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INTRODUCTION |
Nitric-oxide synthases are key proteins that produce NO and
thereby regulate many important biological processes. NO is generated during the oxidation of L-arginine to
L-citrulline by at least three different isoforms of
nitric-oxide synthase. Endothelial and neuronal nitric-oxide synthases
are constitutively expressed, and their activity is Ca2+-
and calmodulin-dependent, whereas the third isoform is
transcriptionally inducible
(iNOS),1 and its activity is
independent of Ca2+ and calmodulin and can produce very
high levels of nitric oxide over a sustained period of time (1, 2). It
has been shown that iNOS is transcriptionally up-regulated in
pathophysiologic conditions such as hypoxia, ischemia-reperfusion
injury, and trauma and by reactive oxygen species (3, 4).
NO is a key central molecule in cellular biochemical processes, as it
is freely diffusible and traverses cell membranes to reach different
targets, alters signaling networks by redox-sensitive modifications,
and transcriptionally regulates multiple gene families (5-10). NO
production following iNOS up-regulation is associated with increased
wound healing and repair in tissue injury (11, 12). NO is also known to
activate multiple gene and cell signaling pathways through processes
such as nitrosation and cGMP production (3, 9). Furthermore, numerous
studies have shown that NO has antitumor effects and that forced
expression of iNOS causes regression of tumors (13-16).
Krüppel-like factor 6 (KLF6) is a ubiquitously expressed member
of the Krüppel-like family of transcription factors, which have
characteristic Cys2/His2 zinc finger motifs and
bind very similar "GC box" or "CACCC element" sites on DNA (17,
18). KLF6 is an immediate-early gene that regulates the expression of
multiple genes and is involved in tissue differentiation (19-23). KLF6
is rapidly induced in cells after acute injury and directly activates
collagen
1 and TGF-
along with TGF-
receptor I and II genes,
thereby mediating wound-healing mechanisms of fibrogenesis and
extracellular matrix formation (18, 24). Recently, KLF6 has been shown
to function as a tumor suppressor gene that was mutated in prostate
cancer (25).
The iNOS promoter defines a number of NF-
B and AP-1 sites dispersed
through out the 16-kb region. A number of iNOS inducers, including cell
injury, heat shock, and various cytokines, have been found to exert
their effect by activating either NF-
B or AP-1 (1, 26-28). Because
KLF6 and iNOS are involved in common processes such as cell injury,
wound repair, embryogenesis, tissue differentiation, and suppression of
tumorigenesis and because the iNOS promoter defines multiple CACCC
sites (KLF6-binding motifs), we hypothesized that KLF6 binds to the
iNOS gene and regulates its expression.
In this study, we have identified a novel transcriptional regulator of
iNOS. Specifically, we demonstrate that KLF6 binds to CACCC sites
within the proximal 0.63-kb region and regulates the expression
of the iNOS promoter in various cell types. We also show that in cells
exposed to stress conditions, the enhanced expression of KLF6 causes a
direct increase in expression of the endogenous iNOS gene and NO.
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MATERIALS AND METHODS |
Cell Culture--
Jurkat cells were cultured in RPMI 1640 medium
(Invitrogen) supplemented with 10% fetal bovine serum. COS-7 cells
were maintained in Dulbecco's modified Eagle's medium (Invitrogen)
supplemented with 10% fetal bovine serum. Peripheral blood mononuclear
cells were obtained from healthy adult volunteers. Primary T
lymphocytes were obtained from peripheral blood mononuclear cells using
a pan T cell isolation kit (Miltenyi Biotec, Auburn, CA) according to
the manufacturer's instructions.
Plasmid Constructs--
The human iNOS-luciferase reporter
constructs 0.63, 1.3, 3.8, 5.8, 7.2, and 16.0 kb upstream have been
previously described (29). The two CACCC sites in the 0.63-kb iNOS
promoter were mutated to AAAAA using the QuikChangeTM XL
site-directed mutagenesis kit (Stratagene, La Jolla, CA). The mutations
were confirmed by sequencing. The promoterless luciferase gene vectors
pXP1 and pXP2 were used as control vectors. The iNOS promoter-thymidine kinase-luciferase constructs have been described previously (29). The KLF6 expression vector pXCPBP and the control vector pX (pBluescript) have been described previously (30). The p50
and p65 NF-
B vectors were a kind gift from Dr. Barbara Rellahan
(Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, MD).
