Departments of Internal Medicine and of Integrative Biology, Pharmacology and Physiology, University of Texas Medical School at Houston, Houston, Texas 77030
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ABSTRACT |
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Nitric oxide
production by nitric oxide synthase 2 (NOS2) has been implicated in
epithelial cell injury from oxidative and immunologic stress. The
NOS2 gene is transcriptionally
activated by lipopolysaccharide (LPS) and cytokines in medullary thick
ascending limb of Henle's loop (MTAL) cells and other cell types. The
5'-flanking region of the NOS2
gene contains a consensus element for CCAAT/enhancer binding proteins
(C/EBP) at 150 to
142 that we hypothesized contributes to
NOS2
trans-activation in the mouse MTAL
cell line ST-1. Gel shift assays demonstrated LPS + interferon-
(IFN-
) induction of C/EBP family protein-DNA complexes in nuclei
harvested from the cells. Supershift assays revealed that the complexes were comprised of C/EBP
, but not C/EBP
, C/EBP
, or C/EBP
.
NOS2 promoter-luciferase genes
harboring deletion or mutation of the C/EBP box exhibited lower
activities in response to LPS + IFN-
compared with
wild-type NOS2 promoter constructs.
Overexpression of a C/EBP-specific dominant-negative mutant limited
LPS + IFN-
activation of the
NOS2 promoter. In
trans-activation assays,
overexpression of C/EBP
stimulated basal
NOS2 promoter activity. Thus C/EBP
appears to be an important
trans-activator of the
NOS2 gene in the MTAL.
gene transcription; kidney; cell signaling; promoter; transcription factors
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INTRODUCTION |
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NITRIC OXIDE is a potent effector molecule involved in numerous physiological processes, including neurotransmission, the control of vascular tone, inflammation, and immunity. In the kidney, NO participates in the regulation of glomerular and medullary hemodynamics, the tubuloglomerular feedback response, renin release, and the extracellular fluid volume (24). NO production from its substrate L-arginine is catalyzed by the multigene NO synthase (NOS) family, which includes three principal members. NOS1 and NOS3 are constantly expressed in selected tissues (although changes in the expression of these genes have been documented under several conditions, see Refs. 23 and 24 for review) but are inactive until intracellular Ca2+ levels increase sufficiently to maintain calmodulin binding (5). NOS2, which binds calmodulin at resting intracellular Ca2+ levels, is quiescent in most tissues until it is transcriptionally activated by immune stimuli to produce large amounts of NO (32). Although this large production of NO by NOS2 promotes host defense, it also contributes to septic shock and injury to tissues, including epithelial cells of the respiratory tract (37), gastrointestinal tract (26), and renal tubules (23). Indeed, high-output NO production has been linked to several forms of glomerular and renal tubular injury.
In virtually all nucleated cells of mammals, bacterial
lipopolysaccharide (LPS) and certain cytokines transcriptionally
activate the NOS2 gene.
Structure-function studies of the murine
NOS2 promoter have demonstrated that
the region 48 to
209 (region
I), serves as a core promoter module, whereas a more
distal region (region II,
780
to
1588; Ref. 4) serves as an LPS- and cytokine-responsive enhancer (4, 29, 41, 49). Numerous response elements reside within the
two regions, and several have been shown to be active. These include
B sites in both regions I and
II, a hypoxia-responsive enhancer
element in region I (31), a novel LPS
response element in region I to which
an Oct-1-like protein binds (47), interferon (IFN) regulatory factor-1
(IRF-1) (30, 42), two sequential IFN-stimulated response elements
(ISREs), and an IFN-
-activated site (GAS) in
region II (13). The responsiveness to
LPS and cytokines and the specific mechanisms involved in
NOS2 transcriptional regulation vary
depending on cell type. For example, our studies in ST-1 cells, a
well-characterized cell line derived from murine medullary thick
ascending limb of Henle (MTAL) cells, established that nuclear factor
for immunoglobulin
chain in B cells (NF-
B) proteins p50 and p65,
but not c-Rel, are critical for LPS + IFN-
inducibility
of the NOS2 gene in these cells
(25), whereas NF-
B p50, c-Rel, and possibly p65 serve
this regulatory role in RAW 264.7 macrophage-like cells (48).
