Public RNA-seq data-based identification and functional analyses reveal that MXRA5 retains proliferative and migratory abilities of dental pulp stem cells.

Dental pulp stem cells (DPSC) usually remain quiescent in the dental pulp tissue; however, once the dental pulp tissue is injured, DPSCs potently proliferate and migrate into the injury microenvironment and contribute to immuno-modulation and tissue repair. However, the key molecules that physiologically support the potent proliferation and migration of DPSCs have not been revealed. In this study, we searched publicly available transcriptome raw data sets, which contain comparable (i.e., equivalently cultured) DPSC and mesenchymal stem cell data. Three data sets were extracted from the Gene Expression Omnibus database and then processed and analyzed. MXRA5 was identified as the predominant DPSC-enriched gene associated with the extracellular matrix. MXRA5 is detected in human dental pulp tissues. Loss of MXRA5 drastically decreases the proliferation and migration of DSPCs, concomitantly with reduced expression of the genes associated with the cell cycle and microtubules. In addition to the known full-length isoform of MXRA5, a novel splice variant of MXRA5 was cloned in DPSCs. Recombinant MXRA5 coded by the novel splice variant potently induced the haptotaxis migration of DPSCs, which was inhibited by microtubule inhibitors. Collectively, MXRA5 is a key extracellular matrix protein in dental pulp tissue for maintaining the proliferation and migration of DPSCs.

PPARγ-Induced Global H3K27 Acetylation Maintains Osteo/Cementogenic Abilities of Periodontal Ligament Fibroblasts.

The periodontal ligament is a soft connective tissue embedded between the alveolar bone and cementum, the surface hard tissue of teeth. Periodontal ligament fibroblasts (PDLF) actively express osteo/cementogenic genes, which contribute to periodontal tissue homeostasis. However, the key factors maintaining the osteo/cementogenic abilities of PDLF remain unclear. We herein demonstrated that PPARγ was expressed by in vivo periodontal ligament tissue and its distribution pattern correlated with alkaline phosphate enzyme activity. The knockdown of PPARγ markedly reduced the osteo/cementogenic abilities of PDLF in vitro, whereas PPARγ agonists exerted the opposite effects. PPARγ was required to maintain the acetylation status of H3K9 and H3K27, active chromatin markers, and the supplementation of acetyl-CoA, a donor of histone acetylation, restored PPARγ knockdown-induced decreases in the osteo/cementogenic abilities of PDLF. An RNA-seq/ChIP-seq combined analysis identified four osteogenic transcripts, RUNX2, SULF2, RCAN2, and RGMA, in the PPARγ-dependent active chromatin region marked by H3K27ac. Furthermore, RUNX2-binding sites were selectively enriched in the PPARγ-dependent active chromatin region. Collectively, these results identified PPARγ as the key transcriptional factor maintaining the osteo/cementogenic abilities of PDLF and revealed that global H3K27ac modifications play a role in the comprehensive osteo/cementogenic transcriptional alterations mediated by PPARγ.

DMP-1 promoter-associated antisense strand non-coding RNA, panRNA-DMP-1, physically associates with EGFR to repress EGF-induced squamous cell carcinoma migration.

Accumulating evidence suggests that specific non-coding RNAs exist in many types of malignant tissues, and are involved in cancer invasion and metastasis. However, little is known about the precise roles of non-coding RNAs in squamous cell carcinoma (SQCC) invasion and migration. Recently, the dentin matrix protein-1 (DMP-1) gene locus was identified as a transcriptionally active site in squamous cell carcinoma (SQCC) tissue and cells. However, it is unclear whether RNA associated with cell migration exist at the DMP-1 gene locus in SQCC cells. We identified a novel promoter-associated non-coding RNA in the antisense strand of DMP-1 gene locus, promoter-associated non-coding RNA (panRNA)-DMP-1, by the RACE method in SQCC cells and tissues, and characterized the functions of panRNA-DMP-1 in EGF-driven SQCC cell migration. The inhibition of endogenous panRNA-DMP-1 expression by specific siRNAs and exogenous over-expression of panRNA-DMP-1 resulted in increased and suppressed cellular migration toward EGF in SQCC cells, respectively, and nuclear expression of panRNA-DMP-1 was induced by EGF stimulation. Mechanistically, suppression of panRNA-DMP-1 expression increased EGFR nuclear localization upon EGF treatment and nuclear panRNA-DMP-1 physically interacted with EGFR, which was confirmed by RNA immunoprecipitation assay using a bacteriophage-delivered PP7 RNA labeling system. Furthermore, co-immunoprecipitation assay revealed that suppression of panRNA-DMP-1 stabilized EGFR interaction with STAT3, a known co-transcription factors of EGFR, to induce migratory properties in many cancer cells. Based on these findings, panRNA-DMP-1 is an EGFR-associating RNA that inhibits the EGF-induced migratory properties of SQCC possibly by regulating EGFR nuclear localization and EGFR binding to STAT3.

