From the Department of Medicine and Sealy Center for
Molecular Science and the ¶ Department of Pediatrics,
University of Texas Medical Branch, Galveston, Texas 77555-1060
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ABSTRACT |
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The alveolar macrophage-derived peptide tumor
necrosis factor- (TNF
) initiates pulmonary inflammation through
its ability to stimulate interleukin-8 (IL-8) synthesis in alveolar
epithelial cells through an incompletely described transcriptional
mechanism. In this study, we use the technique of ligation-mediated
polymerase chain reaction (LMPCR) to record changes in transcription
factor occupancy of the IL-8 promoter after TNF
stimulation of A549 human alveolar cells. Using dimethylsulfate/LMPCR, no detectable proteins bind the TATA box in unstimulated cells. By contrast, TNF
rapidly induces protection of G residues at
79 and
80 coincident with endogenous IL-8 gene transcription. Using DNase I/LMPCR, we
observe inducible protection of nucleotides
60 to
99 (the TNF
response element) and nucleotides
3 to
32 (containing the TATA
box). Surprisingly, extensive TATA box protection is only seen after
TNF
stimulation. Using a two-step microaffinity isolation/Western immunoblot DNA binding assay, we observe that the NF-
B subunits Rel
A, NF-
B1, and c-Rel inducibly bind the TNF response element; these
proteins undergo rapid TNF
-inducible increases in nuclear abundance as a consequence of I
B
proteolysis. Furthermore, the peptide aldehyde N-acetyl-Leu-Leu-norleucinal, an agent
that blocks both I
B
proteolysis and NF-
B subunit
translocation, abrogates recombinant human TNF
-inducible IL-8 gene
transcription. These studies demonstrate that IL-8 is activated by a
promoter recruitment mechanism in alveolar epithelial cells, where
NF-
B subunit translocation is required for (and coincident with)
binding of the constitutively active TATA box-binding proteins.
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INTRODUCTION |
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The respiratory epithelium contributes to normal pulmonary
function in its ability to clear inhaled particulates through
mucociliary action, ensure alveolar patency through surfactant
secretion, and facilitate bacterial opsonization through secretory
immunoglobulin production. A large body of evidence now supports an
additional role for the airway epithelial cell to amplify cytokine
signals from pathogen-activated alveolar macrophages into secretion of chemokines, arachidonic acid metabolites, and phospholipid-molecules that recruit additional inflammatory cells into the airway mucosa (1,
2). Of the potent alveolar macrophage-derived cytokines studied, the
secretion of tumor necrosis factor (TNF
),1 in particular, has
been implicated in the pathophysiology of neutrophilic-infiltrating
disorders including acute lung injury from sepsis, silica-induced
pulmonary fibrosis, allograph rejection, and acute respiratory tract
infection (3-6).
The peptide hormone TNF activates signaling cascades by inducing
trimerization of the airway epithelial cell-expressed 55-kDa TNF
receptor type I (TNFR1). Liganded trimeric TNFR1, in turn, recruits TNF
receptor-associated proteins (TRADD, FADD, TRAF2, and others) to its
intracytoplasmic domain to generate intracellular signaling cascades
via second messengers including ceramide, 1,2-diacylglycerol, and
arachidonic acid metabolites (see Refs. 7-9, and references therein).
TNF
-inducible gene activation occurs subsequently through the action
of mitogen-activated protein kinase/Erk kinase kinase-1 (MEKK1; Ref.
10) and/or the NF-
B-inducing kinase (11).
IL-8, a potent 8.5-kDa chemoattractant for neutrophils, is one of the
most potent neutrophilic chemoattractant activities in stimulated
epithelial supernatants (2, 12). In airway epithelium, IL-8 secretion
is potently induced by alveolar macrophage-derived cytokines
TNF, IL-1
/
(2, 13), bacterial cell wall products (14-16), and
respiratory virus infection (13, 17, 18), via a mechanism that, at
least in part, involves enhanced gene transcription.
