Tumor necrosis factor receptor 1-mediated signaling is required for skin cancer development induced by NF-kappaB inhibition
ABSTRACT NF-kappaB signaling plays an important role in skin development and epidermal growth control. Moreover, inhibition of NF-kappaB signaling in murine epidermal keratinocytes in vivo, by expression of a keratin 5 (K5)-directed superrepressor form of inhibitor of NF-kappaB (IkappaBalpha), results in an inflammatory response characterized by a massive dermal infiltration of neutrophils, epidermal hyperplasia, and a rapid development of aneuploid squamous cell carcinomas (SCC). We now show that by crossing K5-IkappaBalpha mice onto a tumor necrosis factor receptor 1(Tnfr1)-null background, both the inflammatory and the tumorigenic responses are blocked. The specificity of the block is illustrated by the fact that K5-IkappaBalpha mice lacking the IL-1 receptor type 1 (Il1r1) develop inflammation and squamous cell carcinomas. Reconstitution of lethally irradiated K5-IkappaBalpha/Tnfr1(-/-) mice with Tnfr1(+/-) bone-marrow does not induce the inflammatory or the tumorigenic phenotype, indicating a critical dependence on Tnfr1-mediated signaling in skin cells or nonimmune cells. Our results suggest a critical role of local Tnfr1-mediated signaling and associated inflammatory response cooperating with repressed keratinocyte NF-kappaB signaling in driving skin cancer development.
- SourceAvailable from: Jeremías Galletti[Show abstract] [Hide abstract]
ABSTRACT: Purpose: To evaluate the role of nuclear factor kappa B (NF-κB) activation in eye drop preservative toxicity and the effect of topical NF-κB inhibitors on preservative-facilitated allergic conjunctivitis. Methods: Balb/c mice were instilled ovalbumin (OVA) combined with benzalkonium chloride (BAK) and/or NF-κB inhibitors in both eyes. After immunization, T cell responses and antigen-induced ocular inflammation were evaluated. NF-κB activation and associated inflammatory changes were also assessed in murine eyes and in an epithelial cell line after BAK exposure. Results: BAK promoted allergic inflammation and leukocyte infiltration of the conjunctiva. Topical NF-κB inhibitors blocked the disruptive effect of BAK on conjunctival immunological tolerance and ameliorated subsequent ocular allergic reactions. In line with these findings, BAK induced NF-κB activation and the secretion of interleukin 6 and granulocyte-monocyte colony-stimulating factor in an epithelial cell line and in the conjunctiva of instilled mice. In addition, BAK favored major histocompatibility complex (MHC) II expression in cultured epithelial cells in an NF-κB-dependent fashion after interaction with T cells. Conclusion: BAK triggers conjunctival epithelial NF-κB activation, which seems to mediate some of its immune side effects, such as proinflammatory cytokine release and increased MHC II expression. Breakdown of conjunctival tolerance by BAK favors allergic inflammation, and this effect can be prevented in mice by topical NF-κB inhibitors. These results suggest a new pharmacological target for preservative toxicity and highlight the importance of conjunctival tolerance in ocular surface homeostasis.Investigative Ophthalmology & Visual Science 09/2014; · 3.66 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Immune responses in the skin are important for host defence against pathogenic microorganisms. However, dysregulated immune reactions can cause chronic inflammatory skin diseases. Extensive crosstalk between the different cellular and microbial components of the skin regulates local immune responses to ensure efficient host defence, to maintain and restore homeostasis, and to prevent chronic disease. In this Review, we discuss recent findings that highlight the complex regulatory networks that control skin immunity, and we provide new paradigms for the mechanisms that regulate skin immune responses in host defence and in chronic inflammation.Nature Reviews Immunology 04/2014; 14(5). · 33.84 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The nuclear factor kappa B (NF-κB) signalling pathway exhibits both tumour-promoting and tumour-suppressing functions in different tissues and models of carcinogenesis. In particular in epidermal keratinocytes, NF-κB signalling was reported to exert primarily growth inhibitory and tumour-suppressing functions. Here, we show that mice with keratinocyte-restricted p65/RelA deficiency were resistant to 7, 12-dimethylbenz(a)anthracene (DMBA)-/12-O-tetra decanoylphorbol-13 acetate (TPA)-induced skin carcinogenesis. p65 deficiency sensitized epidermal keratinocytes to DNA damage-induced death in vivo and in vitro, suggesting that inhibition of p65-dependent prosurvival functions prevented tumour initiation by facilitating the elimination of cells carrying damaged DNA. In addition, lack of p65 strongly inhibited TPA-induced epidermal hyperplasia and skin inflammation by suppressing the expression of proinflammatory cytokines and chemokines by epidermal keratinocytes. Therefore, p65-dependent NF-κB signalling in keratinocytes promotes DMBA-/TPA-induced skin carcinogenesis by protecting keratinocytes from DNA damage-induced death and facilitating the establishment of a tumour-nurturing proinflammatory microenvironment.EMBO Molecular Medicine 06/2014; · 7.80 Impact Factor
Tumor necrosis factor receptor 1-mediated signaling
is required for skin cancer development induced by
Maria H. Lind*, Bjo ¨rn Rozell†, Robert P. A. Wallin‡, Max van Hogerlinden*, Hans-Gustaf Ljunggren‡, Rune Toftgård*§,
and Inderpreet Sur*
*Department of Bioscience and†Unit for Morphological Phenotype Analysis, Clinical Research Center, and Department of Laboratory Medicine Division of
Pathology, Karolinska Institutet, Novum, SE-141 57 Huddinge, Sweden; and‡Center for Infectious Medicine, Department of Medicine, Karolinska Institutet,
Huddinge University Hospital, SE-141 86, Huddinge, Sweden
Edited by David V. Goeddel, Tularik, Inc., South San Francisco, CA, and approved February 2, 2004 (received for review November 3, 2003)
NF-?B signaling plays an important role in skin development and
epidermal growth control. Moreover, inhibition of NF-?B signaling
in murine epidermal keratinocytes in vivo, by expression of a
keratin 5 (K5)-directed superrepressor form of inhibitor of NF-?B
(I?B?), results in an inflammatory response characterized by a
massive dermal infiltration of neutrophils, epidermal hyperplasia,
and a rapid development of aneuploid squamous cell carcinomas
(SCC). We now show that by crossing K5-I?B? mice onto a tumor
necrosis factor receptor 1(Tnfr1)-null background, both the inflam-
matory and the tumorigenic responses are blocked. The specificity
of the block is illustrated by the fact that K5-I?B? mice lacking the
mice with Tnfr1?/?bone marrow does not induce the inflamma-
tory or the tumorigenic phenotype, indicating a critical depen-
Our results suggest a critical role of local Tnfr1-mediated signaling
and associated inflammatory response cooperating with repressed
keratinocyte NF-?B signaling in driving skin cancer development.