Surgical Sealing of Laterally Localized Accessory Root Canal with Resin Containing S-PRG Filler in Combination with Non-Surgical Endodontic Treatment: A Case Report.

The spread of root canal infection to surrounding periodontal tissue through accessory root canals reduces the success rate of endodontic treatment. In this case, cone-beam computed tomography revealed a lesion (4 mm from the apex) resulting from an accessory root canal of the maxillary left central incisor. First, non-surgical endodontic treatment was conducted but the sinus tract remained. Surgical preparation of the root cavity was then conducted to remove potentially infected dentin surrounding the accessory root canal. The cavity was filled and the foramen was sealed with resin containing bioactive surface pre-reacted glass (S-PRG) filler. The photopolymerized resin was then contoured and polished. In combination with subsequent supportive non-surgical endodontic treatment, a good clinical outcome with the disappearance of the sinus tract and clinical symptoms such as discomfort and pressure pain and the regeneration of the alveolar bone hanging over the cavity was obtained. In this case, the good clinical outcome may have been due to the dentin-adhesive property and durability of the pre-adhesive system and composite resin. The better biocompatibility of S-PRG fillers presumably facilitated periodontal tissue healing.

A small nuclear acidic protein (MTI-II, Zn2+-binding protein, parathymosin) attenuates TNF-α inhibition of BMP-induced osteogenesis by enhancing accessibility of the Smad4-NF-κB p65 complex to Smad binding element.

Pro-inflammatory cytokines prevent bone regeneration in vivo and activation of nuclear factor-κB (NF-κB) signaling has been proposed to lead to suppression of bone morphogenetic protein (BMP)-induced osteogenesis via direct binding of p65 to Smad4 in vitro. Application of a small nuclear acidic protein (MTI-II) and its delivered peptide, MPAID (MTI-II peptide anti-inflammatory drug) has been described to elicit therapeutic potential via strong anti-inflammatory action following the physical association of MTI-II and MPAID with p65. However, it is unclear whether MTI-II attenuates tumor necrosis factor (TNF)-α inhibition of BMP-induced osteogenesis. Herein, we found that TNF-α-mediated suppression of responses associated with BMP4-induced osteogenesis, including expression of the osteocalcin encoding gene Ocn, Smad binding element (SBE)-dependent luciferase activity, alkaline phosphatase activity, and alizarin red S staining were largely restored by MTI-II and MPAID in MC3T3-E1 cells. Mechanistically, MTI-II and MPAID did not inhibit nuclear translocation of p65 or disassociate Smad4 from p65. Further, results from chromatin immunoprecipitation (ChIP) analyses revealed that Smad4 enrichment in cells over-expressing MTI-II and treated with TNF-α was equivalent to that in cells without TNF-α treatment. Alternatively, Smad4 enrichment was considerably decreased following TNF-α treatment in control cells. Moreover, p65 enrichment in the Id-1 promoter SBE was detected only when cells over-expressing MTI-II were stimulated with TNF-α. Overall, our study concludes that MTI-II restored TNF-α-inhibited suppression of BMP-Smad-induced osteogenic differentiation by enhancing accessibility of the Smad4-p65 complex to the SBE rather than by liberating Smad4 from p65.

Dentin phosphoprotein inhibits lipopolysaccharide-induced macrophage activation independent of its serine/aspartic acid-rich repeats.

