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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.

An azimuthal-rotation sample holder, with improvements in the rotation-angle accuracy and rotation-axes displacement during azimuthal rotation, was developed for molecular-orientation analysis using scanning transmission X-ray microscopy., In this study, an azimuthal-rotation sample holder compatible with scanning transmission X-ray microscopy was developed. This holder exhibits improvement in the accuracy of rotation angles and reduces the displacement of the rotation axes during azimuthal rotation by using a crossed roller bearing. To evaluate the performance of the holder, the authors investigated the dependence of the optical density around the C K-edge absorption of π-orbital-oriented domains in natural spherical graphite on the rotational angle by using linearly horizontally and vertically polarized undulator radiation. Based on the dependence of the optical density ratio between C 1s → π* and 1s → σ* excitation on the polarization angle of the X-rays, the average two-dimensional orientation angle of the π orbital in each position in a natural spherical graphite sample was visualized.

In vitro studies on adherent cells require a process of passage to dissociate the cells from the culture substrate using enzymes or other chemical agents to maintain cellular activity. However, these proteolytic enzymes have a negative influence on the viability and phenotype of cells. The mesenchymal stem cell (MSC)-like cell line, C3H10T1/2, adhered, migrated, and proliferated to the same extent on newly designed microporous titanium (Ti) membrane and conventional culture dish, and spontaneous transfer to another substrate without enzymatic or chemical dissociation was achieved. The present study pierced a 10 μm-thick pure Ti sheet with 25 μm square holes at 75 μm intervals to create a dense porous structure with biomimetic topography. The pathway of machined holes allowed the cells to access both sides of the membrane frequently. In a culture with Ti membranes stacked above- and below-seeded cells, cell migration between the neighboring membranes was confirmed using the through-holes of the membrane and contact between the membranes as migration routes. Furthermore, the cells on each membrane migrated onto the conventional culture vessel. Therefore, a cell culture system with enzyme-free passaging was developed.

Due to their multi-differentiation potential, periodontal ligament fibroblasts (PDLF) play pivotal roles in periodontal tissue regeneration in vivo. Several in vitro studies have suggested that PDLFs can transmit mechanical stress into favorable basic cellular functions. However, the application of mechanical force for periodontal regeneration therapy is not expected to exhibit an effective prognosis since mechanical forces, such as traumatic occlusion, also exacerbate periodontal tissue degeneration and loss. Herein, we established a standardized murine periodontal regeneration model and evaluated the regeneration process associated with cementum remodeling. By administering a kinase inhibitor of YAP/TAZ suppressor molecules, such as large tumor suppressor homolog 1/2 (LATS1/2), we found that the activation of YAP/TAZ, a key downstream effector of mechanical signals, accelerated periodontal tissue regeneration due to the activation of PDLF cell proliferation. Mechanistically, among six kinds of MAP4Ks previously reported as upstream kinases that suppressed YAP/TAZ transcriptional activity through LATS1/2 in various types of cells, MAP4K4 was identified as the predominant MAP4K in PDLF and contributed to cell proliferation and differentiation depending on its kinase activity. Ultimately, pharmacological activation of YAP/TAZ by inhibiting upstream inhibitory kinase in PDLFs is a valuable strategy for improving the clinical outcomes of periodontal regeneration therapies.

Glutamate is one of the signaling molecules responsible for transmission in the central nervous system. Periodontal ligament (PDL) cells were recently reported to express metabotropic glutamate receptors (mGluRs). However, the functions of mGluR signaling in PDL cells or PDL-related cells remain largely unknown. The aim of this study was to investigate the expression and function of mGluRs in PDL-related cells.OCCM-30 cells, immortalized murine cementoblasts, were stimulated with l-glutamate or mGluRs antagonists. The cells' proliferative response was evaluated using a colorimetric assay and gene expression was assessed using real-time polymerase chain reaction. The nuclear translocation of cyclin D1 was evaluated by immunohistochemistry.l-Glutamate promoted the proliferation of OCCM-30 cells, which expressed mGluR1, but not mGluR5. Dihydroxyphenylglycine (DHPG), an agonist of group I mGluRs (mGluR1 and mGluR5), also promoted cell proliferation, and this was inhibited by LY456236, an mGluR1 antagonist. DHPG increased the expression of cyclin D1, a key regulator of cell proliferation, and its nuclear translocation. DHPG also increased the expression of Bcl2A1, an antiapoptotic oncogene and simultaneously reduced the expression of Bax, a pro-apoptotic marker. Furthermore, the DHPG-induced proliferation of OCCM-30 cells was reduced by pretreatment with SB203580, SP600125, and PD98059, inhibitors of p38, JNK, and ERK1/2, respectively.These findings indicate that activation of mGluR1 expressed by OCCM-30 cells induces cell proliferation in a manner that is dependent on mitogen-activated protein kinase pathways and that cyclin D1 and Bcl2A1/Bax may be involved. Our results provide useful information for elucidating the mechanisms underlying cementum homeostasis and regeneration.

