From the Department of Cell and Molecular Biology,
CIEMAT, Ave. Complutense 22, Madrid E-28040, Spain and the
¶ Instituto de Biomedicina de Valencia, Jaume Roig 11, Valencia
46010, Spain
Received for publication, August 9, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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Both the diversity and the precisely regulated
tissue- and differentiation-specific expression patterns of keratins
suggest that these proteins have specific functions in epithelia
besides their well known maintenance of cell integrity. In the search for these specific functions, our previous results have demonstrated that the expression of K10, a keratin expressed in postmitotic suprabasal cells of the epidermis, prevents cell proliferation through
the inhibition of Akt kinase activity. Given the roles of Akt in
NF- The keratins are the main components of the intermediate filament
cytoskeleton in epithelial cells. The functions of these proteins were
clarified when human epithelial fragility syndromes were attributed to
mutations of epidermal keratin genes (for reviews see Refs. 1-4).
However, this shared function does not clearly explain the great
diversity of these proteins, which suggests they may have additional
family member-specific functions.
In the search for possible specific keratin functions, a study was made
of keratin K10. This protein is expressed in postmitotic differentiating keratinocytes in epidermis in vivo (5), and its expression is severely reduced in hyperproliferative situations, including skin tumors (6, 7). It has been shown that the expression of
this keratin inhibits cell proliferation in cultured cells and in
transgenic mice (8-10). The modulation of cell growth by keratin K10
is linked to the retinoblastoma (pRB) protein and the molecular
machinery controlling cell cycle progression during G1, in particular cyclin D1 expression (8, 9). This
activity appears to be promoted by the sequestration of Akt to the
keratin cytoskeleton, mediated by keratin K10 through its amino
terminus. This leads to decreased Akt kinase activity (9). More
recently these results have been amplified to include in
vivo situations (10). Transgenic mice were generated in which
human keratin K10 gene expression was targeted to the basal layer of
the epidermis by using the bovine keratin K5 promoter (11). These
animals displayed severe alterations in the epidermis, including
decreased proliferation, which results in hypoplastic epidermis,
associated with impaired activation of Akt kinase activity in the skin.
Finally, by using chemical skin carcinogenesis protocols, it was
demonstrated that K10 expression reduces the formation of tumors
in vivo (10). These results are in agreement with the
recently described fundamental roles of Akt kinase in mouse skin
carcinogenesis (12).
Akt is a serine/threonine kinase that plays an important role not only
in tumorigenesis but also in many aspects of cell physiology (reviewed
in Refs. 13-16). This kinase phosphorylates many cellular substrates
involved in the control of cell proliferation, apoptosis, and
metabolism. Its role in activating Akt in NF- The canonical activation of NF- The importance of NF- Another line of information on the roles of NF- Given the above importance of Akt modulation of NF- Transgene Construction and Generation of Transgenic
Mice--
The plasmid bK5hK10 was used to generate transgenic mice in
a (C57 BL/10 × BALB/c) F1 and (C57BL/10 × DBA/2) F1 genetic background as previously described (10).
The presence of the transgene was analyzed by Southern blots.
Homozygous and heterozygous mice were identified using a PhosphorImager
scanner (Bio-Rad) as reported (10). Primary keratinocyte cultures were
established isolating keratinocytes from newborn mice cultured in
Eagle's minimal medium containing 8% Chelex-treated serum and 0.03 mM Ca2+ as previously described (43).
Histological Analysis--
Dorsal skin samples and tumors were
fixed either in formalin or ethanol and embedded in paraffin prior to
sectioning. 4-µm sections were cut and stained with
hematoxylin-eosin. Immunodetection of dermal inflammatory cells
including T lymphocytes (anti-CD3e), granulocytes (anti-Ly-6G/Gr-1),
and macrophages (anti-CD11b/Mac1) was performed in frozen sections
using fluorescein isothiocyanate-labeled specific rat anti-mouse
monoclonal antibodies (145-2C11, RB6-8C5, and M1/70 clones,
respectively; Amersham Biosciences). Sections were also stained for
the expression of K10 as previously described (10). The localization of
TNF Plasmids and Transfection--
The 8x Retroviral Constructs and Infection--
Retrovirus coding for
wt Akt or dominant negative Akt were generated by subcloning the
corresponding cDNAs into pStMCS vector (kindly provided by Dr.
J. C. Segovia, CIEMAT). These plasmids were transfected
into Nxe cells and the supernatants were collected 48 h after
transfection. For infections, exponentially growing PB keratinocytes
were cultured in the presence of retroviral supernatants for 3 h,
and afterward fresh medium was added and the cultures were further
incubated overnight. Protein extracts were collected after 48 h
and used in Western blotting analyses as commented below.
