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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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Related in: MedlinePlus

C/EBPβ binding element is crucial for RUNX2 to regulate transcriptional activity of Ihh.(A) Deletion constructs were co-transfected with 0.2 µg of RUNX2 or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05. (B) Mutation constructs of C/EBPβ and RUNX2 binding elements in pDel3 were co-transfected with 0.2 µg of RUNX2 or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05. (C) EMSA for specific binding of RUNX2 to the C/EBPβ binding site of Ihh promoter. Wild-type (WT) probe, which harbors C/EBPβ binding site, was incubated with nuclear extract from C/EBPβ-transfected ATDC5 cells. Supershift experiment using RUNX2 antibody was also performed. Data are representative of two independent experiments performed in duplicate. (D) A ChIP assay for RUNX2 using ATDC5 cells cultured for 3 weeks. Semi-quantitative RT-PCR was performed using same primers as indicated in Figure 5B. Data are representative of two independent experiments performed in duplicate. (E) Immunoprecipitation (IP) and Immunoblotting were performed. Nuclear extract was obtained from C/EBPβ-transfected ATDC5 cells. Immunoprecipitated proteins with C/EBPβ, RUNX2 or IgG antibody were subjected to SDS-PAGE and immunoblotting using C/EBPβ or RUNX2 antibody.
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pone-0104547-g006: C/EBPβ binding element is crucial for RUNX2 to regulate transcriptional activity of Ihh.(A) Deletion constructs were co-transfected with 0.2 µg of RUNX2 or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05. (B) Mutation constructs of C/EBPβ and RUNX2 binding elements in pDel3 were co-transfected with 0.2 µg of RUNX2 or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05. (C) EMSA for specific binding of RUNX2 to the C/EBPβ binding site of Ihh promoter. Wild-type (WT) probe, which harbors C/EBPβ binding site, was incubated with nuclear extract from C/EBPβ-transfected ATDC5 cells. Supershift experiment using RUNX2 antibody was also performed. Data are representative of two independent experiments performed in duplicate. (D) A ChIP assay for RUNX2 using ATDC5 cells cultured for 3 weeks. Semi-quantitative RT-PCR was performed using same primers as indicated in Figure 5B. Data are representative of two independent experiments performed in duplicate. (E) Immunoprecipitation (IP) and Immunoblotting were performed. Nuclear extract was obtained from C/EBPβ-transfected ATDC5 cells. Immunoprecipitated proteins with C/EBPβ, RUNX2 or IgG antibody were subjected to SDS-PAGE and immunoblotting using C/EBPβ or RUNX2 antibody.

Mentions: It has been reported that C/EBPβ regulates transcriptional activity of various genes by interacting with RUNX2 [17], [20], [21] and that RUNX2 directly regulates Ihh through its promoter region [23]. Therefore, we investigated the cooperative binding of C/EBPβ and RUNX2 in the regulation of Ihh expression. Similar to the results with C/EBPβ, RUNX2 stimulated the promoter activity of the Ihh deletion constructs until the promoter sequence was deleted to −400 bp (Figure 6A). A previous study reported that there were three RUNX2 binding sites in the Ihh promoter [23]. The pDel3 construct contains one functional binding site for RUNX2, which is located nearest to the transcription start site (Figure 4A). Interestingly, RUNX2 could not enhance the promoter activity of pDel3-C/EBPβmut even with a functional RUNX2 binding site (Figure 6B). In contrast, a point mutation introduced into the RUNX2 binding element in pDel3 (pDel3-RUNX2mut) had a weak effect on the promoter activity by exogenous RUNX2 (Figure 6B). As expected, RUNX2 could not stimulate the promoter activity of pDel3-Dmut, which had mutations in both the C/EBPβ and RUNX2 binding elements (Figure 6B). An EMSA revealed that the band intensity of the DNA probe for the sequence of the C/EBPβ binding site and protein complex was decreased when adding RUNX2 antibody (Figure 6C). In addition, a ChIP assay revealed binding of endogenous RUNX2 to the Ihh promoter located between −259 bp and −160 bp (Figure 6D). To confirm the interaction between C/EBPβ and RUNX2, IP was performed (Figure 6E). Immunoblotting with C/EBPβ antibody showed positive bands for C/EBPβ-LAP and –LIP on the sample immunoprecipitated with RUNX2 antibody. Immunoblotting with RUNX2 was also positive on the sample immunoprecipitated with C/EBPβ antibody. This result demonstrated that RUNX2 forms heterodimer complex with both of C/EBPβ-LAP and -LIP in the nuclei of chondrocytes. Together, these results indicated that the C/EBPβ binding site is also important for RUNX2 to regulate transcriptional activation of Ihh.


