Molecules mimicking Smad1 interacting with Hox stimulate bone formation

Bone morphogenetic proteins (BMPs) induce osteoblast differentiation and bone formation. Smads, a group of functionally and structurally related intracellular effectors, mediate signaling initiated by BMPs and regulate cell definite commitment. Previously, we showed that Smad1 activates osteopontin and osteoprotegerin gene expression by dislodging Hoxc-8 from its DNA binding sites. A domain of Smad1, termed Smad1C, was characterized as interacting with Hoxc-8 and then crippling its DNA-binding ability. Ectopic expression of Smad1C is able to bypass BMP signaling in the induction of osteoblast differentiation and bone formation in vitro. To test the function of Smad1C on osteogenesis in vivo, we generated transgenic mice in which Smad1C expression was induced with doxycycline and localized in bone by using a tetracycline-inducible expression system (Tet-on) modified with a bone-specific gene promoter, type I collagen alpha1. The mice expressing Smad1C showed increased skeletal bone mineral density compared with their littermates. Bone histomorphometric analysis of mouse tibiae showed that Smad1C significantly increases trabecular bone area and length of trabecular surface covered with osteoid and up-regulates bone marker gene (OPN, Cbfa1, Col I alpha1, BSP, ALP) expression in vivo. Moreover, stromal cells isolated from mice expressing Smad1C displayed a higher potential for differentiating into osteoblasts than the other mice. These results indicate that Smad1C mimics BMPs in the induction of osteogenesis in vivo. Most important, using a high throughput screening assay based on mimicking Smad1C's displacement of Hoxc-8 binding to DNA, we identified chemical entities that exhibit bone anabolic activity in cell and bone organ cultures, suggesting the possibility that the compounds may be used as bone anabolic agents to treat bone pathologies.     

Characterization of a bone morphogenetic protein-responsive Smad-binding element

Bone morphogenetic proteins (BMPs) are pleiotropic growth and differentiation factors belonging to the transforming growth factor-beta (TGF-beta) superfamily. Signals of the TGF-beta-like ligands are propagated to the nucleus through specific interaction of transmembrane serine/threonine kinase receptors and Smad proteins. GCCGnCGC has been suggested as a consensus binding sequence for Drosophila Mad regulated by a BMP-like ligand, Decapentaplegic. Smad1 is one of the mammalian Smads activated by BMPs. Here we show that Smad1 binds to this motif upon BMP stimulation in the presence of the common Smad, Smad4. The binding affinity is likely to be relatively low, because Smad1 binds to three copies of the motif weakly, but more repeats of the motif significantly enhance the binding. Heterologous reporter genes (GCCG-Lux) with multiple repeats of the motif respond to BMP stimulation but not to TGF-beta or activin. Mutational analyses reveal several bases critical for the responsiveness. A natural BMP-responsive reporter, pTlx-Lux, is activated by BMP receptors in P19 cells but not in mink lung cells. In contrast, GCCG-Lux responds to BMP stimulation in both cells, suggesting that it is a universal reporter that directly detects Smad phosphorylation by BMP receptors.     

Development and optimization of a cell‐based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein‐2 activity

The requirement of large amounts of the recombinant human bone morphogenetic protein‐2 (BMP‐2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP‐2. In this study, we describe the development and optimization of a cell‐based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP‐2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP‐responsive Smad1‐driven luciferase reporter gene. LIM mineralization protein‐1 (LMP‐1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP‐2. Our previous report elucidated that the binding of LMP‐1 with the WW2 domain in Smad ubiquitin regulatory factor‐1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell‐based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP‐1, and its mutant (LMP‐1ΔSmurf1) that lacks the Smurf1‐WW2 domain‐binding motif. Both the wild type and the mutant proteins were engineered to contain an 11‐amino acid HIV‐TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell‐based reporter assay confirmed that LMP‐1 potentiates the BMP‐induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP‐1 was significantly reduced when a specific‐motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and reliability of our cell‐based assay. Direct delivery of synthesized protein can be limited by high cost, instability or inadequate post‐translational modifications. Thus, there would be a clear benefit for a low cost, cell penetrable chemical compound. We successfully used our gene expression‐based assay to choose an active compound from a select group of compounds that were identified by computational screenings as the most likely candidates for mimicking the function of LMP‐1. Among them, we selected SVAK‐3, a compound that showed a dose‐dependent potentiation of BMP‐2 activity in inducing osteoblastic differentiation of C2C12 cells. We show that either the full length LMP‐1 protein or its potential mimetic compound consistently exhibit similar potentiation of BMP‐2 activity even when multiple markers of the osteoblastic phenotype were parallely monitored. Published in 2009 by John Wiley & Sons, Ltd.     

Tumor Necrosis Factor �� Represses Bone Morphogenetic Protein (BMP) Signaling by Interfering with the DNA Binding of Smads through the Activation of NF-��B

Bone morphogenetic proteins (BMPs) induce not only bone formation in vivo but also osteoblast differentiation of mesenchymal cells in vitro. Tumor necrosis factor α (TNFα) inhibits both osteoblast differentiation and bone formation induced by BMPs. However, the molecular mechanisms of these inhibitions remain unknown. In this study, we found that TNFα inhibited the alkaline phosphatase activity and markedly reduced BMP2- and Smad-induced reporter activity in MC3T3-E1 cells. TNFα had no effect on the phosphorylation of Smad1, Smad5, and Smad8 or on the nuclear translocation of the Smad1-Smad4 complex. In p65-deficient mouse embryonic fibroblasts, overexpression of p65, a subunit of NF-κB, inhibited BMP2- and Smad-induced reporter activity in a dose-dependent manner. Furthermore, this p65-mediated inhibition of BMP2- and Smad-responsive promoter activity was restored after inhibition of NF-κB by the overexpression of the dominant negative IκBα. Although TNFα failed to affect receptor-dependent formation of the Smad1-Smad4 complex, p65 associated with the complex. Chromatin immunoprecipitation and electrophoresis mobility shift assays revealed that TNFα suppressed the DNA binding of Smad proteins to the target gene. Importantly, the specific NF-κB inhibitor, BAY11-7082, abolished these phenomena. These results suggest that TNFα inhibits BMP signaling by interfering with the DNA binding of Smads through the activation of NF-κB.