From Boehringer Ingelheim Pharmaceuticals,
Ridgefield, Connecticut 06877-0368 and the § Department of
Biochemistry and Cell Biology and ¶ Genetics Graduate Program,
Institute for Cell and Developmental Biology, State University of New
York, Stony Brook, New York 11794-5215
Received for publication, January 30, 2001
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ABSTRACT |
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The I NF- I Major issues about the IKK signaling pathway remain unexplored,
including the short versus long term effects of NF- Tissue Culture--
70Z/3 and 70Z/3-1.3E2 cells (19, 29) and
CH12-I
Stimulations to activate the IKK signalsome pathway in 70Z/3 and
70Z/3-1.3E2 cells were performed by supplementing growth media with 15 µg/ml LPS and 10 ng/ml PMA (both from Sigma Chemical Co.) for 12 h, or LPS alone for 2 or 12 h or 20 ng/ml recombinant murine IL-1
(Life Technologies, Inc.) for 2 or 12 h prior to isolating total
cellular RNAs. In some experiments, cellular protein synthesis was
inhibited by co-incubation with 100 µM anisomysin (Sigma) to block translational initiation.
An I
CH12-I Probe Preparation, Chip Hybridization, and Data
Analyses--
Total cellular RNAs were extracted from 70Z/3 and 1.3E2
cells with Triazol reagent (Roche Molecular Biochemicals).
Poly(A)+ RNAs were isolated from total RNAs of unstimulated
and LPS+PMA-stimulated cells with Oligotex (Qiagen). Purified RNAs were
converted to double-stranded cDNA with a SuperScript kit (Life
Technologies, Inc.) and an oligo-dT primer containing a T7 RNA
polymerase promoter (Genset). Biotin-labeled cRNAs were generated from
the cDNA samples by an in vitro transcription with T7
RNA polymerase (Enzo kit, Enzo Diagnostics). The labeled cRNAs were
fragmented to an average size of 35-200 bases by incubation at
94 °C for 35 min. Hybridization (16 h), washing, and staining
protocols have been described previously (Affymetrix Gene Chip
Expression Analysis technical manual (33)). Affymetrix murine chips
(mouse 11K set, subA and subB) were used for hybridization. Chips were
stained with streptavidin-phycoerythrin (Molecular Probes) and read
with a Hewlett-Packard GeneArray scanner.
DNA microarray chip data analysis was performed using GENECHIP 3.2 software (Affymetrix). The quantitation of each gene expression was
obtained from the hybridization intensities of 20 perfectly matched and
mismatched control probe pairs (34). The average of the differences
(perfectly matched minus mismatched) for each gene-specific probe
family was calculated. The software computes a variety of different
parameters to determine if an RNA molecule is present or absent
(Absolute Call) and whether each transcript's expression level has
changed between the baseline and experimental samples (Difference
Call). In this work, all chip files were scaled to a uniform intensity
value (1500) for all probe sets. For a comparative chip file (such as
stimulated Wt. versus stimulated Mut.), the experimental
file (stimulated Wt.) was compared with the baseline file (stimulated
Mut.). To minimize false positives, the following criteria were
selected for significant changes for each primary screen: 1) the change
in the average difference across all probe sets was >3-fold; 2) for
induced genes, a difference call of "increase" or "marginal
increase" should be present, and an absolute call of "presence"
should be associated with the experimental file; 3) for suppressed
genes, a difference call of "decrease" or "marginal decrease"
should be present, and an absolute call of "presence" should be
associated with the baseline file.
Hierarchical clustering was performed with the Cluster program
(available at the Stanford Web site) as described previously (35). Genes that showed >3-fold changes in at least two of the Wt.+/Mut.+ comparisons (i.e. IL-1, 2 h; IL-1, 12 h; LPS, 2 h; or LPS, 12 h) were subjected to clustering
analysis for eight stimulated Wt. and Mut. samples (see Fig. 2). Genes
that were detected as absent in all eight arrays were removed. The
average difference values (representing the quantity of mRNA, see
above) of the selected genes (360) were median-centered by subtracting the median-observed value, normalized by genes to the magnitude (sum of
the squares of the values) of a row vector to 1.0. The normalized data
were clustered through one cycle of K-means clustering (K = 5) and then further clustered by average linkage
clustering analysis of Y axis (genes) using an uncentered
correlation similarity metric, as described in the program Cluster.
Average difference values of 50 or less were set to 50 before median
centering and normalization. The clustered data were visualized by the
program TreeView (available at the Stanford Web site).
RT-PCRs and TaqMan Real-time Quantitative PCR--
RT-PCRs were
performed as previously described (36). To establish their relative
qualities, serial dilutions of cDNAs were amplified with
TaqMan Real-time quantitative PCR is based on a fluorogenic 5'-nuclease
assay (37). The same total RNA samples that were used to prepare probes
for microarray hybridization were treated with Dnase I followed by
RNeasy Mini protocol for RNA cleanup (Qiagen). The TaqMan probe
consists of an oligonucleotide with a 5'-reporter dye (FAM) and a
3'-quencher dye (TAMRA). To measure the gene copy numbers of the target
transcript, cloned plasmid DNA or mouse genomic DNA was serially
diluted and used to produce a standard curve as described elsewhere
(38). Data from TaqMan PCR analyses were normalized based on mRNA
copy numbers of GAPDH using the TaqMan rodent
GAPDH control reagents (Applied Biosystems).
