1 Keratinocyte Laboratory, Cancer Research UK London Research Institute, 44
Lincoln's Inn Fields, London, WC2A 3PX, UK
2 Institute of Human Genetics, The Bartholin Building, University of Aarhus,
DK-8000 Aarhus C, Denmark
3 Cancer Research UK Skin Tumour Laboratory, The Royal London Hospital, 2 Newark
Street, London, E1 2AT, UK
Author for correspondence (e-mail:
fiona.watt{at}cancer.org.uk)
Accepted 28 August 2003
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SUMMARY |
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Key words: Melanoma chondroitin sulphate proteoglycan, Epidermal stem cells, Patterning
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Introduction |
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The clustering of stem cells in specific regions of the epidermis is
interesting for two reasons. First, it suggests that there might be aspects of
those regions (the stem cell microenvironment or niche) that are particularly
conducive to the maintenance of the stem-cell phenotype
(Watt and Hogan, 2000;
Spradling et al., 2001
).
Second, it implies that movement of stem progeny within the epidermal basal
layer occurs as part of the process of differentiation.
Cell migration has long been thought to play a role in maintaining the hair
follicle through the hair growth cycle
(Panteleyev et al., 2001), and
lineage marking has unequivocally demonstrated the movement of cells from the
bulge to the IFE and sebaceous glands
(Oshima et al., 2001
).
Classically, movement of individual keratinocytes in IFE was thought to be
restricted to the movement of cells upwards from the basal layer as they
initiate terminal differentiation (Watt
and Green, 1982
), whereas lateral movement was restricted to the
migration of intact sheets of keratinocytes during wound healing
(Martin, 1997
). However, the
clustering of stem cells in IFE implies lateral movement of stem cell
daughters as they become committed progenitors (also known as transit
amplifying cells). Evidence for the movement of individual keratinocytes
relative to one another within the basal layer of IFE has come from analysis
of green fluorescent protein (GFP)-marked clones in cultured sheets of human
epidermis; here, GFP-positive committed progenitors are found intermingled
with their unmarked neighbours, and GFP-labelled stem cells remain as cohesive
groups (Jensen et al., 1999
;
Lowell et al., 2000
).
The molecular basis of epidermal stem cell clustering is poorly understood.
In both human and mouse epidermis, stem cells are more adhesive to
extracellular matrix proteins than their nonstem progeny
(Jones and Watt, 1993;
Bickenbach and Chism, 1998
)
and, in human epidermis, this is due at least in part to high expression of
ß1 integrins (Jones and Watt,
1993
; Jones et al.,
1995
; Jensen et al.,
1999
). There is also high ß1-integrin expression in the bulge
region of human hair follicles (Jones et
al., 1995
; Lyle et al.,
1998
; Akiyama et al.,
2000
). Elevated integrin expression results in reduced motility of
both human keratinocytes (Jensen et al.,
1999
) and other cell types
(Huttenlocher et al., 1995
),
and would therefore contribute to stem-cell clustering
(Jensen et al., 1999
). Human
IFE stem cells also express higher levels of the Notch ligand Delta 1 than
other basal cells, and this decreases keratinocyte motility via an unknown
mechanism (Lowell et al.,
2000
; Lowell and Watt,
2001
). One would predict that stem-cell clustering might also
reflect increased intercellular adhesiveness, but although heterogeneity in
expression of E-cadherin has been reported in vivo
(Molès and Watt, 1997
),
this has not been confirmed in vitro (Zhu
et al., 1999
).
In a search for potential markers of human IFE stem cells, we noted that
heterogeneous expression of melanoma chondroitin sulphate proteoglycan (MCSP)
has been observed in the basal layer of human epidermis and in the outer root
sheath of hair follicles (Kupsch et al.,
1995). MCSP is so named because it is expressed in most human
melanomas (Pluschke et al.,
1996
). MCSP is a cell surface proteoglycan that plays a role in
the spreading, migration and invasion of melanoma cells
(de Vries et al., 1986
),
stimulating
4ß1-integrin-mediated cell adhesion and spreading via
activation of the Rho family GTPase Cdc42
(Eisenmann et al., 1999
). The
rat homologue of MCSP is NG2 (Nishiyama et
al., 1991
); ligation of NG2 also results in cell attachment,
migration and spreading, and reorganization of the actin cytoskeleton
(Fang et al., 1999
;
Majumdar et al., 2003
).
Epidermal expression of MCSP is therefore of interest both as a putative stem
cell marker and as a potential regulator of the adhesive properties, and thus
spatial organization, of IFE keratinocytes.
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Materials and methods |
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Retroviral packaging lines were generated by a two-step procedure as
follows. pBp, pBp-CD8 and pBp-CD8/MCSP DNAs were first transfected using a
standard calcium phosphate transfection procedure
(Sambrook et al., 1989) into
the Phoenix ecotropic packaging line [obtained from ATCC with kind permission
of G Nolan, Stanford University School of Medicine, Stanford, CA, USA
(Lorens et al., 2000
)].
Retroviral supernatant from the transfectants was used to infect the
amphotropic retroviral producer line AM12 using a standard infection procedure
(Morgenstern and Land, 1991
).
Infected cells were selected in the presence of 2 µg ml1
puromycin (Sigma).
For retroviral infection, keratinocytes were cultured in the presence of
AM12 producer lines which had been pre-treated with 4 µg
ml1 mitomycin C (Levy et
al., 1998). The producer cells were removed after 3 days and
replaced with puromycin-resistant J2-3T3 feeder cells, and 2 µg
ml1 puromycin was added to the medium. Infected
keratinocytes were used for experiments immediately or following passage on
puromycin-resistant J2-3T3 cells in medium supplemented with 2 µg
ml1 puromycin. Expression of constructs encoding CD8 in
keratinocytes was determined by flow cytometry following labelling with
monoclonal antibody (mAb) OKT8 (Zhu et
al., 1999
).
Keratinocyte culture
Normal human epidermal keratinocytes from neonatal foreskin (strain km;
passages 2-8) were cultured as described previously
(Watt, 1998) on a feeder layer
of J2-3T3 feeder cells pre-treated with 4 µg ml1
mitomycin C (Sigma). The culture medium (FAD+FCS+HICE) consisted of one part
Ham's F12 medium and three parts Dulbecco's modified Eagle's medium,
supplemented with 1.8x104 M adenine (FAD), 10% foetal
calf serum (FCS), 0.5 µg ml1 hydrocortisone, 5 µg
ml1 insulin, 1010 M cholera toxin and 10
ng ml1 epidermal growth factor (HICE). Cells were grown at
37°C in a humid atmosphere containing 5% CO2. The culture
medium was changed every 2 days. Cells were harvested by first removing the
feeders with EDTA and then treating the keratinocytes with trypsin/EDTA. SCC4
cells, derived from a human squamous cell carcinoma of the tongue, were
cultured as described previously (Evans et
al., 2003
). Preconfluent or newly confluent cultures were used for
all experiments.
Antibodies and probes
MCSP was detected using mAb 9.2.27 (PharMingen), which recognizes both the
nonglycanated and proteoglycan forms of the molecule
(Morgan et al., 1981;
Bumol et al., 1984
), or mAb
LHM2 (Kupsch et al., 1995
).
For staining of whole mounts, mAb LHM2 was directly conjugated to Alexa-594.
ß1-Integrins were detected using either a FITC-conjugated anti-CD29 mAb
(DAKO), mAb AIIB2 (Bohnsack et al.,
1990
) or P5D2 (Dittel et al.,
1993
). E-Cadherin was detected using mAb HECD-1
(Shimoyama et al., 1989
;
Braga et al., 1995
), CD8 was
detected using mAb OKT8 (Reinherz et al.,
1980a
; Reinherz et al.,
1980b
) and involucrin using mAb SY5
(Hudson et al., 1992
). The
anti-Ki67 antigen Ab was a rabbit polyclonal antiserum (Dako). Biotinylated
anti-mouse Ig, RPE-Cy5-streptavidin and mouse serum were purchased from DAKO.
TRITC-phalloidin was purchased from Sigma. Secondary antibodies conjugated to
Alexa 488 and Alexa 594 were purchased from Molecular Probes.
Alexa-633-conjugated streptavidin was purchased from Jackson ImmunoResearch
Laboratories.
Preparation of whole mounts
Normal human skin was obtained from adult plastic surgery operations
(breast). Whole mounts were prepared as described previously
(Jensen et al., 1999), except
that dispase was avoided because of the sensitivity of the MCSP epitopes. The
skin was prepared using a dermatome in order to cut very thin pieces, then
divided into 1 cm2 pieces and incubated at 37°C in S-MEM
(Gibco), 15 mM EDTA for 2-3 hours, depending on the donor. Using forceps, the
epidermis was gently removed from the underlying dermis as an intact sheet and
fixed immediately in normal buffered formalin, pH 7.2 (Sigma), for 2 hours at
room temperature. Fixed epidermal sheets were stored in PBS containing 0.2%
sodium azide at 4°C for up to 4 weeks before staining.
Immunostaining and confocal microscopy
Frozen sections of human foreskin or scalp were air dried, fixed with 3%
paraformaldehyde and blocked with PBSABC containing 0.5% bovine serum albumin
(BSA), 0.2% fish skin gelatin (Sigma) and 10% FCS. Colonies of keratinocytes
were cultured in the presence of J2-3T3 feeder cells on glass coverslips,
rinsed in PBSABC, fixed in 3% paraformaldehyde and blocked with PBSABC, 0.5%
BSA and 10% FCS. When necessary, cultured cells were permeabilized by the
addition of 0.2% saponin (Sigma) to the block solution. Cells from mixing
experiments to analyse keratinocyte cohesion were fixed in 3% paraformaldehyde
and blocked with PBSABC containing 0.2% saponin (Sigma), 0.5% BSA and 10% FCS.
The preparation of whole mounts is described above.
All antibodies used for immunolabelling were diluted in block buffer. Where MCSP was detected using secondary biotinylated anti-mouse Ig and tertiary 633-conjugated streptavidin, an additional blocking step with mouse serum was performed before detection of ß1 integrin with a directly FITC-conjugated anti-CD29 mAb (Dako). Incubation times were 1 hour at room temperature and samples were washed three times in block buffer between incubations. Samples were rinsed in distilled water and mounted in Gelvatol (Monsanto, St Louis, MO) containing 0.5% 1,4-diazabicyclo[2.2.2]octane (DABCO) (Sigma).
Images were acquired using a Zeiss 510 confocal microscope. Objectives used were Zeiss 10/NA 0.45, Zeiss 20/NA 0.75 and Zeiss 63/NA 1.4.
FACS and flow cytometry
For detection of CD8 with mAb OKT8 or involucrin with mAb SY5, single cell
suspensions were prepared by incubation with trypsin/EDTA. OKT8 immunostaining
was performed without fixation at 4°C and differentiating cells were gated
out as described by Jones and Watt (Jones
and Watt, 1993). For detection of involucrin, cells were fixed at
room temperature using 3% paraformaldehyde and resuspended in block buffer
(PBSABC, 0.5% BSA, 10% FCS) supplemented with 0.2% saponin (Sigma) for 30
minutes. Primary and secondary antibodies were diluted in block buffer and
samples were washed three times in block buffer between incubations. Cells
were resuspended in PBSABC and analysed in a FACScan (BD Biosciences).
Double labelling of keratinocytes for MCSP and ß1 integrins was
performed as follows. Confluent cultures of keratinocytes were harvested using
trypsin/EDTA and plated in 10 cm diameter type-I-collagen-coated dishes (BD
Biosciences) at a density of 5x105 cells per dish for 5-8
hours at 37°C. Cells were removed using EDTA and resuspended in
HEPES-buffered DMEM (pH 7.5), 10% FCS and 0.5% BSA for 30 minutes at 4°C.
Cells were then incubated sequentially with mAb 9.2.27, biotinylated
anti-mouse Ig, RPE-Cy5-conjugated streptavidin, mouse serum and
FITC-conjugated CD29 mAb. Samples were washed three times between incubations
and finally resuspended in PBSABC containing 0.1% FCS. Fluorescence-activated
cell sorting (FACS) was performed using a MoFlo® High-Performance Cell
Sorter (DakoCytomation). Differentiating cells were gated out as described by
Jones and Watt (Jones and Watt,
1993) to leave populations enriched for basal cells. Further gates
were then applied to select populations expressing high and low levels of MCSP
and ß1 integrins. FACS-selected populations were analysed using a FACScan
to check sorting efficiency and to quantify the expression levels of MCSP and
ß1 integrins in sorted populations.
Clonogenicity assays
Populations of uninfected keratinocytes selected on the basis of MCSP and
ß1 integrin expression were used directly from the FACS procedure.
Retrovirus-infected keratinocyte populations were harvested using
trypsin/EDTA. Keratinocytes were plated at 200, 500 and 1000 cells per dish,
in triplicate, onto 6 cm diameter tissue-culture dishes containing mitotically
inactivated J2-3T3 feeders and cultured for 10-14 days. Cells were fixed using
3% paraformaldehyde, permeabilized with 0.2% saponin and stained with 1%
Rhodamine B and 1% Nile Blue (BDH). Colonies were viewed using a Wild 3BZ
dissection microscope and scored as abortive or actively growing on the basis
of size and proportion of terminally differentiated cells, as described by
Jones and Watt (Jones and Watt,
1993).
Function-blocking experiments with antibodies and pharmacological
inhibitors
Keratinocytes expressing CD8 or CD8/MCSP were incubated with the following
pharmacological inhibitors for 6 hours at 37°C: 5 µM Y-27632
(Calbiochem) (Narumiya et al.,
2000); 5 µg ml1 C3 exoenzyme from
Clostridium botulinum (Calbiochem)
(Udagawa and McIntyre, 1996
);
and 50 µM LY294002 (Sigma-Aldrich)
(Pullen and Thomas, 1997
).
Antibody crosslinking experiments were performed by incubating keratinocytes expressing either CD8 or CD8/MCSP with 50 µg ml1 of mAb OKT8 for 30 minutes, followed by 1 hour at 37°C with 50 µg ml1 of anti-mouse IgG. Control cultures were incubated with OKT8 alone, anti-mouse IgG alone or no antibody for 1 hour at 37°C.
To examine the effects on cell-cell adhesion of interfering with endogenous
MCSP, intact sheets of SCC4 were cultured in low-calcium medium for 2 days to
inhibit intercellular adhesion (Hodivala
and Watt, 1994). Cells were then transferred to standard medium
and incubated for 2 hours in medium alone, medium supplemented with
anti-E-cadherin (80 µg ml1 HECD-1) and anti-MCSP (80
µg ml1 9.2.27 or LHM2) antibodies (alone or in
combination), or with rat NG2 ectodomain (30 µg ml1;
generous gift of W. B. Stallcup). Cells were fixed, permeabilized, stained
with TRITC-phalloidin and viewed by confocal microscopy, as described
above.
Motility assays
Retrovirus-infected keratinocyte populations were harvested with
trypsin/EDTA and plated at a density of 1x105 cells per dish
onto type-I-collagen-coated 6 cm diameter tissue culture dishes (BD
Biosciences) in FAD+FCS+HICE. Cell migration was recorded overnight at a 5
minute time lapse-interval using a 10x objective and the images were
acquired using AQM Kinetic Acquisition Manager software (Kinetic Imaging) or
by video microscopy. Analysis of motility was performed by manually tracking
cells within each field over the sequence of time-lapse digital images using
Motion Analysis software (Kinetic Imaging). All the individual cell
trajectories were then pooled and mean speed of cells at each time point
during the assay calculated. The average speed of cell motility was calculated
for each assay and compared over four independent experiments. Results were
compared using analysis of variance.
Time-lapse video microscopy of keratinocyte colonies
Retrovirus-infected keratinocyte populations were harvested with
trypsin/EDTA, plated onto mitotically inactivated feeder cells in 6 cm dishes
and cultured until small colonies appeared. Feeder cells were removed with
EDTA and fresh culture medium added. Cells were allowed to recover for 30
minutes and then analysed by time-lapse video microscopy. Images were acquired
at time lapse intervals of 5 minutes over a period of 68 hours.
Assays of keratinocyte cohesiveness
Populations of retrovirus-infected keratinocytes were harvested with
trypsin/EDTA. 200 CD8- or CD8/MCSP-expressing keratinocytes were mixed with
1x105 keratinocytes infected with the empty retroviral
vector, plated on mitotically inactivated feeder cells in six-well
tissue-culture plates and cultured for 10-14 days to reconstitute confluent
epidermal sheets. Clones of CD8 or CD8/MCSP-expressing keratinocytes within
the sheets were detected by immunostaining with the anti-CD8 mAb OKT8.
Fluorescence and phase-contrast images of these clones and surrounding
unlabelled keratinocytes were acquired using a Nikon Diaphot 200 microscope
attached to a Hammamatsu digital camera and AQM Kinetic Acquisition Manager
software (Kinetic Imaging). Clones were scored as cohesive if they contained
more than 70% of cells in contact with one another, whereas noncohesive clones
contained fewer than 70% of cells in contact.
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Results |
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To evaluate MCSP expression further, we used the technique of whole mount
immunofluoresence labelling, in which the epidermis is detached as an intact
sheet from the underlying dermis, fixed, incubated with the relevant
antibodies and then viewed en face with a confocal microscope. Visualized by
this technique, ß1 integrin-bright keratinocytes appear as discrete
clusters surrounded by more differentiated, integrin-dull cells
(Fig. 1C)
(Jensen et al., 1999). Double
labelling for MCSP showed that the clusters of MCSP-positive basal cells
co-expressed high levels of ß1 integrins
(Fig. 1C). Actively cycling
basal cells have previously been shown to lie in the integrin-dull basal
compartment (Jensen et al.,
1999
). As predicted, MCSP-positive keratinocyte clusters tended to
be negative for the proliferation marker Ki67 and, conversely, Ki67-positive
basal cells were largely MCSP negative
(Fig. 1D).
The expression of MCSP by clusters of ß1-integrin-bright, nonproliferating keratinocytes suggested that MCSP might be a novel marker of stem cells in human IFE.
Heterogeneous expression of MCSP by primary human keratinocytes in
culture
When primary human keratinocytes are cultured at low density on a feeder
layer of mitotically inactivated 3T3 cells, different types of clone can be
observed (Barrandon and Green,
1987; Jones and Watt,
1993
; Jones et al.,
1995
). At 14 days after plating, large clones consisting of a
mixture of dividing and terminally differentiated cells can be distinguished
from small, abortive colonies in which all or most cells have initiated
terminal differentiation (Jones and Watt,
1993
). Stem cells found the former type of colony, whereas the
abortive clones can be attributed to transit amplifying (committed progenitor)
cells (reviewed by Watt,
1998
). We therefore examined MCSP expression in clones of primary
human IFE keratinocytes.
As in IFE, MCSP expression was heterogeneous in culture (Fig. 2A-C). Abortive, terminally differentiated colonies usually lacked any MCSP, even though the basal cells expressed ß1 integrins (Fig. 2B). By contrast, proliferative keratinocyte colonies contained a mixture of MCSP-positive and -negative cells, the positive cells expressing higher levels of ß1 integrins than the negative cells (Fig. 2A). In the youngest colonies (<7 days after plating), MCSP expression was largely concentrated in the peripheral basal cells (Fig. 2A), whereas in more mature clones MCSP-positive cells were scattered throughout the basal layer (Fig. 2C).
|
To compare the expression of MCSP and ß1 integrins quantitatively, we
performed flow cytometry on disaggregated cultures of human keratinocytes
(Fig. 3). The extracellular
domain of MCSP was sensitive to the trypsinization procedure used to generate
single cell suspensions (data not shown)
(Nishiyama et al., 1995).
Therefore, it was necessary to replate the cells on collagen-coated dishes for
5-8 hours to allow newly synthesized MCSP to reach the cell surface, and then
to detach the cells with EDTA alone. The MCSP profile obtained from basal
keratinocytes was more heterogeneous than the ß1 integrin profile
(Fig. 3B,C, `Total'). This
correlates with the greater variation in MCSP levels between cells
(Fig. 2A), although it might
also reflect different rates of export of newly synthesized MCSP to the
surface of cells following replating.
|
We conclude that the correlation between high MCSP expression and high ß1 integrin expression observed in IFE (Fig. 1) extends to cultured IFE keratinocytes, and that it is possible to use FACS to select keratinocytes that are MCSP positive or negative.
Selection of high expressing keratinocytes gives no further
enrichment for clonogenic cells
We used FACS to investigate whether selection of MCSP-positive, ß1
integrin bright keratinocytes gave more enrichment for clonogenic human
keratinocytes than selection on the basis of ß1 integrin expression
alone. Terminally differentiated cells were gated out as described previously
(Jones and Watt, 1993) and
basal keratinocytes were fractionated into the following four populations: the
20% of cells with the lowest ß1-integrin expression (ß1-Low); the
cells in the highest 20% of ß1-integrin expression and negative for MCSP
(MCSP-Low/ß1-High); cells within the highest 20% of ß1-integrin
expression and with high expression of MCSP (MCSP-High/ß1-High); and, as
a control, unselected basal keratinocytes (Total)
(Fig. 4A).
|
We conclude that selection on the basis of high ß1 integrin and MCSP expression does not provide any further enrichment for clonogenic keratinocytes than selection for high ß1 integrin levels alone. We also conclude that, given its trypsin sensitivity, MCSP does not provide a useful selectable cell surface marker for stem cell enrichment in vitro.
MCSP cytoplasmic domain does not regulate terminal differentiation of
human keratinocytes in vitro
The cytoplasmic domain of MCSP is highly conserved between species
(Fig. 5A) and mediates several
functions of the proteoglycan, including modulation of integrin-mediated
adhesion (Iida et al., 1995;
Eisenmann et al., 1999
;
Fang et al., 1999
;
Stallcup and Dahlin-Huppe,
2001
). In order to interfere with the function of MCSP in
keratinocytes, we generated a chimeric construct consisting of the MCSP
cytoplasmic domain fused to the extracellular and transmembrane domains of
CD8, which is not endogenously expressed by keratinocytes, in the retroviral
expression vector pBabepuro (pBp) (Fig.
5B). Populations of primary human keratinocytes or the human
squamous cell carcinoma line SCC4 were then generated that expressed this
construct (CD8/MCSP), full-length CD8 (CD8) or empty vector alone
(Fig. 5C,D). Expression of CD8
or CD8/MCSP had no effect on the levels of endogenous MCSP on the cell surface
(Fig. 5D). There was no
detectable shedding of the MCSP ectodomain
(Nishiyama et al., 1995
) in
untransduced keratinocytes and SCC4 cells, nor cells expressing CD8 or
CD8/MCSP (data not shown).
|
|
CD8/MCSP decreases keratinocyte cohesiveness without affecting
cell-ECM adhesion or motility
When primary keratinocytes infected with CD8/MCSP were grown at clonal
density, the morphology of the resulting colonies was strikingly different
from that of colonies containing cells expressing full-length CD8 or the empty
retroviral vector (Fig. 7A). CD8/MCSP-expressing colonies had an irregular shape, compared with the smooth,
rounded borders of colonies formed by the controls. Individual cells within
colonies often appeared to be trying to detach from the colonies, although
cell-cell contact was usually maintained, and cells tended to have an
elongated/polarized/`motile' appearance, compared with the typical
keratinocyte `cobblestone' morphology. These observations were confirmed by
time-lapse microscopy of CD8/MCSP colonies; in addition, the time-lapse movies
revealed that cells around the periphery of colonies were more motile and
there was more movement within colonies (see Movies 1 and 2 at
http://dev.biologists.org/supplemental/).
The effects of CD8/MCSP were more obvious in small colonies and were less
apparent upon confluence, although confluent cultures of CD8/MCSP-expressing
keratinocytes frequently appeared to be disorganized (data not shown).
|
CD8/MCSP perturbs intercellular adhesion and the actin cytoskeleton,
effects that can be mimicked by inhibiting Rho and are independent of ligation
of the CD8 extracellular domain
We next investigated whether cell-cell adhesion was disrupted by CD8/MCSP
expression. Immunofluorescence analysis of cells at the edges of large
keratinocyte colonies showed that both CD8/MCSP and CD8 were localized to
actin-based plasma membrane structures such as protrusions and microvilli, and
were concentrated at cell-cell contacts
(Fig. 8A). CD8/MCSP colocalized
with endogenous MCSP on the cell surface (data not shown). The appearance of
cell-cell contacts was markedly different in CD8/MCSP-expressing cells
compared with controls. Whereas control cells had well-defined, discrete
borders, the junctions of CD8/MCSP-expressing cells appeared more diffuse
because of CD8/MCSP-positive protrusions extending between adjacent cells
(Fig. 8A).
|
The perturbation in E-cadherin and actin observed in keratinocytes
expressing CD8/MCSP suggested that the chimera might modulate the activity of
Rho GTPases (Etienne-Manneville and Hall,
2002). We examined this by incubating keratinocytes that expressed
CD8 or CD8/MCSP with different pharmacological inhibitors and examining the
effects on the distribution of E-cadherin (data not shown) and polymerized
actin (Fig. 9A). We treated
cells with the C3 exoenzyme from Clostridium botulinum, which
inhibits the function of RhoA, RhoB and RhoC; the other Rho family proteins
Rac-1 and Cdc42 are poor C3 substrates
(Udagawa and McIntyre, 1996
).
We also used Y-27632, a specific inhibitor of the Rho-associated
coiled-coil-forming protein serine/threonine kinase (ROCK) family of protein
kinases (Narumiya et al.,
2000
). In addition, we treated cells with LY294002 to inhibit
phosphatidylinositol 3-kinase, which is a downstream effector of Rac
(Pullen and Thomas, 1997
).
|
Ligation of NG2 with antibodies can trigger changes in the actin
cytoskeleton (Fang et al.,
1999; Majumdar et al.,
2003
), so we investigated the effect of ligating CD8/MCSP. We
induced clustering of the CD8 extracellular domain by incubating transduced
keratinocytes with mouse anti-CD8 followed by anti-mouse IgG
(Fig. 9B). There was no
detectable effect of inducing capping of the CD8 extracellular domain either
in cells expressing CD8 or CD8/MCSP. Capped cells were compared with untreated
cells (Fig. 9A) and cells
treated either with anti-CD8 alone (Fig.
9B) or anti-mouse IgG alone (data not shown). We conclude that the
effects of CD8/MCSP on the actin cytoskeleton are independent of ligand
induced clustering.
Role for MCSP in stem-cell cohesiveness
To examine whether interference with endogenous MCSP had any effect on
cell-cell adhesion, we cultured SCC4 cells in low-calcium medium and
transferred them to standard medium for 2 hours. This treatment induces the
assembly of adherens junctions and desmosomes, and the concentration of actin
at cell-cell borders (Hodivala and Watt,
1994; Braga et al.,
1995
). As previously reported
(Hodivala and Watt, 1994
),
anti-E-cadherin partially inhibited calcium-induced cell-cell adhesion and the
accumulation of polymerized actin at cell-cell borders
(Fig. 10A). The NG2 ectodomain
(data not shown) or antibodies to MCSP
(Fig. 10A) did not prevent
cell-cell adhesion or actin reorganization. However, the combination of
anti-MCSP and anti-E-cadherin perturbed these processes more than
anti-E-cadherin alone (Fig.
10A).
|
Whereas most clones formed by CD8-expressing keratinocytes were cohesive with few detached cells, clones formed by CD8/MCSP-expressing keratinocytes were more dispersed (Fig. 10B). Single cells were frequently observed to have detached from the edges of CD8/MCSP-positive clones, whereas this was rarely observed in CD8-positive clones. No differences between the sizes of CD8- or CD8/MCSP-expressing clones were observed, consistent with the finding that CD8/MCSP did not affect keratinocyte differentiation or clonogenicity (Fig. 6).
All CD8- and CD8/MCSP-positive clones were scored for cohesiveness
(Lowell et al., 2000;
Lowell and Watt, 2001
). Clones
in which fewer than 30% of cells were detached from the main clone were scored
as cohesive and clones in which greater than 30% of cells were not associated
with the clone were scored as non-cohesive. Expression of CD8/MCSP strongly
inhibited cohesiveness of clones, with 78.5% of CD8-expressing clones being
cohesive compared with 26% of CD8/MCSP-expressing clones
(Fig. 10C). We conclude that
MCSP regulates keratinocyte cohesiveness by a mechanism that is independent of
differentiation and motility.
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Discussion |
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When the cytoplasmic domains of ß integrin subunits or classical
cadherins are coupled to an irrelevant extracellular domain, such as CD8, the
resultant chimeras have a dominant negative effect on
cell/extracellular-matrix adhesion (Chen
et al., 1994; LaFlamme et al.,
1994
; Lukashev et al.,
1994
) or cell-cell adhesion
(Kintner, 1992
;
Fujimori and Takeichi, 1993
),
respectively. Such constructs have been expressed in human epidermal
keratinocytes via retroviral infection. A ß1 integrin chimera reduces
extracellular-matrix adhesion and activation of mitogen-activated protein
kinase, thereby promoting exit from the stem-cell compartment
(Zhu et al., 1999
). A cadherin
cytoplasmic domain chimera inhibits cell-cell adhesion
(Zhu and Watt, 1996
) but also
stimulates differentiation by depleting the cytoplasmic pool of ß-catenin
available for signalling (Zhu and Watt,
1999
). Based on these observations, we believe that CD8/MCSP is
acting as a dominant negative mutation. This conclusion is supported by the
findings that inhibition of Rho or ROCK mimics the effect of CD8/MCSP
(Fig. 9A) and that anti-MCSP in
combination with anti-E-cadherin can perturb intercellular adhesion
(Fig. 10A). Because
full-length MCSP is too large to be efficiently expressed via a retroviral
vector, we cannot formally exclude the possibility that CD8/MCSP acts as a
gain-of-function mutation. Nevertheless, the high expression of MCSP in
stem-cell clusters and the dispersion of cells expressing CD8/MCSP fit well
with the model that CD8/MCSP has a dominant negative mechanism of action.
The adhesive function of NG2/MCSP has been attributed to
extracellular-matrix adhesion and collagen types V and VI are known ligands
(Tillet et al., 1997). Several
cell surface proteoglycans, including MCSP, have been reported to modulate
integrin function (Iida et al.,
1995
; Couchman and Woods,
1999
; Eisenmann et al.,
1999
) by lateral association with integrins and other cell surface
receptors within the plasma membrane
(Woods and Couchman, 2000
).
NG2/MCSP collaborates with the
4ß1 integrin in mediating adhesion
and spreading on fibronectin via activation of the Rho-family GTPase Cdc42
(Iida et al., 1995
;
Eisenmann et al., 1999
).
NG2-mediated adhesion does not, however, depend on integrins, because
introduction of NG2 into ß1 integrin-negative cells promotes attachment
to collagen types V and VI (Tillet et al.,
2002
). In the absence of ß1 integrins, engagement of NG2 by
collagen type VI triggers cell spreading and rearrangement of the actin
cytoskeleton (Tillet et al.,
2002
). MCSP forms an association with membrane-type-3 matrix
metalloproteinase, and this interaction might play a role in triggering
invasion through extracellular matrix
(Iida et al., 2001
).
Although MCSP was expressed by those keratinocytes with the highest levels
of ß1 integrins, we did not obtain any evidence that MCSP-mediated
keratinocyte/extracellular-matrix adhesion. Keratinocytes do not express
4ß1 integrin (Watt,
2002
) and we did not see convincing localization of MCSP to focal
adhesions. Keratinocytes did not adhere to type-VI collagen regardless of
whether they expressed CD8/MCSP (data not shown), and CD8/MCSP had no effect
on adhesion to type I collagen or on motility of individual cells. It is
nevertheless interesting that FRAS1, the gene that is mutated in
Fraser syndrome, has sequence similarity to NG2 and there is epidermal
detachment from the basement membrane in Fraser syndrome patients
(McGregor et al., 2003
).
In addition to its extracellular-matrix adhesive functions, NG2 modulates
cellular responses to growth factors, a common property of cell surface
proteoglycans (Kresse and Schonherr,
2001). NG2 is required for the responsiveness of aortic smooth
muscle cells to platelet-derived growth factor (PDGF) AA
(Grako and Stallcup, 1995
;
Grako et al., 1999
). NG2 and
the PDGF
receptor are co-expressed in immature oligodendrocyte
progenitors and antibodies to NG2 block their response to PDGF
(Nishiyama et al., 1996
).
Basic fibroblast growth factor also binds NG2
(Goretzki et al., 1999
). NG2
binds to plasminogen and to angiostatin, which contains plasminogen kringle
domains; soluble NG2 enhances the activation of plasminogen by urokinase-type
plasminogen activator and inhibits the antagonistic effect of angiostatin on
proliferation of endothelial cells
(Goretzki et al., 2000
). In
our experiments, there was no effect of CD8/MCSP on keratinocyte growth or
terminal differentiation but this does not exclude a potential role for MCSP
in modulating the local concentration of growth factors within the
epidermis.
Although no role has previously been reported for NG2/MCSP in cell-cell
adhesion, our data are consistent with the known association of NG2 with the
actin cytoskeleton (e.g. Fang et al.,
1999). The localization of MCSP and CD8/MCSP on keratinocytes is
also in good agreement with an earlier report that NG2 is found on cell
surface microspikes and microvilli
(Garrigues et al., 1986
). The
cytoplasmic domain of NG2 binds a multiple-PDZ-domain-containing protein
called MUPP1; MUPP1 might be involved in linking NG2 to actin filaments or to
cytoplasmic signalling cascades (Barritt et
al., 2000
). NG2 also regulates Rho-dependent mechanisms in the
trailing processes of motile cells
(Stallcup and Dahlin-Huppe,
2001
) and can signal via Rac
(Majumdar et al., 2003
). The
effect of CD8/MCSP on the actin cytoskeleton of keratinocytes could be
mimicked by inhibiting Rho or ROCK (Fig.
9A), in keeping with the conclusion that inhibition of Rho
prevents adherens junction assembly in epithelial cells
(Etienne-Manneville and Hall,
2002
). A role for MCSP in intercellular adhesion is also plausible
given that the central region of the MCSP extracellular domain has a series of
repeats that show weak similarity to, and might have a similar structural role
to, repeats in cadherin extracellular domains
(Staub et al., 2002
). CD8/MCSP
perturbed cadherin-mediated adhesion, and the interaction of another
chondroitin-sulphate proteoglycan (neurocan) with its receptor coordinately
inhibits both N-cadherin and ß1 integrin-mediated adhesion
(Li et al., 2000
). It is
interesting that neurocan is proposed to play a role in preventing cell and
neurite migration across boundaries (Li et
al., 2000
), given that we are proposing a similar function,
stem-cell clustering, for MCSP in the epidermis.
The finding that MCSP expression was confined to the basal layer of the
epidermis agrees with the observation that NG2 is downregulated with the onset
of terminal differentiation in a range of cell types
(Grako and Stallcup, 1995;
Dawson et al., 2000
). However,
whereas MCSP is a marker for the mitotically inactive stem cell compartment in
the epidermis, NG2 is expressed on mitotically active progenitor populations
(the progeny of stem cells that are committed to differentiate) in the central
nervous system. For example, NG2 is widely used as a marker for
oligodendrocyte progenitors in the adult mammalian central nervous system
(Dawson et al., 2000
;
Hartmann and Maurer, 2001
).
NG2/MCSP is not expressed by normal haematopoietic cells but is expressed on
the surface of leukaemic blasts in certain childhood leukaemias
(Behm et al., 1996
;
Smith et al., 1996
). MCSP is
expressed on melanoma but not normal melanocytes, and we have noted
upregulation of MCSP expression in squamous cell carcinoma lines (J.L. and
F.M.W., unpublished; see also Fig.
2E). The overall significance of MCSP expression in different cell
types is, however, difficult to assess because of the diverse potential
functions of the proteoglycan in adhesion and growth factor
responsiveness.
In conclusion, MCSP expression is a new marker of stem cells in human IFE.
It is not particularly useful for purifying stem cells from cultured epidermis
because it is trypsin sensitive and does not enrich for clonogenic cells any
more than ß1 integrins. Nevertheless, it does appear to be functionally
important in promoting stem-cell clustering. NG2-null mice exhibit no gross
phenotypic abnormalities (Grako et al.,
1999), but it would be interesting to see whether the location of
stem cells is altered in the epidermis. MCSP might contribute to the stem-cell
microenvironment by influencing the accessibility of growth factors to
keratinocytes and modulating cell/extracellular-matrix adhesion in ways that
we have yet to uncover.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
* Present address: Cambridge Antibody Technology, Milstein Building, Granta
Park, Cambridge, CB1 6GH, UK
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