Genome sequencing identified a novel exonic microdeletion in the RUNX2 gene that causes cleidocranial dysplasia

A transcription factor, Cbfa1, which belongs to the runt-domain gene family, is expressed restrictively in fetal development. To elucidate the function of Cbfa1, we generated mice with a mutated Cbfa1 locus. Mice with a homozygous mutation in Cbfa1 died just after birth without breathing. Examination of their skeletal systems showed a complete lack of ossification. Although immature osteoblasts, which expressed alkaline phophatase weakly but not Osteopontin and Osteocalcin, and a few immature osteoclasts appeared at the perichondrial region, neither vascular nor mesenchymal cell invasion was observed in the cartilage. Therefore, our data suggest that both intramembranous and endochondral ossification were completely blocked, owing to the maturational arrest of osteoblasts in the mutant mice, and demonstrate that Cbfa1 plays an essential role in osteogenesis.

We have generated Cbfa1-deficient mice. Homozygous mutants die of respiratory failure shortly after birth. Analysis of their skeletons revealed an absence of osteoblasts and bone. Heterozygous mice showed specific skeletal abnormalities that are characteristic of the human heritable skeletal disorder, cleidocranial dysplasia (CCD). These defects are also observed in a mouse Ccd mutant for this disease. The Cbfa1 gene was shown to be deleted in the Ccd mutation. Analysis of embryonic Cbfa1 expression using a lacZ reporter gene revealed strong expression at sites of bone formation prior to the earliest stages of ossification. Thus, the Cbfa1 gene is essential for osteoblast differentiation and bone formation, and the Cbfa1 heterozygous mouse is a paradigm for a human skeletal disorder.

Vascular calcification is a hallmark of atherosclerosis, a major cause of morbidity and mortality in the United States. We have previously reported that the osteogenic transcription factor Runx2 is an essential and sufficient regulator of calcification of vascular smooth muscle cells (VSMC) in vitro. To determine the contribution of osteogenic differentiation of VSMC to the pathogenesis of vascular calcification and the function of VSMC-derived Runx2 in regulating calcification in vivo. SMC-specific Runx2-deficient mice, generated by breeding SM22α-Cre mice with the Runx2 exon 8 floxed mice, exhibited normal aortic gross anatomy and expression levels of SMC-specific marker genes. Runx2 deficiency did not affect basal SMC markers, but inhibited oxidative stress-reduced expression of SMC markers. High-fat-diet-induced vascular calcification in vivo was markedly inhibited in the Runx2-deficient mice in comparison with their control littermates. Runx2 deficiency inhibited the expression of receptor activator of nuclear factor κB ligand, which was accompanied by decreased macrophage infiltration and formation of osteoclast-like cells in the calcified lesions. Coculture of VSMC with bone marrow-derived macrophages demonstrated that the Runx2-deficient VSMC failed to promote differentiation of macrophages into osteoclast-like cells. These data have determined the importance of osteogenic differentiation of VSMC in the pathogenesis of vascular calcification in mice and defined the functional role of SMC-derived Runx2 in regulating vascular calcification and promoting infiltration of macrophages into the calcified lesion to form osteoclast-like cells. Our studies suggest that the development of vascular calcification is coupled with the formation of osteoclast-like cells, paralleling the bone remodeling process.