Although inducible IL-8 gene expression has been investigated
intensively, controversy still exists as to mechanism responsible for
TNF-inducible transcription in epithelial cells. In human gastric
adenocarcinoma cells, TNF
activates IL-8 through a synergistic effect on two regions, one cis element located at nucleotides (nt)
126 to
120 that contains a putative AP-1 binding site, and a second
element at nt
80 to
71 that contains a nuclear factor-
B
(NF-
B) binding site (19). In human bronchial epithelium-derived HS-24 and BET-1a cell lines, an element encompassing nt
130 to
112
was shown to be essential for TNF-inducible IL-8 promoter activity
(20). Finally, in HeLa epithelial cells, TNF
activates IL-8 through
a mechanism involving cooperative binding of nuclear factor-IL6
(NF-IL6) and NF-
B to a cis element encompassing nt
97 to
69 (21,
22). Given the highly tissue-restricted pattern of NF-IL6 expression,
the relevance of this latter study to alveolar epithelium is uncertain
(23, 24). Moreover, these studies have solely relied on transient
transfection assays, a technique that does not always faithfully
reproduce phenomenon of inducible gene expression within chromatin
contexts (25); thus, whether these observations are relevant to
endogenous IL-8 gene expression is problematic.
Therefore, in this study, we examine the mechanism for TNF-inducible
gene transcription in human alveolar epithelial (A549) cells using the
technique of ligation-mediated PCR (LMPCR) to record changes in binding
to the endogenous IL-8 promoter. LMPCR in recombinant human TNF
(rhTNF
)-treated cells demonstrates inducible DMS protection of
guanine (G) bases at
79 and
80 and a DNase I footprint over nt
60
to
99 of the endogenous IL-8 promoter, occurring simultaneously with
the peak in its endogenous transcription (at 0.5-1 h). Surprisingly,
although no stable TATA box binding could be observed in unstimulated
cells, an additional extended footprint from nt
3 to
32, spanning
the TATA box, was observed in rhTNF
-stimulated cells. Transient
transfection assays of 5
-deleted and site mutations of IL-8
promoter/luciferase plasmids demonstrate nt
60 to
99 is a
TNF-response element (TNFRE). Inducible TNFRE DNA binding activity is
composed of the NF-
B subunits Rel A, NF-
B1, and c-Rel. These
subunits undergo rapid increases in nuclear abundance after rhTNF
treatment via a mechanism involving I
B
proteolysis. Finally, the
peptide aldehyde N-acetyl-Leu-Leu-norleucinal, an agent that
blocks I
B
proteolysis and inducible NF-
B binding, completely
blocks endogenous IL-8 expression. We conclude that the NF-
B
transcription factor complex, Rel A, c-Rel and NF-
B1, activate
endogenous IL-8 gene expression through a promoter recruitment mechanism.
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EXPERIMENTAL PROCEDURES |
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Materials--
rhTNF and
N-acetyl-Leu-Leu-norleucinal were obtained from Calbiochem
(San Diego, CA). Lysolecithin
(D-L-
-lysophosphatidylcholine) DNase I and
dimethyl sulfate (DMS) were obtained from Sigma-Aldrich. Thermostable
Vent polymerase was obtained from New England BioLabs (Beverly,
MA).
Cell Culture and Treatment--
A549 human alveolar type II-like
epithelial cells (American Type Culture Collection) were grown in
minimal essential medium containing 10% (v/v) fetal bovine serum, 10 mM glutamine, 100 IU penicillin/ml, and 100 µg
streptomycin/ml. rhTNF was added to growth medium at a final
concentration of 20 ng/ml. For in vitro modification of DNA,
medium containing 1:1200 (v/v) DMS (diluted immediately prior to
addition to cell culture) for 1 min at 37 °C. Afterward, cells were
washed in phosphate-buffered saline and genomic DNA was extracted using
ion-exchange chromatography (A.S.A.P. kit, Boehringer Mannheim) for
piperidine cleavage using previously published protocols (26, 27). For
in vivo treatment with DNase I, A549 cells in 100-mm dishes
were first permeabilized for 1.5 min by 0.5 µg/ml lysolecithin in
buffer A (150 mM sucrose, 80 mM KCl, 35 mM HEPES (pH 7.4), 5 mM MgCl2, 5 mM CaCl2). The permeabilized cells were washed
in 4 ml of buffer A, and treated with DNase I either at 10 µg/ml or
50 µg/ml in solution B (buffer A with 80 mM NaCl
substituted for KCl) for 2 min. The DNase I-treated cells were then
washed with phosphate-buffered saline, and genomic DNA was isolated by
ion exchange chromatography.
