Institute of Physiological Chemistry and Bonner Forum Biomedizin, University of Bonn, Nussallee 11, 53115 Bonn, Germany
* Author for correspondence (e-mail: t.magin{at}uni-bonn.de )
Accepted 12 April 2002
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Summary |
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Key words: Keratins, c-Myc, 14-3-3 proteins, Epidermis, Cell proliferation
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Introduction |
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c-Myc is an oncoprotein involved in both the regulation of cell cycle
progression and growth control (Elend and
Eilers, 1999). Expression of c-Myc in suprabasal cell layers of
transgenic mice induced hyperproliferation and neoplastic changes
(Waikel et al., 1999
;
Pelengaris et al., 1999
). If
restricted to the basal epidermis of transgenic mice, it was recently shown
that c-Myc overexpression resulted in hyperproliferation but did not interfere
with interfollicular terminal differentiation
(Arnold and Watt, 2001
). The
hyperproliferation seemed to result from an increase in the number of transit
amplifying cells, following depletion of the stem cell compartment
(Arnold and Watt, 2001
;
Waikel et al., 2001
). In
agreement with this observation, the transgenic expression of cyclin D1, D2 or
D3, which represent c-Myc target genes
(Bouchard et al., 1999
;
Obaya et al., 1999
), was also
reported to stimulate epidermal proliferation upon overexpression in basal
cells (Robles et al., 1996
;
Rodriguez-Puebla et al., 1999
;
Rodriguez-Puebla et al.,
2000
).
14-3-3 is a member of the 14-3-3 protein family, which is
predominantly expressed in epithelial cells (Leffers, 1993). Members of the
14-3-3 family of proteins bind to conserved phosphoserine-containing motifs of
many proteins (Yaffe et al.,
1997
), including keratins (Ku
et al., 1998
). By sequestration of proteins involved in cell cycle
progression 14-3-3
is able to induce a G2 block
(Hermeking et al., 1997
). The
depletion of 14-3-3
in primary human keratinocytes using antisense
technology led to the activation of telomerase and to the downregulation of
p16, and forced these cells into continuous proliferation
(Dellambra et al., 2000
). It
was argued that 14-3-3
or one of its interaction partners could expand
the proliferative potential of epidermal keratinocytes.
The differentiation state of epidermal keratinocytes is reflected by the
intricate expression pattern of keratins
(Fuchs and Green, 1980;
Moll et al., 1982
). Keratins
are members of the large intermediate filament gene family
(Hesse et al., 2001
) and form
the intermediate filament (IF) cytoskeleton in all epithelia including the
epidermis. Basal keratinocytes express keratins (K) K5, K14 and K15, which are
replaced by K1 and K10 once the cells lose contact with the basement membrane
and move to the suprabasal compartment
(Fuchs and Green, 1980
). The
functional significance of this change in keratin expression is not well
understood. Given that keratins are primarily regarded as cytoskeletal
proteins, changes in their expression have been regarded as a result of
differentiation but not as a means to influence the differentiation state of a
cell or a tissue.
During the past decade it became evident that mutations in epidermal
keratins interfere with the generation of a normal IF cytoskeleton, lead to
epidermal fragility and cause blistering skin diseases including epidermolysis
bullosa simplex and epidermolytic hyperkeratosis
(Corden and McLean, 1996;
Fuchs and Cleveland, 1998
).
Based on the patient data and from the knowledge that came from the study of
transgenic mice, it is widely accepted that keratins are important structural
proteins (Fuchs et al., 1992
;
Lloyd et al., 1995
;
Bickenbach et al., 1996
;
Porter et al., 1996
;
Hesse et al., 2000
;
Tamai et al., 2000
;
Arin et al., 2001
;
Cao et al., 2001
;
Peters et al., 2001
).
Recently, however, we demonstrated that K10-/- mice are fully
viable and exhibit an intact skin that resists mechanical stress due to a
compensatory suprabasal persistence of the basal keratins 5 and 14
(Reichelt et al., 2001
).
In an attempt to understand the role of K10, we went on to analyse adult
mice, given that in neonatal mice it was dispensable as a cytoskeletal
protein. In adult mice, which still presented with an intact epidermis, we
noted a considerable hyperproliferation of basal keratinocytes. This pointed
to a specific suprabasal function of K10, or K1/K10 IF, that may not be
fulfilled by other keratins. In fact, several studies in mice and man have
shown that K10 is downregulated in most carcinomas
(Ivanyi et al., 1989;
Roop et al., 1988
;
Toftgard et al., 1985
;
Winter et al., 1983
;
Maddox et al., 1999
).
