1 Department of Cell and Molecular Biology and Gene Therapy, CIEMAT, Madrid
E28040, Spain
2 Division of Molecular Genetics and Centre of Biomedical Genetics, The
Netherlands Cancer Institute, Plesmanlaan, 121 Amsterdam, 1086 CX, The
Netherlands
Authors for correspondence (e-mail:
a.berns{at}nki.nl
and
jesusm.paramio{at}ciemat.es)
Accepted 25 February 2004
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SUMMARY |
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Key words: Mouse, Rb1, Rbl1, Rbl2, Epidermis, Stem cell, Differentiation, Cre/loxP
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Introduction |
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Rb encodes a nuclear phosphoprotein that actively represses genes
required for the G1-S transition mainly through the binding to members of the
E2F transcription factor family (Dyson,
1998). As cell cycle progresses, pRb is phosphorylated by
cyclin/CDK complexes, resulting in the release of bound E2Fs and allowing the
G1 to S progression. Accordingly, forced expression of pRb leads to growth
arrest, and suppresses pRb-deficient tumor growth in mice
(Riley et al., 1996
). pRb
inactivation is further regulated by two families of CDK inhibitors (CKIs)
that regulate the CDK kinase activity
(Harper and Elledge,
1996
).
Most of the insights into the role of the Rb family in proliferation and
differentiation have been obtained from knockout models. Mice lacking pRb die
in utero displaying defects in erythroid, neuronal and lens fiber cell
differentiation (Clarke et al.,
1992; Jacks et al.,
1992
; Lee et al.,
1992
). Whereas mice with null mutations in either p107
(Rbl1 Mouse Genome Informatics) or p130
(Rbl2 Mouse Genome Informatics) are viable and normal, mice
lacking both p107 and p130 die at birth with defects in
endochondral bone development associated with inappropriate cell cycle exit
(Cobrinik et al., 1996
;
Lee et al., 1996
). This
indicates that p107 and p130 perform overlapping functions that cannot be
carried out by pRb. Similarly, pRb may have overlapping functions with p130
that are not shared with p107 (Ciarmatori
et al., 2001
). Finally, mice deficient for both pRb and p107 show
an aggravated Rb-null phenotype
(Lee et al., 1996
), and mouse
embryo fibroblasts lacking all three Rb family members are immortal and do not
respond to senescence inducing signals
(Dannenberg et al., 2000
;
Sage et al., 2000
).
The epidermis is well suited to study proliferation and differentiation as
both processes are compartmentalized. The proliferative cells are confined to
a single basal layer and the non-proliferative differentiating cells are
located in the suprabasal layers. At their final stage of differentiation,
epidermal cells are shed from the skin. This process requires a continuous
replenishment of cells, which is fulfilled by epidermal stem cells. These stem
cells display a low proliferative rate and give rise to all the epidermal cell
subtypes (Fuchs et al., 2001;
Watt, 2001
; Alonson and Fuchs,
2003).
The induction and maintenance of cell cycle arrest is crucial for normal
tissue homeostasis and regulators of the cell cycle also modulate epidermal
differentiation (Di Cunto et al.,
1998; Paramio et al.,
1998
; Ruiz et al.,
2003
; Zhang et al.,
1998
; Zhang et al.,
1999
). Recently, the Rb family has been implicated in several
aspects of differentiation processes such as terminal cell cycle exit,
maintenance of the post-mitotic state and induction of tissue-specific gene
expression (Lipinski and Jacks,
1999
).
We and others have found a role for Rb family members during the in vitro
differentiation of keratinocytes (Martinez
et al., 1999; Paramio et al.,
1998
), involving the specific interaction with distinct E2F
factors (D'Souza et al., 2001
;
Paramio et al., 2000
).
However, little is known about the function of this family during normal skin
development and epidermal homeostasis in vivo. Transgenic mice expressing the
viral oncoprotein HPV-E7, which binds and inactivates retinoblastoma family
members, in epidermal cells develop epithelial cancer
(Arbeit et al., 1994
;
Griep et al., 1993
;
Herber et al., 1996
). In
addition, ink4
2,3:p21waf doubly-deficient mice
display severe alterations in epidermal differentiation
(Paramio et al., 2001b
).