Transient Transfections and Luciferase Activity
Assays--
Plasmid DNA transfections of Jurkat T cells and COS-7
cells were carried out in 24-well plates (Corning Inc., Corning, NY) using LipofectAMINETM 2000 reagent (Invitrogen) following
the manufacturer's protocol. The day before transfection, 6 × 104 COS-7 cells or 0.6 × 106 Jurkat T
cells were plated in 0.5 ml of medium/well. For each well,
LipofectAMINE reagent (2-3 µl) was mixed with plasmid DNA (1.5 µg)
in serum-free Opti-MEM to allow DNA-LipofectAMINE reagent complexes to
form. The complexes were added to respective wells and mixed by gently
rocking the plate back and forth. The cells were incubated in a
CO2 incubator at 37 °C for 48 h and then lysed with
60 µl of reporter lysis buffer (Promega, Madison, WI). Luciferase activity was assayed with 20 µl of lysate and 80 µl of luciferase assay reagent (Promega) in a TD20/20 luminometer (Promega).
Transfection efficiency was determined in all samples by cotransfection
with 0.5 µg of plasmid encoding the cytomegalovirus promoter-driven
-galactosidase gene, and the luciferase activity was normalized to
the
-galactosidase activity.
Electrophoretic Mobility Shift Assay (EMSA)--
COS-7 cells
(2 × 106) were transfected with the KLF6 expression
vector overnight. Briefly, the transfected cells were detached using
trypsin/EDTA and washed with phosphate-buffered saline, and nuclear
extracts were prepared as described previously (31). The sequences of
the oligonucleotides used for EMSA are as follows: probe 2, 5'-GAT CAG GTC ACC CAC AGG CCC-3', and its complementary sequence,
5'-GGG CCT GTG GGT GAC CTG ATC-3'; and probe 4, 5'-AGC AGC CAC CCT GCT
GAT GAA C-3', and its complementary sequence, 5'-GTT CAT CAG CAG GGT
GGC TGC T-3'. The oligonucleotides were synthesized by Genosys
Biotechnologies, Inc. (The Woodlands, TX). Complementary
single-stranded oligonucleotides were annealed, end-labeled with
[
-32P]ATP (PerkinElmer Life Sciences) and T4
polynucleotide kinase (Roche Molecular Biochemicals, Mannheim,
Germany), purified by Centri-Sep columns (Princeton Separations,
Adelphia, NJ), and used as probes in EMSA. In all experiments,
unlabeled oligonucleotides were used as unlabeled competitors. In each
experiment, 5-10 µg of nuclear extracts were incubated for 20 min at
room temperature with the labeled oligonucleotide (2-3 ng) in 20 µl
of buffer containing 20 mM HEPES (pH 7.4), 1 mM
MgCl2, 10 µM ZnSO4, 20 mM KCl, 15% Ficoll, and 2 µg of poly(dI-dC). For
supershift experiments, 3 µg each of control anti-KLF4 (clone T-16)
and anti-KLF6 (clone R-173) antibodies (Santa Cruz Biotechnology, Santa
Cruz, CA) were added and incubated for 45 min at room temperature prior
to the addition of the radiolabeled probe. The DNA-protein complex was separated on a 4% nondenaturing polyacrylamide gel in 0.5× Tris borate/EDTA buffer. The data were analyzed using the PhosphorImager system (Amersham Biosciences) and Quantity One software (Bio-Rad).
Chromatin Immunoprecipitation (ChIP) Assay--
COS-7 cells
(10 × 106) were transfected with the KLF6 expression
vector or were non-transfected (control). Both the transfected and
control cells were divided into two subgroups. One group was left
untreated, and the other was treated with 20 mM NaCN for 3 h; both subgroups were used for the ChIP assay. Jurkat cells and
T lymphocytes (10 × 106) were also treated similarly
to COS-7 cells; however, only the COS-7 cells were subjected to KLF6
transfection. Moreover, all the cells were also treated with PMA/A23187
or heat-shocked or serum-starved and subjected to ChIP analysis. The
ChIP assay was performed following the recommendations of Upstate
Biotechnology, Inc. (Lake Placid, NY) and previously published
protocols (32-34). Briefly, KLF6 was cross-linked to DNA by adding
formaldehyde to a final concentration of 1%. The chromatin samples for
studying phosphorylated KLF6 were incubated with anti-phosphoserine
(clone PSR-45, Sigma) and anti-phosphotyrosine (clone 4G10, Santa Cruz Biotechnology) antibodies overnight at 4 °C and immunoprecipitated with salmon sperm DNA-bovine serum albumin-Sepharose beads, followed by
treatment with 10 mM phenyl phosphate for 15 min. The
supernatants were used for further steps. All the samples (for
phosphorylated as well as non-phosphorylated KLF6) were then incubated
overnight at 4 °C with anti-KLF6 antibody, followed by incubation
with Sepharose beads. The immunocomplexes were treated with DNase- and
RNase-free proteinase K, and DNA was purified using a DNA purification
kit (QIAGEN Inc., Santa Clara, CA). PCR was performed with primers flanking the proximal as well as distal KLF6-binding sites in the proximal 0.63-kb iNOS promoter (5'-CAG AGA GCT CCC TGC TGA GGA
AA-3' and 5'-GAG AGT TGT TTT TGC ATA AAG GTC TC-3') (see Fig. 5).