The murine NOS2 promoter/enhancer
contains a nucleotide sequence 150 TGATGTAAT
142 that
conforms to the consensus CCAAT/enhancer binding protein (C/EBP) box
TKNNGYAAK (1) and that neighbors the
B site (
88 to
74)
important for LPS inducibility. In vivo footprinting studies of the
5'-flanking region of the NOS2
gene in RAW 264.7 cells demonstrated LPS-induced protection of guanine
146 within this C/EBP box (15), suggesting transcription factor occupation of this site. In addition, correlative changes in C/EBP
[also known as NF-IL6 (2) and LAP, for liver-associated
transcriptional activator protein (11)] DNA binding activity and
NOS2 gene expression in cultured
cardiac myocytes (22) and vascular smooth muscle cells (17) provided
inferential evidence to suggest a role for C/EBP
in
trans-activation of the
NOS2 gene. Most recently, work by
Eberhardt et al. (12) suggested the involvement of a similarly positioned C/EBP site in the rat NOS2
promoter/enhancer in the cAMP-mediated induction of NOS2 in cultured
mesangial cells.
The C/EBP proteins comprise a family of bZIP (basic region leucine
zipper) transcription factors that participate in the regulation of
genes involved in the acute phase response, inflammation, cell growth,
and differentiation (3, 7, 46). The C/EBP proteins ,
,
,
,
and
function as activators of transcription, whereas C/EBP homology
protein (CHOP) and the alternative translation products
liver-enriched transcriptional inhibitory protein (LIP) and C/EBP-30
serve as repressors (46). Of these, C/EBP
and C/EBP
are known to
be inducible by LPS or cytokines. Induction of C/EBP
expression has
been demonstrated in kidneys from LPS-treated (3) or hypoxic (50) mice
and the MTAL of rats subjected to ischemic injury (36), as well as in
macrophages (40), cardiac myocytes (22, 50), lung (50), and vascular
smooth muscle cells (17) subjected to immune or hypoxic challenge.
Since we observed residual LPS + IFN- induction of NOS2
in ST-1 cells even when NF-
B activity was completely inhibited by pyrrolidine dithiocarbamate (25), we hypothesized that
transcription factors other than NF-
B must be important for NOS2
induction in this setting. Given the emerging evidence that C/EBP
family proteins might be important for NOS2 induction in other cell
types (12) and the preliminary report of C/EBP
induction in the
postischemic MTAL of the rat (36), we hypothesized that C/EBP
contributes to NOS2 induction in ST-1 cells. In this report, using ST-1
cells as a model epithelium, we show that LPS + IFN-
induces nuclear expression of C/EBP
and that binding of C/EBP
to
the
150 to
142 C/EBP box in the
NOS2 promoter is necessary for maximal
LPS + IFN-
-mediated induction of the
NOS2 gene in these cells.
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MATERIALS AND METHODS |
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Reagents.
L-Glutamine, fetal bovine serum
(FBS), penicillin-streptomycin, and DMEM were from Life Technologies
(Grand Island, NY). LPS from Escherichia
coli O111:B4 was from Sigma Chemical (St. Louis, MO).
Mouse recombinant IFN- was from Genzyme (Cambridge, MA).
Radiochemicals were purchased from Amersham (Arlington Heights, IL).
