Ablation of the desmosomal plaque component plakophilin 1 underlies the autosomal recessive genodermatosis, skin fragility-ectodermal dysplasia syndrome (OMIM 604536). Skin from affected patients is thickened with increased scale, and there is loss of adhesion between adjacent keratinocytes,which exhibit few small, poorly formed desmosomes. To investigate further the influence of plakophilin 1 on keratinocyte adhesion and desmosome morphology,we compared plakophilin 1-deficient keratinocytes (vector controls) with those expressing recombinant plakophilin 1 introduced by retroviral transduction. We found that plakophilin 1 increases desmosomal protein content within the cell rather than enhancing transcriptional levels of desmosomal genes. Re-expression of plakophilin 1 in null cells retards cell migration but does not alter keratinocyte cell growth. Confluent sheets of plakophilin 1-deficient keratinocytes display fewer calcium-independent desmosomes than do plakophilin 1-deficient keratinocytes expressing recombinant plakophilin 1 or keratinocytes expressing endogenous plakophilin 1. In addition electron microscopy studies show that re-expression of plakophilin 1 affects desmosome size and number. Collectively, these results demonstrate that restoration of plakophilin 1 function in our culture system influences the transition of desmosomes from a calcium-dependent to a calcium-independent state and this correlates with altered keratinocyte migration in response to wounding. Thus,plakophilin 1 has a key role in increasing desmosomal protein content, in desmosome assembly, and in regulating cell migration.

Introduction

Desmosomes (DMs) are specialised cadherin-mediated adhesion complexes that impart stability and rigidity to tissues under mechanical stress(Garrod et al., 1999). These structures are dynamic and continual renewal of stratifying epidermis involves both rapid synthesis and degradation of cellular junctions. Such characteristics are important in epidermal homeostasis and wound healing(Moll et al., 1999; Wallis et al., 2000). Desmosomal adhesion may also be compromised in invading and metastasising transitional cell carcinomas, leading to speculation regarding possible tumour suppressor function of desmosomal attachment(Tselepis et al., 1998). DMs are formed later than adherens junctions during embryogenesis(Fleming et al., 1991) but their biological importance is underlined by the embryonic lethal mouse knockouts of the desmosomal components plakoglobin(Bierkamp et al., 1996; Ruiz et al., 1996) and desmoplakin (Dp) (Gallicano et al.,1998). Recently evidence has emerged demonstrating the importance of DMs in epithelial morphogenesis and cell positioning(Runswick et al., 2001; Vasioukhin et al., 2001).

Desmosomal cell-cell attachment is mediated through two types of transmembrane glycoproteins of the cadherin superfamily, desmoglein and desmocollin (Collins et al.,1991; Schafer et al.,1994). There are currently three known isoforms of both these molecules and each display differentiation-specific and tissue-specific expression patterns (Koch et al.,1992). Indirect attachment of desmosomal cadherins to intermediate filaments (IFs) is facilitated by plaque and armadillo proteins including Dp,plakoglobin and plakophilins (for review, see Green and Gaudry, 2001). Dp and plakoglobin are ubiquitous components of DMs whereas the plakophilins have multiple members displaying tissue-specific expression patterns that vary with epithelial differentiation (Hatzfeld et al., 1994; Heid et al.,1994; Mertens et al.,1996; Schmidt et al.,1997; Schmidt et al.,1999).

Understanding the precise role of DMs in cell biology has been difficult owing to the heterogeneity of their molecular composition. However,considerable insight has been gained from human and mouse model mutants. Specifically, naturally occurring monogenic disorders involving dysfunction or absence of desmosomal components have resulted in loss of cell-cell adhesion and striking clinical phenotypes (reviewed by Green and Gowdry, 2000; Norgett et al., 2000). These observations are supported by knockout and transgenic mice studies (reviewed by Green and Gaudry, 2000; Chidgey et al., 2001; Vasioukhin et al., 2001).

The first monogenic human disorder involving a desmosomal component to be described was the skin fragility-ectodermal dysplasia syndrome (OMIM 604536)characterised by complete ablation of plakophilin 1 (PKP1)(McGrath et al., 1997). Although PKP1 had been identified previously as a desmosomal component(Kapprell et al., 1988; Hatzfeld et al., 1994; Heid et al., 1994), it was not until the correlation of its absence with a human skin fragility disease that the importance of plakophilins in desmosomal adhesion was recognised. To date,three patients displaying complete ablation of PKP1(McGrath et al., 1997; McGrath et al., 1999; Whittock et al., 2000) have been reported. These naturally occurring mutations provide valuable resources for the investigation of human PKP1 function.

PKP1 is a major component of DMs in stratifying and complex epithelia but also is expressed widely in the nuclei of cells devoid of DMs(Schmidt et al., 1997),leading to speculation of an, as yet, unidentified nuclear signalling function. Other plakophilins, including plakophilin 2(Mertens et al., 1996) and plakophilin 3 (Schmidt et al.,1999), display dual localisation to DMs and nuclei but have differentiation-specific distributions.

Models of DM structure and macromolecular interactions have been derived from in vitro domain mapping, reconstitution studies (for review, see Green and Gaudry, 2001) and immunoelectron microscopy(North et al., 1999). Keratin IFs have been shown to bind to Dp(Stappenbeck and Green, 1992; Kouklis et al., 1994; Bornslaeger et al., 1996; Gallicano et al., 1998) as well as to PKP1 (Kapprell et al.,1988; Smith and Fuchs,1998; Hofmann et al.,2000). Dp has been shown to bind to plakoglobin (Steppenbeck et al., 1993; Smith and Fuchs,1998) and PKP1 (Kowalczyk et al., 1999; Hatzfeld et al.,2000; Hofmann et al.,2000), while plakoglobin binds to desmosomal cadherins(Kowalczyk et al., 1994; Troyanovsky et al., 1994). Plakophilin has also been found to bind desmosomal cadherins in vitro(Smith and Fuchs, 1998; Kowalczyk et al., 1999; Hatzfeld et al., 2000) but the significance of this interaction, as well as the interaction between PKP1 and keratin IFs in vivo, is unclear and a model of PKP1 providing lateral interactions for Dp has been suggested(Kowalczyk et al., 1999) which is supported by other immunoelectron microscopy data(North et al., 1999).

To analyse the role of PKP1 in keratinocyte cell biology we have characterised cell lines derived from the unique resource of PKP1-deficient keratinocytes. Specifically, we have retrovirally delivered recombinant PKP1 to cells derived from two separate PKP1-deficient patients and then compared these modified keratinocytes with appropriate vector controlled cells. We also have compared these cells with unaffected keratinocytes from two separate control individuals.

We demonstrate that PKP1 increases desmosomal components within cells but does not enhance expression of key desmosomal components at the transcriptional level. We also demonstrate that the level of the adherens junction proteins E-cadherin and β-catenin, and that of keratin 14 and keratins identified with a pan-keratin antibody, are not altered significantly. Levels of cellular plakophilin 2 and plakophilin 3 are not altered significantly relative to the other desmosomal proteins studied,indicating that in cultured PKP1-deficient cells there is no compensatory up-regulation of plakophilin 2 or plakophilin 3. Furthermore, PKP1 does not affect keratinocyte cell growth but does influence cell migration. We demonstrate that PKP1 has a role in the transition of DMs from a calcium-dependent state to a calcium-independent state and that PKP1 also affects the size and number of keratinocyte DMs.

Materials and Methods

Cell culture and construction of cell lines

After gaining appropriate consent, biopsy samples were obtained from thigh skin of one 3-month-old PKP1-deficient male patient (patient 1), buttock skin from one five-year-old PKP1-deficient male patient (patient 2), breast skin from one 31-year-old healthy female (control 1), and leg skin from one 45-year-old healthy female (control 2). Primary keratinocytes from these sources were isolated and grown in the presence of an irradiated 3T3 feeder layer (Rheinwald and Green,1975). Cells were immortalised with HPV16 (E6^E7) as described previously (Storey et al.,1988). All experiments were carried out between 15-25 passages after immortalisation. Cell lines with the prefix null indicate immortalised PKP1-deficient keratinocytes, while cell lines with the prefix norm indicate immortalised breast- or leg-derived keratinocytes derived from the controls.

Cells under normal Ca2+ conditions (approx. 1.2 mM) were cultured in DMEM:Ham's F12 medium (Invitrogen BV) at a ratio 3:1 and 10%foetal calf serum (FCS; ICN Biomedicals), supplemented with 1% penicillin and streptomycin (Sigma-Aldrich), 0.4 μg/ml hydrocortisone (Sigma-Aldrich),10–10 M cholera toxin (Sigma-Aldrich) and 5 μg/ml insulin(Sigma-Aldrich) at 37°C in 10% CO2. For low Ca2+experiments, cells were cultured in keratinocyte SFM (serum free; Invitrogen BV) at a Ca2+ concentration of 0.09 mM. For Ca2+ switch experiments (see below) controls were first performed using keratinocyte SFM supplemented with 1.2 mM CaCl2. Cell numbers were determined using a CASY® 1 (Scharfe System GmbH) cell counter.

