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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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Runx2 directly regulates the activation of the type X collagen gene. (A) Runx2 is upregulated in hypertrophic MCT cells. When MCT cells were shifted from 32°C to 37°C, Runx2 showed two-fold upregulation as assayed by real time RT-PCR (left). Meanwhile, Col10a1 showed more than 20-fold up-regulation in hypertrophic MCT cells (right). Each real time PCR experiment was performed in triplicate and the standard deviations are shown by the error bars. Similar results were obtained from three independent experiments. (B) Endogenous downregulation of Col10a1 in Runx2+/− and Runx2−/− mouse limbs. Runx2 expression was also decreased by 45% in Runx2 heterozygotes (left). Endogenous Col10a1 expression was also decreased by 50% in Runx2 heterozygotes and barely detectable in Runx2−/− mice as compared to that of the wild-type littermate control by real time RT-PCR assay (right). Similar results were obtained from five wild-type, eight Runx2+/− and two Runx2- mice. One representative set of results is presented here with the standard deviations shown by the error bars.
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fig6: Runx2 directly regulates the activation of the type X collagen gene. (A) Runx2 is upregulated in hypertrophic MCT cells. When MCT cells were shifted from 32°C to 37°C, Runx2 showed two-fold upregulation as assayed by real time RT-PCR (left). Meanwhile, Col10a1 showed more than 20-fold up-regulation in hypertrophic MCT cells (right). Each real time PCR experiment was performed in triplicate and the standard deviations are shown by the error bars. Similar results were obtained from three independent experiments. (B) Endogenous downregulation of Col10a1 in Runx2+/− and Runx2−/− mouse limbs. Runx2 expression was also decreased by 45% in Runx2 heterozygotes (left). Endogenous Col10a1 expression was also decreased by 50% in Runx2 heterozygotes and barely detectable in Runx2−/− mice as compared to that of the wild-type littermate control by real time RT-PCR assay (right). Similar results were obtained from five wild-type, eight Runx2+/− and two Runx2- mice. One representative set of results is presented here with the standard deviations shown by the error bars.

Mentions: To quantify the correlation between Runx2 and Col10a1 expression, quantitative real time RT-PCR was used to determine their respective levels of expression in both the MCT cells and in Runx2+/+, Runx2+/−, and Runx2−/− mice (Otto et al., 1997; Livak and Schmittgen, 2001; Pfaffl, 2001). Runx2 was weakly expressed in MCT cells grown at 32°C by Northern and qualitative RT-PCR analysis (unpublished data). However, when cells were terminally differentiated by growth at nonpermissive temperatures (from 32°C to 37°C), Col10a1 was upregulated (Fig. 2 B, left, and Fig. 6 A, right) in conjunction with a two fold up-regulation of Runx2 (Fig. 6 A, left). In limbs from newborn mice, real time RT-PCR assay showed less Col10a1 expression in Runx2 heterozygotes (∼50%) compared to that of the wild-type littermate controls (Fig. 6 B, right). Furthermore, Col10a1 expression was at a level barely detectable in Runx2−/− mice limbs (Fig. 6 B, right; Inada et al., 1999). As expected, Runx2 expression was decreased by 45% in Runx2 heterozygotes (Fig. 6 B, left). Thus, our data suggest that Runx2 might directly regulate the activation of the type X collagen gene during chondrocyte maturation in vivo.


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)

Runx2 directly regulates the activation of the type X collagen gene. (A) Runx2 is upregulated in hypertrophic MCT cells. When MCT cells were shifted from 32°C to 37°C, Runx2 showed two-fold upregulation as assayed by real time RT-PCR (left). Meanwhile, Col10a1 showed more than 20-fold up-regulation in hypertrophic MCT cells (right). Each real time PCR experiment was performed in triplicate and the standard deviations are shown by the error bars. Similar results were obtained from three independent experiments. (B) Endogenous downregulation of Col10a1 in Runx2+/− and Runx2−/− mouse limbs. Runx2 expression was also decreased by 45% in Runx2 heterozygotes (left). Endogenous Col10a1 expression was also decreased by 50% in Runx2 heterozygotes and barely detectable in Runx2−/− mice as compared to that of the wild-type littermate control by real time RT-PCR assay (right). Similar results were obtained from five wild-type, eight Runx2+/− and two Runx2- mice. One representative set of results is presented here with the standard deviations shown by the error bars.
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fig6: Runx2 directly regulates the activation of the type X collagen gene. (A) Runx2 is upregulated in hypertrophic MCT cells. When MCT cells were shifted from 32°C to 37°C, Runx2 showed two-fold upregulation as assayed by real time RT-PCR (left). Meanwhile, Col10a1 showed more than 20-fold up-regulation in hypertrophic MCT cells (right). Each real time PCR experiment was performed in triplicate and the standard deviations are shown by the error bars. Similar results were obtained from three independent experiments. (B) Endogenous downregulation of Col10a1 in Runx2+/− and Runx2−/− mouse limbs. Runx2 expression was also decreased by 45% in Runx2 heterozygotes (left). Endogenous Col10a1 expression was also decreased by 50% in Runx2 heterozygotes and barely detectable in Runx2−/− mice as compared to that of the wild-type littermate control by real time RT-PCR assay (right). Similar results were obtained from five wild-type, eight Runx2+/− and two Runx2- mice. One representative set of results is presented here with the standard deviations shown by the error bars.
Mentions: To quantify the correlation between Runx2 and Col10a1 expression, quantitative real time RT-PCR was used to determine their respective levels of expression in both the MCT cells and in Runx2+/+, Runx2+/−, and Runx2−/− mice (Otto et al., 1997; Livak and Schmittgen, 2001; Pfaffl, 2001). Runx2 was weakly expressed in MCT cells grown at 32°C by Northern and qualitative RT-PCR analysis (unpublished data). However, when cells were terminally differentiated by growth at nonpermissive temperatures (from 32°C to 37°C), Col10a1 was upregulated (Fig. 2 B, left, and Fig. 6 A, right) in conjunction with a two fold up-regulation of Runx2 (Fig. 6 A, left). In limbs from newborn mice, real time RT-PCR assay showed less Col10a1 expression in Runx2 heterozygotes (∼50%) compared to that of the wild-type littermate controls (Fig. 6 B, right). Furthermore, Col10a1 expression was at a level barely detectable in Runx2−/− mice limbs (Fig. 6 B, right; Inada et al., 1999). As expected, Runx2 expression was decreased by 45% in Runx2 heterozygotes (Fig. 6 B, left). Thus, our data suggest that Runx2 might directly regulate the activation of the type X collagen gene during chondrocyte maturation in vivo.

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