Moreover, the targeted expression of human K10 under the control of the K6
promoter showed a delay in tumor formation in mice
(Santos at al., 1997
). While
the downregulation of K10 can be taken as a consequence of an altered
differentiation known to occur in most tumors, another study pointed to an
active involvement of K10 in cell cycle regulation
(Paramio et al., 1999
). Most
recently, the same authors demonstrated that, in cultured human HaCaT
keratinocytes, Akt kinase was sequestered by K10, which impaired its
translocation to the cell membrane where it normally becomes activated by PI
3-kinase (Paramio et al.,
2001a
). The authors reasoned that K10 can directly inhibit cell
proliferation via sequestration of Akt
(Paramio et al., 2001a
).
Here we show that the targeted deletion of K10 leads to epidermal hyperproliferation in adult mice. Our data imply that the altered composition of the suprabasal cytoskeleton is able to alter the proliferation state of basal cells through the induction of c-Myc. We discuss the possible links between K10, cell growth and proliferation.
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Materials and Methods |
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For the preparation of epidermal sheets, ears were excised immediately after the animals were killed. The ears were then torn with forceps to separate the outer epithelial and the inner side. Both halves were then immersed in 0.5 M ammonium thiocyanate (Sigma) solution in 0.1 M phosphate buffer, pH 6.8, for 20 minutes at 37°C. The epidermis was separated from the dermis with forceps and washed three times for 5 minutes with PBS. The epidermal sheets were subsequently immersed in PBS containing 0.5 µg/ml DAPI (Sigma) for 60 minutes followed by washing in PBS. After a brief rinse in double distilled water and ethanol, the sheets were mounted and embedded in Mowiol (Calbiochem, Bad Soden, Germany).
BrdU labelling
Mice were injected with 1 ml BrdU labelling reagent/100 g mouse weight i.p.
(RPN 201, Amersham Pharmacia Biotech, Freiburg, Germany) 2 or 24 hours before
they were sacrificed. Skin samples were fixed, embedded and sectioned as
described above. After deparaffination, the sections were placed in 2 N HCl
for 30 minutes at 37°C and subsequently rinsed thoroughly in PBS. This was
followed by a 30 minute treatment with 0.1% (w/v) trypsin (Invitrogen,
Karlsruhe, Germany) in PBS at 37°C. After rinsing in PBS, the sections
were covered with the monoclonal anti-BrdU antibody BU 33 (Sigma), 1:1000.
After 1 hour, the slides were washed three times for 5 minutes in PBS and
incubated with an Alexa-488-conjugated anti-mouse secondary antiserum
(Molecular Probes, Leiden, The Netherlands), 1:400, for 40 minutes. After
washing as before, sections were briefly rinsed once with double-distilled
water and once with ethanol and mounted in Mowiol (Calbiochem, Germany).
Northern blotting
Skin samples were immediately frozen in liquid nitrogen. RNA was extracted
with Trizol (Invitrogen) according to the manufacturer's instructions. Thirty
micrograms were loaded per lane, separated, transferred and hybridized as
described before (Reichelt et al.,
1999). Wnt-probes for Wnt-3, Wnt-4, Wnt-5a (a gift of Rolf Kemler,
Freiburg, Germany), a 720 bp probe for 14-3-3
and a K5 probe used for
normalization were derived from cDNA clones and labelled with Decalabel (MBI,
St Leon-Rot, Germany).
