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