The 70Z/3 pre-B line (Wt.) and its 1.3E2 NF-B kinase (IKK) signaling complex is
responsible for activating NF-
B-dependent gene
expression programs. Even though NF-
B-responsive genes are known to
orchestrate stress-like responses, critical gaps in our knowledge
remain about the global effects of NF-
B activation on cellular
physiology. DNA microarrays were used to compare gene expression
programs in a model system of 70Z/3 murine pre-B cells
versus their IKK signaling-defective 1.3E2 variant with
lipopolysaccharide (LPS), interleukin-1 (IL-1), or a combination of LPS + phorbol 12-myristate 13-acetate under brief (2 h) or long term (12 h)
stimulation. 70Z/3-1.3E2 cells lack expression of
NEMO/IKK
/IKKAP-1/FIP-3, an essential positive effector of the IKK
complex. Some stimulated hits were known NF-
B target genes, but
remarkably, the vast majority of the up-modulated genes and an
unexpected class of repressed genes were all novel targets of this
signaling pathway, encoding transcription factors, receptors,
extracellular ligands, and intracellular signaling factors. Thirteen
stimulated (B-ATF, Pim-2, MyD118,
Pea-15/MAT1, CD82, CD40L,
Wnt10a, Notch 1, R-ras,
Rgs-16, PAC-1, ISG15, and CD36) and five repressed (CCR2,
VpreB,
5, SLPI, and
CMAP/Cystatin7) genes, respectively, were bona
fide NF-
B targets by virtue of their response to a
transdominant I
B
SR (super repressor). MyD118 and
ISG15, although directly induced by LPS stimulation, were unaffected by IL-1, revealing the existence of direct NF-
B target genes, which are not co-induced by the LPS and IL-1 Toll-like receptors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B transcription factors are established nuclear regulators
of gene expression programs culminating in a host of cellular stress-like responses that play important roles in an organism's acquired and innate immune responses (reviewed in Refs. 1-4). A
variety of extracellular and endogenous stimuli, including viral and
bacterial infections, oxidative and DNA-damaging agents, hyperosmotic shock, chemotherapeutics, and pro-inflammatory cytokines all result in
NF-
B activation (1, 3-5). NF-
B factors bind to DNA as heterodimers assembled from five known proteins (RelA, c-Rel, RelB,
p50, and p52) with each subunit contacting one-half of a conserved
10-base pair consensus motif (GGGRNWTYCC) (1, 5). NF-
B is generally
held in an inactive state, tethered in the cytoplasm to inhibitory
factors termed inhibitors of NF-
B
(I
Bs)1 (1, 5). Activators
of NF-
B cause the specific phosphorylation of pairs of
amino-terminal serines in the I
Bs, which mark them for
ubiquitination and subsequent proteasomal destruction. NF-
B then
becomes available to activate its nuclear target genes (1, 5).
B phosphorylation is mediated by a high molecular weight
signalsome complex comprising at least two direct I
B kinases
(IKK
and IKK
, also called IKK1/CHUK and IKK2) and a regulatory,
docking/adapter protein (NEMO, NF-
B
essential modulator, also called
IKK
/IKKAP-1/FIP-3) (reviewed in Refs. 3, 4, 6). IKK
and IKK
are atypical serine/threonine kinases possessing an amino-terminal
catalytic domain and two carboxyl-proximal interaction motifs
resembling leucine zipper and helix-loop-helix domains (7-12). The
essential role of the IKK signalsome in NF-
B activation has been
demonstrated in mice lacking IKK
or IKK
(13-18). NEMO is an
essential non-catalytic, adapter/docking component of the IKK
signalsome. Loss of NEMO in cultured cells resulted in a complete lack
of signal-induced NF-
B activation (19-22). More importantly, murine
embryos that were genetically null for NEMO, akin to IKK
KO mice,
succumbed to severe liver apoptosis due to a virtually complete block
in NF-
B activation (23).
B
activation to program cellular gene expression on a genomic scale.
Because NF-
B regulates a variety of ubiquitous and
cell-type-specific gene products in different cellular contexts, we
elaborated the signal-induced, NEMO-dependent gene
expression program in the context of the 70Z/3 murine pre-B lymphoma
line. 70Z/3 pre-B cells recapitulate aspects of the pre-B to immature B
cell transition in response to NF-
B activation (24-26). In response
to LPS, they uniformly differentiate into an immature B lymphocyte-like
state by activating the transcription of a pre-rearranged
-light-chain allele (24-27). By employing immunoselection against
surface-bound IgM, Mains and Sibley (28) isolated spontaneously arising
mutants of 70Z/3 that were completely unresponsive to LPS treatment,
failing to express
light chains (28, 29). Molecular and biochemical analyses subsequently revealed that the 70Z/3-1.3E2 variant was defective in a crucial NF-
B signaling step, making the cells refractory to all NF-
B-activating stimuli, with the exception of
anti-oxidant-insensitive pathways and the HTLV-Tax-1 gene product (19).
More recently, Yamaoka et al. (22) showed that, unlike the
70Z/3 parental line, the 70Z/3-1.3E2 variant lacked NEMO protein expression but their wild type phenotype was rescued by NEMO. With high
density oligonucleotide arrays, we have performed a genomic analysis of
signal-induced, NEMO-dependent NF-
B induction in 70Z/3
versus 1.3E2 cells. A large number of novel induced and repressed NEMO/IKK target genes were revealed. Experiments with an
I
B
(SS/AA) super repressor revealed that many of these genes are
novel and direct targets of NF-
B.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
AA1A2 cells (30) were routinely cultured in growth media
consisting of RPMI 1640 supplemented with 50 µM
-mercaptoethanol, 2 mM glutamine, 10% fetal bovine
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
B
(S32A/S36A) super repressor gene, with serines 32 and
36 mutated to alanines (a kind gift of Dr. Dean Ballard) (31), was
introduced into 70Z/3 cells by retroviral infection. Stably infected
and neomycin- or puromycin-resistant populations of 70Z/3 cells (>1000
clones) were obtained after 12 days of selection in 800 µg/ml
Geneticin (Life Technologies, Inc.) or 1 µg/ml puromycin following
infection with recombinant murine retroviruses harboring I
B
(S32A/S36A)-IRES-Neo or I
B
(S32A/S36A)-IRES-Puro
expression cassettes.
B
AA1A2 cells, a derivative of the CH12.LX line harboring a
constitutively expressed LacI repressor and an IPTG-regulated I
B
(S32A/S36A) super-repressor, was maintained in growth media supplemented with 200 + 400 µg/ml hygromycin and Geneticin,
respectively (30). After inducing their transfected I
B
(S32A/S36A)
gene with 200 µM IPTG for 24 h (30), cells were
stimulated for 2 h with 2.7 µg/ml plasma membranes from
Sf21 insect cells, which had been stably infected with a murine
CD40L-expressing recombinant baculovirus (32).
-actin and GAPDH-specific primers for internal standardization. Similarly, linear response ranges were determined for
each gene to semi-quantify their levels of expression as a function of
LPS stimulation in 70Z/3 and 1.3E2 mutant cells. The sizes of PCR
products corresponded to those expected for each gene. PCR primer pairs
were 22- to 24-mers, and their nucleotide sequences are available from
the authors upon request.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
signaling-defective mutant (Mut.) were initially exposed to a
combination of 15 µg/ml LPS and 100 ng/ml PMA (phorbol 12-myristate
13- acetate) for ~12 h (equivalent to one to two cell generations) to
simulate a condition of long term, constitutive NF-
B activation.
