Loss of function of the Wnt co-receptor, lipoprotein receptorrelated protein 5, decreases bone formation, and a point mutation in this gene results in high bone mass, indicating the importance of this signaling pathway in bone formation. However, the exact mechanism is currently unknown. We examined a potential role for Wnt signaling and functional cross-talk of bone morphogenetic protein 2 (BMP-2) in osteoblast differentiation. To assess the contribution of Wnt, we generated C2C12 cells over-expressing Wnt3a or Wnt5a and treated these with BMP-2. We showed that expression of matrix extracellular phosphoglycoprotein was induced by BMP-2 in Wnt3a over-expressing C2C12 cells but not in Wnt5a over-expressing C2C12 cells. Over-expression of Wnt3a blocked BMP-2-induced inhibition of myotube formation in C2C12 cells when switched to low mitogen medium. In these cultures, expression of inhibitor of DNA binding/differentiation (Id) 1, a helix-loop-helix protein induced by BMP-2, decreased in stable Wnt3a-but not in Wnt5a-expressing cells. This suppression is mediated by a GC-rich region of the BMP-2-responsive element of the Id1 gene promoter, and interaction between Smad1/4 and ␤-catenin is crucial for Wntmediated suppression of the BMP-2 response in C2C12 cells. Overexpression of the inhibitor of canonical Wnt signaling, Dickkopf, inhibits this suppression. In contrast, BMP-2 or Smad1/4 up-regulated Wnt3a or activated ␤-catenin-induced lymphoid-enhancing factor 1/T cell factor-dependent transcriptional activity. These findings identify functional cross-talk of Id1 expression between Wnt and BMP signaling and demonstrate a novel mechanism for Wnt regulation of the BMP-2 response, linking Id1 expression to Wnt/␤-catenin signaling.

Wnt/β-catenin signaling plays an important role in the developing skeletal system. Our previous studies demonstrated that Wnt/β-catenin signaling inhibits the ability of bone morphogenetic protein (BMP)-2 to suppress myotube formation in the multipotent mesenchymal cell line C2C12 and that this inhibition is mediated by Id1. In this study, we examined the role of intracellular signaling by Wnt/β-catenin and BMP-2 in regulating the expression of osteoprotegerin (OPG) and of the receptor activator of NFκB ligand (RANKL). OPG expression was induced by Wnt/ β-catenin signaling in C2C12 cells and osteoblastic MC3T3-E1 cells. Silencing of glycogen synthase kinase-3β also increased OPG expression. In contrast, R expression was suppressed by Wnt/β-catenin signaling. In a transfection assay, β-catenin induced the activity of a reporter gene, a 1.5 kb fragment of the 5′-flanking region of the OPG gene. Deletion and mutation analysis revealed that Wnt/β-catenin signaling regulates transcription of OPG via a promoter region containing two Wnt/β-catenin responsive sites. BMP-2 enhanced Wnt/β-catenin-dependent transcriptional activation of the OPG promoter. In response to BMP-2 stimulation, Smad 1 and 4 interacted with Wnt/β-catenin responsive sites. These results show that the regulation of OPG expression is mediated through two transcription pathways that involve the OPG promoter.

We have been developing a unique system for the downregulation of a gene expression through cutting a specific mRNA by the long form of tRNA 3 0 -processing endoribonuclease (tRNase Z L ) under the direction of small-guide RNA (sgRNA). However, the efficacy of this system and the involvement of tRNase Z L in the living cells were not clear. Here we show, by targeting the exogenous luciferase gene, that the efficacy of the sgRNA/tRNase Z L method can become comparable to that of the RNA interference technology and that the gene silencing is owing to tRNase Z L directed by sgRNA not owing to a simple antisense effect. We also show that tRNase Z L together with sgRNA can downregulate expression of the endogenous human genes Bcl-2 and glycogen synthase kinase-3b by degrading their mRNAs in cell culture. Furthermore, we demonstrate that a gene expression in the livers of postnatal mice can be inhibited by an only seven-nucleotide sgRNA. These data suggest that sgRNA might be utilized as therapeutic agents to treat diseases such as cancers and AIDS.

ObjectiveThe periodontal ligament (PDL) is a fibrous connective tissue composed of heterogeneous cell types, including PDL fibroblasts. It is not clear whether cells within the PDL fibroblast population retain the potency to differentiate into other cell types. DesignIn the present study, clonal cell lines, derived from Clawn miniature swine PDLs, were established by gene transfection for a human telomerase reverse transcriptase, and characterized. ResultsThese cell lines, denoted TesPDL1-4, had PDL fibroblasts that showed fibroblastic morphology and expressed procollagen α1(I), osteopontin, periostin and alkaline phosphatase mRNA. Under the specific culture conditions, TesPDL3 cells also have the ability to express CD31, vascular endothelial cadherin, von Willebrand factor, osteocalcin, and to form extracellular mineralized nodules. ConclusionsOur data indicate that TesPDL3 cells have unique properties of expressing several phenotype of fibroblasts, vascular endothelial cells and osteoblasts in cultures.2