Zoledronic Acid Enhances Lipopolysaccharide-Stimulated Proinflammatory Reactions through Controlled Expression of SOCS1 in Macrophages

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious side effect of nitrogen-containing bisphosphonate (NBP) use. Many studies have shown that BRONJ is limited to the jawbone and does not occur in the other bones. We hypothesized that BRONJ is related to local bacterial iections and involves the innate immune system. To examine the relationship between BRONJ and innate immunity, we examined the effects of NBPs on macrophages, one of the important cell types in innate immunity. The expression of toll-like receptor-4 (TLR4) in cells after pretreatment with zoledronic acid (ZOL) did not considerably differ from that in untreated control cells. However, cytokine levels and nitric oxide (NO) production increased after pretreatment with ZOL. Furthermore, ZOL induced NF-κB activation by enhancing IκB-α degradation. Lipopolysaccharide (LPS)-induced apoptosis also increased after pretreatment with ZOL. This effect was mediated by a reduction of suppressor of cytokine signaling-1 (SOCS1), which is a negative regulator of myeloid differentiation primary response gene 88 (MyD 88)-dependent signaling. These results suggest that ZOL induced excessive innate immune response and proinflammatory cytokine production and that these processes may be involved in the bone destruction observed in BRONJ

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

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

Effects of ZOL on LPS-induced cell apoptosis.

<p>RAW264.7 cells were incubated for the indicated times (0, 24, 48, or 72 h) with control medium, LPS (100 ng/mL), ZOL (10 µM), or ZOL+LPS. annexin V and PI were added to the cultures prior to flow cytometry. See the methods for a detailed explanation of the contour plots. (B) Plot of apoptosis in (▴) controls, (•) LPS-treated, (□) ZOL-treated, and (▪) ZOL+LPS-treated cells. After stimulation with LPS, apoptosis increased compared to that in unstimulated controls. A similar amount of LPS-stimulated apoptosis was observed after ZOL pretreatment, and a significant increase in apoptosis was observed after further LPS stimulation. Significant differences from the cikb ontrols that were not treated with ZOL are indicated by an asterisk (*<i>P</i><0.01).</p

Effects of ZOL on LPS-induced TLR4-mediated activation of NF-κB cytokines.

<p>(A) RAW264.7 cells were cultured with or without ZOL (10 µM) for 24 h and then with LPS (100 ng/mL) for the indicated times (0, 15, 30, or 60 min). The levels of phospho-IκB-α, IκB-α, and ERK2 were analyzed by western blotting. These data indicated that ZOL treatment increased the levels of phosphorylated IκB-α and enhanced the degradation of IκB-α. (B) RAW264.7 cells were cultured with ZOL for the indicated times (0, 1, 4, 8, or 12 h). STAT1 protein levels decreased over time after ZOL treatment. The levels of Phospho-STAT1 increased 1 h after ZOL treatment and then decreased in a time-dependent manner. The levels of SOCS1, which suppresses TLR4 signaling, increased 1 h after ZOL treatment and then decreased in a time-dependent manner. The levels of MyD88 protein increased with time after ZOL treatment.</p

Effects of ZOL on NO release from LPS-stimulated macrophages.

<p>RAW264.7 cells were cultured with or without ZOL for 24 h and then with LPS for an additional 0, 1, 2, 4, or 6 h. After treatment, iNOS expression was examined by real-time PCR. LPS-stimulated production of iNOS from RAW264.7 cells increased significantly after ZOL pretreatment. Significant differences from the negative controls that were not treated with ZOL are indicated by an asterisk (*<i>P</i><0.05). (B) RAW264.7 cells were cultured with or without ZOL (10 µM) and LPS (100 ng/mL) for 24 h. NO release was measured by the Griess method. NO production was significantly higher in ZOL-pretreated cells than in LPS-treated positive controls. ZOL had no effect on the release of NO. Significant differences from the negative controls that were not treated with ZOL are indicated by an asterisk (*<i>P</i><0.05).</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