From the Laboratories of Gene Regulation and
¶ Molecular Pathogenesis of Human Immunodeficiency
Virus, The Picower Institute for Medical Research,
Manhasset, New York 11030
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ABSTRACT |
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The synthesis of tumor necrosis factor- has
been suggested to be regulated at both the transcriptional and
translational levels in response to stimulation by bacterial
lipopolysaccharide, although the relative contribution of these two
mechanisms has not been quantitatively evaluated. Here, using the
murine monocytic cell line RAW 264.7 as a model system, we show that
steady-state TNF-
mRNA levels increase ~77-fold following
treatment with lipopolysaccharide for 2 h and to a maximum of
164-fold after 8 h as measured by an RNase protection assay. The
TNF-
gene transcription rate increases ~5-fold following exposure
to lipopolysaccharide for 2 h as measured by a nuclear run-on
assay. TNF-
mRNA stability did not change in the presence of
lipopolysaccharide. A ribosomal sedimentation assay and an RNA
transfection assay revealed that the translation rate of endogenous as
well as transiently transfected TNF-
mRNAs increases only
~2-3-fold after stimulation with lipopolysaccharide for 2 h.
Taken together, these results suggest that the large increase in the
level of secreted TNF-
protein in RAW 264.7 cells is due primarily
to activation of TNF-
gene transcription.
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INTRODUCTION |
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Tumor necrosis factor-
(TNF-
)1 is a well
characterized cytokine that plays an important role in many
inflammatory diseases caused by endotoxins produced by Gram-negative
bacteria (1-3). Overproduction of TNF-
contributes significantly to
the pathological complications observed in these diseases; therefore,
the molecular mechanism of TNF-
induction is of considerable medical
interest, and its elucidation will likely contribute to the development of new pharmacological reagents that can prevent TNF-
overproduction. TNF-
is produced mainly in cells of
reticuloendothelial origin (e.g. macrophages) and is induced
by many external stimuli, the strongest of which is lipopolysaccharide
(LPS), a component of the cell wall of Gram-negative bacteria (4, 5).
Although TNF-
was first described over 20 years ago, its regulation
on the molecular level remains controversial. Depending on the specific study, LPS has been shown to activate TNF-
synthesis primarily through a translational (6-9), or transcriptional (10-14) mechanism. The differences in conclusions reached in those studies may be due to
the use of different cell types and experimental conditions that induce
TNF-
synthesis by different mechanisms (e.g. mouse versus human macrophages or primary monocytes
versus monocytic cell lines). However, it is also possible
that the use of different experimental methods to measure transcription
and translation accounts for the conflicting conclusions.
LPS-induced changes in the transcription rate of the endogenous TNF-
gene have been measured using nuclear run-on assays and observed to be
relatively small (7, 8); however, relatively large changes in the rate,
as derived indirectly from measurements of steady-state mRNA levels
by Northern blot analysis, have also been observed (8). Translation and
transcription changes in the activity of artificial TNF-
promoter-reporter constructs have been measured after either stable
(6-9) or transient (10-14) transfection and have likewise yielded
conflicting conclusions. On the one hand, using primary mouse
macrophages, it has been established that LPS increases the
steady-state level of TNF-
mRNA derived from a transiently
transfected TNF-
promoter-reporter construct, suggesting a
transcriptional activation mechanism. Functional mapping of the TNF-
promoter in this system revealed that LPS induction is dependent on the
presence of intact binding sites for the transcription factor NF-
B
(10-12). Conversely, using the murine monocytic cell line RAW 264.7, it has been shown that a heterologous stably transfected TNF-
-CAT
fusion gene can be strongly induced by LPS at the translational (but
not transcriptional) level (7). In this case, LPS has been suggested to
function through the Raf/Ras signaling pathway (15) that ultimately
targets sequence elements present in the 3
-untranslated region (UTR) of TNF-
mRNA (6, 7).
In this study, we have attempted to measure more precisely the relative
contribution of transcription and translation to the induction by LPS
of endogenous TNF- mRNA in RAW 264.7 cells. To determine the
relative contributions of transcription and translation quantitatively
and to minimize the risk of experimental artifacts, we utilized several
functionally distinct transcription and translation assays measuring
both endogenous and exogenous TNF-
mRNA levels. We show that it
is possible to measure the relative contributions of translation and
transcription to the induction not only of transfected TNF-
promoter-reporter constructs, but also of the endogenous TNF-
gene.
