Carbonate apatite increases gene expression of osterix and bone morphogenetic protein 2 in the alveolar ridge after socket grafting.

PURPOSE: After tooth extraction, preservation of the alveolar ridge by socket grafting attenuates bone resorption. Runt-related transcription factor 2 (RUNX2) and SP7/Osterix (OSX) are transcription factors playing an important role in osteoblast differentiation. The purpose of this study was to evaluate the effects of carbonate apatite (CO3Ap) on osteoblast-related gene and protein expression after socket grafting. METHODS: Alveolar bone and new bone after CO3Ap grafting were collected at the time of implant placement. Levels of mRNA for RUNX2, SP7/OSX, bone morphogenetic protein 2 (BMP2), BMP7 and platelet derived growth factor B were determined by real-time PCR. Immunostaining was performed using antibodies against RUNX2, SP7/OSX, vimentin and cytokeratin. To evaluate bone resorption rates, cone-beam CT (CBCT) imaging was performed after socket grafting and before implant placement. RESULTS: CBCT imaging showed that the average degree of bone resorption at the CO3Ap graft site was 7.15 ± 3.79%. At the graft sites, levels of SP7/OSX and BMP2 mRNA were significantly increased. Replacement of CO3Ap with osteoid was evident histologically, and in the osteoid osteoblast-like cells were stained for SP7/OSX and vimentin. CONCLUSION: These results show that gene expression of both SP7/OSX and BMP2 can be induced by CO3Ap, suggesting that increased expression of SP7/OSX and vimentin may be involved in the BMP pathway.

Comparison of early wound healing using modified papilla preservation technique between enamel matrix derivative and recombinant human fibroblast growth factor.

PURPOSE: Enamel matrix derivative (EMD) has demonstrated beneficial effects on wound healing following surgery. However, the effects of recombinant human fibroblast growth factor 2 (rhFGF-2) in periodontal regeneration therapy have not been extensively studied. This retrospective study was conducted to compare the wound healing outcomes of the modified papilla preservation technique (mPPT) between EMD and rhFGF-2 therapies. METHODS: A total of 79 sites were evaluated for early wound healing using the modified early wound healing index (mEHI), which included 6 items: incision, fibrin clotting, step, redness, swelling, and dehiscence. A numeric analog scale, along with postoperative images of the 6 mEHI items, was established and used for the evaluations. The inter-rater reliability of the mEHI was assessed via intraclass correlation coefficients (ICCs). After adjusting for factors influencing the mPPT, the differences in mEHI scores between the EMD and rhFGF-2 groups were statistically analyzed. Additionally, radiographic bone fill (RBF) was evaluated 6 months after surgery. RESULTS: The ICC of the mEHI was 0.575. The mEHI, redness score, and dehiscence scores were significantly higher in the rhFGF-2 group (n=33) than in the EMD group (n=46). Similar results were observed in the subgroup of patients aged 50 years or older, but not in those younger than 50 years. In the subgroup with non-contained bone defects, related results were noted, but not in the subgroup with contained bone defects. However, early wound healing did not correlate with RBF at 6 months after surgery. CONCLUSIONS: Within the limitations of this study, the findings suggest that early wound healing following the use of mPPT with rhFGF-2 is somewhat superior to that observed after mPPT with EMD. However, mEHI should be improved for use as a predictive tool for early wound healing and to reflect clinical outcomes after surgery.

Role of Macrophage Colony-Stimulating Factor for Staphylococcal Infection in the Oral Cavity.

OBJECTIVE: There are few valid indicators of oral infection owing to the complexity of pathogenic factors in oral diseases. Salivary markers are very useful for scrutinizing the symptoms of disease. To provide a reliable and useful predictive indicator of infection for opportunistic pathogens in individuals with compromised immune systems, such as those with periodontal diseases and Human Immunodeficiency Virus (HIV), this study examines opportunistic pathogens such as C. albicans and staphylococci and macrophage colony-stimulating factor (M-CSF) and CA125/MUC16 in saliva. The aim was to explore the correlations investigated among these factors. METHODS: Samples were divided into two groups (based on patient sex, the absence and presence of dentures in elderly, or HIV-positive patients and healthy subjects), and the correlation was analyzed in two groups of elderly patients with periodontal disease (64.5 ± 11.2 years old) and HIV-infected patients (41.9 ± 8.4 years old). Healthy subjects (33.8 ± 9.1 years old) were also analyzed as a control. Levels of C. albicans, staphylococci, and M-CSF, which is an immunological factor for the differentiation of macrophage, and CA125/MUC16, which provides a protective lubricating barrier against infection, were investigated. RESULTS: A significant and positive correlation between the levels of M-CSF and staphylococci was found in elderly individuals and HIV-positive patients treated with antiretroviral therapy. A significant and positive correlation between the levels of M-CSF and CD125/MUC16 was also found in both patients. These correlations were enhanced in both patients as compared with healthy subjects. CONCLUSION: Salivary M-CSF might be useful as a new indicator of opportunistic infection caused by staphylococci and a defense against infection in immunocompromised hosts.

