Background: RIPK4 and IRF6 are important for epidermal development. However, whether they function together to regulate keratinocyte differentiation has not been addressed. Results: RIPK4 directly activates IRF6, resulting in expression of the transcriptional regulators GRHL3 and OVOL1. Conclusion: RIPK4 and IRF6 promote keratinocyte differentiation by functioning as a signaling axis. Significance: This study reveals how mutations in RIPK4 may cause epidermal disorders.

IFN regulatory factors (IRFs) help to shape the immune response to pathogens by imparting signaling specificity to individual TLRs. We recently demonstrated that IRF6 provides specificity to TLR2 signaling in oral epithelial cells. TLR2 plays an important role in eliciting inflammation to Porphyromonas gingivalis, a keystone pathogen in periodontitis. Therefore, we investigated a role for IRF6 in mediating the inflammatory cytokine response of oral epithelial cells to P. gingivalis. IRF6 expression was strongly upregulated when human oral epithelial cells were challenged with P. gingivalis. Moreover, gene silencing and gene promoter experiments indicated that IRF6 acts downstream of IL-1R-associated kinase 1 to stimulate the expression of the IL-1 family cytokine IL-36γ in response to P. gingivalis. IRF6 and IL-1R-associated kinase 1 also regulated the stimulation of IL-36γ expression by a TLR2 agonist. IL-36γ was shown to elicit inflammatory responses by human monocyte-derived dendritic cells and macrophages, including the expression of the neutrophil chemokines IL-8 and CXCL1, as well as the Th17 chemokine CCL20. IL-36γ similarly stimulated their expression by human oral epithelial cells. Significantly, the Th17 cytokine IL-17 not only stimulated the expression of important regulators of neutrophil recruitment and survival by oral epithelial cells, but IL-17 also stimulated them to express IL-36γ. Thus, our findings suggest that IRF6 is likely to promote inflammation to P. gingivalis through its regulation of IL-36γ.

Porphyromonas gingivalis is a keystone pathogen in chronic periodontitis. Its expression of gingipain proteases (Kgp and RgpA/B) is central to the stimulation of chronic inflammation. In this study, we investigated the inflammatory response of oral epithelial cells to P. gingivalis. The cells responded by upregulating the expression of the orphan chemokine CXCL14. The stimulation of CXCL14 expression was largely triggered by the gingipain proteases and was dependent on the host protease-activated receptor PAR-3. Significantly, CXCL14 expression was transcriptionally repressed in response to epidermal growth factor (EGF)-induced activation of the MEK-ERK1/2 pathway. P. gingivalis overcomes the repression of CXCL14 via the gingipain protease-mediated degradation of EGF. Therefore, P. gingivalis not only directly stimulates CXCL14 expression via PAR-3 but also promotes its expression by antagonising EGF signalling. In addition to chemotactic activity, some chemokines also have antimicrobial activities. CXCL14 was demonstrated to have bactericidal activity, against commensal oral streptococci associated with health. Notably though, P. gingivalis was not susceptible to killing by CXCL14, potentially because the gingipain proteases can degrade CXCL14. This suggests that the stimulation of dysregulated CXCL14 expression by P. gingivalis may help promote dysbiosis and the development of chronic periodontitis.

Interleukin-36 (IL-36) cytokines are important regulators of mucosal homeostasis and inflammation. We have previously established that oral epithelial cells upregulate IL-36γ expression in response to the bacterial pathogen Here, we have established that IL-36γ can stimulate the gene expression of mechanistically distinct antimicrobial proteins, including the peptidoglycan amidase PGLYRP2, in oral epithelial cells (e.g., TIGK cells). PGLYRP2 gene expression was not stimulated by either IL-17 or IL-22, thus demonstrating selectivity in the regulation of PGLYRP2 by IL-36γ. The IL-36γ-inducible expression of PGLYRP2 was shown to be mediated by IRAK1- and p38 mitogen-activated protein (MAP) kinase-dependent signaling. Furthermore, our finding that IL-36γ-inducible PGLYRP2 expression was reduced in proliferating TIGK cells but increased in terminally differentiating cells suggests that control of PGLYRP2 expression is associated with the maturation of the oral epithelium. PGLYRP2 expression in TIGK cells can also be directly stimulated by oral bacteria. However, the extracellular gingipain proteases (Kgp and RgpA/B) produced by, which are critical virulence factors, can antagonize PGLYRP2 expression. Thus, the expression of IL-36γ by oral epithelial cells in response to might enable the subsequent autocrine stimulation of PGLYRP2 expression. In summary, our data identify how IL-36γ may promote oral mucosal homeostasis by regulating PGLYRP2 expression.

We recently demonstrated that the expression of the interferon regulatory factor 6 (IRF6) transcription factor in oral keratinocytes was stimulated by the periodontal pathogen Porphyromonas gingivalis. Here, we have established that IRF6 promotes the differentiation of oral keratinocytes in response to P. gingivalis. This was evidenced by the IRF6-dependent upregulation of specific markers of keratinocyte terminal differentiation (e.g., involucrin [IVL] and keratin 13 [KRT13]), together with additional transcriptional regulators of keratinocyte differentiation, including Grainyheadlike 3 (GRHL3) and Ovo-like zinc finger 1 (OVOL1). We have previously established that the transactivator function of IRF6 is activated by receptor-interacting protein kinase 4 (RIPK4). Consistently, the silencing of RIPK4 inhibited the stimulation of IVL, KRT13, GRHL3, and OVOL1 gene expression. IRF6 was shown to also regulate the stimulation of transglutaminase-1 (TGM1) gene expression by P. gingivalis, as well as that of small proline-rich proteins (e.g., SPRR1), which are covalently cross-linked by TGM1 to other proteins, including IVL, during cornification. The expression of the tight junction protein occludin (OCLN) was found to also be upregulated in an IRF6-dependent manner. IRF6 was demonstrated to be important for the barrier function of oral keratinocytes; specifically, silencing of IRF6 increased P. gingivalis-induced intercellular permeability and cell invasion. Taken together, our findings potentially position IRF6 as an important mediator of barrier defense against P. gingivalis. KEYWORDS differentiation, GRHL3, IRF6, keratinocyte, Porphyromonas gingivalis, RIPK4, barrier function T he oral epithelium is an important physical and immunological barrier to pathogens (1). The integrity of the epithelium is maintained through continuous cycles of keratinocyte proliferation and differentiation, whereby basal keratinocytes intermittently exit the cell cycle and undergo terminal differentiation as they migrate toward the epithelium surface, concomitantly upregulating the expression of proteins that confer mechanical strength and elasticity to the epithelium (1-3). Depending on the anatomical location, terminally differentiated keratinocytes may also become cornified through the covalent cross-linking of IVL and other cornified envelope proteins by transglutaminases (e.g., TGM1) (1, 2).IRF6 is a critical transcriptional regulator of keratinocyte differentiation (4-7). IRF6 promotes keratinocyte differentiation in part by inducing the expression of additional transcriptional regulators of differentiation, including GRHL3 and OVOL1 (5-7). GRHL3 promotes keratinocyte differentiation through its interaction with other transcription factors (e.g., LMO4) (8, 9) and by recruiting the Trithorax complex to the promoters of differentiation-associated genes (10). OVOL1 can promote keratinocyte differentiation by repressing the transcription of target genes, including c-Myc (11).The transactivator function of IRF6 can be activated...