Antibodies and Reagents--
The antibodies used included rabbit
polyclonal antibodies to p50 (sc-114), p65/RelA (sc-372), I Western Blots--
Protein extracts (20 µg) were boiled in
Laemmli buffer and separated on 10% SDS-PAGE and transferred to
nitrocellulose filters (Hybond ECL, Amersham Biosciences, Aylesbury,
UK). Filters were blocked with 5% nonfat dry milk in PBS/0.1% Tween
20 at 4 °C overnight, washed three times in PBS/0.1% Tween 20, and
incubated with the indicated antibodies. After washing, membranes were
incubated with a peroxidase-conjugated secondary antibody, washed
again, and analyzed using the enhanced chemiluminescence method (West Picosignal, Pierce), according to the manufacturer's instructions. Membranes were stripped by incubation with 62.5 mM Tris-HCl
(pH 6.7)/2% SDS/100 mM Band Shift Analysis--
Electrophoretic mobility shift assays
(EMSA) were performed by incubating whole cell extracts from mouse skin
with a labeled oligonucleotide corresponding to a palindromic
Complexes were separated on 5.5% native polyacrylamide gels in 0.25×
Tris borate-EDTA buffer, dried, and exposed to Hyperfilm-MP (Amersham
Biosciences) at In Vitro Kinase Assays--
Whole skin extracts from newborn
mice were obtained in buffer A (1% Triton X-100, 10% glycerol, 137 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 µg/ml
aprotinin and leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM disodium
pyrophosphate, and 1 mM Na3VO4). The kinase activity for Akt and IKK complexes was determined by immunoprecipitation of the endogenous kinase proteins using anti Akt1/2
antibody or a mixture of different IKK Northern Blots--
Total RNA from freshly harvested mouse
epidermis and frozen tumors was isolated by guanidine
isothiocyanate-phenol-chloroform extraction. Northern blots containing
total RNA (15 µg/lane) were probed for expression of TNF TNF Determination--
The quantification of TNF Decreased NF-
Experiments were then performed to see whether this decreased NF- Ectopic Expression of K10 Leads to Decreased IKK
The decreased protein levels of the IKK components and the concomitant
inhibition of IKK activity are very striking. It was therefore, through
experiments similar to those shown in Fig. 1 (C and
C'), determined whether expression vectors for IKK Inhibition of Akt Leads to Decreased IKK Phenotypic Alterations in Epidermis of Transgenic Mice Are
Different to Those of IKK
To further confirm that keratinocytes are the source of TNF
As a second approach, the possible presence of inflammatory cells in
the dermis of transgenic and non-transgenic skin samples was monitored
using antibodies against T lymphocytes (CD-3), granulocytes (GR-1), and
macrophages (Mac-1) in frozen skin samples. No increase in any of these
cell populations was observed in the transgenic samples (Fig.
7, compare A, B,
and C with A', B', and C',
respectively). In fact, the dermis of transgenic mice displayed lower
number of these cell types as compared with non-transgenic littermates. Given that the increased production of cytokines in the transgenic skin
(Fig. 6) would allow for the recruitment of inflammatory cells, the
observed decrease would reflect a possible alteration in the production
and/or maturation of these cells in transgenic mice. In agreement, we
have observed defective thymus development in bK5hK10 homozygous
transgenic mice (not
shown).2
Together, these results demonstrate that the expression of K10 in the
basal layer of the epidermis leads to the production of higher levels
of TNF- Activation of the JNK Pathway in Transgenic Mouse
Epidermis--
The pro-inflammatory cytokine TNF-
The binding of TNF- The functional diversity of the keratins is a matter of debate.
This report focuses on the possible functions of keratin K10, a protein
expressed in non-proliferative suprabasal skin keratinocytes, which in
addition is down-regulated during hyperproliferative situations. Using
cultured cells and transgenic mouse models we have previously
demonstrated that the expression of keratin K10 inhibits cell cycle
progression through its ability to bind and inhibit the activation of
Akt and PKC Decreased NF- The present data clearly indicate that decreased NF- The phenotype of the high expressing bK5hK10 transgenic mice was,
however, clearly different to that reported for IKK The increased production of TNF This report provides evidence that the specific expression of keratins
in epithelia may affect the transcriptional program executed by these
cells, probably through the modulation of signaling molecules. This may
lead to abnormal production of cytokines, which results, not only in a
cell autonomous effect but in a complete disturbance of tissue
homeostasis. In this regard, it is worth mentioning that the recently
described effect of K10 loss in adult mice results in
hyperproliferation in basal layer of epidermis (64). This observation
points to a paracrine effect of keratin expression. Whether this might
be attributed to altered expression of cytokines as those described
here is an attractive possibility that merits future investigation.
B signaling and the importance of these processes in the
epidermis, a study was made into the possible alterations of the
NF-
B pathway in transgenic mice expressing K10 in the proliferative
basal layer. It was found that the inhibition of Akt, mediated by K10
expression, leads to impaired NF-
B activity. This appears to occur
through the decreased expression of IKK
and IKK
. Remarkably,
increased production of tumor necrosis factor
and concomitant JNK
activation was observed in the epidermis of these transgenic mice.
These results confirm that keratin K10 functions in
vivo include the control of many aspects of epithelial physiology, which affect the cells not only in a cell autonomous manner
but also influence tissue homeostasis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B signaling has
recently been demonstrated, which may be an essential antiapoptotic event. However, the mechanism responsible for the activation of NF-
B
by Akt remains uncertain and may depend on the cell type analyzed
(17-19).