SDS-PAGE and western blotting
Total skin protein was extracted, blotted and stained as described before
(Reichelt et al., 1999). Gels
of 8, 10 and 18% polyacrylamide were used to separate proteins (see
Fig. 5 legend). Primary
antibodies were diluted as follows: K1 antiserum AF 109 (Babco, Richmond, CA),
1:400,000; K5 antiserum AF 138 (Babco), 1:100,000; K14 antiserum (a gift of
Manfred Blessing, Mainz, Germany), 1: 50,000; K15 antiserum (a gift of Elaine
Fuchs, Chicago, IL), 1:5000; K6 antiserum (a gift of Manfred Blessing),
1:10,000; K16 antiserum (a gift of Rebecca Porter, Dundee, UK), 1:25,000;
14-3-3
antiserum N14 (Santa Cruz Biotechnology) 1:1000; p53 antiserum
CM.5, 1:50,000; p21 antiserum C-19 (Santa Cruz Biotechnology), 1:500; p27
antiserum (Biosource, Nivelles, Belgium), 1:2000; ß-catenin monoclonal
antibody (Beckton Dickinson-Transduction Laboratories, Heidelberg, Germany),
1:10,000; Rb monoclonal antibody G3-245 (Beckton Dickinson-Pharmingen,
Heidelberg, Germany), 1:4000; phospho-Rb (Ser780) antiserum (Cell Signaling
Technology, Beverly, MA), 1:8000; Akt antiserum (Cell Signaling Technology),
1:2000; phospho-Akt (Thr308) antiserum (Cell Signaling Technology), 1:2000;
phospho-Akt (Ser473) antiserum (Cell Signaling Technology), 1:2000.
HRP-conjugated secondary antisera (Dianova, Hamburg, Germany) were diluted
1:30,000. For detection, Super Signal (Pierce, Rockford, Illinois) was
used.
|
Immunofluorescence analysis
Immunofluorescence analysis was performed as described before
(Reichelt et al., 1999). The
K1 antiserum AF 109 (Babco), 1:5000; K5 antiserum AF 138 (Babco), 1:5000; K6
antiserum (a gift of Manfred Blessing), 1:1000; K14 antiserum (a gift of
Manfred Blessing), 1:5000; K15 antiserum (a gift of Elaine Fuchs), 1:200; K16
antiserum (a gift of Rebecca Porter), 1:500; the Alexa 488-conjugated
secondary antiserum (Molecular Probes, The Netherlands) was diluted 1:400.
Allele-specific PCRs
To detect allelic variants of p16 exon 1 we used a previously published
protocol (Zhang et al., 1998)
with minor modifications. Briefly, exon 1 was amplified from DNA preparations
from BALB/c, 129/Ola (both inbred strains) and K10-/- mice as well
as wild-type mice, which were used for our studies (the two latter are on a
mixed BALB/cx129/Ola background) using the following primers: (forward)
5'-ACTGAATCTCCGCGAGGAAAGCGAACT-3' and (reverse)
5'-GAATCGGGGTACGACCGAAAGAGT-3'. Cycling conditions were 94°C
for 1 minute, followed by 35 cycles of 94°C for 1 minute, 62°C for 45
seconds, and 72°C for 2 minutes. The PCR products were digested with
NlaIII (New England Biolabs, Frankfurt, Germany). Exon 2 was
amplified from the same DNA preparations using primers: (forward)
5'-GTGATGATGATGGGCAACGTTCA-3' and (reverse)
5'-GGGCGTGCTTGAGCTGAAGCTA-3'. Cycling conditions were 94°C for
1 minute, followed by 35 cycles of 94°C for 1 minute, 68°C for 30
seconds, 72°C for 1 minute. The PCR products were digested with
BsaAI (New England Biolabs).
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Results |
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A columnar organization of epidermal keratinocytes has been documented for
many species ranging from rodents to humans (for a review, see
Potten, 1981). This ordered
pattern of keratinocytes with normally regularly spaced epidermal
proliferative units (EPUs) was impaired in K10-/- mice as
demonstrated by DAPI staining of sections and epidermal sheets. Epidermal
sheets viewed from the top as well as transversal sections showed that the
spacing of nuclei in K10-/- epidermis appeared unordered
(Fig. 1D,F,H), while wild-type
epidermis (Fig. 1C,E,G) showed
a regular spacing of nuclear columns. The observed alterations in the normal
structural organization of K10-/- epidermis suggested a
deregulation of cell proliferation.
The amounts of K1 (Fig. 2A),
K5 (Fig. 2B) and K14
(Fig. 2C) protein were slightly
increased while K15 (Fig. 2D)
was unaltered, as revealed by western blotting. The distribution of K1 protein
was unaltered in K10-/- epidermis
(Fig. 2F) compared with that in
the wild-type, while the basal keratins K5 and K14 were also present in
suprabasal cells in K10-/- epidermis
(Fig. 2H and K, respectively, G and
I, wild-type) as described before for neonatal epidermis
(Reichelt et al., 2001). In
contrast to K5 and K14, another basal keratin, namely K15, remained restricted
to basal keratinocytes (Fig.
