ZEB2-transgene expression in the epidermis compromises the integrity of the epidermal barrier through the repression of different tight junction proteins

Abstract

Epithelial homeostasis within the epidermis is maintained by means of multiple cell-cell adhesion complexes such as adherens junctions, tight junctions, gap junctions, and desmosomes. These complexes co-operate in the formation and the regulation of the epidermal barrier. Disruption of the epidermal barrier through the deregulation of the above complexes is the cause behind a number of skin disorders such as psoriasis, dermatitis, keratosis, and others. During epithelial-to-mesenchymal transition (EMT), epithelial cells lose their adhesive capacities and gain mesenchymal properties. ZEB transcription factors are key inducers of EMT. In order to gain a better understanding of the functional role of ZEB2 in epidermal homeostasis, we generated a mouse model with conditional overexpression of Zeb2 in the epidermis. Our analysis revealed that Zeb2 expression in the epidermis leads to hyperproliferation due to the combined downregulation of different tight junction proteins compromising the epidermal barrier. Using two epidermis-specific in vivo models and in vitro promoter assays, we identified occludin as a new Zeb2 target gene. Immunohistological analysis performed on human skin biopsies covering various pathogeneses revealed ZEB2 expression in the epidermis of pemphigus vulgaris. Collectively, our data support the notion for a potential role of ZEB2 in intracellular signaling of this disease.

Fig. 1

Immunohistochemical analysis of ZEB2 in human samples. Immunohistochemical analysis using an antibody recognizing human ZEB2 was performed on a series of human biopsies covering various diseases (summarized in Table S1). This analysis showed that ZEB2 protein is present in epithelial cells of pemphigus vulgaris (c, d), but not in epithelial cells of non-affected skin (a), lupus (b), eczema (e), and lichen planus (f). Bars 40 μm

Fig. 2

Expression of Zeb2 in the epidermis of K14Cre;ROSA26-Zeb2 ^tg/tg and K5Cre;ROSA26-Zeb2 ^tg/+mice. Hematoxylin-eosin staining on wild-type and transgenic skin revealed that Zeb2 expression in the epidermis results in hyperproliferation in adult K14Cre;ROSA26-Zeb2 ^tg/tg mice (a) and in K5Cre;ROSA26-Zeb2 ^tg/+ P4.5 neonates (e). Zeb2 is present in all epidermal layers in both strains (b and f) as seen by immunohistochemistry using a mouse Zeb2-specific antibody. Analysis for the proliferation marker Ki67 revealed that the basal layer is actively proliferating in both strains (c and g). Quantification of the number of Ki67-positive cells confirmed this observation (d and h). The graphs depict the average percentage ± SD of Ki67-positive cells in the basal layer (100 %) counted in four different low magnification photographs (p < 0.001 in both d and h). Bars 40 μm

Fig. 3

Expression of Zeb2 in the epidermis of K5Cre;ROSA26-Zeb2 ^tg/+mice affects the functionality of the epidermal barrier. Trans-epidermal water loss (TEWL) (a) and stratum corneum hydration level (b) of P4.5 K5Cre;ROSA26-Zeb2 ^tg/+ and wild-type neonates was measured using specialized equipment described in the Materials and methods section. The graphs show the average ± SD of five mice per group. The K5Cre;ROSA26-Zeb2 ^tg/+ mice exhibited significantly higher water loss through their skin than their wild-type littermates (a and b) (p < 0.001). The evaluation of outside-to-inside barrier was done using two different methods both in utero and also after birth. Toluidine Blue penetration assay was performed on E16.5 and E18.5 embryos (c). No difference in the establishment of the epidermal barrier between K5Cre;ROSA26-Zeb2 ^tg/+ and wild-type embryos is seen, although Zeb2 is already present in the epidermis of the embryos at E16.5 as shown by immunohistochemistry (d). No difference in Lucifer Yellow penetration between transgenic and wild-type P4.5 neonates (e). Bars 20 μm (d), 10 μm (e)

Fig. 4

Occludin is absent from the epidermis of K5Cre;ROSA26-Zeb2 ^tg/+neonates, leading to loss of function of the inside-outside epidermal barrier. a Intradermal injection of biotin and subsequent tracing with fluorescently labeled streptavidin shows that biotin diffusion in wild-type epidermis stops at the tight junctions of the granular layer, whereas in the K5Cre;ROSA26-Zeb2 ^tg/+ epidermis biotin passes through and reaches the outer surface of the epidermis. The “free ends” (arrows) marking the tight junctions formed between the stratum granulosum and the cornified envelope are absent in the Zeb2 transgenic epidermis. b Occludin is missing in the epidermis of P4.5 K5Cre;ROSA26-Zeb2 ^tg/+ pups. Claudin-1 is disturbed in the lower epidermal layers (white arrow) but not in the stratum granulosum, while ZO-1 is only mildly disturbed. Bars 20 μm (a) and 10 μm (b)

Fig. 5

Analysis of the RNA and protein levels of occludin and claudin-1 in the inducible cell line A431-ZEB2 and in Zeb2-expressing epidermis. a, b Quantitative RT-PCR analysis of ZEB2 and occludin levels in the inducible cell line A431-ZEB2 upon doxycycline administration for different time points (24, 48, and 96 h). The quantitative data are normalized against the average of two reference genes and the bars represent relative levels compared to the non-induced cells. This analysis clearly shows that the upregulation of ZEB2 expression leads to a downregulation of occludin mRNA levels. c, d Western-blot analysis of the protein expression levels of occludin and claudin-1 in the A431-ZEB2 cell line (c) and in P4.5 K5Cre;ROSA26-Zeb2 ^tg/+ epidermis (d). This protein analysis confirms the qPCR data and suggests that the occludin downregulation is an effect of ZEB2 expression

Fig. 6

Analysis of the hOccludin promoter in MCF7/AZ cells. a Graphic overview of the human occludin promoter. The promoter fragment is approximately 440 bp and contains one of the two transcription initiation sites. The mutant construct carries the mutation of the E-box from CAGGTG to CAGTTG. b The ZEB2 expression plasmid was co-transfected in MCF7/AZ cells together with wild-type or mutant occludin promoter constructs. The graph depicts luciferase activity measured 48 h after transfection and the luciferase values are normalized against β-galactosidase expressed from a co-transfected plasmid. The values are scaled relative to the wild-type promoter activity. c The E-cadherin promoter was used as an experimental control. The bars represent the average of two independent transfection experiments in both b and c