sion, apoptosis, and regulation of the immune response (1).
Under nonstimulating conditions, NF-?B is retained in the
cytoplasm in an inactive form because of its interaction with the
inhibitory proteins I?Bs. In response to an activating stimulus,
I?B is phosphorylated by an I?B kinase (IKK) complex, which
targets it for degradation by the proteasome, releasing NF-?B,
which translocates to the nucleus, where it regulates the tran-
scription of target genes.
Evidence gathered from studies during recent years have
shown that NF-?B has a growth inhibitory function in the skin.
Initial studies showed that overexpression of the NF-?B subunits
p50 or p65 in the basal layer of the murine epidermis by using
a keratin 14 (K14) promoter leads to hypoplasia of the epider-
mis, whereas expression of a superrepressor form of the inhibitor
of NF-?B type ? (I?B?) results in hyperplasia (2). Although
mice with keratin 5 (K5)-directed expression of I?B? (K5-I?B?)
to the basal layer of the epidermis survive to adulthood (3). Not
only do these mice develop hyperplasia of the epidermis, but the
skin phenotype of K5-I?B? mice is also characterized by an
intense neutrophil-dominated inflammation of the skin and an
early development of squamous cell carcinomas (SCC).
Recent data suggest that human sporadic SCC may show a
block in NF-?B signaling based on nuclear exclusion of the RelA
NF-?B subunit (ref. 4 and M.v.H., unpublished results). Fur-
thermore, it was recently shown that coexpression of a super-
repressor form of I?B? and Ha-Ras results in neoplasia resem-
bling invasive SCC in human keratinocytes transplanted to
severe combined immunodeficient (scid?scid) mice, supporting
he Rel?NF-?B transcription factors have a central role in
several cellular processes, including proliferation, cell adhe-
the relevance of a NF-?B block in the induction of human
Inflammation of the skin with neutrophil infiltration and
hyperproliferation is also seen in female heterozygous Ikk?-
deficient mice, which develop a condition similar to the human
X-linked disorder Incontinentia Pigmenti (IP) (5, 6). IKK? is the
regulatory subunit of the IKK complex, which, in addition,
contains the two catalytic subunits IKK? (IKK1) and IKK?
(IKK2) (1). It is now well established that human IP results from
loss-of-function mutations in the IKK? gene (7). Interestingly,
subungual keratoacanthoma-like tumors and cases of SCC have
been reported as late manifestations of IP (8–11).
The catalytic IKK? subunit is necessary for activation of
NF-?B in response to proinflammatory stimuli (1). Skin-specific
deletion of the IKK? gene, K14-Cre?Ikk2FL/FL, was recently
shown to result in an inflammatory phenotype with concomitant
hyperproliferation of the epidermis (12), very similar to what is
seen in the K5-I?B? mice. Because the K14-Cre?Ikk2FL/FLmice
die between day 7 and day 9 after birth, it is currently not known
whether they, similar to K5-I?B? mice, are prone to develop
Besides inflammation and hyperproliferation, an up-
regulation of the proinflammatory cytokine tumor necrosis
factor type ? (Tnf?) in the skin is commonly observed in
K5-I?B?, Ikk? heterozygous females and K14-Cre?Ikk2FL/FL
mice (3, 5, 12). Here, we show that the Tnf? response is a major
driving force of the disease in the K5-I?B? mice. Removal of
Tnfr1 prevents both the development of inflammation and the
hyperproliferation of the skin, in line with what was recently
reported for K14-Cre?Ikk2FL/FLmice (12). Importantly, we also
show that the development of SCC in K5-I?B??Tnfr1?/?mice is
abolished. Reconstitution of lethally irradiated K5-I?B??
cause reinduction of the inflammatory phenotype, hyperprolif-
eration, or cancer development. Up-regulation of Tnf? can be
detected in the skin of K5-I?B??Tnfr1?/?mice when compared
with Tnfr?/?mice, indicating that both the primary cells
responsible for the up-regulation of Tnf? and the critical
Tnfr1-responding cells reside in the skin. Our data show that
local Tnfr1-mediated signaling and an associated inflamma-
tory response cooperate with repressed keratinocyte NF-?B
signaling in driving SCC development.
