Transcriptional regulation of human odontogenic ameloblast-associated protein gene by tumor necrosis factor-α.

OBJECTIVE: Odontogenic ameloblast-associated protein (ODAM) is produced by maturation stage ameloblasts and junctional epithelium (JE). The function of ODAM is thought to be involved in the attachment of teeth and JE. To elucidate transcriptional regulation of human ODAM gene in inflamed gingiva, we have analyzed the effects of TNF-α on the expression of ODAM gene in Ca9-22 and Sa3 gingival epithelial cells. MATERIALS AND METHODS: Total RNAs were extracted from Ca9-22 and Sa3 cells after stimulation by TNF-α (10 ng/ml). ODAM mRNA and protein levels were analyzed by qPCR and Western blotting. Luciferase (LUC) analyses were performed using LUC constructs inserted in various lengths of ODAM gene promoter. Gel shift and chromatin immunoprecipitation (ChIP) assays were carried out. RESULTS: TNF-α increased ODAM mRNA and protein levels at 3 to 24 h. TNF-α induced LUC activities of the ODAM gene promoter constructs, and the activities were inhibited by protein kinase A, tyrosine kinase, MEK1/2, PI3-kinase and NF-κB inhibitors. Gel shift and ChIP assays revealed that TNF-α increased CCAAT/enhancer-binding protein (C/EBP) ß and Yin Yang1 (YY1) binding to three kinds of C/EBPs and YY1 elements. CONCLUSION: These results demonstrate that TNF-α stimulates ODAM gene transcription via C/EBPs and YY1 elements in the human ODAM gene promoter.

Examination of Suprahyoid Muscle Resection and Other Factors Affecting Swallowing Function in Patients With Advanced Oral Cancer After Surgical Resection and Reconstruction.

Dysphagia is one of the most common adverse effects associated with oral cancer therapy and could greatly impair postoperative quality of life. The objective of this study was to analyze postoperative swallowing outcomes and factors influencing postoperative swallowing function in patients with advanced oral cancer who underwent primary reconstruction after surgical resection to identify patients at risk of experiencing severe dysphagia after immediate reconstruction of surgical defects, and to determine an ideal approach to provide appropriate perioperative interventions. The swallowing status was evaluated at 4 week postoperatively using the Functional Oral Intake Scale. We also analyzed the effects of patient, tumor, surgical, and other factors on postoperative swallowing function. The study included 67 patients. At 4 weeks postoperatively, 11 patients showed reduced swallowing function, whereas 56 patients showed good swallowing function. The number of resected suprahyoid muscles (odds ratio, 1.55; 95% confidence interval, 1.03-2.32; P=0.035) was an independent factor influencing postoperative swallowing function. Thus, among patients who underwent radical resection of oral cancer with primary reconstruction, those with extensive resection of the suprahyoid muscles were at higher risk of developing postoperative dysphagia. These findings are expected to facilitate increased vigilance for dysphagia, better counseling, and appropriate rehabilitation interventions.

Interleukin-1ß regulates odontogenic ameloblast-associated protein gene transcription in human gingival epithelial cells.

Junction epithelium (JE) is located apical to the bottom of the gingival sulcus and binds enamel to hemidesmosomes to protect the periodontal tissue from bacterial infection. Function of odontogenic ameloblast-associated protein (ODAM) is suggested by its expression sites (JE and maturation stage ameloblasts) to be involved in the adhesion between the JE and enamel, and odontogenesis. To analyze the changes in ODAM gene and protein expressions in inflamed gingiva, Ca9-22 gingival epithelial cells were stimulated with 1 ng/ml interleukin-1ß (IL-1ß) for 3-24 h, and ODAM mRNA and protein levels were analyzed by real-time PCR and Western blotting. Luciferase (LUC) constructs were made ligating various lengths of human ODAM gene promoters and performed LUC analyses in Ca9-22 cells. Gel shift and chromatin immunoprecipitation (ChIP) assays were performed. IL-1ß induced ODAM mRNA and protein levels at 6-24 h. IL-1ß increased LUC activities of the ODAM gene promoter constructs from - 85 to - 950. These activities were blocked by protein kinase A, tyrosine kinase, mitogen-activated protein (MAP) kinase kinase and phosphoinositide 3-kinase inhibitors. Gel shift and ChIP assays showed that IL-1ß induced CCAAT/enhancer-binding protein (C/EBP) ß and Yin Yang1 (YY1) binding to C/EBP1, 2, 3, and YY1 elements. These data indicate that IL-1ß stimulates ODAM gene transcription mediated through C/EBP1, C/EBP2, C/EBP3, and YY1 elements in the human ODAM gene promoter.