OBJECTIVE: The objective of this study was to investigate the effects of dentin phosphoprotein (DPP) on lipopolysaccharide-induced inflammatory responses of macrophages in vitro. DESIGN: Wildtype and mutant recombinant dentin phosphoprotein (rDPP) proteins were generated using a mammalian expression system. Macrophages, phorbol 12-myristate 13-acetate-differentiated THP-1 cells, were stimulated with lipopolysaccharide in the absence or presence of rDPP proteins. After the 24-hr incubation, the inflammatory gene expression levels were examined by quantitative reverse-transcription polymerase chain reaction and the amount of secreted TNF-α protein was evaluated by enzyme-linked immunosorbent assay. Furthermore, the subcellular localization of exogenously added rDPP was examined by immunocytochemistry, and the direct binding of rDPP to lipopolysaccharide was quantified by solid-phase binding assay. RESULTS: rDPP dose-dependently reduced the expression of lipopolysaccharide-induced inflammatory genes, such as TNFα, IL-1ß, and IL-8, and TNF-α protein secretion from the macrophages. Furthermore, mutant rDPP having a shortened serine/aspartic acid-rich repeats (SDrr) was also able to inhibit lipopolysaccharide-induced inflammatory responses of macrophages. rDPP was localized adjacent to the cellular membrane rather than in the cytoplasm, and rDPP was able to bind to lipopolysaccharide. These results suggested that rDPP inhibited lipopolysaccharide-induced inflammatory responses by binding to lipopolysaccharide. CONCLUSIONS: In addition to the well-known functions of DPP for dentin mineralization that depend on the SDrr, we demonstrated that DPP possesses anti-inflammatory effects on lipopolysaccharide-stimulated macrophages that are independent of the SDrr.

The Role of NF-κB in Physiological Bone Development and Inflammatory Bone Diseases: Is NF-κB Inhibition "Killing Two Birds with One Stone"?

Nuclear factor-κB (NF-κB) is a transcription factor that regulates the expression of various genes involved in inflammation and the immune response. The activation of NF-κB occurs via two pathways: inflammatory cytokines, such as TNF-α and IL-1ß, activate the "classical pathway", and cytokines involved in lymph node formation, such as CD40L, activate the "alternative pathway". NF-κB1 (p50) and NF-κB2 (p52) double-knockout mice exhibited severe osteopetrosis due to the total lack of osteoclasts, suggesting that NF-κB activation is required for osteoclast differentiation. These results indicate that NF-κB may be a therapeutic target for inflammatory bone diseases, such as rheumatoid arthritis and periodontal disease. On the other hand, mice that express the dominant negative form of IκB kinase (IKK)-ß specifically in osteoblasts exhibited increased bone mass, but there was no change in osteoclast numbers. Therefore, inhibition of NF-κB is thought to promote bone formation. Taken together, the inhibition of NF-κB leads to "killing two birds with one stone": it suppresses bone resorption and promotes bone formation. This review describes the role of NF-κB in physiological bone metabolism, pathologic bone destruction, and bone regeneration.

Dental pulp cell-derived powerful inducer of TNF-α comprises PKR containing stress granule rich microvesicles.

It is well known that dental pulp tissue can evoke some of the most severe acute inflammation observed in the human body. We found that dental pulp cells secrete a factor that induces tumor necrosis factor-α production from macrophages, and designated this factor, dental pulp cell-derived powerful inducer of TNF-α (DPIT). DPIT was induced in dental pulp cells and transported to recipient cells via microvesicles. Treatment of dental pulp cells with a PKR inhibitor markedly suppressed DPIT activity, and weak interferon signals were constitutively activated inside the cells. In recipient macrophages, stimulation with DPIT-containing supernatants from pulp cells resulted in activation of both nuclear factor-κB and MAP kinases like JNK and p38. Proteomics analyses revealed that many stress granule-related proteins were present in supernatants from dental pulp cells as well as microvesicle marker proteins like GAPDH, ß-actin, HSPA8, HSPB1, HSPE1, and HSPD1. Furthermore, giant molecule AHNAK and PKR were detected in microvesicles derived from dental pulp cells, and gene silencing of AHNAK in dental pulp cells led to reduced DPIT activity. Thus, it appeared that the core protein of DPIT was PKR, and that PKR was maintained in an active state in stress granule aggregates with AHNAK and transported via microvesicles. The activity of DPIT for TNF-α induction was far superior to that of gram-negative bacterial endotoxin. Therefore, we, report for the first time, that active PKR is transported via microvesicles as stress granule aggregates and induces powerful inflammatory signals in macrophages.

A peptide that blocks the interaction of NF-κB p65 subunit with Smad4 enhances BMP2-induced osteogenesis.