Retroviral transduction

Full-length PKP1 was cloned into the pBabe puro retroviral vector(Morgenstern and Land, 1990)using standard molecular biology techniques. PKP1 constructs were fully sequenced to confirm retention of the wild-type open reading frame. The Phoenix amphotrophic retroviral packaging cell line, ΦNX-Ampho Cells(obtained from G.P. Nolan, Stanford, CA), was used to generate viral particles as described previously (Kinsella and Nolan, 1996). Cells were transduced with retroviral vectors at a low multiplicity of infection to prevent multiple integrations. Cells were selected using 2 μg/ml puromycin 48-72 hours post-transduction for a maximum of 14 days prior to expansion. Recombinant PKP1 expression in selected populations of transduced cells was verified to >99% using immunofluorescence microscopy.

Antibodies and immunofluorescence microscopy

Cells grown on glass coverslips were fixed using the following methods.

  • Cells membranes were made permeable by treatment with 0.2% Triton X-100 in PBS for 5 minutes before fixation for 20 minutes in methanol at–20°C or 4% formaldehyde at room temperature, followed by three 5-minute washes in PBS at room temperature.

  • Cells were fixed for 20 minutes at –20°C in 1:1 methanol:acetone followed by three 5-minute washes in PBS at room temperature.

PKP1 was detected with the monoclonal antibody PP1-5C2 (Progen) using method (i) (for nuclear and membrane localisation) or (ii) (for membrane only localisation) (Schmidt et al.,1997). Method (ii) was used with all other primary antibodies/antisera, which were as follows: 11-5F [Dps I and II(Parrish et al., 1987)], PG5.1(plakoglobin; Cymbus Biotechnology Ltd); Dsc-3-U114 (Desmocollin 3; Cymbus Biotechnology Ltd), AHP319 (desmoglein 3; Serotec). Secondary antibodies used were Alexa 488 goat antimouse (Molecular Probes, Inc.), Alexa 568 goat anti-rabbit (Molecular Probes, Inc.), FITC goat anti-mouse (DAKO) or FITC swine anti-rabbit (DAKO) IgG. Actin filaments were visualised by TRITC-coupled phalloidin (Molecular Probes, Inc.) after fixation using method (i). Counterstaining was performed using DAPI (Molecular Probes, Inc.) and microscopy was carried out using a Nikon Optiphot microscope (Nikon) with Kodak Microscopy Document System 290 (Kodak). Antibodies not used for immunfluorescence but for immunoblotting were as follows: sc-7298(β-actin; Autogen Bioclear), ab6276 (HSC-70; Abcam Ltd), AHP322(pan-desmocollin; Serotec), AHP321 (pan-desmoglein; Serotec), 33-3D[desmoglein 2 (Vilela et al.,1995)], PP2/62/86/150 multi-epitope cocktail (plakophilin 2;Progen), 310.9.1 (plakophilin 3; Progen), HECD-1 (E-cadherin), 6F9(β-catenin; Sigma-Aldrich), and LL001 [Keratin 14(Purkis et al., 1990)], LP34[reactive with Keratins 1, 5, 6 and 18(Lane and Alexander, 1990)]. Antibodies used for flow cytometry were as follows: FB12 (integrin α1;Chemicon International), P1E6 (integrin α2; Chemicon International),P1B5 (integrin α3; Chemicon International), P1D6 (integrin α5;Chemicon International), G0H3 (integrin α6; Serotec), Y9A2 (integrinα9; Chemicon International), L230 (integrin αV;prepared from hybridoma cells obtained from ATCC), LM609 (integrinα Vβ3; Chemicon International), P1F6 (integrinα Vβ5; Chemicon International), 10D5 (integrinα Vβ6; Chemicon International), 3E1 (integrin β4;Chemicon International), and IgG1 (mouse control; DAKO).

Immunoblotting

Total protein extracts were obtained from cells seeded at high density(5×105 cells/35 mm Petri dish) and grown for 72 hours under normal Ca2+ conditions as previously described(Wan et al., 2003). Internal controls of heat-shock protein HSC-70 or β-actin were used to reprobe each blot to demonstrate equivalent protein loading. All blots were subject to densitometry analysis using the NIH Image 1.61 software Gel Plotting macro.

Electron microscopy

Cells grown in 35 mm plastic Petri dishes were fixed in 2% formaldehyde with 2.5% glutaraldehyde in 0.1 M Sorrenson's phosphate buffer pH 7.4 followed by post-fixation in aqueous 1.3% osmium tetroxide and en-bloc staining with 2%uranyl acetate in 50% ethanol. Cells were not detached from the plastic Petri dishes. The samples were then processed using standard techniques and embedded in TAAB 812 (medium hardness) epoxy resin(Eady, 1985). Ultrathin sections (60-90 nm) were stained with 2% uranyl acetate in 50% ethanol and Reynold's lead citrate before observation in a JEOL 100CX transmission electron microscope (JEOL). To measure DM density, electron micrographs were taken at 5,000× magnification and 6-8 arbitrary fields per sample were examined. The negatives were scanned into a personal computer using Adobe Photoshop image software. Only DMs displaying recognisable opposing plaques were scored. To estimate the DM density relative to cell cytoplasm we applied a coherent single square lattice over each EM negative to produce a total of 252 test points (at a distance of 0.5 cm, corresponding to 10 μm on the section). Estimation of DM density was achieved by counting the total number of DMs per negative relative to the total number of test points falling on keratinocyte cytoplasm only. From these counts the DM density was calculated and expressed as a ratio of DM per cytoplasm test points. To measure DM size,electron micrographs were taken at 13,000× magnification of several arbitrary fields that contained DMs in the ultrathin sections used for DM counting. The length of the extracellular space between two clear, opposing plaques was measured using the Photoshop measurement tool.

RNase protection assays

Total RNA was isolated from cultured cells grown for 72 hours post-confluency. RNA antisense probes were prepared as follows. Fragments of PKP1, plakoglobin, Dp, desmoglein 2, desmoglein and GAPDHwere PCR amplified from keratinocyte cDNA and cloned using the TOPO TA Cloning® Kit Dual Promoter (Invitrogen BV). RNA probes generated using[32P]dUTP (NEN Life Science Products, Inc.) and T7 RNA polymerase(Promega Corporation) were then purified from unincorporated nucleotides and DNase treated to remove plasmid DNA using RNeasy® Mini Kit columns(Qiagen). Between 4 and 8 μg of total RNA was used for RNase protection assays using an RNase Protection Kit (Roche) according to the manufacturer's specifications. Each RNA probe representing a desmosomal gene was mixed with GAPDH probe as an internal control in each separate assay. Protected products were resolved using 5% TBE-urea ready gels and a mini-PROTEAN® 3 cell(Bio-Rad), then dried, exposed with a phosphorimager and analysed using ImageQuant™ software (Amersham Pharmacia Biotech).

Time-lapse microscopy and image analysis

Migration study

Cells were seeded at equal density (7×105/35 mm Petri dish) and grown for 48 hours. Mitomycin C (Sigma-Aldrich) was used to arrest mitosis before confluent cultures were washed with medium and wounded by scraping the blunt end of a 1 ml micro-Gilson pipette tip across the centre of the cell sheet, removing cells in a linear fashion to a width between 3 and 6 mm. The cultures were then washed three times and transferred to a specially constructed two-piece circular aluminium housing that had a glass lid and an epicentric hole in the base through which the cells were observed. The chamber was gassed with 10% CO2 in humidified air and was placed onto the stage of an Olympus IMT-2 inverted phase contrast microscope (Olympus Microscopes), fitted with a Fujitsu TC2-336P charge coupled device camera (EOS Electronics AV Ltd) and surrounded by a Perspex temperature-regulated jacket that was adjusted to 37°C. Images were collected every 6 minutes to a Power Mac 7100 computer by use of Adobe Premiere software. After calibration using Optilab Pro 2.6.1 software the area of cells occupying each image at the given time points was calculated and expressed in μm moved/hour.

Adhesion study

Cells were seeded at equal density (7×105/35 mm Petri dish) and grown for 72 hours in 1.2 mM Ca2+ medium. Cells seeded at this density were 100% confluent after 16 hours. 1.2 mM Ca2+ medium was replaced with 0.09 mM Ca2+ medium after washing three times with 0.09 mM Ca2+ medium (low Ca2+ switch) and the cells were transferred to the time-lapse apparatus detailed above. When cells change from a flattened morphology towards a rounded one they become more refractile. We monitored changes in adhesiveness using image analysis. Images were recorded every 6 minutes. At given time points, 256 grey scale (0=black,255=white) images were collected and analysed. The average grey for image 1(time=0 minutes) of all samples was calculated and used as a background threshold. For each image analysed the number of pixels above this threshold was calculated as a percentage of total pixels. Control samples were grown in calcium-supplemented low Ca2+ medium (1.2 mM) for 72 hours and monitored in this fashion. In control experiments no difference was noted in the percentage pixels above the given grey threshold for each cell line tested(data not shown).

Flow cytometry

Integrin complement of the cell lines was assessed using flow cytometry. Confluent sheets of cells were harvested, washed and incubated with primary antibody for 40 minutes at 4°C. Secondary antibody was applied for 30 minutes at 4°C after washing 4 times. Cells were then washed twice with PBS and resuspended in 0.4 ml PBS. Labelled cells were scanned on a FACSCalibur cytometer (Becton Dickinson) and analysed using Cellquest software, acquiring 1×105 events. Flow cytometry was performed in triplicate on two separate occasions. For each antibody the geometric mean was noted and the value of IgG1 control was subtracted to give a value for level of expression.

Sequential detergent extractions

Cells were seeded at equal high density (7×105/35 mm Petri dish) and grown for 72 hours in 1.2 mm Ca2+ before being either extracted, subject to 1 hour of a low Ca2+ switch and extracted, or subject to 24 hours of a low Ca2+ switch followed by extraction. Extractions were performed as described(Palka and Green, 1997). Equal volumes of each extracted pool were resolved and immunoblotted as described above.