Amplified fragments (321 bp) were analyzed on a 2% agarose gel by
staining with SYBR Green (FMC Corp. BioProducts, Rockland, ME).
The no-antibody immunoprecipitation samples served as negative controls.
Real-time Quantitative PCR--
Total RNA was isolated from
5 × 106 control cells using an RNeasy minikit (QIAGEN
Inc.), treated with DNase I, and reverse-transcribed using avian
myeloblastosis virus reverse transcriptase and oligo(dT) primer
(Promega). The PCR primers synthesized by Genosys Biotechnologies, Inc.
were as follows: KLF6, 5'-AGA GCG AGC CCT GCT ATG TTT CAG-3' (forward) and 5'-CGC TGG TGT GCT TTC AAG TGG GAG-3' (reverse); GAPDH,
5'-CAA CTA CAT GGT TTA CAT GTT CC-3' (forward) and 5'-GGA CTG TGG TCA
TGA GTC CT-3' (reverse); and iNOS, 5'-ACC TAC CAC ACC CGA GAT GGC
CAG-3' (forward) and 5'-AGG ATG TCC TGA ACA TAG ACC TTG GG-3'
(reverse). Quantitative PCR was performed by monitoring in real
time the increase in fluorescence of the SYBR Green dye on a
SmartCyclerTM (Cepheid, Sunnyvale, CA) according to the
manufacturer's instructions. The relative expression level of the
target gene in KLF6-transfected and NaCN-treated cells was plotted as
-fold change compared with control vector-transfected and
non-NaCN-treated cells, respectively. GAPDH gene expression was used
for normalization. Each real-time quantitative PCR assay was performed
twice using triplicate samples.
SDS-PAGE and Immunoblotting--
KLF6-transfected and control
COS-7 cells (0.5 × 106 cells) were lysed in cold 1%
Nonidet P-40 lysis buffer (Sigma) with protease inhibitors as described
previously (35), and protein was assayed using a Bio-Rad protein assay
kit. Seventy micrograms of the protein were resolved by 4-12%
BisTris-NuPAGE and transferred to polyvinylidene difluoride
membrane. The blot was probed with control anti-
-actin (clone 54, BD
Biosciences), anti-KLF6, and anti-iNOS (BD Biosciences) antibodies. The
membranes were washed and incubated with the corresponding horseradish
peroxidase-conjugated secondary antibody (Bio-Rad). Protein bands were
detected by ECL enhanced chemiluminescence reagents (Amersham
Biosciences). The Western blot bands were quantitated by densitometry
using GelProTM software (Media Cybernetics, Silver Spring, MD).
Estimation of Nitrite + Nitrate--
Briefly, 50 µl of cell
culture medium (Dulbecco's modified Eagle's medium) were treated with
nitrate reductase (N-7265, Sigma) in the presence of NADPH (N-7505,
Sigma) to convert nitrate to nitrite (36, 37). Upon the addition of
2,3-diaminonaphthalene (Sigma), which reacts with nitrite under acidic
conditions to form a fluorescent product
(1H-naphthotriazole), fluorescence intensity was measured
with a fluorescence microplate reader with excitation at 365 nm and
emission at 450 nm, and nitrite was quantitated by comparison with a
standard curve of NaNO2.
Quantitation and Statistical Analysis--
Statistical analysis
of the data was carried out with Minitab Version 14 using Student's
t test, and p values <0.05 were considered as significant.
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RESULTS |
KLF6 Induces iNOS Promoter Activity in COS-7 Cells--
A
schematic diagram showing putative KLF6-binding motifs in the
5'-flanking region of the human iNOS gene is shown in Fig. 1A. To investigate the effect
of KLF6 on the expression of human iNOS, we performed luciferase
reporter assays in COS-7 cells transfected with the full-length 16-kb
iNOS promoter construct and the KLF6 expression vector. As shown in
Fig. 1B, transfection of COS-7 cells with KLF6 induced the
luciferase activity by 3-fold compared with the control vector. To
compare the induction of iNOS with a well characterized inducer, we
studied the effect of transcription factor NF-
B subunits p50 and p65
on the iNOS promoter. NF-
B has been reported to bind multiple sites
on the 7.3-kb iNOS promoter and to induce its activity by 4.1-fold in
AKN-1 and by 3.9-fold in A549 cells following cytokine stimulation (1).
Transfection of COS-7 cells with p65 and p50 with the full-length iNOS
promoter construct induced promoter activity by 2- and 4-fold,
respectively (Fig. 1B). These results indicate that KLF6 can
induce the transcription of the iNOS promoter at levels comparable to
those induced by NF-
B (4, 26, 29, 38, 39).