2M, K10-/-, L, wild-type).
|
Increased proliferation and induction of K6 and K16 in
K10-/- epidermis
The reason for the thickening and the disordered architecture of the
K10-/- epidermis became evident after Ki-67 labelling. In addition
to an increased number of basal cells, a considerable number of suprabasal
cells were Ki-67-positive (Fig.
1K). Scoring revealed that the knockout epidermis exhibited 32%
stained cells (Fig. 1K),
whereas in the wild-type, only 8% of all keratinocytes were Ki-67-positive
(Fig. 1I). We reasoned that the
increase in the labelling index could be directly or indirectly linked to the
loss of K10. Of note, several groups reported the downregulation of K10 in
mouse and human skin carcinomas (Nelson
and Slaga, 1982; Roop et al.,
1988
; Toftgard et al.,
1985
; Winter et al.,
1980
; Winter et al.,
1983
), which led to the assumption that K10 might normally inhibit
cell proliferation. In fact, it was recently reported that forced expression
of K10 inhibited cell proliferation in the human epithelial cell line HaCaT,
possibly involving Rb (Paramio et al.,
1999
). In activated keratinocytes, the loss or downregulation of
K10 is accompanied by an increase in the so-called wound healing keratins K6
and K16 (McGowan and Coulombe,
1998
; Freedberg et al.,
2001
). In contrast to neonatal K10-/- mice, adult
animals displayed a strong increase in K6 and K16 as indicated by western
blotting (Fig. 3A,B). While it
is often assumed that K6 and K16 are not only coexpressed but also colocalize,
immunofluorescence analysis showed this not to be the case (see also
Porter et al., 1998
). Using an
antiserum that recognizes both K6a and K6b, K6 was localized both in basal and
in suprabasal keratinocytes (Fig.
3D). This is in agreement with data from knockout mice, according
to which K6a is present both in basal and suprabasal keratinocytes, while K6b
is restricted to the latter (Wojcik et
al., 2000
; Wojcik et al.,
2001
; Wong et al.,
2000
). By contrast, K16 was confined to the spinous and granular
layer (Fig. 3E).
|
The alterations in the suprabasal keratin pattern may contribute to the increased suprabasal cell size observed in adult K10-/- mice. BrdU-labelling revealed that the increase in Ki-67-positive basal and suprabasal cells in K10-/- epidermis resulted from a decreased transition time of proliferating basal keratinocytes through the epidermal layers (Fig. 4). When skin samples were taken 2 hours after BrdU injection we found incorporation only in basal cells in both the wild-type and the knockout tissue, although the number of cells was markedly increased in the latter (Fig. 4A,B). Twenty-four hours after BrdU injection many basal cells in the wild-type had finished one round of division and entered the next cycle, which is shown by the frequent labelling of two adjacent cells (Fig. 4C). In contrast, in K10-/- mice, proliferating cells have cycled more than once and a large number of suprabasal cells were labelled (Fig. 4D), similar to the observed Ki-67 staining pattern (Fig. 1K). Most basal cells lost the BrdU label 24 hours after the injection, which underlines the increased transition time of proliferating cells in K10-/- mice (Fig. 4D). These results established that the loss of K10 stimulated primarily basal cells to proliferate.
|
c-Myc and 14-3-3 induction in K10-/- epidermis
To investigate the molecular mechanism underlying the increase in basal
cell proliferation, we investigated changes in a number of
proliferation-associated proteins.
c-Myc and one of its targets, cyclin D1, are cell cycle-associated proteins
that in epidermis are normally expressed in a subset of basal cells
(Hurlin et al., 1995).
Targeted transgenic overexpression of each of the proteins resulted in a
strong increase in cell growth and epidermal proliferation
(Arnold and Watt, 2001
;
Waikel et al., 2001
;
Robles et al., 1996
;
Rodriguez-Puebla et al.,
1999
). We show here that both proteins were upregulated in the
epidermis of K10-/- mice, where they were detected in almost all
basal cells and in some suprabasal cells
(Fig. 5A-D). Given that c-Myc
and cyclin D1 have been identified as targets of the Wnt signalling pathway
(He et al., 1998
;
Shtutman et al., 1999
), we
monitored Wnt expression by RNA analysis. Using probes for Wnt-3, Wnt-4 and
Wnt-5a, northern blotting did not provide evidence for an activation of Wnt
signalling (Fig. 5E). In
agreement with the drastic phenotype resulting from transgenic overexpression
of ß-catenin in mouse epidermis (Gat
et al., 1998
), we conclude that the increase in c-Myc and cyclin
D1 was not mediated by Wnt signalling. In line with that notion, the
distribution of ß-catenin in the epidermis [i.e. at sites of cell to cell
contact (Fig. 5E, wild-type; F,
K10-/-)] as well as its overall level as judged by western blotting
(Fig. 7D) were unchanged.
