Bone loss induced by Runx2 Over‐expression in mice is blunted by osteoblastic over‐expression of TIMP‐1

The Runx2 gene is essential for osteoblast differentiation and function. In vivo over‐expression of Runx2 in osteoblasts increases bone resorption, and blocks terminal osteoblast differentiation. Several lines of evidence suggest that osteoblastic matrix metalloproteinases (MMPs) could contribute to the increased bone resorption observed in mice over‐expressing Runx2 (Runx2 mice). The goal of our study was to use a transgenic approach to find out whether the inhibition of osteoblastic MMPs can reduce the bone loss induced by the over‐expression of Runx2. We analyzed the effect of the in vivo over‐expression of the TIMP‐1 in osteoblasts on the severe osteopenic phenotype in Runx2 mice. Females with the different genotypes (WT, Runx2, TIMP‐1 and TIMP‐1/Runx2) were analyzed for bone density, architecture, osteoblastic and osteoclastic activity and gene expression using qPCR. TIMP‐1 over‐expression reduces the bone loss in adult Runx2 mice. The prevention of the bone loss in TIMP‐1/Runx2 mice was due to a combination of reduced bone resorption and sustained bone formation. We present evidence that the ability of osteoblastic cells to induce osteoclastic differentiation is lower in TIMP‐1/Runx2 mice than in Runx2 mice, probably due to a reduction in the expression of RANK‐L and of the Runx2 transgene. Osteoblast primary cells from TIMP‐1/Runx2 mice, but not from Runx2 mice, were able to differentiate into fully mature osteoblasts producing high osteocalcin levels. In conclusion, our findings suggest that osteoblastic MMPs can affect osteoblast differentiation. Our work also indicates that osteoblastic MMPs are partly responsible for the bone loss observed in Runx2 transgenic mice. J. Cell. Physiol. 222:219–229, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

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.     

GATA4 negatively regulates osteoblast differentiation by downregulation of Runx2

Osteoblasts are specialized mesenchymal cells that are responsible for bone formation. In this study, we examine the role of GATA4 in osteoblast differentiation. GATA4 was abundantly expressed in preosteoblast cells and gradually down-regulated during osteoblast differentiation. Overexpression of GATA4 in osteoblastic cells inhibited alkaline phosphatase activity and nodule formation in osteogenic conditioned cell culture system. In addition, overexpression of GATA4 attenuated expression of osteogenic marker genes, including Runx2, alkaline phosphatase, bone sialoprotein, and osteocalcin, all of which are important for osteoblast differentiation and function. Overexpression of GATA4 attenuated Runx2 promoter activity, whereas silencing of GATA4 increased Runx2 induction. We found that GATA4 interacted with Dlx5 and subsequently decreased Dlx5 binding activity to Runx2 promoter region. Our data suggest that GATA4 acts as a negative regulator in osteoblast differentiation by downregulation of Runx2. [BMB Reports 2014; 47(8): 463-468]     

Adenovirus-mediated transfer of siRNA against Runx2/Cbfa1 inhibits the formation of heterotopic ossification in animal model

The heterotopic ossification of muscles, tendons, and ligaments is a common problem faced by orthopaedic surgeons. Runx2/Cbfa1 plays an essential role during the osteoblast differentiation and is considered as a molecular switch in osteoblast biology. RNA interference technology is a powerful tool for silencing endogenous or exogenous genes in mammalian cells. In this study, we investigated the effect of Runx2/Cbfa1-specific siRNA on osteoblast differentiation and mineralization in osteoblastic cells, and then constructed adenovirus containing siRNA against Runx2/Cbfa1 (Ad-Runx2-siRNA) to inhibit the formation of heterotopic ossification induced by BMP4, demineralized bone matrix, and trauma in animal model. Our results showed that the Runx2/Cbfa1-specific siRNA could inhibit the expression of Runx2/Cbfa1 at the level of mRNA and protein. Analysis of the expression of osteoblast maturation genes including type I collagen, osteopontin, bone sialoprotein, and osteocalcin, alkaline phosphatase activity, and matrix mineralization (von kossa) revealed that osteoblast differentiation was inhibited in cultured primary mouse osteoblasts transduced with Ad-Runx2-siRNA. Furthermore, adenovirus-mediated transfer of siRNA against Runx2/Cbfa1 could inhibit the formation of heterotopic ossification induced by BMP4, demineralized bone matrix, and trauma in animal model. It is likely that the inhibition of Runx2/Cbfa1 by RNAi could be developed as a powerful approach to prevent or treat heterotopic ossification.     

Selective Runx2-II deficiency leads to low-turnover osteopenia in adult mice

Runx2 transcribes Runx2-II and Runx2-I isoforms with distinct N-termini. Deletion of both isoforms results in complete arrest of bone development, whereas selective loss of Runx2-II is sufficient to form a grossly intact skeleton with impaired endochondral bone development. To elucidate the role of Runx2-II in osteoblast function in adult mice, we examined heterozygous Runx2-II (Runx2-II(+/-)) and homozygous Runx2-II (Runx2-II(-/-))-deficient mice, which, respectively, lack one or both copies of Runx2-II but intact Runx2-I expression. Compared to wild-type mice, 6-week-old Runx2-II(+/-) had reduced trabecular bone volume (BV/TV%), cortical thickness (Ct.Th), and bone mineral density (BMD), decreased osteoblastic and osteoclastic markers, lower bone formation rates, impaired osteoblast maturation of BMSCs in vitro, and significant reductions in mechanical properties. Homozygous Runx2-II(-/-) mice had a more severe reduction in BMD, BV/TV%, and Ct.Th, and greater suppression of osteoblastic and osteoclastic markers than Runx2-II(+/-) mice. Non-selective Runx2(+/-) mice, which have an equivalent reduction in Runx2 expression due to the lack one copy of Runx2-I and II, however, had an intermediate reduction in BMD. Thus, selective Runx2-II mutation causes diminished osteoblastic function in an adult mouse leading to low-turnover osteopenia and suggest that Runx2-I and II have distinct functions imparted by their different N-termini.