RNAzol II was acquired from TEL-TEST "B" (Friendswood, TX). Rabbit polyclonal IgGs specific for C/EBP
, C/EBP
, C/EBP
, and C/EBP
were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Other oligonucleotides were custom synthesized by Genosys (The
Woodlands, TX). Poly(dI-dC)-poly(dI-dC) was purchased from Pharmacia-LKB Biotech. The Dual-Luciferase Reporter Assay System and
the luciferase vectors pGL3-Basic and pRL-TK were from Promega. The
bicinchoninic acid (BCA) protein estimation kit was from Pierce Chemical.
Plasmids and site-directed
mutagenesis. The C/EBP expression plasmid
pMSV-C/EBP
was kindly provided by Dr. Steven McKnight (Tularik).
This plasmid was designed to express selectively C/EBP
-LAP under the
control of the Moloney murine sarcoma virus long terminal repeat (7,
51). Excision of the encoding DNA for C/EBP
-LAP from this plasmid,
followed by religation of the ends, produced the recombinant molecule
pMSV-BS, which was used as a vector control in some experiments.
pRG-GBF-F, a C/EBP-specific, dominant-negative expression plasmid (34),
was a gift from Drs. Charles Vinson and Michelle Olive (National Cancer
Institute). The GBF-F protein contains the basic DNA binding region of
the plant bZIP protein GBF-1 joined to a leucine zipper that
preferentially heterodimerizes with the leucine zippers of C/EBP family
members (34). GBF-F inhibits the activity of all known C/EBP proteins
(34). The murine NOS2
promoter/enhancer and a portion of exon 1 (nucleotides
1486 to
+145) were amplified from 100 ng mouse genomic DNA by PCR using
oligonucleotides based on the published sequence (49). The forward
primer (P1) was
5'-TCAGCTCTTGTTTCCCAGGTT-3' (
1486 to
1466)
and the reverse primer (P3) was
5'-GGAGTGAACAAGACCCAAGCGTG-3' (+133 to +145). The product
was cloned into pCR2.1 (Invitrogen) and sequenced (Sequenase,
Amersham). The sequence was identical to that previously reported (29).
The NOS2 promoter fragment was then
excised with Hind III and
Xho I and cloned into these sites in
pGL3-Basic to create pNOS2-luc.
Deletion and site-directed mutation of the 150 to
142
C/EBP box in pNOS2-luc was accomplished by PCR splicing by overlap extension (18), using the wild-type
NOS2 promoter/enhancer cDNA as
template. For deletion of the C/EBP box, forward
primer P1 was used with mutagenic
reverse primer P3
(5'-TACTCCTATGTGGTGTCTCGTTCGTGTGTCTGATCC-3') in the
upstream reaction, and mutagenic forward primer
P4
(5'-ATGAGGATACACCACAGAGCAAGCACACAGACTAGG-3') and reverse
primer P2 were used in the downstream
PCR. For mutation of the C/EBP box (
150 TGATGTAAT
142
replaced with
T), the upstream reaction contained forward primer
P1 and mutagenic reverse primer
P5
(5'-GTGTGCT
CTCTGTGGTGTATCC-3'),
whereas mutagenic forward primer P6
(5'-GGATACACCACAG
CAAGCACAC-3') and reverse primer P2 were used
in the downstream reaction. The full-length, site-deleted or -mutated
NOS2 promoter/enhancer was then
constructed in a PCR containing 50-fold dilutions of the upstream and
downstream PCR products from the initial PCR together with
primers P1 and
P2. The mutated
P1-P2 promoter fragments PCR products were first cloned into pCR2.1, sequenced to verify the presence of the desired mutations and the absence of spurious mutations, and then subcloned into pGL3-Basic to create the recombinant molecules pNOS2-C/EBPdel-luc (C/EBP box deleted) and pNOS2-
C/EBP-luc (C/EBP box mutated).
Cell culture and transfection. ST-1
cells, a gift from Drs. Adam Sun and Steve Hebert, were maintained in
DMEM supplemented with 10% FBS, 50 U/ml penicillin, 50 µg/ml
streptomycin, and 2 mM
L-glutamine (complete medium).