|
Previous work established that 14-3-3 is involved in cell cycle
regulation (Hermeking et al.,
1997
; Chan, T. A. et al., 1999) and in the control of keratinocyte
proliferation and differentiation
(Dellambra et al., 2000
). By
immunohistochemistry, we found a prominent staining of 14-3-3
in the
cytoplasm of suprabasal cells of K10-/- mice compared with that of
wild-type epidermis (Fig.
6A,B). The increase in protein
(Fig. 6C) was accompanied by an
equally strong increase in the mRNA as revealed by northern blotting
(Fig. 6D). In basal
keratinocytes, 14-3-3
was unaltered in comparison with wild-type mice,
and appeared very weakly expressed in K10-/- mice
(Fig. 6A,B).
|
Additional proteins have been linked to epidermal proliferation and
differentiation. Here we analysed the expression and proliferation status of
some candidate proteins by western blotting. Rb, one of the key regulators of
the cell cycle, is controlled by phosphorylation
(Kitagawa et al., 1996;
Lundberg and Weinberg, 1998
).
Therefore, we included a phosphorylation-specific antibody against Rb in our
studies. The experiment revealed that neither the overall amount of Rb
(Fig. 7E) nor of Rb
phosphorylated at serine 780 (Fig.
7F), was changed in total protein extracts from skin. Whether the
subfraction of cells expressing c-Myc was also positive for phospho-Rb could
not be resolved, as the antibody available was not suitable for
immunocytochemistry. Akt kinase is a key component of the PI 3-K/Akt kinase
cascade. One of its major tasks is to promote cell survival via
phosphorylation of various target proteins
(Datta et al., 1999
). In this
function, Akt might be linked to the hyperproliferation observed in
K10-/- epidermis. The kinase is activated by phosphorylation on two
domains, both of which are essential for maximal activation
(Alessi et al., 1996
). While
multiple stimuli are known to trigger Akt phosphorylation
(Vanhaesebroeck and Alessi,
2000
), Dimmeler and co-workers reported an activation of Akt
kinase in response to shear stress in endothelial cells
(Dimmeler et al., 1998
).
Therefore, we presumed that the replacement of K1/K10 by K1/K5/14 in the
K10-/- mice may render suprabasal keratinocytes more susceptible to
mechanical strain, which could lead to an activation of Akt. Additionally, in
cell culture transfection experiments it has been shown that unphosphorylated
Akt colocalized with K10-containing IF
(Paramio et al., 2001a
). The
authors concluded from their findings that Akt was sequestered by K10, which
impaired its translocation to the cell membrane, where it is normally
activated through phosphorylation, thereby causing cell cycle arrest. To
identify activated Akt kinase, antibodies specific for each of the essential
phosphorylated residues were used in addition to an antibody that recognizes
total Akt protein. We found the amount of unphosphorylated Akt kinase
unaltered (Fig. 7G, lanes 8,9,
upper band), whereas phosphorylated Akt at threonine 308
(Fig. 7G, lanes 10,11) and
serine 473 (Fig. 7G, lanes
12,13) was strongly reduced. This makes it unlikely that Akt kinase becomes
activated following the loss of K10. The lower band in
Fig. 7G, lane 8 represents
phosphorylated Akt, as the antibody we used here detects total Akt
(phosphorylation-state independent). As phosphorylated Akt was reduced in
K10-/- mice the lower band was strongly reduced in the knockout
(Fig. 7G, lanes 8,9). Finally,
the amounts of the tumor suppressor p53
(Fig. 7A), and of the cell
cycle inhibitors p21 (Fig. 7B)
and p27 (Fig. 7D) remained
unaltered. At present, the significance of p53 post-translational
modifications on its activation and on its subcellular localization in vivo is
not yet fully determined (Meek,
1999
; Liang and Clarke,
2001
) and has not yet been examined in our mice.