Employing DNA microarrays of 11,800 known cellular genes and expressed
sequence tags (Affymetrix Mu11KsubA and Mu11KsubB Arrays), 1.3% of
genes displayed greater than 3-fold increases whereas 0.9% revealed greater than 3-fold decreases in expression in comparisons of 12-h
stimulated 70Z/3 wild type versus 1.3E2 mutant cells.
Independent microarray screenings of both Wt. and Mut. cells that were
either unstimulated or stimulated by 15 µg/ml LPS or 20 ng/ml IL-1
were also performed. Genes affected 3-fold or more in the primary
screening (Wt.+/Mut.+, 12-h LPS+PMA) were confirmed in a 12-h LPS
screen with only occasional variations. The -fold changes of these
selected hits in various comparisons between Wt. and Mut. stimulated
and unstimulated cells were visualized as a two-color image (Fig. 1). Most hits were also confirmed in
independent microarray screens of stimulations for 12 h with IL-1
or LPS (Fig. 1), indicating that the different stimuli (LPS+PMA, LPS,
and IL-1) regulate these genes by a common mechanism.
View larger version (62K):
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Fig. 1.
Image of -fold change values from various
chip screenings. IKK/NEMO-regulated genes are listed in
order of their -fold changes in 12-h LPS+PMA-stimulated 70Z/3
(Wt.) versus stimulated 1.3E2 (Mut.)
cells. The induced and repressed genes are shown in red and
green, according to the listed color scale. Genes (178 induced and 78 repressed) were selected based on >3-fold changes in
the Wt.+/Mut.+ (LPS+PMA, 12 h) and confirmed with >2-fold changes
in the Wt.+/Mut.+ (+LPS, 12 h) screening. All comparisons were
performed with Wt. and Mut. cells that were stimulated under the same
conditions as indicated. Wt. and Mut.
represent unstimulated Wt. and Mut. samples, respectively. Enlarged
images of genes exhibiting the strongest effects are displayed.
Given that IKK/NF-B activation mediated by LPS, PMA, and IL-1 are
all defective in the NEMO null 1.3E2 line, genes identified in the
primary Wt.+ versus Mut.+ screens should represent direct and indirect targets of the IKK/NF-
B pathway, provided that
IKK/NF-
B-independent NEMO signaling pathways do not exist. Even
though NEMO/IKK
has been clearly established to function as an
essential non-catalytic component of the IKK complex in
vivo, this physiological role need not constitute its only
cellular raison d'être. Because the IKK/NF-
B pathway is
latent in unstimulated cells, similar IKK/NF-
B-dependent
gene expression changes in stimulated versus unstimulated
70Z/3 Wt. cells would be anticipated. Consistently, the majority of the
up-regulated and repressed genes identified in the Wt.+/Mut.+, LPS+PMA
comparison in Fig. 1 were also identified in another microarray screen
comparing 12-h LPS+PMA stimulated to unstimulated 70Z/3 wild type cells
(see Wt.+/Wt.
, LPS+PMA in Fig. 1). As expected, genes affected in the
Wt.+ versus Wt.
screen were not observed in a Mut.+
versus Mut.
screen (see Fig. 1). However, a fraction of
the genes identified in the primary Wt.+ versus Mut.+ screen
were inversely affected in the Mut.+ versus Mut.
screen
(i.e. stimulated genes being repressed and repressed
genes being stimulated) (see Fig. 1). The results indicate that these
latter genes can only be effected by extracellular signals in the
absence of NEMO or NF-
B but not in the presence of NEMO or
NF-
B.
We classified the novel target genes into eight functional categories
in Tables I and II. To ensure that genes identified in the primary Wt.+
versus Mut.+ screens have a higher probability of belonging
to the IKK/NF-B signaling pathway and not to an unknown,
NEMO-dependent, IKK-independent pathway, we employed an
additional selection criteria to assemble the relevant genes. Thus, all
genes exhibiting inverse Mut.+ versus Mut.
effects of
2-fold or more in at least two independent screens were filtered out of
Tables I and II. Table I displays the
known genes identified by the initial screen of 70Z/3 Wt.
versus 1.3E2 Mut. cells stimulated with LPS+PMA for 12 h. In addition to co-stimulating with LPS and PMA for 12 h, we
also performed chip screens of cells stimulated with LPS or IL-1 alone
for 2 and 12 h. Most genes that were identified by the additional
screenings are the same as those identified by the initial LPS+PMA
screen (Table I). Table II displays genes that were revealed by the 2-h LPS, 2-h IL-1, and 12-h IL-1 screens, which were not modulated more than 3-fold in the primary 12-h LPS+PMA
screen. Genes such as Etl-1, TNF-
,
Bcl-2, N-myc, PAC-1, PLA2,
and 2B4 were only affected in the 2-h stimulation time
(Table II). Genes presented in Tables I and II also showed minimal
expression changes in a subsequent screen of Wt.
versus
Mut.
cells (Fig. 1 and Tables I and II), providing additional
confidence that the activated IKK pathway targets them. Taken together,
these results are consistent with most of the genes in Tables I and II
being co-dependent on NEMO and the IKKs to activate the
NF-
B signaling pathway.
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We used the hierarchical clustering method to identify gene expression
patterns in Wt. and Mut. samples stimulated by either IL-1 or LPS for 2 or 12 h. As shown in Fig. 2, most
genes are commonly up-regulated or repressed by IL-1 or LPS. The 2- and 12-h screens showed a remarkable degree of correspondence (Fig. 2),
indicating that the effect of the IKK pathway was sustained between 2 and 12 h for most genes. However, there were classes of genes
whose expression was dramatically up- or down-regulated only in the
LPS12-h sample (Fig. 2). A rapidly repressed class of genes is also
evident that contains smaller subclusters of coordinately regulated
genes (see pre-BCR components VpreB and 5, and the protease
inhibitors SLPI and PN-1 in Fig. 2). Hierarchical clustering also
revealed coordinately controlled classes of 2-h LPS and 2-h
IL-1-specific genes (Fig. 2), representing immediate-early response
genes, that were not detected after 12 h of exposure to either
stimulus (Fig. 2 and Table II). The presence of known NF-
B targets
among the stimulated 70Z/3 up-regulated genes (such as
I
B
, the RANTES chemokine,
lymphotoxin-
, Ig
light chain, macrophage inflammatory protein,
NF-
B P100, C4b binding protein, Bcl-2, and
TNF-
) (reviewed in Ref. 39) verify the efficacy of the
screen. A number of the novel target genes were also revealed in
independent microarray screens of genes in other cell types (monocytes,
macrophages, and mature B cells), which are up- or down-modulated by
multiple NF-
B-activating stimuli, indicating that a significant
portion of these target genes are not unique to the 70Z/3 pre-B cell
background (data not shown). A selected set of novel targets was chosen
for TaqMan real-time quantitative RT-PCRs to verify the microarray data
and to determine their mRNA copy numbers before and after
stimulation (see 12 representative examples in Fig.
