1 Department of Cell and Molecular Pathology, St John's Institute of
Dermatology, Guy's, King's and St Thomas' School of Medicine, London, UK
2 Richard Dimbleby/Cancer Research UK Department of Cancer Research, Guy's,
King's and St Thomas' School of Medicine, London, UK
3 Centre for Cutaneous Research, St Bartholomew's and the Royal London School of
Medicine and Dentistry, London, UK
* Author for correspondence (e-mail: andrew.south{at}kcl.ac.uk)
Accepted 28 April 2003
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Summary |
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Key words: Desmosomes, Keratinocyte, Calcium, Plakophilin 1, Skin
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Introduction |
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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 ß-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.
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Materials and Methods |
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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), 1010 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,
NX-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.
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
(ß-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
(ß-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
1;
Chemicon International), P1E6 (integrin
2; Chemicon International),
P1B5 (integrin
3; Chemicon International), P1D6 (integrin
5;
Chemicon International), G0H3 (integrin
6; Serotec), Y9A2 (integrin
9; Chemicon International), L230 (integrin
V;
prepared from hybridoma cells obtained from ATCC), LM609 (integrin
Vß3; Chemicon International), P1F6 (integrin
Vß5; Chemicon International), 10D5 (integrin
Vß6; Chemicon International), 3E1 (integrin ß4;
Chemicon International), and IgG1 (mouse control; DAKO).
Immunoblotting
Total protein extracts were obtained from cells seeded at high density
(5x105 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 ß-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 5,000x 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 13,000x 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 GAPDH
were 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
ImageQuantTM software (Amersham Pharmacia Biotech).
Time-lapse microscopy and image analysis
Migration study
Cells were seeded at equal density (7x105/35 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 (7x105/35 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 1x105 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 (7x105/35 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.
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Results |
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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).
|
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 13,000x 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, ß-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. 2A
shows 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 ß-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).
|
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, nullPKP
and 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 nullPKP2
cells 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 1, 2, 3, 5, 6, 9,
V,
Vß5,
Vß6 and ß4 (data not shown) between nullpB, nullPKP
and normpB cells. No
Vß3 was detected in any of the cell
lines (data not shown).
|
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 null
keratinocytes 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 nullpB
cell lines became more refractile than nullPKP and normpB
cell 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 nullpB
cells, data not shown).
|
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 nullPKP
and 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 normpB
cells for all desmosomal components examined (examples of Dp and desmocollin 3
shown in Fig. 5B and 5C, right
hand panels).
|
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).
|
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).
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Discussion |
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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 or
ß-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 nullpB2
cells 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,
ß-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 with
ß-actin at the edge of wounded HaCat cells
(Hatzfeld et al., 2000
), but
we saw no differences in ß-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.
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Acknowledgments |
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