title: α5β1 integrin induces the expression of noncartilaginous procollagen gene expression in articular chondrocytes cultured in monolayers.
abstract: BACKGROUND
  Articular chondrocytes undergo an obvious phenotypic change when cultured in monolayers. During this change, or dedifferentiation, the expression of type I and type III procollagen is induced where normal chondrocytes express little type I and type III procollagen. In this study, we attempted to determine the mechanism(s) for the induction of such procollagen expression in dedifferentiating chondrocytes.
  
  
  METHODS
  All experiments were performed using primary-cultured human articular chondrocytes under approval of institutional review boards. Integrin(s) responsible for the induction of type I and type III procollagen expression were specified by RNAi experiments. The signal pathway(s) involved in the induction were determined by specific inhibitors and RNAi experiments. Adenovirus-mediated experiments were performed to identify a small GTPase regulating the activity of integrins in dedifferentiating chondrocytes. The effect of inhibition of integrins on dedifferentiation was investigated by experiments using echistatin, a potent disintegrin. The effect of echistatin was investigated first with monolayer-cultured chondrocytes, and then with pellet-cultured chondrocytes.
  
  
  RESULTS
  In dedifferentiating chondrocytes, α5β1 integrin was found to be involved in the induction of type I and type III procollagen expression. The induction was known to be mediated by v-akt murine thymoma viral oncogene homolog (AKT) signaling. Among the three AKT isoforms, AKT1 seemed to be most involved in the signaling. Elated RAS viral (r-ras) oncogene homolog (RRAS) was considered to regulate the progression of dedifferentiation by modulating the affinity and avidity of α5β1 integrin to ligands. Echistatin inhibited dedifferentiation of monolayer-cultured chondrocytes. Furthermore, the matrix formed by pellet-cultured chondrocytes more closely resembled that of normal cartilage compared with the controls.
  
  
  CONCLUSIONS
  The result of this study has shown, for the first time, that α5β1 integrin may be responsible for the induction of non-cartilaginous collagen expression in chondrocytes undergoing dedifferentiation. Again, this study has shown that the inhibition of ligand ligation to integrins may be an effective strategy to inhibit phenotypic change of cultured chondrocytes, and to improve the quality of matrix synthesized by primary cultured chondrocytes.
authors.forename: Nobuho
authors.initials: N
authors.lastname: Tanaka
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Ikeda
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Furukawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Mitomi
authors.forename: Takumi
authors.initials: T
authors.lastname: Nakagawa
authors.forename: Shigeto
authors.initials: S
authors.lastname: Tohma
authors.forename: Naoshi
authors.initials: N
authors.lastname: Fukui
mesh_terms: D015153:Blotting, Western; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism; D054337:Cell Dedifferentiation / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D024042:Collagen Type I / Q000235:genetics* / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D024061:Collagen Type III / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D039081:Integrin alpha5beta1 / Q000235:genetics* / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D024061:Collagen Type III; D039081:Integrin alpha5beta1; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D019869:Phosphatidylinositol 3-Kinases; D010975:Platelet Aggregation Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D018631:ras Proteins; C439971:COL3A1 protein, human; C057986:echistatin; C494918:AKT1 protein, human; C085929:RRAS protein, human