Endochondral ossification is regulated by various transcription factors in a temporo-spatial manner. Identification of novel transcription factors involved in chondrogenesis would facilitate to uncover the molecular basis of endochondral ossification. To approach this, we generated transgenic mice in which chondrocytes were specifically fluorescence-labeled with Venus gene using Col2a1 gene promoter, thereby allowing us to isolate Venus-positive chondrogenic cells using FACS Aria. Differential microarray between Venus-positive and -negative cells identified Foxc1 (Forkhead Box c1) as a candidate transcription factor selectively expressed in chondrogenic cells. Immunohistochemical analysis showed that Foxc1 was expressed not only in resting and proliferating chondrocytes but also hypertrophic chondrocytes. Overexpression of Foxc1 induced alcian blue-positive chondrogenesis in limb bud cells in micromass culture.
We next studied endochondral ossification in spontaneous Foxc1-inactivated mice (Foxc1ch). Foxc1ch mice showed shorter limbs and thinner ribs than WT mice. Of note, ossification center of sternum was completely absent and the length of Col10-positive hypertrophic zone was significantly shorter in newborn Foxc1ch mice compared to WT mice.
We then examined the molecular mechanism by which Foxc1 regulates endochondral ossification. Overexpression of Foxc1 in primary chondrocytes increased PTHrP and Col10a1 expression. DNA pull-down and ChIP assays displayed direct binding of Foxc1 to the PTHrP and Col10a1 gene promoter. Foxc1 physically associated with Gli2 and cooperatively increased the expression of Ihh/Gli2 target genes including PTHrP, Gli1 and Ptch1. Moreover, Foxc1 also co-immunoprecipitated with Runx2 and synergistically increased the expression of Col10a1 and MMP13 gene, which are the target genes of Runx2 in chondrogenesis.
In conclusion, we identified a transcription factor Foxc1 which directly regulates PTHrP and Col10a1 via complex formation with Ihh/Gli2 and Runx2. Our findings suggest that Foxc1 is a multifaceted transcription factor that contributes to the promotion of endochondral ossification.
17 May 2014 - 20 May 2014