Another cell cycle regulator that has been shown to be involved in mouse
epidermal differentiation and proliferation is p16Arf4a
(Paramio et al., 2001b). This
protein inhibits Cdk4 and Cdk6, thereby maintaining Rb in its active form,
arresting cells in G1 phase. The K10-/- population as well as the
wildtype animals presented here were on a mixed BALB/cx129/Ola mouse
strain background. Based on an in vitro kinase assay, Zhang et al. concluded
that BALB/c allelic variants in exons 1 and 2 of p16Ink4a are
inefficient inhibitors of Rb phosphorylation
(Zhang et al., 1998
). Using
PCR and subsequent specific restriction of the PCR products as previously
described (Zhang et al.,
1998
), we found that K10-/- mice as well as the
corresponding wild-type mice carry both allelic variants of exon 1
(Fig. 8A) and both allelic
variants described for exon 2 (Fig.
8B). The fact that the p16Ink4a variants represent
polymorphisms, as indicated by the presence of these variants in wild-derived
strains of mice (Zhang et al.,
1998
), makes it unlikely that in vivo these alleles lead to the
formation of an inefficient form of p16Ink4a. In addition, the
knockout of p16 did not show an epidermal phenotype
(Krimpenfort et al., 2001
;
Sharpless et al., 2001
).
Moreover, our results demonstrated that the mice we used for this study
carried both allelic variants and therefore could express p16Ink4a
protein, which may efficiently inhibit Rb phosphorylation.
|
Collectively, the analysis of adult K10-/- mice suggested that K10 is indirectly involved in the regulation of cell proliferation in mice, possibly via c-Myc (Fig. 9).
|
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Discussion |
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Is K10 excluded from proliferating cells?
Among keratins, K10 appears to be unique because its expression is
restricted to postmitotic cells (Moll et
al., 1982), and because it can form highly bundled IF typical of
the upper epidermis. Given their abundance and the fact that their head and
tail include glycine-rich domains, K10 and K1 have underscored the view that
keratins represent the prototype cytoskeletal protein. The downregulation of
K10 upon wound healing, where it becomes replaced by K6 and K16 in activated
keratinocytes, has been taken as further evidence that the presence of K10 is
not compatible with the state of a migratory or proliferating cell
(McGowan and Coulombe, 1998
).
In line with this notion, a considerable number of publications has
demonstrated the absence of K10 from carcinomas in mice
(Roop et al., 1988
;
Toftgard et al., 1985
;
Winter et al., 1980
;
Nelson and Slaga, 1982
) and
humans (Winter et al., 1983
).
Although K10 has been found in rare cells throughout tumors
(Moll et al., 1983
;
Moll, 1998
), it was not
resolved whether these were actively proliferating or rather resting cells.
These in vivo situations, in which K10 is restricted to postmitotic
keratinocytes and is absent in a number of malignant cancers, have prompted
studies that intended to show an antiproliferative potential of K10
(Santos et al., 1997
;
Paramio et al., 1999
).
However, the expression of K10 under control of a K6 promoter, which is
activated in hyperproliferative cells
(Takahashi and Coulombe,
1997
), could neither inhibit tumor formation nor influence the
degree of malignancy in animals subjected to a skin carcinogenesis protocol
but merely delayed tumor formation (Santos
et al., 1997
). Moreover, the forced expression of K10 in
ß-cells of the pancreas, using an insulin promoter, was compatible with
the development of a normal organ (Blessing
et al., 1993
).
In another attempt to study the correlation between K10 and cell
proliferation, it was transfected into the human keratinocyte cell line HaCaT,
where it was found to inhibit cell proliferation. This block was shown to
depend on the inactivation of Rb and seemed to require the non-helical end
domains of K10 (Paramio et al.,
1999). In this setting, the cell cycle block imposed by K10 could
be released by K16. Most recently it has been shown by the same authors, that
K10 can directly interact via its non-helical N-terminal domain with Akt
kinase and PKC
, thereby impairing their translocation and subsequent
activation (Paramio et al.,
2001a
). Furthermore, they showed that this inhibition impeded Rb
phosphorylation and reduced the expression of cyclins D1 and E
(Paramio et al., 2001a
).