Results

Production of cell lines expressing recombinant PKP1 and appropriate vector controls using retroviral transduction

Keratinocytes isolated from two PKP1-deficient patients and two healthy control individuals were immortalised as described in Materials and Methods. Cells were cultured after crisis for over 10 passages with no obvious gross differences in morphology or behaviour (differentiation, proliferation, cell turnover).

The pBabe-puro vector and Phoenix amphotrophic packaging cell line were used to introduce recombinant PKP1 into both normal and PKP1-deficient cell lines as described. A vector-only construct was also used in order to provide control populations. Viral particles were collected from transiently transfected packaging cells as described and target cells were selected for puromycin resistance, transmitted by the puromycin resistance gene within the vector. Viral titre was not calculated or optimised and infection rates were low, less than 5% as judged by the initial growth of puromycin-resistant cells, indicating that multiple integrations (more than two) were unlikely(Garlick et al., 1991). PKP1 was detected in >99% of selected null cells expressing recombinant PKP1 2 weeks post-infection, as judged by immunofluorescence(Fig. 1A-C).

Fig. 1.

Expression of PKP1 and DM formation in cultured keratinocyte cell lines derived from PKP1-deficient and control individuals. (A-C) The presence of PKP1 was determined using PP1-5C2 antibody in (A) nullpB1 cells (left panel; right panel shows DAPI counterstain), (B) nullPKP2 cells, and(C) normpB1 cells. Recombinant PKP1 (B) and endogenous PKP1 (C), both localise in nuclei and to the cell membrane. (D-F) Lack of PKP1 has no discernible effect on DM ultrastructure after 8 days of confluent culture in nullpB1 cells (D), nullPKP1 cells (E) and normpB1 cells (F). Arrows indicate two DM examples per micrograph.(G) Immunoblotting of total cell extracts for PKP1 (upper panel) and Hsc-70 as loading control (lower panel) from (lane 1) null cells, (lane 2) nullpB1, (lane 3) nullpB2, (lane 4) nullPKP1, (lane 5) nullPKP2, (lane 6) normpB1. No PKP1 is detectable in null and nullpB cells. Scale bar: 10 μm (A-C); 1 μm(D-F).

Fig. 1.

Expression of PKP1 and DM formation in cultured keratinocyte cell lines derived from PKP1-deficient and control individuals. (A-C) The presence of PKP1 was determined using PP1-5C2 antibody in (A) nullpB1 cells (left panel; right panel shows DAPI counterstain), (B) nullPKP2 cells, and(C) normpB1 cells. Recombinant PKP1 (B) and endogenous PKP1 (C), both localise in nuclei and to the cell membrane. (D-F) Lack of PKP1 has no discernible effect on DM ultrastructure after 8 days of confluent culture in nullpB1 cells (D), nullPKP1 cells (E) and normpB1 cells (F). Arrows indicate two DM examples per micrograph.(G) Immunoblotting of total cell extracts for PKP1 (upper panel) and Hsc-70 as loading control (lower panel) from (lane 1) null cells, (lane 2) nullpB1, (lane 3) nullpB2, (lane 4) nullPKP1, (lane 5) nullPKP2, (lane 6) normpB1. No PKP1 is detectable in null and nullpB cells. Scale bar: 10 μm (A-C); 1 μm(D-F).

Cells expressing pBabe-puro vector only are referred to with the suffix pB while cells expressing pBabe-puro containing and expressing PKP1 are referred to with the suffix PKP. Null indicates null cells and norm indicates normal cells. Where needed, 1 indicates cells derived from patient 1 or control 1 while 2 indicates cells derived from patient 2 or control 2 (see Materials and Methods). Therefore nullpB1 refers to vector control null cells derived from patient 1 while nullPKP2 refers to null cells derived from patient 2 expressing recombinant PKP1.

PKP1 was detected in nullPKP cells but not in nullpB or null only populations as determined by immunoblotting(Fig. 1G). PKP1 was detected in immortalised normal keratinocytes as well as in derived populations expressing vector controls. All experiments and observations of transduced cell populations were made using passages 5-10 after introduction of retroviruses.

Electron micrographs at 13,000× magnification from nullpB1,nullPKP1 and normpB1 cell lines fixed after 8 days growth were analysed blind and a representative DM field from each sample set (5 negatives for each line) is presented in Fig. 1D-F. No noticeable difference is seen in DM ultrastructure from these fields.

Lack of PKP1 reduces the level of cellular desmosomal components but not E-cadherin, β-catenin, or Keratin IFs

Using western blotting we determined whether de novo expression of PKP1 affected expression of other desmosomal proteins, adherens junctional proteins, or keratins. Fig. 2Ashows that, in comparison with the retroviral control lines (nullpB),re-expression of PKP1 in the nullPKP lines resulted in significant increases in desmoplakin, desmocollin and plakoglobin, but not E-cadherin. Increases were also observed in desmoglein 3, while levels of desmoglein 1,plakophilin 2 and plakophilin 3 were not significantly different when comparing nullpB with nullPKP western blots(Fig. 2A). No discernible difference was observed in the level of β-catenin expression between cell lines tested. Using antibodies against keratin 14 and a pan-keratin antibody recognising keratins 1, 5, 6 and 18, we could not identify any differences between nullpB, nullPKP and normpB cell lines(Fig. 2A).

Fig. 2.

Characterisation of junctional proteins and keratin content of cell lines used in this study, as well as RNase protection assays of PKP1, Dp,plakoglobin, desmoglein 2 and desmoglein 3 mRNA levels. (A) Lack of PKP1 is associated with reduced levels of the desmosomal components but not E-cadherin, β-catenin, or keratins. Desmoglein 1, PKP2 and PKP3 are not significantly up- or down-regulated in PKP1 deficient cells. Immunoblotting of total cell extracts from nullpB1, nullpB2, nullPKP1, nullPKP2,normpB2 and normpB2 are shown and labelled nullpB,nullPKP and normpB. The graph beneath represents the levels of protein, determined by densitometry, with respect to the loading control and normalised against normpB protein level (density=1±s.e.m. for 2-3 replica experiments). Pg, plakoglobin; Dscs, pan desmocollin (presence of the single band shown is dependent on resolution of gel used as antibody recognises both isoforms), Dsg1, desmoglein 1; Dsg2, desmoglein 2; Dsg3,desmoglein 3; E-cad, E-cadherin; β-cat, β-catenin; PKP2, plakophilin 2; PKP3, plakophilin 3; K14, keratin 14; LP34, antibody reactive to keratins 1, 5, 6, 18. (B) Lack of PKP1 does not affect mRNA expression levels of Dp,plakoglobin, desmoglein 2 or desmoglein 3 when comparing nullpB1 with nullPKP1. RNase protection assay using; (lane 1) yeast tRNA (negative control); (lane 2) PCR fragments used as template to generate probe (positive control); (lane 3) nullpB1 RNA; (lane 4) nullPKP1 RNA; (lane 5) normpB1 RNA; to protect [32P]dUTP-labelled RNA probes. The graph beneath represents mRNA expression levels relative to GAPDH internal probe and normalised to normpB1 levels (density=1±s.e.m. for 2-3 replica experiments).

Fig. 2.

Characterisation of junctional proteins and keratin content of cell lines used in this study, as well as RNase protection assays of PKP1, Dp,plakoglobin, desmoglein 2 and desmoglein 3 mRNA levels. (A) Lack of PKP1 is associated with reduced levels of the desmosomal components but not E-cadherin, β-catenin, or keratins. Desmoglein 1, PKP2 and PKP3 are not significantly up- or down-regulated in PKP1 deficient cells. Immunoblotting of total cell extracts from nullpB1, nullpB2, nullPKP1, nullPKP2,normpB2 and normpB2 are shown and labelled nullpB,nullPKP and normpB. The graph beneath represents the levels of protein, determined by densitometry, with respect to the loading control and normalised against normpB protein level (density=1±s.e.m. for 2-3 replica experiments). Pg, plakoglobin; Dscs, pan desmocollin (presence of the single band shown is dependent on resolution of gel used as antibody recognises both isoforms), Dsg1, desmoglein 1; Dsg2, desmoglein 2; Dsg3,desmoglein 3; E-cad, E-cadherin; β-cat, β-catenin; PKP2, plakophilin 2; PKP3, plakophilin 3; K14, keratin 14; LP34, antibody reactive to keratins 1, 5, 6, 18. (B) Lack of PKP1 does not affect mRNA expression levels of Dp,plakoglobin, desmoglein 2 or desmoglein 3 when comparing nullpB1 with nullPKP1. RNase protection assay using; (lane 1) yeast tRNA (negative control); (lane 2) PCR fragments used as template to generate probe (positive control); (lane 3) nullpB1 RNA; (lane 4) nullPKP1 RNA; (lane 5) normpB1 RNA; to protect [32P]dUTP-labelled RNA probes. The graph beneath represents mRNA expression levels relative to GAPDH internal probe and normalised to normpB1 levels (density=1±s.e.m. for 2-3 replica experiments).