Lateral roots, in addition to adventitious roots, form a barrier to radial oxygen loss in Zea nicaraguensis and a chromosome segment introgression line in maize.

Plants typically respond to waterlogging by producing new adventitious roots with aerenchyma and many wetland plants form a root barrier to radial O2 loss (ROL), but it was not known if this was also the case for lateral roots. We tested the hypothesis that lateral roots arising from adventitious roots can form a ROL barrier, using root-sleeving electrodes and O2 microsensors to assess ROL of Zea nicaraguensis, the maize (Zea mays ssp. mays) introgression line with a locus for ROL barrier formation (introgression line (IL) #468) from Z. nicaraguensis and a maize inbred line (Mi29). Lateral roots of Z. nicaraguensis and IL #468 both formed a ROL barrier under stagnant, deoxygenated conditions, whereas Mi29 did not. Lateral roots of Z. nicaraguensis had higher tissue O2 status than for IL #468 and Mi29. The ROL barrier was visible as suberin in the root hypodermis/exodermis. Modelling showed that laterals roots can grow to a maximum length of 74 mm with a ROL barrier, but only to 33 mm without a barrier. Presence of a ROL barrier in lateral roots requires reconsideration of the role of these roots as sites of O2 loss, which for some species now appears to be less than hitherto thought.

Optimal Rectification without Forward-Current Suppression by Biological Molecular Motor.

We experimentally show that biological molecular motor F_{1}-ATPase (F_{1}) implements an optimal rectification mechanism. The rectification mechanism hardly suppresses the synthesis of adenosine triphosphate by F_{1}, which is F_{1}'s physiological role, while inhibiting the unfavorable hydrolysis of adenosine triphosphate. This optimal rectification contrasts highly with that of a simple ratchet model, where the inhibition of the backward current is inevitably accompanied by the suppression of the forward current. Our detailed analysis of single-molecule trajectories demonstrates a novel but simple rectification mechanism of F_{1} with parallel landscapes and asymmetric transition rates.

MiR-200b suppresses TNF-α-induced AMTN production in human gingival epithelial cells.

Amelotin (AMTN) is an enamel protein that is localized in junctional epithelium (JE) of gingiva and suggested to be involved in the attachment between JE and tooth enamel. MicroRNA is a small non-coding RNA that regulates gene expression at post-transcriptional level by binding to the 3'-untranslated region (3'-UTR) of target mRNAs. In this study, we have analyzed the effects of miR-200b on the expression of AMTN in human gingival epithelial (Ca9-22) cells. Total RNAs and proteins were extracted from Ca9-22 cells transfected with miR-200b expression plasmid or miR-200b inhibitor and stimulated by TNF-α (10 ng/ml, 12 h). AMTN and inhibitor of kappa-B kinase beta (IKKß) mRNA and protein levels were measured by qPCR and Western blot. Human AMTN 3'-UTR that contains putative miR-200b target sites were cloned downstream of -353AMTN luciferase (LUC) plasmid. Ca9-22 cells were transfected with -353AMTN 3'-UTR LUC constructs and miR-200b expression plasmid, and LUC activities were measured with or without stimulation by TNF-α. TNF-α-induced AMTN mRNA levels were partially inhibited by miR-200b overexpression and enhanced by miR-200b inhibitor. TNF-α-induced IKKß mRNA and protein levels were almost completely inhibited by miR-200b. Transcriptional activities of -353AMTN 3'-UTR LUC constructs were induced by TNF-α and partially inhibited by miR-200b. IKKß inhibitor IMD0354 and NF-κB inhibitor triptolide decreased TNF-α-induced LUC activities. Furthermore, both inhibitors reduced AMTN mRNA levels in the presence or absence of TNF-α. These results suggest that miR-200b suppresses AMTN expression by targeting to AMTN and IKKß mRNAs in the human gingival epithelial cells.