While these findings suggested a direct and negative involvement of K10 in
the regulation of human keratinocyte proliferation, the analysis of adult
K10-deficient mice allows another conclusion. Most importantly, the in vivo
ablation of K10 in suprabasal keratinocytes in mice did not induce
proliferation of the same cells but rather acted on neighbouring cells
residing in the basal layer. Therefore, the hyperproliferation is most likely
an indirect consequence following the absence of K10. With respect to the role
of Rb suggested by cell transfection studies
(Paramio et al., 1999), we
cannot completely rule out its contribution in vivo, given that we adressed
its phosphorylation state in total skin extracts. To resolve this issue, a
phospho-Rb antibody suitable for immunocytochemistry would be useful. Given
the extent of proliferative changes in K10-/- epidermis, we would
argue, however, that Rb does not appear to be a major candidate in mice. It is
also possible that human and mouse cells use different mechanisms involving
different pathways to regulate proliferation.
The changes in K10-/- epidermis are compatible with the
increase in c-Myc
c-Myc expression is normally very low in the epidermis
(Hurlin et al., 1995). We show
here that the loss of K10 led to an increase in c-Myc predominantly in the
basal epidermal layer. Collectively, the histological changes in the epidermis
of adult K10-/- mice, namely hyperproliferation and an increase in
cell size, were strikingly similar to those exhibited by transgenic mice
overexpressing c-Myc in basal (Arnold and
Watt, 2001
; Waikel et al.,
2001
) or in suprabasal keratinocytes
(Pelengaris et al., 1999
;
Waikel et al., 1999
).
Remarkably, all of these transgenic lines shared an impairment of the ordered
arrangement of keratinocytes, while other aspects of terminal differentiation
proceeded unimpaired.
In addition to the increase in basal cell proliferation, K10-/-
mice displayed an increase in cell size and in the size of keratohyalin
granules. This was similar to transgenic mice generated by Pelengaris et al.,
which also displayed a distortion of the normal epidermal architecture, an
increase in granular cell size accompanied by the failure of these cells to
flatten, and an increase in keratohyalin
(Pelengaris et al., 1999). The
change of cell size is compatible with the induction of c-Myc which, in
addition to its established involvement in cell-cycle progression and
oncogenesis (Pelengaris et al.,
2000
), is known to regulate cell growth
(Elend and Eilers, 1999
;
Johnston et al., 1999
). Given
that basal cells show a different morphology than suprabasal cells, it is also
possible that the replacement of the normal suprabasal K1/K10 cytoskeleton by
one that contains the basal keratins K5/K14 in addition to K1 may influence
suprabasal cell size and morphology in K10-/- epidermis.
K10-/- mice differ from c-Myc transgenic mice
(Arnold and Watt, 2001;
Waikel et al., 2001
) in
various aspects. Our mice did neither display ulcerated lesions nor an
impairment of wound healing (Reichelt and Magin, unpublished). This is best
explained by the higher level of c-Myc in transgenic mouse lines where it is
present in all basal cells including stem cells. As previously argued
(Waikel et al., 2001
;
Arnold and Watt, 2001
), the
expression of c-Myc resulted in a depletion of the stem cell compartment and
stimulated the proliferation of transit amplifying cells. In due course, this
could lead to the alterations seen in c-Myc transgenic mice. In contrast to
the situation in c-Myc transgenic mice, c-Myc need not necessarily be induced
in epidermal stem cells of K10-/- mice. Indeed, not all basal cells
were labelled in the latter (data not shown); this would explain the
morphological differences.
The induction of cyclin D1 in K10-/- mice may be caused by c-Myc
(Daksis et al., 1994).
Although cyclin D1 expression is present in multiple malign tumors, transgenic
expression of cyclin D1 in the basal layer of the epidermis did not result in
tumor formation but only in hyperproliferation
(Robles et al., 1996
), which
is in line with our observations that K10-/- mice do not develop
spontaneous skin tumors. Skin carcinogenesis experiments on K10-/-
mice will reveal whether the susceptibility of these mice to skin tumors is
increased. Given that both c-Myc and cyclin D1 are among the few known target
genes of the Wnt/ß-catenin-signalling pathway
(He et al., 1998
;
Shtutman et al., 1999
), we
investigated the activity of different Wnt genes known to be active in skin
(Millar et al., 1999
;
Saitoh et al., 1998
). In
agreement with the normal pattern of epidermal differentiation, no alterations
in the transcription of Wnt genes were detectable by northern blotting.