Lack of PKP1 does not alter levels of Dp, Plakoglobin, Desmoglein 2 or Desmoglein 3 mRNA expression

In order to assess whether the different levels of protein identified in Fig. 2A were a result of increased mRNA expression, RNA probes for GAPDH, PKP1, Dp, plakoglobin,desmoglein 2 and desmoglein 3 were used in RNase protection assays of total RNA isolated from nullpB1, nullPKP1 and normpB1. No PKP1 mRNA was detected in nullpB1 cells but it was evident in normpB1 and nullPKP1 cells(Fig. 2B). No significant difference in Dp, desmoglein 3 or plakoglobin expression(Fig. 2B) was seen. A slight decrease was seen in desmoglein 2 mRNA levels between null and norm cells, but there were no observed differences between nullpB1 and nullPKP1 cells. Overall we demonstrated no differences in levels of mRNA expression of desmosomal components between cells lacking PKP1 (nullpB1) and matched cells expressing PKP1(nullPKP1).

No difference in growth rates are seen between transduced populations of normal or PKP1 null keratinocyte cell lines.

PKP1-deficient skin demonstrates hyperkeratosis and thickening compared with normal skin (McGrath et al.,1997; McGrath et al.,1999). We investigated whether PKP1 expression influences in vitro cellular growth rates. Cells were seeded at equal density in identical 6-well plates, harvested at indicated time intervals, and counted as described. No significant differences were observed in the growth rate of null and norm populations expressing recombinant PKP1 or vector controls in either 1.2 mM Ca2+ or 0.09 mM Ca2+ (data not shown).

PKP1 null cells migrate at a faster rate in response to an in vitro wounding model.

Patients lacking PKP1 are characterised by skin fragility and an altered pattern of wound healing (chronic erosions and excessive scale-crust after trauma). We compared in vitro wound healing between nullpB, nullPKPand normpB cells. We noted repeatedly that cells lacking PKP1 migrate at a faster rate than cells expressing PKP1 in response to wounding(Fig. 3). Two sets of experimental data were generated on two different sets of time-lapse apparatus(A and B). Between 8 and 16 hours post-wounding in data set A, nullpB1 cells migrated at speeds ranging between 161.8 μm/hour and 189.85 μm/hour, while nullPKP1 cells migrated at speeds ranging between 106.49 μm/hour and 139.09 μm/hour (n=3 in both cases,see Fig. 3). Between 8 and 16 hours post-wounding in data set B, nullpB2 cells migrated at speeds ranging between 120 μm/hour and 193.3 μm/hour, while nullPKP2cells migrated at speeds ranging between 73.6 μm/hour and 105.5 μm/hour(n=3 in both cases, data not shown). We considered that a reorganisation of the actin cytoskeleton into a more migratory phenotype (less stress fibres, more ruffling membranes) might also characterise PKP1 deficiency. However, examination of actin filament organisation revealed no obvious differences between nullpB and nullPKP cells at the wound edge or in a monolayer (data not shown). We also considered that the integrin complement could be altered between nullpB and nullPKP cells but could not show any significant difference in the levels of integrins α1, 2, 3, 5, 6, 9, αV, αVβ5,αVβ6 and β4 (data not shown) between nullpB, nullPKPand normpB cells. No αVβ3 was detected in any of the cell lines (data not shown).

Fig. 3.

Lack of PKP1 increases migration rates in response to in vitro wounding. Keratinocyte cell lines were seeded at high density and grown for 48 hours in 35 mm dishes prior to wounding. Time-lapse microscopy monitored migration over 24 hours and image analysis determined movement (μm/hour) between the given time points. Graph represents mean±s.e.m. (*P<0.05) using patient 1 (3-month-old male) and control 1 (31-year-old female) cells. Lower phase images taken after 16 hours, Black line indicates wound edge at time 0. Scale bar: 100 μm.

Fig. 3.

Lack of PKP1 increases migration rates in response to in vitro wounding. Keratinocyte cell lines were seeded at high density and grown for 48 hours in 35 mm dishes prior to wounding. Time-lapse microscopy monitored migration over 24 hours and image analysis determined movement (μm/hour) between the given time points. Graph represents mean±s.e.m. (*P<0.05) using patient 1 (3-month-old male) and control 1 (31-year-old female) cells. Lower phase images taken after 16 hours, Black line indicates wound edge at time 0. Scale bar: 100 μm.

Less cell-cell contacts are retained in confluent sheets of cells lacking PKP1 when exposed to low calcium concentrations.

Light and electron microscopy of PKP1-deficient skin sections revealed widening of the intercellular spaces between keratinocytes in the suprabasal layers but not in the basal or upper granular layers(McGrath et al., 1997; McGrath et al., 1999). Keratinocytes lacking PKP1 growing in culture under high (1.2 mM)Ca2+ concentrations showed no obvious morphological differences from keratinocytes expressing PKP1 (Fig. 4, upper panel). We examined the response of nullkeratinocytes to a low Ca2+ switch. In low Ca2+concentrations, cell-cell zonula occludens and zonula adherens junctions are disassembled and inhibited from forming whereas DMs are only disassembled in culture if they have not reached a state of maturity known as calcium-independence (Garrod,1996) (discussed below). It was observed that, after a low Ca2+ switch, nullpB cells appeared to round up more and become more refractile under phase contrast light microscopy than nullPKP or normpB cells. We quantified this finding using phase contrast time-lapse microscopy in two separate experimental data sets using six cell lines derived from four different individuals. Cells seeded at high density and grown for 72 hours in high Ca2+ medium were then switched to low Ca2+ medium and photographed every 6 minutes for 24 hours. Using image analysis software it was determined that nullpBcell lines became more refractile than nullPKP and normpBcell lines (Fig. 4). This difference was not observed if the cells were grown for less than 48 hours(nullPKP and normpB became as refractile as nullpBcells, data not shown).

Fig. 4.

Cells lacking PKP1 become more refractile in response to low Ca2+ concentrations compared to those expressing PKP1. Cells were seeded at high density and grown for 72 hours under normal Ca2+concentrations (1.2 mM) before being subjected to a low Ca2+ switch and monitored using time-lapse imaging. (A) Phase contrast images at 10×magnification show confluent cell sheets after a low Ca2+ switch at time 0 (left panels), after 48 minutes (middle panels), and after 24 hours(right panels). Tight cell-cell contacts are lost in nullpB1 cells after 24 hours (top right panel), while areas of tight cell-cell contacts can be seen in nullPKP1 in addition to refractile cell edges (bottom right panel). Scale bar: 100 μm. (B,C) Digital images were analysed to determine the percentage of pixels above the mean background threshold at time 0. Graphs show the mean±s.e.m. Graph B compares cells derived from patient 1 (3-month-old male) with those derived from control 1 (31-year-old female), while graph C compares cells derived from patient 2 (5-year-old-male)with those derived from control 2 (45-year-old female) using separate time-lapse equipment (*P<0.05, ** P<0.01,***P<0.005).

Fig. 4.

Cells lacking PKP1 become more refractile in response to low Ca2+ concentrations compared to those expressing PKP1. Cells were seeded at high density and grown for 72 hours under normal Ca2+concentrations (1.2 mM) before being subjected to a low Ca2+ switch and monitored using time-lapse imaging. (A) Phase contrast images at 10×magnification show confluent cell sheets after a low Ca2+ switch at time 0 (left panels), after 48 minutes (middle panels), and after 24 hours(right panels). Tight cell-cell contacts are lost in nullpB1 cells after 24 hours (top right panel), while areas of tight cell-cell contacts can be seen in nullPKP1 in addition to refractile cell edges (bottom right panel). Scale bar: 100 μm. (B,C) Digital images were analysed to determine the percentage of pixels above the mean background threshold at time 0. Graphs show the mean±s.e.m. Graph B compares cells derived from patient 1 (3-month-old male) with those derived from control 1 (31-year-old female), while graph C compares cells derived from patient 2 (5-year-old-male)with those derived from control 2 (45-year-old female) using separate time-lapse equipment (*P<0.05, ** P<0.01,***P<0.005).

NullpB cells retain less membrane-bound desmosomal components after low calcium switch compared with nullPKP and normpB cells

Using immunofluorescence microscopy, we examined the distribution of the desmosomal components PKP1 (Fig. 5A), Dp (Fig. 5B),plakoglobin (data not shown), desmoglein 3 (data not shown) and desmocollin 3(Fig. 5C) in cells grown on coverslips in high Ca2+ medium(Fig. 5A-C, left hand panels),cells that were grown in high Ca2+ medium and subject to a low Ca2+ switch for 1 hour (Fig. 5A-C, middle panels), and cells grown in high Ca2+medium and subject to a low Ca2+ switch for 24 hours(Fig. 5A-C, right hand panels)prior to fixation. No differences in desmosomal protein distribution could be seen between nullpB, nullPKP and normpB cell lines grown in high Ca2+ medium (examples of Dp and desmocollin 3 shown in the left hand panels of Fig. 5B,C). However, 1 hour after a low Ca2+ switch the distribution of Dp markedly changed in nullpB cell lines compared with nullPKPand normpB cell lines (Fig. 5B, right hand panels). Far less Dp was localised to the cell membrane in nullpB cells after 1 hour of a low Ca2+switch, although a small proportion of cells did retain membrane bound Dp,compared to nullPKP and normpB cells. This difference was not obvious for plakoglobin (data not shown), desmoglein 3 (data not shown) or desmocollin 3 (Fig. 5C, middle panels) at the one-hour time point. However, 24 hours after a low Ca2+ switch, we noted a large reduction in membrane bound Dp,plakoglobin (data not shown), desmocollin 3 and desmoglein 3 (data not shown)in nullpB cells compared to nullPKP and normpBcells for all desmosomal components examined (examples of Dp and desmocollin 3 shown in Fig. 5B and 5C, right hand panels).