Materials and Methods
Transgenic Mice. FVB?N K5-I?B? mice were generated as de-
scribed in ref. 3. These mice were crossed with C57BL?6
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: I?B?, inhibitor of NF-?B type ?; IKK, I?B kinase; SCC, squamous cell carci-
noma; Tnf, tumor necrosis factor; K14, keratin 14; K5, keratin 5; JNK, c-jun N-terminal
kinase; Cdk4, cyclin-dependent kinase 4; MAPK, mitogen-activated protein kinase; Il1r1,
IL-1 receptor type 1; scid, severe combined immunodeficient.
§To whom correspondence should be addressed. E-mail: email@example.com.
© 2004 by The National Academy of Sciences of the USA
April 6, 2004 ?
vol. 101 ?
no. 14 www.pnas.org?cgi?doi?10.1073?pnas.0307106101
Tnfr1?/?mice (TnfrsflatmlMakkindly provided by Tak Mak,
Advanced Medical Discovery Institute, Toronto, Canada). In-
tercross of F1 K5-I?B??Tnfr1?/?generated F2 K5-I?B??
Tnfr1?/?and K5-I?B??Tnfr1 wild-type (Tnfr1?/?and Tnfr1?/?)
littermates. Mice were killed by cervical dislocation at 3.5 weeks
of age, and skin samples were taken. A total of 65 mice were
analyzed: 15 K5-I?B??Tnfr1?/?, 15 K5-I?B??Tnfr1?/?, 15 K5-
I?B??Tnfr1?/?, 10 Tnfr1?/?, and 10 Tnfr1?/?. No phenotypic
differences were observed between K5-I?B??Tnfr1?/?and K5-
I?B??Tnfr1?/?mice; hence, they were treated as one group:
K5-I?B?. The cross between FVB?N K5-I?B? mice and IL-1
receptor type 1 (Il1r1)?/?mice (C57BL?6 Il1r1tmllmx) was per-
formed as described for the Tnfr1?/?cross above. F2 littermates
from two different litters were analyzed (n ? 2). Mice were kept
according to Swedish national requirements, and ethical per-
mission was obtained for all animal manipulations.
Histology and Immunohistochemistry. Skin samples were fixed in
10% neutral buffered formalin overnight and embedded in
paraffin. Sections were stained with hematoxylin?eosin for his-
tological analysis. For immunohistochemistry, paraffin sections
were deparaffinized in xylene and passed through a graded
alcohol series. In most cases, the sections were microwaved in 10
mM sodium citrate buffer (SCB; pH 6.0) before incubation with
antibody (indicated below). Antibodies and dilutions used were
as follows: monoclonal rat anti-CD3 (NovoCastra, Newcastle
upon Tyne, U.K.), 1:200 (SCB); polyclonal rabbit anti-Cdk4
(Santa Cruz Biotechnology), 1:200 (SCB); monoclonal rat anti-
CD-45R?B220 (Pharmingen), 1:100; polyclonal goat anti-IL-1?
(R & D Systems), 0.5 ?g?ml (SCB); rabbit polyclonal anti-Ki67
(NovoCastra), 1:1000 (SCB); rabbit polyclonal anti-myeloper-
oxidase (DAKO), 1:4000 (SCB); polyclonal rabbit anti-phospho-
Technologies, Beverly, MA), 1:100 (SCB); polyclonal rabbit
anti-active c-Jun N-terminal kinase (JNK) (pTPpY, Promega),
1:100 (SCB); and polyclonal goat anti-Pax-5 (Santa Cruz Bio-
technologies), 1:2000 (SCB). Bound antibodies were visualized
by diaminobenzidine, and sections were counterstained with
Cytokine mRNA Arrays, RT-PCR, and Real-Time PCR Analysis. Total
RNA was prepared from skin biopsies by using RNABee
solution (Tel-Test, Friendswood, Texas) and was treated with
RQ1 RNase-Free DNase (Promega) to remove contaminating
genomic DNA. Five micrograms of RNA was used as a template
for32P-labeled cDNA probe synthesis that was subsequently
used in hybridization to a GEArray Q Series Mouse Common
Cytokine Gene Array (SuperArray, Bethesda) according to the
manufacturer’s instructions. Radioactive signal intensities were
analyzed on a PhosphorImager. The signal from expression of
each cytokine gene was normalized to the signal derived from
?-actin on the same array. One microgram of RNA was used for
cDNA preparation by using SuperScript RNase H?Reverse
Transcriptase (Promega), which was subsequently used as a
template for PCR or Real-Time PCR quantification. For PCR
amplification, Taq Gold (Promega) was used. The primers and
procedures are as follows: Il-1?: 5?-TGCCATTGAC-
CATCTCTCTCTG-3? and 5?-TGGCAACTCCTTCAGCAA-
CACG-3?; 94°C for 30 seconds, 54°C for 1 min, 60°C for 2 min,
25 cycles; Il-1?: 5?-GCAACTGTTCTGAACTCA-3? and
CTCGGAGCCTGTAGTGCAG-3?; 94°C for 30 seconds, 49°C
for 1 min, and 60°C for 2 min, 37 cycles. PCR for mouse ?-actin
was run as a control as described in ref. 3. Real-Time PCR
analysis for Tnf? expression was done by using an Assays-on-
Demand gene expression kit (Assay ID Mm00443258-m1, Ap-
plied Biosystems). GAPDH was used as endogenous control
(TaqMan Rodent GAPDH reagents, Applied Biosystems).
