Figure 7.

Regulation of Ihh expression by Runx2 and Runx3. (A,B) Real-time RT–PCR analysis of Ihh (A) and Col10a1 (B) expression using RNA extracted from wild-type (wt), Runx2^–/– (2^–/–), and Runx2^–/–3^–/– (2^–/–3^–/–) limbs at E18.5. The mean of two to three embryos is shown. The values in Runx2^–/– limbs were defined as 1, and relative values are shown. (C–F) Real-time RT–PCR analysis of Runx2^–/– chondrocyte cultures. Runx2^–/– chondrocytes were infected with EGFP-expressing (○), Runx2-and-EGFP-expressing (□), or Runx3-and-EGFP-expressing (▵) adenovirus. Infected cells were harvested at the indicated times after the onset of viral infection, and Runx2 (C), Runx3 (C), Ihh (D), Mmp13 (E), and Col10a1 (F) expression were examined by real-time RT–PCR. The value of Runx2 mRNA expression at 48 h was defined as 1, and the relative values of Runx2 and Runx3 mRNA expression are shown in the same graph (Runx2 and Runx3). In the cells infected with EGFP-expressing adenovirus, the values of Runx2 mRNA expression were nearly zero and the values of Runx3 mRNA expression were very low; therefore, they were not included in the graph. The value of Ihh, Mmp13, or Col10a1 mRNA expression in Runx2-and-EGFP-expressing adenovirus infection at 48 h was defined as 1, and relative values are shown. Adenoviral introduction of Runx2 strongly up-regulated Ihh expression, whereas adenoviral introduction of Runx3 did not up-regulate Ihh expression. In the cells infected with any of the three adenoviruses, the level of Col10a1 expression was at the background level during the culture period examined. Data represent the mean of three to six wells, and representative data of three independent experiments are shown.