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INTRODUCTION |
Epidermis is our first line of defense from the environment and
must often respond to various types of injury. Keratinocyte is the
predominant cell type in the epidermis. When an injury occurs
keratinocytes can become hyperproliferative, e.g. in wound healing, or they can become inflammatory, e.g. in contact
dermatitis, or both, e.g. in psoriasis (1, 2). These
responses are coordinated and orchestrated by growth factors and
cytokines that carry signals from cells to cells (3). Keratinocytes
produce paracrine signals to alert fibroblasts, endothelial cells,
melanocytes, and lymphocytes, as well as autocrine signals targeted at
neighboring keratinocytes. In response to such signals keratinocytes
produce and release additional signaling molecules, increase the levels
of their cell surface receptors, and change their cytoskeleton.
The most prominent cytoskeletal proteins in keratinocytes are keratins.
The basal layer of the epidermis produces keratins K5 and K14; the
differentiating, suprabasal layers produce K1 and K10, and the
activated keratinocytes express keratins K6, K16, and K17 (4, 5). These
three keratins are markers of inflammatory and hyperproliferative
processes. Healthy interfollicular epidermis does not contain K6, K16,
or K17; however, these keratins are constitutively expressed in the
outer root sheet of the hair follicle, the nail bed, oral epithelia,
and some other tissues (reviewed in Ref. 6). The expression of keratins
K6 and K16 can be disconnected from proliferation per se,
and cultured keratinocytes express K6 even when their proliferation is
inhibited (7). Thus, the presence of K6 and K16 keratins could be a
marker of inflammation.
In vitro, when grown in cell culture as monolayers,
keratinocytes are already activated and hyperproliferative, expressing copious amounts of K6 keratin (8). However, convenient in
vivo systems for analysis of the effects of TNF
and other
growth factors and cytokines in human skin have not been described.
Therefore, we developed an elegant and convenient "propior
vivo" experimental system, using organ culture explants of human
skin specimens otherwise discarded during surgery (9). We incubated
such biopsies of human skin for relatively short times in minimal
keratinocyte culture medium to which we added
TNF.1 In this system we
demonstrated the specific induction of K6 keratin protein and mRNA
expression by TNF, without the frequently concomitant proliferative signals.
The specific function of the K6 keratin is not fully understood, but
curiously, there are multiple K6 genes in mammals, encoding virtually identical proteins (10, 11). Mutations in K6a
keratin lead to pachyonychia congenita Type I,
whereas those in K6b lead to pachyonychia congenita
Type II, reflecting the slightly different expression patterns of
the two genes (12, 13). The paralogous genes are differentially
expressed in human and in murine tissues, although their expression is
generally restricted to the suprabasal layers of stratified epithelia
(11, 14-16).
This poses an interesting conundrum: if both proliferative and
proinflammatory signals induce K6 expression, is the same K6 protein
induced by both, or does one of the K6 genes respond to the
proliferative and the other to the proinflammatory stimuli? Our
previous results have demonstrated that EGF or TGF, the
proliferative signals, induce the expression of keratins K6b and K16
(17, 18). Therefore, focusing on the K6b keratin, we decided to
determine whether TNF, which is a strong proinflammatory but not a
proliferative signal in keratinocytes (19), affects its expression.
TNF is produced by a wide variety of cells in response to infection
or injury, primarily macrophages and monocytes but also by epithelial
cells including epidermal keratinocytes. A low level of TNF is
present in the upper layers of the healthy epidermis, but its synthesis
and release from keratinocytes is greatly augmented in allergic and
irritant contact dermatitis, infection, UV irradiation, etc. (20, 21).
In these pathological conditions, TNF activates immune responses
through inducing production of proteins such as amphiregulin, TGF,
IL-1, IL-1 receptor antagonist, EGF receptor, and ICAM1 (22-26).
Mice lacking TNF develop normally but have delayed and prolonged
inflammatory responses, confirming the role of TNF in inflammation
(27).
The signaling cascades mediating cellular responses to TNF have been
partly elucidated (28-30). There are two TNF receptors, but
keratinocytes express mainly the 55-kDa receptor, type 1 (31-33). The
most direct TNF effect involves proteins TRADD and TRAF2 and
activates transcription factors NF
B and C/EBP
. The NFB family
includes the proteins p65, p50, and c-Rel, which both homo- and
heterodimerize among themselves (34). These proteins are stored latent
in the cytoplasm, bound to the inhibitory protein, IB. TNF causes
activation of IKKs, kinases that phosphorylate IB and induce its
degradation, which results in activation and nuclear translocation of
the NFB protein (28, 35-37). Knockout of IKK has severe
epidermal phenotype causing incomplete epidermal differentiation (38,
39). On the other hand a knockout of IKK is defective in signaling
from TNF to NFB (40, 41). NFB proteins can interact with
C/EBP, AP1, and other transcription factors to regulate gene
expression (42, 43). In keratinocytes in vitro
overexpression of NFB inhibits proliferation. In epidermis in
vivo NFB is present in all layers but is nuclear only in the suprabasal ones; this suggests a role for NFB in epidermal
differentiation (44). On the other hand, constitutive activation of
NFB in IB knockout mice results in normal epidermal development
and differentiation but a widespread, lethal dermatitis in the first few days of life (45).