Tight Chemomechanical Coupling of the F1 Motor Relies on Structural Stability.

The F1 motor is a rotating molecular motor that ensures a tight chemomechanical coupling between ATP hydrolysis/synthesis reactions and rotation steps. However, the mechanism underlying this tight coupling remains to be elucidated. In this study, we used electrorotation in single-molecule experiments using an F1ßE190D mutant to demonstrate that the stall torque was significantly smaller than the wild-type F1, indicating a loose coupling of this mutant, despite showing similar stepping torque as the wild-type. Experiments on the ATPase activity after heat treatment and gel filtration of the α3ß3-subcomplex revealed the unstable structure of the ßE190D mutant. Our results suggest that the tight chemomechanical coupling of the F1 motor relies on the structural stability of F1. We also discuss the difference between the stepping torque and the stall torque.

Follicular dendritic cell-secreted protein gene expression is upregulated and spread in nifedipine-induced gingival overgrowth.

Follicular dendritic cell-secreted protein (FDC-SP) is secreted protein expressed in follicular dendritic cells, periodontal ligament and junctional epithelium (JE). Its expression could be controlled during inflammatory process of gingiva; however, responsible mechanism for gingival overgrowth and involvement of FDC-SP in clinical condition is still unclear. We hypothesized that JE-specific genes are associated with the initiation of drug-induced gingival enlargement (DIGE) called gingival overgrowth, and investigated the changes of JE-specific gene's expression and their localization in overgrown gingiva from the patients. Immunohistochemical analysis revealed that the FDC-SP localization was spread in overgrown gingival tissues. FDC-SP mRNA levels in GE1 and Ca9-22 cells were increased by time-dependent nifedipine treatments, similar to other JE-specific genes, such as Amelotin (Amtn) and Lamininß3 subunit (Lamß3), whereas type 4 collagen (Col4) mRNA levels were decreased. Immunocytochemical analysis showed that FDC-SP, AMTN, and Lamß3 protein levels were increased in GE1 and Ca9-22 cells. Transient transfection analyses were performed using luciferase constructs including various lengths of human FDC-SP gene promoter, nifedipine increased luciferase activities of -345 and -948FDC-SP constructs. These results raise the possibility that the nifedipine-induced FDC-SP may be related to the mechanism responsible for gingival overgrowth does not occur at edentulous jaw ridges.

Negative feedback by SNAI2 regulates TGFß1-induced amelotin gene transcription in epithelial-mesenchymal transition.