B requires the phosphorylation of the
inhibitory I
B protein by a high molecular weight complex that
includes IKK
, IKK
, and
IKK
1 proteins. This allows
the NF-
B to move to the nucleus and activate target genes. In this
context, it has been shown that Akt may increase the transactivation of
the p65 subunit of NF-
B, without interfering with its nuclear
localization (20-22). Similarly, the increased transactivation of p50
subunit by Akt phosphorylation has been reported (23). Furthermore, the
phosphorylation of IKK proteins by Akt, leading to increased
phosphorylation and thus degradation of I
B proteins, has been widely
reported (24-27). Finally, in breast cancer lines, it has been
suggested that Akt mediates the calpain degradation of I
B protein
instead of the consensus ubiquitin pathway (28). Collectively, all
these studies demonstrate the importance of Akt in activating NF-
B
signaling, but also emphasize the controversy in identifying the
molecular mechanisms responsible for such activation.
B signaling in epidermis has been highlighted
in recent years (reviewed in Ref. 29). This stems from findings
obtained in transgenic mice ectopically expressing a non-degradable
I
B
protein (I
B
M), or which overexpress p50 and/or p65
subunits in the epidermis (30). In these animals, increased NF
B
activity leads to epidermal hypoplasia and growth inhibition, probably
in association with p21waf/cip1 expression (30, 31). The
repression of NF-
B activity, on the other hand, produces epidermal
hyperplasia (30) and may cause spontaneous squamous cell carcinomas in
transgenic mice (32). Nevertheless, we have observed increased
endogenous NF-
B activity during chemically induced mouse skin
tumorigenesis or upon UV treatment of mouse skin (33, 34). This
apparent discrepancy might be explained in that the overexpression of
regulatory molecules used in earlier transgenic studies may differ from
those involved in endogenous activity. In this regard, one has to take
into account that dominant negative I
B
expression does not
generate a true NF-
B null phenotype and that nuclear accumulation of
Bcl-3 and its interaction with other NF-
B dimers is independent of
inhibition by other I
B proteins and IKK stimulation. In agreement
with this, neither tumors nor phenotypic alterations were observed in
the absence of I
BM overexpression in epidermis (32). This suggests that a threshold in the level of expression of this protein is required
to produce the phenotype. More recently, using a knock in
approach, which does not produce massive overexpression, it has been
shown that the repression of NF-
B promotes severe epidermal defects
affecting hair follicle development and increased apoptosis but not
increased proliferation or abnormalities in differentiation (35).
Finally, the epidermal-specific ablation of IKK
, which is
accompanied by a deficiency in NF-
B activation, results in a
TNF-mediated inflammatory skin disease but does not led to
hyperproliferation or impaired keratinocyte differentiation (36).
B signaling in
keratinocytes comes from the strong phenotypes observed in the
epidermis of mice lacking the IKK
or IKK
subunits of the IKK
complex (37-41). In the case of IKK
, the abnormalities are not only
due to the possible alterations of NF-
B signaling but also to the
decreased production of a yet-unidentified soluble factor capable of
inducing keratinocyte differentiation (42).
B activity in a
range of systems, and the importance of NF-
B in epidermal physiology, an investigation was made into the possible alterations in
this pathway that might arise as a consequence of keratin K10 ectopic
expression in the basal layer of the epidermis. A dramatic decrease was
seen in NF-
B activity in both transgenic skin and cultured mouse
keratinocytes. This decrease is attributed to the inhibition of IKK
activity associated with decreased expression of IKK
and IKK
.
Remarkably, the transgenic animals showed aberrant overproduction of
TNF
, and concomitant increased JNK activity, which may account for
some of the phenotypic characteristics observed in bK5hK10 transgenic mice.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and IL-6 was carried out in formalin-fixed, paraffin-embedded
sections from transgenic and non-transgenic littermates using specific
rabbit polyclonal antibodies purchased from Calbiochem and diluted
1/100, followed by horseradish-peroxidase-labeled anti-rabbit antibody
(Jackson ImmunoResearch Laboratory, diluted 1:2000). Positive staining
was determined using diaminobenzidine as a substrate (diaminobenzidine
kit, Vector, Burlingame, CA) following the manufacturer's
recommendations. Sections were then counterstained with hematoxylin and
mounted. For ultrastructural analysis, skin samples were fixed in 2.5%
glutaraldehyde in PBS and postfixed in 1% osmium tetroxide prior to
dehydration and embedding in Epon 812 resin. Ultrathin sections were
stained with uranyl acetate and lead citrate.
B-Tk-CAT plasmid
reporter gene used for transfection assays contained eight copies of
the Ig
enhancer NF-
B binding site. The expression vectors for
IKK
, IKK
, and IKK
containing the open reading frame of the
indicated genes, and cloned in pCDNA3 under the cytomegalovirus
promoter/enhancer were a generous gift of Dr. A. Israël. The
plasmid coding for hK10 has been described previously (8, 9). Plasmids
coding for wt and dominant-negative Akt were a generous gift of Dr.
J. S. Gutkind. Chloramphenicol acetyltransferase assays were
performed using an ELISA kit (Roche Molecular Biochemicals) following
the manufacturer's instructions.
B
(sc-371), IKK
(sc-7182), IKK
(sc-8032) (Santa Cruz Biotechnology,
Santa Cruz, CA), and IKK
(Imgenex IMG-19). Antibodies against JNK,
RIP, TRAF2, and TRADD were obtained from Transduction Laboratories.
Antibodies against phosphorylated ATF2 and JNK were purchased from Cell
Signaling. An anti-rabbit immunoglobulin G and anti-mouse
peroxidase-conjugated (Amersham Biosciences, Aylesbury, UK) were used
for immunoblotting. IL-1
, was purchased from Endogen and IL-1
and
TNF
from Sigma. All cytokines were used at 10 ng/ml.