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 binding element is crucial for RUNX2 to regulate transcriptional activity of Ihh.(A) Deletion constructs were co-transfected with 0.2 µg of RUNX2 or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05. (B) Mutation constructs of C/EBPβ and RUNX2 binding elements in pDel3 were co-transfected with 0.2 µg of RUNX2 or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05. (C) EMSA for specific binding of RUNX2 to the C/EBPβ binding site of Ihh promoter. Wild-type (WT) probe, which harbors C/EBPβ binding site, was incubated with nuclear extract from C/EBPβ-transfected ATDC5 cells. Supershift experiment using RUNX2 antibody was also performed. Data are representative of two independent experiments performed in duplicate. (D) A ChIP assay for RUNX2 using ATDC5 cells cultured for 3 weeks. Semi-quantitative RT-PCR was performed using same primers as indicated in Figure 5B. Data are representative of two independent experiments performed in duplicate. (E) Immunoprecipitation (IP) and Immunoblotting were performed. Nuclear extract was obtained from C/EBPβ-transfected ATDC5 cells. Immunoprecipitated proteins with C/EBPβ, RUNX2 or IgG antibody were subjected to SDS-PAGE and immunoblotting using C/EBPβ or RUNX2 antibody.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104547-g006: C/EBPβ binding element is crucial for RUNX2 to regulate transcriptional activity of Ihh.(A) Deletion constructs were co-transfected with 0.2 µg of RUNX2 or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05. (B) Mutation constructs of C/EBPβ and RUNX2 binding elements in pDel3 were co-transfected with 0.2 µg of RUNX2 or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. *p<0.05. (C) EMSA for specific binding of RUNX2 to the C/EBPβ binding site of Ihh promoter. Wild-type (WT) probe, which harbors C/EBPβ binding site, was incubated with nuclear extract from C/EBPβ-transfected ATDC5 cells. Supershift experiment using RUNX2 antibody was also performed. Data are representative of two independent experiments performed in duplicate. (D) A ChIP assay for RUNX2 using ATDC5 cells cultured for 3 weeks. Semi-quantitative RT-PCR was performed using same primers as indicated in Figure 5B. Data are representative of two independent experiments performed in duplicate. (E) Immunoprecipitation (IP) and Immunoblotting were performed. Nuclear extract was obtained from C/EBPβ-transfected ATDC5 cells. Immunoprecipitated proteins with C/EBPβ, RUNX2 or IgG antibody were subjected to SDS-PAGE and immunoblotting using C/EBPβ or RUNX2 antibody.
Mentions: It has been reported that C/EBPβ regulates transcriptional activity of various genes by interacting with RUNX2 [17], [20], [21] and that RUNX2 directly regulates Ihh through its promoter region [23]. Therefore, we investigated the cooperative binding of C/EBPβ and RUNX2 in the regulation of Ihh expression. Similar to the results with C/EBPβ, RUNX2 stimulated the promoter activity of the Ihh deletion constructs until the promoter sequence was deleted to −400 bp (Figure 6A). A previous study reported that there were three RUNX2 binding sites in the Ihh promoter [23]. The pDel3 construct contains one functional binding site for RUNX2, which is located nearest to the transcription start site (Figure 4A). Interestingly, RUNX2 could not enhance the promoter activity of pDel3-C/EBPβmut even with a functional RUNX2 binding site (Figure 6B). In contrast, a point mutation introduced into the RUNX2 binding element in pDel3 (pDel3-RUNX2mut) had a weak effect on the promoter activity by exogenous RUNX2 (Figure 6B). As expected, RUNX2 could not stimulate the promoter activity of pDel3-Dmut, which had mutations in both the C/EBPβ and RUNX2 binding elements (Figure 6B). An EMSA revealed that the band intensity of the DNA probe for the sequence of the C/EBPβ binding site and protein complex was decreased when adding RUNX2 antibody (Figure 6C). In addition, a ChIP assay revealed binding of endogenous RUNX2 to the Ihh promoter located between −259 bp and −160 bp (Figure 6D). To confirm the interaction between C/EBPβ and RUNX2, IP was performed (Figure 6E). Immunoblotting with C/EBPβ antibody showed positive bands for C/EBPβ-LAP and –LIP on the sample immunoprecipitated with RUNX2 antibody. Immunoblotting with RUNX2 was also positive on the sample immunoprecipitated with C/EBPβ antibody. This result demonstrated that RUNX2 forms heterodimer complex with both of C/EBPβ-LAP and -LIP in the nuclei of chondrocytes. Together, these results indicated that the C/EBPβ binding site is also important for RUNX2 to regulate transcriptional activation of Ihh.

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