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Fig. 1.
KLF6 induces iNOS expression in COS-7
cells. A, shown is a schematic diagram of the
full-length 16-kb human iNOS promoter. The positions of the
KLF6-binding sites (CACCC) identified in the 8-kb sequenced region are
indicated by arrows. The position of the beginning
nucleotide of each CACCC site according to Spitsin et al.
(41) is indicated. B, the 16-kb iNOS-luciferase construct
was transfected with KLF6 or the p50 or p65 subunit of NF- B into
COS-7 cells, and the luciferase activity was determined. Data are
representative of three independent experiments performed in
triplicate.
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The Proximal 0.63-kb iNOS Promoter Is Sufficient for the Induction
of Activity by KLF6--
After ascertaining that KLF6 can activate the
full-length 16-kb iNOS promoter, we performed transfection experiments
to define the region of the iNOS promoter that is required for the
induction of transcription by KLF6 (29). COS-7 and Jurkat cells
transfected with serial deletion constructs of the 16-kb iNOS
promoter-luciferase reporter compared with cells transfected with the
control vector demonstrated that the induction of iNOS promoter
activity remained similar in all constructs compared with the
full-length 16-kb iNOS promoter (Fig. 2,
A and B). Previous studies have shown that KLF6
binds to the CACCC motifs of DNA (40). The 0.63-kb iNOS construct has
two CACCC motifs, and there are a total of 10 CACCC binding sites in
the first 8 kb of the 16-kb iNOS promoter. Optimum induction (3-fold)
of the 0.63-kb iNOS promoter by KLF6 (Fig. 2, A and
B) suggests that additional upstream KLF6-binding sites do
not further enhance the iNOS promoter activity in the presence of CACCC
sites in the proximal 0.63-kb region. However, when the iNOS
core promoter with the CACCC sites (within 0.63 kb) was replaced with
the thymidine kinase (thymidine kinase-luciferase) core promoter, a
20% increase in the luciferase activity with the KLF6 expression vector (compared with transfection with the control pX vector) was observed with the 3.8-5.8-kb iNOS- and 7.2-16-kb iNOS-thymidine kinase-luciferase constructs, whereas there was no luciferase induction
with the iNOS promoter region from 5.8 to 7.0 kb (Fig. 2C).
The significance of this finding is presently unknown.

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Fig. 2.
Effect of KLF6 on iNOS promoter activity in
cells transfected with various deletion constructs of the iNOS
promoter. Shown is a schematic view of the iNOS promoter
indicating the restriction enzymes used to generate various deletion
constructs with luciferase. A and B, COS-7 and
Jurkat cells were transfected with the KLF6 expression vector (pXCPBP,
1.5 µg) and iNOS promoter deletion constructs (1.5 µg) or with the
empty KLF6 vector (1.5 µg) and iNOS promoter deletion constructs (1.5 µg) along with a -galactosidase-expressing vector (0.5 µg; as a
transfection efficiency control). pXP1 and pXP2 are empty vectors of
iNOS transfected with KLF6 or the empty KLF6 vector (pX). The
luciferase activity was determined 24-48 h post-transfection. The
effect of KLF6 on the respective constructs is indicated as -fold
induction of luciferase activity over that of the control pX vector.
C, COS-7 cells were transfected with the KLF6 expression
vector (pXCPBP, 1.5 µg) and iNOS promoter-thymidine kinase
(TK)-luciferase (Luc) deletion constructs or with
the empty KLF6 vector (1.5 µg) and iNOS promoter deletion constructs
(1.5 µg) along with a -galactosidase-expressing vector (0.5 µg;
as a transfection efficiency control). The luciferase activity was
determined 24-48 h post-transfection. The effect of KLF6 on the
respective constructs is indicated as percent increase in luciferase
activity over that of the control pX vector. Data are representative of
five independent experiments performed in triplicate.
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Mutational Analysis of the Two CACCC Sites in the 0.63-kb iNOS
Promoter--
To understand the contribution of KLF6 to the activation
of the 0.63-kb iNOS promoter by the two CACCC sites, we created single mutation constructs in which we mutated the proximal and distal CACCC
sites individually and a dual mutation construct with both CACCC sites
mutated. Transfection studies with these mutants revealed that the two
sites, which are separated by 92 bases, had an additive effect on the
stimulation of the 0.63-kb iNOS promoter. The distal CACCC site
contributed to 1.6- and 3.2-fold induction of luciferase activity in
COS-7 cells (Fig. 3A) and
Jurkat cells (Fig. 3B), respectively. Similarly, the
proximal CACCC site contributed to 1.3- and 2-fold induction in COS-7
cells (Fig. 3A) and Jurkat cells (Fig. 3B),
respectively. Mutation of both CACCC sites completely abolished the
KLF6-induced iNOS promoter activity, suggesting that the CACCC motifs
are necessary for the interaction of KLF6 with the 0.63-kb iNOS
promoter. These studies demonstrate that the proximal and distal
CACCC sites in the 0.63-kb iNOS promoter are necessary for optimum
basal promoter activity.