TNF as well as other extracellular stimuli activate C/EBP
(46-48). The mechanisms that activate C/EBP have not been fully characterized. C/EBP, also known as NF-IL6 or LAP, interacts with
many other transcription factors, such as the RB protein, the
glucocorticoid receptor, Myc and, importantly for our studies, with AP1
and NFB (42, 49-54). In epidermis the C/EBP proteins are
differentially expressed during differentiation (55, 56). Whereas
knockout mice lacking C/EBP have no cutaneous phenotype (57),
overexpression of C/EBP in keratinocytes causes growth arrest and
induction of early differentiation markers (58).
To determine the roles of TNF-activated transcription factors in
regulating K6b keratin gene expression, we have used the clone containing the promoter of the human K6b keratin gene
(59, 60). The promoter contains several sites that bind transcription factors responsive to extracellular stimuli (5, 59, 60). By using
transfection experiments, gel shifts, and footprinting, we have mapped
the TNF-responsive element. We determined that both NFB and
C/EBP act through the same DNA sequence. Only C/EBP binds this
DNA directly and NFB does not. By using specific inhibitors and
antisense oligonucleotides, we have shown that both NFB and C/EBP
are essential for the regulation by TNF, and we propose that a
complex containing NFB and C/EBP binds the K6b
promoter through the C/EBP DNA binding domain to convey the TNF
signal. Finally, we physically separated the DNA element responsive to TNF, NFB, and C/EBP from the element responsive to EGF and AP1, thus showing that the inflammation and hyperproliferation in
keratinocytes are distinct and independent processes.
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EXPERIMENTAL PROCEDURES |
Organ Culture Explants of Normal Human Skin--
Pieces of
normal human skin were obtained immediately after surgery. They were
cut into pieces approximately 5 mm3 and incubated in
keratinocyte basal medium, KBM, (keratinocyte-SFM, Life Technologies,
Inc.) with or without TNF (50 ng/ml, Intergen), in a humidified
incubator at 37 °C for 24 h. Generally, we use 24-well culture
dishes with up to 5 pieces in the same well and enough medium to just
cover the explants. The explants were mounted in tissue Tec OCT
compound (Sakura Finetek) and frozen. Sections, 4-6 µm thick, were
obtained with a cryostat (Miles Laboratories), fixed with
methanol/acetone for 10 min, incubated with anti-keratin K6 antibody
(Progen Biotechnik GMBH) at 4 °C overnight, treated with
peroxidase-conjugated anti-mouse IgG secondary antibody (Vecstatin ABC-mouse IgG kit from Vector Laboratories), at room temperature for
1 h, incubated with ABC complex (Vector Laboratories) at room temperature for 1 h, and treated with
3,3'-diaminobenzidine-tetrahydrochloride (Dojindo Corp.) and 0.01%
H2O2 in Tris, pH 7.6, for 2 min. The samples
were observed and photographed under the light microscope (Microphot-FXA, Nikon). Additional antibodies used were from Monosan, Uden Holland; antibodies specific for keratins K5, K8, K10, and K18
were from Progen, Heidelberg, Germany; antibodies specific for
keratins K19 and K17 were from Neomarkers, Freemont, CA; and antibodies specific for K14 and for NFB and C/EBP were
from Santa Cruz Biotechnology.
RT-PCR from Explant Tissue--
Explanted skin samples were
incubated with or without TNF for 16 h and harvested, and total
RNAs were isolated utilizing RNeasy RNA extraction kit from Qiagen
(Santa Clarita, CA). Between 1 and 15 µg of RNA were subjected to
RT-PCR, with Access RT-PCR system from Promega (Madison, WI). By
optimizing the number of cycles (30 cycles) and application amount of
total RNA (1-9 µg), we achieved linear correlation between the
amount of RNA added and the density of bands. We used commercial
primers for glyceraldehyde-3-phosphate dehydrogenase
(CLONTECH, Palo Alto, CA), and the K6 keratin
primers are given in Table I. The PCR products were subjected to
agarose-gel electrophoresis, visualized with ethidium bromide (Sigma)
with a transilluminator from Ultraviolet Products (Upland, CA), and photographed with a photographing unit from Polaroid (Germany). The
densities of bands were quantified by utilizing an image scanner (GT-9000 from Epson, Tokyo, Japan).
Immunofluorescence of Cultured Keratinocytes--
Human
epidermal keratinocytes in the fourth passage were plated on glass
coverslips and grown for 24 h in KBM. The cells were then treated
with TNF, washed twice with phosphate-buffered saline, and then
fixed and methanol/acetone (1:1) for 5 min. The coverslips were stained
with NFB- and C/EBP-specific antibodies. As the secondary
antibodies we used anti-mouse immunoglobulin G-fluorescein isothiocyanate conjugate absorbed with human serum proteins or anti-rabbit immunoglobulin G-fluorescence isothiocyanate conjugate absorbed with human serum proteins (both from Sigma).
DNA Constructs--
The plasmids containing keratin promoters
and the control plasmids pRSVZ have been described previously (17, 18,
60). The plasmids containing the IL-8 promoter and (NFB)3-CAT were gifts from J. Vilcek (52); and those expressing NFB proteins were
from A. Beg and D. Baltimore (35); those expressing C/EBP were
from S. Chen-Kiang (61); IB and His-NFB were from S. Ghosh
(62, 63), and CHOP was from D. Ron (64).
