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.

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.

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.

Impact of adjunctive procedures on recombinant human fibroblast growth factor-2-mediated periodontal regeneration therapy: A retrospective study.

BACKGROUND: Human fibroblast growth factor-2 (rhFGF-2) therapy has been used for periodontal tissue regeneration. However, few studies have reported their adjunctive procedures based on strategy of tissue engineering. The aim of this retrospective study is to assess the adjunctive effects of modified papilla preservation technique (mPPT) and combination with autogenous bone grafts (AG) on the rhFGF-2 therapy. METHODS: Total of 44 sites underwent rhFGF-2 therapies and the evaluations in the survey periods. The primary outcome was set to the radiographic bone fill by radiographic examinations at 6 and 12 months after surgeries. We analyzed the correlation between influencing factors and the primary outcome, and differences of therapeutic effect by combination therapy with mPPT and that with AG. RESULTS: After surgeries, probing depth (PD), clinical attachment level (CAL) and bone defects significantly improved. The improvements of radiographic bone fill were significantly positive correlated with a number of bone walls, combination with mPPT, and AG at 6 months after surgeries, and with combination with mPPT and AG at 12 months after surgeries. The significant differences of improvements of radiographic bone fill were demonstrated between combination with or without mPPT at 12 months after surgeries, and with or without AG at 6 and 12 months after surgeries. Moreover, the multiple linear regression analysis for the radiographic bone fill indicated the significant regression coefficient with conducts of mPPT. CONCLUSIONS: mPPT and AG had powerfully adjunctive effects on rhFGF-2 therapy. Further studies are needed in order to verify by randomized clinical trials.

C/EBPß and YY1 bind and interact with Smad3 to modulate lipopolysaccharide-induced amelotin gene transcription in mouse gingival epithelial cells.

Junctional epithelium (JE) develops from reduced enamel epithelium during tooth formation and is critical for the maintenance of healthy periodontal tissue through ensuring appropriate immune responses and the rapid turnover of gingival epithelial cells. We have previously shown a relationship between inflammatory cytokines and expression of JE-specific genes, such as amelotin (AMTN), in gingival epithelial cells. Here, we elucidated the effects of Porphyromonas gingivalis-derived lipopolysaccharide (Pg LPS) on Amtn gene transcription and the interaction of transcription factors. To determine the molecular basis of transcriptional regulation of the Amtn gene by Pg LPS, we performed real-time PCR and carried out luciferase assays using a mouse Amtn gene promoter linked to a luciferase reporter gene in mouse gingival epithelial GE1 cells. Gel mobility shift and chromatin immunoprecipitation assays were performed to identify response elements bound to LPS-induced transcription factors. Next, we analyzed protein levels of the LPS-induced transcription factors and the interaction of transcription factors by western blotting and immunoprecipitation. LPS increased Amtn mRNA levels and elevated luciferase activities of constructs containing regions between -116 and -238 of the mouse Amtn gene promoter. CCAAT/enhancer-binding protein (C/EBP) 1-, C/EBP2- and Ying Yang 1 (YY1)-nuclear protein complexes were increased by LPS treatment. Furthermore, we identified LPS-modulated interactions with C/EBPß, YY1 and Smad3. These results demonstrate that Pg LPS regulates Amtn gene transcription via binding of C/EBPß-Smad3 and YY1-Smad3 complexes to C/EBP1, C/EBP2 and YY1 response elements in the mouse Amtn gene promoter.

IL-1ß and TNF-α regulate mouse amelotin gene transcription in gingival epithelial cells.

Amelotin (AMTN) is an enamel protein expressed in maturation-stage ameloblasts and junctional epithelium. To clarify the transcriptional regulation of the AMTN gene by interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), we conducted real-time PCR, Western blotting, transient transfection analyses with luciferase constructs including various lengths of the mouse AMTN gene promoter, and gel shift and chromatin immunoprecipitation assays using mouse gingival epithelial GE1 cells. The levels of AMTN mRNA and protein in GE1 cells were increased after 6 h of stimulation with IL-1ß (1 ng/mL) and TNF-α (10 ng/mL). IL-1ß and TNF-α induced luciferase activities of the constructs between -116AMTN and -705AMTN including the mouse AMTN gene promoter. Transcriptional activation by IL-1ß and TNF-α was partially inhibited in -460AMTN including 3-bp mutations in the CCAAT-enhancer-binding protein 1 (C/EBP1), C/EBP2 and Yin Yang 1 (YY1) elements. Transcriptional activities induced by IL-1ß and TNF-α were inhibited by tyrosine kinase, MEK1/2 and PI3-kinase inhibitors. Results of ChIP assays showed that IL-1ß and TNF-α increased C/EBPß and YY1 binding to the C/EBP1, C/EBP2 and YY1 elements. These results demonstrate that IL-1ß and TNF-α increase AMTN gene transcription via the C/EBP1, C/EBP2 and YY1 elements in the mouse AMTN gene promoter.