However, these studies have only addressed the function of pRb family members
in an indirect manner. More recently, Balsitis et al.
(Balsitis et al., 2003
) have
shown that Rb ablation in the epidermis mimics many of the features
displayed by transgenic mice expressing E7 protein in the skin. However,
concomitant expression of E7 in pRb-deficient skin aggravates this phenotype,
indicating that E7 exerts Rb-independent activities
(Balsitis et al., 2003
),
possibly by inactivating p107 and/or p130.
We have begun to dissect the specific and overlapping roles of pRb family
in skin by analyzing mouse mutants deficient for one or more members. We
recently demonstrated that the concomitant absence of p107 and p130 impairs
terminal differentiation of skin in mice, whereas the onset of differentiation
was unaltered (Ruiz et al.,
2003). The embryonic lethality of Rb-null mutant mice
precludes the phenotypic analysis of pRb in the epidermis, whose morphogenesis
starts at 14.5 dpc (Byrne et al.,
1994
). Using a conditional knockout approach based on the
Cre/loxP system (Sauer and
Henderson, 1988
) we have examined the role of pRb and p107 in
epithelial proliferation and differentiation by targeted deletion of
Rb in stratified epithelia using keratin K14-driven Cre
recombinase (K14cre) (Jonkers et
al., 2001
).
Our results demonstrate that pRb plays an essential role as a regulator of epidermal homeostasis. Importantly, the consequences of Rb loss differ dramatically between the closely related follicular and interfollicular epidermis. Finally, Rb and p107 have shared roles in the epidermis in which p107 can compensate for loss of Rb function in differentiation and proliferation.
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Materials and methods |
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Antibodies and immunofluorescence
Immunofluorescence was performed using standard protocols on deparaffinized
sections using antibodies against K10 (K8.60 mAb, 1/500 dilution, Sigma), K5
and K6 (both at 1/500 dilution, rabbit polyclonal antibody, Covance). BrdU
incorporation was monitored in formalin-fixed sections as described
(Santos et al., 2002).
Horseradish-peroxidase, Texas-Red- and FITC-conjugated secondary antibodies
were purchased from Jackson and used 1/4000, 1/500 and 1/50 dilution,
respectively. Apoptosis was monitored using Apoptag kit (Oncor). Peroxidase
was visualized using DAB kit (Vector). Control slides were obtained by
replacing the primary antibody with PBS (data not shown). For lacZ
staining, adult mouse tissues were briefly fixed in 0.2% PFA and equilibrated
in 30% sucrose/PBS at 4°C and embedded in OCT compound. Cryosections (10
µm) were stained for ß-galactosidase activity as described
(Jonkers et al., 2001
).
Primary keratinocytes cultures
Primary keratinocytes were obtained from newborn mice skin and cultured in
EMEM medium supplemented with 4% Chelex-treated fetal bovine serum
(Biowhitaker), 0.05 mM of CaCl2, EGF (10 ng/ml, Serono) and
antibiotics. Differentiation was induced by adding Ca2+ up to 1.2
mM in the culture medium. BrdU was added to the medium (10 µm) and cells
were incubated for 1 hour after which cells were fixed and processed for
immunohistochemistry. Three independent experiments were carried out and at
least 1000 cells were scored on each. Keratinocyte growth analyses were
performed by plating 105 cells and counting cell numbers daily.
Adenoviral infections were performed by incubating primary keratinocytes with
Adeno-cre or Adeno-GFP supernatants for 2 hours in the presence of Polybrene
in mixed DMEM/Optimem (50%) and 2.5% FCS. Afterwards, the medium was changed
to standard low Ca2+ medium.
Western blot analysis
Primary keratinocytes were lysed in 20 mM Tris-HCl (pH 7.5), 137 mM NaCl,
10% glycerol, 1% Triton X-100, 1 mM PMSF, 20 mM NaF, 1 µg/ml aprotinin and
leupeptin, 1 mM disodium pyrophosphate and 1 mM Na3VO4.
