CCAAT/Enhancer Binding Protein β Regulates Expression of Indian Hedgehog during Chondrocytes Differentiation | Zendy

Takahiro Ushijima | Zendy; Ken Okazaki | Zendy; Hidetoshi Tsushima | Zendy; Kohei Ishihara | Zendy; Toshio Doi | Zendy; Yukihide Iwamoto | Zendy

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CCAAT/Enhancer Binding Protein β Regulates Expression of Indian Hedgehog during Chondrocytes Differentiation

Author(s) -

Takahiro Ushijima,

Ken Okazaki,

Hidetoshi Tsushima,

Kohei Ishihara,

Toshio Doi,

Yukihide Iwamoto

Publication year - 2014

Publication title -

plos one

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.99

H-Index - 332

ISSN - 1932-6203

DOI - 10.1371/journal.pone.0104547

Subject(s) - indian hedgehog , runx2 , chromatin immunoprecipitation , chondrocyte , transcription factor , microbiology and biotechnology , electrophoretic mobility shift assay , ccaat enhancer binding proteins , hedgehog , gene knockdown , enhancer , biology , transactivation , immunoprecipitation , cellular differentiation , sox9 , promoter , chemistry , gene expression , nuclear protein , signal transduction , cartilage , cell culture , gene , genetics , anatomy

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. Methodology/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.

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