Effects of interleukin-1ß on human follicular dendritic cell-secreted protein gene expression in periodontal ligament cells.

Follicular dendritic cell-secreted protein (FDC-SP) is expressed in FDCs, human periodontal ligament (HPL) cells, and junctional epithelium. To evaluate the effects of interleukin-1 beta (IL-1ß) on FDC-SP gene expression in immortalized HPL cells, FDC-SP mRNA and protein levels in HPL cells following stimulation by IL-1ß were measured by real-time polymerase chain reaction and Western blotting. Luciferase (LUC), gel mobility shift, and chromatin immunoprecipitation (ChIP) analyses were performed to study the interaction between transcription factors and promoter regions in the human FDC-SP gene. IL-1ß (1 ng/mL) induced the expression of FDC-SP mRNA and protein levels at 3 h, and reached maximum levels at 12 h. IL-1ß increased LUC activities of constructs (-116FDCSP - -948FDCSP) including the FDC-SP gene promoter. Transcriptional inductions by IL-1ß were partially inhibited by 3-base-pair (3-bp) mutations in the Yin Yang 1 (YY1), GATA, CCAAT-enhancer-binding protein2 (C/EBP2), or C/EBP3 in the -345FDCSP. IL-1ß-induced -345FDCSP activities were inhibited by protein kinase A, tyrosine-kinase, mitogen-activated protein kinase (MEK)1/2, and PI3-kinase inhibitors. The results of gel shift and ChIP assays revealed that YY1, GATA, and C/EBP-ß interacted with the YY1, GATA, C/EBP2, and C/EBP3 elements that were increased by IL-1ß. These studies demonstrate that IL-1ß increases FDC-SP gene transcription in HPL cells by targeting YY1, GATA, C/EBP2, and C/EBP3 in the human FDC-SP gene promoter.

Transcriptional regulation of human amelotin gene by interleukin-1ß.

One of the major causes of tooth loss is chronic inflammation of the periodontium, the tissues surrounding the tooth. Amelotin (AMTN) is a tooth enamel protein which is expressed in maturation-stage ameloblasts and also in the internal basal lamina of junctional epithelium, a unique epithelial structure attached to the tooth surface which protects against the constant microbiological challenge to the periodontium. Localization of AMTN suggests that its function could be involved in the dentogingival attachment. The purpose of this study was to investigate the effect of interleukin-1ß (IL-1ß) on AMTN gene transcription in human gingival epithelial Ca9-22 cells. IL-1ß increased AMTN mRNA and protein levels at 3 h, and the levels reached maximum at 6 and 12 h. IL-1ß induced luciferase activities of human AMTN gene promoter constructs (-211, -353, -501, -769, and -950AMTN), but these activities were partially inhibited in -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 IL-1ß were abrogated by protein kinase A (PKA), tyrosine kinase, mitogen-activated protein kinase kinase (MEK1/2), and phosphatidylinositol 3-kinase (PI3K) inhibitors. Gel shift and ChIP assays showed that IL-1ß increased C/EBPß binding to C/EBP1 and C/EBP2, and YY1 binding to YY1 elements after 3 h, and that these DNA-protein interactions were inhibited by PKA, tyrosine kinase, MEK1/2, and PI3K inhibitors. These results demonstrated that IL-1ß increases AMTN gene transcription in human gingival epithelial cells mediated through C/EBP1, C/EBP2, and YY1 elements in the human AMTN gene promoter.

TGFß1-induced Amelotin gene expression is downregulated by Bax expression in mouse gingival epithelial cells.

Amelotin (AMTN) is induced upon initiation of apoptosis by transforming growth factor beta1 (TGFß1) and is mediated by Smad3 in gingival epithelial cells (GE1 cells). This upregulation of AMTN gene expression is temporary, and the mechanism responsible is still unclear. The present study investigated the transcriptional downregulation of TGFß1-induced AMTN gene expression in GE1 cells during the progression of apoptosis. To examine time-dependent changes in the levels of AMTN, Smad3 and Bax mRNA induced by TGFß1, real-time PCR analyses were performed. Immunocytochemistry was carried out to detect the expression of Smad3 and Bax. Transient transfection analyses were performed using mouse AMTN gene promoter constructs of various lengths including Smad response elements (SBEs), in the presence or absence of TGFß1. Changes in Smad3 binding to SBEs resulting from overexpression of Bax were examined using ChIP assays. Overexpression of Bax dramatically downregulated the levels of TGFß1-induced AMTN mRNA and transcription of the AMTN gene. Smad3 binding to SBEs in the mouse AMTN gene promoter was induced by overexpression of Smad3 or TGFß1, and this was inhibited by Bax overexpression. These results show that the levels of AMTN mRNA induced by TGFß1 and Smad3 are decreased by robust expression of Bax in gingival epithelial cells.