p63 directly induces expression of Alox12, a regulator of epidermal barrier formation

Abstract

Epidermal development and differentiation are tightly controlled processes that culminate in the formation of the epidermal barrier. A critical regulator of different stages of epidermal development and differentiation is the transcription factor p63. More specifically, we previously demonstrated elsewhere that p63 is required for both the commitment to stratification and the commitment to terminal differentiation. We now demonstrate that DeltaNp63alpha, the predominantly expressed p63 isoform in postnatal epidermis, also plays a role in the final stages of epidermal differentiation, namely the formation of the epidermal barrier. We found that DeltaNp63alpha contributes to epidermal barrier formation by directly inducing expression of ALOX12, a lipoxygenase which contributes to epidermal barrier function. Our data demonstrate that DeltaNp63alpha directly interacts with the promoter of Alox12 in the developing epidermis. Furthermore, we found that the induction of Alox12 expression by DeltaNp63alpha depends on intact p63 binding sites in the Alox12 promoter. Finally, we found that DeltaNp63alpha can induce Alox12 expression only in differentiating keratinocytes, consistent with the role of ALOX12 in epidermal barrier formation.

Figure 1. ΔNp63α induces Alox12 expression in mouse skin

(A) Real Time RT-PCR analysis for Alox12 on skin RNA isolated from three newborn ΔNp63 i-kd and three control littermates after 5 days of topical RU486 treatment. Error bars represent standard deviations of data obtained from three mice per genotype (p<0.05). (B) Western blot analysis for ALOX12 and ΔNp63α on protein extracts isolated from newborn ΔNp63 i-kd and control skin after 5 days of topical RU486 treatment. β-Actin (ACTB) was used as a loading control. (C) Real Time RT-PCR analysis for Alox12 on RNA isolated from primary keratinocytes transduced with adenoviruses expressing ΔNp63α (Ad-ΔNp63α) or GFP (Ad-GFP) as a control. Keratinocytes were cultured in media containing 0.05mM Ca²⁺ to maintain the keratinocytes in a proliferative state or in 0.1mM Ca²⁺ to induce differentiation. All expression values were normalized to Ad-GFP transduced keratinocytes cultured under low Ca²⁺ conditions. Error bars represent standard deviations of data obtained from three individual cultures of keratinocytes per condition (low Ca²⁺ cultures: p=0.22; high Ca²⁺ cultures: p<0.05). (D) Real Time RT-PCR for Alox12 on RNA isolated from embryonic skin at different developmental stages. Each embryonic skin was processed individually, except for E10.5–E12.5 embryos, for which several individual skins were combined and processed as one sample. RNA was isolated from three independent samples per developmental stage. The Alox12 expression level at E13.5 was arbitrarily set to 1. C_t values between E10.5 and E12.5 were >32, and were considered background. Error bars represent standard deviations of data obtained from three independent samples (p<0.05 for E13.5–E14.5, E14.5–E15.5 and E15.5–E16.5). For (A), (C), and (D), mRNA expression levels were normalized to the amount of 18S mRNA in each sample.

Figure 2. p63 directly interacts with the Alox12 promoter

Chromatin immunoprecipitation (ChIP) analysis on E14.5 and E15.5 mouse skin using an anti-p63α antibody and primers surrounding p63RE-Alox12. Anti-K14 antibody was used as a negative control and primers specific for GAPDH were used as loading control.

Figure 3. ΔNp63α transactivates a reporter construct containing p63RE-Alox12

(A) A putative p63RE was identified in the Alox12 promoter (p63RE-Alox12) using Consite (http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite). To identify p63RE, we searched for p53 binding sites (>65% conservation of core sequence) which were located within a region of the promoter that was at least 75% conserved. The core sequences of p63RE-Alox12 are underlined. Mutations generated in p63RE-Alox12 are indicated in italics and underlined with a wavy line. (B) Reporter constructs containing p63RE-Alox12 and p63RE-Alox12-mut were nucleofected into primary keratinocytes with or without a ΔNp63α expression vector. Keratinocytes were cultured in high Ca²⁺ media to induce differentiation. Error bars represent standard deviations of three independent experiments (p<0.05).