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Fig. 3.
CACCC motifs in the 0.63-kb iNOS promoter are
necessary for activation by KLF6. The two CACCC sites in the
0.63-kb iNOS promoter were mutated to AAAAA by PCR-based mutagenesis.
A and B, the wild-type and mutant reporter genes
(1.5 µg) were cotransfected with either the empty vector (pX, 1.5 µg) or the KLF6 expression vector (pXCPBP, 1.5 µg) in the presence
of a -galactosidase-expressing vector (0.5 µg) in COS-7 or Jurkat
cells. The luciferase activity was measured 24-48 h post-transfection
and normalized to -galactosidase levels. Data are presented as -fold
induction over that of the empty vector. Data are representative of
three similar experiments performed in triplicate. N, no
mutations; *, mutation.
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KLF6 Binds to the CACCC Sites in the 0.63-kb iNOS Promoter--
To
demonstrate that KLF6 binds to the human iNOS promoter, we designed two
oligonucleotides, each defining the CACCC motif regions at positions
164 to
168 and
261 to
265 in the 0.63-kb iNOS promoter,
respectively (41). The primers were end-labeled, and EMSA was performed
using nuclear extracts from KLF6-transfected COS-7 cells. Nuclear
extracts from these cells bound to both oligonucleotides defined by the
0.63-kb iNOS promoter (Fig. 4,
A and B). To demonstrate the specificity of the
binding, supershift assays were performed using an antibody specific to
KLF6. As shown in Fig. 4 (A and B, lanes
4), the shifted band in lane 1 was supershifted by
anti-KLF6 antibody, demonstrating that KLF6 directly interacts with the iNOS promoter in vitro. The absence of a supershifted band
with anti-KLF4 antibody (Fig. 4, A and B,
lanes 2) further confirmed the specificity of KLF6 for the
CACCC binding sites.

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Fig. 4.
KLF6 binds to oligonucleotides containing the
two CACCC motifs in the 0.63-kb iNOS promoter. Nuclear extracts
were prepared from KLF6-transfected COS-7 cells and incubated with
labeled oligonucleotides from the iNOS promoter, followed by EMSA as
described under "Materials and Methods." A, EMSA was
done using the oligonucleotide defining the proximal CACCC site in
lane 1. A nonspecific antibody (Ab; anti-KLF4)
was added in lane 2, and a hundredfold excess of specific
unlabeled competitor was added in lane 3. Lane 4 depicts a supershifted band with anti-KLF6 antibody. Antibody (2 µg)
was added to the oligonucleotide and nuclear extract. B,
EMSAs were repeated as described for A with an
oligonucleotide defining the distal CACCC site.
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In Vivo Binding of KLF6 to the iNOS Promoter--
Next, to
determine whether KLF6 interacts with iNOS in vivo, we
performed ChIP analysis using primary T, Jurkat, and COS-7 cells. The
DNA-KLF6 complexes were immunoprecipitated with anti-KLF6 antibody,
followed by reversal of cross-linking and PCR amplification using
primers flanking the proximal and distal CACCC binding sites in the
0.63-kb iNOS promoter (Fig.
5A). In transfected COS-7
cells, the intensity of the PCR product was significantly higher
compared with the non-transfected cells (Fig. 5B, lane
2 versus lane 4), suggesting that increased expression
of KLF6 increases its binding to the iNOS promoter.

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Fig. 5.
In vivo binding of KLF6 to the
iNOS promoter in COS-7 and Jurkat cells and T lymphocytes. COS-7
cells (10 × 106/sample) were treated as per protocol,
fixed with formalin, washed, lysed, and sonicated. The DNA-protein
complexes were immunoprecipitated (IP) with anti-KLF6
antibody (Ab) and extracted with protein A-agarose beads.
The DNA was purified and amplified with primers flanking the
iNOS promoter. A, a schematic view of the 0.63-kb
iNOS promoter depicting the positions of the primers flanking the CACCC
sites that were used for PCR of the ChIP DNA. Primer sequences are
indicated by arrows. The two KLF6-binding CACCC
sites are shown. B, ChIP analysis of the iNOS
promoter in KLF6-transfected COS-7 cells and non-transfected COS-7,
Jurkat, and T cells after treatment with sodium cyanide. Lane
1, the PCR product from the input (positive control) DNA;
lanes 2 and 3, KLF6-transfected COS-7 cells;
lanes 4 and 5, 6 and 7, and
8 and 9, non-transfected COS-7, Jurkat, and
primary T cells, respectively. Cells in lanes 3,
5, 7, and 9 were treated with NaCN.
