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

Ushijima T, Okazaki K, Tsushima H, Ishihara K, Doi T, Iwamoto Y - PLoS ONE (2014)

Bottom Line: Conversely, knockdown of C/EBPβ by lentivirus expressing shRNA significantly repressed Ihh and Runx2 in ATDC5 cells.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

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.

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C/EBPβ up-regulated transcriptional activity of Ihh.(A) The Ihh reporter construct containing −1224 to +43 bp of the Ihh promoter, and various deletion constructs were generated. Gray and black boxes indicate RUNX2 binding elements reported by a previous study and C/EBPβ binding motif, respectively. Mutation construct of each element was also generated. (B) The Ihh reporter construct (pFull) was co-transfected with pCMV-LAP and GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. (C) The Ihh reporter construct (pFull) was co-transfected with 0.1 µg of pCMV-LAP and various amounts of A-C/EBP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. (D) Deletion constructs were co-transfected with 0.1 µg of pCMV-LAP or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05. (E) A mutation construct of C/EBPβ binding motif in pDel3 was co-transfected with 0.1 µg of pCMV-LAP or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05.
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pone-0104547-g004: C/EBPβ up-regulated transcriptional activity of Ihh.(A) The Ihh reporter construct containing −1224 to +43 bp of the Ihh promoter, and various deletion constructs were generated. Gray and black boxes indicate RUNX2 binding elements reported by a previous study and C/EBPβ binding motif, respectively. Mutation construct of each element was also generated. (B) The Ihh reporter construct (pFull) was co-transfected with pCMV-LAP and GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. (C) The Ihh reporter construct (pFull) was co-transfected with 0.1 µg of pCMV-LAP and various amounts of A-C/EBP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. (D) Deletion constructs were co-transfected with 0.1 µg of pCMV-LAP or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05. (E) A mutation construct of C/EBPβ binding motif in pDel3 was co-transfected with 0.1 µg of pCMV-LAP or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05.

Mentions: To confirm the transcriptional regulation of Ihh by C/EBPβ, a luciferase reporter construct containing −1224 to +43 bp of the Ihh promoter was generated (Figure 4A) and it was co-transfected with various expression vectors into HeLa cells. C/EBPβ up-regulated Ihh promoter activity in a dose-dependent manner (Figure 4B). In contrast, A-C/EBP, which inhibits binding of C/EBP family members to specific binding sites by forming a heterodimeric complex [27], reversed the up-regulation of Ihh promoter activity caused by C/EBPβ in a dose-dependent manner (Figure 4C). These results suggest that C/EBPβ regulates the expression of Ihh at the transcriptional level.


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

Ushijima T, Okazaki K, Tsushima H, Ishihara K, Doi T, Iwamoto Y - PLoS ONE (2014)

C/EBPβ up-regulated transcriptional activity of Ihh.(A) The Ihh reporter construct containing −1224 to +43 bp of the Ihh promoter, and various deletion constructs were generated. Gray and black boxes indicate RUNX2 binding elements reported by a previous study and C/EBPβ binding motif, respectively. Mutation construct of each element was also generated. (B) The Ihh reporter construct (pFull) was co-transfected with pCMV-LAP and GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. (C) The Ihh reporter construct (pFull) was co-transfected with 0.1 µg of pCMV-LAP and various amounts of A-C/EBP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. (D) Deletion constructs were co-transfected with 0.1 µg of pCMV-LAP or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05. (E) A mutation construct of C/EBPβ binding motif in pDel3 was co-transfected with 0.1 µg of pCMV-LAP or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126692&req=5

pone-0104547-g004: C/EBPβ up-regulated transcriptional activity of Ihh.(A) The Ihh reporter construct containing −1224 to +43 bp of the Ihh promoter, and various deletion constructs were generated. Gray and black boxes indicate RUNX2 binding elements reported by a previous study and C/EBPβ binding motif, respectively. Mutation construct of each element was also generated. (B) The Ihh reporter construct (pFull) was co-transfected with pCMV-LAP and GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. (C) The Ihh reporter construct (pFull) was co-transfected with 0.1 µg of pCMV-LAP and various amounts of A-C/EBP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. (D) Deletion constructs were co-transfected with 0.1 µg of pCMV-LAP or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05. (E) A mutation construct of C/EBPβ binding motif in pDel3 was co-transfected with 0.1 µg of pCMV-LAP or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05.
Mentions: To confirm the transcriptional regulation of Ihh by C/EBPβ, a luciferase reporter construct containing −1224 to +43 bp of the Ihh promoter was generated (Figure 4A) and it was co-transfected with various expression vectors into HeLa cells. C/EBPβ up-regulated Ihh promoter activity in a dose-dependent manner (Figure 4B). In contrast, A-C/EBP, which inhibits binding of C/EBP family members to specific binding sites by forming a heterodimeric complex [27], reversed the up-regulation of Ihh promoter activity caused by C/EBPβ in a dose-dependent manner (Figure 4C). These results suggest that C/EBPβ regulates the expression of Ihh at the transcriptional level.

Bottom Line: Conversely, knockdown of C/EBPβ by lentivirus expressing shRNA significantly repressed Ihh and Runx2 in ATDC5 cells.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

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.

Show MeSH
Related in: MedlinePlus