These cells express phenotypic properties of the MTAL in vivo,
including expression of Tamm-Horsfall glycoprotein, and the
5-HT1A receptor (25). Vehicle or
LPS (100 ng/ml) + IFN- (0.5 U/ml) were added to the cells as
indicated in the text and legends to Figs. 1-3. These
concentrations have been shown to activate maximally
NOS2 transcription in ST-1 cells (25).
Plasmid preparations were made using the Endotoxin-free Plasmid
Maxi-prep kit (Qiagen, Santa Clarita, CA). For transient transfections, ST-1 cells were seeded in 6-well plates and grown to 70-90%
confluence in DMEM + 10% FBS without antibiotics and
transfected the following day using the LipoFectamine PLUS reagent
following the manufacturer's protocol and a total of 5 µg/well of
plasmid DNAs. For comparative purposes between reporter gene
constructs, transfection efficiencies were normalized by cotransfection
with 0.5 µg/well of the Renilla luciferase expression plasmid pRL-TK.
Trans-activation/trans-repression experiments used 1 µg of pNOS2-luc and 3 µg of pMSV-C/EBP,
pRG-GBF-F, or insertless expression vector. Twenty-four hours after
transfection, the medium was replaced with complete medium and vehicle
or LPS + IFN-
. Sixteen hours later, cell lysates for
measurement of firefly and Renilla
luciferase activities were prepared using Passive Lysis Buffer
(Promega) according to the manufacturer's directions. Protein content
of the lysates was determined using the BCA Assay kit (Pierce). Firefly
and Renilla luciferase activities in
100-µl lysate samples were measured in a Turner Systems 20/20 luminometer using the Dual-Luciferase Reporter Assay System according to the manufacturer's protocol. After background subtraction, firefly
luciferase activities were normalized for
Renilla luciferase activity and
protein content of the lysates and recorded as "normalized firefly
luciferase activity." The value for the vector-transfected cells
(see pGL3-Basic and pMSV-BS in Figs. 2 and 3, respectively) was
arbitrarily set at 1.0. Each observation represents the mean of
duplicate determinations using a new plasmid preparation.
Electrophoretic mobility shift assays
(EMSA). Nuclear extracts were prepared from time-paired
control and LPS + IFN--treated (4 h) ST-1 cells as
detailed in our earlier work (25). Double-stranded oligonucleotides
(1.75 pmol) corresponding to nucleotides
157 to
136
(sense strand,
5'-CACAG
CAAGCA-3',
C/EBP box is underscored) of the native murine
NOS2 promoter were end labeled with
[
-32P]ATP (3,000 Ci/mmol) using T4 polynucleotide kinase. Binding reactions were
performed in 20 µl of solution for 30 min at room temperature by
incubating 20 µg nuclear extract protein with 1.75 pmol of duplex DNA
probe (~2 × 105 cpm) in
reaction buffer [25 mM HEPES, pH 8.0, 50 mM KCl, 0.1 mM EDTA, 1 mM MgCl2, 1 mM dithiothreitol,
10% glycerol, and 50 µg/ml poly(dI-dC)-poly(dI-dC)] in the
presence or absence of a 50-fold molar excess of nonradiolabeled
competitor oligonucleotides. For supershift assays, antibodies specific
for individual transcription factors were added to the binding reaction
and incubated at room temperature for 30 min. Aliquots of the reactions
were resolved on 5% native polyacrylamide gels in 0.5× Tris
borate-EDTA buffer. The gels were dried and exposed to X-ray film with
an enhancing screen at
70°C to detect the DNA-protein and
DNA-protein-antibody complexes. Each observation represents a binding
reaction performed on a new nuclear extract preparation. Experiments
were replicated a minimum of three times as indicated in the legends to
Figs. 1-3.
Data analysis. The number of independent observations (n) for each experiment is indicated in the legends to Figs. 1-3. Quantitative data are presented as means ± SE and were analyzed by analysis of variance. Significance was assigned at P < 0.05.