-mercaptoethanol at 55 °C for
30 min and reprobed with different antibodies. No cross-reactivity
with other NF-
B/I
B/IKK proteins was detected.
B site
as previously described (44). The sequence of the
B
oligonucleotide coding strand was:
5'-GATCCAACGGCAGGGGAATTCCCCTCTCCTTA-3' (44).
70 °C. The composition of the
B complexes in
newborn mouse skin has been previously described (34).
antibodies (Santa Cruz
Biotechnology). Histone H2B was used as a substrate for Akt, and
full-length I
B
(Santa Cruz Biotechnology) for IKK in in vitro kinase assays (essentially as described in Refs. 12 and 45).
Jun kinase assay was performed essentially as described (46) upon
immunoprecipitation of JNK using GST-c-Jun (kindly provided by Dr.
J. S. Gutkind) as substrate.
, IL-6,
and IL-1 employing DNA probes prepared by random primed reactions using
the complete sequences. The membranes were also hybridized with a
keratin K14 cDNA probe to verify that equal amounts of mRNA
were loaded and transferred.
in mouse
serum and in the culture supernatant from primary keratinocytes was
performed using an ELISA kit (R&D) following the manufacturer's recommendations.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B Activity in bK5hK10 Transgenic
Mice--
Transgenic mice ectopically expressing K10 in the basal
layer of the epidermis display severe epidermal abnormalities
associated with decreased proliferation in close conjunction with Akt
kinase activity inhibition (10). Given the importance of Akt in the activation of NF-
B (see the introduction), the present study was
designed to investigate NF-
B binding activity in the epidermis of
transgenic mice through electrophoretic mobility shift analysis using a
B-labeled oligonucleotide. In non-transgenic samples, two retarded
complexes were observed (Fig.
1A, lanes 1 and
2) that were identified as p50-p50 homodimers and p50-p65
heterodimers by supershift experiments using specific antibodies (not
shown; see also Ref. 34). Severely decreased DNA binding activity was observed in heterozygous transgenic mice (Fig. 1A,
lanes 3 and 4). The
B binding activity in
homozygous animals was barely detectable (Fig. 1A,
lanes 5 and 6). Interestingly, the inhibition of
Akt kinase activity runs in parallel with the increased K10 expression observed in heterozygous and homozygous transgenic mice (Fig. 1A') (not shown, see also Ref. 10). Given that we used whole skin extracts for these experiments, and to rule out the possibility that other cell types were actually responsible for the observed effect, similar analysis were performed using primary keratinocytes derived from wt and bK5hK10 transgenic mice. In addition, we also tested whether stimulation with IL-1
could induce increased DNA binding in these cells. IL-1
treatment led to increased NF-
B activity in non-transgenic keratinocytes but not in keratinocytes derived from transgenic animals (Fig. 1B). Western blot
analysis against keratin K14 (Fig. 1B') also demonstrated
that this effect was not due to different amounts of keratinocyte
protein in the assays. These data show that the expression of K10 leads
to a dramatic reduction in NF-
B activity in keratinocytes and
impedes the activation of this complex on stimulation.
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Fig. 1.
Expression of human keratin K10 in the
epidermis of transgenic mice results in impaired
NF- B signaling. A, whole cell
extracts from non-transgenic, heterozygous, and homozygous bK5hK10
transgenic mice were analyzed by EMSA using a labeled
B
oligonucleotide. The composition of the complexes is indicated on the
right. A', in vitro Akt kinase
activity of the same protein extracts. B, cultured primary
keratinocytes obtained from non-transgenic and homozygous transgenic
mice were analyzed by EMSA as in A. Where indicated, the
keratinocytes were also stimulated by incubation (10 min) in the
presence of IL-1
(10 ng/ml). Note that in transgenic epidermis and
primary keratinocytes there is a dramatic inhibition of NF-
B binding
activity. No stimulation was observed. B', the same protein
extracts shown in B were analyzed by Western blot against
K14 to rule out the possibility that the observed differences were due
to different protein concentrations. C and C',
the K10-mediated inhibition of Akt is responsible for impaired NF-
B
signaling. PB keratinocytes were transfected with the quoted plasmids.
48 h after transfection cells were stimulated as indicated with
TNF
or IL-1
(both 10 ng/ml for 10 min). Chloramphenicol
acetyltransferase assays were performed using ELISA (Roche Molecular
Biochemicals), and the activities were normalized to
-galactosidase
activity. Data were taken from duplicate experiments and are shown as
mean ± S.D.