Junctional epithelium (JE) demonstrates biological responses with the rapid turnover of gingival epithelial cells. The state occurs in inflammation of gingiva and wound healing after periodontal therapy. To understand the underlying mechanisms and to maintain homeostasis of JE, it is important to investigate roles of JE-specific genes. Amelotin (AMTN) is localized at JE and regulated by inflammatory cytokines and apoptotic factors that represent a critical role of AMTN in stabilizing the dentogingival attachment, which is an entrance of oral bacteria. In this study, we demonstrated that the AMTN gene expression was regulated by SNAI2 and transforming growth factor ß1 (TGFß1)-induced epithelial-mesenchymal transition (EMT) that occurs in wound healing and fibrosis during chronic inflammation. SNAI2 downregulated AMTN gene expression via SNAI2 bindings to E-boxes (E2 and E4) in the mouse AMTN gene promoter in EMT of gingival epithelial cells. Meanwhile, TGFß1-induced AMTN gene expression was attenuated by SNAI2 and TGFß1-induced SNAI2, without inhibition of the TGFß1-Smad3 signaling pathway. Moreover, SNAI2 small interfering RNA (siRNA) rescued SNAI2-induced downregulation of AMTN gene expression, and TGFß1-induced AMTN gene expression was potentiated by SNAI2 siRNA. Taken together, these data demonstrated that AMTN gene expression in the promotion of EMT was downregulated by SNAI2. The inhibitory effect of AMTN gene expression was an independent feedback on the TGFß1-Smad3 signaling pathway, suggesting that the mechanism can be engaged in maintaining homeostasis of gingival epithelial cells at JE and the wound healing phase.

Tumor necrosis factor-α regulates human follicular dendritic cell-secreted protein gene transcription in gingival epithelial cells.

Follicular dendritic cell-secreted protein (FDC-SP) is a secreted protein expressed in follicular dendritic cells, periodontal ligament and junctional epithelium. To elucidate the transcriptional regulation of the human FDC-SP gene by tumor necrosis factor-α (TNF-α), we conducted real-time PCR, Western blotting, transient transfection analyses with chimeric constructs of the FDC-SP gene promoter linked to a luciferase reporter gene, gel mobility shift and chromatin immunoprecipitation assays using Ca9-22 gingival epithelial cells. TNF-α (10 ng/ml) induced FDC-SP mRNA and protein levels at 3 hr and reached maximum at 12 hr. In transient transfection assays, TNF-α (12 hr) increased the LUC activities of constructs between -116FDCSP and -948FDCSP including the human FDC-SP gene promoter. Transcriptional stimulations by TNF-α were partially inhibited in the -345FDCSP constructs that included 3-bp mutations in the YY1, GATA, CCAAT enhancer-binding protein 2 (C/EBP2) and C/EBP3. Transcriptional activities induced by TNF-α were inhibited by tyrosine kinase, MEK1/2 and phosphoinositide 3-kinase inhibitors. The results of ChIP assays showed that YY1, GATA and C/EBPß transcription factors interacted with the YY1, GATA, C/EBP2 and C/EBP3 elements that were increased by TNF-α. These studies show that TNF-α stimulates human FDC-SP gene transcription by targeting YY1, GATA, C/EBP2 and C/EBP3 in the FDC-SP gene promoter.

Tumor necrosis factor-α stimulates human amelotin gene transcription in gingival epithelial cells.

OBJECTIVE: Amelotin (AMTN) is an enamel protein that is localized in the basal lamina of ameloblasts in their maturation stage and the internal basal lamina of junctional epithelium (JE) and it is suggested that AMTN could be involved in the dentogingival attachment. To elucidate the transcriptional regulation of human AMTN gene in inflamed gingiva, we have analyzed the effect of tumor necrosis factor-α (TNF-α) on the expression of AMTN gene in Ca9-22 and Sa3 human gingival epithelial cells. MATERIALS AND METHODS: Total RNAs were extracted from Ca9-22 and Sa3 cells after stimulation by TNF-α (10 ng/ml). AMTN mRNA and protein levels were measured by real-time PCR and Western blotting. Transient transfection analyses were completed using the various lengths of human AMTN gene promoter constructs with or without TNF-α. Gel mobility shift and chromatin immunoprecipitation assays were performed to investigate the transcription factors bindings to the human AMTN gene promoter by TNF-α. RESULTS: TNF-α (10 ng/ml) increased AMTN mRNA and protein levels after 12 h. TNF-α induced luciferase activities of human AMTN gene promoter constructs (- 211AMTN, - 353AMTN, and - 501AMTN). TNF-α-induced luciferase activities were partially inhibited in the mutation - 353AMTN constructs that included 3-bp mutations in CCAAT enhancer-binding protein 1 (C/EBP1), C/EBP2 and Ying Yang 1 (YY1) elements. Transcriptional activities induced by TNF-α were inhibited by protein kinase A, Src-tyrosine kinase, MEK1/2, p38 kinase, NF-κB, and PI3-kinase inhibitors. Gel shift assays showed that TNF-α increased nuclear proteins binding to two types of C/EBP elements (C/EBP1 and C/EBP2) and YY1 element. The results of the chromatin immunoprecipitation assays showed that C/EBPß binding to C/EBP1 and C/EBP2, and YY1 binding to YY1 were increased by TNF-α. CONCLUSIONS: These findings demonstrated that TNF-α stimulates AMTN gene transcription in human gingival epithelial cells via C/EBP1, C/EBP2, and YY1 elements in the human AMTN gene promoter.

