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.     

Requirement for ErbB2/ErbB signaling in developing cartilage and bone

During endochondral ossification, the skeletal elements of vertebrate limbs form and elongate via coordinated control of chondrocyte and osteoblast differentiation and proliferation. The role of signaling by the ErbB family of receptor tyrosine kinases, which consists of ErbB1 (epidermal growth factor receptor or EGFR), ErbB2, ErbB3 and ErbB4, has been little studied during cartilage and bone development. Signaling by the ErbB network generates a diverse array of cellular responses via formation of ErbB dimers activated by distinct ligands that produce distinct signal outputs. Herstatin is a soluble ErbB2 receptor that acts in a dominant negative fashion to inhibit ErbB signaling by binding to endogenous ErbB receptors, preventing functional dimer formation. Here, we examine the effects of Herstatin on limb skeletal element development in transgenic mice, achieved via Prx1 promoter-driven expression in limb cartilage and bone. The limb skeletal elements of Prx1-Herstatin embryos are shortened, and chondrocyte maturation and osteoblast differentiation are delayed. In addition, proliferation by chondrocytes and periosteal cells of Prx1-Herstatin limb skeletal elements is markedly reduced. Our study identifies requirements for ErbB signaling in the maintenance of chondrocyte and osteoblast proliferation involved in the timely progression of chondrocyte maturation and periosteal osteoblast differentiation.     

Induction of osteoclast differentiation by Runx2 through receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin regulation and partial rescue of osteoclastogenesis in Runx2-/- mice by RANKL transgene

Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), and macrophage-colony stimulating factor play essential roles in the regulation of osteoclastogenesis. Runx2-deficient (Runx2-/-) mice showed a complete lack of bone formation because of maturational arrest of osteoblasts and disturbed chondrocyte maturation. Further, osteoclasts were absent in these mice, in which OPG and macrophage-colony stimulating factor were normally expressed, but RANKL expression was severely diminished. We investigated the function of Runx2 in osteoclast differentiation. A Runx2-/- calvaria-derived cell line (CA120-4), which expressed OPG strongly but RANKL barely, severely suppressed osteoclast differentiation from normal bone marrow cells in co-cultures. Adenoviral introduction of Runx2 into CA120-4 cells induced RANKL expression, suppressed OPG expression, and restored osteoclast differentiation from normal bone marrow cells, whereas the addition of OPG abolished the osteoclast differentiation induced by Runx2. Addition of soluble RANKL (sRANKL) also restored osteoclast differentiation in co-cultures. Forced expression of sRANKL in Runx2-/- livers increased the number and size of osteoclast-like cells around calcified cartilage, although vascular invasion into the cartilage was superficial because of incomplete osteoclast differentiation. These findings indicate that Runx2 promotes osteoclast differentiation by inducing RANKL and inhibiting OPG. As the introduction of sRANKL was insufficient for osteoclast differentiation in Runx2-/- mice, however, our findings also suggest that additional factor(s) or matrix protein(s), which are induced in terminally differentiated chondrocytes or osteoblasts by Runx2, are required for osteoclastogenesis in early skeletal development.