Periodontal tissue susceptibility to glycaemic control in type 2 diabetes.

AIM: To assess the direct effect of intensive glycaemic control on periodontal tissues in patients with diabetes mellitus. MATERIALS AND METHODS: Twenty-nine patients with type 2 diabetes were enrolled and hospitalized to receive a 2-week intensive glycaemic control regimen. We observed and analysed the systemic and oral disease indicators before and after treatment and clarified the indicators related to periodontal inflammation. RESULTS: A significant reduction in glycaemic and periodontal parameters, including glycated albumin levels and periodontal inflamed surface area (PISA), was observed after treatment. The changes in PISA per tooth, indicative of periodontal healing, exhibited a bimodal distribution; the patients were divided into two groups on this basis. Correlations were observed between the changes in PISA per tooth and fasting plasma glucose, acetoacetic acid, and beta-hydroxybutyrate levels in the PISA-improved group. Significantly lower levels of C-peptide, coefficient of variation of R-R interval, and ankle-brachial pressure index were observed before treatment in the PISA non-improved group. CONCLUSIONS: Glycaemic control treatment can effectively improve periodontitis in patients with type 2 diabetes, even in the absence of specific periodontal treatments. However, the periodontal responsiveness to glycaemic control treatment depends on the systemic condition of the patient.

Estimated economic costs of dental implants among older South Korean adults, 2015-2018.

OBJECTIVE: Dental implants are a considerable financial burden for elderly people and their caregivers. This study aimed to calculate the estimated economic costs of dental implants on the Korean older population. MATERIALS AND METHODS: The economic costs of dental implants for adults aged 75 years and older were estimated from a societal perspective, considering both direct and indirect expenditures. We used data from the Korean National Health Insurance Service for the period 2015-2018 to estimate the medical costs. Noninsured medical costs were estimated from research on medical expenses by the National Health Insurance Review and Assessment Service. Indirect costs related to transportation fees and time loss were obtained. The Cochran Armitage trend test was performed to examine the trend of the economic burden of dental implants. RESULTS: The estimated economic costs showed from 2015 to 2018 (total costs: $26.54-55.66 million, total costs after discount: $17.11-39.56 million). Direct costs, including insured and noninsured medical costs, represented from $25.81 to $47.03 million. Indirect costs, including transportation and time costs, ranged from $0.73 to $1.63 million. The impact of the total dental implant costs was 0.0018%-0.0034% of the Korean annual gross domestic product and 1.03%-1.59% of the annual total costs of dental care benefits. CONCLUSIONS: The estimated economic burden of dental implants significantly increased from 2015 to 2018 in older South Korean adults. These results will provide a foundation and guidance for further health economic studies on the burden of dental implants in the elderly population.

Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of coxsackievirus and adenovirus receptor.

Porphyromonas gingivalis is a major pathogen of human periodontitis and dysregulates innate immunity at the gingival epithelial surface. We previously reported that the bacterium specifically degrades junctional adhesion molecule 1 (JAM1), causing gingival epithelial barrier breakdown. However, the functions of other JAM family protein(s) in epithelial barrier dysregulation caused by P. gingivalis are not fully understood. The present results show that gingipains, Arg-specific or Lys-specific cysteine proteases produced by P. gingivalis, specifically degrade coxsackievirus and adenovirus receptor (CXADR), a JAM family protein, at R145 and K235 in gingival epithelial cells. In contrast, a gingipain-deficient P. gingivalis strain was found to be impaired in regard to degradation of CXADR. Furthermore, knockdown of CXADR in artificial gingival epithelium increased permeability to dextran 40 kDa, lipopolysaccharide and peptidoglycan, whereas overexpression of CXADR in a gingival epithelial tissue model prevented penetration by those agents following P. gingivalis infection. Together, these results suggest that P. gingivalis gingipains breach the stratified squamous epithelium barrier by degrading CXADR as well as JAM1, which allows for efficient transfer of bacterial virulence factors into subepithelial tissues. TAKEAWAYS: P. gingivalis, a periodontal pathogen, degraded coxsackievirus and adenovirus receptor (CXADR), a JAM family protein, in gingival epithelial tissues. P. gingivalis gingipains, cysteine proteases, degraded CXADR at R145 and K235. CXADR degradation by P. gingivalis caused increased permeability to lipopolysaccharide and peptidoglycan through gingival epithelial tissues.