Total protein (25 µg) was used for SDS-PAGE, transferred to nitrocellulose
(Amersham) and probed with antibodies against pRb (Pharmingen), p107 and p130
(Santa Cruz) all used at 1/1000 dilution. Actin (Santa Cruz) was used for
normalization. Detection was performed using ECL (Pierce).
Luciferase assays
Plasmid pSV40-Renilla was obtained from Promega. pE2F-Luc was a generous
gift from Dr X. Lu. Primary mouse keratinocytes were transfected using
Superfect (Quiagen) with pSV-Renilla (0.1 µg) and pE2F-Luc (2.4 µg).
Lysates were prepared and analyzed with the Dual Luciferase Reporter Assay
system (Promega). Relative luciferase expression was determined as the ratio
of firefly to Renilla luciferase activity. Transfections were
performed in triplicate, and the mean and standard error were calculated for
each condition. Two independent transfection experiments were performed and
luciferase activity was normalized to the values obtained with control,
RbF19/F19, cells cultured in low calcium.
Label-retaining cell analysis
Ten-day-old pups were injected with BrdU (20 µl of a 12.5 mg/ml dilution
in NaCl 0.9%) every 12 hours for a total of four injections. Skin sections
were collected at 30 and 75 days after the last injection, and BrdU
incorporation was measured as percentage of hair follicles containing positive
cells. Four different animals of RbF19/F19 and
RbF19/F19;K14cre were used to count at least 100
follicles in each time point.
Dye penetration assay
The epidermal permeability barrier function was monitored as described
previously (Hardman et al.,
1998). Briefly, unfixed and freshly isolated new born mice were
rinsed in PBS then immersed in
5-bromo-4-chloro-3-indolyl-ßD-galactopyranoside (X-gal) reaction mix at
pH 4.5 [100 mM NaPO4, 1.3 mM MgCl2, 3 mM
K3Fe(CN)6, 3 mM K4Fe(CN)6 and 1
mg/ml X-gal] and incubated at room temperature overnight.
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Results |
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To determine whether K14cre expression in the skin of
RbF19/F19 mice resulted in pRb loss, we analyzed total
cell lysates from primary keratinocytes derived from neonate skin by
immunoblotting (Fig. 1H).
Whereas keratinocytes derived from RbF19/F19 mice
expressed normal levels of pRb, RbF19/F19;K14cre,
keratinocytes showed complete loss of pRb
(Fig. 1H). To substantiate this
observation, we isolated RbF19/F19 keratinocytes and
infected them with adenoviruses encoding Cre or GFP
(Akagi et al., 1997). Lysates
from Cre-but not GFP-infected RbF19/F19 cells showed
complete absence of pRb protein within 48 hours of infection
(Fig. 1G). Antibodies
recognizing either the C- or the N-terminal of pRb failed to detect any
protein, indicating that the pRb
19 protein or mRNA is very
unstable in cultured cells. We did not detect any Cre-mediated deletion in
parallel cultured dermal fibroblasts of
RbF19/F19;K14cre mice (not shown), consistent
with the absence of dermal staining in K14cre:R26R mice.
Lysates from Rb deficient keratinocytes showed a strong increase
in p107 levels (Fig. 1H),
similar to that seen pRb-null fibroblasts
(Dannenberg et al., 2000;
Hurford et al., 1997
;
Sage et al., 2003
). By
contrast, in p107-null keratinocytes, we detected no increase in pRb protein
levels (Fig. 1H). The
upregulation of p107 in pRb-deficient keratinocytes suggested that p107 might
functionally compensate for Rb loss. To address this aspect, we
generated RbF19/F19;K14cre mice lacking one or
both alleles of p107 (i.e. p107+/ and
p107/). As expected, neither pRb nor p107
protein could be detected in
RbF19/F19;p107/;K14cre
keratinocytes. We observed no changes in p130 expression among the different
genotypes.
Phenotypic consequences of Rb and/or p107 loss in the skin
RbF19/F19;K14cre mice were born at the
expected Mendelian ratio and were indistinguishable from
RbF19/+ littermates in terms of external appearance until
postnatal day 8 (P8), when RbF19/F19;K14cre start
showing a slight reduction in pelage hair and scaling (data not shown).
