Urara Tanaka

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.

Mutation of Spry2 Induces Proliferation and Differentiation of Osteoblasts but Inhibits Proliferation of Gingival Epithelial Cells

Sprouty was identified as an inhibitor of the fibroblast growth factor (FGF) receptor, and Sprouty2 (Spry2) functions as a negative regulator of receptor tyrosine kinase signaling. In this study, we investigated how inhibition of Spry2 affects osteoblasts and gingival epithelial cells in periodontal tissue regeneration in vitro. Transduction of a dominant‐negative mutant of Spry2 (Y55A‐Spry2) enhanced basic fibroblast growth factor (bFGF)‐ and epidermal growth factor (EGF)‐induced ERK activation in MC3T3‐E1 osteoblastic cells. In contrast, it decreased their activation in GE1 cells. Consistent with these observations, Y55A‐Spry2 increased osteoblast proliferation with bFGF and EGF stimulation, whereas the proliferation of Y55A‐Spry2‐introduced GE1 cells was decreased via the ubiquitination and degradation of EGF receptors (EGFRs). In addition, Y55A‐Spry2 caused upregulation of Runx2 expression and downregulation of Twist, a negative regulator of Runx2, with treatment of bFGF and EGF, resulting in enhanced osteoblastogenesis accompanied by alkaline phosphatase activation and osteocalcin expression in MC3T3‐E1 cells. These data suggest that suppression of Spry2 expression induces proliferation and differentiation of osteoblastic cells after the addition of a bFGF and EGF cocktail but inhibits proliferation in gingival epithelial cells. These in vitro experiments may provide a molecular basis for novel therapeutic approaches in periodontal tissue regeneration. Taken together, our study proposes that combined application of an inhibitor for tyrosine 55 of Spry2, bFGF, and EGF may effectively allow alveolar bone growth and block the ingrowth of gingival epithelial cells toward bony defects, biologically mimicking a barrier effect in guided tissue regeneration, with in vivo investigation in the future. J. Cell. Biochem. 116: 628–639, 2015.

Grp78 Is Critical for Amelogenin-Induced Cell Migration in a Multipotent Clonal Human Periodontal Ligament Cell Line

Periodontal ligament stem cells (PDLSCs) are known to play a pivotal role in regenerating the periodontium. Amelogenin, which belongs to a family of extracellular matrix (ECM) proteins, is a potential bioactive molecule for periodontal regenerative therapy. However, its downstream target molecules and/or signaling patterns are still unknown. Our recent proteomic study identified glucose‐regulated protein 78 (Grp78) as a new amelogenin‐binding protein. In this study, we demonstrate, for the first time, the cellular responses induced by the biological interaction between amelogenin and Grp78 in the human undifferentiated PDL cell line 1‐17, which possesses the most typical characteristics of PDLSCs. Confocal co‐localization experiments revealed the internalization of recombinant amelogenin (rM180) via binding to cell surface Grp78, and the endocytosis was inhibited by the silencing of Grp78 in 1‐17 cells. Microarray analysis indicated that rM180 and Grp78 regulate the expression profiles of cell migration‐associated genes in 1‐17 cells. Moreover, Grp78 overexpression enhanced rM180‐induced cell migration and adhesion without affecting cell proliferation, while silencing of Grp78 diminished these activities. Finally, binding of rM180 to Grp78 promoted the formation of lamellipodia, and the simultaneous activation of Rac1 was also demonstrated by NSC23766, a widely accepted Rac1 inhibitor. These results suggest that Grp78 is essential for enhancing amelogenin‐induced migration in 1‐17 cells. The biological interaction of amelogenin with Grp78 offers significant therapeutic potential for understanding the biological components and specific functions involved in the signal transduction of amelogenin‐induced periodontal tissue regeneration. J. Cell. Physiol. 231: 414–427, 2016.

Inhibition of Sprouty2 polarizes macrophages toward an M2 phenotype by stimulation with interferon γ and Porphyromonas gingivalis lipopolysaccharide

Periodontitis is a chronic inflammatory disorder caused by specific bacteria residing in the biofilm, particularly Porphyromonas gingivalis (Pg). Sprouty2 (Spry2) functions as a negative regulator of the fibroblast growth factor (FGF) signaling pathway. We previously demonstrated that sequestration of Spry2 induced proliferation and osteogenesis in osteoblastic cells by basic FGF (bFGF) and epidermal growth factor (EGF) stimulation in vitro, but diminished cell proliferation in gingival epithelial cells. In addition, Spry2 knockdown in combination with bFGF and EGF stimulation increases periodontal ligament cell proliferation and migration accompanied by prevention of osteoblastic differentiation. In this study, we investigated the mechanisms through which Spry2 depletion by interferon (IFN) γ and Pg lipopolysaccharide (LPS) stimulation affected the physiology of macrophages in vitro. Transfection of macrophages with Spry2 small‐interfering RNA (siRNA) promoted the expression of genes characteristic of M2 alternative activated macrophages, induced interleukin (IL)‐10 expression, and enhanced arginase activity, even in cells stimulated with IFNγ and Pg LPS. In addition, we found that phosphoinositide 3‐kinase (PI3K) and AKT activation by Spry2 downregulation enhanced efferocytosis of apoptotic cells by increasing Rac1 activation and decreasing nuclear factor kappa B (NFκB) p65 phosphorylation but not signal transducer and activator of transcription 1 (STAT1) phosphorylation. Collectively, our results suggested that topical administration of Spry2 inhibitors may efficiently resolve inflammation in periodontal disease as macrophage‐based anti‐inflammatory immunotherapy and may create a suitable environment for periodontal wound healing. These in vitro findings provide a molecular basis for new therapeutic approaches in periodontal tissue regeneration.