Our results suggest that in RAW 264.7 cells, the endogenous TNF-
promoter is induced by LPS through a different molecular mechanism as
described in studies that used stably transfected TNF-
promoter-CAT
constructs (6-9). For the endogenous TNF-
message, we demonstrate a
77-fold enhancement of steady-state mRNA levels, an ~5-fold
increase in the transcription rate, and only a 2-3-fold increase in
the translation rate following LPS treatment for 2 h. These
results suggest that activation of transcription is the predominant
mechanism of LPS-mediated TNF-
induction in RAW 264.7 cells. We
therefore suggest that endogenous TNF-
is induced by LPS in both
monocytic cell lines and primary monocytes primarily through a
transcriptional mechanism.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- Murine RAW 264.7 cells were grown as adherent cells in RPMI 1640 medium (Life Technologies, Inc.) with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT) and gentamicin (Life Technologies, Inc.) unless indicated otherwise. For RNA isolation, cells were grown in 6-, 10-, or 15-cm tissue culture dishes to 50% confluency and harvested as described below.
Recombinant DNA and Plasmids--
TNF-CAT hybrid vectors were
constructed to permit the in vitro synthesis of capped and
polyadenylated TNF--CAT hybrid RNAs that were used in
electroporation experiments. A TNF-
-CAT construct containing the 5
-
and 3
-UTRs of TNF-
(7) was used as template in a polymerase
chain reaction with the synthesized primers
5
-CCCGAATTCAGCAGAAGCTCCCTCAGCGAG-3
and
5
-TTTGGATCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAATTAAAGTCACGGCTCC-3
(Life Technologies, Inc.) to generate a polymerase chain reaction product that represents a full-length cDNA copy of murine TNF-
mRNA. The TNF-
polymerase chain reaction product begins at the TNF-
transcription start site and ends with a 30-nucleotide poly(A) tail that begins several nucleotides upstream of the natural TNF-
polyadenylation site. Thus, this product contains the entire 5
- and
3
-UTRs; however, in the hybrid constructs, the TNF-
coding region
is exchanged for the bacterial gene coding for CAT. This polymerase
chain reaction product was cloned into the EcoRV site of the
Bluescript SK
vector (Stratagene, La Jolla, CA). The
resulting vector, pBSTNF-CAT, was used for in vitro
transcription of TNF-
-CAT mRNA. The deletion mutations shown in
Fig. 5 were derived from this vector by standard techniques (16).
In Vitro RNA Synthesis--
Typically, 1 µg of pBSTNF--CAT
DNA was linearized with BamHI, BstEII, or
PacI and added to an in vitro transcription
reaction using T7 bacterial RNA polymerase (Promega, Madison, WI)
according to the manufacturer. The CAP structure analogue used,
7mGpppG, was obtained from New England Biolabs Inc.
(Beverly, MA). After the transcription reaction, RNA synthesis was
verified using an RNase-free nondenaturing agarose gel. One µg of DNA
typically yielded ~3 µg of transcribed RNA, which was stored in
70% ethanol at
70 °C. TNF-
and GAPDH antisense RNAs were
generated from the plasmid pGEM3Zf+ TNF (10) and the
commercially available GAPDH plasmid (Life Technologies, Inc.),
respectively.
RNA Isolation and RNase Protection Assay--
Isolation of total
cytoplasmic RNA and RNase protection assays were performed according to
standard procedures (16). Murine TNF- was detected with a
212-nucleotide RNA probe derived from the EcoRI-linearized
template plasmid pGEM3Zf+ TNF. This probe is complementary
to the natural 5
-end of the TNF-
transcript and yields a protected
fragment of 169 nucleotides. Murine GAPDH mRNA was detected using a
135-nucleotide RNA probe. This probe is complementary to an internal
coding region of human GAPDH and yields a protected fragment of 95 nucleotides. However, since human and mouse GAPDH DNA sequence are not
completely identical, incomplete hybridization leads to the generation
of a shorter protected fragment of ~50 nucleotides following RNase
treatment as measured by denaturing gel electrophoresis.
TNF- ELISA--
Human TNF-
protein levels were determined
using a specific sandwich ELISA (R&D Systems, Minneapolis, MN) as
described by the supplier.
