A regulatory feedback loop involving p63 and IRF6 links the pathogenesis of 2 genetically different human ectodermal dysplasias
Francesca Moretti, … , Luisa Guerrini, Antonio Costanzo
Francesca Moretti, … , Luisa Guerrini, Antonio Costanzo
Published April 26, 2010
Citation Information: J Clin Invest. 2010;120(5):1570-1577. https://doi.org/10.1172/JCI40267.
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Research Article

A regulatory feedback loop involving p63 and IRF6 links the pathogenesis of 2 genetically different human ectodermal dysplasias

Abstract

The human congenital syndromes ectrodactyly ectodermal dysplasia-cleft lip/palate syndrome, ankyloblepharon ectodermal dysplasia clefting, and split-hand/foot malformation are all characterized by ectodermal dysplasia, limb malformations, and cleft lip/palate. These phenotypic features are a result of an imbalance between the proliferation and differentiation of precursor cells during development of ectoderm-derived structures. Mutations in the p63 and interferon regulatory factor 6 (IRF6) genes have been found in human patients with these syndromes, consistent with phenotypes. Here, we used human and mouse primary keratinocytes and mouse models to investigate the role of p63 and IRF6 in proliferation and differentiation. We report that the ΔNp63 isoform of p63 activated transcription of IRF6, and this, in turn, induced proteasome-mediated ΔNp63 degradation. This feedback regulatory loop allowed keratinocytes to exit the cell cycle, thereby limiting their ability to proliferate. Importantly, mutations in either p63 or IRF6 resulted in disruption of this regulatory loop: p63 mutations causing ectodermal dysplasias were unable to activate IRF6 transcription, and mice with mutated or null p63 showed reduced Irf6 expression in their palate and ectoderm. These results identify what we believe to be a novel mechanism that regulates the proliferation-differentiation balance of keratinocytes essential for palate fusion and skin differentiation and links the pathogenesis of 2 genetically different groups of ectodermal dysplasia syndromes into a common molecular pathway.

Authors

Francesca Moretti, Barbara Marinari, Nadia Lo Iacono, Elisabetta Botti, Alessandro Giunta, Giulia Spallone, Giulia Garaffo, Emma Vernersson-Lindahl, Giorgio Merlo, Alea A. Mills, Costanza Ballarò, Stefano Alemà, Sergio Chimenti, Luisa Guerrini, Antonio Costanzo

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Figure 1

Irf6 is a direct p63 target.

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Irf6 is a direct p63 target. (A) Microarray analysis of Irf6 RNA ex...
(A) Microarray analysis of Irf6 RNA expression in mouse primary keratinocytes transfected with ΔNp63- or TAp63-specific siRNAs. Representative heat map and raw value data are shown. (B) Irf6 mRNA expression in mouse primary keratinocytes transfected with ΔNp63- or TAp63-specific siRNA. Data are presented as mean ± SEM. *P = 0.001. (C) p63 REs present within the Irf6 gene. The p63 RE consensus sequence is shown at the bottom. Arrowheads indicate the position of primers used in ChIP analysis. Putative p53/p63 REs were identified by PathSearch algorithm. (D) ChIP analysis to detect p63 occupying the Irf6 promoter in differentiating mouse primary keratinocytes. PCR was performed using the indicated primers (Supplemental Methods). (E) IRF6 expression in human primary keratinocytes by RT-qPCR. Data are presented as mean ± SEM. (F) Immunoblot analysis of human primary keratinocyte protein extracts to detect IRF6, ΔNp63, K1, and PCNA proteins. (G) IRF6 expression in human primary keratinocytes transfected with the indicated expression vectors and induced to differentiate. ΔNp63α induces IRF6 mRNA. Data are presented as mean ± SEM. cons., consensus sequence; ex., exon; mIrf6, mouse Irf6; siCtr., control siRNA; siΔNp63, ΔNp63-specific siRNA; siTAp63, TAp63-specific siRNA.