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.

Utility of a haemoglobin test of gingival crevicular fluid: A multicentre, observational study.

OBJECTIVE: The purpose of this study was to verify the accuracy and utility of clinical parameters (plaque index, gingival crevicular fluid volume, probing depth, clinical attachment level, bleeding on probing and gingival index) and biochemical parameters (aspartate aminotransferase, protein and haemoglobin) in a longitudinal analysis during the supportive periodontal therapy period. SUBJECTS AND METHODS: A total of 279 test sites of 128 patients were investigated clinically and biochemically. After the first examination of clinical and biochemical parameters, periodontal support treatments were administered immediately and performed once every three months up to the second examination. RESULTS: All of the clinical and biochemical parameters were significantly lower at the second examination than at the first, except for the plaque index and bleeding on probing. Of these parameters, in particular, aspartate aminotransferase and haemoglobin in the gingival crevicular fluid were significantly reduced compared to those of the first examination in both the ≤4 and ≥5 mm probing depth groups, and they clearly suggested that periodontitis tended to recover. CONCLUSION: Adding the haemoglobin test to the bleeding on probing test strongly improves the accuracy of measurement of clinical parameters after periodontal treatment.

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.

Epstein-Barr Virus Promotes the Production of Inflammatory Cytokines in Gingival Fibroblasts and RANKL-Induced Osteoclast Differentiation in RAW264.7 Cells.

Periodontitis is an inflammatory condition that causes the destruction of the supporting tissues of teeth and is a major public health problem affecting more than half of the adult population worldwide. Recently, members of the herpes virus family, such as the Epstein-Barr virus (EBV), have been suggested to be involved in the etiology of periodontitis because bacterial activity alone does not adequately explain the clinical characteristics of periodontitis. However, the role of EBV in the etiology of periodontitis is unknown. This study aimed to examine the effect of inactivated EBV on the expression of inflammatory cytokines in human gingival fibroblasts (HGFs) and the induction of osteoclast differentiation. We found that extremely high levels of interleukin (IL)-6 and IL-8 were induced by inactivated EBV in a copy-dependent manner in HGFs. The levels of IL-6 and IL-8 in HGFs were higher when the cells were treated with EBV than when treated with lipopolysaccharide and lipoteichoic acid. EBV induced IκBα degradation, NF-κB transcription, and RAW264.7 cell differentiation into osteoclast-like cells. These findings suggest that even without infecting the cells, EBV contributes to inflammatory cytokine production and osteoclast differentiation by contact with oral cells or macrophage lineage, resulting in periodontitis onset and progression.

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.

Prospective Longitudinal Changes in the Periodontal Inflamed Surface Area Following Active Periodontal Treatment for Chronic Periodontitis.

Periodontal disease is a chronic inflammatory disease of the periodontal tissue. The periodontal inflamed surface area (PISA) is a proposed index for quantifying the inflammatory burden resulting from periodontitis lesions. This study aimed to investigate longitudinal changes in the periodontal status as evaluated by the PISA following the active periodontal treatment. To elucidate the prognostic factors of PISA, mixed-effect modeling was performed for clinical parameters, tooth-type, and levels of periodontal pathogens as independent variables. One-hundred-twenty-five patients with chronic periodontitis who completed the active periodontal treatment were followed-up for 24 months, with evaluations conducted at 6-month intervals. Five-times repeated measures of mean PISA values were 130+/-173, 161+/-276, 184+/-320, 175+/-417, and 209+/-469 mm2. Changes in clinical parameters and salivary and subgingival periodontal pathogens were analyzed by mixed-effect modeling. Plaque index, clinical attachment level, and salivary levels of Porphyromonas gingivalis were associated with changes in PISA at the patient- and tooth-level. Subgingival levels of P. gingivalis and Prevotella intermedia were associated with changes in PISA at the sample site. For most patients, changes in PISA were within 10% of baseline during the 24-month follow-up. However, an increase in the number of bleeding sites in a tooth with a deep periodontal pocket increased the PISA value exponentially.