3). Each gene responded to 2 and 12 h of LPS and IL-1 signaling in agreement with the primary chip screen
data (see Fig. 3).
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Novel Target Genes Directly Induced or Repressed by
NEMO-dependent Signaling--
Thirty-five up-regulated and
repressed genes (32 novel and 3 known NF-B targets) were selected
for a combination of semi-quantitative RT-PCR and quantitative TaqMan
real-time PCRs to validate their direct or indirect NEMO target status
and to compare their expression time courses as a function of NEMO
activation by different inducers. As internal reference controls,
RT-PCR reactions were performed for Gapdh and
-Actin with limiting amounts of cDNA
templates, prepared from total cellular RNAs of stimulated 70Z/3 and
1.3E2 cells (see GAPDH and
-Actin results for LPS+PMA-stimulated
cells in Fig. 4, A and
B, and Gapdh for IL-1-stimulated cells in Fig. 4C). All but five (Lef-1, Stat1,
Gas2, PtpN8, and Mkp-3) of these 32 novel genes were confirmed as direct NEMO targets of LPS+PMA signaling,
because they were modulated independently of de novo protein
synthesis (Fig. 4B). CD36, CD40L,
CCR7, Wnt10a, BID, B-ATF, Pim-2, MyD118, PAC-1,
Ich-3, Pea-15/MAT1, CD82/KAI1,
Notch 1, Rgs16, UCRP/ISG15,
Mapk/Erk-1, Mirf5, Cyclin D2,
Hexokinase II, and R-ras were directly induced.
In contrast, CCR2, Cyclin D3, CMAP/Cystatin7, PN-1,
5,
VpreB, and SLPI were directly repressed. All but
two (MyD118 and UCRP/ISG15) of these 27 LPS
inducible NEMO-dependent genes were also found to be
similarly affected by IL-1 signaling (see Figs. 3 and
4C).
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Identification of Bona Fide NF-B Targets among NEMO-responding
Genes--
We derived stable populations of 70Z/3 cells expressing a
transdominant I
B
(S32A/S36A) mutant by retroviral infection to determine which of the novel NEMO target genes were also new targets of
NF-
B. I
B
(S32A/S36A) functions like a dominant negative protein that remains tethered to NF-
B subunits even in response to
extracellular NF-
B-activating stimuli, because amino-terminal serine
residues 32 and 36 are mutated to alanines thereby preventing
signal-induced I
B phosphorylation and its subsequent ubiquitination
and proteasomal degradation (31, 40, 41). To enforce expression of the
I
B
SR in large populations of retrovirally infected cells, we
generated bicistronic expression cassettes of I
B
SR in
retroviral vectors, where it was inserted 5' of IRES sequences fused to
either neomycin or puromycin resistance genes. Stable populations of
70Z/3 cells were initially obtained expressing the I
B
SR-IRES-Neo
cassette (70Z/3-IBIN cells). To increase the penetrance of the super
repressor in cells, the 70Z/3 IBIN population was sequentially infected with the I
B
SR-IRES-Puro virus (70Z/3-INIP cells).
70Z/3, 70Z/3-1.3E2, 70Z/3-IBIN, and 70Z/3-INIP cells were stimulated
with LPS for 2 h, and each of the 27 novel genes exhibiting direct
NEMO responses by RT-PCR in Fig. 4B were similarly
re-evaluated. Expression of the IB
SR protein in LPS-stimulated
70Z/3-IBIN and 70Z/3-INIP populations were verified by Western blotting
(data not shown). Similar results were obtained with 70Z/3 cells
harboring either one or two copies of I
B
SR. As shown in Fig.
5A, 18 genes responded to LPS
stimulation in 70Z/3-IBIN and 70Z/3-1.3E2 cells in a similar fashion,
substantiating their NF-
B target status. EBI-3, which was
previously shown to be an NF-
B-dependent gene target in
response to the EBV (Epstein-Barr virus) LMP-1 (latent membrane
protein-1) gene product in human lymphoblastoid cells (42), was
included as a positive control in Fig. 5A. 12 of these 19 genes (including EBI-3) were selected for
quantitative TaqMan real-time PCR with two independent populations of
70Z/3-INIP cells yielding similar results (see representative examples
in Fig. 6). Unlike the RT-PCRs, the
quantitative TaqMan real-time analyses also permitted a comparison of
the relative mRNA copy numbers for each IKK/NF-
B regulated gene
(see Figs. 3 and 6). Individual genes were affected to varying degrees
by the I
B
SR, revealing differential dependencies on
NF-
B for their regulated expression. Nevertheless, the NEMO null
1.3E2 cells exhibited greater penetrance over the 70Z/3-INIP
populations in a number of cases. This was not unexpected, because the
expression of the I
B
SR is likely to be somewhat heterogeneous,
and its ubiquitination and proteasomal destruction are not completely
abolished by its dual serine to alanine mutations (31, 40, 41). The
remaining nine genes, which were direct targets of NEMO in Fig.
4B (CCR7, Mapk/Erk1, PN-1,
Mirf5, CycD2, CycD3, HexII,
BID, and Ich-3), were not significantly affected
in either 70Z/3-IBIN or -INIP cells implying that: 1) they are
regulated by low levels of NF-
B that escaped I
B
SR sequestration or 2) they represent a class of
NEMO-dependent, NF-
B-independent target genes.
Additional experiments are in progress to address these two
possibilities.
