30 research outputs found
Molecular Mechanisms Regulating Epithelial Barrier Function and Inflammatory Response
The long-term goal of this study is to develop mode of treatment for inflammation in the oral cavity. To this end, we sought to investigate both (1) the mechanism regulating the integrity of the mucosal barrier, as well as (2) the epigenetic mechanisms by which inflammatory response is elicited and regulated. Epithelial tissue serves as an important barrier against infection. In response to physical injury or infection, this tissue undergoes significant phenotypic changes for eliciting its barrier function. For example, epithelial cells, major components of epithelial tissue, upregulate the expression of TGF-β when the tissue is encountered by inflammation or injury in the human oral cavity. TGF-β induces cellular proliferation and differentiation, and also initiates a reversible process known as epithelial-mesenchymal transition (EMT) for wound healing processes. During EMT, epithelial cells exhibit phenotypic changes, loss of cell-cell adhesion, enhanced migratory capacity, and disruption of epithelial integrity. We have demonstrated that transcription factors Grainyhead-like 2 (GRHL2) and p63 regulate epithelial proliferation and differentiation, and may regulate EMT in human keratinocytes. Thus, to explore the molecular mechanism of TGF-β-dependent EMT, we investigated the effects of p63 and Grainyhead-like 2 (GRHL2) modulation on epithelial plasticity. We found that TGF-β leads to downregulation of GRHL2 and p63 expression, and facilitation of EMT molecular phenotype. Knockdown of all p63 isoforms by transfection of p63 Si-RNA was sufficient to induce EMT phenotype in normal human keratinocytes (NHK), and EMT in NHK accompanied loss of GHRL2 and miR-200 family gene expression, both of which play crucial roles in determining epithelial phenotype. Modulation of GRHL2 in NHK also led to congruent changes in p63 expression. Lastly, conditional knockout of GRHL2 resulted in significant phenotypic changes affecting the epithelial barrier and led to enhanced Porphyromonas gingvalis (P.g.) bacterial load within the bloodstream. These findings indicate that GRHL2 and p63 play an important role in inhibiting TGF-β-dependent EMT in epithelial cells, and that loss of GRHL2 expression induces phenotypic changes altering epithelial barrier function and facilitates accumulation of P.g. bacteria in the bloodstream. These bacteria are known to release lipoglycan endotoxin lipopolysaccharide (LPS) that triggers the expression of pro-inflammatory cytokines. Although previous literature has identified an association between dynamic demethylation of distinct histone marks and cytokine transcriptional activation, the role of histone lysine demethylases in the epigenetic regulation of inflammatory response is not well understood. Thus, to explore the epigenetic regulation of P.g. lipopolysaccharide (P.g. LPS) induced inflammatory response, we discovered a novel histone lysine demethylase KDM3C that regulates pro-inflammatory cytokine induction and inflammatory response. We found that P.g. LPS culture led to KDM3C upregulation and enrichment on the promoter regions of several inflammatory cytokines, driving their transcriptional activation by demethylating H3K9me2. Overexpression of histone methyltransferase G9a maintained the H3K9me2 repressive mark and prevented inflammatory cytokine induction. Knockout of KDM3C also prevented induction of inflammatory signaling molecules, including pro-inflammatory cytokines, by P.g. LPS. These findings indicate that KDM3C plays an important functional role in the epigenetic regulation of inflammatory response. Collectively, these data demonstrate the effect that injury or infection in the oral cavity can have on epithelial integrity and resistance against pathogenic bacteria, and the epigenetic mechanisms that trigger the inflammatory response to these bacteria. As a result, we have identified the potential of KDM3C as novel anti-inflammatory therapeutic target, and our understanding of the mechanisms regulating epithelial barrier function and inflammatory response will be useful in the management and treatment of inflammatory diseases affecting oral tissues
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Preexisting Periapical Inflammatory Condition Exacerbates Tooth Extraction-induced Bisphosphonate-related Osteonecrosis of the Jaw Lesions in Mice.