Fig. 5.

Loss of DM adhesion in response to a low Ca2+ switch is more prominent in PKP1-deficient cells. (A-C) Fixed cells with immunofluorescent staining for PKP1 (A), Dp (B) and desmocollin 3 (C). In each case (A-C) cells were grown for 72 hours in high Ca2+ medium (1.2 mM; High, left hand panels), 72 hours in high Ca2+ medium followed by 1 hour of a low Ca2+ switch (Low 1 hour, middle panels), or 72 hours in high Ca2+ medium followed by 24 hours of a low Ca2+ switch(Low 24 hours, right hand panels). (A) PKP1 staining is negative in nullpB cells and is membrane bound after a low Ca2+ switch in nullPKP and normpB cells for all time courses (lack of nuclear staining reflects fixation method used). (B) Dp staining after growth in 1.2 mM Ca2+ without low Ca2+ switch is linear and membrane bound in all three cell lines. Dp staining after 1 hour of a low Ca2+ switch is linear and membrane localised in the majority of nullPKP and normpB cells (middle and lower middle panels)but linear, membrane localised, Dp staining is only seen in a small proportion of nullpB cells (upper middle panel). Dp staining after 24 hours of a low Ca2+ switch is membrane localised in the majority of nullPKP and normpB cells (middle right hand and lower right hand panels) but membrane localised Dp staining is absent in the majority of nullpB cells (upper middle panel). (C) Desmocollin 3 staining after growth in 1.2 mM Ca2+ for 72 hours and following a subsequent low Ca2+ switch for 1 hour displays a linear, membrane localised distribution in the majority of cells for cell lines tested (all left hand and middle panels). After 72 hours of growth in 1.2 mM Ca2+ and a subsequent low Ca2+ switch for 24 hours, desmocollin 3 staining is membrane localised in the majority of nullPKP and normpBcells (middle right hand and lower right hand panels) while virtually no membrane localised desmocollin 3 was detected in nullpB cells (upper right hand panel). Scale bar: 10 μm. (D) Subcellular distribution of plakoglobin (Pg) and desmoglein 3 (Dsg 3) in nullpB2, nullPKP2 and normpB2 cells following a low Ca2+ switch for 1 or 24 hours. Lanes 1, nullpB2 1.2 mM Ca2+; 2, nullpB2 1 hour low Ca2+ switch; 3, nullpB2 24 hours low Ca2+ switch; 4, nullPKP2 1.2 mM Ca2+; 5, nullPKP2 1 hour low Ca2+ switch; 6, nullPKP2 24 hours low Ca2+ switch; 7, normpB2 1.2 mM Ca2+;8, normpB2 1 hour low Ca2+ switch; 9, normpB2 24 hours low Ca2+ switch. Overall there is less plakoglobin and desmoglein 3 in nullpB2 cells (lanes 1-3) compared with nullPKP2 (lanes 4-6) and normpB2 cells (lanes 7-9). After 24 hours of a switch to low Ca2+ there is proportionally less membrane bound and junctional/cytoskeletal-associated protein in nullpB2 (lane 3) compared to nullPKP2 (lane 6) and normpB2 (lane 9).

Fig. 5.

Loss of DM adhesion in response to a low Ca2+ switch is more prominent in PKP1-deficient cells. (A-C) Fixed cells with immunofluorescent staining for PKP1 (A), Dp (B) and desmocollin 3 (C). In each case (A-C) cells were grown for 72 hours in high Ca2+ medium (1.2 mM; High, left hand panels), 72 hours in high Ca2+ medium followed by 1 hour of a low Ca2+ switch (Low 1 hour, middle panels), or 72 hours in high Ca2+ medium followed by 24 hours of a low Ca2+ switch(Low 24 hours, right hand panels). (A) PKP1 staining is negative in nullpB cells and is membrane bound after a low Ca2+ switch in nullPKP and normpB cells for all time courses (lack of nuclear staining reflects fixation method used). (B) Dp staining after growth in 1.2 mM Ca2+ without low Ca2+ switch is linear and membrane bound in all three cell lines. Dp staining after 1 hour of a low Ca2+ switch is linear and membrane localised in the majority of nullPKP and normpB cells (middle and lower middle panels)but linear, membrane localised, Dp staining is only seen in a small proportion of nullpB cells (upper middle panel). Dp staining after 24 hours of a low Ca2+ switch is membrane localised in the majority of nullPKP and normpB cells (middle right hand and lower right hand panels) but membrane localised Dp staining is absent in the majority of nullpB cells (upper middle panel). (C) Desmocollin 3 staining after growth in 1.2 mM Ca2+ for 72 hours and following a subsequent low Ca2+ switch for 1 hour displays a linear, membrane localised distribution in the majority of cells for cell lines tested (all left hand and middle panels). After 72 hours of growth in 1.2 mM Ca2+ and a subsequent low Ca2+ switch for 24 hours, desmocollin 3 staining is membrane localised in the majority of nullPKP and normpBcells (middle right hand and lower right hand panels) while virtually no membrane localised desmocollin 3 was detected in nullpB cells (upper right hand panel). Scale bar: 10 μm. (D) Subcellular distribution of plakoglobin (Pg) and desmoglein 3 (Dsg 3) in nullpB2, nullPKP2 and normpB2 cells following a low Ca2+ switch for 1 or 24 hours. Lanes 1, nullpB2 1.2 mM Ca2+; 2, nullpB2 1 hour low Ca2+ switch; 3, nullpB2 24 hours low Ca2+ switch; 4, nullPKP2 1.2 mM Ca2+; 5, nullPKP2 1 hour low Ca2+ switch; 6, nullPKP2 24 hours low Ca2+ switch; 7, normpB2 1.2 mM Ca2+;8, normpB2 1 hour low Ca2+ switch; 9, normpB2 24 hours low Ca2+ switch. Overall there is less plakoglobin and desmoglein 3 in nullpB2 cells (lanes 1-3) compared with nullPKP2 (lanes 4-6) and normpB2 cells (lanes 7-9). After 24 hours of a switch to low Ca2+ there is proportionally less membrane bound and junctional/cytoskeletal-associated protein in nullpB2 (lane 3) compared to nullPKP2 (lane 6) and normpB2 (lane 9).

The observation that Dp seems to move from the cell membrane in response to low Ca2+ earlier than other desmosomal components analysed is interesting in light of the fact that, of the desmosomal components examined in this study, only Dp showed gross differences in distribution in PKP1-deficient skin compared with control skin(Fig. 6A,B). In order to assess whether Dp staining moved from membrane to cytoplasm before that of the other desmosomal components examined, nullpB cells were double stained for Dp and desmoglein 3 after 1 hour of a low Ca2+ switch. Cells that contained only membrane-bound desmoglein 3, rather than colocalised Dp and desmoglein 3, were readily detected (Fig. 6C, upper panels). Co-localised membrane staining of Dp and desmoglein 3 was also readily detectable as expected, but membrane bound Dp in the absence of desmoglein 3 expression was not detected. This was not the case with double staining for Dp and desmoglein 3 in the nullPKP cells,where co-localisation was readily seen in the majority of cells(Fig. 6C, lower panels).

Fig. 6.

Dp staining is altered in PKP1-deficient epidermis and also in PKP1-deficient keratinocytes following a low Ca2+ switch. (A)Examples of 5 μm thickness skin sections from normal epidermis (top panels)and PKP1-deficient epidermis (bottom panels) stained with antibodies against plakoglobin (PG5.1, left panels) and Dp (11-5F, right panels). Plakoglobin staining is unaltered in PKP1-deficient epidermis (bottom left) compared with normal epidermis (top left), while Dp displays a more diffuse, intercellular staining pattern in PKP1 epidermis (bottom right, arrow) compared to normal control (top right). (B) Confocal microscopy imaging of Dp (11-5F, green) and desmoglein (AHP321, red) staining of a region of suprabasal normal breast epidermis (upper panels) and a region of suprabasal PKP1-deficient epidermis(lower panels). Right column displays merged images. Normal epidermis shows diffuse and linear staining for both Dp and desmogleins (upper left and upper middle) while PKP1-deficient epidermis shows linear cell membrane staining for desmogleins (lower middle) but Dp staining shows wide, peripheral membrane staining (lower left, arrow). (C) Double staining of nullpB cells(upper panels) and nullPKP cells (lower panels), cultured for 72 hours in high Ca2+ medium and switched to low Ca2+medium for 1 hour, for Dp (11-5F, left panels) and desmoglein 3 (AHP319, right panels). The majority of Dp staining is non-membrane localised in nullpB cells (upper left panel, arrow indicates cell-cell junction with reduced Dp reactivity), while the majority of desmoglein 3 staining is membrane bound (upper right panel). Areas of membrane-bound desmoglein 3 but not Dp were readily identified. In nullPKP cells the majority of Dp staining is membrane localised (lower left panel), while the majority of desmoglein 3 is also membrane bound (lower right panel). Scale bars: 100 μm(A); 10 μm (B,C).

Fig. 6.