Bone Marrow Transplantation. FVB?N K5-I?B? mice were crossed
with C57BL?6 Tnfr1?/?mice. The F1 K5-I?B??Tnfr1?/?were
backcrossed to C57BL?6 Tnfr1?/?giving K5-I?B??Tnfr1?/?,
K5-I?B??Tnfr1?/?, Tnfr1?/?, and Tnfr1?/?mice. K5-I?B??
Tnfr1?/?mice developed inflammation of the skin within 1
month of age and could not be included in the experiment (data
not shown). MHC class I typing for H-2Dband H-2Dq?H-2Lq
alleles was performed by fluorescent-activated cell sorter anal-
ysis on peripheral blood cells (KH95 and KH117, respectively;
Pharmingen). Bone marrow-derived cells from Tnfr1?/?or
Tnfr1?/?littermates were obtained by flushing femurs and tibias
with PBS and transplanting 1 ? 106cells i.v. into 1-month-old
MHC-matched, natural killer (NK) cell-depleted and lethally
irradiated mice (6.5 ? 5.5 Gy, 5-h interval; n ? 7 per group).
Successful engraftment was verified by genotyping peripheral
blood DNA. Skin biopsies for histopathology were taken 3
months after engraftment. NK cell depletion was performed by
i.p. administration of 200 ?g of purified anti-NK1.1 antibody
(PK136; Pharmingen) in 200 ?l of PBS 2 days before initiation
of the experiment.
Tnfr1 Signaling Is Required for Development of Inflammation and
Epidermal Hyperplasia in K5-I?B? Mice. The development of in-
flammation and epidermal hyperplasia in K5-I?B? transgenic
mice is associated with a strong up-regulation of Tnf? (3). To
evaluate the role of Tnf? in the development of this disease, we
crossed the K5-I?B? mice onto a Tnfr1-null background. The
K5-I?B? mice developed macroscopic skin changes within 3
weeks of age, seen as verrucous keratinized lesions on the back
(Fig. 1a). Histological analysis of dorsal skin from 3.5-week-old
K5-I?B? mice showed severe focal changes with hyperplasia?
dysplasia and hyperkeratosis of the epidermis also involving the
hair follicles with a massive inflammatory dermal infiltrate
consisting mainly of polymorphonuclear granulocytes (Fig. 1c).
In contrast, age-matched K5-I?B??Tnfr1?/?mice showed no
inflammatory skin changes (Fig. 1 a and c).
including suprabasally located cells, was observed by immuno-
histochemical staining for the proliferation marker Ki67 (Fig.
1d). Wild-type and K5-I?B??Tnfr1?/?skin only showed sparse
staining of basal keratinocytes (Fig. 1d). Thus, the inflammation
and the epidermal hyperplasia observed in the K5-I?B? mice
appears secondary to the Tnfr1-mediated signaling, similar to
what was recently reported for K14-Cre?Ikk2FL/FLmice (12).
Removal of Tnfr1 Abolishes the Development of SCC in K5-I?B? Mice.
In ?90% of the 3.5-week-old K5-I?B? mice analyzed, dysplastic
changes were seen, which, in several animals, developed into
infiltrative SCC (Fig. 1b). However, 1-year-old K5-I?B??
Tnfr1?/?mice (n ? 12) still showed no signs of tumor develop-
ment. Our data show that not only does transfer of the K5-I?B?
transgene onto a Tnfr1?/?background prevent the inflammation
and hyperproliferation of the skin, but it also abolishes the
development of SCC. Data obtained here were from a mixed
FVB?N-C57BL?6 background. We have also repeated the ex-
periment on a FVB?N background (six backcrosses) with the
same results (data not shown). It should be noted that the
penetrance for SCC in K5-I?B? mice of either genetic back-
ground is 100% (3).
Massive Neutrophil Invasion and Presence of Plasmacytoid Dendritic
Cells in Lesional Skin of K5-I?B? Mice. The inflammatory infiltrate
observed in K5-I?B? mice is dominated by polymorphonuclear
leukocytes, mainly neutrophils, which increase in number during
disease progression (Fig. 2 and M.v.H., unpublished data). In
contrast, T lymphocytes are only present in low numbers and do
not change in number over time (Fig. 2 and M.v.H., unpublished
Lind et al.PNAS ?
April 6, 2004 ?
vol. 101 ?
no. 14 ?
data). In K5-I?B??Tnfr1?/?mice, no immune cell infiltration
can be detected, which confirms the results from the histological
analysis (Fig. 2). Positive staining for T cells (CD3) in the
epidermis of K5-I?B??Tnfr1?/?mice labels the resident den-
as in wild-type mice (data not shown).
When staining K5-I?B? mice for the B lymphocyte marker
B220, two different cell populations are seen (Fig. 2): one with
typical lymphocyte morphology that can be found deep in the
dermis and hypodermal fat layer of inflamed skin and the other
with a dendritic morphology that is seen throughout the dermis,
invading also the epidermal cell layers. Staining with another B
lymphocyte marker, Pax5, only stains the lymphocyte population
(data not shown). Although the B220(?) lymphocyte population
remains stable, the B220(?) Pax5(?) dendritic cells increase in
number with progression of the disease (data not shown).
