Dual usage of a stage-specific fluorescent reporter system based on a helper-dependent adenoviral vector to visualize osteogenic differentiation.

We developed a reporter system that can be used in a dual manner in visualizing mature osteoblast formation. The system is based on a helper-dependent adenoviral vector (HDAdV), in which a fluorescent protein, Venus, is expressed under the control of the 19-kb human osteocalcin (OC) genomic locus. By infecting human and murine primary osteoblast (POB) cultures with this reporter vector, the cells forming bone-like nodules were specifically visualized by the reporter. In addition, the same vector was utilized to efficiently knock-in the reporter into the endogenous OC gene of human induced pluripotent stem cells (iPSCs), by homologous recombination. Neural crest-like cells (NCLCs) derived from the knock-in reporter iPSCs were differentiated into osteoblasts forming bone-like nodules and could be visualized by the expression of the fluorescent reporter. Living mature osteoblasts were then isolated from the murine mixed POB culture by fluorescence-activated cell sorting (FACS), and their mRNA expression profile was analyzed. Our study presents unique utility of reporter HDAdVs in stem cell biology and related applications.

In Vitro Study of the Effects of Denosumab on Giant Cell Tumor of Bone: Comparison with Zoledronic Acid.

Giant cell tumor of bone (GCTB) is a locally aggressive primary bone tumor that contains numerous osteoclasts formed from marrow-derived precursors through receptor activator of nuclear factor κ-B ligand (RANKL), an osteoclast differentiation factor expressed in neoplastic cells of GCTB. Denosumab, a fully human monoclonal antibody targeting RANKL, has recently been used for the treatment of GCTB, and superior treatment effects have been reported. The aim of this work was to elucidate the mechanism of action of denosumab, and the differences between denosumab and zoledronic acid at the level of GCTB cells. We isolated GCTB cells from 3 patients and separated them into osteoclasts, osteoclast precursors and proliferating spindle-shaped stromal cells (the true neoplastic component), and examined the action of denosumab on differentiation, survival and bone resorption activity of osteoclasts. Denosumab and zoledronic acid inhibited osteoclast differentiation from mononuclear cells containing osteoclast precursors. Zoledronic acid inhibited osteoclast survival, whereas an inhibitory effect of denosumab on osteoclast survival was not observed. The inhibitory effect on bone resorption by both agents was confirmed in culture on dentin slices. Furthermore, zoledronic acid showed dose-dependent inhibition of cell growth of neoplastic cells whereas denosumab had no inhibitory effect on these cells. Denosumab has an inhibitory effect on osteoclast differentiation, but no inhibitory effects on survival of osteoclasts or growth of neoplastic cells in GCTBs.

Mutant activin-like kinase 2 in fibrodysplasia ossificans progressiva are activated via T203 by BMP type II receptors.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disorder characterized by progressive heterotopic ossification in soft tissues, such as the skeletal muscles. FOP has been shown to be caused by gain-of-function mutations in activin receptor-like kinase (ALK)-2, which is a type I receptor for bone morphogenetic proteins (BMPs). In the present study, we examined the molecular mechanisms that underlie the activation of intracellular signaling by mutant ALK2. Mutant ALK2 from FOP patients enhanced the activation of intracellular signaling by type II BMP receptors, such as BMPR-II and activin receptor, type II B, whereas that from heart disease patients did not. This enhancement was dependent on the kinase activity of the type II receptors. Substitution mutations at all nine serine and threonine residues in the ALK2 glycine- and serine-rich domain simultaneously inhibited this enhancement by the type II receptors. Of the nine serine and threonine residues in ALK2, T203 was found to be critical for the enhancement by type II receptors. The T203 residue was conserved in all of the BMP type I receptors, and these residues were essential for intracellular signal transduction in response to ligand stimulation. The phosphorylation levels of the mutant ALK2 related to FOP were higher than those of wild-type ALK2 and were further increased by the presence of type II receptors. The phosphorylation levels of ALK2 were greatly reduced in mutants carrying a mutation at T203, even in the presence of type II receptors. These findings suggest that the mutant ALK2 related to FOP is enhanced by BMP type II receptors via the T203-regulated phosphorylation of ALK2.