Dp staining is altered in PKP1-deficient epidermis and also in PKP1-deficient keratinocytes following a low Ca2+ switch. (A)Examples of 5 μm thickness skin sections from normal epidermis (top panels)and PKP1-deficient epidermis (bottom panels) stained with antibodies against plakoglobin (PG5.1, left panels) and Dp (11-5F, right panels). Plakoglobin staining is unaltered in PKP1-deficient epidermis (bottom left) compared with normal epidermis (top left), while Dp displays a more diffuse, intercellular staining pattern in PKP1 epidermis (bottom right, arrow) compared to normal control (top right). (B) Confocal microscopy imaging of Dp (11-5F, green) and desmoglein (AHP321, red) staining of a region of suprabasal normal breast epidermis (upper panels) and a region of suprabasal PKP1-deficient epidermis(lower panels). Right column displays merged images. Normal epidermis shows diffuse and linear staining for both Dp and desmogleins (upper left and upper middle) while PKP1-deficient epidermis shows linear cell membrane staining for desmogleins (lower middle) but Dp staining shows wide, peripheral membrane staining (lower left, arrow). (C) Double staining of nullpB cells(upper panels) and nullPKP cells (lower panels), cultured for 72 hours in high Ca2+ medium and switched to low Ca2+medium for 1 hour, for Dp (11-5F, left panels) and desmoglein 3 (AHP319, right panels). The majority of Dp staining is non-membrane localised in nullpB cells (upper left panel, arrow indicates cell-cell junction with reduced Dp reactivity), while the majority of desmoglein 3 staining is membrane bound (upper right panel). Areas of membrane-bound desmoglein 3 but not Dp were readily identified. In nullPKP cells the majority of Dp staining is membrane localised (lower left panel), while the majority of desmoglein 3 is also membrane bound (lower right panel). Scale bars: 100 μm(A); 10 μm (B,C).

We also investigated the levels of cytosolic, membrane or junctional/cytoskeletal associated plakoglobin and desmoglein 3 using a sequential detergent extraction method described by Palka and Green(Palka and Green, 1997)(Fig. 5D). The overall level of plakoglobin and desmoglein 3 is less in nullpB2 cells than in nullPKP2 and normpB2 cells over all three fractions. This is in agreement with the corresponding protein levels in the total cell lysates presented in Fig. 2A. Comparing the Triton X-100 soluble pool (membrane associated) at 24 hours after a low Ca2+ switch, the level of plakoglobin and desmoglein 3 in the nullpB2 pool is virtually undetectable compared with nullPKP2 and normpB2 pool, supporting the immunofluorescence data which showed less desmosomal membrane-bound retaining cells in the nullpB2 population after 24 hours of a low Ca2+ switch(Fig. 5A-C).

DMs are smaller in cells lacking PKP1

To investigate the effect of re-expressing PKP1 in PKP1-deficient cells on DM number and length, we compared EM micrographs of nullpB1 and nullPKP1 lines. Results are shown for cells in culture after 3 days in 1.2 mM Ca2+ as well as cells grown for the same time period and subjected to a low Ca2+ switch for 1 hour(Fig. 7). In total, we counted 130 DMs from 31 EM micrographs for nullpB1 and 354 DMs from 32 EM micrographs of nullPKP1. We measured the length of 74 DMs from 33 EM micrographs of nullpB1 and 91 DMs from 27 EM micrographs of nullPKP1. Fig. 7 shows that PKP1 influences number and size of DMs in culture after 3 days of confluent growth (Fig. 7A,B).

Fig. 7.

Ultrastructural analysis of nullpB1 and nullPKP1 cells grown for 3 days and subjected to a Ca2+ switch of 1 hour identifies differences in DM length and number. In all cases columns are: 1, nullpB1 no switch; 2, nullpB1 low Ca2+ switch; 3, nullPKP1 no switch; 4, nullPKP1 low Ca2+ switch.(A) Relative DM density (mean±s.e.m.) in cells after 3 days of confluent culture before and after a 1-hour Ca2+ switch. Graph displays the DM number from 14-17 micrographs, taken at 5,000×magnification, from sections of cells in culture expressed as density relative to cytoplasm. Data show a reduced DM number in nullpB1 cells compared to nullPKP1 cells. (B) DM length in cells after 3 days of confluent culture before and after a 1-hour Ca2+ switch. Bar chart shows mean±s.e.m. of DM length from 13-17 micrographs taken at 13,000×magnification from sections of cells in culture for 3 days. Data show a reduction of nullpB1 DM length when compared to nullPKP1. Lower images are representative DMs from nullpB1 and nullPKP1. Scale bar: 1 μm.

Fig. 7.

Ultrastructural analysis of nullpB1 and nullPKP1 cells grown for 3 days and subjected to a Ca2+ switch of 1 hour identifies differences in DM length and number. In all cases columns are: 1, nullpB1 no switch; 2, nullpB1 low Ca2+ switch; 3, nullPKP1 no switch; 4, nullPKP1 low Ca2+ switch.(A) Relative DM density (mean±s.e.m.) in cells after 3 days of confluent culture before and after a 1-hour Ca2+ switch. Graph displays the DM number from 14-17 micrographs, taken at 5,000×magnification, from sections of cells in culture expressed as density relative to cytoplasm. Data show a reduced DM number in nullpB1 cells compared to nullPKP1 cells. (B) DM length in cells after 3 days of confluent culture before and after a 1-hour Ca2+ switch. Bar chart shows mean±s.e.m. of DM length from 13-17 micrographs taken at 13,000×magnification from sections of cells in culture for 3 days. Data show a reduction of nullpB1 DM length when compared to nullPKP1. Lower images are representative DMs from nullpB1 and nullPKP1. Scale bar: 1 μm.

Discussion

Re-expression of PKP1 does not alter cell growth or the transcriptional levels of the key desmosomal components Dp, plakoglobin,desmoglein 2 and desmoglein 3

The localisation of PKP1 to nuclei of cells indicates a possible signalling function, as demonstrated for other members of the armadillo gene family(Peifer et al., 1994). Plakophilin 2 has been shown to complex with nuclear particles, specifically RNA polymerase III, suggesting an important nuclear role for this family member (Mertens et al., 2001). It has also been shown that plakophilin 2 can associate with β-catenin, a critical downstream effector of the Wnt signalling pathway(Chen et al., 2002). One possible nuclear role of PKP1 then could be enhanced transcriptional levels of desmosomal proteins at sites where more adhesive strength is needed. We have demonstrated that this is not the case for Dp, plakoglobin, desmoglein 2 or desmoglein 3 (Fig. 2B).

Patients lacking PKP1 show a marked thickening of the skin, even in non-lesional sites, indicating a possible role for PKP1 in regulating keratinocyte proliferation. In our in vitro model, we have shown that re-expression of PKP1 in PKP1-deficient keratinocyte lines or over-expression of PKP1 in control keratinocyte lines does not affect cellular growth rates(data not shown).

PKP1 increases desmosomal protein content within the cell but does not affect the level of adherens junction proteins E-cadherin orβ-catenin and does not affect the levels of keratin 14, or those seen with a pan-keratin antibody

Previous work has demonstrated that PKP1 concentrates endogenously and ectopically expressed desmosomal components, in particular Dp, to the plasma membrane (Smith and Fuchs,1998; Kowalcyzk et al., 1999; Hatzfeld et al., 2000). In this study, we identified an increase in levels of desmosomal proteins within cells expressing PKP1 compared with PKP1-deficient cells(Fig. 2A), although this difference was not a result of transcriptional regulation(Fig. 2B). We were unable to demonstrate a notable difference in the amount of membrane bound desmosomal proteins in cells lacking PKP1 compared with those expressing PKP1 using immunofluorescence analysis (for example Fig. 5B,C, left panels). When we looked biochemically at membrane bound desmosomal protein levels for plakoglobin and desmoglein 3 (Fig. 5D, Triton X-100 soluble, lanes 1, 4 and 7) we noted less protein in cells lacking PKP1 (in agreement with the data seen from total cell lysate levels, Fig. 2A) but that the proportion of cytosol-membrane-cytoskeletal pool protein level was similar. After 24 hours of a switch to low Ca2+ we noted that membrane bound plakoglobin and desmoglein 3 were virtually undetectable in nullpB2cells compared with nullPKP2 and normpB2 cells. Turnover of desmosomal proteins is high (Penn et al.,1987; Pasdar and Nelson, 1989) and the data presented here are consistent with the concept that PKP1 stabilises desmosomal proteins within the cell (Kowalcyzk et al., 1999), although we cannot rule out post-translational modification as a mode of action for increasing the levels of desmosomal proteins in cells expressing PKP1 compared to those lacking PKP1. We did not detect a significant difference in levels of E-cadherin,β-catenin or keratins between cell lines tested.

Lack of PKP1 increases keratinocyte motility

Calcium-dependence of DMs has been shown to correlate with wounding of confluent cell sheets (Wallis et al.,2000). Specifically, calcium-independence can be reversed upon wounding and this reversal is propagated to cells hundreds of micrometers from the wound edge (Wallis et al.,2000). We have shown that PKP1 influences not only the Ca2+ responsiveness of DMs but also keratinocyte migration in our culture system. We cannot rule out that PKP1 also acts on cell motility through other mechanisms, as it has been reported that PKP1 co-localises withβ-actin at the edge of wounded HaCat cells(Hatzfeld et al., 2000), but we saw no differences in β-actin organisation (data not shown) or integrin complement (data not shown). The data presented here demonstrate for the first time, that keratinocyte motility is affected by the lack of a specific desmosomal protein.