B
activity was due to K10 expression and K10-mediated Akt inhibition. PB
mouse keratinocytes were transfected with a NF
B reporter element
(8x
BCAT) plus empty vector, K10, or K10 plus an expression vector
for wt Akt (Fig. 1C). It has previously been shown that
co-expression of wt Akt is sufficient to override K10-induced cell
cycle arrest in keratinocytes (10). The expression of K10 produced a
severe inhibition of NF-
B transcriptional activity induced either by
IL-1
or TNF
(Fig. 1, C and C',
respectively), whereas co-expression of Akt almost completely abolished
such inhibition (Fig. 1, C and C'). Finally, to
determine whether the inhibition of Akt activity might be sufficient to
account for the decreased NF-
B activity, the reporter plasmid was
co-transfected with a dominant negative form of Akt, resulting in
almost complete inhibition of NF-
B activity in response to TNF
or
IL-1
(Fig. 1, C and C'). Together, these
results demonstrate that K10-mediated Akt inhibition leads to impaired
NF-
B basal activity and NF-
B activation in cultured keratinocytes
and in epidermis in vivo.
and IKK
Expression in Skin--
A study was then undertaken to determine
whether the decreased NF-
B activity found in the transgenic mouse
epidermis was due to altered expression of different NF-
B family
members (Fig. 2A). No major
alterations in p65 (RelA) or I
B
protein levels were detected by
Western blotting (Fig. 2A), and a decrease in only p50 was
observed in whole skin extracts from homozygous transgenic mice (Fig.
2A). Because the NF-
B activity is dependent on the activity of the IKK complex, the expression of IKK
, IKK
, and IKK
was analyzed. Surprisingly, in homozygous transgenic mice there
was a significant decrease in IKK
and IKK
levels (Fig. 2A'). Because these two components of the IKK complex are
essential for I
B phosphorylation and for NF-
B activation (47,
48), I
B kinase activity was evaluated in the transgenic mouse
extracts (45). Almost complete inhibition of the I
B kinase activity was seen in homozygous mouse extracts (Fig. 2A"), and,
surprisingly, a significant inhibition of this activity in extracts
from heterozygous animals despite the fact these show no decrease in
IKK
or IKK
levels. These data clearly suggest that the decrease
in Akt activity elicited by K10 also results in a dramatic inhibition
of I
B kinase activity.
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Fig. 2.
Decreased expression of
IKK and IKK
in the
epidermis of bK5hK10 transgenic mice. A, protein extracts
from non-transgenic, heterozygous, and homozygous bK5hK10 transgenic
mice epidermis were probed by Western blot for the expression of p65,
p50, and I
B
. A', the same protein extracts shown in
A were probed by Western blot for the expression of IKK
,
IKK
, and IKK
. Note than in homozygous mice there is a dramatic
reduction of IKK
and IKK
levels. A", in
vitro kinase assay following immunoprecipitation of IKK
from
the same protein extracts shown in A and A' and
using I
B
as substrate. B and B',
co-expression of IKK
and IKK
restores the NF
B activity.
Chloramphenicol acetyltransferase assays were performed essentially as
in Fig. 1 (C and C') in PB keratinocytes
transfected with the quoted plasmids. Data were taken from duplicate
experiments and are shown as mean ± S.D.
or IKK
were able to rescue the K10-induced inhibition of NF-
B
activity. IKK
co-expression partially restored the NF-
B
inhibition elicited by K10, whereas IKK
co-expression totally
rescued it (Fig. 2, B and B'). These results
strongly indicate that the reduction of IKK
and IKK
in transgenic
epidermis is probably responsible for the K10-induced impaired
activation of NF-
B activity. In addition, the partial rescue
observed with IKK
also indicates that a residual amount of IKK
must be present upon K10 expression, because this protein is essential
for I
B kinase activity (48).
and IKK
Expression--
The above commented results prompted us to analyze if
the observed decrease in Akt activity might be responsible of the
decrease in IKK
and IKK
proteins. To this, PB keratinocytes were
infected with retrovirus coding for dominant negative Akt, wt Akt, or
empty retroviral backbone. Forty-eight hours after infection protein extracts were obtained and used for Western blot determination of
IKK
, IKK
, IKK
, and Akt as well as Akt phosphorylated in Ser-473. The results (Fig. 3)
demonstrate that the inhibition of Akt
leads to a significant decrease in IKK
and IKK
proteins, whereas
IKK
remained unaffected. This indicates that the observed decrease
in these two proteins in skin extracts from bK5hK10 transgenic mice can
be attributed to the inhibition of Akt mediated by the expression of
K10. In addition, these data are of great importance, because, to our
knowledge, this is the first evidence suggesting that Akt activity
might regulate the protein level of these two components of the IKK
complex.
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Fig. 3.
Decreased Akt activity leads to decreased
IKK and IKK
protein
levels. PB keratinocytes were infected with retrovirus coding for
wt or dominant negative Akt or empty backbone (mock).
Protein extracts were obtained and used to analyze the expression of
Akt, Akt phosphorylated in Ser-473, IKK
, IKK
, and IKK
by
Western blotting as in Fig. 3. Note that the expression of dominant
negative Akt leads to a decreased expression of IKK
and IKK
,
whereas IKK
remains unaffected.