Estimation of the Periodontal Inflamed Surface Area by Simple Oral Examination.

The periodontal inflamed surface area (PISA) is a useful index for clinical and epidemiological assessments, since it can represent the inflammation status of patients in one contentious variable. However, calculation of the PISA is difficult, requiring six point probing depth measurements with or without bleeding on probing on 28 teeth, followed by data input in a calculation program. More simple methods are essential for screening periodontal disease or in epidemiological studies. In this study, we tried to establish a convenient partial examination method to estimate PISA. Cross-sectional data of 254 subjects who completed active periodontal therapy were analyzed. Teeth that represent the PISA value were selected by an item response theory approach. The maxillary second molar, first premolar, and lateral incisor and the mandibular second molar and lateral incisor were selected. The sum of the PISAs of these teeth was significantly correlated with the patient's PISA (R2 = 0.938). More simply, the sum of the maximum values of probing pocket depth with bleeding for these teeth were also significantly correlated with the patient's PISA (R2 = 0.6457). The simple model presented in this study may be useful to estimate PISA.

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.

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.

Optimal Examination Sites for Periodontal Disease Evaluation: Applying the Item Response Theory Graded Response Model.

Periodontal examination data have a complex structure. For epidemiological studies, mass screenings, and public health use, a simple index that represents the periodontal condition is necessary. Periodontal indices for partial examination of selected teeth have been developed. However, the selected teeth vary between indices, and a justification for the selection of examination teeth has not been presented. We applied a graded response model based on the item response theory to select optimal examination teeth and sites that represent periodontal conditions. Data were obtained from 254 patients who participated in a multicenter follow-up study. Baseline data were obtained from initial follow-up. Optimal examination sites were selected using item information calculated by graded response modeling. Twelve sites-maxillary 2nd premolar (palatal-medial), 1st premolar (palatal-distal), canine (palatal-medial), lateral incisor (palatal-central), central incisor (palatal-distal) and mandibular 1st premolar (lingual, medial)-were selected. Mean values for clinical attachment level, probing pocket depth, and bleeding on probing by full mouth examinations were used for objective variables. Measuring the clinical parameters of these sites can predict the results of full mouth examination. For calculating the periodontal index by partial oral examination, a justification for the selection of examination sites is essential. This study presents an evidence-based partial examination methodology and its modeling.

Effects of Initial Periodontal Therapy on Heat Shock Protein 70 Levels in Gingival Crevicular Fluid from Periodontitis Patients.

Periodontitis is an inflammatory disease of periodontium which is caused by periodontopathic bacteria. Moreover, various cytokines such as interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and IL-6 are expressed in the inflamed periodontium. Heat shock proteins (HSPs) protect cells from abnormal conditions including inflammation, microbial infection and diseases. The 70-kDa HSPs (HSP70s) are major HSPs that express in the inflamed tissues. In this study, an enzyme-linked immunosorbent assay was applied to measure the levels of HSP70 in gingival crevicular fluid (GCF) from two periodontal pockets in each of 10 patients with Stage III, Grade B periodontitis. Sites with probing pocket depth (PPD) of ≤3 mm were named the healthy control (HC) sites, and sites with PPD of ≥5 mm were named the diseased sites. HSP70 levels in GCF were expressed higher at diseased sites than at HC sites, and decreased after initial periodontal therapy at diseased sites. These results suggest the association of HSP70 with the stage of periodontitis.

Anti-heat shock protein 70 levels in gingival crevicular fluid of Japanese patients with chronic periodontitis.

Periodontitis is an inflammatory disease involving complex tripartite cross-interactions among bacterial, host and environment factors. Heat shock proteins (Hsps) are a protein family produced in response to stress conditions. Hsps protect cells under adverse circumstances such as infection, inflammation and disease. One of the causes of periodontal disease is thought to be an imbalance in the expression of Hsps and anti-Hsp antibodies. Hsps are classified according to their molecular weight, and one of the major ones is Hsp70. In the present study, enzyme-linked immunosorbent assay was used to measure the levels of anti-Hsp70 antibody in gingival crevicular fluid (GCF) from two gingival sulci in each of nine patients with chronic periodontitis (CP): one healthy control (HC) site with a probing pocket depth (PPD) of ≤3 mm and one CP site with a PPD of >5 mm. Anti-Hsp70 antibody levels in GCF were higher at HC sites than at CP sites. Moreover, the anti-Hsp70 antibody levels were found to increase after initial periodontal therapy at both HC and CP sites. These results suggest an association of anti-Hsp70 antibody with periodontitis.