Additionally, we did not find alterations in ß-catenin cellular
distribution or protein levels. Consistent with this, hair follicle
morphogenesis seemed completely normal in our mice (data not shown). It has
been described recently that Wnt-signalling has a major impact on hair
follicle development (Gat et al.,
1998
) and that the stabilization of ß-catenin may lead to de
novo hair follicle morphogenesis and malignant transformation
(Chan et al., 1999a
). The
turnover of cyclin D1 can be upregulated by phosphorylation through
GSK-3ß, which in turn is negatively regulated by activated Akt kinase
(Diehl et al., 1998
). Although
we found a decrease in phosphorylated Akt in skin extracts, we observed an
increase in cyclin D1-positive nuclei in the basal cell layer and in some
suprabasal cells in K10-/- epidermis. An explanation for this may
be that the increase in cyclin D1 is restricted to a minor number of
keratinocytes, while the decrease in activated Akt may concern the major cell
population. Moreover, the relative contribution of c-Myc and Akt to cyclin D1
regulation has not been examined in an in vivo setting.
The increase in 14-3-3 may explain the lack of malignant
changes in K10-/- epidermis
Despite a strong increase in basal cell proliferation, combined with the
disturbance of epidermal proliferative units, we have failed to observe any
signs of malignant transformation in adult K10-/- epidermis, even
in mice older than 18 months. 14-3-3 is a p53-inducible gene which is
involved in G2 arrest (Fu et al.,
2000
). In tumors including breast cancer, downregulation of
14-3-3
has been reported to involve methylation of CpG islands and may
represent an epigenetic event critical for the accumulation of mutations
(Ferguson et al., 2000
).
Interestingly, the experimental treatment of breast cancer cell lines with
5-aza-2'-deoxycytidine, which demethylates DNA, leads to the
transcriptional activation of the 14-3-3
gene
(Ferguson et al., 2000
).
Remarkably, we detected a significant increase of 14-3-3
mRNA and
protein in the epidermis of adult K10-/- mice. While at present we
have no data to support whether the upregulation of 14-3-3
involves
demethylation of its gene, our histochemical localization of 14-3-3
in
suprabasal, postmitotic epidermal keratinocytes is consistent with the absence
of malignant changes.
This localization is also in agreement with a recent report suggesting that
14-3-3 is a key regulator of keratinocyte stem cells
(Dellambra et al., 2000
).
Based on gene inactivation studies in a human colon carcinoma cell line it
became evident that 14-3-3
is required to sequester cdc2-cyclin B1
complexes in the cytoplasm to initiate a G2/M block
(Hermeking et al., 1997
;
Chan et al., 1999b
). Such a
feature is consistent with the increased size of suprabasal keratinocytes that
we observed in K10-/- mice, which might point to an arrest at this
cell cycle stage. Moreover, c-Myc, when transfected into cultured
keratinocytes, can also cause a G2/M block and initiate cell growth
(Gandarillas et al.,
2000
).
In addition to their obvious role in cell cycle regulation (for a review,
see Fu et al., 2000), 14-3-3
proteins can associate with K8/K18 tetramers, mainly through binding of
phosphorylated K18 during the S/G2/M phase of the cell cycle. By keratin
sequestration, they may be albe to influence keratin distribution and
intermediate filament organization (Liao
and Omary, 1996
; Ku et al.,
1998
; Toivola et al.,
2001
). Whether 14-3-3
can interact with epidermal keratins
in an analogous way and whether such an interaction is of significance in the
epidermis has yet to be explored.
In our work, we have demonstrated for the first time that the deletion of a
keratin in one cell type leads to the stimulation of cell proliferation in
neighbouring cells, involving an increase in c-Myc. The stimulation of basal
cell proliferation upon the loss of K10 must involve transmission of a signal
from the suprabasal to the basal compartment of the epidermis. This could
involve paracrine-acting cytokines such as KGF, GM-CSF, IL-1 and/or members of
the TGF-ß superfamily (for a review, see
Werner and Smola, 2001).
Alternatively, the altered composition of the suprabasal IF cytoskeleton in
K10-/- epidermis could render suprabasal cells more susceptible to
mechanical stress, providing an appropriate signal
(Takei et al., 1997
;
Kippenberger et al., 2000
).
With respect to the large number of keratins, it will be interesting to see
whether K10 is unique.
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Acknowledgments |
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References |
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