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Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte-specific expression in vivo.

Zheng Q, Zhou G, Morello R, Chen Y, Garcia-Rojas X, Lee B - J. Cell Biol. (2003)

Bottom Line: In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites.When the 4-kb Col10a1 promoter transgene was bred onto a Runx2(+/-) background, the reporter was expressed at lower levels.Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The alpha1(X) collagen gene (Col10a1) is the only known hypertrophic chondrocyte-specific molecular marker. Until recently, few transcriptional factors specifying its tissue-specific expression have been identified. We show here that a 4-kb murine Col10a1 promoter can drive beta-galactosidase expression in lower hypertrophic chondrocytes in transgenic mice. Comparative genomic analysis revealed multiple Runx2 (Runt domain transcription factor) binding sites within the proximal human, mouse, and chick Col10a1 promoters. In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites. When the 4-kb Col10a1 promoter transgene was bred onto a Runx2(+/-) background, the reporter was expressed at lower levels. Moreover, decreased Col10a1 expression and altered chondrocyte hypertrophy was also observed in Runx2 heterozygote mice, whereas Col10a1 was barely detectable in Runx2- mice. Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

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Genetic interaction of Runx2 and Col10a1. (A) Runx2 and Col10a1 promoter/ reporter transgene. The 4-kb Col10a1 promoter-βgeo transgene (Tg) was bred onto Runx2+/+ and Runx2+/− backgrounds and compared to Runx2+/− mice without Tg. X-gal blue staining is much weaker in the lower hypertrophic zones of ulna of Tg/Runx2+/− mice (middle) than in that of Tg/Runx2+/+ mice (left), whereas no staining is observed in the hypertrophic zone of Runx2+/− mice (right). The hypertrophic zone (HZ) appeared shorter in sections from mice with Runx2+/− than that of Runx2+/+ background. (B) Transgene expression in Tg/Runx2+/+ versus Tg/Runx2+/− mice. (left) Semi-quantitative densitometric analysis of transgene expression in mouse limb sections by comparing the gray values generated for blue-staining cells as described in Materials and methods. Sum, total gray value of blue cells; SD, standard deviation. (right) Transgene expression was decreased by 40% in Runx2 heterozygotes as compared to that of the wild-type littermate control by real time RT-PCR assay. Similar results were obtained from two Tg/Runx2+/+ and three Tg/Runx2+/− mice. One representative set of results is presented here with the standard deviations shown by the error bars. (C) Col10a1 expression pattern in Runx2+/− mice. Runx2+/+ mice showed signal of Col10a1 transcripts throughout the hypertrophic zone of femur by in situ hybridization (left), whereas Runx2+/− mice also have shortened hypertrophic zone as indicated by Col10a1 expression pattern (right).
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fig7: Genetic interaction of Runx2 and Col10a1. (A) Runx2 and Col10a1 promoter/ reporter transgene. The 4-kb Col10a1 promoter-βgeo transgene (Tg) was bred onto Runx2+/+ and Runx2+/− backgrounds and compared to Runx2+/− mice without Tg. X-gal blue staining is much weaker in the lower hypertrophic zones of ulna of Tg/Runx2+/− mice (middle) than in that of Tg/Runx2+/+ mice (left), whereas no staining is observed in the hypertrophic zone of Runx2+/− mice (right). The hypertrophic zone (HZ) appeared shorter in sections from mice with Runx2+/− than that of Runx2+/+ background. (B) Transgene expression in Tg/Runx2+/+ versus Tg/Runx2+/− mice. (left) Semi-quantitative densitometric analysis of transgene expression in mouse limb sections by comparing the gray values generated for blue-staining cells as described in Materials and methods. Sum, total gray value of blue cells; SD, standard deviation. (right) Transgene expression was decreased by 40% in Runx2 heterozygotes as compared to that of the wild-type littermate control by real time RT-PCR assay. Similar results were obtained from two Tg/Runx2+/+ and three Tg/Runx2+/− mice. One representative set of results is presented here with the standard deviations shown by the error bars. (C) Col10a1 expression pattern in Runx2+/− mice. Runx2+/+ mice showed signal of Col10a1 transcripts throughout the hypertrophic zone of femur by in situ hybridization (left), whereas Runx2+/− mice also have shortened hypertrophic zone as indicated by Col10a1 expression pattern (right).