PCR products were resolved on a 2% agarose gel. C, ChIP
analysis of control and treated Jurkat, primary T, and COS-7 cells.
D, ChIP analysis of serine- and tyrosine-phosphorylated KLF6
in PMA/A23187-treated, sodium cyanide-treated, and control primary T
cells. Data are representative of two similar experiments.
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It has been demonstrated in independent studies that iNOS and KLF6 are
up-regulated during cell stress (3, 4, 18, 24). To examine whether
up-regulation of iNOS is mediated by KLF6 under conditions of stress,
we treated COS-7 and Jurkat cells and primary T lymphocytes with NaCN.
NaCN blocks mitochondrial respiration and induces cellular hypoxia
(42). As shown in Fig. 5B (lanes 3, 5,
7, and 9), treatment with NaCN strongly increased the binding of KLF6 to the iNOS promoter as evidenced by the increased intensity of the PCR product.
We also subjected COS-7, Jurkat, and primary T cells to heat stress,
serum starvation, and PMA/A23187 and analyzed the binding of KLF6 to
the iNOS promoter by ChIP assay. As shown in Fig. 5C,
compared with the control cells, KLF6 binding to the iNOS promoter was
increased in a similar fashion in all cell types subjected to these
conditions. These data conclusively show that the association of KLF6
with the iNOS promoter is increased in cells subjected to conditions of
stress and stimulation.
Next, we investigated whether the phosphorylation status of KLF6 could
play a role in its binding to the iNOS promoter. To ascertain whether
serine and tyrosine phosphorylation of KLF6 plays a role in binding to
the iNOS promoter, we performed ChIP analysis involving a two-step
immunoprecipitation process using anti-phosphoserine or
anti-phosphotyrosine antibody, followed by treatment with phenyl
phosphate (to separate the phosphorylated protein from the bound
antibody) and further immunoprecipitation with anti-KLF6 antibody.
The data show that the KLF6 that bound to the iNOS promoter was
serine-phosphorylated in resting T cells, which was increased in
PMA/A23187- and NaCN-treated cells (Fig. 5D). Interestingly,
however, we observed that tyrosine-phosphorylated KLF6 bound to the
iNOS promoter only in NaCN-treated cells. The absence of any PCR
products for the no-antibody and mouse IgG immunoprecipitation controls
further confirmed the specificity of our findings (data not shown).
Thus, differential phosphorylation of KLF6 mediates differential
binding to iNOS in various conditions.
Induction of iNOS mRNA and Protein and NO in KLF6-transfected
Cells--
To establish the functional association between KLF6 and
iNOS expression, we measured the iNOS mRNA and protein and NO
production in KLF6-transfected COS-7 cells. We performed real-time PCR
to estimate the -fold induction of mRNA based on the fluorescence cycle threshold differences between various time points compared with
controls. As shown in Fig. 6A,
the production of KLF6 mRNA was induced by up to 14-fold 24 h
post-transfection and by 13-fold 48 h post-transfection. In tandem
with the KLF6 mRNA, there was a corresponding increase in the iNOS
mRNA, which showed a steady increase ranging from 3.5-fold (24 h)
to 6-fold (48 h) (Fig. 6A). There was no increase in the
control (GAPDH) mRNA.

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Fig. 6.
KLF6 transfection up-regulates iNOS mRNA
and protein and NO in COS-7 cells. A, COS-7 cells were
transfected with KLF6 and incubated for various time periods. Total RNA
was isolated; cDNA was synthesized; and KLF6, iNOS, and GAPDH
(control) mRNAs were amplified by real-time quantitative PCR.
B and C, KLF6, iNOS, and -actin protein levels
were estimated by Western blotting. D, NO production was
estimated by assay for NO metabolites (nitrite + nitrate) from the same
samples. Data are representative of three similar experiments.
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Next, to study the kinetics of iNOS protein induction by KLF6 following
KLF6 expression vector transfection, we performed Western blot
experiments at earlier time points (0-18 h) (Fig. 6B) and
pursued the kinetics of KLF6 and iNOS expression over 72 h (Fig.
6C). KLF6 protein levels increased starting at 6 h post-transfection, whereas iNOS protein levels increased starting at
12 h post-transfection (Fig. 6B), suggesting that KLF6
is able to initiate transcription and to produce iNOS protein within
6 h. It was previously observed that an increase in KLF6 mRNA
in culture-activated cells is accompanied by an even greater increase in KLF6 protein; moreover, the rate of degradation of KLF6 protein is
also lower (18). In light of these data, we believe that the induction
of KLF6 protein seen beyond 48 h could be attributed to
accumulation of KLF6 as a result of decreased protein degradation (Fig.