Validity of a combination of periodontal pathogens and salivary biomarkers as predictors of periodontitis.

OBJECTIVE: Chronic periodontitis is caused by multiple risk factors. To predict chronic periodontitis in older people, we evaluated the association between a combination of major periodontal pathogens and salivary biomarkers and the presence of periodontitis. METHODS: Stimulated saliva samples were collected to analyze the prevalence of Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Prevotella intermedia, as well as four biomarkers: interleukin (IL)-1ß, IL-6, tumor necrosis factor-α (TNF-α), and prostaglandin E2 (PGE2). A total of 201 Japanese patients were recruited. Oral examinations ware performed to determine chronic periodontitis as measured by Community Periodontal Index. The sociodemographic and behavioral characteristics were also obtained, and the parameters were adjusted as potential confounders to employ statistical models. RESULTS: The odds ratio (OR) for the presence of P. gingivalis and the third tertile level of IL-1ß as compared with the absence of P. gingivalis and the lowest tertile of IL-1ß was highest in individuals with periodontitis (OR = 13.98; 95% confidence interval [CI] 3.87-50.52) with the best level (0.79) of area under the curve (AUC) based on the receiver operating characteristic curve. The OR for the presence of P. gingivalis and the third tertile of PGE2 was 7.76 (CI 1.89-31.91) with an AUC of 0.78. The coexistence of more than two periodontal bacteria and the third tertile of PGE2 was also strongly associated with chronic periodontitis (OR = 9.23, 95% CI 2.38-35.79) with an AUC of 0.76. CONCLUSIONS: The combined information of the presence of P. gingivalis in stimulated saliva, and higher levels of salivary IL-1ß may play a vital role in the detection and prediction of chronic periodontitis in older adults.

Supplemental or dietary intake of omega-3 fatty acids for the treatment of periodontitis: A meta-analysis.

AIM: To evaluate the intervention effect of omega-3 fatty acids on changes in periodontal parameters. MATERIALS AND METHODS: This meta-analysis included studies published in English language between 2010 and 2020, which were extracted from the Cochrane Library, EMBASE, and PubMed databases. The effects of omega-3 fatty acid intervention were investigated using the amount of omega-3 intake, periodontal pocket depth (PPD), clinical attachment loss (CAL), and bleeding on probing (BOP). The random-effects model was generated for data analysis. To obtain robustness of the model, sensitivity analysis was implemented. Subgroup analyses were performed based on the intervention period for each parameter. RESULTS: All 13 studies included in the meta-analysis were interventional, randomized controlled trials. Two studies implemented omega-3 fatty acid-rich diets, while 11 studies used supplements. Risk of bias was low, and publication bias was not shown. Meta-analysis showed a statistically significant PPD reduction (standardized mean difference [SMD] = -0.81, absolute mean difference [MD] = -0.44 mm), CAL gain (SMD = -0.77, MD = -0.51 mm), and BOP reduction (SMD = -0.65, MD = -9.45%) for the omega-3 fatty acid intervention overall. CONCLUSION: This study suggests that supplemental or dietary intake of omega-3 fatty acids for the treatment of periodontitis may have a positive impact on the disease.

Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of junctional adhesion molecule 1.