Ligation-mediated PCR (LMPCR)--
hIL-8 gene-specific primers
synthesized to amplify the noncoding strand using the downstream (d)
primers were: d1, 5-CACACAGTGAGAATGGTTCCT-3
; d2,
5
-CCGGTGGTTTCTTCCTGGCTC-3
; and d3,
5
-CCGGTGGTTTCTTCCTGGCTCTTGTCCTAGAAG-3
. Due to the presence of an
adenine-thymine-rich tract upstream of nt
170, it was not
possible to synthesize upstream primers with the appropriate annealing
temperature for LMPCR (28) to footprint the noncoding strand.
Unidirectional "linker primers" were synthesized as described (29).
LMPCR was performed as reported, using the following modifications. For
the composition of the first strand synthesis reaction, 2-4 µg of
modified genomic DNA was extended with 0.3 pmol primer d1 in a 30-µl
reaction volume with 2 units of Vent polymerase. Extension parameters
were: denaturation, 5 min at 95 °C; annealing, 30 min at 60 °C;
and extension, 10 min at 76 °C. After ligation of undirectional
linkers, DNA was ethanol-precipitated and amplified using 10 pmol of
primer d2, 10 pmol of linker primer, and 2 units of Vent polymerase in
a total volume of 100 µl. PCR cycling parameters were: denaturation, 1 min at 95 °C; annealing, 2 min at 66 °C; and extension, 3 min at 76 °C for 18 cycles (with 5 s added to extension step with each successive cycle). Samples were iced immediately and labeled using
2.3 pmol of [
-32P]ATP/T4 polynucleotide kinase
end-labeled primer d3 by PCR (denaturation, 3.5 min at 95 °C;
annealing, 2 min at 67 °C; extension, 10 min at 76 °C for 2 cycles). Radiolabeled DNA was ethanol- precipitated, resuspended in
formamide loading dye, and fractionated on a 6% polyacrylamide, 8 M urea sequencing gel. The gel was dried and exposed to
Kodak XAR film using an intensifying screen at
70 °C.
Plasmid Construction and Transient Transfections--
5
deletion constructs of the hIL-8 promoter were produced using the PCR
with
1498/+44 hIL-8/Luc reporter plasmid (18) as a template and a
downstream oligonucleotide hybridizing +86 to +55 (30) of the
luciferase cDNA. The following upstream primers were used to
produce 5
deletions (5
nt indicated by minus; underline indicates BamHI restriction site):
162 hIL-8:
5
-AACTTTGGATCCACTCCGTATTTGATAAGG-3
;
132 hIL-8:
5
-AACAAAGGATCCTGTGATGACTCAGGTTTG-3
;
99 hIL-8:
5
-TGAAGGGGATCCGCCATCAGTTGCAAATCG-3
;
54 hIL-8: 5
CATAATGGATCCATGAGGGTGCATAAGTTC-3
. The PCR products were
restricted with BamHI and HindIII, gel-purified,
and subcloned into the poLUC reporter vector (31). Site-directed
mutagenesis of the NF-
B site in the context of
162/+44 hIL-8 was
introduced using the technique of PCR "SOEING" (18) with the
mutagenic primers (mutations underlined):
5
-TTCATTATGTCAGATTAAATTAAACGATTT-3
and
5
-TTGCAAATCGTTTAATTTAATCTGACAATA-3
. For the
NF-IL6 binding site-mutation, the primers 5
-
GCCATCAGCTACGAGTCGTGGAATTTCCTCTGA-3
and
5
-GAAATTCCACGACTCGTAGCTGATGGCCCATCC-3
were used. hIL-8/LUC plasmids were purified by ion exchange
(Qiagen, Chatsworth, CA) and sequenced to verify authenticity.
Transient transfections were performed in logarithmically growing A549
cells using 8 µg of IL-8/LUC reporter and 1 µg of
CMV-
-galactosidase internal control plasmid (per 60-mm dish) using
DEAE-dextran (18). After 32 h, cells were stimulated in presence
or absence of 20 ng/ml rhTNF
for 6 h. Cytoplasmic lysates were
prepared and independently assayed for luciferase and
-galactosidase
activity (18, 32). Luciferase activity is presented as normalized to
-galactosidase activity to control for plate-to-plate variations in
transfection efficiency.