Putatively, these cells are identified as plasmacytoid dendritic
cells, which have recently been suggested to be involved both in
inflammation and in the development of cancer (13, 14).
Il1r1 Signaling Is Not Required for Development of Inflammation,
Epidermal Hyperplasia, or SCC in K5-I?B? Mice. By using array
analysis for cytokine expression, we detected strong up-
regulation of Il-1? and Il-1? in lesional skin from K5-I?B? mice
(data not shown) and confirmed this detection by RT-PCR
analysis (Fig. 3a). Immunohistochemical staining for Il-1?
showed strong up-regulation in inflamed skin in a mixed pop-
ulation of dermal cells; the majority of which were identified as
polymorphonuclear granulocytes based on their morphology
(Fig. 3b). To assess a potential role for Il-1 in the inflammatory
process and the development of cancer, we transferred the
K5-I?B? transgene onto an Il1r1-null background. Il1r1 is the
main Il-1 receptor responsible for signal transduction induced by
both Il-1? and Il-1? (15). No difference was seen in development
of inflammation or cancer between K5-I?B??Il1r1?/?mice and
K5-I?B? mice (Fig. 3c). Thus, Il1r1 signaling is not required
either for development of inflammation or SCC in this model.
Interestingly, the up-regulation of Il-1? and Il-1? is downstream
of Tnfr1 signaling, which is also indicated by the absence of Il-1
up-regulation in the K5-I?B??Tnfr1?/?mice (Fig. 3a).
Tumor Development Driven by Tnfr1-Mediated Signaling and Inflam-
mation. It was recently reported that coexpression of a superre-
invasive SCC in human keratinocytes transplanted to scid?scid
mice (4). In this setting, expression of I?B? was shown to cause
an up-regulation of cyclin-dependent kinase 4 (Cdk4). We have
previously shown that the tumors in the K5-I?B? mice do not
display mutations in Ha-Ras (16). Nor could we detect any
compared to wild-type skin (Fig. 4 a and b). Fig. 4a shows the
staining pattern for active MAPK in the tail skin of 3.5-week-old
wild-type and K5-I?B? mice, with a strong labeling of cells in the
suprabasal cell layers. In most tumors, no positive staining can
be seen, except for a fraction of well differentiated tumors that
show occasional staining in suprabasal cell layers (Fig. 4b).
Furthermore, we could not find any up-regulation of Cdk4 either
in primary keratinocytes from K5-I?B? mice in vitro (Fig. 4c) or
in vivo where Cdk4 is abundantly expressed in the basal cell
layers with no evident difference between wild-type and trans-
genic skin (Fig. 4d). In the inflamed, severely hyperplastic
K5-I?B? skin of older mice, the expression of Cdk4 is increased
compared with wild-type mice, probably reflecting the increased
proliferation (Fig. 4e).
Tnf? Is Up-Regulated in the Skin of K5-I?B? Independent of the
Inflammation. Real-Time PCR analysis showed that Tnf? expres-
with wild-type skin. Interestingly, a moderate up-regulation of
compared with Tnfr1?/?mice. Because the K5-I?B??Tnfr1?/?
animals do not show any inflammation in the skin, our inter-
(Lower) mice. Mice were 3.5 weeks old. (b) Hematoxylin?eosin-stained SCC in a 3.5-week-old K5-I?B? mouse. Overview shows the histology of a clinically
and follicular epithelium with concomitant hyperkeratosis. In the deep portion of the follicular structures, progression into infiltrative SCC occurs (boxed areas).
eosin-stained dorsal skin sections from wild-type, K5-I?B??Tnfr1?/?, and K5-I?B? mice. (Scale bar, 100 ?m.) (d) Ki-67 staining, labeling proliferating cells in
wild-type, K5-I?B??Tnfr1?/?, and K5-I?B? dorsal skin. (Scale bar, 50 ?m.)
Tnfr1 signaling is required for development of inflammation and epidermal hyperplasia in K5-I?B? mice. (a) K5-I?B??Tnfr1?/?(Upper) versus K5-I?B?
www.pnas.org?cgi?doi?10.1073?pnas.0307106101Lind et al.
pretation is that the moderate up-regulation of Tnf? seen in the
K5-I?B??Tnfr1?/?animals depends on the inhibition of NF-?B
in the keratinocytes. This up-regulation of Tnf? may be the first
priming event of the disease, preceding the development of
inflammation and SCC.
Development of Inflammation, Epidermal Hyperplasia, and SCC Can-
Derived Cells. If up-regulation of Tnf? is the initiating event
driving the development of disease in the K5-I?B? mice, the next
crucial question that follows is which cells are responding to
Tnf?: the immune cells or the nonbone-marrow-derived cells in
the skin. To address this question, Tnfr1?/?or Tnfr1?/?bone
marrow cells were transplanted to lethally irradiated K5-I?B??