Porphyromonas gingivalis is a major pathogen in severe and chronic manifestations of periodontal disease, which is one of the most common infections of humans. A central feature of P. gingivalis pathogenicity is dysregulation of innate immunity at the gingival epithelial interface; however, the molecular basis underlying P. gingivalis-dependent abrogation of epithelial barrier function remains unknown. Gingival epithelial cells express junctional adhesion molecule (JAM1), a tight junction-associated protein, and JAM1 homodimers regulate epithelial barrier function. Here we show that Arg-specific or Lys-specific cysteine proteases (gingipains) secreted by P. gingivalis can specifically degrade JAM1 at K134 and R234 in gingival epithelial cells, resulting in permeability of the gingival epithelium to 40 kDa dextran, lipopolysaccharide (LPS), and proteoglycan (PGN). A P. gingivalis strain lacking gingipains was impaired in degradation of JAM1. Knockdown of JAM1 in monolayer cells and a three-dimensional multilayered tissue model also increased permeability to LPS, PGN, and gingipains. Inversely, overexpression of JAM1 in epithelial cells prevented penetration by these agents following P. gingivalis infection. Our findings strongly suggest that P. gingivalis gingipains disrupt barrier function of stratified squamous epithelium via degradation of JAM1, allowing bacterial virulence factors to penetrate into subepithelial tissues.

Association between mastication-related factors and the prevalence of dementia in Korean elderly women visiting senior centres.

OBJECTIVE: The purpose of this study was to evaluate the mastication ability of elderly women by assessing the number of their remaining teeth, subjective mastication comfort, subjective chewing ability of five food items, relative occlusion balance and mastication performance involving in chewing gum. BACKGROUND: Korea has already entered an aged society, issues related to the elderly are also growing; for example, dementia is emerging as a social problem. In addition, oral health of the elderly is very important because it is directly related to nutrient intake. A total of 101 subjects aged ≥65 who attended senior citizen centres in Daegu city provided consent and were included in this study. MATERIALS AND METHODS: The Korean version of the Mini-Mental State Examination (MMSE-DS) was used to evaluate cognitive function. To assess the degree of objective mastication, we measured colour changes using a chewing gum and posterior occlusion force using a T-scan Ⅲ® system. RESULTS: There was an association between mastication ability and cognitive function, indicated by the colour changes in the chewing gum (P < .05). The participants with greater relative posterior occlusion forces had higher MMSE-DS scores than those with stronger relative anterior occlusion forces. There was a positive correlation between cognitive function and posterior occlusion force. CONCLUSION: The elderly having more occlusion force of posterior teeth rather than anterior teeth were associated with better cognitive ability. Therefore, it may be important for the elderly to restore the masticatory function for the posterior part to prevent against deterioration of cognitive function.

Design and fabrication of portable continuous flow PCR microfluidic chip for DNA replication.

The reasons for restricting continuous flow polymerase chain reaction (CF-PCR) microfluidic chip from lab to application are that it is not portable and requires costly external precision pumps for sample injection. Herein, we employed water as the substitute for PCR solution, and investigated the effect of the cross-section, width-to-depth ratio, and the length ratio for three temperature zones of the micro channel on the thermal and flow distribution of fluid in micro tube by finite element analysis. Results show that the central velocity is uniform and stable velocity occupies the most if the cross-section is rectangular. The deviation between predefined temperature and theoretical temperature is slight and the fluid flux is the most if width-to-depth ratio is 1:1. It is suitable for the short DNA replication if the high temperature zone Wh is larger than the low temperature zone Wl, and vice versa. Then a portable CF-PCR microfluidic chip was fabricated and an automatic sample injection system was developed. As an application, we have successfully amplified the DNA of Treponema denticola in the chip within 8 min. Such a study may offer new insight into the design of CF-PCR microfluidic chip and promote it from lab-scale research to full-scale application.

Autophagosomes form at ER-mitochondria contact sites.