Effect of topical administration of propolis in chronic periodontitis.

To investigate the effect of topical administration of propolis (a honeybee product) or curry leaf (an herbal product) into the periodontal pockets of periodontitis patients, a double-blind controlled clinical trial was conducted with 24 subjects including one drop-out diagnosed with moderate-to-advanced chronic periodontitis who completed initial periodontal therapy. They were randomly allocated to the following treatments: placebo, propolis, curry leaf, and minocycline. Gingival crevicular fluid (GCF) samples collected before and after the intervention were analyzed to quantify the number of total bacteria and number of six major periodontopathic bacteria by real-time PCR. Periodontitis-related clinical parameters were also analyzed. Among the six propolis-treated patients whose GCF samples were P. gingivalis-positive, three patients converted to be P. gingivalis-negative after the intervention. The minocycline-treated group exhibited a decrease in probing pocket depth (PPD) with statistically significant improvement, but not gain of clinical attachment level (CAL). Both PPD and CAL have been improved in the propolis-treated group at a statistically significant level, but not the curry leaf-treated group. In conclusion, treatment with propolis significantly improved both PPD and CAL, together with a tendency towards reduced P. gingivalis burden in GCF. It is likely that a propolis-based therapy becomes an alternative treatment option for chronic periodontitis during supportive periodontal therapy.

IL-1ß enhances cell adhesion through laminin 5 and ß4 integrin in gingival epithelial cells.

The junctional epithelium and dental enamel adhere because of hemidesmosomes containing laminin 5 and α6ß4 integrin, which are important adhesion molecules in the internal basal lamina. Interleukin (IL)-1 is important in the pathogenesis of periodontal disease. IL-1ß induces bone resorption by activating osteoclasts; however, its effects on adhesion of epithelial cells remain to be clarified. Laminin ß3, ß4 integrin, and focal adhesion kinase mRNA levels were higher after 1 h and 3 h of stimulation with IL-1ß (1 ng/mL), and IL-1ß, type I α1, and type IV α1 collagen mRNA levels were higher after 1 h and lower after 3 h of stimulation with IL-1ß. After IL-1ß stimulation, colocalization of laminin 5 and ß4 integrin was increased after 1 h, colocalization of ß4 integrin and plectin was increased after 1 h and decreased after 3 h, and colocalization of ß4 integrin and type IV collagen was decreased after 3 h. Wound healing assays showed that IL-1ß treatment (3 h) delayed wound healing. These results suggest that IL-1ß enhances cell adhesion by altering localization of epithelial adhesion molecules.

Induction of chondrogenic or mesenchymal stem cells from human periodontal ligament cells through inhibition of Twist2 or Klf12.

Periodontitis leads to destruction of periodontal ligament, cementum and alveolar bone. Regeneration of periodontal tissue is dependent on mesenchymal stem cells (MSC) present in the periodontal ligament, and transcription factors determine the direction of MSC differentiation. The present study was conducted to investigate the transcription factors that are crucial for maintaining the characteristics of the periodontal ligament. The mRNA levels of several transcription factors were measured in cultured human periodontal ligament (HPDL) cells, human gingival fibroblasts and osteoblast-like Saos2 cells. HPDL cells were transfected for 72 h with siTwist2, siKlf12, or siMix (siTwist2, siPax9, and siKlf12). The cells were then harvested and subjected to real-time PCR and Western blotting. siTwist2 suppressed the levels of Twist2, Sox2 and Col1a1 mRNAs, and increased those of Sox5 and aggrecan mRNAs. siKlf12 decreased the mRNA levels of Klf12, Runx3, Zfp521, and Stab2, and increased those of Sox2, Klf4, and the MSC markers CD90 and CD105. These results suggest that transfection with siMix and siTwist2 induced chondrogenesis, and that siKlf12 induced the differentiation of MSC in HPDL cells. Thus, inhibition of Twist2 or Klf12 induced the differentiation of chondrogenic or mesenchymal stem cells in this setting, suggesting that the characteristics of HPDL cells may be altered by inhibition of specific transcription factors.

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.

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.