Figure 6: Nrf2 regulates the expression of DSC2 in the epidermis and controls formation of hyper-adhesive desmosomes.

(a) Levels of Dsc2 mRNA in total skin from K5Cre-CMVcaNrf2 mice and control littermates (N=3) at P2.5 and P32. Levels in control mice were set as 1 for each time point (dashed line). (b) Levels of Dsc2 mRNA in primary keratinocytes from K5Cre-CMVcaNrf2 mice and control littermates (N=4) isolated from epidermis at P4.5. (c) Tail skin of K5Cre and K5Cre-CMVcaNrf2 mice was stained with antibodies against Dsc2 (green; left and right panel) and desmoplakin (Dsp, red, left panel). Yellow colour indicates co-localization of Dsc2 and Dsp in desmosomes at the cell–cell border in K5Cre epidermis. Scale bars, 20 μm. (d) HPKs were transfected with DSC2-YFP or GFP (control) expression vectors and exposed to 2 mM CaCl₂ for 1–3 h to induce the formation of desmosomes. After removal of CaCl₂, the remaining hyper-adhesive, calcium-insensitive desmosomes were stained with a desmoplakin (DSP) antibody. The percentage of keratinocytes connected by calcium-insensitive, hyper-adhesive desmosomes among all keratinocytes was determined. Ten fields per dish with at least 100 cells per field were counted in three independent dishes for every time point. Representative images of keratinocytes 2 h after exposure to high CaCl₂ concentrations stained with a DSP antibody. DSP in hyper-adhesive desmosomes is indicated with arrowheads. (e) DSC2-YFP (green) co-localizes with DSP (red) at the site of desmosome formation (arrowheads). Note that transfection with a GFP expression vector does not affect formation/function of desmosomes and that GFP does not co-localize with DSP at the cell membrane (arrow). (f) Formation or hyper-adhesive desmosomes by HaCaT cells under high calcium concentrations following transfection with scrambled (Scr) or DSC2 siRNA. Desmoplakin staining indicates hyper-adhesive desmosomes (arrowheads). Arrows point to single cells disconnected from neighbouring cells as a result of loss of hyper-adhesive desmosomes. (g) Quantification of disconnected cells following transfection with Scr or DSC2 siRNA and after treatment with ethylene glycol tetraacetic acid. At least 2,000 cells were counted in three independent dishes. (h) Total number of cells transfected with Scr or DSC2 siRNA and incubated in high or low calcium medium. Error bars represent s.d., t-test P value *<0.05; **<0.01, ***<0.001.