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Because these screens were performed in the context of 70Z/3 pre-B
cells, a number of these NF-B target genes might be expected to be
more specific to the pre-B to immature B cell stages of B cell
development. Nine of nineteen bona fide NF-
B target genes were also affected in a similar fashion by an IPTG-inducible I
B
SR in response to CD40 ligand engagement of the CH12 mature B cell line
(see RT-PCRs for R-ras, Pim-2, MyD118,
Pea-15/MAT1, CD36, CD40L,
Rgs16, CD82/KAI1, and EBI-3 in Fig.
5B).
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DISCUSSION |
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Distinct Categories of NEMO-responsive Genes Reveal Differential
Modes of Gene Expression Programming--
Many genes were up- or
down-modulated in a sustained fashion throughout the 2- to 12-h
stimulation regimens, but brief and long term stimulations also
revealed transiently responsive categories, immediate-early genes, and
a delayed responder class (see hierarchical clustering in Fig. 2 and
Tables I and II). Genes including N-myc, PAC-1,
PLA2, and the NF-B target genes Bcl-2 and
TNF-
were more significantly up-modulated within 2 h
of stimulation (Table II). In contrast, Lef-1,
Gas2, PtpN8, and Mkp-3 were repressed
and STAT1 was stimulated by 12 h but not within 2 h of LPS or
IL-1 stimulation (Table I and RT-PCR analyses not shown). Thus, LPS and
IL-1 signaling also established a delayed program of gene expression
with indirect dependence on NEMO. Interestingly, expression levels of
several direct NEMO/NF-
B targets continued to rise throughout the
12-h time course, suggesting that prolonged LPS and IL-1 signaling
could maintain NF-
B activity.
After 2 h of stimulation, six NF-B target genes
(CD36, EBI-3, CD40L,
UCRP/ISG15, and Rgs16) were induced in the 70Z/3
wild type line and were virtually undetectable in the 1.3E2 mutant, indicating that a NEMO-regulated signalsome was absolutely essential for their activation (see Figs. 3 and 4). Levels of each activated gene
remained fairly constant throughout the time course. In the CH12LX
mature B cell line (43), which constitutively expresses nuclear
NF-
B, considerably higher levels of MyD118,
EBI-3, CD40L, and Rgs16 expression
were observed. The expression of these genes was further induced in
response to NF-
B-activating stimuli (30) (see Fig. 5B).
These observations would be consistent with the notion that these genes
turn on at the pre-B/immature B cell transition at the time of NF-
B activation.
Unexpected Classes of Novel NEMO/NF-B-dependent
Repressed Target Genes--
The NEMO-dependent NF-
B
signaling pathway is well known to orchestrate the transcriptional
activation of genes encoding mediators of stress-like responses,
although its potential ability to repress gene expression has not been
examined. Surprisingly, our global screen for novel target genes, which
were regulated by the NEMO signaling pathway, revealed several classes
of repressed genes. Akin to many of the up-modulated targets,
signal-induced repression of a portion of these repressed genes was
also found to be independent of de novo protein synthesis,
implying a direct, NEMO-dependent repression phenomenon.
However, unlike the induced class of target genes, repression was
usually incomplete or partial. In a number of cases, repressed targets
were down-modulated in unstimulated wild type versus mutant
cells and further down-modulated by NEMO-dependent signal
transduction. Some genes were only repressed after prolonged stimulation implying an indirect link to the NEMO-initiated expression program. In addition, expressions of some repressed genes also exhibited more complex regulation being partially up-modulated by LPS
or IL-1 stimulation of 1.3E2 cells, suggesting that they could be
positively influenced by other signaling pathways in the absence of
NEMO. Repression of CCR2/Mcp-1R, CMAP/Cystatin7, SLPI, VpreB, and
5 in
70Z/3 Wt. cells were shown to be dependent on NF-
B, as their levels
of expression in the presence of an I
B
SR rose to those observed
in the 1.3E2 line. The mechanisms of NF-
B-mediated repression in
each of these examples remain to be determined. Conceivably, some
control elements of these genes may preferentially bind to dimers of
p50 and/or p52 NF-
B subunits, excluding the binding of the p65 and
c-Rel transactivation competent subunits. Alternatively, this
phenomenon could be caused by physiological competition between NF-
B
and other transcriptional activators for the same limiting pool of the
p300 or CREB-binding protein transcriptional co-activators (44) or
other limiting co-factors necessary for their optimal transcriptional
activity. Indeed such negative, transcriptional cross-talk between
NF-
B and p53 has recently been described (44).
Not All Direct NF-B Targets Are Induced by Both LPS and IL-1
Signaling--
Interestingly, RT-PCR and quantitative TaqMan PCR
analyses confirmed that MyD118 and UCRP/ISG15
were strongly induced by LPS but unaffected by IL-1 (Figs. 3
4B and data not shown). UCRP/ISG15 and
MyD118 were also tightly clustered in hierarchical
expression analysis (Fig. 2), and both were also found to be direct
targets of NF-
B (Fig. 5A). Similarly, the RANTES
chemokine (Scya5), an established NF-
B target gene (45),
which was dramatically induced by stimulation with LPS or LPS+PMA, was
only marginally affected in response to IL-1 (see Scya5 in
Table I). MyD118 is a mediator of growth suppression and
apoptosis in differentiating cells (46). UCRP/ISG15, a novel
15-kDa ubiquitin homologue, is activated as part of the cellular
response to viral infection, type I interferons, and TNF-
(47,
48).
It is important to note in this context that the Toll family
receptors, responding to LPS and IL-1, both employ MyD88 as a common
adapter protein. MyD88 funnels signals to other adapters and associated
kinases, including IRAK, TRAF6, ECSIT, and MEKK-1, which
bifurcate to activate NF-B via the NEMO/IKK complex and other
mediators of mitogenic and stress responses via parallel MAPK/ERK
cascades (49-51). Indeed MyD88 KO mice presented a severe deficit in
their physiological responses to IL-1 and LPS and a marked delay in the
activation of NF-
B and MAP kinases (52). Our findings demonstrate
that LPS signaling can lead to direct activation of some NF-
B target
genes that do not respond to IL-1. These results imply that unlike the
IL-1 Toll-like receptor, the LPS Toll-like-receptor 4 can also directly
respond via an MyD88-independent mechanism leading to IKK activation.
Alternatively, MyD118, UCRP/ISG15, and
Scya5 could also belong to a unique class of
MyD88-dependent NF-
B target genes requiring the
coordinate, post-translational activation of other transcription
factors, which respond to LPS but not IL-1. In contrast, all
immediate-early IL-1 target genes revealed in this genomic screen also
responded to LPS signaling in agreement with published reports that
MyD88 knockout mice were completely defective in NF-
B
activation by IL-1 signaling.
