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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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Knockdown of C/EBPβ repressed the expression of Ihh and Runx2 in ATDC5 cells.(A) Western blot of nuclear extracts obtained from stable ATDC5 cells was performed to investigate the expression of C/EBPβ. Densitometric scanning of C/EBPβ expression was performed. Each density of C/EBPβ was normalized with that of LAMIN A/C and the ratio by corrected densities of C/EBPβ to control on the 4th day was calculated. Data are representative of two independent experiments performed in duplicate. *p<0.05 vs. control. (B) ATDC5 cells stably expressing shRNA for Cebpb were differentiated for 2 weeks. Expression of Cebpb, Runx2, Ihh and Pthrp mRNA was determined by real-time RT-PCR. Each value was normalized to 18S in the same sample. The value of each mRNA expression relative to that of control on the 4th day was indicated. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05 vs. control.
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pone-0104547-g003: Knockdown of C/EBPβ repressed the expression of Ihh and Runx2 in ATDC5 cells.(A) Western blot of nuclear extracts obtained from stable ATDC5 cells was performed to investigate the expression of C/EBPβ. Densitometric scanning of C/EBPβ expression was performed. Each density of C/EBPβ was normalized with that of LAMIN A/C and the ratio by corrected densities of C/EBPβ to control on the 4th day was calculated. Data are representative of two independent experiments performed in duplicate. *p<0.05 vs. control. (B) ATDC5 cells stably expressing shRNA for Cebpb were differentiated for 2 weeks. Expression of Cebpb, Runx2, Ihh and Pthrp mRNA was determined by real-time RT-PCR. Each value was normalized to 18S in the same sample. The value of each mRNA expression relative to that of control on the 4th day was indicated. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05 vs. control.

Mentions: Next, we investigated the effect of C/EBPβ knockdown on the expression of Ihh. ATDC5 cells were transfected with lentivirus expressing shRNA targeting Cebpb and stably infected cells were differentiated with ITS for 2 weeks. Knockdown of Cebpb was confirmed with nuclear extracts and mRNA in cells transfected with shRNA compared to the controls at all differentiation stages (Figure 3A, B). Ihh and Runx2 expression was significantly repressed by shRNA for Cebpb on the 14th day (Figure 3B). However, the expression of Pthrp was markedly increased by shRNA on the 4th day (Figure 3B). These results suggest that C/EBPβ is involved in the regulation of Ihh expression at the endogenous level during chondrocyte differentiation.


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)

Knockdown of C/EBPβ repressed the expression of Ihh and Runx2 in ATDC5 cells.(A) Western blot of nuclear extracts obtained from stable ATDC5 cells was performed to investigate the expression of C/EBPβ. Densitometric scanning of C/EBPβ expression was performed. Each density of C/EBPβ was normalized with that of LAMIN A/C and the ratio by corrected densities of C/EBPβ to control on the 4th day was calculated. Data are representative of two independent experiments performed in duplicate. *p<0.05 vs. control. (B) ATDC5 cells stably expressing shRNA for Cebpb were differentiated for 2 weeks. Expression of Cebpb, Runx2, Ihh and Pthrp mRNA was determined by real-time RT-PCR. Each value was normalized to 18S in the same sample. The value of each mRNA expression relative to that of control on the 4th day was indicated. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05 vs. control.
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Related In: Results  -  Collection

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pone-0104547-g003: Knockdown of C/EBPβ repressed the expression of Ihh and Runx2 in ATDC5 cells.(A) Western blot of nuclear extracts obtained from stable ATDC5 cells was performed to investigate the expression of C/EBPβ. Densitometric scanning of C/EBPβ expression was performed. Each density of C/EBPβ was normalized with that of LAMIN A/C and the ratio by corrected densities of C/EBPβ to control on the 4th day was calculated. Data are representative of two independent experiments performed in duplicate. *p<0.05 vs. control. (B) ATDC5 cells stably expressing shRNA for Cebpb were differentiated for 2 weeks. Expression of Cebpb, Runx2, Ihh and Pthrp mRNA was determined by real-time RT-PCR. Each value was normalized to 18S in the same sample. The value of each mRNA expression relative to that of control on the 4th day was indicated. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05 vs. control.
Mentions: Next, we investigated the effect of C/EBPβ knockdown on the expression of Ihh. ATDC5 cells were transfected with lentivirus expressing shRNA targeting Cebpb and stably infected cells were differentiated with ITS for 2 weeks. Knockdown of Cebpb was confirmed with nuclear extracts and mRNA in cells transfected with shRNA compared to the controls at all differentiation stages (Figure 3A, B). Ihh and Runx2 expression was significantly repressed by shRNA for Cebpb on the 14th day (Figure 3B). However, the expression of Pthrp was markedly increased by shRNA on the 4th day (Figure 3B). These results suggest that C/EBPβ is involved in the regulation of Ihh expression at the endogenous level during chondrocyte differentiation.

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