Expression of PKP1 alters calcium stability of DMs in PKP1-deficient cell lines

The adhesive properties of DMs within confluent sheets of epithelial cells differ from those within sub-confluent sheets of epithelial cells(Watt et al., 1984; Mattey and Garrod, 1986). At sub-confluency, DMs are said to be calcium dependent as their formation and internalisation is influenced by increasing, or decreasing (<0.1 mM),Ca2+ concentration. Conversely, cells at confluency possess DMs that are mostly calcium independent since neither depleting the Ca2+ concentration of the medium nor adding chelating agents promote DM disruption. However, calcium-independent DMs are not permanent structures, since they retain the capacity to revert to calcium-dependence in certain situations, such as following wounding(Wallis et al., 2000). Using phase-contrast microscopy and time-lapse image capture, we found that PKP1-deficient cells became significantly more refractile in response to a low Ca2+ switch. This implies that cells are less adherent to neighbouring cells in response to a low Ca2+ switch over 24 hours(Fig. 4).

Calcium-independent DMs have been identified by immunofluorescent staining of desmosomal components after Ca2+ depletion(Watt et al., 1984; Mattey and Garrod, 1986; Wallis et al., 2000). Using this approach we have shown that, after 24 hours of a low Ca2+switch, far fewer calcium-independent DMs are present in nullpB cells than in nullPKP and normpB cells. These differences can be identified using an antibody to Dp after only 1 hour of a low Ca2+switch (Fig. 6C). We also demonstrated, using biochemical methods, that the proportion of membrane bound plakoglobin and desmoglein 3 was far less after a low Ca2+ switch for 24 hours in the nullpB population than in nullPKP and normpB populations.

Collectively these data suggest that, firstly, PKP1 has a role in the transition of DMs from a calcium-dependent to a calcium-independent state and,secondly, in cells lacking PKP1, Dp is displaced from the membrane in response to low Ca2+ before other desmosomal proteins studied here. This indicates a key function for PKP1 in stabilising Dp within DMs and is consistent with Dp immunostaining in patient skin samples(Fig. 6A,B).

Re-expression of PKP1 affects DM size and number

Ultrastructural comparison of PKP1-deficient cells expressing recombinant PKP1 or vector controls shows that the reexpression of PKP1 increases the size of DMs in our culture system (Fig. 7B). This observation supports other models that predict PKP1 enhances desmosomal cohesiveness through lateral interactions with Dp,providing additional links with the DM and the IF network (Kowalcyzk et al.,1999). Ultrastructural analysis of cells under Ca2+ switch conditions demonstrates that after 3 days of confluent culture, nullpB cells retained fewer DMs than nullPKP(Fig. 7A), supporting the immunofluorescence data identifying a lower proportion of calcium-independent DMs. These data, when assessed with those showing a reduction of membrane bound desmosomal components in nullpB cells compared to nullPKP and normpB after Ca2+ switching, suggest that calcium-independence may take longer to form in cells lacking PKP1.

The role of PKP1 in keratinocyte cell biology

We have examined cellular and biochemical characteristics of keratinocytes derived from two patients with skin-fragility ectodermal dysplasia syndrome. For ethical reasons our two control populations of keratinocytes were derived from adult, rather than aged matched, donors. Nonetheless these `older' cells behaved no differently than did PKP1-deficient keratinocytes to which we restored PKP1 expression. The most direct comparisons though were with vector alone controls and our results suggest that PKP1 influences DM stability and organisation. Specifically, restoration of PKP1 expression stabilises the aggregation/assembly of other DM components, especially Dp, although this process does not involve changes in DM gene transcription. Lack of PKP1 leads to epidermal thickening and hyperkeratosis but our study shows that PKP1 does not affect in vitro cell growth. PKP1 does have a role in regulating cell migration since lack of PKP1 increases keratinocyte migration after wounding. PKP1 also influences the transition of DM from a calcium-dependent state to a calcium-independent state, which may be important in restoration and maintenance of an intact epithelial barrier during wound healing, a function that is compromised in patients with inherited ablation of this DM component.

Acknowledgements

We thank Fiona Keane for providing immortalised normal breast keratinocytes, David R. Garrod for providing the antibody 11-5f, Gary P. Nolan and ATCC for providing the ΦNX-Ampho Cells and Mark Morgan for assistance with flow cytometry. This work was supported by DEBRA UK, the Wellcome Trust,the FH Muirhead Charitable Trust, the National Foundation for Ectodermal Dysplasia (NFED), and Action Research.

