title: Identification of the core element responsive to runt-related transcription factor 2 in the promoter of human type X collagen gene.
abstract: OBJECTIVE
  Type X collagen and runt-related transcription factor 2 (RUNX-2) are known to be important for chondrocyte hypertrophy during skeletal growth and repair and development of osteoarthritis (OA) in mice. Aiming at clinical application, this study was undertaken to investigate transcriptional regulation of human type X collagen by RUNX-2 in human cells.
  
  
  METHODS
  Localization of type X collagen and RUNX-2 was determined by immunohistochemistry, and their functional interaction was examined in cultured mouse chondrogenic ATDC-5 cells. Promoter activity of the human type X collagen gene (COL10A1) was examined in human HeLa, HuH7, and OUMS27 cells transfected with a luciferase gene containing a 4.5-kb promoter and fragments. Binding to RUNX-2 was examined by electrophoretic mobility shift assay and chromatin immunoprecipitation.
  
  
  RESULTS
  RUNX-2 and type X collagen were co-localized in mouse limb cartilage and bone fracture callus. Gain and loss of function of RUNX-2 revealed that RUNX-2 is essential for type X collagen expression and terminal differentiation of chondrocytes. Human COL10A1 promoter activity was enhanced by RUNX-2 alone and more potently by RUNX-2 in combination with the coactivator core-binding factor beta in all 3 human cell lines examined. Deletion, mutagenesis, and tandem repeat analyses identified the core responsive element as the region between -89 and -60 bp (termed the hypertrophy box [HY box]), which showed specific binding to RUNX-2. Other putative RUNX-2 binding motifs in the human COL10A1 promoter did not respond to RUNX-2 in human cells.
  
  
  CONCLUSIONS
  Our findings indicate that the HY box is the core element responsive to RUNX-2 in human COL10A1 promoter. Studies on molecular networks related to RUNX-2 and the HY box will lead to treatments of skeletal growth retardation, bone fracture, and OA.
authors.affiliation: Sensory and Motor System Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Akiro
authors.initials: A
authors.lastname: Higashikawa
authors.forename: Taku
authors.initials: T
authors.lastname: Saito
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Ikeda
authors.forename: Satoru
authors.initials: S
authors.lastname: Kamekura
authors.forename: Naohiro
authors.initials: N
authors.lastname: Kawamura
authors.forename: Akinori
authors.initials: A
authors.lastname: Kan
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Oshima
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Ohba
authors.forename: Naoshi
authors.initials: N
authors.lastname: Ogata
authors.forename: Katsushi
authors.initials: K
authors.lastname: Takeshita
authors.forename: Kozo
authors.initials: K
authors.lastname: Nakamura
authors.forename: Ung-il
authors.initials: UI
authors.lastname: Chung
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawaguchi
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019902:Chondrocytes / Q000166:cytology / Q000502:physiology*; D024121:Collagen Type X / Q000235:genetics*; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D007763:Lac Operon; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016296:Mutagenesis / Q000502:physiology; D010012:Osteogenesis / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology
chemical_list: D024121:Collagen Type X; D050659:Core Binding Factor Alpha 1 Subunit; C493709:RUNX2 protein, human