Runx2 haploinsufficiency ameliorates the development of ossification of the posterior longitudinal ligament

Ossification of the Posterior Longitudinal Ligament (OPLL) is a disease that is characterized by the ectopic calcification of the ligament; however, the pathogenesis of OPLL remains to be investigated. We attempted to identify the in vivo role of Runx2, a master regulator of osteoblast differentiation and skeletal mineralization, in the pathogenesis of OPLL. The expression of Runx2 in the ligament was examined using in situ hybridization and immunohistochemistry and by monitoring the activity of a LacZ gene that was inserted into the Runx2 gene locus. To investigate the functional role of Runx2, we studied ENPP1(ttw/ttw) mice, a mouse model of OPLL, that were crossed with heterozygous Runx2 mice to decrease the expression of Runx2, and we performed histological and quantitative radiological analyses using 3D-micro CT. Runx2 was expressed in the ligament of wild-type mice. The induction of Runx2 expression preceded the development of ectopic calcification in the OPLL-like region of the ENPP1(ttw/ttw) mice. Runx2 haploinsufficiency ameliorated the development of ectopic calcification in the ENPP1(ttw/ttw) mice. Collectively, this study demonstrated that Runx2 is expressed in an OPLL-like region, and its elevation is a prerequisite for developing the complete OPLL-like phenotype in a mouse model of OPLL.     

Cbf beta regulates Runx2 function isoform-dependently in postnatal bone development

Runx2 and Cbfbeta are essential for skeletal development during the embryonic stage. Runx2 has two isoforms with different N-termini. We examined the functions of the Runx2 isoforms and Cbfbeta in postnatal bone development. On luciferase and electrophoretic mobility shift assays, Runx2-I was less active than Runx2-II in the absence of Cbfb, but the two Runx2 isoforms had similar activity levels in the presence of Cbfb. We generated Runx2-I transgenic mice under the control of Col1a1 promoter and Runx2-I/Cbfb and Runx2-II/Cbfb double transgenic mice. Runx2-I transgenic mice showed less severe osteopenia and fragility than Runx2-II transgenic mice due to milder inhibition of both osteoblast maturation and transition to osteocytes, even though the former mice showed higher transgene expression. However, Runx2-I/Cbfb and Runx2-II/Cbfb double transgenic mice had enhanced inhibition of osteoblast maturation, resulting in similar severity of osteopenia and fragility, although the latter mice had less osteocytes. These findings indicate that (1) Runx2-II more strongly inhibits osteoblast maturation and transition to osteocytes than Runx2-I; (2) Cbfbeta regulates Runx2 function isoform-dependently; and (3) Runx2-I activity is highly dependent on Cbfbeta. These findings demonstrate that Runx2 isoforms exert their functions through at least partly different mechanisms and Cbfbeta regulates bone development by regulating Runx2 function isoform-dependently.     

Role of Runx genes in chondrocyte differentiation

Runx2/Cbfa1 plays a central role in skeletal development as demonstrated by the absence of osteoblasts/bone in mice with inactivated Runx2/Cbfa1 alleles. To further investigate the role of Runx2 in cartilage differentiation and to assess the potential of Runx2 to induce bone formation, we cloned chicken Runx2 and overexpressed it in chick embryos using a retroviral system. Infected chick wings showed multiple phenotypes consisting of (1) joint fusions, (2) expansion of carpal elements, and (3) shortening of skeletal elements. In contrast, bone formation was not affected. To investigate the function of Runx2/Cbfa1 during cartilage development, we have generated transgenic mice that express a dominant negative form of Runx2 in cartilage. The selective inactivation of Runx2 in chondrocytes results in a severe shortening of the limbs due to a disturbance in chondrocyte differentiation, vascular invasion, osteoclast differentiation, and periosteal bone formation. Analysis of the growth plates in transgenic mice and in chick limbs shows that Runx2 is a positive regulator of chondrocyte differentiation and vascular invasion. The results further indicate that Runx2 promotes chondrogenesis either by maintaining or by initiating early chondrocyte differentiation. Furthermore, Runx2 is essential but not sufficient to induce osteoblast differentiation. To analyze the role of runx genes in skeletal development, we performed in situ hybridization with Runx2- and Runx3-specific probes. Both genes were coexpressed in cartilaginous condensations, indicating a cooperative role in the regulation of early chondrocyte differentiation and thus explaining the expansion/maintenance of cartilage in the carpus and joints of infected chick limbs.     

Haploinsufficiency of Runx2 results in bone formation decrease and different BSP expression pattern changes in two transgenic mouse models

Runx2 has been identified as "a master gene" for the differentiation of osteoblasts and Runx2-deficient mice has demonstrated a complete absence of mature osteoblast and ossification. To further characterize the Runx2 responsive elements within the bone sialoprotein (BSP) promoter and further investigate into the role of Runx2 haploinsufficiency in osteoblast differentiation, mBSP9.0Luc mice and mBSP4.8Luc mice were crossed with Runx2-deficient mice respectively. Luciferase assay, micro CT scan, and histological analysis were performed using tissues isolated from mBSP9.0luc/Runx2+/- mice, mBSP4.8luc/Runx2+/- mice and their corresponding Runx2+/+ littermates. Alkaline phosphatase activity, mineralization assays and RT-PCR analysis using calvarial osteoblasts isolated from these transgenic mice were also performed. Luciferase assay demonstrated an early increase in luciferase expression in mBSP9.0luc/Runx2+/- mice before the expression level of luciferase dramatically decreased and turned lower than that in their control littermates in later stages. In contrast, luciferase expression in mBSP4.8luc/Runx2+/- failed to show such an early increase. Micro CT scan and histological analysis showed that BMD and trabecular bone volume were decreased and bone formation was delayed in Runx2+/- mice. Furthermore, mineralization assay and semi-quantitative RT-PCR assay demonstrated a gene-dose-dependent decrease in bone nodule formation and bone marker genes expression levels in cultured calvarial osteoblasts derived from Runx2 knockout mice. Reconstitution of Runx2-null cells with Runx2 vector partially rescued the osteoblast function defects. In conclusion, the 9.0 kb BSP promoter demonstrated a higher tissue-specific regulation of the BSP gene by Runx2 in vivo and full Runx2 gene dose is essential for osteoblast differentiation and normal bone formation.