MiR-200b attenuates IL-6 production through IKKß and ZEB1 in human gingival fibroblasts.

OBJECTIVE: MicroRNAs (miRNAs) play important roles in biological processes such as cell differentiation, development, infection, immune response, inflammation and tumorigenesis. We previously reported that the expression of miR-200b was significantly increased in inflamed gingiva compared with non-inflamed gingiva. To elucidate the roles of miR-200b in the inflamed gingiva, we have analyzed the effects of miR-200b on the expression of IL-6 in human gingival fibroblasts (HGF). MATERIALS AND METHODS: Total RNA and protein were extracted from HGF after stimulation by interleukin-1ß (IL-1ß; 1 ng/ml) or tumor necrosis factor-α (TNF-α; 10 ng/ml) and transfected with miR-200b expression plasmid or miR-200b inhibitor. IL-6, IL-1ß, inhibitor of nuclear factor kappa-B kinaseß (IKKß), Zinc-finger E-box-binding homeobox 1 (ZEB1) and E-cadherin mRNA and protein levels were analyzed by real-time PCR and Western blot. RESULTS: IL-1ß and TNF-α increased IL-6 mRNA and protein levels, and they were significantly suppressed by miR-200b overexpression, whereas they were further increased by miR-200b inhibitor in HGF. IKKß and ZEB1 which are target genes of miR-200b negatively regulate E-cadherin. MiR-200b suppressed the expression of IKKß and ZEB1 and increased E-cadherin mRNA and protein levels in HGF. CONCLUSIONS: These results suggest that miR-200b attenuates inflammatory response via IKKß and ZEB1 in periodontal tissue.

Clinical usability of aspartate aminotransferase to evaluate the prognosis of periodontal regeneration therapies: prospective, longitudinal study.

To evaluate the degree of periodontal tissue destruction, aspartate aminotransferase (AST) levels in the gingival crevicular fluid (GCF) are utilized as a predictor of periodontal therapy. We have previously shown that the usefulness of AST activities [periodontal tissue monitor (PTM) values] using a PTM-kit to evaluate the effects of initial periodontal therapy and periodontal regeneration therapy by enamel matrix derivative (EMD). This prospective, longitudinal study was conducted using 38 healthy and 80 periodontitis sites with probing depth (PD) of 5-10 mm for guided tissue regeneration (GTR) and EMD from 36 patients. GCF samples were used to evaluate PTM values at base line (BL) and after 6 months of surgeries (re-evaluation: RE), and periodontal examinations were performed concurrently. PTM values at BL were statistically improved at RE, accompanied by the improvement of periodontal parameters in both groups. PTM values and PD, and the clinical attachment level (CAL) showed high correlations. PD, CAL and bleeding on probing (BOP) were highly correlated with PTM values in both groups, whereas only PD showed a significant correlation with PTM values at RE in the GTR group. Change in the amounts of PD, CAL and BOP between BL and RE in both groups showed no correlation with PTM values. In the negative PTM value sites at BL in EMD group, the mean PD was significantly reduced at RE compared with positive PTM sites at BL. PTM values are able to be utilized as the biochemical predictor of prognosis after periodontal regeneration therapy.