Sprouty2 inhibition promotes proliferation and migration of periodontal ligament cells

OBJECTIVES We previously demonstrated that a dominant-negative Sprouty2 (Spry2) mutation promotes osteoblast proliferation and differentiation after basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulation, whereas it diminishes proliferation of gingival epithelial cells, thereby inducing favourable conditions for periodontal tissue regeneration. In this study, we investigated how Spry2 inhibition affects the cellular physiology of periodontal ligament (PDL) cells. METHODS A total of 1-17 PDL cells (multipotent clonal human PDL cell line) were stimulated with bFGF and EGF after transfection of Spry2 siRNA. Cell proliferation, migration, ALP staining, real-time PCR, Western blot and immunofluorescence assays were performed. RESULTS ERK1/2 activation and proliferation of 1-17 PDL cells were significantly upregulated by the addition of Spry2 siRNA in the presence of bFGF and EGF. In addition, Spry2 siRNA reduced transcription of osteogenesis-related genes and ALP staining relative to control cells. Furthermore, it increased AKT/phosphatidylinositol 3-kinase (PI3K) phosphorylation; consequently, Rac1 but not Cdc42 was activated, thereby promoting lamellipodia formation, cell proliferation and migration after stimulation by bFGF and EGF. CONCLUSION Spry2 combined with bFGF and EGF stimulation reduced PDL cell migration and proliferation with inducing osteoblastic differentiation. These in vitro findings may provide a molecular basis for novel therapeutic approaches for establishing periodontal tissue regeneration.

Glucose-Regulated Protein 78: A Novel Therapeutic Target for Amelogenin-Induced Periodontal Tissue Regeneration

Amelogenin, the major component of a commercial enamel matrix derivative (Straumann® Emdogain), is commonly employed in periodontology. It is mainly used in periodontal surgery to stimulate the regeneration of periodontal tissues, including the cementum, periodontal ligament, and alveolar bone. However, the precise molecular mechanisms underlying amelogenin-induced regeneration have not yet been elucidated. Thus, to gain further insight into how amelogenin induces periodontal tissue regeneration, we performed a protein interaction screen using recombinant full-length amelogenin (rM180) as bait. Coupling affinity chromatography with proteomic analysis identified glucose-regulated protein 78 (Grp78) as a new amelogenin-binding protein. We further demonstrated that the interaction between amelogenin and Grp78 not only contributes to cell proliferation in osteoblastic cells but also enhances cell migration in periodontal ligament stem/progenitor cells. Although the potentiation effects of Grp78 should be further examined in vivo, our findings indicate the significant therapeutic potential for amelogenin-induced periodontal tissue regeneration.

Adhesion attenuates respiratory burst induced by different modes of triggering in resting or LPS-primed neutrophils

The effects of adherence on neutrophil superoxide anion (O2-) generation triggered by surface, soluble ligand, or adherence were studied. Resting-neutrophils adhered to the uncoated tubes resulting in O2- generation, but not on plasma-, fibrinogen-, vitronectin-, fibronectin-, laminin-, collagen-, or poly HEMA-coated surfaces. Enhanced N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated O2- generation by LPS-primed-neutrophils was induced by the incubation on plasma, fibrinogen, vitronectin, fibronectin, or laminin in the absence of Mg2+. In the presence of Mg2+, this response was observed in cells on collagen or poly HEMA. LPS-primed-neutrophils adhered to uncoated, BSA- or IgG-coated tubes and did not respond to fMLP, indicating that the fMLP-response of LPS-primed-neutrophils was suppressed by adherence. Upon incubation on plasma, fibrinogen, vitronectin, fibronectin in the presence of Mg2+, LPS-primed-neutrophils showed O2- generation. Upon incubation on collagen or poly HEMA, the primed-neutrophils neither generated O2- nor adhered. We found that O2- response of LPS-primed-neutrophils was attenuated depending on the time of exposure to plasma-coated surface. This attenuation was evident on plasma or fibrinogen, but not on collagen in the presence of Mg2+, indicating that O2- generation by LPS-primed-neutrophils was attenuated dependent on adherence but not on Mg2+. Thus, adhesion attenuated the O2- generation triggered by both soluble (fMLP) and insoluble (surface) stimuli.