Northern Blot Analysis--
Total RNA was extracted from control
or rhTNF-treated A549 monolayers by the acid guanidium
thiocyanate-phenol chloroform method (Tri-Solv; Biotecx, Houston, TX)
and RNA abundance quantitated spectrophotometrically. Twenty micrograms
of RNA was then fractionated on a 1.2% agarose-formaldehyde gel and
transferred to nylon-reinforced nitrocellulose membrane (MSI, Westboro,
MA). The gel was then hybridized using body-labeled cDNA
probe generated by PCR either using IL-8 primers (sense,
5
-ATGACTTCCAAGCTGGCCGTGGCT-3
; antisense, 5
-TCTCAGCCCTCTTCAAAAACTTCTC-3
), producing a 302-base pair probe or
actin primers (sense, 5
-ATCTGGCACCACACCTTCTACAAT-3
; antisense 5
-CGTCATACTCCTGCTTGCTGATCC-3
) using standard PCR conditions (18). Blots hybridized at 1 × 106 cpm/ml probe and
were washed and exposed to a phosphorimager screen as described
(33).
EMSAs and Microaffinity Purification--
Nuclear extracts (NE)
of A549 monolayers were prepared using our previously published
protocol (18, 24, 34). Duplex oligonucleotides corresponding to 96 to
69 base pairs of hIL-8 promoter are listed below.
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Western Immunoblot--
Proteins were fractionated by SDS-PAGE
and transferred onto polyvinylidene difluoride membranes as described
(24, 34). Affinity-purified rabbit polyclonal antibodies to Rel A,
c-Rel, NF-B1, and Rel B were obtained commercially (Santa Cruz
Biotechnology, Santa Cruz, CA) and used as primary antibody according
to the supplier's recommendations. Secondary detection was using
horseradish peroxidase-coupled donkey anti-rabbit antibody in the ECL
enhanced chemiluminescence assay (Amersham Life Sciences) as described (18, 24).
Nuclear Run-on Transcriptional Rate Assay--
Nuclear
suspensions from 107 control or rhTNF-treated A549 cells
were used to incorporate [
-32P]UTP into nascent RNA at
30 °C for 45 min (18). After purification of RNA by sequential
trichloroacetic acid and ethanol precipitation, a constant 5 × 106 cpm/ml from each treatment was used to hybridize
immobilized single-stranded plasmid DNAs corresponding to hIL-8,
-actin, and cyclophilin cDNAs. Membranes were washed, and
quantitated by exposure to a phosphorimager screen.
IL-8 Enzyme-linked Immunosorbent Assay (ELISA)-- Immunoreactive IL-8 was quantitated in cell culture supernatants by a double-antibody ELISA kit (R&D Systems) following the manufacturer's protocol.
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RESULTS |
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Kinetics of rhTNF-inducible IL-8 Transcription in A549
Cells--
Human alveolar epithelial A549 cells were selected for
study because they maintain features of type II pneumocytes through their morphological appearance, ability to secrete surfactant, and
susceptibility to respiratory syncytial virus infection, all of which
are features of human alveolar cells (2, 18, 35). Immunoreactive IL-8
was quantified by ELISA in A549 cell culture supernatants after
stimulation with 20 ng/ml rhTNF
(Fig.
1A). A statistically
significant 3-fold increase in IL-8 production was detected after
2 h (as compared with control supernatants); immunoreactive IL-8
continued to accumulate to a 53-fold increase at 24 h. Northern
blot assays were performed to determine changes in steady state IL-8
mRNA abundance at times preceding the changes in protein secretion
(0-8 h; Fig. 1B). In the absence of rhTNF
, IL-8 mRNA
was barely detectable. However, accumulation of a single 1.8-kilobase
IL-8 transcript was strongly induced by rhTNF
treatment, starting at
0.5 h and peaking at 2 h (a 110-fold increase). To determine
the transcriptional component for induction of IL-8 mRNA, the
kinetics of IL-8 gene transcription was measured by nuclear run-on
assay for 0-4 h after rhTNF
treatment (Fig. 1C). Slightly preceding the maximal changes in mRNA, IL-8 transcription rapidly peaked at 0.5-1 h (a 20-fold induction). Although IL-8 transcription declined thereafter, it still remained elevated compared
with control levels at 2 h (7-fold) and 4 h (3-fold). The
slight lag in mRNA accumulation is probably reflective of the time
required for the longer-lived IL-8 mRNA levels to reach steady
state within the cell.