Tnfr1?/?mice. Surprisingly, reconstitution with Tnfr1?/?bone
marrow did not recover the inflammatory phenotype, epidermal
hyperplasia, or tumor development (animals were monitored for
6 months after transplantation) (Fig. 5). This suggests that
critical Tnfr1 signaling occurs within the resident skin cells or
nonimmune cells. Recent data supports this as it was shown that
Tnfr1 signaling and downstream JNK activity drives hyperpro-
liferation of RelA?/?epidermis transplanted onto scid?scid mice
(17). Similar to the reported increase in the levels of active JNK
in RelA?/?epidermis, we also observe a strong increase in JNK
activity in K5-I?B? hyperplastic epidermis, which is abolished in
K5-I?B??Tnfr1?/?mice (Fig. 6, which is published as supporting
information on the PNAS web site).
We have earlier shown that selective inhibition of Rel?NF-?B
signaling in the murine skin by targeted expression of a super-
repressor form of I?B? results in inflammation, hyperprolifera-
tion, increased apoptosis, and spontaneous early development of
SCC, with a penetrance of 100% (3). In this study, we show that
the inflammation and hyperproliferation seen in the K5-I?B?
mice depend on Tnfr1 signaling, similar to what was recently
reported for K14-Cre?Ikk2FL/FLmice, also having a defect in
NF-?B signaling (12). More importantly, no spontaneous cancer
development was observed in K5-I?B??Tnfr1?/?mice. The
unique role of Tnf? for the development of the disease in these
mice is further demonstrated by the fact that removal of the
receptor for the proinflammatory cytokines Il-1? and Il-1?, both
of which are shown to be highly overexpressed in affected skin,
does not influence the development of disease.
Our data show that inhibiting NF-?B in keratinocytes in vivo
leads to an up-regulation of Tnf? in the skin independent of
inflammation, given that it can also be seen in K5-I?B??
Tnfr1?/?mice. This supports a model in which up-regulation of
Tnf? in the skin is the first event in the disease process that
precedes the development of inflammation and SCC. The cel-
lular source of the increase in Tnf? expression is currently not
known. Keratinocytes from K5-I?B? mice cultured in vitro do
not show up-regulation of Tnf? compared with wild-type (data
not shown). However, that keratinocytes do not show up-
regulation in vitro does not exclude them from being responsible
for the up-regulation in vivo.
Even more intriguing, our data strongly indicate that the
Tnf?-responding cells also reside in the skin, given that recon-
stitution of lethally irradiated K5-I?B??Tnfr1?/?mice with
Tnfr1?/?bone marrow-derived cells could not recover the
inflammatory phenotype, epidermal hyperplasia, or tumor de-
in K5-I?B??Tnfr1?/?mice. Shown is staining for neutrophils (myeloperoxi-
dase), T lymphocytes (CD3), B lymphocytes, and plasmacytoid dendritic cells
(B220?CD-45R). Arrows indicate positive staining in K5-I?B? mice. Note the
two populations seen by B220?CD-45R staining. (Scale bar, 100 ?m.)
The immune-cell infiltration in the skin of K5-I?B? mice is abolishedFig. 3.
epidermal hyperplasia, or cancer in K5-I?B? mice. (a) RT-PCR showing up-
regulation of Il-1? and Il-1? in K5-I?B? mice. (b) Immunohistochemical stain-
ing for IL-1? in wild-type and K5-I?B? mice. (c) Hematoxylin?eosin staining of
dorsal skin from K5-I?B??Il1r1?/?versus K5-I?B? mice. (Scale bars, 100 ?m.)
Il1r1 signaling is not required for development of inflammation,
Lind et al. PNAS ?
April 6, 2004 ?
vol. 101 ?
no. 14 ?
velopment. These results support a two-step model in which the
Tnfr1 response in resident skin cells leads to induction of other
cytokines?chemokines that in turn attract the inflammatory cells
to the skin. However, additional experiments are needed to
verify this notion. Bone-marrow reconstitution is limited to
experiments on adult mice, and at the present time, we cannot
definitely exclude the possibility of a different result in a case in
which Tnfr1 had been present in the bone-marrow cells from
birth. It is also possible that Tnfr1 is needed both on the
keratinocytes and on the immune cells to induce the inflamma-
tion. Additional experiments will allow further investigation of
these questions. Support for an important role of Tnfr1 signaling
in the keratinocytes is provided by the recent report that Tnfr1
and downstream JNK activity drives hyperproliferation in
RelA?/?epidermis (17). Similar to RelA?/?epidermis, hyper-
plastic skin in K5-I?B? mice shows a strong up-regulation of
active JNK (Fig. 6).
There is increasing evidence for a role of Tnf? in tumor
promotion in skin carcinogenesis. Removal of the Tnf? response
in mice by deletion of Tnf? or Tnfr1 has been shown to attenuate
and confers resistance in two-stage 7,12-dimethylbenz(a)anthra-
cene?12-O-tetradecanoylphorbol-13-acetate carcinogenesis ex-
periments (18, 19).
Although critical, increased expression of Tnf? in the skin is
not likely to be the only explanation for the phenotype observed
in the K5-I?B? mice. Transgenic overexpression of TNF? in the
murine epidermis leads to inflammation of the skin but without
hyperplasia and development of SCC; rather, such mice develop
hypoplasia of the epidermis (20). In contrast, the extremely fast
development of SCC in K5-I?B? mice, in which the earliest
tumors were seen within 3 weeks of birth, suggests an intricate
interplay between the inflammatory response, Tnfr1 signaling,
and blockade of NF-?B signaling in the target keratinocytes.
We propose that inhibition of NF-?B in epidermal?follicular
keratinocytes disturbs the stress-induced growth arrest response.