RbF19/F19, RbF19/+;K14cre and
K14cre mice showed no detectable pathology, and served as controls
(see below). This indicates that, in our experimental settings, the forced
expression of Cre by itself does not contribute to any detectable phenotype
(Loonstra et al., 2001).
The external appearance of the RbF19/F19;K14cre mice was aggravated with concurrent loss of one or both p107 alleles. Specifically, RbF19/F19;p107+/;K14cre mice showed a clear reduction in pelage hair and started developing an obvious scaling, whereas RbF19/F19;p107/;K14cre showed essentially no hair, severe growth retardation and died between postnatal day 8 (P8) and P11 of an unknown cause (Fig. 2A). Importantly, whereas RbF19/F19;K14cre mice showed a p107 copy-number dependent aggravation of the external phenotype, a single wild-type Rb allele in RbF19/+;p107/;K14cre mice was sufficient to suppress this phenotype as these animals appear healthy, fertile and indistinguishable from wild type (Fig. 2A).
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No obvious histological abnormalities were observed in tongue, palate, stomach or esophagus from RbF19/F19;K14cre and RbF19/F19;p107+/;K14cre adult animals; hence, we focused our analysis on the skin.
Proliferation defects in pRb-deficient skin
The increase in epidermal thickness and the hyperkeratosis suggest a defect
in the regulation of proliferation and/or differentiation. To address this, we
monitored BrdU incorporation at P10 in the basal layer of control mice, and
RbF19/F19;K14cre,
RbF19/F19;p107+/;K14cre
and RbF19/F19;
p107/;K14cre littermates
(Fig. 3A-D). We observed a
progressive increase in BrdU incorporation in the basal layer of
RbF19/F19,
RbF19/F19;p107/,
RbF19/F19;K14cre and
RbF19/F19;p107+/;K14cre
(Fig. 3E). Remarkably, we found
no significant increase in BrdU incorporation between
RbF19/F19;
p107+/;K14cre (15.0±1.1) and
RbF19/F19;p107/;K14cre
(15.6±0.2) (Fig. 3E).
This was unexpected because the epidermal thickness significantly differed
between these genotypes (Fig.
2H). A possible explanation for this observation could be the
continued proliferation of pRb/p107-deficient suprabasal cells that normally
arrest and undergo terminal differentiation
(Fig. 3D). To test this
hypothesis, we also determined the number of BrdU-positive suprabasal cells in
each genotype. A significant increase in the number of proliferating cells
dependent on the number of p107 alleles was observed
(Fig. 3E'). These results
suggest that p107, in a dose-dependent manner, plays a crucial role
in regulating the cell cycle exit in the absence of Rb in vivo but
only plays a modest role in controlling basal cell proliferation in wild-type
cells.
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Alterations in epidermal stem cells in the absence of pRb
The transiently amplifying cells ensure the continuous cell renewal
required in the epidermis as cells are shed from the surface. One prediction
that stems from this is that increased cellular turnover caused by pRb loss
would gradually deplete these progenitors as well as the epidermal stem cell
compartment. Therefore, we determined whether the epidermal stem cell
compartment was affected by pRb loss by using a label-retaining technique
(Cotsarelis et al., 1990;
Taylor et al., 2000
). In
Rb-deficient skin, we found a consistent reduction in the number of
labeled cells after 30 (Fig.
3N) and 75 days (Fig.
3N') post BrdU administration, compared with control
littermates. This could indicate a reduction in stem cell number as a
consequence of pRb loss.
Differentiation defects in pRb-deficient skin
The abnormal proliferation of suprabasal cells in the skin of
RbF19/F19;K14cre prompted us to investigate
whether there was any defect in differentiation. In normal epidermis,
expression of keratin K5 is restricted to the basal layer, whereas expression
of keratin K10 is found in the non-proliferative differentiated suprabasal
layers (Fuchs and Green,
1980). Their expression patterns rarely overlap in normal mice
(Fig. 4A). By contrast, in the
RbF19/F19;K14cre epidermis both the K5- and the
K10-expressing cell layers are expanded, and cells co-expressing both K5 and
K10 are regularly observed (Fig.