Nuclear Run-on Analysis--
RAW 264.7 nuclei were isolated and
stored frozen as described previously (17). For run-on analysis, nuclei
(0.2 ml) were thawed in 120 mM KCl, 1 mM
MgCl2, 2.5 mM dithiothreitol, and 0.3 mCi of
[-32P]UTP (3000 Ci/mmol; DuPont) in a final volume of
0.4 ml. After 1 h at 30 °C, total RNA was extracted with 3.2 ml
of 4 M guanidinium thiocyanate, 25 mM sodium
acetate, pH 5.0, and 10 mM dithiothreitol and separated
from proteins and DNA by centrifugation on a 1.3-ml cushion of 5.7 M CsCl and 100 mM EDTA, pH 6.5, at 30,000 rpm
in an SW 41 rotor for 22 h at 18 °C. The RNA pellet was
dissolved in 10 mM Tris-HCl, pH 8.5. RNA was
ethanol-precipitated; dissolved in 10 mM EDTA, pH 8.0, adjusted with 0.2 M NaOH; incubated for 10 min at 0 °C;
neutralized with HEPES; and precipitated with ethanol. RNA was then
dissolved in water, and an equal amount of labeled RNA was hybridized
to slot blots as described previously (18). Hybridization was performed
for 48 h in 50% formamide, 750 mM NaCl, 75 mM sodium citrate, 2 mg/ml Ficoll, 2 mg/ml
polyvinylpyrrolidone, 2 mg/ml bovine serum albumin (Fraction V; Life
Technologies, Inc.), 1 mM EDTA, 10 mM Tris-HCl,
pH 7.4, 0.2% SDS, and 0.1 mg/ml salmon sperm DNA at 52 °C with
2 × 106 cpm/ml [32P]RNA. Blots were
washed as described with an RNase step (18) and autoradiographed for
48 h at
70 °C. Signals were quantitated with a Molecular
Dynamics PhosphorImager.
Measurement of Ribosomes Bound to Native TNF-
mRNA--
One 15-cm plate of logarithmically growing RAW 264.7 cells was placed on ice and rinsed with 10 ml of ice-cold 1 × phosphate-buffered saline. The adherent cells were scraped off the
plate with a plastic scraper, yielding ~1-1.5 ml of cell suspension,
which was transferred to an RNase-free Eppendorf tube. Cells were
pelleted at 3000 rpm for 4 min at 4 °C. The tubes were immediately
placed on ice, and the cells were lysed by gentle resuspension in 0.5 ml of cold RNase-free lysis buffer (0.5% Nonidet P-40, 150 mM NaCl, 1.5 mM MgCl, 10 mM
Tris-HCl, pH 8.6, and 20 mM vanadyl-ribonucleoside complexes) followed by a 3-5-min incubation on ice. The lysate was
centrifuged at 14,000 rpm in a refrigerated centrifuge at 4 °C for 2 min, and the supernatant was carefully removed and layered directly on
top of a prepared and precooled 20-50% sucrose gradient containing
150 mM NaCl, 1.5 mM MgCl, 10 mM
Tris-HCl, pH 8.6, and 50 µg/ml cycloheximide. The gradient tube was
placed into an SW 40 Ti swinging bucket rotor (Beckman Instruments) and centrifuged at 20,000 rpm for 3 h in a Beckman ultracentrifuge. The gradient was subsequently analyzed with a Hoefer gradient analyzer,
and gradient fractions (1.5 ml) were immediately frozen. To isolate RNA
from the gradient fractions, 20 µg of yeast tRNA and 15 µl of 3 M sodium acetate, pH 5.0, were added, and the mixture was
extracted with phenol and chloroform. The RNA was precipitated with
ethanol, washed once with 70% ethanol, dried briefly under vacuum, and
used in RNase protection assays.
Electroporation of TNF- mRNA into RAW 264.7 Cells--
RAW 264.7 cells (5 × 105) were grown in
20 ml of RPMI 1640 medium supplemented with 10% FBS for 14 h and
then scraped off. After washing, the cells were resuspended in 0.4 ml
of RPMI 1640 medium at room temperature, with or without LPS (1 µg/ml), and divided into two electroporation cuvettes (Bio-Rad).
Immediately prior to electroporation, RNA (freshly dissolved in 50 µl
of 1 × phosphate-buffered saline) or phosphate-buffered saline
alone was added to the cuvettes. Electroporation was performed at 250 V
and 960 microfarads. Similar results were obtained when LPS was added
after electroporation. The cuvettes were put on ice, and 0.8 ml of RPMI
1640 medium containing 10% FBS was mixed with the cells until the
suspension became homogeneous; 0.5-ml aliquots were plated per well in
a Primaria six-well plate (Becton Dickinson Labware, Franklin Lakes,
NJ), and 3 ml of RPMI 1640 medium containing 10% FBS was added. After
4 h, >70% of the cells readhered to the plate, and the cells
were washed once with medium and then harvested for CAT activity.