The cellular stress in the K5-I?B? mouse is induced by inflam-
mation of the skin, which depends on the Tnfr1-mediated
signaling, and we predict that other origins of stress may have
similar effects. In this context, it is interesting to note that
coexpression of a superrepressor form of I?B? and Ha-Ras
results in neoplasia resembling invasive SCC in human keratin-
ocytes transplanted to scid?scid mice (4). The expression of
Ha-Ras in human keratinocytes may be seen as a stress factor,
similar to the inflammation in the K5-I?B? model, and the
requirement of both Ras and I?B? to induce neoplasia in the
skin grafts likely reflects the absence of an intact immune system
in scid?scid mice. We have previously shown that tumors in
K5-I?B? mice do not display mutations in Ha-Ras (16), and we
now find that there is no increase in activated MAPK in the skin
or tumors of K5-I?B? mice, essentially ruling out Ras as a factor
critical to cancer development in this model. Interestingly, both
Ras and Tnf? induce growth arrest in keratinocytes in vitro (4,
21), and an attractive possibility is that keratinocytes with
blocked NF-?B signaling have lost the growth arrest response to
both Ras and cytokines.
We have previously reported that keratinocytes in K5-I?B?
mice show an abnormal cell cycle arrest in response to gamma
irradiation (16). The effect is only seen in hyperplastic, inflamed
skin whereas primary keratinocytes isolated from K5-I?B? mice
grown in vitro respond normally to ? irradiation (data not
shown), suggesting that inflammation and?or increased expres-
sion of cytokines contribute to the altered DNA-damage re-
sponse in the K5-I?B? mice.
Expression of I?B? in human keratinocytes has also been
mice and K5-I?B? mice with SCC stained for active MAPK. (Scale bar, 100 ?m.)
(c) Whole-cell extracts from keratinocytes isolated from wild-type or K5-I?B?
mice, blotted and stained for Cdk4 and ?-actin. (d) Tail skin section from
adult mice stained for Cdk4. (Scale bar, 100 ?m.)
Activation of the Ras-pathway or overexpression of Cdk4 does not
cannot be recovered in K5-I?B??Tnfr1?/?mice by Tnfr1?/?bone marrow-
figure. (Scale bar, 50 ?m.)
Development of inflammation, epidermal hyperplasia, and SCC
www.pnas.org?cgi?doi?10.1073?pnas.0307106101 Lind et al.
find such an up-regulation in primary keratinocytes from K5-
I?B? mice in vitro or in K5-I?B? skin in vivo before the
skin, the expression of Cdk4 is increased. However, this increase
likely only reflects increased proliferation (Fig. 4e). Cdk4 is
expressed in proliferating cells of the basal cell layer in normal
skin (Fig. 4d). Although not excluding a role for Cdk4 up-
regulation, our data indicate that other I?B?-dependent alter-
ations contribute to the phenotype.
The massive infiltration of granulocytes, with a domination of
neutrophils that is seen in the skin of K5-I?B? mice, is striking
(Fig. 2 and M.v.H., unpublished observations). A critical role of
neutrophils in driving the hyperproliferation in the flaky skin
mouse mutant has been demonstrated, and it is possible that
It is known that neutrophils produce reactive oxygen species,
lipid mediators, and proteolytic enzymes that potentially can
affect keratinocyte proliferation (23–25). Of interest is also the
which we identify as plasmacytoid dendritic cells. Plasmacytoid
dendritic cells have recently been shown to play a role both in
inflammation of the skin and in cancer development, where they
have been proposed to be attracted by the tumor cells and inhibit
tumor-specific immunity (13, 14).
Although only a minor fraction of human SCC harbor Ras
mutations (26, 27), it is interesting to note that recent data
suggest that human SCC may show a block in NF-?B signaling
based on nuclear exclusion of the RelA NF-?B subunit (ref. 4
and M.v.H., unpublished results). Furthermore, inflammation
and chronic wounds are associated with development of human
SCC, as in the development of SCC in burn scars, chronic venous
stasis ulcers, and discoid lupus erythematosus (28–30). Progres-
sion of actinic keratosis to SCC has recently also been shown to
be associated with an inflammatory stage (31).
Taken together, these findings imply that the combination of
inflammatory stress and inhibition of NF-?B signaling is of
relevance for SCC development, creating a cellular environment
enhancing generation of DNA alterations [e.g., induced by
oxidative stress (32)], and?or selecting cells with preexisting
mutations (33). The K5-I?B? mouse model we describe will be
valuable for unraveling the detailed molecular mechanisms and
signals involved in the interplay between perturbed NF-?B
signaling in keratinocytes and inflammation in SCC develop-
ment. Furthermore, the critical role of local NF-?B and Tnfr1-
mediated signaling provides the rationale for novel approaches
to treat SCC and prevent progression of premalignant lesions.
We thank Åsa Bergstro ¨m for expert technical assistance and Peter
Zaphiropoulos and Susan Warner for helpful suggestions and comments.
This study was supported by a grant from the Swedish Cancer Fund and
National Institutes of Health Grant P01 AR47898-02. B.R. is supported
by grants from the Karolinska Institutet and Wallenberg Consortium
North for Functional Genomics.
1. Karin, M. & Ben-Neriah, Y. (2000) Annu. Rev. Immunol. 18, 621–663.
2. Seitz, C. S., Lin, Q., Deng, H. & Khavari, P. A. (1998) Proc. Natl. Acad. Sci.
USA 95, 2307–2312.