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RESULTS |
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LPS and IFN- promote
C/EBP
binding activity in ST-1 cells.
To determine whether LPS + IFN-
induces expression of
C/EBP family proteins, EMSAs with double-stranded oligonucleotides containing the
150 to
142 C/EBP box of the native murine
NOS2 promoter and nuclear extracts
prepared from control and LPS + IFN-
-treated ST-1 cells
were performed. As seen in Fig.
1A, a major gel shift band was evident in nuclear extracts prepared from both
control and LPS + IFN-
-treated cells. The approximate abundance of the gel shift band was consistently greater in
LPS + IFN-
-treated cells compared with the controls
(n = 4). Sequence specificity of the
protein-DNA complex was verified in competition experiments: the gel
shift band was not evident in the presence of a 50-fold molar excess of
unlabeled C/EBP box oligomers but was apparent when a 50-fold molar
excess of unlabeled activator protein-2 (AP-2) site oligomers were
included in the reaction (Fig.
1A).
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Mutation of the 150 to
142 C/EBP box
limits
LPS + IFN-
induction of NOS2 promoter activity.
To determine whether the
150 to
142 C/EBP box is
functional within the structural context of the
NOS2 promoter, structure-function analyses were performed. Three constructs were made: one contained the
wild-type NOS2 promoter (pNOS2-luc), a
second contained the NOS2 promoter
lacking the
150 to
142 C/EBP box (pNOS2-C/EBP
del-luc), and a third contained the NOS2
promoter bearing a six-nucleotide mutation in the
150 to
142 C/EBP box (pNOS2-
C/EBP
-luc). These constructs or the
parent vector pGL3-Basic were transfected together with pTK-RL (to
control for transfection efficiency) into ST-1 cells, and the cells
were later stimulated with LPS + IFN-
. Multiple plasmid
preps were used to verify reproducibility of the findings. After
vehicle treatment alone, cells transfected with each vector exhibited
comparable, background-level activity of the luciferase reporter gene
(Fig. 2). ST-1 cells transfected with
pNOS2-luc together with pTK-RL and treated with
LPS + IFN-
exhibited roughly an eightfold increase in
normalized firefly luciferase activity (Fig. 2). In contrast, cells
transfected with pNOS2-C/EBPdel-luc or pNOS2-
C/EBP-luc together with
pTK-RL generated only fourfold increases in normalized firefly
luciferase activities after LPS + IFN-
stimulation (Fig.
2). These results indicated that the
150 to
142 C/EBP box
is functional in regulating murine
NOS2 gene expression in response to
these stimuli.
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Effects of overexpression of C/EBP
or a C/EBP dominant-negative mutant on the NOS2 promoter.
To determine whether overexpression or inhibited expression of C/EBP
in ST-1 cells could alter basal or LPS + IFN-
-induced NOS2 promoter activity, transient
transfections were performed with the wild-type
NOS2 promoter construct pNOS2-luc
together with pTK-RL and either empty expression vector or expression
plasmids for C/EBP
(pMSV-C/EBP
) or the C/EBP-specific,
dominant-negative mutant (pRG-GBF-F). ST-1 cells cotransfected with
pMSV-C/EBP
exhibited normalized
NOS2 promoter activity that was
approximately twofold greater than that of vector-transfected controls
after exposure to vehicle alone (Fig. 3).
In the presence of LPS + IFN-
, comparable levels of
induction of normalized NOS2 promoter
activity were observed in the pMSV-C/EBP
and vector-transfected
controls, suggesting that induction of endogenous C/EBP
was
sufficient to promote maximal NOS2
promoter activity. In contrast, cells cotransfected with pRG-GPF-F
exhibited basal, normalized NOS2 promoter activity that was comparable to that of controls and LPS + IFN-
-stimulated levels that were roughly two- to
threefold less than the vector controls treated with
LPS + IFN-
(Fig. 3).