Novel Effectors of Development and Cellular
Differentiation--
The IKK complex modulated gene products that
represent regulators of cell fate determination and differentiation.
Important extracellular factors or receptors orchestrating the
acquisition of novel cell fates in embryonic development like
Wnt10a (53) and Notch1 (54) were up-regulated
with a direct dependence on NF-B activity (Figs. 5A and
6). Interestingly, Jagged 1, a Notch 1 receptor ligand, has also
recently been shown to be a direct target of NF-
B (55).
Lef-1, an important transcriptional regulator of early T and
B cell development (56), which responds to the Wnt signaling pathway
regulating cell fate and proliferation (57), was repressed in response
to prolonged IKK activation. Furthermore, the VpreB and
5 genes, encoding the proteins of the pre-B
cell receptor that are required during the early phase of B lymphoid cell development, were coordinately repressed in response to both brief
and long term LPS stimulation. Their levels of partial repression were
similar after 2- or 12-h LPS or IL-1 stimulation, suggesting the
recruitment of other regulatory factors or post-transcriptional effects
occurs during the 12-h time course. Notably, the partial repression of
both pre-B cell receptor genes was abrogated by enforced expression of
an I
B
(S32A/S36A) super repressor, identifying them as direct
NF-
B repression targets. VpreB and
5 are positively regulated by early B cell
factor and the E47 helix-loop-helix protein during early stages of B
cell development, but their expressions are extinguished between the
pre-B to immature B cell transition. However, factors involved in their
coordinate, developmentally regulated repression have not been
identified. Our results suggest that NF-
B activation could play a
direct, albeit partial, role in programming the disappearance of early
B cell regulators, which would also be consistent with NF-
B
acquiring constitutive activity during later stages of B cell
differentiation. Experiments with normal, differentiating pro-B and
pre-B cells will be necessary to address the physiological significance
of this interesting phenomenon. In contrast, the RelB NF-
B subunit
was positively autoregulated (Table I), in agreement with the
appearance of constitutively elevated levels of RelB in maturing B
cells (58). The B-ATF activator was also directly induced by NF-
B
activation (Fig. 5A). B-ATF encodes a member of
the ATF/CREB family that is up-regulated in Epstein-Barr
virus-stimulated mature human B cells and likely functions as a
cell-type, tissue-specific modulator of the AP-1 transcription factor
complex (59).
Novel Immunomodulatory Genes--
CD40L induces NF-B responses
upon engaging the CD40 receptor, a member of the TNF receptor
superfamily (reviewed in Ref. 39). Our results now show that CD40
ligand is itself a new direct target of IKK/NF-
B induction. In fact,
CD40L gene expression was virtually undetectable in
unstimulated 70Z/3 and stimulated 1.3E2 mutant cells and was
dramatically induced by LPS and IL-1 stimulation in an
NF-
B-dependent fashion, making its expression critically
dependent on NF-
B signaling in this cellular context. CD40L was also an NF-
B target in the CH12 mature B cell
background (Fig. 5B). CD40L/CD40 binding is critical for the
growth, survival, and differentiation of maturing B lymphocytes (60).
Future experiments will be directed to determining the importance of
NF-
B control for functional CD40L expression by immunoregulatory cells.
Two chemokine receptor genes (CCR7/EBI-1 and
CCR2/Mcp-1R) were respectively identified as novel
stimulated and repressed NEMO signaling targets, with CCR2
repression also being dependent on NF-B activation.
CCR7/EBI-1 was originally identified as a B cell-specific
gene that is rapidly induced by EBV infection and also by a
conditionally activated EBNA2 protein, a known NF-
B activator (61).
CCR7/EBI-1 encodes a seven transmembrane-spanning G
protein-coupled chemokine receptor that has recently been shown to be
essential for the physiologically appropriate seeding of mature
dendritic cells (DCs) and resting T and B cells within the
micro-environments of secondary lymphoid organs (62). Resting T cells
and mature, antigen-presenting DCs up-regulate CCR7/EBI-1 to facilitate
their migration to the periarteriolar lymphoid sheaths of secondary
lymphoid organs (reviewed in Refs. 63-65). Antigen-bearing DCs
emigrate from the epithelia, where they first encounter and become
activated by foreign antigen as immature DCs. Interestingly, maturing,
activated DCs produce large quantities of pro-inflammatory response
chemokines like MIP-1, MCP-1, IL-8, and RANTES (all products by known
NF-
B target genes) at the site of antigen encounter. These
chemokines are thought to maintain the recruitment of immature DCs
expressing cognate receptors (such as CCR1, CCR2, CXCR1, and CCR5)
(64). Interestingly, NF-
B-activating, pro-inflammatory stimuli like
IL-1, TNF-
, and LPS facilitate DC maturation by down-modulating the
expression of inflammatory response receptors (like CCR2/MCP-1R), while
up-modulating the expression of CCR7/EBI-1 (64, 65). Our results in the
context of 70Z/3 pre-B cells suggest that coordinate activation of
CCR7 and repression of CCR2 transcription could
in part be controlled by the NEMO signaling pathway.
EBI-3 was first identified as an EBV (Epstein-Barr
Virus)-induced gene in lymphoblastoid cells and encodes an
immunomodulatory, 34-kDa secreted glycoprotein with homology to the p40
subunit of interleukin 12 and the ciliary neurotrophic factor receptor (66). EBI-3 protein associates with the p35 IL-12 subunit in vivo suggesting that it may function as a modulator of
cell-mediated immune responses (66). EBI-3 was also shown to be
up-modulated by the EBV latent membrane protein-1 (LMP-1),
dependent on NF-B activation (42). Akin to CD40L, quantitative
TaqMan PCR revealed that EBI-3 expression was also critically dependent
on NF-
B activation by either LPS or IL-1 NEMO-dependent
signaling in the 70Z/3 pre-B line (see Figs. 3 and 6).
UCRP/ISG15 is a ubiquitin-like polypeptide that is transcriptionally
induced by IFN-dependent antiviral responses and TNF-
(47, 48). We found that NF-
B activation was essential for UCRP/ISG15 expression by 70Z/3 pre-B cells (Figs.