Autophagy is a tightly regulated intracellular bulk degradation/recycling system that has fundamental roles in cellular homeostasis. Autophagy is initiated by isolation membranes, which form and elongate as they engulf portions of the cytoplasm and organelles. Eventually isolation membranes close to form double membrane-bound autophagosomes and fuse with lysosomes to degrade their contents. The physiological role of autophagy has been determined since its discovery, but the origin of autophagosomal membranes has remained unclear. At present, there is much controversy about the organelle from which the membranes originate--the endoplasmic reticulum (ER), mitochondria and plasma membrane. Here we show that autophagosomes form at the ER-mitochondria contact site in mammalian cells. Imaging data reveal that the pre-autophagosome/autophagosome marker ATG14 (also known as ATG14L) relocalizes to the ER-mitochondria contact site after starvation, and the autophagosome-formation marker ATG5 also localizes at the site until formation is complete. Subcellular fractionation showed that ATG14 co-fractionates in the mitochondria-associated ER membrane fraction under starvation conditions. Disruption of the ER-mitochondria contact site prevents the formation of ATG14 puncta. The ER-resident SNARE protein syntaxin 17 (STX17) binds ATG14 and recruits it to the ER-mitochondria contact site. These results provide new insight into organelle biogenesis by demonstrating that the ER-mitochondria contact site is important in autophagosome formation.

Cell Cycle Arrest and Apoptosis Induced by Porphyromonas gingivalis Require Jun N-Terminal Protein Kinase- and p53-Mediated p38 Activation in Human Trophoblasts.

Porphyromonas gingivalis, a periodontal pathogen, has been implicated as a causative agent of preterm delivery of low-birth-weight infants. We previously reported that P. gingivalis activated cellular DNA damage signaling pathways and ERK1/2 that lead to G1 arrest and apoptosis in extravillous trophoblast cells (HTR-8 cells) derived from the human placenta. In the present study, we further examined alternative signaling pathways mediating cellular damage caused by P. gingivalis. P. gingivalis infection of HTR-8 cells induced phosphorylation of p38 and Jun N-terminal protein kinase (JNK), while their inhibitors diminished both G1 arrest and apoptosis. In addition, heat shock protein 27 (HSP27) was phosphorylated through both p38 and JNK, and knockdown of HSP27 with small interfering RNA (siRNA) prevented both G1 arrest and apoptosis. Furthermore, regulation of G1 arrest and apoptosis was associated with p21 expression. HTR-8 cells infected with P. gingivalis exhibited upregulation of p21, which was regulated by p53 and HSP27. These results suggest that P. gingivalis induces G1 arrest and apoptosis via novel molecular pathways that involve p38 and JNK with its downstream effectors in human trophoblasts.

Zinc-finger transcription factor Odd-skipped related 1 regulates cranial bone formation.

Knowledge of the molecular mechanisms of bone formation has been advanced by novel findings related to genetic control. Odd-skipped related 1 (Osr1) is known to play important roles in embryonic, heart, and urogenital development. To elucidate the in vivo function of Osr1 in bone formation, we generated transgenic mice overexpressing full-length Osr1 under control of its 2.8-kb promoter, which were smaller than their wild-type littermates. Notably, abnormalities in the skull of Osr1 transgenic mice were revealed by analysis of X-ray, skeletal preparation, and morphological findings, including round skull and cranial dysraphism. Furthermore, primary calvarial cells obtained from these mice showed increased proliferation and expression of chondrocyte markers, while expression of osteoblast markers was decreased. BMP2 reduced Osr1 expression and Osr1 knockdown by siRNA-induced alkaline phosphatase and osteocalcin expression in mesenchymal and osteoblastic cells. Together, our results suggest that Osr1 plays a coordinating role in appropriate skull closure and cranial bone formation by negative regulation.

Relationship between the burden of major periodontal bacteria and serum lipid profile in a cross-sectional Japanese study.

