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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 stimulated the expression of Ihh and Runx2 in ATDC5 cells.(A) Western blot of nuclear extracts obtained from ATDC5 cells, which were transfected with LacZ or C/EBPβ-LAP, 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. LacZ. (B) ATDC5 cells were differentiated for 2 weeks after transfection with adenovirus vectors expressing C/EBPβ-LAP and LacZ control. Expression of 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 LacZ on the 4th day was indicated. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05 vs. LacZ.
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pone-0104547-g002: C/EBPβ stimulated the expression of Ihh and Runx2 in ATDC5 cells.(A) Western blot of nuclear extracts obtained from ATDC5 cells, which were transfected with LacZ or C/EBPβ-LAP, 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. LacZ. (B) ATDC5 cells were differentiated for 2 weeks after transfection with adenovirus vectors expressing C/EBPβ-LAP and LacZ control. Expression of 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 LacZ on the 4th day was indicated. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05 vs. LacZ.

Mentions: To investigate the effect of C/EBPβ on Ihh expression, ATDC5 cells were transfected with adenovirus vectors expressing C/EBPβ-LAP or LacZ control and the cells were differentiated for 2 weeks. Increase of the mRNA (not shown) and nuclear protein of C/EBPβ-LAP by infection of adenovirus vector demonstrated that transfection of C/EBPβ was effectively performed (Figure 2A). We previously reported that in the same model, exogenous C/EBPβ significantly increased the expression of Runx2 on the 4th and 7th days [19]. The expression of Ihh was significantly increased at all the differentiation stages (Figure 2B). The expression of Pthrp, which is regulated by Ihh, was also stimulated by overexpression of C/EBPβ.


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 stimulated the expression of Ihh and Runx2 in ATDC5 cells.(A) Western blot of nuclear extracts obtained from ATDC5 cells, which were transfected with LacZ or C/EBPβ-LAP, 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. LacZ. (B) ATDC5 cells were differentiated for 2 weeks after transfection with adenovirus vectors expressing C/EBPβ-LAP and LacZ control. Expression of 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 LacZ on the 4th day was indicated. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05 vs. LacZ.
© Copyright Policy
Related In: Results  -  Collection

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pone-0104547-g002: C/EBPβ stimulated the expression of Ihh and Runx2 in ATDC5 cells.(A) Western blot of nuclear extracts obtained from ATDC5 cells, which were transfected with LacZ or C/EBPβ-LAP, 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. LacZ. (B) ATDC5 cells were differentiated for 2 weeks after transfection with adenovirus vectors expressing C/EBPβ-LAP and LacZ control. Expression of 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 LacZ on the 4th day was indicated. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05 vs. LacZ.
Mentions: To investigate the effect of C/EBPβ on Ihh expression, ATDC5 cells were transfected with adenovirus vectors expressing C/EBPβ-LAP or LacZ control and the cells were differentiated for 2 weeks. Increase of the mRNA (not shown) and nuclear protein of C/EBPβ-LAP by infection of adenovirus vector demonstrated that transfection of C/EBPβ was effectively performed (Figure 2A). We previously reported that in the same model, exogenous C/EBPβ significantly increased the expression of Runx2 on the 4th and 7th days [19]. The expression of Ihh was significantly increased at all the differentiation stages (Figure 2B). The expression of Pthrp, which is regulated by Ihh, was also stimulated by overexpression of C/EBPβ.

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