-deficient Mice--
IKK
is an X-linked
gene both in humans and mice. Male mice lacking IKK
are embryonic
lethal (37, 38). Early after birth, heterozygous females show a strong
phenotype in skin closely related to that seen in patients with the
hereditary disorder retinitis pigmentosa (37, 38). In particular, the
epidermis is characterized by a generalized edema with increased
intercellular spaces, severe alterations in differentiation, and
increased proliferation and apoptosis (37, 38). Given the decreased
IKK
protein levels observed in the epidermis of bK5hK10 transgenic
mice, one might expect that they would correlate with phenotypic
alterations similar to those of IKK
-deficient mice. However, as
reported previously (10), heterozygous animals show no overt
alterations, and homozygous mice exhibited severe, although different,
abnormalities to those reported for IKK
-deficient females. By day 21 after birth, homozygous bK5hK10 transgenic mice show an epidermal
phenotype characterized by a clear decrease in epidermal thickness
caused by reduced proliferation as a consequence of K10 expression in
proliferative keratinocytes, along with an increased stratum corneum
(data not shown, see Ref. 10). Electron microscopy analysis also shows
flattened and degenerative basal cells but no intercellular edema in
transgenic mice (Fig. 4A', see
also Ref. 10). In the present work, prominent irregularities of the
basal membrane of the epidermal cells were observed (Fig. 4A"). Another characteristic feature of these animals is
their progressive generalized phenotype. Transgenic animals were
markedly underweight, became very frail, and showed signs of impaired
movement and wasting 3-5 weeks after birth (Fig. 4B). In
addition, the skin of these transgenic mice was characterized by a
severe reduction of the proliferation of the epidermal cells, as
demonstrated by BrdUrd incorporation experiments (Fig.
4C, see also Ref. 10). In contrast, we detected increased
proliferation of the dermal cells in these animals compared with
non-transgenic littermates (Fig. 4C). Finally, a severe
reduction in the amount of dermal adipose tissue was also observed
(Fig. 4, compare D with E). This causes the
bending of hair follicles (arrow in Fig. 4D) due
to the 2- to 4-fold decrease in the distance between the epidermis and
the muscle layer. None of these alterations are present in animals
lacking the IKK
subunit of the I
B kinase complex (37, 38). On the
other hand, most of these features are characteristics of animals
overexpressing TNF
in epidermal cells (49) or as a consequence of
I
B
inactivation (50), which also show increased production of
this cytokine (50). Consequently, the expressions of TNF
and related
cytokines were studied by Northern blotting. Homozygous mice showed a
dramatic increase in the mRNA levels of TNF
, IL-6, and IL-1
(Fig. 5A). To further confirm
this, we measured the levels of TNF
in the serum of these animals
and found a significant increase in circulating levels (Fig.
5B). Finally, experiments were performed to determine
whether the keratinocytes were the source of these increased levels of
TNF
. For this, the production and release of TNF-
by primary
keratinocytes derived from non-transgenic and transgenic mice were
analyzed. Elevated levels of TNF-
were found in the supernatant of
cultured primary transgenic keratinocytes (Fig. 5B).
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Fig. 4.
Phenotypic alterations in bK5hK10 transgenic
mouse skin. A and A', electron
microscopy analysis of non-transgenic (A) and homozygous
(A') transgenic mouse skin showing a single cell layer with
flattened nuclei in the transgenic epidermis. A", higher
magnification of the transgenic epidermis revealed alterations of the
basal membrane. B, weight curve displayed by bK5hK10
transgenic mice compared with non-transgenic littermate.
C, summary of in vivo BrdUrd labeling
experiments demonstrating decreased proliferation in transgenic
epidermis and increased proliferation of dermal cells.
D and E, hematoxylin-eosin-stained skin
sections from non-transgenic (E) and homozygous transgenic
(D) mice at day 6 showing the reduction in the dermal
adipose tissue (double arrows), which causes the bending of
the hair follicles (arrow in D). Bars
in A, A', and A" = 5 µm, in
D and E = 100 µm. Data in B and
C were taken from triplicate experiments and are shown as
mean ± S.D.
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Fig. 5.
Increased production of TNF
in bK5hK10 transgenic mice epidermis.
A, the expression of TNF
, IL-1, IL-6 genes in the quoted
mouse skin RNA extracts were analyzed by Northern blot using the
corresponding cDNA probes. Note the increased expression in the
homozygous bK5hK10 transgenic mice. The loading was normalized by
hybridization with a K14 cDNA probe. B, the levels of
secreted TNF
in serum and primary keratinocyte culture supernatants
were quantified by ELISA.
in
vivo, two experiments were performed. First, we monitored the
localization of TNF
and IL-6 in skin samples from transgenic and
non-transgenic mice using specific antibodies. We observed a positive
TNF
staining in the non-transgenic samples located deep in the
dermis, close to the muscle layer (Fig.
6A), and a few scattered areas
more close to the epidermal-dermal border (Fig. 6A'). On the
contrary, in transgenic samples TNF
was located primarily at the
epidermal-dermal border (Fig. 6B) and in some cases positive
staining was also observed in basal keratinocytes (Fig. 6B',
arrows). Finally, although no IL-6 was detected in non-transgenic mice samples (Fig. 6C), a clear expression
was detected in the epidermis of homozygous littermate samples (Fig. 6D). Interestingly, in this case, a stronger reaction was
observed in the hair follicles (Fig. 6D).
View larger version (103K):
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Fig. 6.
Localization of TNF
and IL-6 in transgenic mice skin. Formalin-fixed,
paraffin-embedded sections from non-transgenic (A,
A', and C) and homozygous bK5hK10 transgenic mice
littermates (B, B', and D) were
stained for TNF
(A, A', B, and
B') or IL-6 (C and D) expression using
specific rabbit polyclonal antibodies. Bars in
A-D = 100 µm; bars in A',
B' = 50 µm.
View larger version (55K):
[in a new window]
Fig. 7.