References

Bierkamp, C., Mclaughlin, K. J., Schwarz, H., Huber, O. and Kemler, R. (
1996
). Embryonic heart and skin defects in mice lacking plakoglobin.
Dev. Biol.
180
,
780
-785.
Bornslaeger, E. A., Corcoran, C. M., Stappenbeck, T. S. and Green, K. J. (
1996
). Breaking the connection, displacement of the desmosomal plaque protein desmoplakin from cell-cell interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly.
J. Cell Biol.
134
,
985
-1001.
Chen, X., Bonne, S., Hatzfeld, M., van Roy, F. and Green, K. J. (
2002
). Protein binding and functional characterisation of Plakophilin 2: Evidence for its diverse roles in desmosomes and β-catenin signaling.
J. Biol. Chem.
277
,
10512
-10522.
Chidgey, M., Brakebusch, C., Gustafsson, E., Cruchley, A., Hail,C., Kirk, S., Merritt, A., North, A., Tselepis, C., Hewitt, J. et al. (
2001
). Mice lacking desmocollin 1 show epidermal fragility accompanied by barrier defects and abnormal differentiation.
J. Cell Biol.
155
,
821
-832.
Collins, J. E., Legan, P. K., Kenny, T. P., MacGarvie, J.,Holton, J. L. and Garrod, D. R. (
1991
). Cloning and sequence analysis of desmosomal glycoproteins 2 and 3 (desmocollins):cadherin-like desmosomal adhesion molecules with heterogeneous cytoplasmic domains.
J. Cell Biol.
113
,
381
-391.
Eady, R. A. J. (
1985
). Transmission electron microscopy. In
Methods in Skin Research
(ed. D. S. C. Skerrow), pp.
1
-36. Chichester: John Wiley and Sons.
Fleming, T. P., Garrod, D. R. and Elsmore, A. J.(
1991
). Desmosome biogenesis in the mouse preimplantation embryo.
Development
112
,
527
-539.
Gallicano, G. I., Kouklis, P., Bauer, C., Yin, M., Vasioukhin,V., Degenstein, L. and Fuchs, E. (
1998
). Desmoplakin is required early in development for assembly of desmosomes and cytoskeletal linkage.
J. Cell Biol.
143
,
2009
-2022.
Garlick, J. A., Katz, A. B., Fenjves, E. S. and Taichman, L. B. (
1991
). Retrovirus-mediated transduction of cultured epidermal keratinocytes.
J. Invest. Dermatol.
97
,
824
-829.
Garrod, D. R. (
1996
). Epithelial development and differentiation: the role of desmosomes.
J. R. Coll. Physicians Lond.
30
,
366
-373.
Garrod, D. R., Tselepis, C., Runswick, S. K., North, A. J.,Wallis, S. R. and Chidgey, M. A. J. (
1999
). Desmosomal adhesion.
Adv. Mol. Cell Biol.
28
,
165
-202.
Green, K. J. and Gaudry, C. A. (
2000
). Are desmosomes more than tethers for intermediate filaments?
Nat. Rev. Mol. Cell. Biol.
1
,
208
-216.
Hatzfeld, M., Kristjansson, G. I., Plessmann, U. and Weber,K. (
1994
). Band 6 protein, a major constituent of desmosomes from stratified epithelia, is a novel member of the armadillo multigene family.
J. Cell Sci.
107
,
2259
-2270.
Hatzfeld, M., Haffner, C., Schulze, K. and Vinzens, U.(
2000
). The function of Plakophilin 1 in desmosome assembly and actin filament organisation.
J. Cell Biol.
149
,
209
-222.
Heid, H. W., Schmidt, A., Zimbelmann, R., Schafer, S.,Winter-Simanowski, S., Stumpp, S., Keith, M., Figge, U., Schnolzer, M. and Franke, W. W. (
1994
). Cell type-specific desmosomal plaque proteins of the plakoglobin family: plakophilin 1 (band 6 protein).
Differentiation
58
,
113
-131.
Hofmann, I., Mertens, C., Brettel, M., Nimmrich, V., Schnolzer,M. and Herrmann, H. (
2000
). Interaction of plakophilins with desmoplakin and intermediate filament proteins, an in vitro analysis.
J. Cell Sci.
113
,
2471
-2483.
Kapprell, H. P., Owaribe, K. and Franke, W. W.(
1988
). Identification of a basic protein of Mr 75,000 as an accessory desmosomal plaque protein in stratified and complex epithelia.
J. Cell Biol.
106
,
1679
-1691.
Kinsella, T. M. and Nolan, G. P. (
1996
). Episomal vectors rapidly and stably produce high-titre recombinant retrovirus.
Hum. Gene Ther.
7
,
1405
-1413.
Koch, P. J., Goldschmidt, M. D., Zimbelmann, R., Troyanovsky, R. and Franke, W. W. (
1992
). Complexity and expression patterns of the desmosomal cadherins.
Proc. Natl. Acad. Sci. USA
89
,
353
-357.
Kouklis, P. D., Hutton, E. and Fuchs, E.(
1994
). Making a connection, direct binding between keratin intermediate filaments and desmosomal proteins.
J. Cell Biol.
127
,
1049
-1060.
Kowalczyk, A. P., Palka, H. L., Luu, H. H., Nilles, L. A.,Anderson, J. E., Wheelock, M. J. and Green, K. J.(
1994
). Posttranslational regulation of plakoglobin expression. Influence of the desmosomal cadherins on plakoglobin metabolic stability.
J. Biol. Chem.
269
,
31214
-31223.
Kowalczyk, A. P., Hatzfeld, M., Bornslaeger, E. A., Kopp, D. S.,Borgwardt, J. E., Corcoran, C. M., Settler, A. and Green, K. J.(
1999
). The head of plakophilin-1 binds to desmoplakin and enhances its recruitment to desmosomes. Implications for cutaneous disease.
J. Biol. Chem.
274
,
18145
-18148.
Lane, E. B. and Alexander, C. M. (
1990
). Use of keratin antibodies in tumor diagnosis.
Semin. Cancer Biol.
1
,
165
-179.
Mattey, D. L. and Garrod, D. R. (
1986
). Calcium-induced desmosome formation in cultured kidney epithelial cells.
J. Cell Sci.
85
,
95
-111.
McGrath, J. A., McMillan, J. R., Shemanko, C. S., Runswick, S. K., Leigh, I. M., Lane, E. B., Garrod, D. R. and Eady, R. A. J.(
1997
). Mutations in the plakophilin 1 gene result in ectodermal dysplasia/skin fragility syndrome.
Nat. Genet.
17
,
240
-244.
McGrath, J. A., Hoeger, P. H., Christiano, A. M., McMillan, J. R., Mellerio, J. E., Ashton, G. H. S., Dopping-Hepenstal, P. J. C.,Lake, B. D., Leigh, I. M., Harper, J. I. and Eady, R. A. J.(
1999
). Skin fragility and hypohidrotic ectodermal dysplasia resulting from ablation of plakophilin 1.
Br. J. Dermatol.
140
,
297
-307.
Mertens, C., Kuhn, C. and Franke, W. W. (
1996
). Plakophilins 2a and 2b: constitutive proteins of dual location in the karyoplasm and the desmosomal plaque.
J. Cell Biol.
135
,
1009
-1025.
Mertens, C., Hofmann, I., Wang, Z., Teichmann, M., Sepehri Chong, S., Schnolzer, M. and Franke, W. W. (
2001
). Nuclear particles containing RNA polymerase III complexes associated with the junctional plaque protein plakophilin 2.
Proc. Natl. Acad. Sci. USA
98
,
7795
-7800.
Moll, I., Houdek, P., Schafer, S., Nuber, U. and Moll, R.(
1999
). Diversity of desmosomal proteins in regenerating epidermis: immunohistochemical study using a human skin organ culture model.
Arch. Dermatol. Res.
291
,
437
-446.
Morgenstern, J. P. and Land, H. (
1990
). Advanced mammalian gene transfer, high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.
Nucl. Acids Res.
18
,
3587
-3596.
Norgett, E. E., Hatsell, S. J., Carvajal-Huerta, L., Cabezas, J. C., Common, J., Purkis, P. E., Whittock, N., Leigh, I. M., Stevens, H. P. and Kelsell, D. P. (
2000
). Recessive mutation in desmoplakin disrupts desmoplakin-intermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma.
Hum. Mol. Genet.
9
,
2761
-2766.
North, A. J., Bardsley, W. G., Hyam, J., Bornslaeger, E. A.,Cordingley, H. C., Trinnaman, B., Hatzfeld, M., Green, K. J., Magee, A. I. and Garrod, D. R. (
1999
). Molecular map of the desmosomal plaque.
J. Cell Sci.
112
,
4325
-4336.
Palka, H. L. and Green, K. J. (
1997
). Roles of plakoglobin end domains in desmosome assembly.
J. Cell Sci.
110
,
2359
-2371.
Parrish, E. P., Steart, P. V., Garrod, D. R. and Weller, R. O. (
1987
). Antidesmosomal monoclonal antibody in the diagnosis of intracranial tumours.
J. Pathol.
153
,
265
-273.
Pasdar, M. and Nelson, W. J. (
1988
). Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells, temporal and spatial regulation of desmoplakin organisation and stabilisation upon cell-cell contact. I. Biochemical analysis.
J. Cell Biol.
106
,
677
-685.
Peifer, M., Berg, S. and Reynolds, A. B.(
1994
). A repeating amino acid motif shared by proteins with diverse cellular roles.
Cell
76
,
789
-791.
Penn, E. J., Burdett, I. D. J., Hobson, C., Magee, A. I. and Rees, D. A. (
1987
). Structure and assembly of desmosome junctions, biosynthesis and turnover of the major desmosome components of Madin-Darby canine kidney cells in low calcium medium.
J. Cell Biol.
105
,
2327
-2334.
Purkis, P. E., Steel, J. B., Mackenzie, I. C., Nathrath, W. B.,Leigh, I. M. and Lane, E. B. (
1990
). Antibody markers of basal cells in complex epithelia.
J. Cell Sci.
97
,
39
-50.
Rheinwald, J. G. and Green, H. (
1975
). Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinising colonies from single cells.
Cell
6
,
331
-343.
Ruiz, P., Brinkmann, V., Ledermann, B., Behrend, M., Grund,C., Thalhammer, C., Vogel, F., Birchmeier, C., Gunthert, U., Franke, W. W. and Birchmeier, W. (
1996
). Targeted mutation of plakoglobin in mice reveals essential functions of desmosomes in embryonic heart.
J. Cell Biol.
135
,
215
-225.
Runswick, S. K., O'Hare, M. J., Jones, L., Streuli, C. H. and Garrod, D. R. (
2001
). Desmosomal adhesion regulates epithelial morphogenesis and cell positioning.
Nat. Cell Biol.
3
,
823
-830.
Schafer, S., Koch, P. J. and Franke, W. W.(
1994
). Identification of the ubiquitous human desmoglein, Dsg2,and the expression catalogue of the desmoglein subfamily of desmosomal cadherins.
Exp. Cell Res.
211
,
391
-399.
Schmidt, A., Heid, H. W., Schafer, S., Nuber, U. A., Zimblemann,R. and Franke, W. W. (
1997
). Plakophilins 1a and 1b:widespread nuclear proteins recruited in specific epithelial cells as desmosomal plaque components.
Cell Tissue Res.
290
,
481
-499.
Schmidt, A., Langbein, L., Pratzel, S., Rode, M., Rackwitz, H. R. and Franke, W. W. (
1999
). Plakophilin 3 – a novel cell-type-specific desmosomal plaque protein.
Differentiation
64
,
291
-306.
Smith, E. A. and Fuchs, E. (
1998
). Defining the interactions between intermediate filaments and desmosomes.
J. Cell Biol.
141
,
1229
-1241.
Stappenbeck, T. S. and Green, K. J. (
1992
). The desmoplakin carboxyl terminus coaligns with and specifically disrupts intermediate filament networks when expressed in cultured cells.
J. Cell Biol.
116
,
1197
-1209.
Stappenbeck, T. S., Bornslaeger, E. A., Corcoran, C. M., Luu, H. H., Virata, M. L. and Green, K. J. (
1993
). Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks.
J. Cell Biol.
123
,
691
-705.
Storey, A., Pim, D., Murray, A., Osborn, K., Banks, L. and Crawford, L. (
1988
). Comparison of the in vitro transforming activities of human papillomavirus types.
EMBO J.
7
,
1815
-1820.
Troyanovsky, S. M., Troyanovsky, R. B., Eshkind, L. G., Leube,R. E. and Franke, W. W. (
1994
). Identification of amino acid sequence motifs in desmocollin, a desmosomal glycoprotein, that are required for plakoglobin binding and plaque formation.
Proc. Natl. Acad. Sci. USA
91
,
10790
-10794.
Tselepis, C., Chidgey, M., North, A. and Garrod, D.(
1998
). Desmosomal adhesion inhibits invasive behavior.
Proc. Natl. Acad. Sci. USA
95
,
8064
-8069.
Vasioukhin, V., Bowers, E., Bauer, C., Degenstein, L. and Fuchs,E. (
2001
). Desmoplakin is essential in epidermal sheet formation.
Nat. Cell Biol.
3
,
1076
-1085.
Vilela, M. J., Hashimoto, T., Nishikawa, T., North, A. J. and Garrod, D. (
1995
). A simple epithelial cell line (MDCK) shows heterogeneity of desmoglein isoforms, one resembling pemphigus vulgaris antigen.
J. Cell Sci.
108
,
1743
-1750.
Wallis, S., Lloyd, S., Wise, I., Ireland, G., Fleming, T. P. and Garrod, D. (
2000
). The alpha isoform of protein kinase C is involved in signalling the response of desmosomes to wounding in cultured epithelial cells.
Mol. Biol. Cell
11
,
1077
-1092.
Wan, H., Dopping-Hepenstal, P. J. C., Gratian, M. J., Stone, M. G., McGrath, J. A. and Eady, R. A. J. (
2003
). Desmosomes exhibit site-specific features in human palm skin.
Exp. Dermatol.
(in press).
Watt, F. M., Mattey, D. L. and Garrod, D. R.(
1984
). Calcium-induced reorganisation of desmosomal components in cultured human keratinocytes.
J. Cell Biol.
99
,
2211
-2215.
Whittock, N. V., Haftek, M., Angoulvant, N., Wolf, F., Perrot,H., Eady, R. A. J. and McGrath, J. A. (
2000
). Genomic amplification of the human plakophilin 1 gene and detection of a new mutation in ectodermal dysplasia/skin fragility syndrome.
J. Invest. Dermatol.
115
,
368
-374.