RRC ID 45224
Author Ushijima T, Okazaki K, Tsushima H, Ishihara K, Doi T, Iwamoto Y.
Title CCAAT/enhancer binding protein β regulates expression of Indian hedgehog during chondrocytes differentiation.
Journal PLoS One
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.
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.
Volume 9(8)
Pages e104547
Published 2014-1-1
DOI 10.1371/journal.pone.0104547
PII PONE-D-14-10954
PMID 25105964
PMC PMC4126692
MeSH Animals CCAAT-Enhancer-Binding Protein-beta / genetics CCAAT-Enhancer-Binding Protein-beta / metabolism* Cell Differentiation Cells, Cultured Chondrocytes / cytology* Chondrocytes / metabolism Chondrogenesis Core Binding Factor Alpha 1 Subunit / metabolism* Gene Expression Regulation Hedgehog Proteins / genetics* Mice Promoter Regions, Genetic Transcriptional Activation*
IF 2.74
Times Cited 3
Human and Animal Cells ATDC5(RCB0565)