4B). This aberrant expression pattern is dramatically enhanced in
RbF19/F19;
p107+/;K14cre and
RbF19/F19;p107/;
K14cre epidermis, respectively
(Fig. 4C,D), where the
K5-expressing cell layer was considerably expanded and few cells could be
found that exclusively expressed K10 in the suprabasal layers. Importantly,
most suprabasal cells co-expressed K10 and K5 and co-expression of these
markers became more prominent with a reduction in p107 (compare
Fig. 4B with D).
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Differentiating epidermal Rb-deficient cells are ectopically dividing
The differentiation program in the epidermis takes place as the committed
cells in the basal proliferative compartment arrest proliferation and move
upwards towards the epidermal surface. This differentiation is accompanied by
changes in keratin expression. Therefore, we monitored BrdU incorporation in
K10-expressing cells, the earliest marker of differentiation that appears in
vivo or in vitro, when primary keratinocytes were induced to differentiate by
calcium (see below). We found that in vivo as well as in cultured
keratinocytes, Rb-deficient K10-expressing cells continued to
proliferate (indicated by arrows in Fig.
5B,D,F,G) a phenomena infrequently seen in wild type keratinocytes
(Fig. 5A,E,G). Because we found
that in pRb-deficient skin both proliferation and differentiation of epidermal
cells was affected (see also below), we investigated whether dividing
suprabasal K10+ cells had lost some of their basal characteristics (i.e. loss
of K5 expression), as would be expected from a normal differentiating cell or
whether: (1) pRb-deficient K5+ basal cells continued to divide and
inappropriately turned on K10 expression; or (2) pRb-deficient basal cells had
failed to turn off K5+ expression when becoming K10+. Using triple
immunofluorescence staining against K5, K10 and BrdU, we found BrdU
incorporation in some K10+, K5cells (arrows in
Fig. 5D). This indicates that
proliferation and differentiation are uncoupled processes in pRb-deficient
skin.
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To address if the Ca2+-induced growth arrest reflected a permanent arrest characteristic of postmitotic terminally differentiated keratinocytes in vivo, wild-type and pRb-deficient keratinocytes treated for 72 hours in high Ca2+, were re-stimulated by low Ca2+ medium (0.05 mM) for 24 hours and assayed for BrdU incorporation. As expected, wild-type keratinocytes did not incorporate BrdU, demonstrating that Ca2+-induced differentiation is associated with a permanent cell cycle withdrawal. By contrast, pRb-deficient keratinocytes could completely overcome this growth arrest and be stimulated to divide to a similar extent as exponentially growing cells within 24 hours after removing Ca2+ (Fig. 6C). In RbF19/F19;p107+/;K14cre keratinocytes, removal of Ca2+ resulted in a threefold increase in the growth fraction; however, only a moderate increase in BrdU incorporation was seen in RbF19/F19;p107/;K14cre keratinocytes.
It has been demonstrated that acute, but not permanent, loss of pRb in
mouse embryo fibroblasts is sufficient to trigger cell cycle re-entry in cells
grown under conditions which induce senescence or quiescence
(Sage et al., 2003). To study
if similar events take place during Ca2+-induced differentiation,
we infected RbF19/F19 keratinocytes with cre-encoding
adenovirus. Cre recombinase expression leads to a complete reduction in pRb
protein levels within 48 hours after infection
(Fig. 1G). Interestingly, in
these cells we could not detect any compensatory increase in p107 or p130
levels, as was seen in primary keratinocytes with constitutive pRb loss (data
not shown). A modest increase in BrdU incorporation was observed by comparing
RbF19/F19 keratinocytes 48 hours after Ad-cre infection
with Ad-GFP infected RbF19/F19 or
RbF19/F19;K14cre primary keratinocytes
(Fig. 6D). As demonstrated
above, RbF19/F19 and
RbF19/F19;K14cre keratinocytes underwent a
similar cell cycle arrest upon Ca2+-induced differentiation. By
contrast, Ad-cre-infected RbF19/F19 keratinocytes were
completely refractory to a calcium induced arrest within 48 or 72 hours. These
results demonstrate that, similar to senescent or quiescent embryo fibroblasts
(Sage et al., 2003
), a
substantial functional compensation by p107 is not immediately installed after
acute loss of pRB.