5A and 6) and that it was also strongly induced by LPS
stimulation of murine RAW 264 macrophages (data not shown). UCRP/ISG15
(ubiquitin cross-reactive protein) is produced by different cell types
and secreted by human monocytes and lymphocytes displaying the
properties of an immune cell modulatory factor. ISG15 was reported to
stimulate the T cell-dependent expansion of B cell-depleted
populations of CD56+ NK (natural killer) cells, to induce IFN
production by T cells and NK cytolytic activity against tumor cell
targets, indicating that it enhances lymphokine-activated killer-like
activity (67, 68). It has been proposed to augment and target the
effects of IFN
or IFN
(67, 68).
Novel Arbitrators of Cellular Growth and Survival--
A number of
the novel target genes attest to the attributes of NF-B activation
to promote cellular growth and survival. Surprisingly, the
N-myc and Pim-2 proto-oncogenes were two of the
most dramatically stimulated genes. N-myc was induced to the
highest degree (more than 70-fold) by LPS or IL-1 signaling in both
Wt.+/Mut.+ and Wt.+/Wt.
comparisons implying that, akin to its
founding family member c-myc, it too is likely to be
regulated by IKK/NF-
B signaling. N-myc functions as an
important transcriptional regulator of cell cycle progression, cellular
growth, and differentiation and has recently been shown to functionally
substitute for c-myc in vivo (69). Unlike
N-myc's transient expression profile, Pim-2
levels were only modestly increased within 2 h of LPS or IL-1
stimulation but climbed to a maximum of 46-fold after 12 h of
exposure to LPS+PMA, at which point N-Myc had turned off
(see Fig. 3 and Table II). Pim-2 and the related
Pim-1 gene encode labile, cytoplasmic serine/threonine
kinases that were discovered by virtue of their ectopic activation via
Moloney Murine Leukemia proviral insertional mutagenesis in T cell
lymphomas (70). Pim-1 and Pim-2 are highly expressed in mitogenically activated hematopoietic cells and are induced by a variety of cytokines that also induce NF-
B (71). Notably, Pim-1 and Pim-2 collaborate with
c-myc to induce neonatal pre-B cell leukemia in doubly
transgenic mice (71). Moreover, Pim-1 and Pim-2
have recently been shown to be targets of gp-130-mediated STAT3
signaling (72). These signals then cooperate with c-myc to
facilitate cell cycle progression by promoting cell survival and
inhibiting the induction of apoptotic pathways (72).
Pea-15/MAT1 also responded in a direct fashion to NF-
B
activation. Pea-15/MAT1 is a microtubule-associated
phosphoprotein containing a death effector domain (73, 74), which has
been reported to elicit cellular survival or cell cycle progression
pathways (73-75). Recent work indicates that Pea-15 binds to
FADD and caspase-8, apical effectors of the TNF apoptotic
pathway (73). In addition, astrocytes of Pea-15 null mice have been
recently shown to be more susceptible to TNF-induced cellular death,
implicating Pea-15 as a cellular survival factor (73). Interestingly,
Bcl-2, a known survival factor and NF-
B target gene, and
the glycolytic enzyme Hexokinase II were also among the
induced genes. The latter two proteins are converging anti-apoptotic
effectors that independently antagonize the opening of mitochondrial
pores, thereby preventing the cytoplasmic release of apoptotic
amplifiers like cytochrome c (76). Finally, a gene encoding
a glucocorticoid-induced leucine zipper (GILZ) factor was
repressed by NEMO signaling (Fig. 1 and Table I). GILZ
is preferentially expressed in lymphoid cells and was reported to
selectively protect T cells from anti-CD3-induced apoptosis in
conjunction with reduced Fas and FasL expression (77).
NEMO Regulates Arbitrators of Cell Cycle Arrest, Apoptosis, and
Metastatic Potential--
The MyD118, BID, and
Ich-3 genes encode proteins that cause cell cycle arrest or
growth suppression; and all were induced in direct response to
NEMO-dependent signaling (see Fig. 4, B and
C). These polypeptides join a growing list of direct or
indirect mediators of cell cycle arrest and cellular death that are
positive targets of NEMO signaling and NF-B activation, including
Fas-ligand, CD95/Fas, p53, Bcl-Xs, c-Myc, and I
B
(reviewed in
Ref. 4). MyD118 was a confirmed NF-
B target in 70Z/3 and
CH12 cells and is a GADD (growth arrest DNA damage) family member,
which regulates growth arrest and apoptosis in differentiating
hematopoietic and non-hematopoietic cells by P53-dependent
and -independent pathways (46). BID encodes a novel death
agonist that heterodimerizes with either agonists (BAX) or antagonists
(Bcl-2) of cell death responses (78). BID counters the
protective effects of Bcl-2 by enhancing mitochondrial permeability to
release cytochrome c and expression of BID, without another
death stimulus, thereby induces ICE-like proteases and apoptosis
(78). Ich-3/Caspase11, a member of the ICE/CED-3
family of cell death genes, induces apoptosis that can be counteracted
by Bcl-2 (79). Ich-3 null mice are resistant to LPS-induced
endotoxicity and are also deficient in IL-1
and IL-1
, because
Ich-3 is essential for ICE activation (79).
Gas2 was also identified among the NEMO-dependent repressed genes, but only after long term LPS stimulation. Gas2, a component of the microfilament system, up-regulates at growth arrest but down-regulates upon cell cycle re-entry (80). Interestingly, in vivo cleavage of Gas2 is believed to lead to some of the microfilament and cell shape changes characteristic of apoptosis; and Gas2 is a known death substrate for Caspase-3 (80, 81).
CD82/KAI1, a member of the tetraspan transmembrane 4 superfamily, was a confirmed NF-B target in 70Z/3 and CH12 cells.
CD82/KAI1 expression was reported to diminish during the
progression of a variety of epithelial malignancies (82, 83) and
appears to function akin to a tumor suppressor by inhibiting pulmonary metastases in experimental metastasis models of prostate cancer and
melanoma (83). It has recently been shown to associate with the EGF
receptor and to suppress EGF-induced lamellipodial extensions and cell
migration by desensitizing EGF-induced signaling (84).