Detection of inflammatory cells in transgenic
mice skin. Frozen skin sections from non-transgenic
(A-C) and homozygous bK5hK10 transgenic mice littermates
(A', B', and C') were stained for T
lymphocytes (CD-3; A and A'), granulocytes (GR-1;
B and B') and macrophages (Mac-1; C
and C') using fluorescein isothiocyanate-labeled rat mAb
antibodies reacting with mouse CD3, GR-1, or Mac-1 antigens,
respectively. Keratin K10 expression was detected using K8.60
monoclonal antibody as previously described (10). Dashed
lines represent the dermal-epidermal border. Bar = 75 µm.
, which might explain some of the features of the phenotype
of these animals. However, it is worth mentioning that such increased
levels were not sufficient to produce liver apoptosis in
vivo (not shown).
plays an
important role in several cellular events such as septic shock,
induction of other cytokines, cell proliferation, differentiation, and
apoptosis. In response to TNF treatment, the transcription factor
NF-
B and c-Jun terminal kinase (JNK) are activated in most cells.
Because it was observed that bK5hK10 transgenic mice keratinocytes do not respond to TNF
either in vivo or in vitro
(Figs. 1 and 2), an investigation was made into the possible
stimulation of JNK as a consequence of increased production of TNF
in these keratinocytes. JNK activity was analyzed by an in
vitro kinase assay using GST-c-Jun as a substrate. The results
showed increased JNK activity in the homozygous mice (Fig.
8A). In agreement, increased
phosphorylation of ATF2 and phosphorylated JNK-1 was also observed
(Fig. 8A).
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Fig. 8.
Increased JNK activity in bK5hK10 transgenic
mouse epidermis. A, in vitro JNK kinase
assay using GST-c-jun as a substrate demonstrating the increased
activity in homozygous bK5hK10 transgenic mice. A', Western
blot analysis of whole skin protein extracts from the quoted genotypes
with the indicated antibodies showing the increase in phosphorylated
ATF2 and phosphorylated JNK in homozygous mouse epidermis.
B, Western blot of the same protein extracts showing similar
expression of TRAF2, RIP, and TRADD in the different genotypes.
to its receptors TNFR-1 or TNFR2 induces
receptor aggregation and results in the recruitment of a number of
cytoplasmic proteins to these complexes. TRAF2 is one of these signaling complexes. It interacts directly with TNFR2 and indirectly through TRADD with TNFR1. TRADD also recruits the death domain kinase
RIP to these complexes. Interestingly, TRAF2 and RIP are differentially involved in the modulation of NF-
B and JNK signals upon TNF
stimulation (51-54). Consequently, we studied the
expression of TRADD, TRAF2, and RIP in the epidermal extracts of
non-transgenic, heterozygous, and homozygous bK5hK10 transgenic mice.
No differences were seen in the expression of these molecules among the
different genotypes (Fig. 8B), therefore, ruling out the
possibility that the different NF-
B and JNK activities were
due to altered expression of these signaling intermediates.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(8-10). More remarkably, transgenic mice expressing
hK10 in the basal proliferative keratinocytes are almost completely
resistant to skin tumorigenesis (10). This is in agreement with our
recent observations indicating a fundamental role for Akt in the
process of mouse skin carcinogenesis (12). Akt kinase is involved in
many aspects of cell physiology, including the regulation of NF-
B
signaling. Because this pathway is also very important in the
regulation of epidermal functions, we sought to analyze the possible
alterations in NF-
B in the epidermis of bK5hK10 transgenic mice.
B activity was seen in the skin and primary
keratinocytes derived from bK5hK10 transgenic mice (Fig. 1). Moreover, such inhibition was dependent on K10-induced repression of Akt activity
(Fig. 1). One of the main phenotypic alterations in these mice is the
decreased proliferation in vivo and in vitro
(10). This seems to be in disagreement with the results shown by other authors who indicate that NF-
B acts in epidermis to arrest cell proliferation (reviewed in Ref. 29). However, it is important to notice
that this antiproliferative function has been inferred from transgenic
mouse models overexpressing different molecules that interfere with
NF-
B activity. It is therefore possible that this effect is owed to
such overexpression. Recent knock-in and epidermal-specific
null animal models have provided data to support this hypothesis (35).
Moreover, increased endogenous NF-
B activity during mouse skin
carcinogenesis has been observed (33).
B activity can
be reversed by Akt co-expression, suggesting that the inhibition of
this kinase is responsible for the observed impaired NF-
B signaling
observed in vitro and in vivo. The experimental evidence suggesting such functional relationship is very ample and
still growing (see the introduction). However, the molecular mechanism
responsible for such a connection has not been fully demonstrated. Here
we show that the inhibition of the I
B kinase complex is involved. In
fact, the co-expression of either IKK
or IKK
was, respectively,
able to rescue NF-
B inhibition partially or totally (Fig. 2). More
strikingly, it was observed that in animals expressing higher amounts
of K10, and thus with the strongest inhibition of Akt kinase and
NF-
B activities, there was a dramatic reduction IKK
and IKK
levels. These data are further confirmed by the use of retroviral
constructs coding for wt or dominant negative Akt. We have found that
the inhibition of Akt activity leads to a decreased expression of
IKK
and IKK
proteins (Fig. 3). To our knowledge, this is the
first evidence suggesting the involvement of Akt in the modulation of
IKK expression. The molecular mechanism underlying this process will be
studied in the future. Finally, the fact that IKK
co-expression can
partially rescue NF-
B activity clearly points to the possibility
that IKK
levels were reduced but not completely absent, because this
protein is absolutely necessary for this process (37, 38).