Mentions: To further delineate the contribution of Runx2 to transactivation of Col10a1 in vivo, we bred the 4-kb promoter-β-galactosidase transgene (Tg) onto a Runx2 heterozygote background to generate four different genotypes for histological analysis: Tg+/−/Runx2+/−, Tg+/−/Runx2+/+, Runx2+/−, and Runx2+/+ (wild type; Otto et al., 1997). Sections of long bones including the distal ulna and proximal tibia from P1 mice showed X-gal staining in hypertrophic chondrocytes in Tg+/−/Runx2+/+ (Fig. 7 A and not depicted). X-gal staining was decreased in Tg+/−/Runx2+/− mice (Fig. 7 A). Although the Runx2 targeted allele does contain a knock-in LacZ reporter, Runx2 expression in hypertrophic chondrocytes is lower compared to its expression in osteoblasts and X-gal blue staining in the Runx2+/−mice was undetectable in hypertrophic chondrocytes with our protocol (Fig. 7 A, right). As expected, X-gal staining was stronger in the bone marrow of Tg+/−/Runx2+/− mice than in Tg+/−/Runx2+/+ mice because of the strong LacZ expression of the Runx2 targeted allele in osteoblast (unpublished data). To quantify and compare the transgene expression in Tg/Runx2+/+ and Tg/Runx2+/−mice, we performed semiquantitative densitometric analysis of transgene expression in mouse limb sections (Ma et al., 2001). 200 blue-stained cells in the hypertrophic zone from twenty limb sections from each of the genotype were chosen for analysis. The gray value, which is inversely related to the intensity of the blue staining, is significantly lower in cells from Tg/Runx2+/+ mice limb sections than that from Tg/Runx2+/−mice (P < 0.01). This result suggested that the transgene is expressed at a lower level in chondrocytes from Tg/Runx2+/− mice compared to that of Tg/Runx2+/+ mice (Fig. 7 B, left). We also performed real time RT-PCR quantification of transgene expression using RNAs from transgenic mice limbs with Runx2 wild-type (Tg/Runx2+/+) or heterozygote background (Tg/Runx2+/−). Transgene expression decreased by approximately 40% in Tg/Runx2+/− compared to that of Tg/Runx2+/+ (Fig. 7 B, right). These data support genetic interaction between the Col10a1 Tg reporter allele and Runx2 expression in hypertrophic chondrocytes. Interpreted in the context of the in vitro and in vivo data, this is most likely explained by direct transactivation of the Col10a1 promoter by Runx2. Interestingly, as shown in the ulna sections in Fig. 7 A, both Runx2+/− and Tg+/−/Runx2+/− mice had a slightly shortened zone of hypertrophy as compared to Runx2+/+ mice. Similar differences were also observed in sections of the growth plates of the humerus and radius (unpublished data). Furthermore, RNA in situ hybridization on distal femur sections of Runx2+/+ or Runx2+/− mice using a Col10a1 riboprobe showed that Col10a1 expression was detected throughout the hypertrophic zone. Moreover, the Runx2+/− mice had a shortened hypertrophic zone in the growth plate region similar to that observed in Tg+/−/Runx2+/− mice (Fig. 7 C).


Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte-specific expression in vivo.

Zheng Q, Zhou G, Morello R, Chen Y, Garcia-Rojas X, Lee B - J. Cell Biol. (2003)