6C). Furthermore, NO production, as measured by assay for NO
metabolites (nitrite and nitrate), indicated a significant increase in
cells transiently transfected with KLF6 (Fig. 6D). There was
no increase in either KLF6 or iNOS mRNA or protein (data not shown)
or NO in control vector-transfected cells (Fig. 6D) and in
cells subjected to LipofectAMINE transfection agent (data not shown),
which were incubated under similar culture conditions as the
KLF6-transfected cells. These data, taken together with the ChIP
results (Fig. 5), demonstrate that KLF6 acts as a transactivator of the
iNOS gene.
NaCN-induced Hypoxia Up-regulates KLF6 and iNOS mRNAs and
Proteins as Well as NO in COS-7 Cells--
ChIP analysis (Fig.
5A) demonstrated increased binding of KLF6 to the iNOS
promoter in cells exposed to NaCN. Therefore, to establish a functional
association between the expression of KLF6 and iNOS in
pathophysiological conditions, we measured the KLF6 and iNOS mRNA
and protein and NO production in NaCN-treated COS-7 cells. The cells
were transiently treated with 20 mM NaCN for 4 h and
then incubated in fresh medium for various time periods. Following
exposure of cells to NaCN, the production of KLF6 mRNA was analyzed
by real-time PCR. As shown in Fig.
7A, a significant induction of
KLF6 mRNA was seen, with levels reaching 4.5- and 8-fold over
control levels by 24 and 48 h, respectively. Consistent with the
KLF6 mRNA results, there was an increase in KLF6 protein by 24 h, and the high amounts were sustained over a 72-h period. iNOS protein
were also induced by 12 h, and the levels reached a peak by
48 h (Fig. 7B). Furthermore, NO production, as assayed by its metabolites (nitrite and nitrate), also indicated a significant increase in the nitrite + nitrate levels starting at 6 h, with a
steady and sustained increase over 72 h (Fig. 7C).
Thus, even though we cannot rule out the additional effect of other
transcription factors in the induction of iNOS following NaCN
treatment, the above data, combined with our ChIP findings (Fig.
5B) and increased iNOS mRNA and protein expression data
following KLF6 transfection (Fig. 6, A and B),
strongly suggest that KLF6 plays a role in iNOS induction following
treatment of cells with NaCN.

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Fig. 7.
NaCN exposure up-regulates KLF6 and iNOS
mRNAs and proteins and NO in COS-7 cells. A, COS-7
cells were exposed to 20 mM NaCN for 4 h, washed, and
incubated for various time periods in normal medium. Total RNA was
isolated; cDNA was synthesized; and KLF6, iNOS, and GAPDH (control)
mRNAs were amplified by real-time quantitative PCR. B,
KLF6, iNOS, and -actin protein levels were estimated by Western
blotting. C, NO production was estimated by assay for NO
metabolites (nitrite + nitrate) from the same samples. Data are
representative of three similar experiments.
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DISCUSSION |
Our study provides the first evidence for the regulation of the
human iNOS promoter by a member of the Krüppel-like family of
transcription factors. Using luciferase reporter gene assays, EMSA, and
ChIP analysis, we have demonstrated that KLF6, a member of the
Krüppel-like family of transcription factors, directly interacts
with the iNOS promoter in resting cells and with greater intensity
following cell stress, injury, and stimulation. Furthermore, we
have shown that up-regulated KLF6 increases both iNOS mRNA and
protein expression, which correlates functionally with the concomitant
nitric oxide expression. The presented evidence strongly suggests that
KLF6 is a major transcriptional regulator of iNOS under conditions of
hypoxia and cell stress.
The human iNOS promoter is 16 kb long and is one of the largest known
promoters (1). Its regulation is complex and occurs at multiple levels,
orchestrated by multiple transcription factors, in response to diverse
conditions in a tissue-specific context. Multiple transcription factors
such as NF-
B, AP-1, STAT1
, and interferon regulatory
factor are known to regulate the iNOS promoter (reviewed in detail in
Ref. 1). NF-
B is ubiquitously expressed and regulates iNOS in
multiple cell types, and the regulation of iNOS by NF-
B is well
characterized (4, 26, 29, 38, 39). Our data demonstrate that the
induction of iNOS by KLF6 is comparable to that by the NF-
B
subunits, suggesting that KLF6 could be an equally important regulator
of iNOS.
