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Comparative Study
. 2007 Feb 27;104(9):3255-60.
doi: 10.1073/pnas.0611376104. Epub 2007 Feb 20.

p63 induces key target genes required for epidermal morphogenesis

Maranke I Koster  1 , Daisy Dai, Barbara Marinari, Yuji Sano, Antonio Costanzo, Michael Karin, Dennis R Roop
Affiliations
Comparative Study

p63 induces key target genes required for epidermal morphogenesis

Maranke I Koster et al. Proc Natl Acad Sci U S A. .

Abstract

Mice lacking p63, a single gene that encodes a group of transcription factors that either contain (TA) or lack (DeltaN) a transactivation domain, fail to develop stratified epithelia as well as epithelial appendages and limbs. DeltaNp63 isoforms are predominantly expressed during late embryonic and postnatal epidermal development, however, the function of these proteins remains elusive. Using an epidermal-specific inducible knockdown mouse model, we demonstrate that DeltaNp63 proteins are essential for maintaining basement membrane integrity and terminal differentiation of keratinocytes. Furthermore, we have identified two DeltaNp63alpha target genes that mediate these processes. We propose that DeltaNp63alpha initially induces expression of the extracellular matrix component Fras1, which is required for maintaining the integrity of the epidermal-dermal interface at the basement membrane. Subsequently, induction of IkappaB kinase-alpha by DeltaNp63alpha initiates epidermal terminal differentiation resulting in the formation of the spinous layer. Our data provide insights into the role of DeltaNp63alpha in epidermal morphogenesis and homeostasis, and may contribute to our understanding of the pathogenic mechanisms underlying disorders caused by p63 mutations.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
In vitro ΔNp63 knockdown. Mouse exon 3′, the only exon unique to ΔNp63, was subcloned into pDECAP as an inverted repeat (pD-E3′). (A) Western blot analysis of Ptk2 cells transfected with a ΔNp63α expression construct (1 μg) and increasing amounts of pD-E3′ (1, 3, and 5 μg) by using an anti-p63 (mAb4A4) antibody. Anti-K18 was used as a loading control. The pD-E3′ construct was placed under control of an RU486 regulatable system to generate ΔNp63 inducible-knockdown (i-kd) mice. (B) RT-PCR for the pD-E3′ transgene on RNA isolated from newborn ΔNp63 i-kd and control skin after 4 days of RU486 treatment. GAPDH was amplified as an internal control. (C) Real-time RT-PCR and (D) Western blot analysis for ΔNp63/ΔNp63α using RNA or protein isolated from the skin of newborn ΔNp63 i-kd and control littermates that were treated with RU486 for 4 days.
Fig. 2.
Fig. 2.
In vivo ΔNp63 knockdown. (A) Gross appearance of 5-day-old ΔNp63 i-kd and control mice after 4 days of topical RU486 treatment. (B) Sections of back skin of newborn ΔNp63 i-kd mice treated for 4 days with RU486 were stained with H&E. Original magnification: ×200. (C) Sections of full-thickness wounds induced on the heads of adult ΔNp63 i-kd and control mice taken 96 h after wounding. Arrows indicate the migrating epithelial tongue. Original magnification: ×100. (D) TUNEL analysis on sections of back skin of newborn ΔNp63 i-kd mice treated for 4 days with RU486. Propidium iodide was used as a nuclear counterstain. Original magnification: ×200. (E) After 4 days of RU486 treatment, newborn mice were injected with BrdU. BrdU incorporation was visualized by immunofluorescence with an anti-BrdU antibody (green). Anti-K14 antibody (red) was used to highlight the epithelial component of the skin. Original magnification: ×200. (F) Immunofluorescence using anti-K1 (green) and anti-K14 (red) antibodies on sections of back skin of newborn ΔNp63 i-kd mice after 4 days of RU486 treatment. Original magnification: ×200. (G) Immunofluorescence using anti-collagen IV (red) and anti-K14 (green) antibodies on sections of back skin of adult ΔNp63 i-kd mice that were treated for 21 days with RU486. Original magnification: ×200.
Fig. 3.
Fig. 3.
Ikkα is a direct transcriptional target of ΔNp63α. (A) Real-time RT-PCR analysis for Ikkα, Ikkβ, and Ikkγ on RNA isolated from primary keratinocytes after a switch in p63 isoform expression from TA- to ΔNp63. (B) Real-time RT-PCR analysis for Ikkα, Ikkβ, and Ikkγ on RNA isolated from newborn ΔNp63 i-kd and control skin after 4 days of topical RU486 treatment. (C) Western blot analysis of protein isolated from E15.5 wild-type skin using a p63 antibody that recognizes all p63 isoforms. (D) ChIP on E15.5 mouse skin using an anti-p63α antibody and primers surrounding p53RE-Ikkα. Anti-K14 antibody was used as a negative control. (E) Reporter constructs (0.5 μg) containing p53RE-Ikkα, p53RE-Ikkα-mut1 and p53RE-Ikkα-mut2 were transfected with or without a ΔNp63α expression vector (0.5 μg). (F) Real-time RT-PCR for Ikkα on RNA isolated from embryonic skin at various developmental stages. (G) One hour before dissecting E16.5 embryos, pregnant females were injected with BrdU. Immunofluorescence using anti-BrdU (green) and anti-K1 (red) antibodies was performed to detect the presence of proliferating cells. Original magnification: ×200. Error bars represent standard deviations of A, B, and F (three independent samples), or E (three independent experiments).
Fig. 4.
Fig. 4.
Fras1 is a direct transcriptional target of ΔNp63α. Real-time RT-PCR analysis for Fras1 on (A) RNA isolated from newborn ΔNp63 i-kd and control skin treated for 4 days with RU486 and (B) RNA isolated from primary keratinocytes after a switch in p63 isoform expression from TA- to ΔNp63. (C) ChIP on E11.5 mouse skin using an anti-p63α antibody and primers surrounding p53RE-Fras1. Anti-K14 antibody was used as a negative control. (D) Real-time RT-PCR for Fras1 on RNA isolated from embryonic skin at various developmental stages. (E) A reporter construct (0.5 μg) containing p53RE-Fras1 was transfected with or without a ΔNp63α expression vector (0.5 μg). Error bars represent standard deviations of A, B, and D (three independent samples) or E (three independent experiments).
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