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

Abstract

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.

Disease-associated mutations in IRF6 and RIPK4 dysregulate their signalling functions

“…In addition to IRF6, RIPK4 can also activate NF-κB and JNK [12,13], and in vivo studies have revealed that appropriately balanced NF-κB and JNK activation is important for normal keratinocyte differentiation [36,37]. However, we found that Ripk4 S378X can activate NF-κB, which is consistent with a recent report that similarly demonstrated normal NF-κB activation by a human RIPK4 S376X mutant [38]. The p.Ser378X mutation also did not appear to affect the ability of Ripk4 to activate JNK.…”

“…These findings were not particularly surprising given that the kinase domain of RIPK4 had previously been shown to be sufficient for NF-κB and JNK activation [12]. Nonetheless, the expression of human RIPK4 S376X in Xenopus embryos failed to rescue the severe gastrulation defects caused by Ripk4 deficiency [38]. Therefore, the mutation may cause BPS by inhibiting other RIPK4-mediated signalling pathways, in addition to impairing IRF6 activation.…”

Genome-wide meta-analyses of nonsyndromic orofacial clefts identify novel associations between FOXE1 and all orofacial clefts, and TP63 and cleft lip with or without cleft palate

“…The GWAS approach has now pointed to several genes involved in the cytoskeleton (Leslie et al 2016a; Leslie et al 2012). Additional molecular evidence on other OFC-related genes further supports regulation of cytoskeletal dynamics in the pathogenesis of OFCs (Biggs et al 2014; Caddy et al 2010; De Groote et al 2015; Leslie et al 2012). Similarly SHROOM3 and NRG1 join a list of genes, including GRHL3, IRF6 , and TFAP2A , required for neurulation that are also implicated in OFCs (Copp and Greene 2013; Kousa et al 2013; Wang et al 2011).…”

Generation of a New Gateway-Compatible Inducible Lentiviral Vector Platform Allowing Easy Derivation of Co-Transduced Cells

“…Time-dependent protein induction was studied in HaCaT cells transduced with our inducible vector pDG2iFlag-puro containing the RIPK4 gene (21). Expression of the Flag-tagged transgene could be detected by WB starting 3 h after treatment with 1 g/mL Dox and reached maximal expression levels 9 h after Dox addition (Figure 4D).…”