CAT Assay-- Cells were scraped off the well; collected in an Eppendorf tube; washed once with 1 × phosphate-buffered saline; and lysed by resuspending in 100 µl of a solution of 0.2 M Tris-HCl, pH 7.5, followed by one freeze-thaw cycle and a 2-min sonication treatment. Cellular debris was removed by centrifugation, and CAT activity (20 µl of each extract) was measured by diffusion of reaction products into scintillation fluid according to standard procedures (16).
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RESULTS |
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The Transcription Rate of the Endogenous TNF- Gene Increases
after LPS Addition--
For our studies, we chose the murine
macrophage cell line RAW 264.7, which secretes large amounts of TNF-
following stimulation with LPS and has been extensively characterized
(6, 7). Using the RNase protection assay, TNF-
mRNA could be
detected in unstimulated RAW 264.7 cells and was rapidly and strongly
induced following addition of LPS (Fig.
1, A and B). The
antisense probe used to detect TNF hybridizes only with the first 169 nucleotides of the TNF-
mRNA molecule that are located at the
initiation site upstream of the first splice site, thereby avoiding
interference from RNA splicing, and therefore is a more direct
measurement of TNF-
promoter strength at early activation time
points. This sensitive RNase protection assay coupled with
densitometric analysis of the signals representing the protected
TNF-
fragment allowed us to more precisely measure the increase in
endogenous TNF mRNA levels. To normalize the measured TNF signal to
the relative amount of cytoplasmic RNA loaded in each lane, we used
GAPDH mRNA as an internal standard since steady-state GAPDH levels
are not affected by LPS addition (Fig. 1A). Quantitation of
the TNF-
and GAPDH signal ratios showed that TNF-
mRNA levels
increased 77-fold following treatment with LPS for 2 h and
164-fold after 8 h (Fig. 1B).
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The Average Number of Ribosomes Bound to an Endogenous TNF-
mRNA Molecule Increases ~2-3-Fold after LPS Addition--
To
obtain an estimate of the translation rate of endogenous TNF-
mRNA (i.e. the amount of TNF-
protein produced per
RNA molecule/time) before and after LPS addition, we determined the average number of ribosomes bound per endogenous TNF-
mRNA
molecule (21). Total cytoplasmic RNA isolated from uninduced or induced RAW 264.7 cells was fractionated on a sucrose gradient (Fig.
3). Under the conditions used for RNA
preparation (i.e. in the presence of the translational
inhibitor cycloheximide), mRNAs already bound to ribosomes in the
intact cells remained complexed with ribosomes, and mRNAs that were
not bound to ribosomes remained free of ribosomes throughout the
fractionation procedure. Conditions used to fractionate sucrose
gradients allowed the resolution of mRNA-ribosome complexes differing by only one bound ribosome. After fractionation, mRNA was
extracted, and the amount of TNF-
mRNA present in each fraction was measured directly by the RNase protection assay (Fig. 3) coupled with densitometry (Table I). The
densitometric analysis showed that in the absence of LPS, 69% of the
total amount of TNF-
mRNA was free of ribosomes, 12% contained
one to three ribosomes, and 19% was bound to four to six ribosomes.
After induction with LPS, 38% of the TNF-
mRNA was free of
ribosomes, 20% contained one to three ribosomes, and 42% was bound to
four to six ribosomes. Therefore, this analysis suggested that the
average number of ribosomes/TNF-
mRNA molecule increased
2-3-fold following addition of LPS.
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The Translation Rate of Transiently Transfected TNF- mRNA
Increases ~2-Fold after LPS Treatment--
Since the experiments
described above did not measure the translation rate of TNF-
mRNA directly, we developed an assay that allows direct measurement
of TNF-
mRNA translation in vivo. For this purpose, a
TNF-
-CAT construct was engineered that allowed in vitro
synthesis of a capped and polyadenylated mRNA molecule that
contained the complete 5
- and 3
-UTRs of TNF-
mRNA, but had the
TNF-
coding region exchanged for the coding region of CAT (Fig.