In March 2011, the Great East Japan Earthquake (GEJE), which was followed by a devastating tsunami, destroyed the societal and the public hygiene systems in Japanese coastal areas. Insomnia, the greatest issue among disaster victims, has detrimental effects on both physical and psychological health. Periodontitis causes chronic discomfort and inflammation, and little is known about its impact on insomnia. Three months after the earthquake, a health panel survey was conducted over four surveys, till September 2013, in which information regarding 8,015 adults was collected and used. In addition to the heath-related questionnaire, other variables including subjective symptoms of oral diseases were recorded, and the Athens Insomnia Scale was used to evaluate the severity of insomnia. The association between insomnia and periodontal disease was examined using multilevel logistic models on the panel data, after adjusting for sex, age, economic status, comorbidities, body mass index, post-traumatic stress reactions, habitual smoking and alcohol drinking, and the Kessler Psychological Distress Scale score. In addition to the higher prevalence of insomnia among GEJE victims, significant association was revealed between insomnia and gum problems (OR = 2.16, 95% CI = 1.43-3.26), and difficulty chewing (OR = 2.22, 95% CI = 1.40-3.51), after adjusting for all covariates. The present study revealed significant association between insomnia and periodontal disease among GEJE victims. This indicated that together, integrated oral health care for disaster victims would contribute not only to prevention of oral infectious diseases, but may also help alleviate other problems caused by these harmful events.

Extracellular adenosine triphosphate (ATP) is sequentially dephosphorylated by two ectoenzymes: CD39/nucleotidase triphosphate dephosphorylase (ENTPD) and CD73/5'-ectonucleotidase (5'-NT). Adenosine, its notable metabolite, may elicit potent anti-inflammatory responses. We examined whether the CD39-adenosinergic axis may exist in gingival fibroblasts and have an effect on the expression of matrix metalloproteinase (MMP)-1, the excess production of which leads to pathological matrix degradation. We showed that transcripts of CD39, CD73, and adenosine receptors A1, A2a, and A2b, but not A3, were expressed by human gingival fibroblasts by RT-PCR. We also identified the expression of CD39 in fibroblastic cells in rat gingiva by immunohistochemistry. ATP inhibited the expression of MMP-1 triggered by interleukin-1 at gene and protein levels. However, ATP-γS, a stable ATP analog, did not. The ATP-mediated MMP-1 inhibition was restored in the presence of POM-1, a specific ENTPD inhibitor, suggesting that CD39/ENTPD was involved in the MMP-1 inhibition. ATP metabolites including adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), and adenosine inhibited MMP-1 expression, but ADP-βS, a stable ADP, did not, suggesting that adenosine converted from ATP by the action of CD39/ENTPD and CD73/5'-NT may contribute to MMP-1 inhibition. Adenosine-mediated MMP-1 inhibition was restored in the presence of H89, a protein kinase A (PKA) inhibitor. Conversely, forskolin, an enhancer of intracellular cAMP, mimicked the effect of adenosine, suggesting that the cAMP/PKA signaling pathway is involved in adenosine-mediated MMP-1 inhibition. The present findings suggest the existence of an endogenous anti-tissue destructive mechanism in gingival tissue via the CD39-adenosinergic axis.

