TNFRSF21 Orchestrates Epithelial Keratinization and Tight Junction Integrity in Periodontitis-Associated Oral Mucosal Repair

Background: The progression of periodontitis is accompanied by destruction of keratinized epithelium, while members of the tumor necrosis factor receptor superfamily (TNFRSF) play critical roles in epithelial repair. This study aimed to elucidate the role of TNFRSF in the pathogenesis and progression of periodontitis. Furthermore, we investigated the mechanisms underlying the repair of epithelial keratinization, with the ultimate aim of translating these insights into clinical therapeutic applications.

Methods: Single-cell RNA Sequencing was used to investigate the TNFRSF expression profiles in the gingival epithelium of patients with severe periodontitis. Gingival tissues were collected from healthy individuals and those with periodontitis. An in vitro model was also established using retinoic acid to inhibit keratinization and BMS493 to promote keratinization. Bulk RNA Sequencing was performed to further substantiate the model and validated the findings by gene knockdown and overexpression experiments. Protein-protein interaction (PPI) analysis and immunoprecipitation identified key protein interactions. In addition, a TNFRSF21 overexpression plasmid and a full-thickness dorsal skin wound mouse model were used to confirm regulatory processes during keratinization.

Results: TNFRSF21 expression, along with epithelial keratinization-related genes were significantly reduced in clinical periodontitis tissues. However, TNFRSF21 increased significantly during epithelial repair following initial periodontal therapy for severe periodontitis, particularly in proliferative keratinocytes and basal layer cells. An in vitro keratinization model revealed that TNFRSF21, Keratin 8 (KRT8), and KRT18, were downregulated during the inhibition of keratinization and upregulated during its promotion. Importantly, the expression levels of KRT8, KRT18, and Claudin-1 were consistently downregulated in the TNFRSF21 knockdown group and upregulated in the TNFRSF21 overexpression group. Single-cell RNA Sequencing combined with PPI analysis revealed a significant interaction between TNFRSF21 and amyloid precursor protein (APP). This was validated by STRING database analysis and immunoprecipitation. Mice treated with TNFRSF21 overexpression plasmids showed accelerated wound healing and increased keratin expression on dorsal skin.

Conclusions: Our findings indicate that TNFRSF21 is a pivotal regulator of epithelial keratinization and tight junction integrity in oral epithelial keratinocytes. Targeting TNFRSF21 may represent a novel therapeutic strategy to restore oral epithelial function.

TNFRSF21 protein; epithelium; keratins; periodontitis; single-cell analysis.