BACKGROUND: The association of periodontal bacteria with lipid profile alteration remains largely unknown, although it has been suggested that chronic periodontitis increases the atherosclerotic risk. This cross-sectional study investigated the relationship between the prevalence and total burden of periodontal bacteria and serum lipid profile. METHODS: Saliva from enrolled participants was collected to detect 4 major periodontal bacteria (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Prevotella intermedia) using Polymerase Chain Reaction method. High-density lipoprotein (HDL) cholesterol, triglycerides (TG), and low-density lipoprotein cholesterol were assessed using blood samples. We compared the averages of each lipid in association with the prevalence of each bacterial species, their burden (low, moderate, and high), and the combination of bacterial burden and periodontal status, defined as periodontitis, using the Community Periodontal Index, after adjustment for other potential confounding factors, by employing general linear models with least square means. RESULTS: A total of 385 Japanese individuals (176 men, 209 women; mean age 69.2 years) were enrolled. The number of bacterial species and their co-existence with periodontitis were significantly related to a decrease in HDL (p for trend < 0.01) and increase in TG (p for trend = 0.04). The adjusted mean HDL levels (mg/dL) in individuals with low, moderate, and high levels of bacterial species were 66.1, 63.0, and 58.9, respectively, and those in the 6 groups defined by combination of the two factors were 67.9, 64.6, 64.3, 65.4, 61.5, and 54.7, respectively. CONCLUSION: Periodontal bacterial burden is suggested to be independently involved in lowering serum HDL level. Our findings suggest that bacterial tests in a clinical setting could be a useful approach for predicting the risk of HDL metabolism dysregulation.

Intracellular periodontal pathogen exploits recycling pathway to exit from infected cells.

Although human gingival epithelium prevents intrusions by periodontal bacteria, Porphyromonas gingivalis, the most well-known periodontal pathogen, is able to invade gingival epithelial cells and pass through the epithelial barrier into deeper tissues. We previously reported that intracellular P. gingivalis exits from gingival epithelial cells via a recycling pathway. However, the underlying molecular process remains unknown. In the present study, we found that the pathogen localized in early endosomes recruits VAMP2 and Rab4A. VAMP2 was found to be specifically localized in early endosomes, although its localization remained unclear in mammalian cells. A single transmembrane domain of VAMP2 was found to be necessary and sufficient for localizing in early endosomes containing P. gingivalis in gingival epithelial cells. VAMP2 forms a complex with EXOC2/Sec5 and EXOC3/Sec6, whereas Rab4A mediates dissociation of the EXOC complex followed by recruitment of RUFY1/Rabip4, Rab4A effector, and Rab14. Depletion of VAMP2 or Rab4A resulted in accumulation of bacteria in early endosomes and disturbed bacterial exit from infected cells. It is suggested that these novel dynamics allow P. gingivalis to exploit fast recycling pathways promoting further bacterial penetration of gingival tissues.

[Periodontal diseases and systemic diseases.]

Accumulated evidence has suggested that the long-term effects of periodontal diseases can be linked to 106 types of systemic conditions such as diabetes and cardiovascular diseases. The molecular bases linking a relationship between the diseases are inflammation and bacteremia due to periodontal diseases. While, the quality of those evidences is very uneven, i.e., from systematic reviews to case reports and basic experiments. Among them, diabetes has been shown to possess the most evident relationship with periodontal diseases. In contrast, other diseases including cardiovascular diseases and articular rheumatism need to gather more evidences for the tight relationship with periodontal diseases and periodontal pathogens.

Arginine-Ornithine Antiporter ArcD Controls Arginine Metabolism and Interspecies Biofilm Development of Streptococcus gordonii.

