A novel regulatory relationship between RIPK4 and ELF3 in keratinocytes

Keratinocytes are central to the barrier functions of surface epithelia, such as the gingiva and epidermis. RIPK4 is a key regulator of keratinocyte differentiation; however, the signalling pathways in which it functions remain poorly defined. In this study, we identified a regulatory relationship between RIPK4 and ELF3, an ETS family transcription factor. RIPK4 was shown to be important for the upregulation of ELF3 gene expression by the PKC agonist PMA in both oral and epidermal keratinocytes. RIPK4 promotes keratinocyte differentiation in part by phosphorylating and thereby activating the IRF6 transcription factor. Significantly, silencing of IRF6 inhibited the PMA-inducible expression of ELF3. A role for the GRHL3 transcription factor, a downstream target gene of IRF6, in the regulation of ELF3 expression was similarly demonstrated. ELF3 has previously been shown to regulate the expression of SPPR1A and SPRR1B, small proline-rich proteins that contribute to the cornification of keratinocytes. Consistently, RIPK4 and IRF6 were important for the PMA-inducible expression of SPRR1A and SPRR1B. They were also important for the upregulation of TGM1, a transglutaminase that catalyses the cross-linking of proteins, including small proline-rich proteins, during keratinocyte cornification. RIPK4 was also shown to upregulate the expression of TGM2 independently of IRF6. Collectively, our findings position RIPK4 upstream of a hierarchal IRF6-GRHL3-ELF3 transcription factor pathway in keratinocytes, as well as provide insight into a potential role for RIPK4 in the regulation of keratinocyte cornification

IRF6 Regulates the Expression of IL-36 gamma by Human Oral Epithelial Cells in Response to Porphyromonas gingivalis

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

Interferon Regulatory Factor 6 Differentially Regulates Toll-like Receptor 2-dependent Chemokine Gene Expression in Epithelial Cells

Fulltext embargoed for: 12 months post date of publicationEpidermal and mucosal epithelial cells are integral to host defense. They not only act as a physical barrier but also utilize pattern recognition receptors, such as the Toll-like receptors (TLRs), to detect and respond to pathogens. Members of the interferon regulatory factor (IRF) family of transcription factors are key components of TLR signaling as they impart specificity to downstream responses. Although IRF6 is a critical regulator of epithelial cell proliferation and differentiation, its role in TLR signaling has not previously been addressed. We show here that IRF6 is activated by IRAK1 as well as by MyD88 but not by TRIF or TBK1. Co-immunoprecipitation experiments further demonstrated that IRF6 can interact with IRAK1. Gene silencing in epithelial cells along with gene promoter reporter assays showed that IRAK1 mediates TLR2-inducible CCL5 gene expression at least in part by promoting IRF6 activation. Conversely, IRAK1 regulated CXCL8 gene expression independently of IRF6, thus identifying a molecular mechanism by which TLR2 signaling differentially regulates the expression of specific chemokines in epithelial cells. Bioinformatics analysis and mutagenesis-based experiments identified Ser-413 and Ser-424 as key regulatory sites in IRF6. Phosphomimetic mutation of these residues resulted in greatly enhanced IRF6 dimerization and trans-activator function. Collectively, our findings suggest that, in addition to its importance for epithelial barrier function, IRF6 also contributes to host defense by providing specificity to the regulation of inflammatory chemokine expression by TLR2 in epithelial cells

A novel RIPK4–IRF6 connection is required to prevent epithelial fusions characteristic for popliteal pterygium syndromes

Receptor-interacting protein kinase 4 (RIPK4)-deficient mice have epidermal defects and fusion of all external orifices. These are similar to Bartsocas-Papas syndrome and popliteal pterygium syndrome (PPS) in humans, for which causative mutations have been documented in the RIPK4 and IRF6 (interferon regulatory factor 6) gene, respectively. Although genetically distinct, these syndromes share the anomalies of marked pterygia, syndactyly, clefting and hypoplastic genitalia. Despite the strong resemblance of these two syndromes, no molecular connection between the transcription factor IRF6 and the kinase RIPK4 was known and the mechanism underlying the phenotype was unclear. Here we describe that RIPK4 deficiency in mice causes epithelial fusions associated with abnormal periderm development and aberrant ectopic localization of E-cadherin on the apical membrane of the outer peridermal cell layers. In Xenopus, RIPK4 depletion causes the absence of ectodermal epiboly and concomitant gastrulation defects that phenocopy ectopic expression of dominant-negative IRF6. We found that IRF6 controls RIPK4 expression and that wild-type, but not kinase-dead, RIPK4 can complement the gastrulation defect in Xenopus caused by IRF6 malfunctioning. In contrast to the mouse, we observed only minor effects on cadherin membrane expression in Xenopus RIPK4 morphants. However, gastrulation defects were associated with a virtual absence of cortical actin in the ectodermal cells that face the blastocoel cavity and this was phenocopied in embryos expressing dominant-negative IRF6. A role for RIPK4 in actin cytoskeleton organization was also revealed in mouse epidermis and in human epithelial HaCaT cells. In conclusion, we showed that in mice RIPK4 is implicated in cortical actin organization and in E-cadherin localization or function, which can explain the characteristic epithelial fusions observed in PPSs. In addition, we provide a novel molecular link between IRF6 and RIPK4 that unifies the different PPSs to a common molecular pathway