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Identification of rhTNF-inducible Binding Sites on the
Endogenous IL-8 Promoter--
To determine whether rhTNF
changes
the composition of proteins interacting with the IL-8 gene promoter,
two variations of the LMPCR (27) technique were used using IL-8
gene-specific primers. First, intact cells were treated with the
membrane-soluble alkylating agent DMS, an agent that methylates exposed
guanine (G) bases in the DNA major groove. The location of G
methylation can be determined following extraction and piperidine
cleavage. Fig. 2A shows the
results of DMS/LMPCR by amplifying the DMS-modified coding strand from
control or rhTNF
-treated cells (at times corresponding to the peak
in IL-8 transcription). As controls, deproteinized genomic DNA was
DMS-treated and amplified in parallel. Surprisingly, in unstimulated
cells, no stable protein-DNA interaction could be detected by DMS/LMPCR
(compare lanes 1 and 2). In contrast, rhTNF
treatment induces a rapid protection of G residues at nt
79 and
80
and the appearance of several hypersensitive sites, the most prominent
at nt
-82 of the coding strand (asterisks and
arrow, respectively, in Fig. 2A). For additional
information on genomic protein binding sites, LMPCR was used to assay
IL-8 promoter occupancy after DNase I treatment in vivo.
DNase I can be introduced into cultured cells after transient
permeabilization of the cell membrane by lysolecithin without
disruption of protein·DNA interaction (36). Fig. 2B
demonstrates DNase I/LMPCR of deproteinized genomic DNA compared with
control or rhTNF
-treated A549 cells. As compared with the DNase I
digestion pattern of deproteinized DNA, in unstimulated cells, a
protected region between nt
55 to
68 and a larger extended
protection could be detected from nt
99 to ~
147 (the upper border
was indistinct) with a hypersensitive site at nt ~
115. Importantly,
no stable protein-DNA interaction was detected over the proximal
promoter from nt
3 to
32 (a region containing the TATA box). By
contrast, after 1 h of rhTNF
treatment, two inducible regions
could be identified: 1) nt
3 to
32 (corresponding to the TATA box),
and 2) nt
60 to
99 (containing the G residues inducibly protected
using DMS/LMPCR assay). Also in the rhTNF
-treated ladder, the
indistinct upstream border of the constitutive protected region became
hypersensitive to DNase I cleavage, probably indicating a
conformational change of the DNA. A summary of the DMS- and DNase
I/LMPCR results is shown in Fig. 2C. That TATA box binding is rhTNF
-inducible implies a promoter-recruitment model in IL-8 gene
activation (see "Discussion").
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The TNF Response Element Maps to the Inducible Contact Sites on the
IL-8 Promoter--
Both techniques of DNA modification indicated
inducible protein binding to region contained in nt 60 to
99, and a
constitutive site containing the putative AP-1 site nt
120 to
126.
To assay for which cis elements are important in inducible activity,
transient transfections of 5
-deletions (Fig.
3A) or site mutations of the IL-8 promoter linked to luciferase reporter genes (Fig. 3B)
were performed.
