Spry2 is a novel therapeutic target for periodontal tissue regeneration through fibroblast growth factor receptor signaling and epidermal growth factor signaling

Sprouty2 (Spry2) inhibits the activation of the extracellular signal-regulated kinase (ERK) pathway via receptor tyrosine kinase signaling. In a recent paper published in Journal of Cellular Biochemistry, we demonstrated that transfection of a dominant-negative mutant of Spry2 enhanced fibroblast growth factor (FGF)- and epidermal growth factor (EGF)-induced ERK activation in osteoblasts. In contrast, it decreased their activation in gingival epithelial cells. Consistent with these observations, the sequestration of Spry2 increased osteoblast proliferation by FGFR and EGFR stimulation, whereas it decreased gingival epithelial cell proliferation via the ubiquitination and degradation of EGF receptors (EGFR). In addition, reduction of Spry2 activity upregulated Runx2 expression and downregulated Twist, a negative regulator of Runx2 through FGFR and EGFR signaling, resulting in enhanced osteoblastogenesis in osteoblasts. Furthermore, we also found that suppression of Spry2 upregulated cell proliferation and migration in human periodontal ligament cell lines when they were stimulated by both FGF and EGF, and led to a shift in macrophage polarization, exerted immunosuppressive and tissue-repairing effects in macrophages. These results suggest that the application of a Spry2 inhibitor may effectively resolve inflammation by periodontitis and allow periodontal ligament and alveolar bone to grow and block the ingrowth of gingival epithelial cells in bony defects, biologically mimicking the barrier effect seen in conventional GTR. This approach has potential for developing a new regeneration strategy

Identification of novel amelogenin-binding proteins by proteomics analysis.

Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology. It is used in periodontal surgery to regenerate cementum, periodontal ligament, and alveolar bone. However, the precise molecular mechanisms underlying periodontal regeneration are still unclear. In this study, we investigated the proteins bound to amelogenin, which are suggested to play a pivotal role in promoting periodontal tissue regeneration. To identify new molecules that interact with amelogenin and are involved in osteoblast activation, we employed coupling affinity chromatography with proteomic analysis in fractionated SaOS-2 osteoblastic cell lysate. In SaOS-2 cells, many of the amelogenin-interacting proteins in the cytoplasm were mainly cytoskeletal proteins and several chaperone molecules of heat shock protein 70 (HSP70) family. On the other hand, the proteomic profiles of amelogenin-interacting proteins in the membrane fraction of the cell extracts were quite different from those of the cytosolic-fraction. They were mainly endoplasmic reticulum (ER)-associated proteins, with lesser quantities of mitochondrial proteins and nucleoprotein. Among the identified amelogenin-interacting proteins, we validated the biological interaction of amelogenin with glucose-regulated protein 78 (Grp78/Bip), which was identified in both cytosolic and membrane-enriched fractions. Confocal co-localization experiment strongly suggested that Grp78/Bip could be an amelogenin receptor candidate. Further biological evaluations were examined by Grp78/Bip knockdown analysis with and without amelogenin. Within the limits of the present study, the interaction of amelogenin with Grp78/Bip contributed to cell proliferation, rather than correlate with the osteogenic differentiation in SaOS-2 cells. Although the biological significance of other interactions are not yet explored, these findings suggest that the differential effects of amelogenin-derived osteoblast activation could be of potential clinical significance for understanding the cellular and molecular bases of amelogenin-induced periodontal tissue regeneration

Japanese Efforts to Promote Steel Reuse in Building Construction

This paper describes the state of the art of structural steel reuse in Japan. A significant part of the material is taken from a document titled the Architectural Institute of Japan (AIJ) Recommendations for Sustainable Steel Building Construction (Draft) available only in the Japanese language. The motivations for and potential benefits of steel reuse are examined. The affinity between seismic design requirements and steel reuse is highlighted through a concept known as damage-control design. Some technologies for disassembly are introduced. The historical development and changes in Japanese structural steel are summarized, followed by a discussion on reusability of historical steel reclaimed from existing buildings. Reuse projects whose details are known to the authors are listed. The heart of the paper is a design procedure that specifies the structural engineer's role and involvement in planning, material procurement, and executing a steel reuse project. The discussion is concluded by six directions that should be pursued to make steel reuse a widely accepted reality in Japan. Among those directions are research needs to establish a procedure to quantify the remaining structural performance of reclaimed steel considering possible exposure to earthquakes, establish connections that allow for easy disassembly, and introduce modularized structural systems. High seismicity and the general practice to adopt full moment frames pose unique challenges for steel reuse in Japan

Decitabine Inhibits Bone Resorption in Periodontitis by Upregulating Anti-Inflammatory Cytokines and Suppressing Osteoclastogenesis.