Bone morphogenetic protein (BMP) potentiates bone formation through the Smad signaling pathway in vitro and in vivo. The transcription factor nuclear factor κB (NF-κB) suppresses BMP-induced osteoblast differentiation. Recently, we identified that the transactivation (TA) 2 domain of p65, a main subunit of NF-κB, interacts with the mad homology (MH) 1 domain of Smad4 to inhibit BMP signaling. Therefore, we further attempted to identify the interacting regions of these two molecules at the amino acid level. We identified a region that we term the Smad4-binding domain (SBD), an amino-terminal region of TA2 that associates with the MH1 domain of Smad4. Cell-permeable SBD peptide blocked the association of p65 with Smad4 and enhanced BMP2-induced osteoblast differentiation and mineralization without affecting the phosphorylation of Smad1/5 or the activation of NF-κB signaling. SBD peptide enhanced the binding of the BMP2-inudced phosphorylated Smad1/5 on the promoter region of inhibitor of DNA binding 1 (Id-1) compared with control peptide. Although SBD peptide did not affect BMP2-induced chondrogenesis during ectopic bone formation, the peptide enhanced BMP2-induced ectopic bone formation in subcortical bone. Thus, the SBD peptide is useful for enabling BMP2-induced bone regeneration without inhibiting NF-κB activity.

Two-year clinical comparison of a flowable-type nano-hybrid composite and a paste-type composite in posterior restoration.

AIM: The purpose of the present study was to compare the clinical efficacy between a flowable-type nano-hybrid composite and a paste-type composite for posterior restoration. METHODS: Of 62 posterior teeth in 33 patients (mean age: 34.1 years), 31 were filled with a paste-type composite (Heliomolar [HM] group), and another 31 with a flowable nano-hybrid composite (MI FIL [MI] group). Clinical efficacy was evaluated at 2 years after the restoration. RESULTS: There were no differences for retention, surface texture deterioration, anatomical form change, deterioration of marginal adaptation, and secondary caries, while a statistical difference was found for marginal discoloration, which was significantly greater in the HM group (P < 0.05). Furthermore, color matching in the MI group was superior to that in the HM group immediately after the restoration throughout the study period. CONCLUSIONS: The present 2-year clinical evaluation of different composites showed that the flowable nano-hybrid composite could be an effective esthetic material for posterior restoration.

A Small Nuclear Acidic Protein (MTI-II, Zn2+ Binding Protein, Parathymosin) That Inhibits Transcriptional Activity of NF-κB and Its Potential Application to Antiinflammatory Drugs.

Nuclear factor-κB (NF-κB) is the most potent proinflammatory transactivator, and an inhibitor of NF-κB is a good antiinflammatory drug. Glucocorticoids (GCs) are the strongest and the most frequently used antiinflammatory drugs. GC-bound glucocorticoid receptor (GR) inhibits the transcriptional activity of NF-κB and thereby suppresses a broad range of inflammatory processes. Concurrently, in whole body outside the inflammation area, the GR exerts a lot of hormone action, which results in severe side effects. There is a long-awaited need for a new NF-κB inhibitor. Previously we found a small nuclear acidic protein (named MTI-II, also known as Zn2+-binding protein or parathymosin), which worked as a coactivator of GR. Here we showed that overexpression of MTI-II inhibited a transcriptional activity of NF-κB independently of GCs and the GR. Vise versa, RNA interference suppression of inherent MTI-II enhanced the transcriptional activity of NF-κB. An immunoprecipitation analysis showed that MTI-II precipitated NF-κB after the stimulation of TNFα. Deletion mutants of MTI-II showed that central acidic region is essential for the inhibition of the transcriptional activity of NF-κB. These results suggest that MTI-II would be an inherent inhibitor that interacts with NF-κB. Next, we constructed MTI-II-based antiinflammatory drugs (three fusion proteins of MTI-II with a protein transduction domain and a fusion peptide of the central acidic region with protein transduction domain). These drugs had significant antiinflammatory effects on acute inflammation models and on animal models of human chronic inflammation diseases without an increase of the blood glucose level and repeated-dose toxicity. The MTI-II-based antiinflammatory drug will be a good alternative of GCs.

The Novel NF-κB Inhibitor, MTI-II Peptide Anti-Inflammatory Drug, Suppresses Inflammatory Responses in Odontoblast-Like Cells.