Additional K6b promoter constructs were prepared by PCR with
Thermus aquaticus DNA polymerase under conditions suggested
by the manufacturer (Perkin-Elmer). All DNA primers, including the phosphorothioate-modified ones used in antisense experiments, were
either synthesized on a Amersham Pharmacia Biotech Gene-Plus Synthesizer or provided by the Kaplan Comprehensive Cancer Center Core
Facility. They are listed in Table I. To
create the deletions of the K6b promoter we used PCR with
K6CAT as a template, a common proximal primer starting just upstream of
the ATG translation initiation codon and a series of nested distal
oligonucleotides (Table I). To create point mutations in the responsive
element, we performed a two-round PCR mutagenesis procedure (65).
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Table I
Oligonucleotides used in PCR, electrophoretic mobility shift assays,
footprinting, RT-PCR, and antisense experiments
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To clone the responsive element into a heterologous vector, we
amplified the DNA using PCR and cloned it into the enhancer trap TK-CAT
vector (Promega). The CAT activity of the resulting plasmid was
compared with that of control plasmid TK-CAT (Promega). The sequences
of the DNAs inserts were confirmed by the Kaplan Comprehensive Cancer
Center Core Facility, using the dideoxy plasmid sequencing method. All
DNAs used in transfections were purified using the Magic Megapreps DNA
purification system (Promega).
Cell Growth and Transfection--
Normal epidermal keratinocytes
from human foreskin were a generous gift from Dr. M. Simon. The
cultures were initiated using 3T3 feeder layers as described (66, 67)
and then frozen in liquid N2 until used. Once thawed, the
keratinocytes were grown without feeder cells in defined serum-free
keratinocyte growth medium, KGM, supplemented with bovine pituitary
extract epidermal growth factor, insulin, thyroid hormone, and
hydrocortisone (keratinocyte-SFM, Life Technologies, Inc.). Cells were
expanded through two 1:4 passages before transfection and transfected
at ~80% confluence. Transfections using Polybrene with
Me2SO shock were performed as described previously
(68). Each transfection contained either 10 µg/dish of K6CAT or 15 µg/dish of deletion and mutant constructs as well as 3 µg/dish of
pRSVZ. The cells were incubated in KGM 18 h after the
transfection, and then TNF was added in combination with various
other agents, as indicated. The cells were usually harvested 24 h later.
HeLa cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% calf serum and transfected using a modified
calcium phosphate precipitation procedure (68). The medium was changed
14-18 h after transfection, and cells were either left untreated for
another 24 h or stimulated with TNF. The cell harvesting,
disruption by repeated freeze-thaw cycles, and -galactosidase assays
have also been described (68). CAT protein concentration in the
supernatant was measured utilizing either the functional assay (68) or
a CAT enzyme-linked immunosorbent assay kit as suggested by the
manufacturer (Roche Molecular Biochemicals). All CAT values were
normalized for transfection efficiency by calculating the ratio of CAT
activity to -galactosidase in each transfected plate. Each
transfection experiment was separately performed three or more times,
with each data point resulting from duplicate or triplicate transfections.
Gel Shift Assay--
Keratinocytes were grown to confluence in
KGM; the medium was switched to KBM overnight, and then the cells were
either left untreated or treated with 50 ng/ml TNF. At each time
point cells from two 100-mm dishes were harvested by scraping,
collected by centrifugation, washed with phosphate-buffered saline, and
resuspended in 100 µl of buffer containing 20 mM HEPES,
pH 7.8, 450 mM NaCl, 0.4 mM EDTA, 0.5 mM dithiothreitol, 25% glycerol, and 0.5 mM
phenylmethylsulfonyl fluoride. The suspension was freeze-thawed in
liquid N2 three times and centrifuged at 4 °C to remove
debris. Approximately 5 µg of protein from keratinocyte whole-cell
extract was initially incubated for 15 min on ice with or without an
excess of unlabeled competitor, in the presence of 1.5 µg of
poly(dI-dC)-nonspecific competitor (Stratagene) in a final
volume of 25 µl. The binding buffer contained 20 mM
Tris-HCl, pH 7.6, 5 mM MgCl2, 100 mM NaCl, 10% glycerol, 1 mM dithiothreitol,
2% polyvinyl alcohol, and 0.1 mM EDTA. The probe was
labeled using the Klenow fragment (Roche Molecular Biochemicals) and
[-32P]dCTP, 50 µCi (Amersham Pharmacia Biotech) per
reaction, and purified by gel filtration using Sephadex G-50 columns
(Chroma-Spin, CLONTECH). 32P-Labeled
oligonucleotide probe (80,000 cpm) was added, and the incubation was
continued for an additional 30 min on ice. The free and the
protein-bound DNAs were separated on 5% polyacrylamide gels (29:1 = acrylamide:bisacrylamide). The gels were pre-run for 30 min in 1×
TBE buffer and then run for 2-2.5 h at 125 V. The gels were
transferred onto filter paper, dried, and exposed to x-ray film
(X-Omat, Eastman Kodak Co.) at 
70 °C for 24-48 h with screen intensifiers.
Antisense Strategies--
Keratinocytes readily take up offered
DNA. Specifically, short oligonucleotides can be introduced into these
cells even in the absence of cationic lipid (69). We targeted the
antisense oligonucleotides, in phosphorothioate form, to the sequences
including and immediately upstream from the initiation codon. The
oligonucleotides were stored at 70 °C in water until use. Their
sequences are given in Table I. Our approach was first to transfect
HeLa cells with a well characterized responding construct,
(NFB)3-CAT, adding the antisense oligonucleotides both to the
transfected DNA and to the culture medium of transfected cells. The
oligonucleotides were added to the medium immediately after the
transfection and 18 h later; these are the times when we normally
change the medium. The medium used with HeLa cells in antisense
experiments contained 1% fetal calf serum. Usually we added 8 µg of
the oligonucleotide into 1 ml of
Ca3(PO4)2 solution with the
transfected DNA and, additionally, 15 mM of the
oligonucleotide into the medium. Subsequently, the same concentrations
and regimens were used with the K6CAT reporter.
