KLF4 transcriptionally activates non-canonical WNT5A to control epithelial stratification

Abstract

Epithelial differentiation and stratification are essential for normal homeostasis, and disruption of these processes leads to both injury and cancer. The zinc-finger transciption factor KLF4 is a key driver of epithelial differentiation, yet the mechanisms and targets by which KLF4 controls differentiation are not well understood. Here, we define WNT5A, a non-canonical Wnt ligand implicated in epithelial differentiation, repair, and cancer, as a direct transcriptional target that is activated by KLF4 in squamous epithelial cells. Further, we demonstrate functionally that WNT5A mediates KLF4 control of epithelial differentiation and stratification, as treatment of keratinocytes with WNT5A rescues defective epithelial stratification resulting from KLF4 loss. Finally, we show that the small GTPase CDC42 is regulated by KLF4 in a WNT5A dependent manner. As such, we delineate a novel pathway for epithelial differentiation and stratification and define potential therapeutic targets for epithelial diseases.

Figure 1. KLF4 transactivates WNT5A in esophageal epithelial cells.

(A,B) By immunofluorescence, control mice had extensive WNT5A staining (red) in the suprabasal and superficial layers of their esophageal epithelia (A). In contrast, WNT5A was nearly absent from esophageal epithelia of ED-L2/Cre;Klf4^loxP/loxP mice (B). DAPI (blue) was used as a counterstain, and the white dashed line represents the approximate location of the basement membrane. Scale bars: 25 μM. (C) By quantitative real-time PCR, Wnt5a mRNA expression was decreased in the esophageal epithelium of each ED-L2/Cre;Klf4^loxP/loxP mouse compared to its littermate control (*p < 0.05). (D) Klf4 knockdown in primary mouse esophageal keratinocytes in culture using either of two shRNA constructs resulted in a 57% decrease in Wnt5a mRNA levels by qPCR. (*p < 0.05) (E) In primary human esophageal keratinocytes, inducible KLF4 knockdown with either of two shRNA constructs led to a 31–39% decrease in WNT5A mRNA expression by qPCR. (*p < 0.05) (F) Right panel: When human primary esophageal keratinocytes were induced to differentiate with CaCl₂, KLF4 bound to the region of WNT5A between −945 to −762 upstream of the transcriptional start site. Left panel: No KLF4 binding to WNT5A was observed in actively proliferating keratinocytes. Lack of binding at −1992 to −1796 (not shown) confirmed specificity. (G) Primary mouse esophageal keratinocytes transfected with pCDNA3-Flag-Klf4 to express Klf4 had an 1148-fold increase in luciferase reporter activity compared to cells transfected with pCDNA3.1 control. (*p < 0.05).

Figure 2. KLF4 knockdown alters esophageal stratification via WNT5A.

(A) In three-dimensional organotypic culture, primary human esophageal keratinocytes form stratified epithelia. Cells in the basal layer were rounded with large nuclear-cytoplasmic ratios, and cells in the suprabasal and superficial layers were flattened with compacted nuclei. (B,C) In contrast, KLF4 knockdown in primary human esophageal keratinocytes in organotypic culture yielded epithelia that were hyperplastic, and cells appeared less mature, with cells outside of the basal layer maintaining a rounded appearance and large nuclei. (D–F) Treatment of the cultures with recombinant WNT5A had little effect on control epithelia (D) but restored normal epithelial stratification of primary esophageal keratinocytes with inducible KLF4 knockdown (E,F). Scale bars, 50 μm.

Figure 3. WNT5A rescues defective esophageal epithelial differentiation that results from KLF4 loss.

(A) Keratin 14 (red), which marks immature keratinocytes, was restricted to the basal layer in organotypic cultures of control keratinocytes. (B,C) When KLF4 was knocked down in primary human esophageal keratinocytes, keratin 14 staining was more extensive, including in cells of the suprabasal layer. (D–F) Treatment of primary human esophageal keratinocytes with recombinant WNT5A had little effect in control cells (D), but restored the normal pattern of keratin 14 expression in cells with KLF4 knockdown, with staining again restricted to the basal layer in these cells (E,F). (G–I) Keratin 4 was expressed in the suprabasal and superficial layers of organotypic cultures of control human esophageal keratinocytes (G) while expression was nearly absent from cells with KLF4 knockdown (B,C). (J–L) Treatment of control primary human esophageal keratinocytes with recombinant WNT5A had minimal effect (J), but WNT5A treatment of organotypic cultures of cells with KLF4 knockdown normalized keratin 4 expression, with keratin 4 staining again seen in the suprabasal and superficial layers (K,L). DAPI (blue) was used as a counterstain. Scale bars, 25 μm.

Figure 4. KLF4 knockdown activates CDC42 in a WNT5A dependent manner.

(A) Quantification of GTPase activation (n = 3) demonstrated increased CDC42 activity with KLF4 knockdown in primary human esophageal keratinocytes induced to differentiate with CaCl₂; activation of CDC42 in cells with KLF4 knockdown was abolished by treatment with recombinant WNT5A. (B) Proposed model for the regulation of squamous epithelial cell differentiation and stratification via KLF4 and WNT5A. Previously described regulators of KLF4 are indicated in gray.