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DISCUSSION |
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The MTAL is the principal nephron segment responsible for urinary
concentration and dilution (38), a major site for the action of
diuretics (14), and an important site for NO-mediated injury related to
toxins, inflammation, and ischemia (6, 23, 33). In previous
work, we demonstrated that LPS + IFN- transcriptionally induces NOS2 in ST-1 cells by both NF-
B-dependent and -independent mechanisms (25). In this study, we provide evidence for the involvement
a C/EBP response element (C/EBP-RE) and C/EBP
in LPS + IFN-
-induced
trans-activation of the murine
NOS2 gene in ST-1 cells. The present
results extend our earlier work, as well as previous in vivo
footprinting, EMSA, and NOS2 gene
expression studies in different cell types that suggested, but did not
confirm, the involvement of C/EBP
in
NOS2 gene activation (12, 15, 17, 22).
Taken together, these data suggest that C/EBP
and perhaps other
C/EBP family members participate in
NOS2 transcriptional activation in a
number of cell types and in response to disparate immune stimuli.
In addition to NOS2, C/EBP is known to
trans-activate several other genes
involved in the inflammatory response, including cell adhesion
molecules (8, 39), integrins (28), proinflammatory cytokines (10, 35),
prostaglandin endoperoxide synthase 2 (20), and manganese superoxide
dismutase (21). C/EBP
is also induced in the kidneys and lungs of
mice subjected to hypoxia (50). The hypoxic induction of C/EBP
, and
consequently or coordinately (31) NOS2, may be particularly relevant to
epithelial cells like those of the MTAL (6) and airways (27) that may
be more frequently exposed to hypoxic stress. Since C/EBP
has also
been implicated in the transcriptional activation of arginase I (16), an enzyme that degrades the substrate for NOS,
L-arginine, C/EBP
appears to
play a pivotal role in coordinating NO biosynthesis. Indeed,
coinduction of NOS2 and arginase I in vivo has been demonstrated in
response to LPS (40) and immune-mediated injury (9, 45). Thus C/EBP
may contribute in some tissues not only to induction of NOS2 expression
but also to inhibition of NOS2 activity by limiting substrate
availability. The latter effect may be important in limiting
cytotoxicity to host cells (45).
The NOS2 promoter fragment used in the
present study contains two other potential C/EBP boxes (474
TGGGGAAAT
466, 9/9 nucleotide match;
387 TGTTGGAAT
379, 8/9 nucleotide match) that we did not specifically examine.
We restricted our focus to the
150 to
142 C/EBP-RE,
because the other two elements do not reside within the
regions I and
II documented in previous deletion
analyses to contain functionally important promoter and enhancer
elements (4, 29, 41, 49), and they were not footprinted after immune
challenge (15). The fact that overexpression of the C/EBP-specific dominant-negative mutant GBF-F (Fig. 3) produced a reduction in NOS2 promoter activity that was
roughly equivalent to the decrement observed with the
NOS2 promoter constructs
bearing mutation or deletion of the
150 to
142 C/EBP-RE (Fig. 2) suggests that the
474 to
466
and
387 to
379 C/EBP boxes did not contribute
significantly to NOS2 induction in our study. It is also
possible that C/EBP-REs located at sites upstream of the 1.6-kb
fragment we examined contribute to C/EBP
trans-activation of the murine
NOS2 gene. Studies of the human
NOS2 promoter, for example, indicated
that cis elements upstream of
4.7 kb contribute to cytokine induction (44). This latter result
highlights important differences in transcriptional regulation of the
human and murine NOS2 genes. Whereas
the first 1.5 kb of the murine NOS2
promoter/enhancer contains important LPS- and cytokine-enhancer
elements (as detailed earlier), the first 4.7 kb of the human
NOS2 5'-flanking region appears
to be devoid of apparent cytokine-enhancer elements (44). Moreover, deletional and mutational analysis of the human
NOS2 promoter/enhancer revealed that
the proximal (
115 to
106) NF-
B element in the human
NOS2 promoter did not limit
cytokine-induced promoter activity in human liver and lung epithelial
cell lines (44), whereas the comparably positioned NF-
B element in
the murine NOS2 promoter was found to
be essential for LPS-inducibility in macrophages (48). Four NF-
B
motifs upstream of 5 kb were, however, found to be important for
maximal cytokine-induced activity of the human NOS2 promoter in these human cell
lines (44). Interestingly, the human
NOS2 promoter contains the sequence
191 TGATGT
183 that differs
at a single nucleotide (underscored) compared with the similarly
positioned
150 TGATGT
142
C/EBP box in the murine NOS2 promoter.