Purification of NFB and C/EBP Proteins--
The plasmid
expressing GST-tagged C/EBP (61) was used to transform BL21(DE3)
Escherichia coli (U. S Biochemical Corp.) which was grown
in LB with ampicillin to A600 of 0.8 and
induced with 1 mM
isopropyl-1-thio--D-galactopyranoside for 3 h. We
used the GST-bulk purification kit that includes glutathione-Sepharose 4B and followed the procedures recommended by the manufacturer (Amersham Pharmacia Biotech). We prepared un-tagged protein using thrombin to remove the GST tag, but we found the tagged and the native
proteins to have indistinguishable properties in gel shift and
footprinting assays. The yield and purity of the proteins were assessed
using standard SDS-polyacrylamide gels. The plasmid expressing
His6-NFB was transfected into the same bacterial host, and its expression was induced the same way. The tagged protein was
purified using the Xpress purification system (Invitrogen). We followed
the manufacturer's recommendations for isolation of both the native
and the denatured-renatured protein, and we found that the native
protein protocol gave significantly higher yields of active protein.
The purified proteins were used in gel shift assays under same
conditions used for the keratinocyte extracts, described above.
DNase I Footprinting Method--
The oligonucleotide containing
the C/EBP binding sequence, 150 ng, was labeled in a kinase reaction
using [
-32P]dATP. Then, 1.5 × 106
cpm of the oligonucleotide was used in the primer extension reaction with Klenow DNA polymerase (Roche Molecular Biochemicals) and purified
by elution from a 2.5% agarose gel into TE buffer, pH 8, at 4 °C
overnight. Two reactions were performed in parallel as follows: A + G
Maxam-Gilbert sequencing (using the reagents and protocols from the NEN
Life Science Products sequencing kit) (70) and DNase I footprinting.
For the footprinting, 25 µl of the binding mix (see the gel shift
protocol above), an amount of purified C/EBP protein, usually 50 ng,
and 50,000 cpm of the probe were incubated at 4 °C. As a control,
the C/EBP protein is omitted from one of the samples. Then, 50 µl
of the solution containing 10 mM MgCl2 and 5 mM CaCl2 was added and incubated 1 min on ice.
Next, 3 µl of the 1:25 dilution of the DNase I (5 units/ml, Roche
Molecular Biochemicals), which we have found optimal for our
conditions, was added, and the incubation continued exactly for 1 min
on ice. The reaction was stopped by adding 90 µl of solution
containing 20 mM EDTA, pH 8.0, 1% SDS, 0.2 M
NaCl and 100 µg/ml of yeast RNA. Next was phenol extraction, followed
by ethanol precipitation. The pellet was resuspended in 1.4 µl of 9 M urea, 1% Nonidet P-40 and after mixing, 4.6 µl of
formamide loading buffer (U. S. Biochemical Corp.) was added. All
samples were heated at 90 °C for 5 min, chilled on ice, and loaded
onto a 12% sequencing polyacrylamide gel. Electrophoresis was run at 2,000 V for 2 h until the blue dye reached bottom of the gel. The
gels were transferred onto filter paper, dried, and exposed to the
x-ray film, as described above.
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RESULTS |
TNF Activates NFB and Induces Expression of Keratin K6b
Protein in Normal Human Skin--
Whereas healthy interfollicular
epidermis does not contain K6 keratin, this protein is present in many
inflammatory and hyperproliferative diseases, where it is induced by
the growth factors and cytokines that orchestrate the inflammatory
responses (14, 59). EGF and TGF cause keratinocytes to
hyperproliferate and can induce the expression of K6b (18), but EGF and
TGF are not inherently proinflammatory. In contrast, TNF does not
cause keratinocytes to proliferate, although it is strongly
proinflammatory (1). Therefore, we decided to determine whether TNF
could induce, in the absence of EGF/TGF, the expression of K6b
keratin in human epidermis. Because convenient systems for analysis of
the effects of TNF and other growth factors and cytokines in human
skin in vivo have not been described, we developed a new and
elegant nearly in vivo experimental system that uses organ
culture of human skin samples otherwise discarded during surgery
(71-74). We obtained 5-mm diameter biopsies of human skin and placed
them in culture medium to which we added TNF. After 24 h the
biopsies were frozen, sectioned, and the presence of K6 determined
using specific antibodies. As a control we used a K17 keratin-specific
antibody; the expression of K17 is induced by interferon but not by
TNF (17, 75). We found that TNF strongly and specifically induced
the expression of K6 in the 24-h period, whereas K17 was not induced
(Fig. 1). Without TNF, the presence of
keratin K6 was detected in the perifollicular and eccrine epithelia,
where K6 is normally seen (Fig. 1). There was no difference in
expression of K8, K18, K5, K14, or K10 between the specimens incubated
with or without TNF (not shown).
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Fig. 1.
TNF activates
NFB and induces expression of keratin K6
protein in explants of normal human skin. Explants of human skin
were incubated in a medium containing TNF, top, or
control medium, bottom. After 24 h, tissue samples were
frozen, sectioned, and stained using antibodies specific for K6 and K17
keratins. Heavy staining with the keratin K6 antibody is present in the
treated but not control samples, whereas K17 is absent from both.