NEMO and NF-B Effects on Cell Cycle Regulators and Other
NF-
B-independent Signaling Molecules--
A number of surprising
cross-talk connections were revealed between the NEMO/NF-
B
activation pathway, components of independent signal transduction
pathways, and regulators of cell cycle progression. Cyclin
D2, known to be up-modulated in B lymphoid malignancies and
preferentially expressed during B cell maturation (85), was a direct
target of the NEMO signalsome (Fig. 4, B and C). TaqMan real-time PCRs also revealed that Cyclin D2
expression was absolutely dependent on NEMO expression (data not
shown). In sharp contrast, Cyclin D3 (86) was directly
repressed, indicating that the NEMO/IKK signaling pathway can have
opposing direct effects on regulators of the G1/S
transition. Interestingly, two pivotal members of independent signaling
pathways, Mapk/Erk1 (87) and Stat1 (88) were
up-regulated as direct and delayed NEMO-dependent responses, respectively. R-ras, a G protein highly related
to the H-Ras proto-oncogene (89), was directly
induced by NF-
B in 70Z/3 and CH12 cells. Rgs16, a
negative regulator of G protein-coupled receptor signaling induced in
response to bacterial infection (90, 91), behaved in a similar fashion
to R-Ras. In addition, Rlf (RalGDS-like
factor), a candidate effector of the Ras and Rap1A GTPases (92), and
Rgs2, a selective negative modulator of Gq signaling (90),
were stimulated and repressed NEMO targets, respectively.
PAC-1, a nuclear dual specificity phosphatase with specificity toward ERK and p38 MAPKs and previously shown to be transiently induced in response to ERK signaling in activated B cells
(93), was directly induced by NF-
B (Figs. 4B,
4C, and 5A). In contrast, Ptpn8
tyrosine phosphatase (94) and Mkp-3 dual specificity protein
phosphatase (95) were repressed as part of the delayed response to
NEMO-dependent signal transduction. A tyrosine kinase
receptor and effectors of intracellular calcium levels were also among
NEMO's immediate-early-responsive genes (see "Signal transduction"
category in Table II). Each of these selected examples points to the
surprising, unpredicted potential of NEMO and NF-
B to alter the
outcomes of diverse intracellular signaling pathways.
Differential Regulation of Pro- and Anti-inflammatory
Mediators--
CD36, a class B scavenger receptor of Ox-LDL
(oxidized low density lipoprotein) (96), was dramatically up-regulated
in response to NEMO-dependent signaling and was also a
confirmed direct NF-B target in 70Z/3 cells and CH12 cells. Ox-LDL
accumulates in cells of atherosclerotic lesions, playing an important
role in foam cell development (96). Interestingly, Ox-LDL has also been
reported to increase CD36 expression itself (97). In
addition, CD36-mediated uptake of Ox-LDL has recently been shown to
contribute to the expression of a variety of inflammatory response
cytokines and to also increase NF-
B activation (98). However, this
is the first direct link between NF-
B signaling and CD36 induction.
In contrast, several protease inhibitors were coordinately repressed in
stimulated 70Z/3 cells, including CMAP/Cystatin 7/Cystatin F
(99), PN-1 (protease nexin-1) (100), and SLPI
(secretory leukocyte protease inhibitor) (101). CMAP/Cystatin 7, a
cystatin-like mediator of liver metastasis that presumably functions as
a protease inhibitor akin to related cystatin proteins (99), was
directly repressed by NF-B activation. PN-1, a serapin class
anti-protease and inhibitor of urokinase-type plasminogen activator
(100), was a NEMO repression target in 70Z/3 cells (Fig. 4,
B and C) and an NF-
B repression target in CH12
cells (data not shown). SLPI is a potent inhibitor of elastase and
cathepsin G serine proteases that suppresses the chronic, destructive
phase of inflammatory reactions and syndromes characterized by overt
tissue destruction. SLPI was a direct NF-
B repression
target in the 70Z/3 line (Figs. 5A and 6). SLPI has also
been reported to suppress NF-
B activity by specifically increasing
I
B
levels in inflamed lung tissue (102). These results suggest
that therapeutic inhibition of NF-
B activation may not only impede
the initiation of inflammatory responses but might also ameliorate the
more deleterious consequences of chronic inflammatory reactions and
diseases by preventing the down-modulation of protective serine
proteases. Counter regulation of protease inhibitors in response
to brief or prolonged NF-
B activation will be necessary in other
cell types to assess the generality of these observations.
In conclusion, we have described the results of the first genomic
screen for genes modulated directly or indirectly by activation of the
NEMO/IKK signaling pathway. More than 100 gene targets were
identified in the context of a NEMO/IKK
null, murine pre-B cell line
stimulated by LPS or IL-1, and many of them represent novel NF-
B
target genes. Our findings allow the establishment and exploration of
many new physiological liaisons between NF-
B signaling and cellular
gene expression programming.
Acknowledgments--
The assistance of Patrick Aro, Sylvia
Samaniego, and Dr. Anne Savitt with vector constructions and Western
blotting is greatly appreciated. We thank Drs. Carol Sibley and Gail
Bishop for their kind gifts of 70Z/3-1.3E2 and CH12 IB
AA1A2 cells
and Dr. Dean Ballard for the I
B
(S32A/S36A) super repressor mutant.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a National Institutes of Health grant (to K. B. M.).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: Tel.:
631-632-8553; Fax: 631-632-9730; E-mail:
kmarcu@ms.cc.sunysb.edu.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M100846200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IB, inhibitors of
NF-
B;
IKK, I
B kinase;
NEMO, NF-
B essential modulator;
LPS, lipopolysaccharide;
PMA, phorbol 12-myristate 13-acetate;
IL-1, interleukin-1;
IPTG, isopropyl-1-thio-
-D-galactopyranoside;
RT-PCR, reverse
transcription-polymerase chain reaction;
TNF-
, tumor necrosis factor
;
SR, super repressor;
EBV, Epstein-Barr virus;
CREB, cAMP-response
element-binding protein;
MAP, mitogen-activated protein;
MAPK, MAP
kinase;
MEKK, MAPK/ERK kinase kinase;
DC, dendritic cells;
IFN, interferon;
ICE, interleukin-1
converting enzyme;
Bcl-2, B-cell
lymphoma 2;
EGF, epidermal growth factor;
Ox-LDL, oxidized low density
lipoprotein;
SLPI, secretory leukocyte protease inhibitor;
STAT, signal transducers and activators of transcription;
ATF, AP-1
transcription factor;
IRAK, IL-1R-associated kinase;
TRAF6, TNF
receptor-associated factor 6;
ECSIT, evolutionarily conserved signaling
intermediate in Toll pathways;
FADD, Fas-associated protein with death
domain.
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