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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 directly bound to Ihh promoter.(A) EMSA for specific binding of C/EBPβ to the Ihh promoter. Consensus oligonucleotide (C), wild-type (WT) and mutant (MT) probes were incubated with nuclear extract from C/EBPβ-transfected ATDC5 cells. Competition and supershift experiments were also performed. Data are representative of two independent experiments performed in duplicate. (B) A ChIP assay for C/EBPβ using ATDC5 cells cultured for 3 weeks. Semi-quantitative RT-PCR was performed using primers as follows: promoter region of Ihh (from −259 to −160) and negative control (from −1274 and −1102 bp). Data are representative of two independent experiments performed in duplicate.
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pone-0104547-g005: C/EBPβ directly bound to Ihh promoter.(A) EMSA for specific binding of C/EBPβ to the Ihh promoter. Consensus oligonucleotide (C), wild-type (WT) and mutant (MT) probes were incubated with nuclear extract from C/EBPβ-transfected ATDC5 cells. Competition and supershift experiments were also performed. Data are representative of two independent experiments performed in duplicate. (B) A ChIP assay for C/EBPβ using ATDC5 cells cultured for 3 weeks. Semi-quantitative RT-PCR was performed using primers as follows: promoter region of Ihh (from −259 to −160) and negative control (from −1274 and −1102 bp). Data are representative of two independent experiments performed in duplicate.

Mentions: To confirm the direct binding of C/EBPβ to the Ihh gene, EMSA was performed (Figure 5A). C/EBPβ bound strongly to the wild-type (WT) probe, but binding to the mutant (MT) probe was weak. Non-labeled WT probe inhibited the binding of C/EBPβ to labeled WT probe, but non-labeled MT probe could not block it. Supershift was observed by addition of a C/EBPβ antibody. Furthermore, a ChIP assay was performed using ATDC5 cells cultured for 3 weeks (Figure 5B). Endogenous C/EBPβ bound to the Ihh promoter region from −259 bp to −160 bp as detected by PCR. These analyses revealed a direct and specific binding of C/EBPβ to the Ihh promoter. Together, these results indicated that C/EBPβ directly stimulates transcriptional activity of Ihh by interacting with its promoter region.


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 directly bound to Ihh promoter.(A) EMSA for specific binding of C/EBPβ to the Ihh promoter. Consensus oligonucleotide (C), wild-type (WT) and mutant (MT) probes were incubated with nuclear extract from C/EBPβ-transfected ATDC5 cells. Competition and supershift experiments were also performed. Data are representative of two independent experiments performed in duplicate. (B) A ChIP assay for C/EBPβ using ATDC5 cells cultured for 3 weeks. Semi-quantitative RT-PCR was performed using primers as follows: promoter region of Ihh (from −259 to −160) and negative control (from −1274 and −1102 bp). Data are representative of two independent experiments performed in duplicate.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104547-g005: C/EBPβ directly bound to Ihh promoter.(A) EMSA for specific binding of C/EBPβ to the Ihh promoter. Consensus oligonucleotide (C), wild-type (WT) and mutant (MT) probes were incubated with nuclear extract from C/EBPβ-transfected ATDC5 cells. Competition and supershift experiments were also performed. Data are representative of two independent experiments performed in duplicate. (B) A ChIP assay for C/EBPβ using ATDC5 cells cultured for 3 weeks. Semi-quantitative RT-PCR was performed using primers as follows: promoter region of Ihh (from −259 to −160) and negative control (from −1274 and −1102 bp). Data are representative of two independent experiments performed in duplicate.
Mentions: To confirm the direct binding of C/EBPβ to the Ihh gene, EMSA was performed (Figure 5A). C/EBPβ bound strongly to the wild-type (WT) probe, but binding to the mutant (MT) probe was weak. Non-labeled WT probe inhibited the binding of C/EBPβ to labeled WT probe, but non-labeled MT probe could not block it. Supershift was observed by addition of a C/EBPβ antibody. Furthermore, a ChIP assay was performed using ATDC5 cells cultured for 3 weeks (Figure 5B). Endogenous C/EBPβ bound to the Ihh promoter region from −259 bp to −160 bp as detected by PCR. These analyses revealed a direct and specific binding of C/EBPβ to the Ihh promoter. Together, these results indicated that C/EBPβ directly stimulates transcriptional activity of Ihh by interacting with its promoter region.

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