Regulation of inflammation is important for pulp wound healing, including protective responses by odontoblast-like cells. However, methods for directly regulating pulp inflammation have not yet been described. The inflammatory response is mediated by a transcription factor, nuclear factor-κB (NF-κB), which activates inflammatory cytokines including tumor necrosis factor (TNF)-α. Macromolecular translocation inhibitor II (MTI-II) was previously demonstrated as an enhancer of the transcriptional activity of glucocorticoid-bound glucocorticoid receptor. Recently, a MTI-II peptide anti-inflammatory drug (MPAID) was bioengineered from the structure of MTI-II as an inhibitor of NF-κB transactivation. Here, we examined the effects of MTI-II and MPAID on the inflammatory responses of odontoblast-like cells. TNF-α inhibited alkaline phosphatase (ALP) activity, a marker of odontoblast/osteogenic differentiation, and induced NF-κB transcriptional activity in KN-3 cells, which are odontoblast-like cells derived from dental papilla cells of rat incisors, without affecting their viability. Exogenous expression of MTI-II suppressed the NF-κB transcriptional activity induced by TNF-α or overexpression of p65 (a main subunit of NF-κB) in the cells, whereas it failed to inhibit degradation of IκBα and nuclear translocation of p65 after TNF-α treatment, suggesting that MTI-II inhibits NF-κB transcriptional activity by modulating the duration of DNA binding by p65. MPAID also inhibited TNF-α-induced NF-κB transcriptional activity, the mRNA expression of IL-6 and IL-8, and IL-6 production. Furthermore, pretreatment of the cells with MPAID restored the inhibitory effect of TNF-α on ALP activity. These results suggest that MPAID may be able to regulate the inflammatory response and maintain a protective response of dental pulp. J. Cell. Biochem. 117: 2552-2558, 2016. © 2016 Wiley Periodicals, Inc.

Effects of 4-META/MMA-TBB Resin at Different Curing Stages on Osteoblasts and Gingival Epithelial Cells.

PURPOSE: To assess the effects of different curing stages of 4-META/MMA-TBB resin on osteoblasts and gingival keratinocytes. MATERIALS AND METHODS: The MC3T3-E1 murine pre-osteoblastic cell line and GE-1 murine gingival epithelial cell line were cultured with mixtures of Super-Bond C&B at different curing stages, and the cell viability was assessed. The alkaline phosphatase (ALP) activity of the MC3T3-E1 cells was also assessed. RESULTS: The majority of the MC3T3-E1 cells died and showed no ALP activity when cultured with 4-META/MMA-TBB resin during the initial curing phase (1 min of curing). A later curing phase of the 4-META/MMA-TBB resin (7 min of curing) showed cytotoxicity at day 1, but the toxic effect was temporary and the proliferative capacity and ALP activity in the cells were similar to control cells at day 7. Completely cured 4-META/MMA-TBB resin (after 1 or 12 h of curing) did not affect the cell viability or ALP activity of the MC3T3-E1 cells. In contrast, 4-META/MMA-TBB resin showed no effect on the GE-1 cells at any stage of curing. CONCLUSION: Although 4-META/MMA-TBB resin during the initial curing phase shows toxic effects on MC3T3-E1 cells, that cytotoxicity is minimal at later curing phases. In contrast, neither the uncured nor cured resins affected the GE-1 cells.

Inhibition of BMP2-induced bone formation by the p65 subunit of NF-κB via an interaction with Smad4.

Bone morphogenic proteins (BMPs) stimulate bone formation in vivo and osteoblast differentiation in vitro via a Smad signaling pathway. Recent findings revealed that the activation of nuclear factor-κB (NF-κB) inhibits BMP-induced osteoblast differentiation. Here, we show that NF-κB inhibits BMP signaling by directly targeting the Smad pathway. A selective inhibitor of the classic NF-κB pathway, BAY11-770682, enhanced BMP2-induced ectopic bone formation in vivo. In mouse embryonic fibroblasts (MEFs) prepared from mice deficient in p65, the main subunit of NF-κB, BMP2, induced osteoblastic differentiation via the Smad complex to a greater extent than that in wild-type MEFs. In p65(-/-) MEFs, the BMP2-activated Smad complex bound much more stably to the target element than that in wild-type MEFs without affecting the phosphorylation levels of Smad1/5/8. Overexpression of p65 inhibited BMP2 activity by decreasing the DNA binding of the Smad complex. The C-terminal region, including the TA2 domain, of p65 was essential for inhibiting the BMP-Smad pathway. The C-terminal TA2 domain of p65 associated with the MH1 domain of Smad4 but not Smad1. Taken together, our results suggest that p65 inhibits BMP signaling by blocking the DNA binding of the Smad complex via an interaction with Smad4. Our study also suggests that targeting the association between p65 and Smad4 may help to promote bone regeneration in the treatment of bone diseases.