New Therapeutic Strategy for Regenerating Periodontal Tissue Based on the Combination of Amelogenin and Reapplications of Existing Grp78 Inducer

The present study aims to develop a highly safe regenerative therapy that effectively induces periodontal tissue regeneration by targeting amelogenin and its newly associated molecule, glucose-regulated protein 78 (Grp78). The enamel matrix derivative (Emdogain® Gel, Straumann) is currently the only bioregeneration tool in use that is approved by the Japanese Ministry of Health, Labour and Welfare. However, in addition to the safety issues that exist owing to it being a foreign protein, a unified opinion on its mechanism of action at the signal transduction level remains unclear. Previously, our laboratory was the first to report the identification of a new amelogenin-associated molecule, Grp78, in osteoblasts. This association was later shown to promote the migration of periodontal ligament stem cells (PDLSCs), which play critical role in periodontal regeneration. Based on these results, we aim to establish the molecular basis for periodontal tissue regeneration via the combined use of recombinant amelogenin and inducers of Grp78 which could be applied by reapplication of existing drugs (drug repositioning).

Roles of serum in innate immune responses of human leukocytes to synthetic lipopeptide

Abstract Tripalmitoyl‐S‐glyceryl‐l‐Cys‐Ser‐(Lys)4 (Pam3CSK4) is a highly conserved molecular motif found in various classes of lipoproteins. The requirement for leukocyte to respond to synthetic Pam3CSK4 were studied. Pam3CSK4 primed neutrophils for a respiratory burst in a serum‐dependent manner. Pam3CSK4 upregulated CD11b, CD14, and cytochrome b558, and downregulated Leu‐8. Treatment of neutrophils with anti‐CD14 antibodies and treatment of serum with anti‐LPS binding protein (LBP) antibodies resulted in the inhibition of priming for respiratory burst by Pam3CSK4. It should be noted that LBP could not replicate the effects of serum in priming of neutrophils for respiratory burst by Pam3CSK4. Serum LBP bound to immobilized Pam3CSK4. Pam3CSK4 induced the interleukin‐8 (IL‐8) production by leukocytes in a serum‐dependent manner. Further, Pam3CSK4‐induced priming of neutrophils for respiratory burst was not inhibited by the LPS antagonists LA‐14‐PP, Rhodobacter sphaeroides LPS, or E5531, and Pam3CSK4‐induced IL‐8 production by leukocytes was not affected by LPS antagonist, E5531, indicating that Pam3CSK4 was recognized by a different receptor than LPS. Thus, Pam3CSK4 and LPS had similar biological activities and similar requirement to act on leukocytes, but were recognized by different receptors. Serum in the action of Pam3CSK4 on leukocytes was not replicated by LBP, suggesting that Pam3CSK4 might be disaggregated by serum to result in the activation of leukocytes. HighlightsPam3CSK4 primed neutrophils for enhanced respiratory burst, upregulated CD11b, and cytochrome b558, and downregulated Leu‐8 in a serum‐dependent manner.Pam3CSK4‐induced production of cytokines was serum‐dependent.Priming by Pam3CSK4 for enhanced respiratory burst was inhibited by anti‐CD14 antibodies or anti‐LBP antibodies.The effects of serum could not be replicated by LBP, suggesting that Pam3CSK4 required serum factors in addition to LBP to act on neutrophils.It was suggested that disaggregation of Pam3CSK4 by serum might be required for the lipopeptide to act on leukocytes.

Amelogenin induces M2 macrophage polarisation via PGE2/cAMP signalling pathway

OBJECTIVES Amelogenin, the major component of the enamel matrix derivative (EMD), has been suggested as a bioactive candidate for periodontal regeneration. Apart from producing a regenerative effect on periodontal tissues, amelogenin has also been reported to have an anti-inflammatory effect. However, the precise molecular mechanisms underlying these effects remain unclear. In the present study, we examined the immunomodulatory effects of amelogenin on macrophages. DESIGN Human phorbol 12-myristate 13-acetate (PMA)-differentiated U937 macrophages and CD14+ peripheral blood-derived monocytes (PBMC)-derived macrophages were stimulated with recombinant amelogenin (rM180). After performing a detailed microarray analysis, the effects of rM180 on macrophage phenotype and signal transduction pathways were evaluated by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, confocal microscopy and flow cytometry. RESULTS The microarray analysis demonstrated that rM180 increased the expression of anti-inflammatory genes in lipopolysaccharide (LPS)-challenged macrophages after 24h, while it temporarily up-regulated inflammatory responses at 4h. rM180 significantly enhanced the expression of M2 macrophage markers (CD163 and CD206). rM180-induced M2 macrophage polarisation was associated with morphological changes as well as vascular endothelial growth factor (VEGF) production. rM180 enhanced prostaglandin E2 (PGE2) expression, and the activation of the cAMP/cAMP-responsive element binding (CREB) signaling pathway was involved in amelogenin-induced M2 macrophage polarisation. Blocking of PGE2 signaling by indomethacin specifically abrogated rM180 with or without LPS-induced M2 shift in PBMC-derived macrophages. CONCLUSION Amelogenin could reprogram macrophages into the anti-inflammatory M2 phenotype. It could therefore contribute to the early resolution of inflammation in periodontal lesions and provide a suitable environment for remodeling-periodontal tissues.