Localization and expression pattern of amelotin, odontogenic ameloblast-associated protein and follicular dendritic cell-secreted protein in the junctional epithelium of inflamed gingiva.

The purpose of this study is to elucidate the localization of amelotin (AMTN), odontogenic ameloblast-associated protein (ODAM) and follicular dendritic cell-secreted protein (FDC-SP) at the junctional epithelium (JE) in Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans infected mice and inflamed and non-inflamed human gingiva. We performed immunostaining to determine the localization and expression pattern of AMTN, ODAM and FDC-SP. AMTN, ODAM and FDC-SP in A. actinomycetemcomitans infected mice did not change dramatically compared with non-infected mice. AMTN and FDC-SP expressions were observed stronger in P. gingivalis infected mice at early stage. However, at the following stage, the coronal part of the AMTN expression disappeared from the JE, and FDC-SP expression decreased due to severe inflammation by P. gingivalis. ODAM expressed internal and external basal lamina, and the expression increased not only at early stage but also at the following stage in the inflammatory JE induced by P. gingivalis. In the human gingival tissues, AMTN was detected at the surface of the sulcular epithelium and JE in the non-inflamed and inflamed gingiva, and the localization did not change the process of inflammation. ODAM and FDC-SP were more widely detected at the sulcular epithelium and JE in the non-inflamed gingiva. In the inflamed gingiva, localization of ODAM and FDC-SP was spread into the gingival epithelium, compared to AMTN. These studies demonstrated that the expression pattern of AMTN, ODAM and FDC-SP at the JE were changed during inflammation process and these three proteins might play an important role in the resistance to inflammation.

Amelotin gene expression is temporarily being upregulated at the initiation of apoptosis induced by TGFß1 in mouse gingival epithelial cells.

Amelotin (AMTN) is expressed and secreted by ameloblasts in the maturation stage of amelogenesis and persist with low levels in the junctional epithelium (JE) of erupted teeth. The purpose of this study is to investigate the transcriptional regulation of the AMTN gene by transforming growth factor beta1 (TGFß1) in gingival epithelial (GE1) cells in the apoptosis phase. Apoptosis was evaluated by the fragmentation of chromosomal DNA and TUNEL staining. A real-time PCR was carried out to examine the AMTN mRNA levels induced by TGFß1 and Smad3 overexpression. Transient transfection analyses were completed using the various lengths of mouse AMTN gene promoter constructs with or without TGFß1. Chromatin immunoprecipitation (ChIP) assays were performed to investigate the Smad3 bindings to the AMTN gene promoter by TGFß1. TGFß1-induced apoptosis in GE1 cells were detected at 24 and 48 h by DNA fragmentation and TUNEL staining. AMTN mRNA levels increased at 6 h and reached maximum at 24 h in GE1 cells. Luciferase activities of the mouse AMTN gene promoter constructs were induced by TGFß1. The results of the ChIP assays showed that there was an increase in Smad3 binding to Smad-binding element (SBE)#1 and SBE#2 after stimulation by TGFß1. Immunohistochemical localization of AMTN was detected in the JE, and the AMTN protein levels in Smad3-deficient mice were decreased compared with wild-type mice. AMTN mRNA levels were induced at the initiation of apoptosis by TGFß1, which mediated through the Smad3 bindings to SBEs in the mouse AMTN gene promoter.

Insulin-like growth factor-II regulates bone sialoprotein gene transcription.