Objective Bone morphogenetic protein (BMP)-2 promotes the osteoblastic differentiation of human periodontal ligament (PDL) cells, which play a pivotal role in periodontal regeneration. Recently, nano-sized hydroxyapatite (nano-HA) has been highlighted due to its advantageous features over micro-sized materials. Design and results We investigated the effect of nano-HA on BMP-2 expression in human PDL cells. Real time PCR analysis revealed that the expression of BMP-2 increased upon stimulation with nano-HA in dose- and time-dependent manners. An immunofluorescence assay demonstrated the synthesis of BMP-2 proteins. Concentrations of Ca 2+ as well as phosphate (Pi) in culture supernatants were unchanged, suggesting that nano-HA functioned as a nanoparticle rather than as a possible source for releasing Ca 2+ and/or Pi extracellularly, which were shown to also enhance the expression of BMP-2. Nano-HA-induced BMP-2 expression was dependent on the p38 MAP kinase pathway because increases in BMP-2 expression were inhibited by treatment with SB203580, a p38 inhibitor, and phosphorylation of p38 was detected by Western blotting. Conclusions This novel mechanism of nano-HA will be important for the rational design of future periodontal regeneration.

Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.

Extracellular phosphate (Pi) is known to play a key role in promoting osteoblastic differentiation by altering gene expression and cellular function. Importantly, it may be possible to use this knowledge as a means to deliver Pi to local sites to regenerate mineralized tissues associated with the oral cavity. Therefore, we determined the ability of Pi to regulate differentiation of pulp cells toward an odontoblast phenotype and further determined if this was in part due to an increase in the expression of bone morphogenetic protein (BMP)-2, a crucial regulator of mineralization. Results showed that Pi increased BMP-2 expression at both mRNA and protein level and BMP-2 promoter activity. Signaling inhibitors revealed that increased BMP-2 expression was dependent on cAMP/protein kinase A but not the protein kinase C signaling pathway. Treatment with 8-Br-cAMP, a cell-permeable analog of cAMP, enhanced Pi-mediated BMP-2 expression, but treatment with 8-Br-cAMP alone did not increase BMP-2, suggesting that cAMP is indispensable but not sufficient for Pi-mediated BMP-2 expression. Pi activated ERK1/2, and treatment with PD98059, an ERK1/2 inhibitor, suppressed Pi-mediated BMP-2 increase, indicating a requirement for activation of ERK1/2. ERK1/2 pathway may operate independently of cAMP-dependent signaling because MDL12,330A, an adenylate cyclase inhibitor, did not inhibit phosphorylation of ERK1/2 in response to Pi. Pulp cells expressed the sodium-dependent Pi transporter (NaPi) III type, but not NaPi-I type or NaPi-II type. Pi-mediated BMP-2 increase was inhibited in the presence of phosphonoformic acid, an inhibitor not only of NaPi transport but also of crystal nucleation. Furthermore, a similar inhibition was observed in the presence of pyrophosphate, a mineralization inhibitor. These findings demonstrate, for the first time, that Pi regulates BMP-2 expression via cAMP/protein kinase A and ERK1/2 pathways in human dental pulp cells.

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Elevated levels of extracellular Ca(2+) have been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of extracellular Ca(2+) signaling in cementogenesis has not been examined. Using RT-PCR, we found that elevated levels of extracellular Ca(2+) increase fibroblast growth factor (FGF)-2 gene expression with a peak at 6h. Pretreatment with a protein kinase A (PKA) inhibitor, H89, or an adenylate cyclase inhibitor, MDL-12,330A, inhibited Ca(2+)-stimulated Fgf-2 expression. In contrast, pretreatment with the protein kinase C (PKC) inhibitor GF-109203X or the phospholipase C (PLC) inhibitor U73122 did not affect the expression of Fgf-2 transcripts, suggesting that the increase in Fgf-2 expression was dependent on the PKA but not the PLC/PKC signaling pathway. Treatment with an activator of adenylate cyclase, forskolin, or a cell-permeable analog of cAMP, 8-Br-cAMP, enhanced Ca(2+)-stimulated Fgf-2 expression, but a single treatment with forskolin or 8-Br-cAMP did not, suggesting that cAMP generation is indispensable but not sufficient for Ca(2+)-stimulated FGF2 expression. Next, we examined the cation specificity of the putative receptor and showed that treatment with trivalent/divalent inorganic ions, Ca(2+), Gd(3+), Sr(2+), or Al(3+), caused a dose-dependent increase in Fgf-2 mRNA levels in a cAMP-dependent fashion, whereas Mg(2+) and the organic ions neomycin and spermine had no effect on Fgf-2 gene expression levels. These findings suggest that an extracellular Ca(2+)-sensing mechanism is present in cementoblasts and its activation leads to FGF-2 stimulation in a cAMP/PKA dependent fashion. Understanding the pathway regulating key genes involved in modulating the regeneration of oral tissues will assist in designing regenerative therapies based on reliable biological principles.