Arginine is utilized by the oral inhabitant Streptococcus gordonii as a substrate of the arginine deiminase system (ADS), eventually producing ATP and NH3, the latter of which is responsible for microbial resistance to pH stress. S. gordonii expresses a putative arginine-ornithine antiporter (ArcD) whose function has not been investigated despite relevance to the ADS and potential influence on inter-bacterial communication with periodontal pathogens that utilize amino acids as a main energy source. Here, we generated an S. gordonii ΔarcD mutant to explore the role of ArcD in physiological homeostasis and bacterial cross-feeding. First, we confirmed that S. gordonii ArcD plays crucial roles for mediating arginine uptake and promoting bacterial growth, particularly under arginine-limited conditions. Next, metabolomic profiling and transcriptional analysis of the ΔarcD mutant revealed that deletion of this gene caused intracellular accumulation of ornithine leading to malfunction of the ADS and suppression of de novo arginine biosynthesis. The mutant strain also showed increased susceptibility to low pH stress due to reduced production of ammonia. Finally, accumulation of Fusobacterium nucleatum was found to be significantly decreased in biofilm formed by the ΔarcD mutant as compared with the wild-type strain, although ornithine supplementation restored fusobacterium biovolume in dual-species biofilms with the ΔarcD mutant and also enhanced single species biofilm development by F. nucleatum. Our results are the first direct evidence showing that S. gordonii ArcD modulates not only alkali and energy production but also interspecies interaction with F. nucleatum, thus initiating a middle stage of periodontopathic biofilm formation, by metabolic cross-feeding.

Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells.

Porphyromonas gingivalis is deeply involved in the pathogenesis of marginal periodontitis, and recent findings have consolidated its role as an important and unique pathogen. This bacterium has a unique dual lifestyle in periodontal sites including subgingival dental plaque (biofilm) and gingival cells, as it has been clearly shown that P. gingivalis is able to exert virulence using completely different tactics in each environment. Inter-bacterial cross-feeding enhances the virulence of periodontal microflora, and such metabolic and adhesive interplay creates a supportive environment for P. gingivalis and other species. Human oral epithelial cells harbor a large intracellular bacterial load, resembling the polymicrobial nature of periodontal biofilm. P. gingivalis can enter gingival epithelial cells and pass through the epithelial barrier into deeper tissues. Subsequently, from its intracellular position, the pathogen exploits cellular recycling pathways to exit invaded cells, by which it is able to control its population in infected tissues, allowing for persistent infection in gingival tissues. Here, we outline the dual lifestyle of P. gingivalis in subgingival areas and its effects on the pathogenesis of periodontitis.

Proteomic and transcriptional analysis of interaction between oral microbiota Porphyromonas gingivalis and Streptococcus oralis.

Porphyromonas gingivalis, a major periodontal pathogen, forms biofilm with other oral bacteria such as streptococci. Here, by using shotgun proteomics, we examined the molecular basis of mixed-biofilm formation by P. gingivalis with Streptococcus oralis. We identified a total of 593 bacterial proteins in the biofilm. Compared to the expression profile in the P. gingivalis monobiofilm, the expression of three proteins was induced and that of 31 proteins was suppressed in the mixed biofilm. Additionally, the expression of two S. oralis proteins was increased, while that of two proteins was decreased in the mixed biofilm, as compared to its monotypic profile. mRNA expression analysis of selected genes using a quantitative reverse transcription polymerase chain reaction confirmed the proteomics data, which included overexpression of P. gingivalis FimA and S. oralis glyceraldehyde-3-phosphate dehydrogenase in association with the biofilm. The results also indicated that S. oralis regulates the transcriptional activity of P. gingivalis luxS to influence autoinducer-2-dependent signaling. These findings suggest that several functional molecules are involved in biofilm formation between P. gingivalis and S. oralis.

The serine phosphatase SerB of Porphyromonas gingivalis suppresses IL-8 production by dephosphorylation of NF-κB RelA/p65.