DNA methylation controls several inflammatory genes affecting bone homeostasis. Hitherto, inhibition of DNA methylation in vivo in the context of periodontitis and osteoclastogenesis has not been attempted. Ligature-induced periodontitis in C57BL/6J mice was induced by placing ligature for five days with Decitabine (5-aza-2'-deoxycytidine) (1 mg/kg/day) or vehicle treatment. We evaluated bone resorption, osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) and mRNA expression of anti-inflammatory molecules using cluster differentiation 14 positive (CD14+) monocytes from human peripheral blood. Our data showed that decitabine inhibited bone loss and osteoclast differentiation experimental periodontitis, and suppressed osteoclast CD14+ human monocytes; and conversely, that it increased bone mineralization in osteoblastic cell line MC3T3-E1 in a concentration-dependent manner. In addition to increasing IL10 (interleukin-10), TGFB (transforming growth factor beta-1) in CD14+ monocytes, decitabine upregulated KLF2 (Krüppel-like factor-2) expression. Overexpression of KLF2 protein enhanced the transcription of IL10 and TGFB. On the contrary, site-directed mutagenesis of KLF2 binding site in IL10 and TFGB abrogated luciferase activity in HEK293T cells. Decitabine reduces bone loss in a mouse model of periodontitis by inhibiting osteoclastogenesis through the upregulation of anti-inflammatory cytokines via KLF2 dependent mechanisms. DNA methyltransferase inhibitors merit further investigation as a possible novel therapy for periodontitis

Effect of Grp78/Bip knockdown on osteoblastic marker expression during amelogenin stimulation.

<p>Negative Control (NC) or Grp78/Bip (siGrp78) siRNA transfected SaOS-2 cells were seeded at a density of 3 × 10⁵ cells / 24-well plate and incubated in growth medium (GM) for 24 h. After confirming the confluence of each wells, the cells were continuously incubated in osteogenic medium (OM) with and without the addition of 30µg/mL rM180 (Am) for 48 h. Total RNA was isolated and subjected to qRT-PCR analysis for the expressions of Runx2, Osterix (Osx), ALP, Type I collagen (Col 1), Osteocalcin (OCN), and Osteopontin (OPN). Expression was normalized to both house keeping genes β-actin and GAPDH and depicted as mRNA concentration relative to Negative Control (NC) siRNA-transfected cells. Values are means ± S.D., <i>n</i> = 3. </p

Proteomic analysis of amelogenin-interacting proteins in osteoblastic cells.

<p>Purified GST-rM180 immobilized on glutathione-Sepharose 4B beads was incubated with no extract (GST-rM180), fractionated soluble protein extract (GST-rM180 + cytoplasm) (<b>A</b>) or membrane-associated protein extract (GST-rM180 + membrane) (<b>B</b>) prepared from SaOS-2 cells. GST control gels for the both extracts ware also shown to exclude the possibility to non-specific bindings (GST + cytoplasm, GST + membrane). To minimize binding of nonspecific proteins, the cell extracts were pre-cleaned with glutathione beads. The proteins bound to affinity matrices were eluted and separated by isoelectric focusing and SDS-PAGE was performed on a 7.5–15% gradient gel. A typical two-dimensional gel is illustrated. The pH gradient of the separation in the first dimension is shown on the top of the gels, and the molecular weight markers are shown in kDa on the left of the gels. Proteins were visualized with Coomassie brilliant blue staining, excised, trypsinized, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078129#pone-0078129-t002" target="_blank">Table <b>2</b></a>, 3. Magnified views of indicated areas were shown to demonstrate the analyzed spots of amelogenin-interacting proteins (Protein spots).</p

Grp78/Bip mediates cellular uptake of amelogenin.

<p>Co-localization of rM180 amelogenin and Grp78/Bip. After incubation at 4°C for 1 h, SaOS-2 cells were incubated with rM180 (30µg/mL) at 37°C for 10 min. For fluorescence microscopy, the cells were stained with amelogenin antibody (A and D; green) and Grp78/Bip antibody (B and E; red): gray is the transmission image. Nuclei were stained with Hoechst dye (blue). The co-localizatoion was illustrated in a merged image (<b>F</b>; yellow). Note that white arrowheads point to membranous localization of Grp78/Bip. Cells were visualized under the Nikon A1 fluorescence microscope using 63×/1.49 NA oil objectives. Images were obtained with the NIS-Elements AR 3.0 software, and the imaging parameters were kept constant whenever the intensity of fluorescence was to be compared. All confocal images are representatives of experiments conducted in triplicates. Scale bars: 10 µm.</p