Insulin-like growth factor-I and -II (IGF-I and IGF-II) have been found in bone extracts of several different species, and IGF-II is the most abundant growth factor stored in bone. Bone sialoprotein (BSP) is a noncollagenous extracellular matrix glycoprotein associated with mineralized connective tissues. In this study, we have investigated the regulation of BSP transcription by IGF-II in rat osteoblast-like ROS17/2.8 cells. IGF-II (50 ng/ml) increased BSP mRNA and protein levels after 6-h stimulation, and enhanced luciferase activities of the constructs pLUC3 (-116 to +60), pLUC4 (-425 to +60), pLUC5 (-801 to +60) and pLUC6 (-938 to +60). Effects of IGF-II were inhibited by tyrosine kinase, extracellular signal-regulated kinase1/2 and phosphatidylinositol 3-kinase inhibitors, and abrogated by 2-bp mutations in cAMP response element (CRE), FGF2 response element (FRE) and homeodomain protein-binding site (HOX). The results of gel shift assays showed that nuclear proteins binding to CRE, FRE and HOX sites were increased by IGF-II (50 ng/ml) at 3 and 6 h. CREB1, phospho-CREB1, c-Fos and c-Jun antibodies disrupted the formation of the CRE-protein complexes. Dlx5 and Runx2 antibodies disrupted the FRE- and HOX-protein complex formations. These studies therefore demonstrated that IGF-II increased BSP transcription by targeting CRE, FRE and HOX elements in the proximal promoter of the rat BSP gene. Moreover, phospho-CREB1, c-Fos, c-Jun, Dlx5 and Runx2 transcription factors appear to be key regulators of IGF-II effects on BSP transcription.

Effects of interleukin-11 on the expression of human bone sialoprotein gene.

Interleukin-11 (IL-11) is a bone marrow stromal fibroblast-derived cytokine with a wide spectrum of activities in different biological systems. IL-11 and IL-6 are two cytokines known to rely on osteoblast-osteoclast communication for their effects on osteoclast differentiation. Bone sialoprotein (BSP) is a mineralized connective tissue-specific protein expressed in differentiated osteoblasts, odontoblasts, and cementoblasts. To determine the molecular basis of the transcriptional regulation of the human BSP gene by IL-11, we conducted real-time polymerase chain reactions (PCR), transient transfection analyses with chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene, gel mobility shift assays, and a chromatin immunoprecipitation assay using human osteoblast-like Saos2 cells. IL-11 (20 ng/ml) increased BSP, Runx2, and Osterix mRNA levels at 6 h and the alkaline phosphatase (ALP) mRNA level at 12 h in osteoblast-like Saos2 cells. In a transient transfection assay, IL-11 (20 ng/ml, 12 h) increased luciferase activities of constructs between -60LUC and -868LUC including the human BSP gene promoter. Transcriptional stimulations by IL-11 were partially inhibited in the constructs that included 2-bp mutations in the cAMP response element 1 (CRE1, -72 to -79) and CRE2 (-667 to -674). When mutations were made in pairs of CRE1 and CRE2 in -868LUC, the effect of IL-11 on luciferase activity was almost totally abrogated. Transcriptional activities induced by IL-11 were inhibited by protein kinase A, tyrosine kinase, ERK1/2, and PI3-kinase inhibitors. Gel mobility shift analyses showed that IL-11 increased nuclear proteins binding to CRE1 and CRE2. CREB1, phospho-CREB1, c-Fos, and c-Jun antibodies disrupted the formation of CRE1 and CRE2 protein complexes. These data demonstrate that IL-11 stimulates BSP gene transcription via CRE1 and CRE2 elements in the human BSP gene promoter.

Transcriptional regulation of amelotin gene by proinflammatory cytokines in gingival fibroblasts.

Amelotin (AMTN) is a secreted protein expressed during the late stages of enamel formation and in the junctional epithelium. Among many differentially expressed genes, we found significantly increased AMTN mRNA level in inflamed gingiva by DNA microarray. The inductions of AMTN mRNA expressions in inflamed gingiva and human gingival fibroblasts (HGF) were confirmed by real-time polymerase chain reaction. To determine the molecular basis of the expression of AMTN and its regulation by proinflammatory cytokines, we have isolated and characterized the promoter region of mouse AMTN gene. Transient transfection assays were performed using luciferase constructs including mouse AMTN gene promoter. Interleukin-1ß, Interleukin-6 and tumor necrosis factor-α induced AMTN mRNA levels in HGF. These cytokines increased the luciferase activities of the AMTN promoter constructs in HGF. The results suggest that proinflammatory cytokines induce AMTN gene transcription and a role for AMTN in gingival inflammation.