Porphyromonas gingivalis is a major pathogen in severe and chronic manifestations of periodontal disease, which is one of the most common infections of humans. A central feature of P. gingivalis pathogenicity is dysregulation of innate immunity at the gingival epithelial interface, including suppression of IL-8 production by epithelial cells. NF-κB is a transcriptional regulator that controls important aspects of innate immune responses, and NF-κB RelA/p65 homodimers regulate transcription of IL8. Phosphorylation of the NF-κB p65 subunit protein on the serine 536 residue affects nuclear translocation and transcription of target genes. Here we show that SerB, a haloacid dehalogenase (HAD) family serine phosphatase secreted by P. gingivalis, is produced intracellularly and can specifically dephosphorylate S536 of p65 in gingival epithelial cells. A P. gingivalis mutant lacking SerB was impaired in dephosphorylation of p65 S536, and ectopically expressed SerB bound to p65 and co-localized with p65 in the cytoplasm. Ectopic expression of SerB also resulted in dephosphorylation of p65 with reduced nuclear translocation in TNF-α-stimulated epithelial cells. In contrast, the p105/50 subunit of NF-κB was unaffected by SerB. Co-expression of a constitutively active p65 mutant (S536D) relieved inhibition of nuclear translocation. Both the activity of the IL8 promoter and production of IL-8 were diminished by SerB. Deletion and truncation mutants of SerB lacking the HAD-family enzyme motifs of SerB were unable to dephosphorylate p65, inhibit nuclear translocation or abrogate IL8 transcription. Specific dephosphorylation of NF-κB p65 S536 by SerB, and consequent inhibition of nuclear translocation, provides the molecular basis for a bacterial strategy to manipulate host inflammatory pathways and repress innate immunity at mucosal surfaces.

Porphyromonas gingivalis promotes invasion of oral squamous cell carcinoma through induction of proMMP9 and its activation.

Recent epidemiological studies have revealed a significant association between periodontitis and oral squamous cell carcinoma (OSCC). Furthermore, matrix metalloproteinase 9 (MMP9) is implicated in the invasion and metastasis of tumour cells. We examined the involvement of Porphyromonas gingivalis, a periodontal pathogen, in OSCC invasion through induced expression of proMMP and its activation. proMMP9 was continuously secreted from carcinoma SAS cells, while P. gingivalis infection increased proenzyme expression and subsequently processed it to active MMP9 in culture supernatant, which enhanced cellular invasion. In contrast, Fusobacterium nucleatum, another periodontal organism, failed to demonstrate such activities. The effects of P. gingivalis were observed with highly invasive cells, but not with the low invasivetype. P. gingivalis also stimulated proteinase-activated receptor 2 (PAR2) and enhanced proMMP9 expression, which promoted cellular invasion. P. gingivalis mutants deficient in gingipain proteases failed to activate MMP9. Infected SAS cells exhibited activation of ERK1/2, p38, and NF-kB, and their inhibitors diminished both proMMP9-overexpression and cellular invasion. Together, our results show that P. gingivalis activates the ERK1/2-Ets1, p38/HSP27, and PAR2/NF-kB pathways to induce proMMP9 expression, after which the proenzyme is activated by gingipains to promote cellular invasion of OSCC cell lines. These findings suggest a novel mechanism of progression and metastasis of OSCC associated with periodontitis.

Involvement of protease-activated receptor 4 in over-expression of matrix metalloproteinase 9 induced by Porphyromonas gingivalis.

Porphyromonas gingivalis, a periodontal pathogen, is epidemiologically associated with oral squamous cell carcinoma (OSCC). Matrix metalloproteinase 9 (MMP9) which degrades the extracellular matrix and basement membrane components has been implicated in invasion and metastasis of tumor cells. We previously reported that P. gingivalis promoted cellular invasion of carcinoma SAS cells, an established cell line from patients with squamous cell carcinoma of the tongue, by induction of MMP9 production via proteinase-activated receptor 2. In this study, we further examined alternative signaling pathways mediating inactive precursor of MMP9 (proMMP9) production induced by P. gingivalis in SAS cells. Following P. gingivalis infection, PAR4 mRNA expression was increased and proMMP9 production was enhanced, leading to acceleration of SAS cell invasion. Small interfering RNA knockdown of PAR4 gene abrogated both proMMP9 expression and cellular invasion induced by P. gingivalis in SAS cells. Moreover, the phosphorylation of p38 and ERK1/2 was reduced in PAR4 gene knockdown cells infected with P. gingivalis, whereas nuclear translocation of NF-kB was not inhibited. These results suggest that P. gingivalis activates PAR4 signaling pathways, leading proMMP9 over-expression and cellular invasion in OSCC cells.