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We note that at the edges of the biopsy after 24 h a weak presence
of K6 can be detected even in the absence of TNF (not shown). This
is presumably due to the release of the endogenous IL-1 by the
peripheral keratinocytes damaged during the surgical procedure. IL-1
can also induce the expression of
K6.2 The expression of K6 at
the edge of the biopsy is most prominent in the first suprabasal layer
of keratinocytes. In contrast, K6 is present in all suprabasal layers
of the TNF-treated samples but most prominently in the granular
layer, the layer of living cells most proximal to the medium that
contains TNF.
In TNF-treated keratinocytes subtle phenotypic changes are observed;
cells treated for 24 h with TNF are flatter, swirled, less
tightly packed, and whereas their nuclei are more prominent, the
cell-cell boundaries are less distinct (Fig.
2).
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Fig. 2.
Transcriptional induction of K6 keratin by
TNF. A, total RNA from
explants of skin samples was extracted, and 1, 3, and 9 µg of RNA
were amplified in RT-PCR with keratin K6 primers and
glyceraldehyde-3-phosphate dehydrogenase (G3PDH)
primers as control. Incubation with TNF induced keratin K6 mRNA
approximately 3-fold compared with untreated samples. B,
densities of PCR bands were calculated using image analyzer and
plotted.
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We performed semi-quantitative RT-PCR with primers specific for K6
mRNA. Incubation of ex vivo skin samples with TNF
resulted in an approximately 3-fold increase of keratin K6 messenger
RNA level (Fig. 3). The linearity of the
assay was confirmed by quantification of the RT-PCR bands obtained with
different amounts of input RNA.
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Fig. 3.
Cultured keratinocytes respond to
TNF. Keratinocytes were grown on
coverslips and incubated in the presence or absence of TNF for 2 days. Note the subtle difference in the appearance of the two
cultures.
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We next examined the effects of TNF on the activation of
transcription factors in skin. Several transcription factors respond to
TNF and transduce its signal to the nucleus; these include NFB,
AP1, and C/EBP (52, 53, 76, 77). Indeed, NFB, which when
activated by TNF enters the nucleus, was predominantly cytoplasmic
in untreated skin samples but exclusively nuclear in the treated ones.
On the other hand, C/EBP was found in the nuclei of both treated and
control skin explants. Both transcription factors behave similarly in
culture and in vivo; C/EBP is always nuclear, whereas
NFB is cytoplasmic in unstimulated cells and enters the nucleus upon
stimulation (not shown).
TNF greatly increased the NFB DNA binding activity (Fig.
4). The activity is detectable 20 min
after addition of TNF, peaks at 1 h, and then returns to the
basal level in the next hour. This time course parallels closely the
one of NFB nuclearization. Within the K6b promoter
sequence we found a cluster of C/EBP sites (see below). By using this
cluster as a probe in gel shift assays, we found a rapid activation of
a DNA binding activity (Fig. 4). Enhanced DNA binding is observed 5 min
after addition of TNF, peaks at 1 h, and then returns to basal
level. These data suggest that addition of TNF activates both NFB
and C/EBP transcription factors in human epidermal keratinocytes.
The AP1 consensus binding activity was fairly high even in the absence
of TNF, most likely due to the EGF in the medium, and did not change
under the influence of TNF (Fig. 4).
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Fig. 4.
Gel shift assays of
NFB, C/EBP, and AP1
proteins using extracts from TNF treated
keratinocytes. Cultured cells were treated with TNF for the
number of minutes, indicated above the lanes, harvested, and
protein extracts prepared. These extracts were allowed to bind to the
NFB consensus, a C/EBP-containing segment of K6b
promoter or the AP1 consensus oligonucleotide (see Table I). The
control lanes contained the 40-min time point with an excess of
unlabeled oligonucleotide. The NFB and C/EBP binding activities
increase in the 1st h, whereas the AP1 binding activity does not.
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TNF Activates the K6b Promoter through NFB and C/EBP
Transcription Factors--
Because the regulation of keratin gene
expression occurs at least partly at the level of transcription, we
transfected keratinocytes and HeLa cells with a DNA construct that
contains the K6b gene promoter driving the CAT reporter and
then incubated the cells in the presence or absence of TNF. We found
that in both cell types TNF activates the K6b promoter
dose-dependently (Fig. 5). The IL-8 gene promoter, used as a positive control, was similarly activated. The effect of TNF is specific for K6b; promoters of several other keratin genes available were not activated by TNF. Although we cannot exclude the possibility that TNF-responsive elements in the other keratin genes lie outside of the available sequences, with the exception of K17, these keratins are not associated with inflamed and proliferative conditions in skin. K17 is induced by
interferon and not by TNF (Fig. 1 and Refs. 17 and 75). The
results of the transfection experiments therefore confirm those
obtained in vivo (Figs. 1 and 3); TNF specifically and dose-dependently activates the promoter of the
K6b keratin gene.
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Fig. 5.
Specific and dose-dependent
activation of the K6b promoter by
TNF. A series of keratin promoter-CAT
constructs was transfected into keratinocytes (A) or HeLa
cells (B), and the cells were incubated with or without
TNF for 24 h, harvested, and the relative CAT levels
determined. The IL-8 gene promoter served as a positive control. The
induction of keratin K6 by TNF is specific because other keratins,
such as K8, K18, K17, K5, K14, or K10, were not induced. C,
the IL-8 and K6b promoters respond with similar
dose curves in keratinocytes. The lengths of the promoter DNAs used are
shown in D. The error bars represent the
differences between duplicate samples in a representative of six or
more transfection experiments.