E2F activity is deregulated in Rb-deficient keratinocytes
During proliferation, growth arrest and differentiation, the activity of
the E2F family of transcription factors is inhibited by interactions with pRb
family members (Lipinski and Jacks,
1999). Because Rb-deficient keratinocytes were impaired
in both the initiation and maintenance of a Ca2+-induced growth
arrest, we investigated whether these defects were a consequence of
deregulated E2F function. We measured E2F function in keratinocytes by
transfection of an E2F-Luciferase reporter construct in wild type and
pRb-deficient keratinocytes. In RbF19/F19;K14cre
keratinocytes E2F activity was increased fivefold over controls and even
further increased in cells lacking both pRb and p107
(Fig. 6E). Upon Ca2+
treatment, E2F activity is downregulated to wild-type levels in
Rb-null keratinocytes closely following to the kinetics of growth
arrest in these cells (Fig.
6C). However, in
RbF19/F19;p107+/;K14cre
and
RbF19/F19;p107/;K14cre
keratinocytes E2F activity remained high up to 48 hours after the addition of
Ca2+ in accordance with their reduced sensitivity to
Ca2+-induced differentiation
(Fig. 6D,E).
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Discussion |
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Similar to pRb-deficient fibroblasts
(Dannenberg et al., 2000;
Hurford et al., 1997
;
Sage et al., 2003
), we
detected increased p107 levels in pRb-null keratinocytes, probably because of
the release of transcriptional repression of p107 by pRb/E2F
complexes (Zhu et al., 1995
).
This suggests that the increase in p107 protein levels could be a mechanism to
compensate for loss of pRb function. We tested this possibility, and found
that concurrent loss of p107 in a Rb-null epidermis
aggravated the `pRb-deficiency' phenotype (Figs
2,
3). Moreover, compensation by
p107 occurs in a dose-dependent manner indicating that p107 levels are
limiting in Rb-deficient cells. It has been demonstrated that E2F
transcription factors that normally bind pRb (i.e. E2F1, E2F2, E2F3) may bind
p107 in Rb-deficient cells (Lee
et al., 2002
). Our results support this observation and further
indicate that functional compensation in Rb-deficient keratinocytes
by p107 crucially depends on the absolute levels of p107 protein.
It is noteworthy to mention that one functional copy of Rb is
sufficient to rescue defects associated with p107 deficiency, as
RbF19/+;p107/;K14cre
keratinocytes behave normally in vitro and in vivo. This may be, in part,
explained by the presence of functional p130 that could compensate for both
pRb and p107 loss. This is in line with our recent finding that p107 and p130
perform crucial overlapping functions in the epidermis in vivo
(Ruiz et al., 2003). The
generation of mice lacking all pocket proteins in the epidermis will be needed
to define the capacity of each of these family members to substitute for the
others.
In the epidermis, the proliferative basal layer and differentiating
suprabasal layers are spatially separated and terminal cell cycle arrest
precedes the onset of differentiation. Our results indicate that, in the
absence of Rb, differentiation and proliferation are uncoupled
processes in vivo, since pRb-deficient cells actively proliferate while
initiating the expression of differentiation markers
(Fig. 5,
Fig. 6C,D). Similarly, the
analysis of pRb-deficiency in telencephalon also revealed that mitotic arrest
is not be required to initiate neural differentiation
(Ferguson et al., 2002).
Epidermal homeostasis requires continuous self-renewal of keratinocytes by
transit amplifying cells that are derived from stem cells
(Watt, 2001; Alonson and
Fuchs, 2003). Most adult stem cells divide infrequently and give rise to
committed progenitors (Watt,
2001
; Alonson and Fuchs, 2003). This feature permits the
quantitation of stem cell niches by identification of label-retaining cells
(Cotsarelis et al., 1990
).