Despite the presence of at least 10 CACCC sites in the 16-kb iNOS
promoter, the KLF6-induced transcriptional activation of the 0.63-kb
construct was similar to that of the 16-kb construct in COS-7 and
Jurkat cells (Fig. 2). The CACCC sites in the 0.63-kb construct are
placed in close proximity at positions
164 and
261 compared with
the other CACCC sites, the nearest of which is much farther upstream at
position
2736. Previous studies have reported similar binding of KLF6
to either tandem sites or sites in close proximity to each other in
other promoters and placed in close proximity to the basal promoter
elements. KLF6 is known to bind the leukotriene C4 synthase
promoter at two tandem CACCC sites located between positions
135 and
149 close to the basal promoter elements (40). In yet another study,
KLF6 was shown to interact with TGF-
1 and both TGF-
receptor I
and II promoters at multiple sites. KLF6 strongly transactivates
TGF-
1 by binding to two tandem Sp1-binding sites between positions
239 and
209 compared with much lesser interactions with promoter
regions including single Sp1-binding sites (24). The transactivation of
the TGF-
receptor II promoter requires the presence of closely
placed GC-rich regions from positions
152 to
127 and
118
to
85 (24). Thus, it is very likely that the two closely placed CACCC
sites, in proximity to the basal promoter elements, in the 0.63-kb iNOS promoter are sufficient for induction of the iNOS gene by KLF6. Our
data suggest that CACCC sites located between 3.8 and 5.8 kb and 7.0 and 16.0 kb upstream in the iNOS promoter may play a role in
KLF6-mediated regulation of the iNOS gene only in the absence of
proximal (0.63 kb) CACCC sites.
The facts that KLF is very rich in serines (30) and that its
transactivation domain contains serines as well as tyrosines (30)
suggest that the activity and binding of KLF6 to the iNOS promoter
could also be regulated by serine and tyrosine phosphorylation. There
are several examples of transcription factors being regulated by their
phosphorylation status. For example, the activation and intermolecular
interactions of KLF1 are known to be dependent on its serine
phosphorylation (43). Similarly, studies have indicated that two
distinct phosphorylation events, i.e. phosphorylation of
tyrosine 701 and serine 727, are necessary for full activation of STAT1
by interferon-
(44, 45). Thus, our observations that
serine-phosphorylated KLF6 binds to the iNOS promoter in resting,
stimulated (PMA/A23187), and stressed (NaCN-treated) cells and that
serine- as well as tyrosine-phosphorylated KLF6 binds to the iNOS
promoter in NaCN-treated cells indicate that differential
phosphorylation of KLF6 could play a very important role in gene
regulation. The precise mechanism of this interaction is currently
under investigation.
Induction of iNOS by KLF6 may have important implications in the
prevention of apoptosis, tissue injury repair, and cancer. Previous
studies have addressed the protective role of NO in apoptosis. NO
blocks apoptosis by multiple mechanisms. First, NO inhibits caspases
(46) by inhibiting interleukin-1
-converting enzyme-like and
cysteine protease protein-32-like proteases (47). Second, NO protects
the mitochondria, lowers cytochrome c release, and inhibits
calcium fluxes (48). Third, NO prevents an increase in Bcl-2 and
induces the expression of heat shock proteins such as HSP70 that
have an anti-apoptotic role. NaCN triggers apoptosis of cells
predominantly by inducing cytotoxic hypoxia by inhibiting cytochrome
c oxidase, the terminal enzyme of the respiratory chain, and
by causing activation of voltage-sensitive calcium channels and calcium
fluxes (49). In NaCN-treated cells, we observed increased binding of
KLF6 to the iNOS promoter (Fig. 5B), in addition to
increased levels of iNOS protein and concomitant NO production (Fig. 7,
B and C). In a similar study, it has been shown
that NO protects NaCN-treated chick embryonic neurons from
cyanide-induced apoptosis (42).
NO plays a role in wound healing and tissue repair. In several studies
on colon anastomosis, bone fracture, and cutaneous wound healing, the
reparative role of NO through up-regulation of iNOS has been well
demonstrated (10, 50, 51). Similarly, KLF6 plays a direct
anti-apoptotic role in conditions of acute injury. KLF6 was found to be
responsible for healing of acutely injured hepatic stellate cells (18)
and aortic endothelial cells (52). Our ChIP data (Fig. 5B)
demonstrate that in heat-shocked and NaCN-treated cells, there is a
strong binding of KLF6 to the iNOS promoter, thereby suggesting that
KLF6 can orchestrate its anti-apoptotic and protective effects by
up-regulating NO through iNOS (Fig. 6).
Recently, KLF6 was demonstrated to be a tumor suppressor gene that was
found mutated in 77% of prostate cancer patients, and it was shown to
act in a p53-independent manner through the p21WAF1/CIP1 pathway
(25). The tumor-suppressive role of iNOS is also well documented (13,
15, 16). NO production by iNOS through its transcriptional
up-regulation by KLF6 could serve as another mechanism for the
antitumor effect of KLF6. In conclusion, our findings provide
evidence that KLF6 binds to the human iNOS promoter and regulates its
expression under conditions of cell stress, injury, and stimulation
with possible implications in the treatment of organ injury and cancer.