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In the promoter of the K6b gene one finds consensus binding
sites for NFB, C/EBP, and AP1 transcription factors. We have shown previously that the co-transfection of vectors expressing NFB
and AP1 strongly induces the K6b promoter (60). Here we show
that co-transfection of C/EBP also strongly induces K6b promoter (Fig. 6). Furthermore, NFB,
C/EBP, and AP1 synergize, providing several hundred-fold higher
promoter activity when overexpressed together.
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Fig. 6.
NFB,
C/EBP, and AP1 activate the K6b
promoter. Co-transfection of K6CAT with vectors
overexpressing NFB (RelA), C/EBP, or AP1 (c-Fos and c-Jun)
increase the CAT levels 30-40-fold, but the combinations have a
synergistic 200-320-fold effect.
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To confirm the involvement of NFB in the induction of keratin
K6b promoter activity, we performed transfection assays
using a vector expressing IB, an inhibitor of NFB. The
co-transfection of IB suppressed the constitutive activity of the
K6b promoter, the effect exactly opposite from that of
NFB (see below, Fig. 7).
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Fig. 7.
Antisense oligonucleotides specifically
regulate NFB and C/EBP
transcription factors. Left, (NFB)3CAT, an
NFB-responsive construct, is inhibited by the AS-NFB
oligonucleotide but induced by the AS-IB oligonucleotide.
Conversely, co-transfection of vectors expressing NFB or IB
enhances and suppresses (NFB)3CAT, respectively. Right,
the effect of co-transfected IB and of antisense oligonucleotides on
K6b promoter activity. TNF induced and the
co-transfection of IB-expressing construct suppressed the
K6b promoter. AS-NFB and AS-C/EBP suppressed both the
constitutive and the TNF-induced activity, whereas AS-IB did not
(compare with the control oligonucleotide, As-NCoR). AS-IB abolished
the suppression by the co-transfected IB.
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We used antisense oligonucleotides targeting NFB, IB, or C/EBP
as an alternative approach to examine the involvement of NFB and
C/EBP in the induction of K6b. The phosphorothioate oligonucleotides
specific for NFB and IB mRNAs were designed to bind the
initiation codon and the sequences immediately upstream, sites that
commonly confer efficient antisense blocking (78). The antisense
oligonucleotides were added to the transfected DNA mixture and
subsequently to the medium of the transfected cells. Including the
antisense DNA into the transfection mixture has the advantage of
ensuring that the cells that received the transfected DNA also received
the antisense oligonucleotides. A major advantage of our choice of
sequences is the fact that NFB and IB have opposing effects on
the reporter. The suppression of NFB synthesis, of course, should
inhibit the NFB function, but the suppression of IB synthesis
should enhance the NFB function because IB is an inhibitor of
NFB. Therefore, the system is internally controlled. Nonspecific
effects of the oligonucleotides, e.g. suppression of
transcription commonly observed in most systems, will be equivalent in
both transfected cultures, so that the two sequences serve as a control
for each other. This approach has been used before in another cell type
(78). As the reporter we initially used the NFB-responsive construct
NFB-TK-CAT, which contains three tandem NFB sites linked to the
TK-CAT responder. The AS-NFB oligonucleotide suppressed while
AS-IB increased 3-fold the reporter activity (Fig. 7). As controls,
we co-transfected DNA constructs overexpressing NFB and IB,
which, respectively, increased and decreased the CAT levels, as expected.
When we tested the effects of the antisense oligonucleotides on the
regulation of the K6b promoter we found that AS-NFB not only reduced the constitutive activity of K6CAT but also greatly inhibited its induction by TNF (Fig. 7). In contrast, AS-IB allowed a substantial induction by TNF and abolished the suppression by co-transfected IB. Thus the AS-IB oligonucleotide effects are
antagonistic to those of AS-NFB, as expected.
Next we examined the effects of the AS-C/EBP oligonucleotide and
found that these were similar to the effects of AS-NFB; AS-C/EBP
reduced the constitutive activity of the K6b promoter and
inhibited the induction by TNF (Fig. 7). We note that antisense oligonucleotides have significant nonspecific effects; they can be
toxic and inhibit the overall effect of TNF, as evidenced in our
control sample, AS-NCoR. In the presence of AS-NCoR (and other
unrelated oligonucleotides, not shown) both the constitutive and the
TNF-induced activity of K6b promoter is reduced by
approximately half. AS-NCoR oligonucleotide specifically blocked the
effects of NCoR (79) in control
experiments.3
IB is a specific inhibitor of the NFB transcription factor, and
CHOP is a specific inhibitor of the C/EBP family proteins (80, 81). We
therefore expected IB to inhibit specifically the effects of NFB
and CHOP to inhibit specifically the effects of C/EBP. To our
surprise, co-transfecting IB abolished the activity of both NFB
and C/EBP, and similarly, co-transfecting CHOP abolished both the
C/EBP and the NFB effects (Fig. 8). The effects are specific, because neither IB nor CHOP abolished the
activity of AP1. IB and CHOP did, however, remove the synergistic effect between AP1 and either NFB or C/EBP. The simplest
explanation of these results is that NFB and C/EBP act in concert
to activate the K6b promoter. Inhibiting either component
abolishes the activation. Furthermore, both transcription factors are
responsible for the induction by TNF because co-transfection of
either IB or CHOP abolished the induction by TNF (Fig. 8).