Genetic interaction of Runx2 and Col10a1. (A) Runx2 and Col10a1 promoter/ reporter transgene. The 4-kb Col10a1 promoter-βgeo transgene (Tg) was bred onto Runx2+/+ and Runx2+/− backgrounds and compared to Runx2+/− mice without Tg. X-gal blue staining is much weaker in the lower hypertrophic zones of ulna of Tg/Runx2+/− mice (middle) than in that of Tg/Runx2+/+ mice (left), whereas no staining is observed in the hypertrophic zone of Runx2+/− mice (right). The hypertrophic zone (HZ) appeared shorter in sections from mice with Runx2+/− than that of Runx2+/+ background. (B) Transgene expression in Tg/Runx2+/+ versus Tg/Runx2+/− mice. (left) Semi-quantitative densitometric analysis of transgene expression in mouse limb sections by comparing the gray values generated for blue-staining cells as described in Materials and methods. Sum, total gray value of blue cells; SD, standard deviation. (right) Transgene expression was decreased by 40% in Runx2 heterozygotes as compared to that of the wild-type littermate control by real time RT-PCR assay. Similar results were obtained from two Tg/Runx2+/+ and three Tg/Runx2+/− mice. One representative set of results is presented here with the standard deviations shown by the error bars. (C) Col10a1 expression pattern in Runx2+/− mice. Runx2+/+ mice showed signal of Col10a1 transcripts throughout the hypertrophic zone of femur by in situ hybridization (left), whereas Runx2+/− mice also have shortened hypertrophic zone as indicated by Col10a1 expression pattern (right).
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fig7: Genetic interaction of Runx2 and Col10a1. (A) Runx2 and Col10a1 promoter/ reporter transgene. The 4-kb Col10a1 promoter-βgeo transgene (Tg) was bred onto Runx2+/+ and Runx2+/− backgrounds and compared to Runx2+/− mice without Tg. X-gal blue staining is much weaker in the lower hypertrophic zones of ulna of Tg/Runx2+/− mice (middle) than in that of Tg/Runx2+/+ mice (left), whereas no staining is observed in the hypertrophic zone of Runx2+/− mice (right). The hypertrophic zone (HZ) appeared shorter in sections from mice with Runx2+/− than that of Runx2+/+ background. (B) Transgene expression in Tg/Runx2+/+ versus Tg/Runx2+/− mice. (left) Semi-quantitative densitometric analysis of transgene expression in mouse limb sections by comparing the gray values generated for blue-staining cells as described in Materials and methods. Sum, total gray value of blue cells; SD, standard deviation. (right) Transgene expression was decreased by 40% in Runx2 heterozygotes as compared to that of the wild-type littermate control by real time RT-PCR assay. Similar results were obtained from two Tg/Runx2+/+ and three Tg/Runx2+/− mice. One representative set of results is presented here with the standard deviations shown by the error bars. (C) Col10a1 expression pattern in Runx2+/− mice. Runx2+/+ mice showed signal of Col10a1 transcripts throughout the hypertrophic zone of femur by in situ hybridization (left), whereas Runx2+/− mice also have shortened hypertrophic zone as indicated by Col10a1 expression pattern (right).
Mentions: To further delineate the contribution of Runx2 to transactivation of Col10a1 in vivo, we bred the 4-kb promoter-β-galactosidase transgene (Tg) onto a Runx2 heterozygote background to generate four different genotypes for histological analysis: Tg+/−/Runx2+/−, Tg+/−/Runx2+/+, Runx2+/−, and Runx2+/+ (wild type; Otto et al., 1997). Sections of long bones including the distal ulna and proximal tibia from P1 mice showed X-gal staining in hypertrophic chondrocytes in Tg+/−/Runx2+/+ (Fig. 7 A and not depicted). X-gal staining was decreased in Tg+/−/Runx2+/− mice (Fig. 7 A). Although the Runx2 targeted allele does contain a knock-in LacZ reporter, Runx2 expression in hypertrophic chondrocytes is lower compared to its expression in osteoblasts and X-gal blue staining in the Runx2+/−mice was undetectable in hypertrophic chondrocytes with our protocol (Fig. 7 A, right). As expected, X-gal staining was stronger in the bone marrow of Tg+/−/Runx2+/− mice than in Tg+/−/Runx2+/+ mice because of the strong LacZ expression of the Runx2 targeted allele in osteoblast (unpublished data). To quantify and compare the transgene expression in Tg/Runx2+/+ and Tg/Runx2+/−mice, we performed semiquantitative densitometric analysis of transgene expression in mouse limb sections (Ma et al., 2001). 200 blue-stained cells in the hypertrophic zone from twenty limb sections from each of the genotype were chosen for analysis. The gray value, which is inversely related to the intensity of the blue staining, is significantly lower in cells from Tg/Runx2+/+ mice limb sections than that from Tg/Runx2+/−mice (P < 0.01). This result suggested that the transgene is expressed at a lower level in chondrocytes from Tg/Runx2+/− mice compared to that of Tg/Runx2+/+ mice (Fig. 7 B, left). We also performed real time RT-PCR quantification of transgene expression using RNAs from transgenic mice limbs with Runx2 wild-type (Tg/Runx2+/+) or heterozygote background (Tg/Runx2+/−). Transgene expression decreased by approximately 40% in Tg/Runx2+/− compared to that of Tg/Runx2+/+ (Fig. 7 B, right). These data support genetic interaction between the Col10a1 Tg reporter allele and Runx2 expression in hypertrophic chondrocytes. Interpreted in the context of the in vitro and in vivo data, this is most likely explained by direct transactivation of the Col10a1 promoter by Runx2. Interestingly, as shown in the ulna sections in Fig. 7 A, both Runx2+/− and Tg+/−/Runx2+/− mice had a slightly shortened zone of hypertrophy as compared to Runx2+/+ mice. Similar differences were also observed in sections of the growth plates of the humerus and radius (unpublished data). Furthermore, RNA in situ hybridization on distal femur sections of Runx2+/+ or Runx2+/− mice using a Col10a1 riboprobe showed that Col10a1 expression was detected throughout the hypertrophic zone. Moreover, the Runx2+/− mice had a shortened hypertrophic zone in the growth plate region similar to that observed in Tg+/−/Runx2+/− mice (Fig. 7 C).

Bottom Line: In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites.When the 4-kb Col10a1 promoter transgene was bred onto a Runx2(+/-) background, the reporter was expressed at lower levels.Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The alpha1(X) collagen gene (Col10a1) is the only known hypertrophic chondrocyte-specific molecular marker. Until recently, few transcriptional factors specifying its tissue-specific expression have been identified. We show here that a 4-kb murine Col10a1 promoter can drive beta-galactosidase expression in lower hypertrophic chondrocytes in transgenic mice. Comparative genomic analysis revealed multiple Runx2 (Runt domain transcription factor) binding sites within the proximal human, mouse, and chick Col10a1 promoters. In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites. When the 4-kb Col10a1 promoter transgene was bred onto a Runx2(+/-) background, the reporter was expressed at lower levels. Moreover, decreased Col10a1 expression and altered chondrocyte hypertrophy was also observed in Runx2 heterozygote mice, whereas Col10a1 was barely detectable in Runx2- mice. Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

Show MeSH
Related in: MedlinePlus