Role of GRHL2 in Oral Mucosal Barrier and Wound Healing

Objectives: Oral mucosal epithelium plays a pivotal role in protecting the integrity of the underlying tissues, including the dentoalveolar structures, by establishing the defending barrier. Grainyhead-Like 2 (GRHL2) is known to determine epithelial phenotypes through regulation of target genes, including adhesion molecules and tight junction (TJ) proteins. We investigated the role of GRHL2 in establishing oral mucosal barriers and wound healing.
Methods: Immortalized human oral keratinocytes were exposed Porphyromonas gingivalis (Pg) or lipopolysaccharide (LPS) from Pg. Protein and mRNA expression levels of GRHL2 and intercellular adhesion molecules were determined by Western blotting and qRT-PCR. We knocked down GRHL2 expression in the cultured cells by shRNA and the level of TJ proteins, e.g., claudins, zona occludins, and occludins, were determined. Epithelial permeability was measured by penetration of Pg or fluorescence dye in cultured HOKs in which GRHL2 was suppressed. Grhl2 conditional knockout (cKO) mice were used for protein expression studies and ligature-induced periodontitis model. Also, oral mucosal wound healing was compared in Grhl2 wt and ko mice after tooth extraction.
Results: Pg inoculation or exposure to Pg LPS strongly suppressed GRHL2 and TJ gene expression in the oral keratinocytes. In the absence of Pg inoculation, GRHL2 knockdown led to strong inhibition of TJ gene expression and increased epithelial permeability. Grhl2 cKO confirmed the suppression of TJ gene expression in the oral mucosal cells and increased bacterial penetration through the oral cavity. Grhl2 ko also led to increased alveolar bone loss in the ligature-induced periodontitis model and impaired mucosal heal after tooth extraction.
Conclusions: GRHL2 determines epithelial barrier function and wound healing in oral mucosa in part by regulation of TJ genes. Loss of GRHL2 expression upon Pg invasion may impair the oral mucosal barrier and lead to increased tissue destruction. This study was supported in part by grants from NIDCR/NIH (R56DE024593; R03DE024259; R21DE028269).