A Runx2 threshold for the cleidocranial dysplasia phenotype

Abstract

Cleidocranial dysplasia (CCD) in humans is an autosomal-dominant skeletal disease that results from mutations in the bone-specific transcription factor RUNX2 (CBFA1/AML3). However, distinct RUNX2 mutations in CCD do not correlate with the severity of the disease. Here we generated a new mouse model with a hypomorphic Runx2 mutant allele (Runx2(neo7)), in which only part of the transcript is processed to full-length (wild-type) Runx2 mRNA. Homozygous Runx2(neo7/neo7) mice express a reduced level of wild-type Runx2 mRNA (55-70%) and protein. This mouse model allowed us to establish the minimal requirement of functional Runx2 for normal bone development. Runx2(neo7/neo7) mice have grossly normal skeletons with no abnormalities observed in the growth plate, but do exhibit developmental defects in calvaria and clavicles that persist through post-natal growth. Clavicle defects are caused by disrupted endochondral bone formation during embryogenesis. These hypomorphic mice have altered calvarial bone volume, as observed by histology and microCT imaging, and decreased expression of osteoblast marker genes. The bone phenotype of the heterozygous mice, which have 79-84% of wild-type Runx2 mRNA, is normal. These results show there is a critical gene dosage requirement of functional Runx2 for the formation of intramembranous bone tissues during embryogenesis. A decrease to 70% of wild-type Runx2 levels results in the CCD syndrome, whereas levels>79% produce a normal skeleton. Our findings suggest that the range of bone phenotypes in CCD patients is attributable to quantitative reduction in the functional activity of RUNX2.

Runx2 Protein Represses Axin2 Expression in Osteoblasts and Is Required for Craniosynostosis in Axin2-deficient Mice*

“…Ϫ/Ϫ single mutants demonstrated reduced skull length, measured from the coronal suture to nose tip (-17%), as compared with wildtype animals (17)(18)(19) (Fig. 4, A, C, and D), which is consistent with previous studies (12).…”

“…Runx2 haploinsufficient mice mimic features of human cleidocranial dysplasia in that they present with hypoplastic clavicles and delayed cranial suture development with persistent fontanels in the calvaria (17)(18)(19). Runx2 is a stimulated by canonical Wnt/␤-catenin signaling (49), and ␤-catenin signaling is enhanced by Axin2 deficiency (10, 12, 16).…”

“…Runx2 is a master osteoblast transcription factor that is essential for proper bone development. Runx2-deficient mice die at birth because of a lack of skeletal ossification, and Runx2 haploinsufficient mice and humans develop an osteopenic phenotype and features of cleidocranial dysplasia, including delayed calvarial development (17)(18)(19). A 30% reduction in Runx2 levels is sufficient to induce a cleidocranial dysplasia phenotype in mice (19).…”

“…Runx2-deficient mice die at birth because of a lack of skeletal ossification, and Runx2 haploinsufficient mice and humans develop an osteopenic phenotype and features of cleidocranial dysplasia, including delayed calvarial development (17)(18)(19). A 30% reduction in Runx2 levels is sufficient to induce a cleidocranial dysplasia phenotype in mice (19). Interestingly, although Runx2 insufficiency impairs cranial suture closure, increased Runx2 expression coincides with the increased osteoblast differentiation that causes premature suture closure in many models of craniosynostosis (20 -24), suggesting convergence on a key regulatory role for Runx2 in cranial suture biology and, in particular, in craniosynostosis etiology.…”

SOX9 determines RUNX2 transactivity by directing intracellular degradation

“…(12) Abnormalities in RUNX2 function can lead to cleidocranial dysplasia (CCD), in which there is defective ossification of the cranial bones with large fontanels and delayed closing of the sutures with complete or partial absence of the clavicles. (13)(14)(15)(16)(17)(18)(19) Mice with homozygous mutations in RUNX2 cannot survive after birth owing to breathing defects caused by the absence of ossification of the ribs. (20,21) The functions of RUNX2 and SOX9 are controlled by transcriptional and posttranscriptional regulation, including ubiquitination, phosphorylation, and acetylation.…”