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Fig. 8.
A complex containing both
NFB and C/EBP conveys
the TNF signal to the K6b
promoter. Left, the keratinocytes were
co-transfected with K6CAT and NFB, C/EBP or AP1 (c-Fos + c-Jun),
either with or without co-transfected IB or CHOP. IB and CHOP
abolished the activities of both NFB and C/EBP but not of AP1.
However the synergism between the co-transfected AP1 and the endogenous
NFB and C/EBP is inhibited by IB or CHOP. Right,
co-transfection of either IB or CHOP abolished the induction of K6b
by TNF.
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Importantly, the AP1 transcription factor acts independently. The
AP1-responsive element can be separated physically from the C/EBP + NFB-responsive one (60), and co-transfection of vectors expressing
IB or CHOP does not abolish the AP1 effect (Fig. 8). This means that
AP1, which is responsive to EGF, and the C/EBP + NFB, which are
responsive to TNF, independently regulate the K6b keratin
gene promoter.
Mapping the TNF-responsive DNA Element--
In the promoter of
the K6b keratin gene we found an NFB site, two AP1 sites,
and a cluster of three C/EBP sites (Fig.
9A). To determine whether
these sites constitute the TNF-responsive element, we prepared a
series of deletion constructs leaving progressively shorter DNA
sequences, and we transfected them into HeLa cells and into
keratinocytes. The deletions that remove the NFB site and one or
both of the AP1 sites were fully responsive to TNF (Fig.
9B). This means that the NFB and AP1 sites do not play a
role in the activation of the K6b promoter by TNF. In
contrast, the deletion D172, which removes the most distal C/EBP site
of the cluster, showed a reduced responsiveness to both TNF and C/EBP, whereas D139, which removes all C/EBP sites, was completely non-responsive. This suggests that the C/EBP sites are essential for
TNF signaling. Virtually identical responses were obtained with
co-transfected vector expressing C/EBP (Fig. 9B). This
means that TNF and C/EBP work through the same DNA elements. We
have shown before similar responses of the deletion constructs to
NFB (60), which means that NFB works through the C/EBP sites as well (Fig. 9B). This finding supports our conclusion that
both C/EBP and NFB are necessary for the induction of K6 keratin expression by TNF.
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Fig. 9.
Mapping the
TNF-responsive site. A, the
DNA sequence of the K6b promoter with the consensus binding
sites for transcription factors marked. The end points of the deletions
and of the insertions into TK-CAT are marked with triangles
labeled D and I, respectively. The transcription
start is at +1, and the initiating methionine codon is
underlined. Below is a line diagram of the promoter region,
the deletion, and the insertion constructs. B, responses of
the deletion constructs to TNF and to co-transfected NFB,
C/EBP, and AP1. Note the parallel between the responses to TNF
and C/EBP, and the very similar response to NFB, whereas the
response to AP1 is distinct. C, the mutants of the
responsive element. On the top M1 and
M2 show the changes made in the K6b promoter
sequence. The C/EBP sites were converted into restriction sites for
easy identification. Both mutations severely diminish the responses to
TNF as well as to NFB and C/EBP. D, the responsive
element can confer responsiveness to a heterologous promoter, in this
case the TK promoter. Note that the EGF responsiveness has not been
conferred with the K6b sequences. The lengths of the K6b DNA inserts
into TK-CAT are indicated in A.
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In contrast, deletions D172 and D139 are fully responsive to AP1 (Fig.
9B (60)), which means that AP1 transcription factors play no
role in the signaling by TNF. Parenthetically, the responsiveness to
EGF parallels the responsiveness to AP1, suggesting that AP1 transcription factors convey the EGF signal (18). Thus, the TNF- and
the EGF-responsive elements in the K6b promoter DNA are
distinct and independent.
To analyze the individual roles of the C/EBP sites, we performed
site-directed mutagenesis. Mutations that disrupt either the proximal
or the distal site reduced simultaneously the responsiveness to TNF,
NFB, and C/EBP. Disrupting both sites abolished the responsiveness completely (Fig. 9C). This means that the
C/EBP sites are necessary for regulation by TNF. To determine
whether they are also sufficient for this regulation, we introduced
them into a construct containing the minimal thymidine kinase gene promoter. Two constructs were prepared, the shorter, IS, received 61 bp, and the longer, IL, received 121 bp of the K6b promoter (Fig. 9A). The parent construct, TK-CAT, is not responsive
to TNF, but both IL and IS were induced by approximately 2-3-fold (Fig. 9D). Note that the responsiveness to EGF has not been
transferred with the K6b promoter sequences, which confirms
that the TNF-responsive element of the K6b keratin gene
is distinct from the EGF-responsive element.
To determine which transcription factors bind to the responsive
element, we prepared NFB- and C/EBP-tagged fusion proteins in
E. coli, purified these proteins, and used them in gel shift assays. As the probe we used a synthetic 81-bp DNA oligonucleotide (K6
footprint long, Table I). We found robust binding of C/EBP to
the probe, but NFB did not bind (Fig.
