title: Sp1 family of transcription factors regulates the human alpha2 (XI) collagen gene (COL11A2) in Saos-2 osteoblastic cells.
abstract: Genes encoding type XI collagen, normally associated with chondrogenesis, are also expressed by osteoblasts. By studying Saos-2 cells, we showed that the transcription factors, Sp1, Sp3, and Sp7 (Osterix), regulate COL11A2 expression through its proximal promoter. The findings indicate both ubiquitous and osteoblast-specific mechanisms of collagen gene regulation.
  
  
  BACKGROUND
  Type XI collagen is essential for skeletal morphogenesis. Collagen XI gene regulation has been studied in chondrocytes but not in osteoblasts.
  
  
  METHODS
  We cultured Saos-2 cells, a human osteosarcoma-derived line of osteoblasts, and analyzed them for alpha2(XI) protein and COL11A2 regulatory mechanisms.
  
  
  CONCLUSIONS
  Although types I and V were the dominant collagens deposited by Saos-2 cells, they expressed COL11A2 mRNA, and alpha2(XI) chains were present in the extracellular matrix. The COL11A2 promoter region (from -149 to -40) containing three Sp1 binding sites was required for promoter activity in transient transfection assays. All three Sp1 sites were critical for binding by nuclear proteins in electrophoretic mobility shift assays. Further analysis using consensus oligonucleotides and specific antibodies as well as chromatin immunoprecipitation assay implicated Sp1 and Sp3 in binding to this promoter region. Overexpressing Sp1 or Sp3 significantly increased COL11A2 promoter activity and endogenous COL11A2 gene expression, an effect that was suppressed by the Sp1-binding inhibitor mithramycin A. Further experiments showed that Sp1, Sp3, CREB-binding protein (CBP), p300, and histone deacetylase (HDAC) were physically associated and HDAC inhibitors (trichostatin A or NaB) upregulated COL11A2 promoter activity and endogenous gene expression. Another Sp1 family member, Sp7 (Osterix), was expressed in Saos-2 cells, but not in chondrocytes, and was shown by chromatin immunoprecipitation to occupy the COL11A2 promoter. Overexpressing Sp7 increased COL11A2 promoter activity and endogenous gene expression, an effect also blocked by mithramycin A. Using siRNA to knockdown Sp1, Sp3, or Sp7, it was shown that depression of any of them decreased COL11A2 promoter activity and endogenous gene expression. Finally, primary cultures of osteoblasts expressed COL11A2 and Sp7, upregulated COL11A2 promoter activity and endogenous gene expression when Sp1, Sp3, or Sp7 were overexpressed, and downregulated them when Sp1, Sp3, or Sp7 were selectively depressed. The results establish that Sp1 proteins regulate COL11A2 transcription by binding to its proximal promoter and directly interacting with CBP, p300, and HDAC.
authors.affiliation: Department of Orthopaedics, Institute of Health Biosciences, The University of Tokushima Graduate School, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Goto
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Matsui
authors.forename: Russell J
authors.initials: RJ
authors.lastname: Fernandes
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Hanson
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kubo
authors.forename: Kiminori
authors.initials: K
authors.lastname: Yukata
authors.forename: Toshimi
authors.initials: T
authors.lastname: Michigami
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Komori
authors.forename: Takashi
authors.initials: T
authors.lastname: Fujita
authors.forename: Liu
authors.initials: L
authors.lastname: Yang
authors.forename: David R
authors.initials: DR
authors.lastname: Eyre
authors.forename: Natsuo
authors.initials: N
authors.lastname: Yasui
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D024161:Collagen Type XI / Q000235:genetics*; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000502:physiology*; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D047468:Immunoprecipitation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism / Q000502:physiology*
chemical_list: C499319:COL11A2 protein, human; D024161:Collagen Type XI; D017931:DNA Primers; D056572:Histone Deacetylase Inhibitors; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor