1 Keratinocyte Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London
WC2A 3PX, UK
2 Pfizer Global Research and Development, Sandwich CT13 9NJ, UK
* Present address: Institute for Genetics, University of Cologne, Cologne
D-50674, Germany
Author for correspondence (e-mail:
fiona.watt{at}cancer.org.uk)
Accepted 4 March 2003
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SUMMARY |
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Key words: Myc, Epidermis, Stem cells, Differentiation, Cell adhesion
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INTRODUCTION |
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Considerable progress has been made in identifying factors that regulate
exit from the epidermal stem cell compartment and differentiation along
specific lineages (Watt, 2001;
Niemann and Watt, 2002
;
Fuchs and Raghavan, 2002
). Wnt
activity controls the choice of differentiation into IFE or hair follicles:
high levels of ß-catenin promote hair follicle morphogenesis
(Gat et al., 1998
), whereas
inhibition of ß-catenin signalling results in conversion of hair
follicles into IFE and sebocytes (Huelsken
et al., 2001
; Merrill et al.,
2001
; Niemann et al.,
2002
). Lineage choice within hair follicles is controlled by the
interplay of several key signalling pathways and transcription factors,
including Notch, Bmp family members and Hox genes
(Niemann and Watt, 2002
;
Fuchs and Raghavan, 2002
).
The majority of the known regulators of epidermal differentiation belong to
well-conserved pathways that are used repeatedly in multiple tissues
throughout development. It is thus surprising that activation of Myc (c-Myc)
also affects epidermal lineage commitment. Myc is a proto-oncogene that is
primarily reported to stimulate proliferation and growth and to induce
apoptosis (Pelengaris et al.,
2000; Grandori et al.,
2000
; Eisenman,
2001
). Activation of Myc in the epidermal basal layer stimulates
exit from the stem cell compartment and differentiation of keratinocytes into
IFE and sebocytes at the expense of the hair lineages
(Gandarillas and Watt, 1997
;
Arnold and Watt, 2001
;
Waikel et al., 2001
). Its mode
of action in promoting epidermal differentiation is unknown and indeed is
counter-intuitive, given that where effects of Myc on differentiation have
been reported in other systems Myc acts as a differentiation inhibitor
(Pelengaris et al., 2000
;
Grandori et al., 2001; Eisenman,
2001
).
Myc expression is tightly regulated within the epidermis. In
interfollicular epidermis it is confined to the basal layer, where the
proliferating keratinocytes, including the stem cells, reside
(Hurlin et al., 1995;
Gandarillas and Watt, 1997
;
Bull et al., 2001
). It is
highly expressed in the stem cells of the hair follicle, which lie in a region
known as the bulge (Bull et al.,
2001
), and is also expressed in keratinocytes undergoing
commitment to hair shaft and inner root sheath differentiation
(Rumio et al., 2000
;
Bull et al., 2001
).
The first evidence that Myc could regulate stem cell fate came from studies
of the proliferative potential of human IFE keratinocytes in culture
(Gandarillas and Watt, 1997).
Primary keratinocytes from neonatal foreskin were infected with a retroviral
vector encoding MycER, a fusion protein in which the ligand-binding domain
(ER) of a mutant estrogen receptor
(Danielian et al., 1993
) is
fused to the carboxy terminus of Myc. ER lacks intrinsic transactivation
activity but responds to the synthetic steroid 4-hydroxytamoxifen (OHT)
(Littlewood et al., 1995
).
When OHT is applied to transduced keratinocytes there is no effect on the
proportion of cycling cells and apoptosis is not stimulated. However, within a
few days there is a marked reduction in growth rate and the cells undergo
terminal differentiation. Activation of Myc causes cells to exit the stem cell
compartment, divide a small number of times as committed progenitors (also
known as transit amplifying cells) and then undergo IFE terminal
differentiation (Gandarillas and Watt,
1997
). In response to Myc keratinocytes can undergo terminal
differentiation at any phase of the cell cycle
(Gandarillas and Watt, 1997
;
Gandarillas et al., 2000
).
When Myc is targeted to the basal layer of the epidermis in transgenic mice
via the keratin 14 promoter proliferation is stimulated, consistent with
recruitment of quiescent stem cells into cycle
(Arnold and Watt, 2001;
Waikel et al., 2001
). The
number of stem cells is reduced, as determined by a reduction in label
retaining cells (Waikel et al.,
2001
). Terminal differentiation of keratinocytes into IFE and
sebocytes is stimulated at the expense of hair lineage differentiation
(Arnold and Watt, 2001
). Using
mice expressing MycER under the control of the keratin 14 promoter it has been
demonstrated that a single application of OHT is as efficient as repeated
doses in inducing the phenotype, even though the activation of Myc is
transient (Arnold and Watt,
2001
). In contrast, when MycER is expressed in terminally
differentiating keratinocytes via the involucrin promoter, the phenotype, one
of preneoplasia, is fully reversible
(Pelengaris et al., 1999
).
It is unclear which Myc target genes, direct or indirect, could be responsible for the observed effects on the epidermal stem cell compartment. Given the role of Myc in other cells, it may be that the relevant genes are not keratinocyte-specific, but that their effects are context-specific. To investigate these issues we have performed a screen of genes that are regulated by Myc in the epidermis in vivo.
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MATERIALS AND METHODS |
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To analyse wound healing in transgenic and wild-type mice, matched according to age and sex, two full thickness wounds were made on the dorsal skin with a 3 mm biopsy punch (Stiefel) under general anaesthetic (Halothane-Vet). Three days after wounding the mice received daily OHT treatment. At different time points after wounding the mice were sacrificed and the wounds collected for histological analysis.
Screen of Affymetrix oligonucleotide arrays and data analysis
Total RNA was prepared using Trizol Reagent (Gibco BRL) and purified using
Qiagen columns according to the manufacturers' instructions. Double stranded
cDNA was generated from 10 µg total RNA using the Superscript Choice kit
(Life Technologies) with a T7-polyT primer. The cDNA was used to generate
biotinylated cRNA by in vitro transcription using a Bioarray High Yield RNA
Transcript Labeling Kit (Enzo). Fragmented cRNA (10 µg) was hybridised in
100 mM MES, 1 M Na+, 20 mM EDTA, 0.01% Tween 20, 0.1 mg/ml herring
sperm DNA, 0.5 mg/ml acetylated BSA plus 50 pM control oligonucleotide and
eukaryotic hybridisation controls to MGU74A GeneChip Oligonucleotide Arrays
(Affymetrix) at 45°C for 16 hours. The probe sets represented 10,043
murine genes and ESTs. Arrays were washed using Affymetrix protocols in
non-stringent buffer (6x SSPE, 0.01% Tween 20, 0.005% antifoam) at
25°C and stringent wash buffer (100 mM MES, 0.1 M Na+, 0.01%
Tween) at 50°C and stained with streptavidin phycoerythrin (10 µg/ml),
including an antibody amplification step. Arrays were scanned using a laser
confocal scanner to generate fluorescence intensities.
The data were analysed using Microarray Analysis Suite version 4.0 (Affymetrix) applying a mask file (MG_U74A.msk) to remove a set of nonfunctional probe sets. Normalized raw data were further analyzed using GeneSpringTM (Silicon Genetics, version 4.1.4) and Excel (Microsoft, version 8.0).
Human keratinocyte culture and retroviral infection
Primary human keratinocytes were isolated from neonatal foreskin (strain
kq) and cultured in the presence of a feeder layer of J2-3T3 cells in FAD
medium [1 part Ham's F12 medium, 3 parts Dulbecco's modified Eagle's medium
(DMEM), 1.8 x 10-4 M adenine] supplemented with 10% fetal
calf serum (FCS) and a cocktail of 0.5 µg/ml hydrocortisone, 5 µg/ml
insulin, 10-10 M cholera toxin and 10 ng/ml epidermal growth factor
(EGF) as described previously (Gandarillas
and Watt, 1997). J2-3T3 cells were cultured in DMEM containing 10%
donor calf serum.
Keratinocytes were infected with the following retroviral vectors: pBabe
puro (empty vector) (Morgenstern and Land,
1990); pBabe-MycER and pBabe106ER
(Littlewood et al., 1995
). In
the 106ER construct amino acids 106-143 have been deleted from Myc
(Littlewood et al., 1995
).
Keratinocytes were infected by co-culture with retroviral producer cells as
described previously and used within one or two passages after infection
(Gandarillas and Watt, 1997
).
Activation of the steroid-inducible constructs was performed by adding 100 nM
OHT (Sigma) to the culture medium.
Mouse keratinocyte culture
Keratinocytes were isolated from three-day-old transgenic and wild-type
mice and cultured on collagen-coated dishes (Becton Dickinson) in low
Ca2+ FAD medium containing 10% chelated FCS and a cocktail of 0.5
µg/ml hydrocortisone, 5 µg/ml insulin, 10-10 M cholera toxin
and 10 ng/ml EGF, as described previously
(Roper et al., 2001).
Keratinocytes were cultured to 80% confluence, incubated for 2 days with 100
nM OHT, harvested and analysed by FACS.
Northern blotting
Total RNA was extracted from mouse skin with TRI Reagent® (Helena
BioScience) according to the manufacturers' instructions. Northern blotting
was performed as described by Gandarillas and Watt
(Gandarillas and Watt, 1995).
The MyBP-H probe was amplified by PCR using the forward primer 5' ATG
ACA GGA AAA GCC ACC TCT G and the reverse primer 5' CTT CCA GAT GCA CGG
TGA GCT C.
Real-time PCR
Total RNA from mouse skin was isolated as described above. RNA
quantification was performed by a two-step RT-PCR procedure. For each sample,
first strand cDNA was prepared using 1-5 µg total RNA, 100 pmol random
nonamers (Sigma) and M-MLV Reverse Transcriptase (Gibco BRL) according to the
manufacturers' instructions. PCR amplification was performed using four
different dilutions (1, 1:2, 1:5, and 1:10) of each first strand cDNA plus
primer and probe master mix containing 900 nM of each primer and 250 nM TaqMan
probe in TaqMan Universal PCR Master Mix (Applied Biosystems). The following
oligonucleotides and TaqMan probes (5' label VIC® and 3'
quencher TAMRA, Applied Biosystems) were used for the analysis: fibronectin
(forward primer 5' GGT TCG GGA AGA GGT TGT GA, reverse primer 5'
TGA GTC ATC TGT AGG CTG GTT CAG, probe 5' TCG CTG ACA GCG TTG CCC ACA),
6 (forward primer 5' TTC CTA CCC CGA CCT TGC T, reverse primer
5' CTG GCC GGG ATC TGA AAA TA, probe 5' TGG GCT CCC TCT CAG ACT
CGG TCA), and adducin (forward primer: 5' CAG CGG TCT CTG CGA TGA A,
reverse primer 5' GCA ACA TCT CCA AGG GAA AGT G, probe 5' TGT GGA
CTC TTG CCT ATC TCC CCG G). Rodent GAPDH Control Reagent (VIC probe, Applied
Biosystems) was used according to the manufacturers' instructions. Real-time
PCR reactions and analysis were performed with a ABI Prism 7700 Sequence
Detection System (Applied Biosystems). Relative quantification of each gene
was determined using the standard curve method. The relative amount of each
mRNA was normalized to the level of GAPDH in each sample.
In situ hybridisation
In situ hybridisation was performed with a plasmid containing a BSSP cDNA
fragment (kindly provided by Peter Angel) as described by Meier et al.
(Meier et al., 1999), using
35S-labeled riboprobes. Hybridisation with a ß-actin antisense
probe served as a positive control. The BSSP sense probe was used as a
negative control.
Antibodies and immunostaining
Antibodies against the following proteins were used: Myc (9E10)
(Gandarillas and Watt, 1997),
murine estrogen receptor (HL7) (Arnold and
Watt, 2001
), keratin 6 (MK6, Covance), keratin 17 (kind gift of P.
Coulombe) (McGowan and Coulombe,
1998
), cornifin (SQ37C, kindly provided by A. M. Jetten)
(Fujimoto et al., 1997
),
filaggrin (AFIII, Covance), fibronectin (Sigma), fibrillin 1 (Fbn-1, Santa
Cruz Biotechnology), laminin (Sigma), E-cadherin (HECD-1, kindly provided by
M. Takeichi), desmoplakin (11-5F, kindly provided by D. R. Garrod)
(Parrish et al., 1987
),
vinculin (V284, Serotec),
6 integrin (MP4F10)
(Anbazhagan et al., 1995
) and
GoH3 (Serotec), ß4 integrin (3E1, Gibco BRL), ß1 integrin (MB1.2)
(Niemann et al., 2002
), CD98
(4F2, BD PharMingen), myosinII (Sigma), adducin (ADD1, kind gift of H.-W.
Kaiser) (Kaiser et al., 1993
)
and actin (AC-40, Sigma).
Tissue samples were either fixed overnight in neutral buffered formalin and
embedded in paraffin or else frozen, unfixed, in OCT compound (Miles) on a
frozen isopentane surface (cooled with liquid nitrogen). Sections (5 µm)
were used for Hematoxylin and Eosin staining and immunofluorescence. Frozen
sections of skin were subjected to indirect immunostaining as described
previously (Carroll et al.,
1995). Paraffin sections were microwaved in antigen retrieval
solution (Bio Genex) for approximately 4 minutes and incubated for another 15
minutes with the retrieval solution. Tissue sections were fixed with 4%
paraformaldehyde for 10 minutes and if necessary treated for 5 minutes with
0.2% Triton X 100. After blocking the sections with 10% FCS in phosphate
buffered saline (PBS) antibodies were incubated for one hour diluted in 10%
FCS in PBS. Secondary antibodies were conjugated with AlexaFluor 488 or 594
(Molecular Probes).
Immunostaining of human cultured keratinocytes was performed as described
above, except when using antibodies against 6 and ß4 integrin
subunits. The distribution of those proteins in hemidesmosomes was examined as
described by Sterk et al. (Sterk et al.,
2000
). Stained preparations were viewed and photographed with a
Zeiss 510 confocal microscope.
Electron microscopy
Back skin from wild-type and transgenic animals that had been treated with
OHT for 9 days was fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in
Sorensen's buffer (pH 7.4). The tissues were embedded in araldite resin and
100 nm sections were cut on a Reichert ultracut S ultramicrotome. Sections
were stained with uranyl acetate and lead citrate and viewed with a JEOL 1010
electron microscope.
Western blotting
Keratinocytes were solubilised in RIPA buffer containing protease inhibitor
cocktail tablets (Roche) and an equal volume of 2% loading buffer [4% sodium
dodecyl sulfate (SDS), 12% glycerol, 50 mM Tris, 2% 2-mercaptoethanol, 0.01%
Serva Blue G, 4 M urea, pH 6.8]. The proteins were resolved by
SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes
(NEN). Blots were incubated overnight at 4°C with primary antibodies.
Primary antibodies were visualized by incubating with anti-mouse or
anti-rabbit IgG horseradish-peroxidase linked antibodies for 1 hour (Amersham
Pharmacia) and by using the ECLä detection kit (Amersham Pharmacia).
FACS analysis
Single cell suspensions of cultured primary mouse keratinocytes were
incubated with antibodies against the ß1 integrin subunit (MB1.2), CD98
(4F2) or the 6 integrin subunit (GoH3) and subsequently with AlexaFluor
488-conjugated secondary antibody directed against rat IgG. FACS analysis was
performed using a Becton-Dickinson FACScan.
Analysis of wound healing in vitro
Human keratinocytes expressing MycER (Kq-MycER), 106ER (Kq-106ER) or the
empty retroviral vector (Kq-pBP) were grown to 90% confluence and incubated
with 100 nM OHT for 24 hours. Cells were treated with mitomycin C in culture
medium for 2 hours to inhibit proliferation, then washed with PBS. A 1
mm-diameter scrape was made across the cultures using a yellow pipette tip
(Fisher). The cells were washed three times with PBS then transferred to
complete culture medium. Wound healing was monitored by photography every 12
hours.
Spreading assay
Spreading assays were performed using Kq-MycER, Kq106ER and Kq-pBP.
Keratinocytes were cultured in complete FAD medium and treated with 100 nM OHT
for 24 hours. Cells were harvested, washed twice with PBS and cultured on
collagen-coated dishes for 3 hours in FAD medium (without serum and growth
factors) or in FAD medium containing 10 ng/ml EGF (Peprotec), 100 ng/ml IGF
(Gibco BRL) or 5 µg/ml cytochalasin D (Sigma). Cells were washed three
times with PBS, fixed with 4% paraformaldehyde and stained with phalloidin
(Sigma). Two-hundred randomly selected cells per treatment group were examined
by microscopy. Photographs were taken using a digital camera (Optronics,
MagnaFire version 1.0). The spreading area of each cell was measured in pixels
using NIH image version 1.58. Each experiment was performed twice. Median
values and standard errors were estimated using Excel.
Motility assay
To analyse the motility of Kq-MycER, Kq-106ER and Kq-pBP, cells were
cultured in complete FAD medium and incubated with OHT for 24 hours, then
harvested and cultured on collagen- or laminin-coated dishes (Becton
Dickinson). The cells were kept humidified at 37°C in 5% CO2
and videotaped for 48 hours. Frames were taken every 4 minutes using Olympus
IMT1 or IMT2 inverted microscopes driven by Broadcast Animation Controllers
(BAC 900) and fitted with monochrome CCD cameras and video recorders (Sony
M370 CE and PVW-2800P, respectively). Recordings were digitised and the
sequence of all frames was run on a PC. Motility was measured using a cell
tracking extension (Cancer Research UK) written for IPLab (Signal Analytics
Inc.), and speed was calculated using a program written in Mathematica by
Daniel Zicha (Cancer Research UK).
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RESULTS |
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The most highly upregulated gene on the arrays (129-fold in 4 days treated
transgenic versus wild-type skin) was the brain and skin serine protease
(BSSP) (Fig. 3A). BSSP is
predominantly expressed in the sebaceous glands of the hair follicle and in
the distal part of the outer root sheath, probably corresponding to the bulge
(Meier et al., 1999). In situ
hybridisation analysis confirmed that BSSP was highly induced on activation of
Myc in transgenic mice. In untreated transgenic animals BSSP expression was
weak and largely confined to the sebaceous glands
(Fig. 3D, tg-0d, arrowheads).
In treated transgenics the expression in sebaceous glands increased and
BSSP-RNA was also highly expressed in patches of interfollicular epidermis
(Fig. 3D, tg-4d, arrowheads).
The increase in BSSP mRNA thus reflected an increase in the number of cells
expressing BSSP.
Most downregulated genes are involved in cell adhesion
When the data from the three individual experiments were compared, 81 genes
were consistently downregulated in 4 days treated transgenic versus wild-type
skin (Table 3). All the
identified downregulated genes were decreased more than twofold, and exhibited
a minimum absolute expression value of 400 (raw data) in 4 days treated
wild-type animals. The 81 genes were grouped according to their functional
roles (Fig. 2B). Most (30%)
were involved in cellular adhesion (Fig.
2B) and included genes mediating cell-cell adhesion and components
of the extracellular matrix (ECM). A further 11% encoded components of the
cytoskeleton or cytoskeleton regulatory factors
(Fig. 2B). Examples of
downregulated genes in 4 days treated transgenic animals are given in
Table 4.
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Myc activation results in delayed wound healing in vivo and in
culture
We performed a range of assays to examine the functional consequences of
Myc-mediated downregulation of genes involved in cell adhesion. Waikel et al.
(Waikel et al., 2001) have
previously reported that constitutive expression of Myc in the basal layer of
the epidermis causes impaired wound healing. We therefore investigated whether
activation of MycER had any effect on wound healing in K14MycER transgenic
mice. Three days after wounding, control mice and transgenic mice received
daily OHT treatment for 4 days. The wounds of wild-type animals were covered
with hyperproliferative epidermis; however, the healing of transgenic wounds
was incomplete (Fig. 5A). The
same results were obtained when OHT treatment was begun immediately after
wounding (data not shown). By 14 days the wounds in transgenic mice had
re-epithelialised (data not shown), indicating that the effect of Myc
activation was to delay rather than to completely inhibit wound healing. The
delay in wound closure did not reflect a defect in proliferation as Ki67 was
highly upregulated at the wound margins in transgenic animals
(Fig. 5A).
|
Re-epithelialisation of skin wounds involves a complex cascade of events that not only requires keratinocyte migration and proliferation, but also deposition of a provisional ECM and an influx of inflammatory cells into the dermis. To examine wound healing in the absence of other cell types and independent of Myc effects on proliferation, confluent sheets of human keratinocytes expressing MycER (Kq-MycER) or the empty retroviral vector (Kq-pBP) were pretreated with OHT for 1 day, then treated with mitomycin C to inhibit proliferation and scraped with a pipette tip to create a wound. The width of the wounds was monitored daily for 3 days. After 24 hours the size of each wound was significantly reduced in control cultures, and by 48 hours keratinocytes on either side of the wound had made contact (Fig. 5B). In contrast, keratinocytes expressing MycER had not moved into the wound by 24 hours and by 48 hours the wounds were reduced in width but contact between the wound edges had not been established (Fig. 5B). The results presented in Fig. 5B are representative of more than five independent experiments.
Motility and spreading are impaired by Myc activation
To examine whether the inhibition of wound healing by Myc activation
reflected decreased migration of keratinocytes, motility assays were performed
using time lapse microscopy. Human keratinocytes (Kq-MycER, Kq-106ER and
Kq-pBP) were pretreated with OHT, plated on collagen- or laminin-coated dishes
and filmed for 36 hours. Fig.
6A shows the results of a single experiment that is representative
of four independent experiments on collagen. The path taken by individual
cells was determined by marking the start (green spot) and end coordinates
(red spot). The average path length of Kq-MycER cells (n=24) on
collagen was decreased compared to controls (Kq-106ER, n=24; Kq-pBP,
n=23). The average speed of all cells in
Fig. 6A is shown in
Fig. 6B: there was no
significant difference in the motility of cells expressing 106ER or the empty
vector, but the speed of cells expressing Myc was decreased three fold. A
similar reduction in motility was observed when Myc was activated in
keratinocytes plated on laminin (data not shown).
|
Effects of Myc on cell-cell and cell-ECM receptors
Myc over-expressing keratinocytes were less motile and spread to a lower
extent than controls, even in the presence of exogenous extracellular matrix
proteins. This suggested that Myc might affect expression of integrin ECM
receptors. Indeed, activation of Myc results in reduced ß1 integrin
expression by human keratinocytes in vitro
(Gandarillas and Watt, 1997)
and in transgenic mice that constitutively express Myc via the K14 promoter
(Waikel et al., 2001
).
However, several of the integrin receptor genes expressed by keratinocytes, in
particular a2, a5 and ß1, were not represented on the Affymetrix arrays;
ß4 RNA levels were unchanged; and
6, though 1.5-fold
downregulated, had a raw expression value of less than 200, placing the result
within the low confidence level category. To establish whether
6 mRNA
was indeed downregulated by Myc, we performed real-time PCR analysis of RNA
extracted from whole skin of transgenic mice
(Fig. 7C).
6 mRNA was
twofold downregulated in transgenic mice after 4 days treatment with OHT
compared to untreated transgenic animals
(Fig. 7C). We therefore used
FACS, immunofluorescence staining and electron microscopy to investigate
whether expression of cell-ECM and cell-cell adhesion receptors was altered
upon Myc activation.
|
Activation of Myc in transgenic mouse keratinocytes led to a marked
reduction in cell surface expression of the 6ß4 integrin
(Fig. 7B). In cultured human
keratinocytes Myc activation resulted in reduced localisation of
6ß4 in the immature hemidesmosomes that are assembled in culture
(Fig. 7A). Reduced assembly of
these junctions was confirmed by immunolabelling for plectin (data not shown).
We conclude that of the major types of cell-cell and cell-ECM adhesive
junctions, only hemidesmosome formation was affected by Myc activation in
cultured keratinocytes.
We next examined 6 expression and hemidesmosome formation in intact
skin. Immunolabelling the skin of transgenic mice after 4 days treatment with
OHT confirmed a decrease in
6 levels at the basement membrane zone
(Fig.
8A,B).
We then performed electron microscopy on the skin of transgenic and wild-type
mice treated for 9 days with OHT (Fig.
8C-F). The number of hemidesmosomes in the epidermis of transgenic
animals was greatly reduced compared to wild-type mice (Fig.
8E,F)
and those hemidesmosomes that were present were smaller than in wild-type
epidermis (Fig.
8C,D,
arrowheads).
|
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Adducin is a membrane skeletal protein that binds to F-actin, modulates the
assembly and disassembly of the spectrin-F-actin network
(Gardner and Bennett, 1987),
and is thought to play a crucial role in cell motility
(Fukata et al., 1999
). Adducin
was localised at the plasma membranes of all cells in the epidermis in
wild-type mice, but was no longer detectable in keratinocytes in the basal
layer of OHT-treated transgenic epidermis
(Fig. 9C, arrowheads).
Real-time PCR confirmed a 1.8-fold downregulation of adducin mRNA in
transgenic skin compared to wild-type skin treated for 4 days with OHT
(Fig. 9F). We also measured
adducin protein levels by western blotting
(Fig. 9G). There was a 3.3-fold
decrease of adducin protein in Kq-MycER cells after 2 days treatment with OHT,
whereas adducin levels were not altered upon OHT treatment of Kq-pBP
(Fig. 9G). Adducin protein was
1.7-fold lower in untreated Kq-MycER cells compared to Kq-pBP, suggesting that
when expressed at high levels in cultured cells the MycER construct is
somewhat `leaky' (see also Gandarillas and
Watt, 1997
). Consistent with the in vivo observations
(Fig. 9C), immunofluorescence
demonstrated that the level of adducin protein at cell-cell borders was
decreased upon activation of Myc in Kq-MycER cells compared to Kq-pBP
(Fig. 9E).
To analyse how activated Myc might influence the cytoskeleton we examined the distribution of actin and myosin in cultured human keratinocytes (Fig. 9D). In Kq-MycER cells treated with OHT the number and size of the leading membrane lamellae were reduced compared to the controls (Kq-pBP) (Fig. 9D, arrowheads). This correlated with reduced polymerisation of actin and myosin at the cell periphery (Fig. 9D).
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DISCUSSION |
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In considering how gene expression is modulated by Myc, direct versus
indirect regulation is an important issue
(Eisenman, 2001). Direct
targets are those genes whose expression is altered by direct Myc binding
(Eisenman, 2001
).
Max-Myc-TRRAP coactivator complexes bind E-boxes, generating acetylation of
histone H4 in the vicinity of the binding site and stimulating induction of
target gene expression (Eisenman,
2001
). Most Myc target genes identified using cultured cells are
involved in growth and metabolism, consistent with the function of Myc in
regulating the growth rate (i.e. size and mass) of cells
(Eisenman, 2001
). Genes
upregulated in response to Myc are involved in ribosome biogenesis, energy and
nucleotide metabolism and translational regulation
(Coller et al., 2000
;
Guo et al., 2000
;
Boon et al., 2001
;
Schuhmacher et al., 2001
).
The major classes of genes that were upregulated in K14MycER mouse skin
were those well documented to be regulated by Myc, including genes that are
known to be direct targets. This is the case for the eukaryotic initiation
factors (eIF) (Table 2), mRNA
cap-binding proteins rate limiting for protein synthesis
(Rosenwald et al., 1993,
Coller et al., 2000
;
Jones et al., 1996
).
Activation of Myc induces protein synthesis by upregulating ribosomal proteins
in fibroblasts (Guo et al.,
2000
), in Drosophila
(Johnston et al., 1999
), human
B cells (Iritani and Eisenman,
1999
) and mice (Schuhmacher et
al., 1999
). Ribosomal proteins were also induced in K14MycER skin,
including the direct Myc target, nucleolin
(Greasley et al., 2000
). Myc
plays an essential role in regulating entry into S-phase by shortening G1;
several proteins upregulated in MycER mouse skin stimulate entry into S-phase
(Table 2), including mCDC47
(MCM7), which has an E-box binding site for Myc in its promoter
(Suzuki et al., 1998
). Another
well documented Myc target gene is Ornithine decarboxylase (ODC)
(Bello-Fernandez et al., 1993
).
Although ODC has been previously described to be upregulated in K14MycER
transgenic mice (Arnold and Watt,
2001
), the mRNA was not regulated on the microarray chips; a
possible explanation for this is a high background value with the control
mismatch probe.
Although our screen was able to detect direct Myc target genes, other identified genes were undoubtedly indirectly regulated. The most obvious categories were genes that were expressed in transgene-negative cells. These include the differentiation markers expressed in the suprabasal epidermal layers (Fig. 3C) and dermal (connective tissue)-specific ECM genes (Table 4). As discussed below, the induction of differentiation genes is most probably a secondary consequence of the Myc-mediated effects on cell adhesion. The mechanism by which dermal gene expression is altered remains to be investigated; however, one possibility is that it is via the effects of Myc on the cytokines expressed by basal keratinocytes (see Fig. 2B).
The induction of cell cycle and differentiation marker genes reflects the
stimulation of proliferation and differentiation by MycER that we have
reported previously (Arnold and Watt,
2001). However, it is the genes that were repressed by Myc that
provide a clue as to the mechanism by which Myc exerts its surprising
differentiation-promoting effects on the epidermis. Myc-mediated repression of
gene expression is incompletely understood. It has been reported to involve
direct binding by Myc-Max complexes to INR elements; alternatively, it may
depend on interaction of Myc with positively acting transcription factors
(reviewed in Orian and Eisenman,
2001
). Myc can also form a complex with the transcription factor
Miz-1 and thereby inhibit Miz-1-mediated transcriptional activation (e.g.
Staller et al., 2001
). There
is also evidence for repression of E-box-dependent transcription via Myc
recruitment of a transcriptional corepressor complex
(Satou et al., 2001
).
The major classes of genes that were downregulated in the transgenics were
those involved in cell adhesion and the cytoskeleton. There is already
evidence from several studies that Myc regulates cell adhesion. Cells
transformed by deregulated expression of Myc or N-Myc are characterised by
reduced expression of cell adhesion molecules, including a range of integrin
subunits (Inghirami et al.,
1990; Judware and Culp,
1995
; Barr et al.,
1998
; Fujimoto et al.,
2001
) and Myc can act directly on certain integrin promoters
(Barr et al., 1998
;
López-Rodríguez et al.,
2000
). Fibronectin, alpha-1 type 3 collagen and tropomyosin alpha
chain genes are downregulated within 9 hours of MycER activation in
fibroblasts (Coller et al.,
2000
) and there is evidence that Myc suppresses collagen genes by
interference with NF-1 (Yang et al.,
1991
; Yang et al.,
1993
), as proposed for the PDGF receptor promoter
(Oster et al., 2000
). It thus
seems probable that at least some of the genes downregulated by Myc are direct
targets.
The categories of adhesion molecules that were repressed by Myc in the skin
included ECM proteins, regulators of the cytoskeleton and membrane proteins
such as the 6 integrin subunit. These changes had profound effects on
the behaviour of keratinocytes, whether assayed in vivo or in vitro. Wound
healing in vivo was impaired, as reported previously
(Waikel et al., 2001
). The
motility of keratinocytes in culture was severely reduced, both within cell
sheets (Fig. 5B) and at the
level of single cells (Fig.
6A,6B).
The spreading of keratinocytes was decreased (Fig.
6C,6D)
and the ability of the cells to form lamellipodia was compromised
(Fig. 9D), reflecting
downregulation of proteins that control the assembly and contractility of the
actin cytoskeleton (Table 4). This was also reflected by the downregulation of adducin, a protein that plays
a crucial role in cell motility (Fig.
9E-G) (Fukata et al.,
1999
). In vivo, there was a substantial reduction in the number of
hemidesmosomes and those hemidesmosomes that were present were reduced in size
(Fig. 8C-F).
Based on the effects of Myc on keratinocyte adhesion and motility we can
now propose a model for the phenotype of K14MycER mouse epidermis. The model
is that reduced cell-ECM adhesion stimulates exit from the stem cell
compartment, whereas reduced motility determines that IFE and sebocyte
differentiation are promoted at the expense of the hair lineages
(Fig. 10). There is good
evidence that epidermal stem cells are more adhesive to ECM than their
differentiating daughters; this is true both for human
(Jones et al., 1995) and mouse
(Bickenbach and Chism, 1998
)
keratinocytes. Furthermore, reduced ECM adhesion is known to promote
differentiation: both human and mouse keratinocytes undergo terminal
differentiation when placed in suspension (reviewed by
Watt, 2001
) and in human
keratinocytes reduced expression of ß1 integrins in vitro stimulates exit
from the stem cell compartment (Zhu et
al., 1999
). High expression of the
6ß4 integrin is
thought to be a marker of stem cells in mouse epidermis
(Tani et al., 2000
) and
6ß4 was markedly downregulated by MycER. It therefore seems
probable that Myc-induced repression of adhesion stimulates epidermal stem
cells to differentiate.
|
The control of stem cell fate in many, if not all, tissues and organisms
involves reciprocal interactions between stem cells and their local
microenvironment or niche (Watt and Hogan, 2001;
Spradling et al., 2001). The
expression and functions of Myc could provide an example of such reciprocity,
because cell-ECM adhesion can regulate Myc. In epithelial cells Myc levels
decrease in suspended cells, correlating with cell cycle arrest or
differentiation (Gandarillas and Watt,
1995
; Benaud and Dickson,
2001a
). Conversely, adhesion of epithelial cells to fibronectin or
collagen induces Myc expression in a concentration-dependent fashion
(Benaud and Dickson, 2001b
).
Pathways implicated in the induction of Myc, namely c-Src, Erk1/2 MAPK and
Ras/Akt (Barone and Courtneidge,
1995
; Sears et al.,
2000
; Benaud and Dickson,
2001b
) are activated by integrin ligation
(Frame et al., 2002
;
Howe et al., 2002
).
In conclusion, we have presented evidence that the effects of Myc on
epidermal differentiation are a consequence of its effects on cell adhesion
and motility. This would explain why Myc promotes differentiation in
keratinocytes (Gandarillas and Watt,
1997; Arnold and Watt,
2001
; Waikel et al.,
2001
) yet suppresses differentiation in non-epithelial cells
(Eisenman, 2001
). Because
inhibition of cell-ECM adhesion promotes apoptosis of certain cell types
(Frisch and Screaton, 2001
),
the repression of cell adhesion genes could also play a role in Myc-induced
apoptosis of some cells. Thus, generic effects of Myc on gene expression have
context-specific outcomes in terms of cell behaviour.
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ACKNOWLEDGMENTS |
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