10). The DNA binding of C/EBP is
specific because it could be inhibited by a C/EBP consensus
oligonucleotide but not by an NFB-specific one (Fig. 10). Our
current hypothesis is that NFB acts via protein-protein interaction
with C/EBP. We tested this hypothesis using bacterially expressed,
His-tagged NFB p65 protein in gel shift assays (63, 82). The
purified His-p65 did not bind to the K6b sequence. In the
presence of both NFB and C/EBP we expected to see a
"supershift" of the C/EBP-generated band by p65, indicating a
direct interaction of the two proteins bound to DNA, but we were unable
to demonstrate it. Under the same conditions p65 bound to its consensus
element (not shown).
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Fig. 10.
Gel shift of the
TNF-responsive element with purified
NFB and C/EBP
proteins. NFB and C/EBP, tagged with His6 and GST,
respectively, were expressed in E. coli and column-purified.
They were then used in gel shift assays with an 81-bp oligonucleotide
containing the responsive element (see Table I). Whereas C/EBP binds
the element, NFB does not. Only the C/EBP consensus DNA competes
for the binding, the NFB consensus does not.
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To identify the exact DNA sequences where C/EBP contacts keratin
K6b promoter, we performed footprinting analysis. Addition of purified C/EBP protein protected the C/EBP sites from cleavage by
DNase I in a dose-dependent manner (Fig.
11A). Importantly, all
C/EBP sites are protected, which is congruent with the result from
mutation analysis. The mutations were designed to affect only one
potential C/EBP site, leaving the other two intact. It was therefore of
interest to examine whether these mutants are still competent to bind
C/EBP. Indeed, when we prepared the corresponding oligonucleotides
and used then in footprinting experiments, we found that the M1 mutant
bound C/EBP in the downstream sequences, and M2 bound it in the
upstream sequences. The binding to the double mutant was greatly
reduced throughout the DNA (Fig. 11B).
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Fig. 11.
Footprinting the
C/EBP-bound sites in the K6b
promoter. A, purified C/EBP protein was bound
to the 81-bp fragment to protect the DNA from DNase. The two
exterior lanes contain the unprotected DNA, and the three
interior lanes contain increasing amounts (from left to
right) of the C/EBP protein. The same footprinting
reactions were loaded onto the gel twice, 3 h apart, to resolve
both the shorter (left) and longer fragments
(right). The thin lines connect the same regions
of the two loadings. Maxam-Gilbert sequencing reactions were run on the
same gel to identify the protected region (not shown). The gray
boxes on the sides of the gel indicate the footprints, the bases
protected from DNase by C/EBP. On the top, the sequence
of the K6b promoter is given, with the gray box
indicating the protected region. B, DNA fragments
corresponding to the mutants M1 and M2 were synthesized, labeled, and
footprinted. The gray boxes on the sides of the gel indicate
the protected areas. The bottom part of the footprint is retained in
M1 and the top part in M2; both are attenuated in
the double mutant M1+2. WT, wild type.
C, footprinting using cell extracts. HeLa cells were treated
with TNF, EGF, serum, or left untreated (control). The
left lane contained unprotected DNA. Note that similar
intensities of the bands were obtained near the top of the gel, in the
unprotected region, indicating similar loading efficiencies. The
extract from TNF-treated cells binds to the DNA better than those
from EGF- and serum-treated or untreated cells.
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In an attempt to correlate the in vitro DNA binding results
with the in vivo effects of TNF, we grew HeLa cultures,
starved them for 16 h, and then treated them with TNF, EGF, or
serum. After 40 min extracts were prepared and used in footprinting
experiments. This approach is significantly more difficult and less
reproducible than the approach using purified proteins because the
extracts contain many DNA-binding proteins, which may obscure the
specific binding of C/EBP. However, we could show an increase of
protein binding to the expected sites in the extracts prepared from
TNF-treated cells. The effects of EGF, if any, were much weaker,
whereas the addition of serum was without effect (Fig.
11C).
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DISCUSSION |
Cutaneous response to injury results in the release of cytokines
and growth factors that are proinflammatory and cause
hyperproliferation. Cytokines and growth factors often use overlapping
signal transducing pathways, which results in shared effects. Here we
show that the proinflammatory cytokine TNF directly induces K6b
keratin expression in normal human skin, describe the mechanism of this
induction, and define the TNF-responsive regulatory element in the
K6b gene promoter. The mechanism of
TNF-dependent induction is completely separate and
independent from the EGF-dependent induction of K6b expression described previously (18). Thus, the proinflammatory signals
induce the expression of the very same keratin, K6b, that the usually
concomitant hyperproliferative signals induce.
We demonstrated the effects of TNF, namely the induction of K6b
expression and the activation of NFB transcription factor, both in
cultured keratinocytes and in explants of human skin, a new
experimental system designed to emulate in vivo conditions (9, 71-74).
We have also shown that TNF induces K6b at the transcriptional
level, and we identified NFB and C/EBP as the responsible transcription factors. Deletions and point mutations that show TNF,
NFB, and C/EBP all act at the same DNA site. The participation of
both transcription factors is obligatory, neither C/EBP nor NFB
can act alone. This conclusion comes from experiments in which the
specific inhibitors of NFB and C/EBP, IB or CHOP, respectively, inhibited both transcription factors and from the use of
antisense oligonucleotides, which by depleting one of the transcription
factor also inhibited the other. Particularly informative are the
antisense oligonucleotide studies because in these overexpression of
regulatory proteins is avoided.
NFB and C/EBP are known to interact in regulating gene
expression; however, usually both transcription factors bind DNA (83,
84). The TNF-responsive element in the K6b gene element binds excl
§¶,
, and
TOP
ABSTRACT
INTRODUCTION