Dental pulp cells, which have been shown to share phenotypical features with osteoblasts, are capable of differentiating into odontoblast-like cells and generating a dentin-like mineral structure. Elevated extracellular Ca(2+)Ca(2+)(o) has been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of Ca(2+)(o) signaling in odontogenesis remains unclear. We found that elevated Ca(2+)(o) increases bone morphogenetic protein (BMP)-2 gene expression in human dental pulp cells. The increase was modulated not only at a transcriptional level but also at a post-transcriptional level, because treatment with Ca(2+) increased the stability of BMP-2 mRNA in the presence of actinomycin D, an inhibitor of transcription. A similar increase in BMP-2 mRNA level was observed in other human mesenchymal cells from oral tissue; periodontal ligament cells and gingival fibroblasts. However, the latter cells exhibited considerably lower expression of BMP-2 mRNA compared with dental pulp cells and periodontal ligament cells. The BMP-2 increase was markedly inhibited by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and partially inhibited by the L-type Ca(2+) channels inhibitor, nifedipine. However, pretreatment with nifedipine had no effect on ERK1/2 phosphorylation triggered by Ca(2+), suggesting that the Ca(2+) influx from Ca(2+) channels may operate independently of ERK signaling. Dental pulp cells do not express the transcript of Ca(2+)-sensing receptors (CaSR) and only respond slightly to other cations such as Sr(2+) and spermine, suggesting that dental pulp cells respond to Ca(2+)(o) to increase BMP-2 mRNA expression in a manner different from CaSR and rather specific for Ca(2+)(o) among cations.

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions; however the role of Wnt signaling on cementogenesis has not been examined. In this study, we have identified a consistent expression profile of Wnt signaling molecules in cementoblasts, in vitro by RT-PCR. Exposure of cells to LiCl, which promotes canonical Wnt signaling by inhibiting GSK-3beta, increased beta-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive promoters, suggesting that an endogenous canonical Wnt pathway functions in cementoblasts. Activation of endogenous canonical Wnt signaling with LiCl suppressed alkaline phosphatase (ALP) activity and expression of genes associated with cementum function; ALP, bone sialoprotein (BSP), and osteocalcin (OCN). Exposure to Wnt3a, as a representative canonical Wnt member, also inhibited the expression of ALP, BSP, and OCN gene. This effect was accompanied by decreased gene expression of Runx2 and Osterix and by increased gene expression of lymphoid enhancer factor-1. Pretreatment with Dickkopf (Dkk)-1, a potent canonical Wnt antagonist, which binds to a low-density lipoprotein-receptor-related protein (LRP)-5/6 co-receptor, attenuated the suppressive effects of Wnt3a on mRNA expression of Runx2 and OCN on cementoblasts. These findings suggest that canonical Wnt signaling inhibits cementoblast differentiation via regulation of expression of selective transcription factors. Wnt3a also increased the expression of cyclin D1, known as a cell cycle regulator, as well as cell proliferation. In conclusion, these observations suggest that Wnt signaling inhibits cementoblast differentiation and promotes cell proliferation. Elucidating the role of Wnt in controlling cementoblast function will provide new tools needed to improve on existing periodontal regeneration therapies.

