title: Paracrine and autocrine signals promoting full chondrogenic differentiation of a mesoblastic cell line.
abstract: The pluripotent mesoblastic C1 cell line was used under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to drive chondrogenesis. Sequential addition of two systemic hormones, dexamethasone and triiodothyronine, permits full chondrogenic differentiation. The cell intrinsic activation of the BMP signaling pathway and Sox9 expression occurring on mesoblastic condensation is insufficient for recruitment of the progenitors. Dexamethasone-dependent Sox9 upregulation is essential for chondrogenesis.
  
  
  BACKGROUND
  Differentiation of lineage stem cells relies on cell autonomous regulations modulated by external signals. We used the pluripotent mesoblastic C1 cell line under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to induce differentiation of a precursor clone along the chondrogenic lineage.
  
  
  METHODS
  C1 cells, cultured as aggregates, were induced toward chondrogenesis by addition of 10(-7) M dexamethasone in serum-free medium. After 30 days, dexamethasone was replaced by 10 nM triiodothyronine to promote final hypertrophic conversion. Mature and hypertrophic phenotypes were characterized by immunocytochemistry using specific antibodies against types II and X collagens, respectively. Type II collagen, bone morphogenetic proteins (BMPs), BMP receptors, Smads, and Sox9 expression were monitored by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and/or Western blot analysis.
  
  
  CONCLUSIONS
  Once C1 cells have formed nodules, sequential addition of two systemic hormones is sufficient to promote full chondrogenic differentiation. In response to dexamethasone, nearly 100% of the C1 precursors engage in chondrogenesis and convert within 30 days into mature chondrocytes, which triggers a typical cartilage matrix. On day 25, a switch in type II procollagen mRNA splicing acted as a limiting step in the acquisition of the mature chondrocyte phenotype. On day 30, substitution of dexamethasone with triiodothyronine triggers the final differentiation into hypertrophic chondrocytes within a further 15 days. The chondrogenic process is supported by intrinsic expression of Sox9 and BMP family genes. Similarly to the in vivo situation, activation of Sox9 expression and the BMP signaling pathway occurred on mesoblastic condensation. After induction, BMP-activated Smad nuclear translocation persisted throughout the process until the onset of hypertrophy. After dexamethasone addition, Sox9 expression was upregulated. Dexamethasone withdrawal reversed the increase in Sox9 expression and stopped differentiation. Thus, Sox9 seems to be a downstream mediator of dexamethasone action.
authors.affiliation: ONRS UPR 1983, Laboratoire de Différenciation cellulaire et Prions, Villejuif, France.
authors.forename: Morgane
authors.initials: M
authors.lastname: Locker
authors.forename: Odile
authors.initials: O
authors.lastname: Kellermann
authors.forename: Marie
authors.initials: M
authors.lastname: Boucquey
authors.forename: Huot
authors.initials: H
authors.lastname: Khun
authors.forename: Michel
authors.initials: M
authors.lastname: Huerre
authors.forename: Anne
authors.initials: A
authors.lastname: Poliard
mesh_terms: D053674:Aggrecans; D000818:Animals; D019898:Autocrine Communication / Q000502:physiology*; D015152:Blotting, Northern; D052004:Bone Morphogenetic Protein Receptors; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D002449:Cell Aggregation / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology*; D045744:Cell Line, Tumor; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D024042:Collagen Type I / Q000032:analysis / Q000378:metabolism; D024043:Collagen Type II / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D024121:Collagen Type X / Q000032:analysis / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D016326:Extracellular Matrix Proteins / Q000032:analysis; D015972:Gene Expression Regulation, Neoplastic; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D037181:Lectins, C-Type; D051379:Mice; D053495:Osteopontin; D019899:Paracrine Communication / Q000502:physiology*; D039904:Pluripotent Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011509:Proteoglycans / Q000032:analysis; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor; D012795:Sialoglycoproteins / Q000032:analysis / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D014284:Triiodothyronine / Q000494:pharmacology; D015854:Up-Regulation / Q000502:physiology
chemical_list: C508320:Acan protein, mouse; D053674:Aggrecans; D019485:Bone Morphogenetic Proteins; D024042:Collagen Type I; D024043:Collagen Type II; D024121:Collagen Type X; D016895:Culture Media, Serum-Free; D016326:Extracellular Matrix Proteins; D006609:High Mobility Group Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D017978:Receptors, Growth Factor; D055758:SOX9 Transcription Factor; D012795:Sialoglycoproteins; C527933:Sox9 protein, mouse; C506896:Spp1 protein, mouse; C508961:Tgfb1 protein, mouse; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014284:Triiodothyronine; D053495:Osteopontin; D007334:Insulin-Like Growth Factor I; D003907:Dexamethasone; D052004:Bone Morphogenetic Protein Receptors