3. van Hogerlinden, M., Rozell, B. L., Ahrlund-Richter, L. & Toftgård, R. (1999)
Cancer Res. 59, 3299–3303.
4. Dajee, M., Lazarov, M., Zhang, J. Y., Cai, T., Green, C. L., Russell, A. J.,
Marinkovich, M. P., Tao, S., Lin, Q., Kubo, Y. & Khavari, P. A. (2003) Nature
5. Makris, C., Godfrey, V. L., Krahn-Senftleben, G., Takahashi, T., Roberts, J. L.,
Schwarz, T., Feng, L., Johnson, R. S. & Karin, M. (2000) Mol. Cell 5, 969–979.
6. Schmidt-Supprian, M., Bloch, W., Courtois, G., Addicks, K., Israel, A.,
Rajewsky, K. & Pasparakis, M. (2000) Mol. Cell 5, 981–992.
7. Smahi, A., Courtois, G., Vabres, P., Yamaoka, S., Heuertz, S., Munnich, A.,
Israel, A., Heiss, N. S., Klauck, S. M., Kioschis, P., et al. (2000) Nature 405,
8. Hartman, D. L. (1966) Arch. Dermatol. 94, 632–635.
9. Simmons, D. A., Kegel, M. F., Scher, R. K. & Hines, Y. C. (1986) Arch.
Dermatol. 122, 1431–1434.
10. Korstanje, M. J. & Bessems, P. J. (1991) Dermatologica 183, 234–236.
11. Sakai, H., Minami, M., Satoh, E., Matsuo, S. & Iizuka, H. (2000) Dermatology
12. Pasparakis, M., Courtois, G., Hafner, M., Schmidt-Supprian, M., Nenci, A.,
Toksoy, A., Krampert, M., Goebeler, M., Gillitzer, R., Israel, A., et al. (2002)
Nature 417, 861–866.
13. Wollenberg, A., Wagner, M., Gunther, S., Towarowski, A., Tuma, E., Moderer,
M., Rothenfusser, S., Wetzel, S., Endres, S. & Hartmann, G. (2002) J. Invest.
Dermatol. 119, 1096–1102.
14. Zou, W., Machelon, V., Coulomb-L’Hermin, A., Borvak, J., Nome, F., Isaeva,
T., Wei, S., Krzysiek, R., Durand-Gasselin, I., Gordon, A., et al. (2001) Nat.
Med. 7, 1339–1346.
15. Sims, J. E., Gayle, M. A., Slack, J. L., Alderson, M. R., Bird, T. A., Giri, J. G.,
Colotta, F., Re, F., Mantovani, A., Shanebeck, K., et al. (1993) Proc. Natl. Acad.
Sci. USA 90, 6155–6159.
16. van Hogerlinden, M., Auer, G. & Toftgård, R. (2002) Oncogene 21, 4969–4977.
17. Zhang, J. Y., Green, C. L., Tao, S. & Khavari, P. A. (2004) Genes Dev. 18,
18. Moore, R. J., Owens, D. M., Stamp, G., Arnott, C., Burke, F., East, N.,
Holdsworth, H., Turner, L., Rollins, B., Pasparakis, M., et al. (1999) Nat. Med.
19. Suganuma, M., Okabe, S., Marino, M. W., Sakai, A., Sueoka, E. & Fujiki, H.
(1999) Cancer Res. 59, 4516–4518.
20. Cheng, J., Turksen, K., Yu, Q. C., Schreiber, H., Teng, M. & Fuchs, E. (1992)
Genes Dev. 6, 1444–1456.
21. Pillai, S., Bikle, D. D., Eessalu, T. E., Aggarwal, B. B. & Elias, P. M. (1989)
J. Clin. Invest. 83, 816–821.
22. Schon, M., Denzer, D., Kubitza, R. C., Ruzicka, T. & Schon, M. P. (2000)
J. Invest. Dermatol. 114, 976–983.
23. Krischel, V., Bruch-Gerharz, D., Suschek, C., Kroncke, K. D., Ruzicka, T. &
Kolb-Bachofen, V. (1998) J. Invest. Dermatol. 111, 286–291.
24. Kragballe, K., Desjarlais, L. & Voorhees, J. J. (1985) Br. J. Dermatol. 113,
25. Rogalski, C., Meyer-Hoffert, U., Proksch, E. & Wiedow, O. (2002) J. Invest.
Dermatol. 118, 49–54.
26. Campbell, C., Quinn, A. G. & Rees, J. L. (1993) Br. J. Dermatol. 128,
27. Rumsby, G., Carter, R. L. & Gusterson, B. A. (1990) Br. J. Cancer 61, 365–368.
28. Copcu, E., Aktas, A., Sisman, N. & Oztan, Y. (2003) Clin. Exp. Dermatol. 28,
29. Baldursson, B., Sigurgeirsson, B. & Lindelof, B. (1995) Br. J. Dermatol. 133,
30. Sulica, V. I. & Kao, G. F. (1988) Am. J. Dermatopathol. 10, 137–141.
31. Berhane, T., Halliday, G. M., Cooke, B. & Barnetson, R. S. (2002) Br. J.
Dermatol. 146, 810–815.
33. Thilly, W. G. (2003) Nat. Genet. 34, 255–259.
Lind et al. PNAS ?
April 6, 2004 ?
vol. 101 ?
no. 14 ?