Volume-based 18F-fluorodeoxyglucose positron emission tomography/computed tomography parameters correlate with delayed neck metastasis in clinical early-stage oral squamous cell carcinoma.

OBJECTIVE: There is no known preoperative marker that can effectively predict the risk of delayed neck metastasis (DNM), which is an important factor that determines the prognosis of early-stage oral cancer. In this study, we examined whether 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET)/computed tomography (CT) uptake parameters of primary cancer can predict the risk of DNM in early-stage oral squamous cell carcinoma (OSCC). METHODS: Data from patients with stage I-II OSCC who underwent surgical resection of the primary tumor without elective neck dissection between January 2009 and December 2016 were retrospectively reviewed. Patient characteristics, histopathological factors, and PET/CT parameters (maximum standardized uptake value [SUVmax], metabolic tumor volume [MTV], and total lesion glycolysis [TLG]) were evaluated for their association with DNM. DNM rates were calculated, and the parameters that were statistically significant in the univariate analysis were used as explanatory variables. Independent factors associated with DNM were identified using multivariate analysis. For all statistical analyses, p-values < 0.05 were considered statistically significant. RESULTS: Data from 71 patients were analyzed in the study. The overall DNM rate among all patients was 21.8%. The univariate analysis showed that the T classification, depth of invasion, pattern of invasion, lymphovascular invasion, SUVmax, MTV, and TLG were significant predictors of DNM. However, the multivariate analysis revealed that only the depth of invasion, MTV, and TLG were independent predictors of DNM. CONCLUSION: This study suggests that, in addition to conventional predictors, volume-based PET parameters are useful predictors of DNM in those with early-stage OSCC.

Changes in the components of salivary exosomes due to initial periodontal therapy.

PURPOSE: Exosomes are membrane vesicles that are present in body fluids and contain proteins, lipids, and microRNA (miRNA). Periodontal tissue examinations assess the degree of periodontal tissue destruction according to the probing depth (PD), clinical attachment loss (CAL), bleeding on probing, and X-ray examinations. However, the accurate evaluation of the prognosis of periodontitis is limited. In this study, we collected saliva from patients before and after initial periodontal therapy (IPT) and compared changes in the clinical parameters of periodontitis with changes in the components of salivary exosomes. METHODS: Saliva was collected from patients with stage III and IV periodontitis at the first visit and post-IPT. Exosomes were purified from the saliva, and total protein and RNA were extracted. Changes in expression levels of C6, CD81, TSG101, HSP70, and 6 kinds of miRNA were analyzed by western blots and real-time polymerase chain reaction. RESULTS: Patients with increased C6 expression after IPT had significantly higher levels of periodontal inflamed surface area (PISA), miR-142, and miR-144 before and after IPT than patients with decreased C6 expression after IPT. Patients with decreased and unchanged CD81 expression after IPT showed significantly higher PD, CAL, and PISA before IPT than after IPT. Patients with decreased and unchanged TSG101 expression after IPT had significantly higher PD before IPT than after IPT. Patients with increased HSP70 expression after IPT had significantly higher PD and PISA before and after IPT than patients with unchanged HSP70 after IPT. The expression levels of miR-142, miR-144, miR-200b, and miR-223 changed with changes in the levels of C6, CD81, TSG101, and HSP70 in the salivary exosomes of periodontitis patients before and after IPT. CONCLUSIONS: The expression levels of proteins and miRNAs in salivary exosomes significantly changed after IPT in periodontitis patients, suggesting that the components of exosomes could serve as biomarkers for periodontitis.