Introduction Surgical interventions such as tooth extraction increase a chance of developing osteonecrosis of the jaw (ONJ) in patients receiving bisphosphonates (BPs) for treatment of bone-related diseases. Tooth extraction is often performed to eliminate pre-existing pathological inflammatory conditions that make the tooth unsalvageable; however, the role of such conditions on bisphosphonate-related ONJ (BRONJ) development following tooth extraction is not clearly defined. Here, we examined the effects of periapical periodontitis on tooth extraction-induced BRONJ development in mice. Methods Periapical periodontitis was induced by exposing the pulp of the maxillary first molar for 3 weeks in C57/BL6 mice that were intravenously administered with BP. The same tooth was extracted, and after 3 additional weeks, the mice were harvested for histological, histomorphometric, and histochemical staining analyses. Results Pulp exposure induced periapical radiolucency as demonstrated by increased inflammatory cells, TRAP+ osteoclasts, and bone resorption. When BP was administered, pulp exposure did not induce apical bone resorption despite the presence of inflammatory cells and TRAP+ osteoclasts. While tooth extraction alone induced BRONJ lesions, pulp exposure further increased tooth extraction-induced BRONJ development as demonstrated by the presence of more bone necrosis. Conclusion Our study demonstrates that pre-existing pathological inflammatory condition such as periapical periodontitis is a predisposing factor that may exacerbate BRONJ development following tooth extraction. Our study further provides a clinical implication whereby periapical periodontitis should be controlled before performing tooth extraction in BP-users in order to reduce the risk of developing BRONJ.

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.

Grainyhead-Like 2 (GRHL2) is an epithelial-specific transcription factor that regulates epithelial morphogenesis and differentiation. Prior studies suggested inverse regulation between GRHL2 and TGF-β in epithelial plasticity and potential carcinogenesis. Here, we report the role of GRHL2 in oral carcinogenesis in vivo using a novel Grhl2 knockout (KO) mouse model and the underlying mechanism involving its functional interaction with TGF-β signaling. We developed epithelial-specific Grhl2 conditional KO mice by crossing Grhl2 floxed mice with those expressing CreER driven by the K14 promoter. After induction of Grhl2 KO, we confirmed the loss of GRHL2 and its target proteins, while Grhl2 KO strongly induced TGF-β signaling molecules. When exposed to 4-nitroquinoline 1-oxide (4-NQO), a strong chemical carcinogen, Grhl2 wild-type (WT) mice developed rampant oral tongue tumors, while Grhl2 KO mice completely abolished tumor development. In cultured oral squamous cell carcinoma (OSCC) cell lines, TGF-β signaling was notably induced by GRHL2 knockdown while being suppressed by GRHL2 overexpression. GRHL2 knockdown or KO in vitro and in vivo, respectively, led to loss of active p-Erk1/2 and p-JNK MAP kinase levels; moreover, ectopic overexpression of GRHL2 strongly induced the MAP kinase activation. Furthermore, the suppressive effect of GRHL2 on TGF-β signaling was diminished in cells exposed to Erk and JNK inhibitors. These data indicate that GRHL2 activates the Erk and JNK MAP kinases, which in turn suppresses the TGF -β signaling. This novel signaling represents an alternative pathway by which GRHL2 regulates carcinogenesis, and is distinct from the direct transcriptional regulation by GRHL2 binding at its target gene promoters, e.g., E-cadherin, hTERT, p63, and miR-200 family genes. Taken together, the current study provides the first genetic evidence to support the role of GRHL2 in carcinogenesis and the underlying novel mechanism that involves the functional interaction between GRHL2 and TGF-β signaling through the MAPK pathways.

Histone Lys‐specific demethylases (KDMs) play a key role in many biological processes through epigenetic mechanisms. However, the role of KDMs in inflammatory responses to oral bacterial infection is poorly understood. Here, we show a novel regulatory role of KDM3C in inflammatory responses to oral bacterial infection. KDM3C expression is transiently suppressed in human and mouse macrophages exposed to LPS from Porphyromonas gingivalis (Pg LPS). Loss of KDM3C in both human and mouse macrophages led to notable induction of proinflammatory cytokines in response to Pg LPS stimulation. Also, KDM3C depletion led to strong induction of p65 phosphorylation and accelerated nuclear translocation in cells exposed to Pg LPS. Kdm3C knockout (KO) in mice led to increased alveolar bone destruction upon induction of experimental periodontitis or pulp exposure compared with those of the wild‐type (WT) littermates. The Kdm3C KO mice also revealed an increased number of osteoclasts juxtaposed to the bony lesions. We also confirmed enhanced osteoclastogenesis by bone marrow–derived macrophages isolated from the Kdm3C KO compared with the WT controls. These findings suggest an anti‐inflammatory function of KDM3C in regulating the inflammatory responses against oral bacterial infection through suppression of NF‐κB signaling and osteoclastogenesis.—Lee, J. Y., Mehrazarin, S., Alshaikh, A., Kim, S., Chen, W., Lux, R., Gwack, Y., Kim, R. H., Kang, M. K. Histone Lys demethylase KDM3C demonstrates anti‐inflammatory effects by suppressing NF‐κB signaling and osteoclastogenesis. FASEB J. 33, 10515–10527 (2019). http://www.fasebj.org

High-risk human papillomavirus (HPV) is a major risk factor for oral and pharyngeal cancers (OPCs), yet the detailed mechanisms by which HPV promotes OPCs are not understood. Forkhead box M1B (FoxM1B) is an oncogene essential for cell cycle progression and tumorigenesis, and it is aberrantly overexpressed in many tumors. We previously showed that FoxM1B was the putative target of an epithelial-specific transcription factor, Grainyhead-like 2 (GRHL2). In the current study, we demonstrate that HPV type 16 (HPV-16) E6 induces FoxM1B in human oral keratinocytes (HOKs) and tonsillar epithelial cells (TECs) in part through GRHL2. FoxM1B was barely detectable in cultured normal human oral keratinocytes (NHOKs) and progressively increased in immortalized HOKs harboring HPV-16 genome (HOK-16B) and tumorigenic HOK-16B/BaP-T cells. Retroviral expression of HPV-16 E6 and/or E7 in NHOKs, TECs, and hypopharyngeal carcinoma cells (FaDu) revealed induction of FoxM1B and GRHL2 by the E6 protein but not E7. Both GRHL2 and FoxM1B were strongly induced in the epidermis of HPV-16 E6 transgenic mice and HPV oral squamous cell carcinomas. Ectopic expression of FoxM1B led to acquisition of transformed phenotype in HOK-16B cells. Loss of FoxM1B by lentiviral short hairpin RNA vector or chemical inhibitor led to elimination of tumorigenic characteristics of HOK-16B/BaP-T cells. Luciferase reporter assay revealed that GRHL2 directly bound and regulated the FoxM1B gene promoter activity. Using epithelial-specific Grhl2 conditional knockout mice, we exposed wild-type (WT) and Grhl2 KO mice to 4-nitroquinolin 1-oxide (4-NQO), which led to induction of FoxM1B in the tongue tissues and rampant oral tumor development in the WT mice. However, 4-NQO exposure failed to induce tongue tumors or induction of FoxM1B expression in Grhl2 KO mice. Collectively, these results indicate that HPV-16 induces FoxM1B in part through GRHL2 transcriptional activity and that elevated FoxM1B level is required for oropharyngeal cancer development.

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