Establishment of a novel model of chondrogenesis using murine embryonic stem cells carrying fibrodysplasia ossificans progressiva-associated mutant ALK2.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disorder characterized by heterotopic endochondral ossification in soft tissue. A mutation in the bone morphogenetic protein (BMP) receptor ALK2, R206H, has been identified in patients with typical FOP. In the present study, we established murine embryonic stem (ES) cells that express wild-type human ALK2 or typical mutant human ALK2 [ALK2(R206H)] under the control of the Tet-Off system. Although wild-type ALK2 and mutant ALK2(R206H) were expressed in response to a withdrawal of doxycycline (Dox), BMP signaling was activated only in the mutant ALK2(R206H)-expressing cells without the addition of exogenous BMPs. The Dox-dependent induction of BMP signaling was blocked by a specific kinase inhibitor of the BMP receptor. The mutant ALK2(R206H)-carrying cells showed Dox-regulated chondrogenesis in vitro, which occurred in co-operation with transforming growth factor-ß1 (TGF-ß1). Overall, our ES cells are useful for studying the molecular mechanisms of heterotopic ossification in FOP in vitro and for developing novel inhibitors of chondrogenesis induced by mutant ALK2(R206H) associated with FOP.

Identification of a novel bone morphogenetic protein (BMP)-inducible transcript, BMP-inducible transcript-1, by utilizing the conserved BMP-responsive elements in the Id genes.

Bone morphogenetic proteins (BMPs) inhibit myogenesis and induce osteoblastic differentiation in myoblasts. They also induce the transcription of several common genes, such as Id1, Id2 and Id3, in various cell types. We have reported that a GC-rich element in the Id1 gene functions as a BMP-responsive element (BRE) that is regulated by Smads. In this study, we analyzed and identified BREs in the 5'-flanking regions of the mouse Id2 and Id3 genes. The core GGCGCC sequence was conserved among the BREs in the Id1, Id2 and Id3 genes and was essential for the response to BMP signaling via Smads. We found a novel BRE on mouse chromosome 13 at position 47,723,740-47,723,768 by searching for conserved sequences containing the Id1 BRE. This potential BRE was found in the 5'-flanking region of a novel gene that produces a non-coding transcript, termed BMP-inducible transcript-1 (BIT-1), and this element regulated the expression of this gene in response to BMP signaling. We found that BIT-1 is expressed in BMP target tissues such as the testis, brain, kidney and cartilage. These findings suggest that the transcriptional induction of the Ids, BIT-1 and additional novel genes containing the conserved BRE sequence may play an important role in the regulation of the differentiation and/or function of target cells in response to BMPs.

R848, a toll-like receptor 7 agonist, inhibits osteoclast differentiation but not survival or bone-resorbing function of mature osteoclasts.

R848, also known as resiquimod, acts as a ligand for toll-like receptor 7 (TLR7) and activates immune cells. In this study, we examined the effects of R848 on differentiation, survival, and bone-resorbing function of osteoclasts. R848 inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) and human peripheral blood-derived monocytes induced by receptor activator of NF-κB ligand in a dose-dependent manner. In addition, it inhibited mouse osteoclast differentiation induced in cocultures of bone marrow cells and osteoblasts in the presence of dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, R848 did not affect the survival or bone-resorbing activity of mouse mature osteoclasts. R848 also upregulated the mRNA expression levels of interleukin (IL)-6, IL-12, interferon (IFN)-γ, and inducible nitric oxide synthase in mouse BMMs expressing TLR7. IFN-ß was consistently expressed in the BMMs and addition of neutralizing antibodies against IFN-ß to the cultures partially recovered osteoclast differentiation inhibited by R848. These results suggest that R848 targets osteoclast precursors and inhibits their differentiation into osteoclasts via TLR7.