Background and Objective: While the primary role of cementoblasts is to synthesize the components of cementum, we have reported that immortalized murine cementoblasts (OCCM-30) express functional Toll-like receptor (TLR)-2 and -4, and these receptors are involved in the alteration of gene expression associated with cementum formation and in the upregulation of osteoclastogenesis-associated molecules, such as receptor activator of nuclear factor-κB (NF-κB) ligand. We hypothesized that cementoblasts express a wide range of pattern recognition receptors in a manner comparable to osteoblasts, which are known to express various functional TLRs and nucleotide-binding oligomerization domain (NOD) proteins. Material and Methods: Murine cementoblasts and pre-osteoblasts were used. The gene and protein levels of TLRs/NODs were analyzed using real-time polymerase chain reaction and flow cytometry. Interleukin-6 (IL-6) and activated NF-κB were measured using enzyme-linked immunosorbent assay. Results: The expressions of TLR-1, -2, -4, -6 and -9, CD14, NOD-1 and -2 were detected in cementoblasts and were upregulated upon differentiation induced by ascorbic acid. Similar patterns were observed in the mouse MC3T3-E1 osteoblast cell line. Synthetic ligands, Pam3CSK4 (TLR-1/2 agonist), Pam2CGDPKHPKSF (TLR-2/6 agonist), lipid A (TLR4 agonist), CpG DNA (TLR-9 agonist), FK565 (NOD1 agonist) and muramyldipeptide (NOD2 agonist), effectively induced NF-κB activation in cementoblasts and/or ascorbic acid-treated cementoblasts. Furthermore, these ligands induced IL-6 production in a NF-κB-dependent manner in cementoblasts and/or ascorbic acid-treated cementoblasts. Conclusion: These results indicate that cementoblasts possess functional TLR and NOD signaling systems and have a similar capacity to osteoblasts in responding to a wide variety of pathogens.

Dendritic cells (DCs) play a critical role in activation of T cells as well as shaping immune responses. DCs express a pattern of Toll-like receptors (TLRs) to recognize invading pathogens and to initiate specific immune responses. In this study, we investigated the effect of Porphyromonas gingivalis fimbriae on the phenotype and function of human peripheral blood DCs (PBDCs) in comparison to TLR2 and TLR4 ligand, peptidoglycan (PGN) from Staphylococcus aureus and Escherichia coli O55:B5 LPS, respectively. P. gingivalis fimbriae and PGN preferentially upregulated CD14 and CD16 expression on immature PBDCs (CD14−CD16−), although E. coli LPS did not alter the expression of these molecules. Fimbriae-stimulated DCs also exhibited a different profile of cytokine production and surface molecule expression as compared with E. coli LPS-stimulated DCs. Pretreatment of PBDCs with anti-TLR2 monoclonal antibody (mAb), but not with anti-TLR4 mAb, abrogated the upregulation of CD14 and CD16 on fimbriae and PGN-treated DCs. These results indicate that different TLR signaling affects the mature DC phenotype and function and is thus crucial to the regulation of immunity to the pathogen.

Human leukocyte elastase, a neutrophil serine protease, is considered to be a potential immunoregulatory protease. Since the PDGF receptor (PDGFR) on periodontal ligament (PDL) cells is a crucial element for various functions, such as wound healing in periodontal tissue, we investigated the effect of elastase on the expression of PDGFR on PDL cells by flow cytometry and Western blotting. We found that PDGFR-α disappeared with an increasing dose of elastase, and PDGFR-β was degraded into several fragments. Elastase degraded both receptors on fixed cells, indicating that the degradation resulted from direct proteolysis on the cell surface. Elastase also then disturbed the phosphorylation of ERK1/2, JNK/SARK, and p38, triggered by PDGF-AA and PDGF-BB, suggesting that elastase inhibited PDGFR-dependent cell activation in PDL cells. These results suggest that elastase may modulate the PDGF-mediated activity of PDL cells during periodontal wound healing.

Background and objectives: Aminopeptidase N (APN)/CD13 is a multifunctional ectoenzyme that is involved in anti-inflammatory reactions, control of immune reactions and differentiation of many cellular systems. Here, we hypothesized that CD13/APN would be expressed on human gingival fibroblasts (hGF) and would contribute to the regulation of immune responses in periodontal tissue. Methods and results: CD13/APN was expressed on hGF at the mRNA and protein levels as determined by reverse transcriptase–polymerase chain reaction (RT–PCR) and flow cytometry, respectively. Enzymatic activities accompanying the expression were assessed by colorimetrical analysis using the synthetic substrate Leu-p-nitroanilide. We examined the possible regulation of CD13/APN expression on hGF in response to T cell-derived cytokines. T helper (Th) 2 cell type cytokines such as interleukin-4 and interleukin-13, but not interleukin-2 or interleukin-15, preferentially increased the expression of proteins as well as the enzymatic activities of CD13/APN in a dose-dependent manner. Receptors for these cytokines, the interleukin-4 receptor α chain, interleukin-13 receptor α1 chain, and interleukin-2R common γ chain, were expressed on hGF assessed by RT–PCR or flow cytometry. hGF exhibited inhibitory effects for formyl-methionyl-leucyl-phenylalanine (FMLP)-induced polymorphonuclear leukocyte-activation that was evaluated by Mac-1 expression, and this inhibitory effect was partially recovered by pre-treatment with the APN-specific inhibitor bestatin. Conclusions: These findings suggested that CD13/APN expressed by hGF could contribute to the anti-inflammatory response in periodontal tissue, and may be involved in disease processes mediated by Th2 cells.

