title: CCAAT/enhancer binding protein β regulates expression of Indian hedgehog during chondrocytes differentiation.
abstract: BACKGROUND
  CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor that promotes hypertrophic differentiation of chondrocytes. Indian hedgehog (Ihh) also stimulates the hypertrophic transition of chondrocytes. Furthermore, runt-related transcription factor-2 (RUNX2) was reported to regulate chondrocyte maturation during skeletal development and to directly regulate transcriptional activity of Ihh. In this study, we investigated whether the interaction of C/EBPβ and RUNX2 regulates the expression of Ihh during chondrocyte differentiation.
  
  
  RESULTS
  Immunohistochemistry of embryonic growth plate revealed that both C/EBPβ and Ihh were strongly expressed in pre-hypertrophic and hypertrophic chondrocytes. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked stimulation of Ihh and Runx2. Conversely, knockdown of C/EBPβ by lentivirus expressing shRNA significantly repressed Ihh and Runx2 in ATDC5 cells. A reporter assay revealed that C/EBPβ stimulated transcriptional activity of Ihh. Deletion and mutation analysis showed that the C/EBPβ responsive element was located between -214 and -210 bp in the Ihh promoter. An electrophoretic mobility shift assay (EMSA) and a chromatin immunoprecipitation (ChIP) assay also revealed the direct binding of C/EBPβ to this region. Moreover, reporter assays demonstrated that RUNX2 failed to stimulate the transcriptional activity of the Ihh promoter harboring a mutation at the C/EBPβ binding site. EMSA and ChIP assays showed that RUNX2 interacted to this element with C/EBPβ. Immunoprecipitation revealed that RUNX2 and C/EBPβ formed heterodimer complex with each other in the nuclei of chondrocytes. These data suggested that the C/EBPβ binding element is also important for RUNX2 to regulate the expression of Ihh. Ex vivo organ culture of mouse limbs transfected with C/EBPβ showed that the expression of Ihh and RUNX2 was increased upon ectopic C/EBPβ expression.
  
  
  CONCLUSIONS
  C/EBPβ and RUNX2 cooperatively stimulate expression of Ihh through direct interactions with a C/EBPβ binding element, which further promotes hypertrophic differentiation of chondrocytes during the chondrocyte differentiation process.
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ushijima
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Okazaki
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Tsushima
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Ishihara
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Doi
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Yukihide
authors.initials: Y
authors.lastname: Iwamoto
mesh_terms: D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D020219:Chondrogenesis; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism*; D005786:Gene Expression Regulation; D053823:Hedgehog Proteins / Q000235:genetics*; D051379:Mice; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D050659:Core Binding Factor Alpha 1 Subunit; D053823:Hedgehog Proteins; C514061:ihh protein, mouse
references.citation: J Cell Biol. 1996 Apr;133(2):457-68
references.pmid: 8609176
references.citation: Development. 2001 Nov;128(22):4523-34
references.pmid: 11714677
references.citation: J Biol Chem. 2002 Jan 11;277(2):1316-23
references.pmid: 11668178
references.citation: Development. 2008 Jun;135(11):1947-56
references.pmid: 18434416
references.citation: Arthritis Rheum. 2009 Mar;60(3):708-16
references.pmid: 19248099
references.citation: Cell. 1991 Nov 1;67(3):569-79
references.pmid: 1934061
references.citation: J Biol Chem. 2002 Aug 30;277(35):31526-33
references.pmid: 12072435
references.citation: Cell. 1995 Jan 27;80(2):353-61
references.pmid: 7530603
references.citation: Clin Orthop Relat Res. 2004 Oct;(427 Suppl):S123-8
references.pmid: 15480054
references.citation: Nat Med. 2009 Dec;15(12):1421-5
references.pmid: 19915594
references.citation: J Cell Sci. 2005 Jun 15;118(Pt 12):2545-55
references.pmid: 15944395
references.citation: Genes Dev. 2002 Apr 15;16(8):908-12
references.pmid: 11959839
references.citation: Arthritis Rheum. 2009 Oct;60(10):3049-60
references.pmid: 19790062
references.citation: J Biol Chem. 2014 Jan 31;289(5):2852-63
references.pmid: 24344131
references.citation: J Biol Chem. 2010 Mar 12;285(11):8395-407
references.pmid: 20048163
references.citation: Nature. 2010 Jan 21;463(7279):318-25
references.pmid: 20032975
references.citation: EMBO J. 1990 Jun;9(6):1897-906
references.pmid: 2112087
references.citation: Arthritis Res. 2001;3(2):107-13
references.pmid: 11178118
references.citation: Nature. 2003 May 15;423(6937):332-6
references.pmid: 12748651
references.citation: J Biol Chem. 1996 Jan 26;271(4):2040-7
references.pmid: 8567657
references.citation: Osteoarthritis Cartilage. 2012 Jul;20(7):755-63
references.pmid: 22469853
references.citation: Ann Rheum Dis. 2012 Jan;71(1):99-107
references.pmid: 21917825
references.citation: Mol Biol Cell. 2009 Nov;20(21):4541-51
references.pmid: 19759178
references.citation: Hum Mol Genet. 2012 Mar 1;21(5):1111-23
references.pmid: 22095691
references.citation: Trends Cell Biol. 2007 Jul;17(7):318-24
references.pmid: 17658261
references.citation: PLoS One. 2009;4(2):e4543
references.pmid: 19229324
references.citation: Cell. 1997 May 30;89(5):755-64
references.pmid: 9182763
references.citation: Science. 1996 Aug 2;273(5275):613-22
references.pmid: 8662546
references.citation: Development. 2009 Dec;136(24):4143-53
references.pmid: 19906842
references.citation: J Biol Chem. 1999 Mar 12;274(11):6972-8
references.pmid: 10066751
references.citation: Mol Biol Cell. 2008 Dec;19(12):5373-86
references.pmid: 18843047
references.citation: Genes Dev. 2004 Apr 15;18(8):952-63
references.pmid: 15107406