The human sequence does not conform to the consensus C/EBP box
TKNNGY
(1) (where K represents G or T),
which might provide at least one explanation why cytokine responsiveness was not conferred by the proximal 4.7 kb of the human
NOS2 promoter in reporter gene assays
(44). Formal tests of the possible functionality of the
191 to
183 sequence in the human NOS2
promoter are clearly needed.
The fact that DNA binding activities for C/EBP, -
, and -
were
not observed in nuclear extracts prepared from
LPS + IFN-
-treated ST-1 cells indicates that these
isoforms were not involved in the NOS2
promoter response. This result does not exclude the possibility that
one or more of these isoforms, or C/EBP
, which was not examined, might contribute to NOS2 regulation in other tissues. For example, redundancy in the activities of C/EBP
, -
, and -
in inducing the expression of IL-6 and monocyte chemoattractant protein-1 was
observed in cultured lymphoblasts (19). Such redundancy might also
account for the finding that peritoneal macrophages harvested from mice
with targeted disruption of the C/EBP
gene exhibited
LPS + IFN-
-induced nitrite production and NOS2 mRNA levels that were interpreted to be comparable to those of wild-type mice (43). In addition, close inspection of the data obtained from the
knockout mice reveals that the nitrite levels of the stimulated cells
from the null mice were numerically lower (statistical analysis was not
reported) than those from the wild-type mice, and the NOS2 mRNA levels,
which were not shown, were only qualitatively analyzed by RT-PCR (43).
Thus more detailed and quantitative analyses of NOS2 induction in
tissues from these knockout mice may reveal deficiencies in NOS2 induction.
Our findings add C/EBP to the growing list of regulatory factors
that contribute to NOS2 biosynthesis and high-output NO generation in
epithelial cells. Given its ability to form functional heterodimers
with other C/EBP family members, cAMP-response element binding protein
(CREB), and NF-
B (46), C/EBP
likely contributes versatility in
the magnitude and cell-specificity of NOS2 induction, at least in mouse
and rat. We did not observe heterodimers consisting of C/EBP
, -
,
or -
or NF-
B proteins, nor is NOS2 induced by cAMP in ST-1 cells
(16). However, cAMP and C/EBP-CREB heterodimers have been implicated in
NOS2 induction in other tissues (22), and thus different cell types
likely exploit distinct signaling cascades to stimulate NOS2 expression.
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ACKNOWLEDGEMENTS |
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We thank Drs. Steven McKnight, Charles Vinson, and Michelle Olive for the gifts of plasmids. We thank Drs. Adam Sun and Steve Hebert for the gift of the ST-1 cell line.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants RO1-DK-50745 and P50-GM-20529 (B. C. Kone) and was conducted during his tenure as an Established Investigator of the American Heart Association. A. K. Gupta was supported by a National Kidney Foundation Research Fellowship.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. C. Kone, Depts. of Internal Medicine and of Integrative Biology, Pharmacology, and Physiology, Univ. of Texas Medical School at Houston, 6431 Fannin, MSB 4.148, Houston, TX 77030 (E-mail: bkone{at}heart.med.uth.tmc.edu).
Received 8 September 1998; accepted in final form 23 December 1998.
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