CCAAT/enhancer binding protein β regulates expression of Indian hedgehog during chondrocytes differentiation

• Published in: PloS one Vol. 9; no. 8; p. e104547
• Main Authors: Ushijima, Takahiro, Okazaki, Ken, Tsushima, Hidetoshi, Ishihara, Kohei, Doi, Toshio, Iwamoto, Yukihide
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 08.08.2014; Public Library of Science (PLoS)
• Subjects: Animals; Arthritis; Assaying; Binding sites; Biology and Life Sciences; Biotechnology; Cbfa-1 protein; CCAAT-Enhancer-Binding Protein-beta - genetics; CCAAT-Enhancer-Binding Protein-beta - metabolism; CCAAT/enhancer-binding protein; Cell culture; Cell Differentiation; Cells, Cultured; Chondrocytes; Chondrocytes - cytology; Chondrocytes - metabolism; Chondrogenesis; Chromatin; Clonal deletion; Collagen; Core Binding Factor Alpha 1 Subunit - metabolism; Differentiation; Electrophoretic mobility; Embryos; Gene deletion; Gene expression; Gene Expression Regulation; Growth plate; Hedgehog protein; Hedgehog Proteins - genetics; Immunohistochemistry; Immunoprecipitation; Laboratories; Mice; Morphology; Mutation; Nuclei; Organ culture; Promoter Regions, Genetic; Proteins; Regulatory sequences; Rodents; Surgery; Transcription factors; Transcriptional Activation; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0104547

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. 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. 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.