title: Transcriptional induction of SOX9 by NF-kappaB family member RelA in chondrogenic cells.
abstract: OBJECTIVE
  Although SOX9 is a key molecule for chondrogenic differentiation, little is known about the upstream signal. The present study attempted to identify transcription factors to induce SOX9 expression and examined the mechanism.
  
  
  METHODS
  Sequences of about 1 kb of 5'-end flanking regions were compared between human and mouse SOX9 genes. In vivo localization was examined by immunohistochemistry in the limb cartilage of fetal mice. Promoter activities of the SOX9, SOX6, and type II collagen (COL2A1) genes were determined in human non-chondrogenic HeLa cells and mouse chondrogenic ATDC5 cells transfected with a luciferase-reporter gene containing the promoter fragments. Protein-DNA binding was examined by electrophoretic mobility shift and chromatin immunoprecipitation assays. The chondrogenic differentiation was assessed by endogenous SOX9, SOX6, and COL2A1 mRNA levels, and by Alcian blue staining and alkaline phosphatase activity.
  
  
  RESULTS
  Among transcription factors whose binding motifs were identified in the highly-conserved regions between human and mouse SOX9 promoters, a nuclear factor kappa B (NF-kappaB) member RelA strongly activated the promoter activity. RelA and SOX9 were co-localized in the limb cartilage. Deletion, mutagenesis, and tandem-repeat analyses identified the core region responsive to RelA at the NF-kappaB binding motif to be around -250bp of the human SOX9 promoter, and this was confirmed to show specific binding to RelA. RelA induced the chondrogenic differentiation parameters in HeLa and ATDC5 cells.
  
  
  CONCLUSIONS
  We have identified RelA as a transcriptional factor for SOX9 induction and chondrogenic differentiation via binding to an NF-kappaB binding motif in the SOX9 promoter.
authors.affiliation: Department of Sensory & Motor System Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Ushita
authors.forename: T
authors.initials: T
authors.lastname: Saito
authors.forename: T
authors.initials: T
authors.lastname: Ikeda
authors.forename: F
authors.initials: F
authors.lastname: Yano
authors.forename: A
authors.initials: A
authors.lastname: Higashikawa
authors.forename: N
authors.initials: N
authors.lastname: Ogata
authors.forename: U
authors.initials: U
authors.lastname: Chung
authors.forename: K
authors.initials: K
authors.lastname: Nakamura
authors.forename: H
authors.initials: H
authors.lastname: Kawaguchi
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D024043:Collagen Type II; D016328:NF-kappa B; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; C527933:Sox9 protein, mouse; D051996:Transcription Factor RelA; D014157:Transcription Factors