162/+44 hIL-8/LUC is 28 ± 5-fold inducible by
20 ng/ml rhTNF
treatment (X ± S.E.,
n = 8 independent transfections). Fig. 3A
shows the results of a representative transfection where -fold
inducibility of
162/+44,
132/+44,
99/+44 and
54/+44 hIL-8/LUC
reporters are compared. -Fold induction of 5
deletions to
99/+44 and
longer promoters are indistinguishable, but deletion to
54 is
significantly less rhTNF
-inducible, indicating the region nt
54 to
99, containing the rhTNF
-inducible LMPCR footprint, plays an
indispensable role in promoter activity. Fig. 3B shows the
kinetics of luciferase reporter induction for the
162/+44 hIL-8/LUC
wild-type (WT) plasmid in comparison to the same promoter containing a
purine to noncomplementary pyrimidine mutation in the G residues
79,
80 and its mirror half-site on nt
72 and
73 on the noncoding
strand (designated
162/+44 hIL-8
/LUC). In the absence of
rhTNF
, the
162/+44 hIL-8
/LUC reporter was consistently less
active than the WT reporter, at 25 ± 17% of the WT activity
(n = 4). Moreover,
162/+44 hIL-8
/LUC was inert to rhTNF
stimulation (Fig. 3B), indicating an absolute
requirement of these residues for rhTNF
-inducible transcription.
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Characterization of rhTNF-inducible Proteins Binding the
TNFRE--
EMSAs were performed to identify the kinetics and
specificity of proteins binding to the TNFRE. As shown in Fig.
4A, rhTNF
rapidly induced
the binding of three complexes (Ci1-Ci3) to
the TNFRE with kinetics similar to those observed for the genomic footprinting (Fig. 2A), and for the transcriptional
activation (Fig. 1C) of endogenous IL-8. (Inducible
complexes Ci2 and Ci3 are so closely
comigrating as not to be separable except under long electrophoresis
conditions (cf. Fig. 4C).) After 15 min, complex
Ci1 increased 13-fold, and complexes Ci2/3
increased 12-fold, without changes in Co. To maximize the resolution of
the individual complexes, EMSAs were fractionated longer (Fig. 4,
B and C). Fig. 4B demonstrates binding
specificity, where either unlabeled wild-type TNFRE, or NF-
B mutated
sequences (
) were used to compete Ci1-3 in EMSA.
TNFRE WT, but not TNFRE
, is able to compete Ci1-3 with high affinity. To again exclude the unlikely possibility that
NF-IL6 was present within the Ci complex, we also competed it with unlabeled NF-IL6 binding sites (37). No effect was seen when
either the wild-type or mutant NF-IL6 binding site was used as a
competitor (Fig. 4B). These data indicate that the
rhTNF
-inducible complexes bind with NF-
B binding specificity.
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Recombinant Rel A Contacts the TNFRE in a Manner Indistinguishable
from That of the Ci1-3 Complex on the Endogenous IL-8
Promoter--
DMS/LMPCR indicates that inducible contacts occur on
79 and
80 nt of the IL-8 promoter. To establish that Rel A makes
similar contacts, recombinant Rel A (p65(12-317); Ref. 18) was used in
methylation interference assays. Recombinant Rel A contacts residues
79 and
80 on the coding strand and symmetrical residues
72 and
73 on the noncoding strand (Fig. 4D).
Two-step Microaffinity/Western Immunoblot Assay Demonstrates
Additional NF-B Members Bind the TNFRE--
Because the supershift
assay depends on the titer and affinity of the antibody used, we could
not exclude the presence of other NF-
B family members in the
heterogeneous Ci1-3 complex. To more rigorously identify
other NF-
B family members binding the TNFRE, we used a two-step
microaffinity isolation/Western immunoblot assay. In this assay,
Bt-TNFRE was used to bind control or rhTNF
-stimulated A549 NE.
TNFRE-binding proteins were captured by the addition of streptavidin
agarose beads, washed, and the presence of bound proteins detected
using Western immunoblot. Fig. 4E shows the result of this
assay where the blot is probed for Rel A. Very little Rel A binding is
detectable prior to rhTNF
stimulation, but its abundance increases
dramatically (6-fold) after stimulation. Rel A is not detected when
nonbiotinylated TNFRE WT is included as competitor in the initial
binding step, but it is detectable when nonbiotinylated TNFRE
mutation is used, indicating sequence-specific binding. Using the same
strategy, the presence of other NF-
B subunits was assayed (Fig.
4F). Both NF-
B1 and c-Rel inducibly bind the TNFRE (we
were unable to detect the expression or binding of Rel B or NF-
B2;
data not shown). These data indicate that Rel A, c-Rel, and NF-
B1
inducibly bind the TNFRE.