4). This mRNA contained most of the
regions that are known to play a role in the regulation of TNF-
mRNA translation, namely the ribosome-binding region in the 5
-UTR,
the 3
-UTR, and the poly(A) tail. This hybrid mRNA and its
derivatives (see below) were synthesized in vitro and
transfected transiently into RAW 264.7 cells using electroporation in
the presence or absence of LPS. Subsequently, CAT assays were performed
to measure directly the amount of translated protein (Fig. 4). Several
controls were performed to confirm that CAT levels in this assay
measure translation of transfected RNA: (i) omission of the CAP
structure in the in vitro RNA synthesis reactions completely
abolished any CAT activity above background levels, indicating that it
is indeed the RNA and not residual amounts of TNF-
DNA that
accounted for CAT activity; and (ii) in addition, the DNA template used
for in vitro transcription cannot by itself induce any CAT
activity because it contains no eukaryotic promoter, but only the
bacterial promoter used for in vitro transcription. In this
system, the CAT activity depends largely on the amount of TNF-
-CAT
mRNA added to the cells; electroporation of 5 µg of TNF-
-CAT
mRNA resulted in an ~5-fold increase in CAT activity (Fig.
4).
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DISCUSSION |
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In this study, we have attempted to re-examine the mechanism of
TNF- induction by LPS. A series of studies investigating LPS-induced
TNF-
synthesis in primary mouse monocytes (10-12) and in the mouse
monocytic cell line P388D1 (13) have indicated primarily a
transcriptional mechanism, whereas another series of studies in the
mouse monocytic cell line RAW 264.7 have provided evidence for a
predominantly translational mechanism (6-9). Our results suggest that
these differences are likely due to the measurement of endogenous or
transiently transfected TNF-
mRNA (10-14), in contrast to
measurements made with cells containing stably integrated TNF-
mRNA (6, 7, 9). Our results support a predominantly transcriptional
mechanism of LPS-mediated TNF-
production in both primary
macrophages and RAW 264.7 cells when endogenous or transiently
transfected TNF-
is measured in contrast to stably transfected
TNF-
. Our results suggest that the transcription rate of the
endogenous TNF-
gene in RAW 264.7 cells, as measured indirectly
using RNase protection assays, increases 77-fold 2 h after
addition of LPS, whereas the transcription rate increases ~5-fold
using the nuclear run-on assay. During the same time period, the
translation rate of endogenous TNF-
mRNA increases ~2-3-fold. We measured the transcription rate additionally through quantitation of
steady-state TNF-
mRNA levels during the early activation phase.
This approach was possible since the stability of steady-state TNF-
mRNA in LPS-stimulated and unstimulated cells treated with actinomycin D did not change (Fig. 1). These results suggested that the
observed rapid increase in steady-state TNF-
levels was due the
accumulation of newly synthesized mRNA, and not due to an increase
in mRNA stability following LPS treatment. Furthermore, low
concentrations of two different transcriptional inhibitors were shown
to completely block the LPS-mediated induction of TNF-
mRNA,
suggesting that induction is due to transcription rather than to an
increase in TNF-
mRNA half-life (Fig. 2). The relatively modest
increase in transcription rate as measured by the nuclear run-on assay
is consistent with previous data observed for the LPS-induced TNF-
gene in RAW 264.7 cells (17). More important, we observed that the
increase in the endogenous TNF-
protein level as measured by ELISA
was only 1.8-fold higher than the increase in the endogenous TNF-
mRNA level, directly suggesting that translation of the endogenous
TNF-
message is not induced more than ~2-fold by LPS.
We have attempted to estimate changes in relative translation rates of
TNF- mRNA after LPS induction in vivo. For this
purpose, we determined the average number of ribosomes bound per
endogenous TNF-
mRNA molecule before and after addition of LPS
(Fig. 3). Assuming that all TNF-
mRNA-bound ribosomes translate
equally well before and after LPS addition, the average number of
ribosomes/TNF-
mRNA molecule should be directly proportional to
the translation rate. We found that the number of ribosomes bound per
TNF-
molecule increased 2-3-fold after addition of LPS; therefore,
we suggest that the translation rate also increases 2-3-fold. This
view is supported by our observation that the translation rate of
transiently transfected TNF-
-CAT mRNA increased ~2-fold after
LPS stimulation (Fig. 4). Since the TNF-
transcription rate
increased 77-fold 2 h after LPS addition and continued to increase
for another 6 h, we conclude that the synthesis of TNF-
is
induced by LPS in RAW 264.7 cells primarily at the transcriptional
level.