-deficient mice.
These animals die in utero while heterozygous IKK
females display severe dermatopathy characterized by keratinocyte
hyperproliferation, skin inflammation, hyperkeratosis, intercellular
edema, and increased apoptosis (37, 38). Of all these characteristics,
however, only hyperkeratosis was observed in bK5hK10 mice along with
some alterations suggestive of increased apoptosis. In addition, the reduced proliferation observed in keratinocytes, in parallel with increased BrdUrd incorporation in the dermal cells, the flattened appearance of the keratinocytes, the reduction in the subepidermal adipose tissue, and the characteristic progressive phenotype (Fig. 4),
were all similar to reported alterations in transgenic mice expressing
TNF
in the epidermis (49). Similar alterations have also been
described in the epidermis of mice lacking I
B (50), which also have
increased levels of circulating TNF
. In agreement with this, the
results show that bK5hK10 keratinocytes produce increased amounts of
this cytokine (Figs. 5 and 6). This increased production may be
responsible for some of the phenotypic alterations observed in bK5hK10
transgenic mice.
is particularly striking. In fact,
it is well established that TNF
, IL-1, and IL-6 genes are
predominantly regulated by NF-
B elements (55). In this regard it is
worth mentioning that similar increases in TNF
, as well as the
increased expression of several genes normally controlled by NF-
B
factor, have been reported in mice lacking IKK
(37), and in
epidermal-specific IKK
-null mice (36). However, in these cases, the
increased production of these cytokines has been attributed to the
inflammatory cells that invade the tissue (36, 37). In bK5hK10 this is
not the main cause, as confirmed by immunofluorescence analysis (Fig.
7). On the other hand, we observed a decreased number of inflammatory
cells in the dermis of bK5hK10 homozygous transgenic mice. This might
be in agreement with our findings indicating that these animals display severe immunodeficiency due to altered thymus development.2
The decreased expression of IKK
observed in bK5hK10 transgenic epidermis clearly indicates the existence of alternative mechanisms for
the production of TNF
, given that this subunit is essential for
NF-
B activation (48). TNF synthesis and secretion are regulated at
several points, including the transcriptional and post-transcriptional levels. Among the elements that may control TNF
gene expression, besides NF-
B sites, are Ets, ATF-2/c-jun, Sp1, Elk1, CBP, and p300
(56-59). This clearly points to a central role of JNK activity in the
positive modulation of TNF
gene expression. Interestingly, JNK
activity is inhibited by Akt kinase through direct binding and
phosphorylation of SEK1/MKK4 (46, 60). Consequently, although we do not
know at present how TNF
is produced in the absence of normal NF-
B
signaling in transgenic keratinocytes, a possible explanation might be
that Akt inhibition promoted by K10 expression can lead to the
activation of JNK. This would allow the production of TNF
, which,
upon binding to TNFR1, might induce increase binding of ATF-2/c-jun to
the TNF
promoter, therefore, inducing increased transcription (57).
Finally, such increased secretion of TNF
may account for the
expression of IL-1 and Il-6 genes. Alternatively, it is tempting to
speculate that differentiation-specific keratins, such as K10, could
directly modulate TNF
. In this regard, it has been demonstrated that
simple epithelial keratins modulate different aspects of TNF
signaling through direct binding with several components of the
TNF-dependent network (61-63). Therefore, K10 and K8
and/or K18 could act in opposite manners. These aspects will be the
subject of future experiments.
![]() |
ACKNOWLEDGEMENTS |
---|
We are greatly indebted to J. Martínez for excellent animal care, I. de los Santos for
histological assistance, A. Bravo for electron microscopy, S. J. Gutkind for his generous gift of wt and dominant-negative Akt
constructs, J. C. Segovia for providing the pStMCS retroviral
vector, and G. Courtois and A. Israël for providing the IKK,
IKK
, and IKK
expression vectors.
![]() |
FOOTNOTES |
---|
* This work was funded by Grants SAF2002-01037 from the Spanish Dirección General de Investigación Científica y Técnica and 08.1/0054/2001 1 from the Comunidad Autónoma de Madrid.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
34-91-346-6438; Fax: 34-91-346-6484; E-mail:
jesusm.paramio@ciemat.es.
Published, JBC Papers in Press, February 1, 2003, DOI 10.1074/jbc.M208170200
2 M. Santos, P. Rio, C. Segrelles, S. Ruiz, J. C. Segovia, and J. M. Paramio, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IKK, IB kinase;
TNF
, tumor necrosis factor a;
JNK, c-Jun terminal kinase;
IL-6, interleukin-6;
PBS, phosphate-buffered saline;
ELISA, enzyme-linked
immunosorbent assay;
wt, wild type;
JNK, c-Jun NH2-terminal
kinase;
TRAF2, TNF receptor-associated factor 2;
EMSA, electrophoretic
mobility shift analysis;
GST, glutathione S-transferase;
BrdUrd, bromodeoxyuridine;
RIP, receptor-interacting protein;
TRADD, tumor necrosis factor-associated death domain.
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