Rel A, NF-B1, and c-Rel Show rhTNF
-inducible Changes in
Nuclear Abundance--
Western immunoblot was performed on cytoplasmic
and sucrose cushion-purified NE taken from rhTNF
-treated A549 cells
(Fig. 5). 65-kDa Rel A was easily
detected in the cytoplasm, but not the nucleus of unstimulated cells;
by 30 min, a significant increase in nuclear Rel A abundance was
detected. The increase in nuclear Rel A was transient and fell below
the limits of detection after 4 h. 75-kDa c-Rel and 50-kDa
NF-
B1 underwent similar rapid increases in nuclear abundance. Like
Rel A, c-Rel nuclear abundance decreases after 4 h, whereas
nuclear NF-
B1 remains detectable. In data not shown, we have
confirmed that the increase in nuclear Rel A, c-Rel, and NF-
B1 occur
in the absence of new protein synthesis, indicating that the mechanism
is via cytoplasmic-nuclear translocation of preformed protein.
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The Peptide Aldehyde, N-Acetyl-Leu-Leu-norleucinal (Nleu), Blocks
Inducible TNFRE binding and Endogenous IL-8 Expression--
NF-B is
maintained in the cytoplasm through association with the I
B family
of inhibitory proteins, I
B
being the most abundantly expressed
(41, 42). In response to signal-induced degradation of I
B
,
NF-
B undergoes nuclear translocation (43). Inhibitors of I
B
proteolysis, then, would allow us to test the requirement of NF-
B
translocation on endogenous IL-8 transcription. The effect of the
protease inhibitor Nleu, a peptide aldehyde with activity for the 26 S
proteasome complex (primarily responsible for I
B
proteolysis;
Refs. 44 and 45), was used to determine the effect of rhTNF
on
steady state I
B
levels. Fig.
6A shows that rhTNF
induces
a rapid and complete disappearance of I
B
within 15 min, followed
by its resynthesis after 1-4 h. By contrast, in the presence of Nleu,
I
B
proteolysis is significantly inhibited (compare 15 min time
points). In addition, Nleu-pretreated A549 cells do not form the
inducible Ci1-3 complexes in response to rhTNF
(Fig.
6B). These data indicate that Nleu blocks inducible I
B
proteolysis and nuclear translocation of Rel A, c-Rel and NF-
B1 (Ci1-3) in pulmonary epithelial cells. Fig. 6C
demonstrates that inducible expression of
162 hIL-8/LUC is completely
blocked by pretreatment with Nleu. Finally, Northern blot assays were used to determine whether inhibition of Ci 1-3 resulted in
inert IL-8 gene expression in rhTNF
-stimulated A549 cells (Fig.
6D). Under these conditions, induction of IL-8 mRNA is
completely abolished.
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DISCUSSION |
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The alveolar macrophage-delivered cytokine, TNF, is a potent
activator of a cytokine cascade in airway epithelial cells. TNF
secretion plays an import aspect of host-defense to respiratory pathogens and may also play a pathophysiological role in neutrophilic infiltrating and fibrotic pulmonary disease. In this study, we have
examined the mechanism used by TNF
to activate expression of the
IL-8 gene, encoding a product that accounts for the majority of
neutrophilic chemotactic activity in pathogen- or cytokine-stimulated airway epithelial cell supernatants. Although IL-8 gene expression has
been intensively studied in a variety of tissue types, the pleiotropic
mechanisms identified for activation of its promoter warrant a
systematic investigation in airway epithelial cells before rational
design of therapeutic agents to block expression of this cytokine in
lung can be undertaken. Our study demonstrates a rapid and transient
burst in IL-8 expression after rhTNF
stimulation that largely
accounts for changes in IL-8 mRNA.