Our results with RAW 264.7 cells differ in several regards from
previous studies with this cell type, which also showed a relatively
small (~3-5-fold) increase in the transcription rate of endogenous
TNF- after LPS induction using nuclear run-on assays (7, 9). While
in our study we measured transcription of the endogenous TNF-
gene
using a TNF-
probe derived near the transcription initiation site in
both steady-state mRNA and nuclear run-on assays, other
investigators have measured transcription rates in isolated nuclei
using TNF-
probes derived farther downstream of the initiation site
(7, 9). In addition, as discussed above, most of the measurement of
transcription and translation rates by these authors was performed with
stably transfected TNF-
-CAT constructs. The random integration of
TNF-
-CAT DNA into the genome may in some cases generate artifactual
effects; indeed, the half-lives of those hybrid mRNAs are much
longer than those of the endogenous TNF message (>3 h
versus 19-24 min, respectively), and the steady-state levels of RNA expressed from various TNF-
-CAT constructs in the absence of LPS vary for unknown reasons up to 17-fold, although the
promoter is identical in all constructs (9). Despite these differences,
a relatively modest increase in TNF-
transcription rate is observed
following LPS treatment in RAW 264.7 cells and in primary murine
monocytes. In an attempt to account for the large increase in both
steady-state TNF-
mRNA and protein levels and this apparent
discrepancy with nuclear run-on measurements, we measured steady-state
mRNA levels at relatively early time points using an RNase probe
derived near the initiation site. Our results suggest that the large
increase in steady-state TNF-
mRNA levels is primarily due to
increased gene transcription and further suggest that these nuclear
run-on experiments do not accurately measure TNF-
gene activity
levels.
Our results with RAW 264.7 cells are consistent with recent data
indicating that LPS induces simultaneously two signaling pathways in
RAW 264.7 cells (22). In that study, induction of the
post-transcriptional Raf-1/mitogen-activated protein
kinase-dependent pathway by itself was observed to only
produce a low level of TNF- protein induction, whereas additional
activation of a putative transcriptional NF-
B-dependent
pathway was observed to result in an ~20-fold enhancement (22). The
primary role of transcription in LPS-induced TNF-
expression is
consistent with the observation that intact NF-
B-binding sites in
the TNF-
promoter are necessary for proper induction of TNF mRNA
in primary bone marrow macrophages (10-12). In this regard, it is
interesting to note that nuclear NF-
B DNA-binding activity is
strongly and rapidly induced in RAW 264.7 cells as early as 15 min
following LPS addition (23); these kinetics are consistent with the
time course we observed for TNF-
mRNA induction. Recently,
treatment of primary human monocytes with LPS for 1 h has been
shown to increase the rate of TNF-
gene transcription ~4-fold, to
increase steady-state TNF-
mRNA levels ~9-fold, to cause an
increase in the fraction of TNF-
mRNA associated with large
polysomes, and to have no effect on TNF-
mRNA stability
(i.e. the measured half-life of TNF-
mRNA is 25 min
in the presence or absence of LPS) (24). Together with our results in
RAW 264.7 cells showing similar TNF-
mRNA half-lives, these
observations suggest that the primary effect of LPS is to rapidly
activate TNF-
gene transcription, which results in an increase in
steady-state TNF-
mRNA levels, the translation of which accounts
for the observed dramatic increase in secreted TNF-
protein.
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ACKNOWLEDGEMENTS |
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We thank Bruce Beutler (University of Texas
Southwestern Medical Center, Dallas, TX) for the TNF--CAT construct
and Christine Metz (Picower Institute for Medical Research) for advice
on the TNF-
ELISA.
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FOOTNOTES |
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* This work was supported by the Picower Institute for Medical Research and by United States Public Health Service Grants AI-38245 from NIAID (to M. B.) and DK-47272 from NIDDK (to R. A. C.), National Institutes of Health.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 reprint requests should be addressed: Dept. of Hematology and Oncology, Ohio State University, Columbus, OH 43210.
To whom correspondence should be addressed: Lab. of Gene
Regulation, Picower Inst. for Medical Research, 350 Community Dr., Manhasset, NY 11030. Tel.: 516-562-9432; Fax: 516-365-5090.
1
The abbreviations used are: TNF-, tumor
necrosis factor-
; LPS, lipopolysaccharide; CAT, chloramphenicol
acetyltransferase; UTR, untranslated region; FBS, fetal bovine serum;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked
immunosorbent assay; DRB, dichloro-
-D-ribofuranosylbenzimidazole.
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REFERENCES |
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