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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- when the tissue is encountered by inflammation or injury in the human oral cavity. TGF- 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- -dependent EMT, we investigated the effects of p63 and Grainyhead-like 2 (GRHL2) modulation on epithelial plasticity. We found that TGF- 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- -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.
This text examines past and current progress on the structural, biochemical and molecular characterization of specialized cell-cell junctions in various types of epithelial tissue. Special attention is devoted to the recent characterization of specific protein components of junctions, and to questions of structure/function relationships in development, disease and phylogeny in epithelial junctions.
The molecular events surrounding TNF-mediated tight junction regulation are the focus of this dissertation. Exposure of cultured epithelial monolayers to TNF in vitro can cause barrier loss. This tight junction regulation occurs within hours and is mediated by myosin light chain kinase (MLCK), which phosphorylates myosin II regulatory light chain (MLC). The mechanisms by which TNF activates MLCK may include initiation of an increase in intracellular Ca2+, but this has not been reported in intestinal epithelium. However, it is clear that TNF induces transcriptional activation of MLCK, both in human intestinal epithelial cell lines in vitro and in mouse enterocytes in vivo. Moreover, this process appears to be active in human inflammatory bowel disease patients in vivo, as intestinal epithelial MLCK expression and MLC phosphorylation are both increased in association with active disease. The data discussed here describe the molecular events in MLCK transcriptional activation and the novel observation that MLCK1 is trafficked to the perijunctional actomyosin ring in response to TNF signaling. Furthermore, I describe here, unique tools for studying MLCK1 trafficking and activity. Targeting MLCK1-specific trafficking with small molecule drugs reverses TNF-induced barrier dysfunction in vitro and in vivo. Therefore, not only are these drugs powerful tools in understanding molecular mechanisms that regulate MLCK1, but may also represent a therapeutic alternative to the current IBD treatment regimen of immune suppression.
In the past thirty years, significant advances have been made in the field of reproductive biology in unlocking the molecular and biochemical events that regulate spermatogenesis in the mammalian testis. It was possible because of the unprecedented breakthroughs in molecular biology, cell biology, immunology, and biochemistry. In this book entitled, Molecular Mechanisms in Spermatogenesis, a collection of chapters has been included written by colleagues on the latest development in the field using genomic and proteomic approaches to study spermatogenesis, as well as different mechanisms and/or molecules including environmental toxicants and transcription factors that regulate and/or affect spermatogenesis. The book begins with a chapter that provides the basic concept of cellular regulation of spermatogenesis. A few chapters are also dedicated to some of the latest findings on the Sertoli cell cytoskeleton and other molecules (e.g., proteases, adhesion proteins) that regulate spermatogenesis. These chapters contain thought-provoking discussions and concepts which shall be welcomed by investigators in the field. It is obvious that many of these concepts will be updated and some may be amended in the years to come. However, they will serve as a guide and the basis for investigation by scientists in the field.
The integrity and barrier properties of intestinal epithelium are determined by specialized adhesive structures known as intercellular junctions; composed of adherens junctions (AJs), tight junctions (TJs) and focal adhesions that mediate cell-cell and cell matrix interactions, respectively. These two types of epithelial cell adhesions regulate each other during disruption and restitution of the epithelial barrier. Inflammatory cytokines such as interferon gamma (IFN[gamma]) and tumor necrosis factor alpha (TNF[alpha]) are elevated during intestinal inflammation. The most notable effects of IFN[gamma] and TNF[alpha] on intestinal epithelial homeostasis involve disruption of apical junctions and attenuation of cell migration. Although molecular mechanisms underlying these effects remain poorly understood, expressional downregulation of different adhesion proteins may play a major role in the cytokine-dependent disruption of the intestinal epithelial barriers. This thesis is based on the hypothesis that inhibition of the protein translation initiation machinery promotes the disruption of the intestinal epithelial barrier and attenuates epithelial restitution during mucosal inflammation. This study was focused on two eukaryotic translation initiation factors, eIF4G1 and eIF4G2, which play essential roles in the regulation of cap-dependent protein translation. Expression of both translation initiation factors was dramatically downregulated in model intestinal epithelial cell monolayers treated with IFNÎđ and TNF[alpha] in parallel to cytokine-induced disruption of the epithelial barrier. siRNA or shRNA-mediated downregulation of either eIF4G1, or eIF4G2 increased permeability of well-differentiated SK-CO15 intestinal epithelial cell monolayers and decreased expression of different adherens junction and tight junction proteins. Furthermore depletion of these translation initiating factors inhibits different modes of migration (wound healing and transfilter migration) of stem-cell like and well-differentiated intestinal epithelial cells. These findings suggest that eukaryotic translation initiation factors of the eIF4G family play unique roles in regulating integrity and restitution of the intestinal epithelial barrier. Downregulation of these translation initiating factors may mediate disruption of the intestinal epithelial barriers during mucosal inflammation.