Inducible Promoter Recruitment as a Mechanism for IL-8 Gene
Transcription--
Inducible gene transcription is due to interactions
between activator proteins and general transcription factors (GTFs)
through one of several experimentally separable mechanisms: 1) promoter recruitment, where activators recruit the GTFs TFIID/TFIIA and TFIIB to
the promoter, or 2) conformational change, where activator binding
alters the conformation of a preexisting preinitiation complex
(reviewed in Ref. 46). To distinguish these mechanisms, we have applied
the technique of LMPCR, a powerful tool used for the identification of
protein-DNA interactions on gene promoters within their native
chromatin context. This study, to our knowledge the first to
demonstrate constitutive and rhTNF-inducible binding of the native
hIL-8 promoter, is surprising because of the paucity of stable
protein-DNA interactions in the proximal IL-8 promoter. LMPCR is, in
part, a population assay that relies on a homogeneous population of
target promoters for its interpretation. Although it is possible that
some proteins engage the TATA box of some, but not all, of the
unstimulated IL-8 promoters in our cultured A549 cells the presence of
which does not result in either a DMS or DNase I footprint, it is clear
that additional protein-TATA interactions occur coincidentally with
NF-
B binding. We interpret these data to mean that IL-8 gene
activation occurs via promoter recruitment, rather than a
conformational change of a preassembled preinitiation complex. In our
data not shown, and in the hands of others (47), TATA binding in
vitro is constitutive and not hormone-regulated.
The TNFRE Is an Essential Cis Element for Inducible IL-8
expression--
Our data in alveolar epithelial cells are not
consistent with previous transient transfection work mapping 130 to
112 as a major site for rhTNF
-inducibility in bronchial cells
(20). In type II A549 cells using both DMS and DNase I protection
assays in LMPCR, we observe a rapid and stable binding of NF-
B-like proteins to a region between nt
60 to
99 that makes specific G
contacts at
79 and
80 indistinguishably from those produced by
recombinant Rel A. Moreover, this NF-
B binding is required for
functional activity of the transiently transfected IL-8 promoter, establishing a direct correlation between genomic binding and transcriptional activation in the same system. In data not shown, we
have demonstrated that rhTNF
-inducible Ci1-3 are also detected in normal human bronchial epithelial cells, indicating that
NF-
B activation is a general phenomenon in pulmonary epithelium.
IB
Degradation and Resynthesis in Controlling IL-8
Expression--
I
B
directly associates with Rel A and serves as
a primary regulator of NF-
B activation through its ability to
inhibit its DNA-binding and nuclear translocation (34, 41). Our Western immunoblots show that inducible I
B
proteolysis occurs in response to rhTNF
in type II pulmonary cells, as has been demonstrated in
other cell lines (34, 43, 55). The role of I
B
degradation in
controlling IL-8 activation is inferred by the inducible rapid proteolysis that exactly coincides with: 1) Rel A, c-Rel, and NF-
B1
nuclear translocation; 2) TNFRE binding of the native IL-8 gene; and 3)
activation of endogenous IL-8 transcription. This association is
further underscored by the effect of the proteasome inhibitor Nleu.
Nleu pretreatment inhibits I
B
proteolysis, NF-
B DNA binding,
and IL-8 gene transcription.
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ACKNOWLEDGEMENT |
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We thank R. Soliz for expert secretarial assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health NIAID Grants 1 R01 AI40218-01A1 (to A. R. B.) and AI/HL 15939-14A1 (to R. G.), NICHD Grant R30HD 27841, and NIEHS Grant P30 ES06676 (to R. S. Lloyd), and by an established investigatorship of the American Heart Association (to A. R. B.).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.
§ To whom correspondence should be addressed: Div. of Endocrinology, MRB 8.138, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1060. Tel.: 409-772-2824; Fax: 409-772-8709; E-mail: arbrasie{at}utmb.edu.
1
The abbreviations used are: TNF, tumor necrosis
factor; Bt, biotinylated; c-Rel, Rel proto-oncogene product; DMS,
dimethylsulfate; EMSA, electrophoretic mobility shift assay; IB,
inhibitor of NF-
B; hIL-8, human interleukin-8; PCR, polymerase chain
reaction; LMPCR, ligation-mediated PCR; NE, nuclear extract; NF-
B1,
nuclear factor-
B 50-kDa subunit; Nleu,
N-acetyl-Leu-Leu-norleucinal (calpain inhibitor I); PAGE,
polyacrylamide gel electrophoresis; Rel A, NF-
B 65-kDa subunit;
rhTNF
, recombinant human tumor necrosis factor
; TNFRE, tumor
necrosis factor response element (of IL-8 gene); ELISA, enzyme-linked
immunosorbent assay; WT, wild-type; TBP, TATA box-binding protein; GTF,
general transcription factors.
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REFERENCES |
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