Background and objective: Periodontal ligament cells are regarded to have the capacity to differentiate into cementoblasts or osteoblasts, and are capable of forming a mineralized nodule in vitro. However, the precise mechanisms are unclear. Here we evaluated the possible involvement of growth factor receptors, such as the platelet-derived growth factor receptor (PDGFR), insulin-like growth factor-I receptor (IGF-IR), and epidermal growth factor receptor (EGFR) on periodontal ligament cells and their ligands during periodontal ligament cells differentiation in vitro. Methods: Human periodontal ligament cells were differentiated via culturing in the presence of dexamethasone, ascorbic acid, and β-glycerophosphate for mineralized nodule formation, characterized by von Kossa staining. Expressions of receptors and their ligands were analyzed by flow cytometry/reverse transcription-polymerase chain reaction. Results: During the differentiation, PDGFR-α was held at a lower level compared with the control. PDGFR-β, however, was maintained at a slightly higher level that was reversed to the control level when mineralized nodules formed. In contrast, IGF-IR and EGFR were not substantially different from the control. The mineralized nodule formation was strongly inhibited by a PDGFR kinase blocker (AG1295 and AG1296), partially inhibited by an IGF-IR kinase blocker (I-Ome-AG538 and AG1024), and not inhibited by an EGFR kinase blocker (AG99). PDGF-A, PDGF-C, PDGF-D, IGF-I, and IGF-II, but not PDGF-B, were expressed on the control as well as dexamethasone/ascorbic acid-treated periodontal ligament cells during mineralized nodule formation; however, the pattern of their expressions was quite different. Conclusion: These findings suggest that a pathway of PDGFs/PDGFR and IGFs/IGF-IR on periodontal ligament cells are involved during mineralized nodule formation, and that PDGFs and IGFs expressed by periodontal ligament cells may contribute to the formation.

A protein mono-ADP-ribosyltransferase (ADPRT), anchored in the cell membrane as a glycosylphosphatidylinositol (GPI)-anchored cell-surface enzyme, was recently described on murine cytotoxic T cells (CTL). Expression of this enzyme was shown to exert regulatory functions on CTL proliferation and cytotoxic activity, presumably by modulating activity of the protein tyrosine kinase p56(lck), which is associated with the CTL co-receptor CD8. Here we report on the molecular cloning and expression of this important regulatory enzyme. The ADPRT coding sequence was derived by making use of ADPRT sequence homologies from different vertebrate species. A cDNA fragment of the enzyme coding sequence was generated by reverse transcription polymerase chain reaction (RT-PCR) from murine T-cell lymphoma SL12, which expresses the cell-surface ADPRT. The cDNA fragment was found to share extensive homology with the corresponding sequences of human and rabbit muscle ADPRT. In Northern blot hybridization, this cDNA fragment generates a strong hybridization signal with RNA from murine heart and skeletal muscle. Weak signals are seen with SL12, thymus, and spleen. Therefore, a murine skeletal muscle cDNA library was used to identify and obtain the coding sequence of the ADPRT gene. It is shown that the nucleic acid open reading frame sequence of the murine skeletal muscle gene shares 80.3% and 76.3% homology with the sequences of the human and rabbit muscle genes, respectively. Semiquantitative RT-PCR with intron-spanning primers shows that the ADPRT mRNA is present in lymphoid organs, cytotoxic T cells, and T-cell lines. Transfection of the ADPRT coding sequence into EL4 cells results in expression of the enzyme as a functional GPI-anchored cell-surface protein, able to ADP-ribosylate the arginine analog agmatine as well as cell-surface molecules.

