Klf4 and corticosteroids activate an overlapping set of transcriptional targets to accelerate in utero epidermal barrier acquisition

Abstract

Premature infants are at an increased risk for infections and dehydration because of incomplete development of the epidermis, which attains its essential function as a barrier only during the last stages of in utero development. When a premature birth is anticipated, antenatal corticosteroids are administered to accelerate lung epithelium differentiation. One pleiotropic, but beneficial, effect of antenatal corticosteroids is acceleration of skin barrier establishment by an unknown mechanism. In mice, the transcription factor Klf4 is both necessary and sufficient, within a developmental field of competence, to establish this skin barrier, as demonstrated by targeted ablation and transgenic expression of Klf4, respectively. Here, we report that Klf4 and corticosteroid treatment coordinately accelerate barrier acquisition in vivo. Transcriptional profiling reveals that the genes regulated by corticosteroids and Klf4 during the critical window of epidermal development significantly overlap. KLF4 activates the proximal promoters of a significant subset of these genes. Dissecting the intersection of the genetic and pharmacological pathways, regulated by KLF4 and corticosteroids, respectively, leads to a mechanistic understanding of the normal process of epidermal development in utero.

Fig. 1.

Ectopic expression of Klf4 and levels of corticosteroids coordinately accelerate developmental barrier acquisition. As visualized with a whole-mount dye penetration assay on E15.5 and E16.5 embryos, corticosteroid injections accelerate barrier acquisition of wild-type (wt) embryos by one-half day. Corticosteroid-treated E15.5 K5-Klf4 mice show greater barrier acceleration than either transgenic untreated littermates or wt corticosteroid-treated embryos.

Fig. 2.

Potential targets of corticosteroids and Klf4 in epidermal development. (A) Venn diagram of genes potentially activated by KLF4 and corticosteroid treatment. Transcriptional profiling identified genes misregulated in corticosteroid-treated, Klf4^−/−, and K5-Klf4 embryonic skin during the critical developmental period of barrier acquisition (E15.5-E16.5). Twenty-eight genes are found up-regulated >2-fold in K5-Klf4, down-regulated >2-fold in Klf4^−/− embryos, and up-regulated >2-fold in corticosteroid-treated mice. (B) Northern blot analysis of representative genes: Fatty acetyl coA reductase 2 (Far2), Dual specificity phosphatase 14 (Dusp14), and Extracellular matrix 1 (Ecm1). (C) Six genes are identified as 2-fold down-regulated in K5-Klf4 and 2-fold up-regulated in Klf4^−/− embryos, and two of these are repressed in corticosteroid-treated mice.

Fig. 3.

Klf4 regulates proximal promoter of target genes. Constructs with proximal promoter (≈1 kb of sequence upstream of TSS) regions cloned upstream of a promoterless luciferase gene are transfected in keratinocytes in the absence or presence of Klf4 and then normalized to vector control. (A) Twelve of the 19 promoters of genes induced by Klf4 show >2-fold activation when cotransfected with Klf4. If promoter level is >100, the value is given above the bar. (B) Two of the five promoters of genes repressed by Klf4 expression show >2-fold repression when cotransfected with Klf4. If promoter level is >10, the value is given above the bar.

Fig. 4.

KLF4 directly binds the proximal promoter of the Far2 gene. (A) Fold activation by Klf4 of the Far2 −0.4 and −0.1 deletion constructs. Location of probes used for EMSA that tile across the Far2 promoter. KLF4 binds to probes 5 and 6. (B) Mutational analysis of possible KLF4-binding sites to determine specificity of KLF4 binding. Probe 6 has three possible KLF4-binding sites, which are all mutated individually and in pairs to demonstrate that only the most 3′ site is required for KLF4 binding to this probe. Mutation of the KLF4-binding site 6 in the 0.4Far2 promoter (0.4Far2 × 6) reduces KLF4 activation from 9.8- to 5.2-fold.