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. 2014 Apr;134(4):1033-1043.
doi: 10.1038/jid.2013.401. Epub 2013 Sep 20.

Plakophilin-1 protects keratinocytes from pemphigus vulgaris IgG by forming calcium-independent desmosomes

Dana K Tucker  1 , Sara N Stahley  1 , Andrew P Kowalczyk  2
Affiliations

Plakophilin-1 protects keratinocytes from pemphigus vulgaris IgG by forming calcium-independent desmosomes

Dana K Tucker et al. J Invest Dermatol. 2014 Apr.

Abstract

Plakophilin-1 (PKP-1) is an armadillo family protein critical for desmosomal adhesion and epidermal integrity. In the autoimmune skin-blistering disease pemphigus vulgaris (PV), autoantibodies (IgG) target the desmosomal cadherin desmoglein 3 (Dsg3) and compromise keratinocyte cell-cell adhesion. Here, we report that enhanced expression of PKP-1 protects keratinocytes from PV IgG-induced loss of cell-cell adhesion. PKP-1 prevents loss of Dsg3 and other desmosomal proteins from cell-cell borders and prevents alterations in desmosome ultrastructure in keratinocytes treated with PV IgG. Using a series of Dsg3 chimeras and deletion constructs, we find that PKP-1 clusters Dsg3 with the desmosomal plaque protein desmoplakin in a manner dependent on the plakoglobin-binding domain of the Dsg3 tail. Furthermore, PKP-1 expression transforms desmosome adhesion from a calcium-dependent to a calcium-independent and hyperadhesive state. These results demonstrate that manipulating the expression of a single desmosomal plaque protein can block the pathogenic effects of PV IgG on keratinocyte adhesion.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors state no conflict of interest.

Figures

Figure 1
Figure 1. PKP-1 promotes desmosome formation
(a–d) Immunofluorescence images and (e, f) quantification of cell-cell border fluorescence intensity from keratinocytes expressing empty vector (EV) or PKP-1.myc in low (0.1mM) or high calcium media (0.6mM) for 24 hours and immunostained for surface Dsg3, total desmoplakin (DP) and myc (PKP-1.myc). Scale bar: 20Hm. (e, f) Mean ± SEM (n= 50 borders per group); *** p < 0.001 compared with EV (Mann Whitney). (g,h,i) Sequential detergent extraction and western blot analysis of Triton X-100 soluble (sol) and insoluble (insol) proteins. Quantification represents the mean of three independent experiments. (j) Electron micrographs and (k) quantification of desmosome lengths (n= 25 desmosomes per group); *** p < 0.001 compared with EV (Mann Whitney). Scale bars 1Hm.
Figure 2
Figure 2. PKP-1 protects desmosomal components from disruption by PV IgG
Keratinocytes expressing empty vector (EV) or PKP-1.myc were treated with NH or PV IgG for 24 hours. (a–d) The mAb AK15 was used live to detect cell surface Dsg3 and total myc was detected after methanol fixation (a′–d′). (e) Quantification of Dsg3 fluorescence intensity at cell-cell borders. Data are mean percentages normalized to EV and PKP-1 NH IgG controls. Mean ± SEM (n= 50 borders per group); *** p < 0.001 compared with EV-NH IgG, ♦ compared with EV-PV IgG and PKP-1.myc-NH IgG (Two-way ANOVA, Holm-Sidak method). (f–i) Cells were briefly pre-extracted prior to fixation and immunostained for DP (green, f′–i′), cytokeratin (keratin, red, f″–i″) and myc (f′″–i′″). Data represent four independent experiments. Scale bars, 20Hm.
Figure 3
Figure 3
PKP-1 protects desmosome ultrastructure and keratinocyte adhesion strength from disruption by PV IgG (a) Electron micrographs and (b) quantification of desmosome lengths from keratinocytes expressing empty vector (EV) or PKP-1.myc treated with NH or PV IgG for 24 hours (n= 25–50 desmosomes per group); *** p < 0.001 compared with EV-NH IgG (Mann Whitney). (c, d) Quantification of monolayer fragmentation after cell dissociation assays using control keratinocytes (no virus, EV, and plakoglobin (PG)) or keratinocytes expressing PKP-1.myc exposed for 24 hours to (c) NH or PV IgG or (d) mAb IL-2R or AK23, a pathogenic Dsg3 mAb. Mean number of fragments ± SEM;*** p < 0.001 compared with no virus and EV (Two-way ANOVA, Holm-Sidak method). Scale bars, 0.2 or 1 Hm as indicated.
Figure 4
Figure 4. PKP-1 clusters the cytoplasmic tail of Dsg3 with DP
(a) Portions of the Dsg3 cytoplasmic tail (grey) were fused to the extracellular and transmembrane domains of the interleukin-2 receptor alpha chain (IL-2R, black) to create chimeric IL-2R-Dsg3 proteins. IA, intracellular anchor; ICS, intracellular cadherin-specific; IPL, proline-rich linker; RUD, repeating unit domain; DTD; desmoglein terminal domain; cyto, entire Dsg3 cytoplasmic tail; cytoΔ866 and cytoΔ715, Dsg3 cytoplasmic tail truncated at residues 866 and 715. (b–e) IL-2R and IL-2R-Dsg3 chimeras expressed in keratinocytes, methanol fixed and immunostained for cell-surface IL-2R (red, b′–e′) and total DP (green, b″–e″). (f–i) IL-2R and IL-2R-Dsg3 chimeras were co-expressed with PKP-1.myc and immunostained for cell surface IL-2R (red, f′–i′), total desmoplakin (DP) (green, f″–i″) and myc (f′″–i′″). Scale bars 20Hm.
Figure 5
Figure 5. Dsg3 cytoplasmic sequences mediate co-localization with DP and differential sensitivity to detergent pre-extraction
(a–h) Images of IL-2R and IL-2RDsg3 chimeras expressed in keratinocytes, with and without PKP-1.myc co-expression. Cells were labeled live with a mAb IL-2R (red and a′–h′) for 30 minutes and then immediately subjected to a 45 second detergent pre-extraction with 0.2% Triton-X100. Following fixation, the cells were immunostained for total DP (green and a″–h″) and myc (blue and e′″–h′″). Scale bars 20Hm.
Figure 6
Figure 6. PKP-1 induces the formation of calcium-independent, hyper-adhesive desmosomes
(a–d) Keratinocytes expressing empty vector (EV) or PKP-1.myc in high calcium (ca++) medium or after 4 hours calcium chelation, immunostained for desmoplakin (DP), PKP-1 and keratin-14 (K14), bar 20Hm. (e) Percent calcium-independent cells per microscope field. Cells were scored as calcium-independent if cell-cell contact sites were positive for DP. Means ± SEM (n=24 fields);*** p < 0.001 compared with EV (Mann Whitney). (f) Percent cells with PKP-1 localization at cell-cell contacts among calcium-independent cells per microscope field. Means ± SEM (n=24 fields). (g) Quantification of cell dissociation assays using monolayers subjected to 4 hours calcium chelation. Means ± SEM;*** p < 0.001 compared with EV (Two-way ANOVA, Holm-Sidak).
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