Using this technique, we found a decrease in the label retaining cell
population in RbF19/F19;K14cre compared with
wild-type mice. This might either indicate a depletion of epidermal stem cells
or an increased rate of cell division of epidermal stem cells in the absence
of pRb, similar to that seen in basal layer keratinocytes. Currently we cannot
distinguish between these possibilities..
Although epidermal differentiation ensues in Rb-deficient skin, it
is highly perturbed, as demonstrated by the ectopic expression of K6,
suprabasal expression of K5 and a reduction in the number of K10-expressing
cells (Fig. 4). These defects
became more pronounced in the absence of p107, which again indicates that p107
can partially compensate for Rb-loss in the epidermis. We detected no
changes in the expression of terminal differentiation markers filaggrin or
loricrin in pRb-deficient epidermis (data not shown). By contrast, in the
epidermis of mice lacking p107 and p130 where the initial steps of
differentiation are unaffected a clear defect in terminal differentiation is
seen (Ruiz et al., 2003).
These functional analyses fit nicely with the overlapping and unique
expressing patterns and functions proposed for pRb family members in skin
(Paramio et al., 1998
).
Our results demonstrate a strong functional overlap between pRb and p107 genes in epidermis and illustrate a dose-dependent effect of p107 in vivo in the context of a pRb deficiency. In addition, these results illuminate functional differences between pRb and p107 in the ability to suppress epidermal hyperplasia and regulate differentiation. To date, we are not able to discriminate if proliferation or differentiation defects are due to the E2F release or to a failure in the active transcriptional repression mediated by pRb/E2F complexes. In addition interactions with transcription factors, different from E2F, might contribute to the resultant phenotype.
The mechanism whereby pRb family members directly or indirectly regulate the onset of differentiation is unknown at present. It is well established that Rb family members regulate the expression of differentiation markers through direct interaction with transcription factors, such as MyoD in muscle (Novitch, 1996) and C/EBPß in lung fibroblasts (Chen, 1996). Interestingly, C/EBPß controls proliferation in keratinocytes and directly regulates the expression of K10 (Zhu, 1999; Maytin, 1999; Charles, 2001). However, we did not find any alterations in C/EBPß activity during differentiation of Rb-deficient keratinocytes compared with control cells (not shown), suggesting that this transcription factor is not deregulated in Rb-deficient keratinocytes.
Among the differentiation alterations observed in absence of pRb, the
reduced expression of K10 (Fig.
4) might be of a particular relevance. Our recent work has
demonstrated that this keratin may impose a cell cycle arrest in keratinocytes
in a pRb-dependent manner (Paramio et al.,
1999; Paramio et al.,
2001a
; Santos et al.,
2002
). Here, we show that K10-expressing cells do not exhibit a
proliferative arrest. Given that K10 induces a cell cycle arrest in vivo and
suppresses tumor development (Santos et
al., 2002
), the possible loop between pRb and K10 merits further
investigation.
The in vitro and in vivo analysis presented here support a dual role for
pRb family members in keratinocyte differentiation. First, pRb is essential to
initiate a growth arrest in response to differentiation signals. However, in
the sustained absence of pRb, p107 is necessary and sufficient to initiate
this proliferative arrest (Fig.
6C), probably owing to the increase in p107 levels that accompany
permanent loss of pRb (Fig.
1H). Second, pRb is necessary and sufficient for the maintenance
of differentiation-induced postmitotic state in keratinocytes
(Fig. 6C). A similar role for
pRb and p107 in the maintenance of terminal differentiation of myocytes has
also been reported (Schneider et al.,
1994). It is currently not understood whether pRb has a unique
role in maintaining terminal postmitotic state or whether p107 levels are
simply not high enough to maintain a growth arrest. Sage and colleagues
recently reported similar findings in fibroblasts with conditionally
inactivated pRb (Sage et al.,
2003
). Maintenance of the quiescent state is essential for normal
development and differentiation. Perturbations in this cellular control lead
to inappropriate proliferation and expansion of cell compartments that are at
increased risk of acquiring cancer prone mutations. These data may help to
explain why Rb, but not p107, is predominantly found mutated
in human tumors.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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