IntroductionSurgical interventions such as tooth extraction increase the chances of developing osteonecrosis of the jaw in patients receiving bisphosphonates (BPs) for the treatment of bone-related diseases. Tooth extraction is often performed to eliminate preexisting pathological inflammatory conditions that make the tooth unsalvageable; however, the role of such conditions on bisphosphonate-related osteonecrosis of the jaw (BRONJ) development after tooth extraction is not clearly defined. Here, we examined the effects of periapical periodontitis on tooth extraction-induced BRONJ development in mice.MethodsPeriapical periodontitis was induced by exposing the pulp of the maxillary first molar for 3 weeks in C57/BL6 mice that were intravenously administered with BPs. The same tooth was extracted, and after an 3 additional weeks, the mice were harvested for histologic, histomorphometric, and histochemical staining analyses.ResultsPulp exposure induced periapical radiolucency as shown by increased inflammatory cells, tartrate-resistant acid phosphatase-positive osteoclasts, and bone resorption. When BPs were administered, pulp exposure did not induce apical bone resorption despite the presence of inflammatory cells and tartrate-resistant acid phosphatase-positive osteoclasts. Although tooth extraction alone induced BRONJ lesions, pulp exposure further increased tooth extraction-induced BRONJ development as shown by the presence of more bone necrosis.ConclusionsOur study demonstrates that a preexisting pathological inflammatory condition such as periapical periodontitis is a predisposing factor that may exacerbate BRONJ development after tooth extraction. Our study further provides a clinical implication wherein periapical periodontitis should be controlled before performing tooth extraction in BP users in order to reduce the risk of developing BRONJ
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Grainyhead-like 2 regulates epithelial plasticity and stemness in oral cancer cells
Grainyhead-like 2 (GRHL2) is one of the three mammalian homologues of Drosophila Grainyhead involved in epithelial morphogenesis. We recently showed that GRHL2 also controls normal epithelial cell proliferation and differentiation. In this study, we investigated the role of GRHL2 in oral carcinogenesis and the underlying mechanism. GRHL2 expression was elevated in cells and tissues of oral squamous cell carcinomas (OSCCs) compared with normal counterparts. Knockdown of GRHL2 resulted in the loss of in vivo tumorigenicity, cancer stemness and epithelial phenotype of oral cancer cells. GRHL2 loss also inhibited oral cancer cell proliferation and colony formation. GRHL2 regulated the expression of miR-200 family and Octamer-binding transcription factor 4 (Oct-4) genes through direct promoter DNA binding. Overexpression of miR-200 genes in the oral cancer cells depleted of GRHL2 partially restored the epithelial phenotype, proliferative rate and cancer stemness, indicating that miR-200 genes in part mediate the functional effects of GRHL2. Taken together, this study demonstrates a novel connection between GRHL2 and miR-200, and supports protumorigenic effect of GRHL2 on OSCCs
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Porphyromonas gingivalis impairs oral epithelial barrier through targeting GRHL2
Oral mucosa provides the first line of defense against a diverse array of environmental and microbial irritants by forming the barrier of epithelial cells interconnected by multiprotein tight junctions (TJ), adherens junctions, desmosomes, and gap junction complexes. Grainyhead-like 2 (GRHL2), an epithelial-specific transcription factor, may play a role in the formation of the mucosal epithelial barrier, as it regulates the expression of the junction proteins. The current study investigated the role of GRHL2 in the Porphyromonas gingivalis (Pg)-induced impairment of epithelial barrier functions. Exposure of human oral keratinocytes (HOK-16B and OKF6 cells) to Pg or Pg-derived lipopolysaccharides (Pg LPSs) led to rapid loss of endogenous GRHL2 and the junction proteins (e.g., zonula occludens, E-cadherin, claudins, and occludin). GRHL2 directly regulated the expression levels of the junction proteins and the epithelial permeability for small molecules (e.g., dextrans and Pg bacteria). To explore the functional role of GRHL2 in oral mucosal barrier, we used a Grhl2 conditional knockout (KO) mouse model, which allows for epithelial tissue-specific Grhl2 KO in an inducible manner. Grhl2 KO impaired the expression of the junction proteins at the junctional epithelium and increased the alveolar bone loss in the ligature-induced periodontitis model. Fluorescence in situ hybridization revealed increased epithelial penetration of oral bacteria in Grhl2 KO mice compared with the wild-type mice. Also, blood loadings of oral bacteria (e.g., Bacteroides, Bacillus, Firmicutes, β-proteobacteria, and Spirochetes) were significantly elevated in Grhl2 KO mice compared to the wild-type littermates. These data indicate that Pg bacteria may enhance paracellular penetration through oral mucosa in part by targeting the expression of GRHL2 in the oral epithelial cells, which then impairs the epithelial barrier by inhibition of junction protein expression, resulting in increased alveolar tissue destruction and systemic bacteremia
Fusobacterium nucleatum secretes amyloid‐like FadA to enhance pathogenicity
Fusobacterium nucleatum (Fn) is a Gram-negative oral commensal, prevalent in various human diseases. It is unknown how this common commensal converts to a rampant pathogen. We report that Fn secretes an adhesin (FadA) with amyloid properties via a Fap2-like autotransporter to enhance its virulence. The extracellular FadA binds Congo Red, Thioflavin-T, and antibodies raised against human amyloid β42. Fn produces amyloid-like FadA under stress and disease conditions, but not in healthy sites or tissues. It functions as a scaffold for biofilm formation, confers acid tolerance, and mediates Fn binding to host cells. Furthermore, amyloid-like FadA induces periodontal bone loss and promotes CRC progression in mice, with virulence attenuated by amyloid-binding compounds. The uncleaved signal peptide of FadA is required for the formation and stability of mature amyloid FadA fibrils. We propose a model in which hydrophobic signal peptides serve as "hooks" to crosslink neighboring FadA filaments to form a stable amyloid-like structure. Our study provides a potential mechanistic link between periodontal disease and CRC and suggests anti-amyloid therapies as possible interventions for Fn-mediated disease processes