BACKGROUND AND OBJECTIVES Retinoic acid (RA) exerts a wide variety of effects on development, cellular differentiation and homeostasis in various tissues. However, little is known about the effects of RA on the differentiation of periodontal ligament cells. In this study, we investigated whether RA can affect the dexamethasone-induced differentiation of periodontal ligament cells. METHODS AND RESULTS Human periodontal ligament cells were differentiated via culturing in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate for mineralized nodule formation, as characterized by von Kossa staining. Continuous treatment with all-trans-RA inhibited the mineralization in a dose-dependent manner, with complete inhibition over 1 microm RA. Other RA analogs, 9-cis-RA and 13-cis-RA, were also effective. Furthermore, addition of RA for just the first 4 days completely inhibited the mineralization; however, as RA was added at later stages of culture, the inhibitory effect was diminished, suggesting that RA had a phase-dependent inhibition of mineralization. RA receptor (RAR)-alpha agonist (AM-580), but not retinoid X receptor agonist (methoprene acid), inhibited the mineralization, and reverse transcription-polymerase chain reaction analysis revealed that RAR-alpha was expressed on the cells, suggesting that RAR-alpha was involved in the inhibitory mechanism. This inhibition was accompanied by inhibition of alkaline phosphatase activity; however, neither expression of platelet-derived growth factor (PDGF) receptor-alpha, PDGF receptor-beta, or epidermal growth factor (EGF) receptor, nor phosphorylation of extracellular signal-regulated kinases triggered by PDGF-ascorbic acid or PDGF-BB was changed, as assessed by flow cytometry or western blot analyses. CONCLUSIONS These findings suggest that RA is a potential negative regulator for differentiation of human periodontal ligament cells.

CD13/aminopeptidase N (APN) is a multifunctional ectoenzyme that is involved in anti-inflammatory reactions, control of immune reactions and differentiation of many cellular systems. In this study, we first demonstrated that CD13/APN was expressed on human gingival fibroblast (hGF). T helper (Th) 2 cell type cytokines such as IL-4 and IL-13, but not IL-2 or IL-15, preferentially increased the expression of CD13/APN, resulting in the inhibition of FMLP-mediated neutrophil activation through the degradation by CD13/APN on HGF. Our data suggested that CD13/APN expressed by hGF could contribute to the anti-inflammatory response in periodontal tissue, and may be involved in disease processes mediated by Th2 cells.

Background The production of chemokines by tissue resident cells during inflammation is considered one of the main mechanisms involved in the formation of inflammatory infiltrates. Fibroblasts are the main resident cell type in gingival and periodontal ligament tissues, and their ability to produce chemokine stromal cell-derived factor 1 alpha (SDF-1α) and its receptor CXCR4 under stimulation by gram negative bacteria, Porphyromonas gingivalis, commonly found in periodontal infections was investigated. Methods Western blots were used to assess SDF-1α and CXCR4 protein expression levels in human gingival fibroblast cells (HGF-1) induced by Lipopolysaccharide (LPS) from P. gingivalis in the presence or absence of LY294002, a highly selective inhibitor of PI-3K/Akt. RT-PCR and quantitative Real-time PCR was performed using gingival mRNAs from periodontitis patients. Immunohistochemistry was performed to analyze the expression and subcellular localization of SDF-1α and CXCR4, together with NF-kβ phosphorylation, in specimens from patients with periodontitis and in an experimental rat periodontitis model. Results We found that P. gingivalis LPS up-regulated SDF-1α and CXCR4 protein levels and elevated phosphorylation of the SDF-1α-responsive NF-kβ and Akt at 24 h in HGF-1 cells. SDF-1α and CXCR4 mRNA and protein expression levels were high in all patients with periodontitis. In the P. gingivalis-induced rat experimental periodontitis model, SDF-1α and CXCR4 immunoreactivity was higher in gingival and periodontal ligament tissues compared to the control. Conclusion Our data showed that PI-3K/Akt is an upstream participant in the P. gingivalis LPS-mediated induction of SDF-1α. Taken together, these results suggest that the chemokine SDF-1α and its receptor CXCR4 contribute to P. gingivalis-induced periodontal inflammation.