signaling proteins (Src-homology collagen [Shc] and extracellular-signal-regulated kinase [Erk]) and the apoptosis marker 'activated' caspase-3 in monolayer or secondary alginate cultures.
  
  
  RESULTS
  The synthesis of cartilage-specific type-II collagen, alpha 3-integrin, Shc and activated Erk1/2 decreased rapidly after four passages in monolayer culture. Up to passage 4, cells redifferentiated in alginate culture. However, between passages 5 and 8, cells began to produce activated caspase-3; these cells not only failed to redifferentiate when recultivated in alginate, but underwent apoptosis.
  
  
  CONCLUSIONS
  We conclude that the loss of chondrogenic potential by chondrocytes maintained in monolayer culture is associated with a decrease in the synthesis of cartilage markers and with a suppressed activation of key signaling proteins in the Ras-mitogen-activated protein kinase pathway (Shc and Erk1/2). These events lead to apoptosis. A decrease in Shc/Erk expression/interaction could serve as a recognition marker for irreversibly dedifferentiated chondrocytes in tissue engineering.
authors.affiliation: Charité University Medical School, Campus Benjamin Franklin, Institute of Anatomy, Department of Cell and Neurobiology, Berlin, Germany.
authors.forename: Gundula
authors.initials: G
authors.lastname: Schulze-Tanzil
authors.forename: Ali
authors.initials: A
authors.lastname: Mobasheri
authors.forename: Philippe
authors.initials: P
authors.lastname: de Souza
authors.forename: Thilo
authors.initials: T
authors.lastname: John
authors.forename: Mehdi
authors.initials: M
authors.lastname: Shakibaei
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000464:Alginates; D017209:Apoptosis / Q000502:physiology*; D015415:Biomarkers / Q000032:analysis; D002358:Cartilage, Articular / Q000166:cytology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000648:ultrastructure; D024043:Collagen Type II / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D006801:Humans; D007124:Immunoenzyme Techniques; D016023:Integrins / Q000378:metabolism; D008875:Middle Aged; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000502:physiology; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000464:Alginates; D015415:Biomarkers; D024043:Collagen Type II; D006603:Hexuronic Acids; D016023:Integrins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine; D020723:Glucuronic Acid; D020928:Mitogen-Activated Protein Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases