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GAPDH Binding to TNF-α mRNA
Contributes to Posttranscriptional
Repression in Monocytes: A Novel
Mechanism of Communication between
Inflammation and Metabolism
Patrick Millet, Vidula Vachharajani, Linda McPhail, Barbara
Yoza and Charles E. McCall
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2016 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2016; 196:2541-2551; Prepublished online 3
February 2016;
doi: 10.4049/jimmunol.1501345
http://www.jimmunol.org/content/196/6/2541
The Journal of Immunology
GAPDH Binding to TNF-a mRNA Contributes to
Posttranscriptional Repression in Monocytes: A Novel
Mechanism of Communication between Inflammation and
Metabolism
Patrick Millet,* Vidula Vachharajani,†,‡ Linda McPhail,x Barbara Yoza,{ and
Charles E. McCall†
T
he link between glycolysis and inflammation is well
established. Many innate immune cell types specifically
require glycolysis to perform their effector functions. When
glycolysis is inhibited, leukocytes show decreased adhesion, mobility, and bacterial clearance (1–4). Monocytes produce less TNF
cytokine when treated with the glycolysis inhibitor 2-deoxyglucose
(2-DG), but not when treated with the mitochondrial inhibitor rotenone (4). Additionally, macrophages express greater levels of
proinflammatory cytokines when forced to rely on glycolysis, but
they express much lower levels when fatty acid oxidation is upregulated (5). This relationship between inflammation and glycolysis appears in certain disease states as well. As the endotoxin
response proceeds to tolerance, monocytes downregulate glycolysis
*Molecular Genetics and Genomics Program, Wake Forest University School of
Medicine, Winston-Salem, NC 27157; †Department of Molecular Medicine, Wake
Forest University School of Medicine, Winston-Salem, NC 27157; ‡Department of
Anesthesiology, Wake Forest University School of Medicine, Winston-Salem, NC
27157; xDepartment of Biochemistry, Wake Forest University School of Medicine,
Winston-Salem, NC 27157; and {Department of General Surgery, Wake Forest University School of Medicine, Winston-Salem, NC 27157
ORCIDs: 0000-0002-0892-7084 (V.V.); 0000-0002-9082-469X (C.E.M.).
Received for publication June 15, 2015. Accepted for publication January 3, 2016.
This work was supported by National Institutes of Health Grants R01AI079144,
R01AI065791, R01GM099807, and R01GM102497.
Address correspondence and reprint requests to Dr. Charles E. McCall, Wake Forest
University School of Medicine, Department of Molecular Medicine, Medical Center
Boulevard, Winston-Salem, NC 27157-1042. E-mail address: Chmccall@wfubmc.
edu
Abbreviations used in this article: ARE, AU-rich element; 2-DG, 2-deoxyglucose;
ECAR, extracellular acidification rate; G3P, glyceraldehyde-3-phosphate; RNA-IP,
RNA immunoprecipitation; RT-qPCR, real-time quantitative PCR; siRNA, small
interfering RNA; UTR, untranslated region.
Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501345
and upregulate fatty acid oxidation (6–8). This shift in metabolism
occurs simultaneously with the onset of immunosuppression.
Recent findings indicate that glycolysis and inflammation communicate in ways not previously appreciated. One of the key
enzymes in glycolysis is GAPDH, which converts glyceraldehyde3-phosphate (G3P) into 1,3-bisphosphoglycerate in the sixth step of
the glycolysis pathway (9). GAPDH also has a lesser known capacity as an RNA-binding protein (10). Specifically, GAPDH
binds to AU-rich elements (ARE) found in the 39 untranslated
region (UTR) of many mRNAs. ARE are present in many inflammatory genes, including cytokines such as IFN-g and TNF
(11–13). GAPDH binding to a generic ARE is inhibited by G3P
(14) and NAD+, a necessary cofactor for its enzymatic activity
(10). Recently, it was shown GAPDH–ARE binding is responsible
for posttranscriptional regulation of IFN-g expression in T cells
(15). This binding is disrupted by the metabolite G3P, making
this mechanism sensitive to cellular metabolism. Some argue that
these types of RNA–enzyme–metabolite interactions broadly affect gene expression (16); however, these mechanisms remain
largely unexplored.
Expression of TNF is tightly regulated in immune cells. During
endotoxin tolerance, much of this regulation occurs at the level of
chromatin (17–24). Tolerant monocytes and other immune cells
fail to generate TNF mRNA in response to an additional stimulus
while they are in the immunosuppressed state. This repression of
TNF expression also occurs at the posttranscriptional level (25–
27). Even when transcription of TNF mRNA is restored to tolerant
monocytes, they continue to show deficiencies in TNF cytokine
production. Posttranscriptional repression mediated by microRNA
accounts for part of this deficiency (25, 26). A number of reports
describe other posttranscriptional mechanisms that regulate TNF
expression (28–32); however, none of these mechanisms suggests
that cellular metabolic state informs the regulation process. In this
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Expression of the inflammatory cytokine TNF is tightly controlled. During endotoxin tolerance, transcription of TNF mRNA is
repressed, although not entirely eliminated. Production of TNF cytokine, however, is further controlled by posttranscriptional
regulation. In this study, we detail a mechanism of posttranscriptional repression of TNF mRNA by GAPDH binding to the TNF
39 untranslated region. Using RNA immunoprecipitation, we demonstrate that GAPDH–TNF mRNA binding increases when
THP-1 monocytes are in a low glycolysis state, and that this binding can be reversed by knocking down GAPDH expression or by
increasing glycolysis. We show that reducing glycolysis decreases TNF mRNA association with polysomes. We demonstrate that
GAPDH–TNF mRNA binding results in posttranscriptional repression of TNF and that the TNF mRNA 39 untranslated region is
sufficient for repression. Finally, after exploring this model in THP-1 cells, we demonstrate this mechanism affects TNF expression
in primary human monocytes and macrophages. We conclude that GAPDH–TNF mRNA binding regulates expression of TNF
based on cellular metabolic state. We think this mechanism has potentially significant implications for treatment of various immunometabolic conditions, including immune paralysis during septic shock. The Journal of Immunology, 2016, 196: 2541–2551.
2542
Materials and Methods
Cell culturing
THP-1 cells were grown in RPMI 1640 with 10% FBS, L-glutamine,
and penicillin/streptomycin media. Cells were kept in a 5% CO 2 incubator at 37˚C and subcultured every 1–3 d to maintain a density of
20–80 3 104 cells/ml (34). THP-1 cells were maintained in an un-
differentiated state. Galactose-fed cells were taken from standard
glucose-fed cultures, spun down, washed with PBS, and grown in
RPMI 1640 (no glucose, 2 g/l galactose) for $5 d before use in any
experiments.
THP-1 cells were tolerized with addition of 1 mg/ml LPS for 24 h. For
experiments involving second-dose exposure of LPS, cells were spun down
and resuspended in fresh media for 1 h before proceeding with second
doses of LPS, also at 1 mg/ml.
Preparation of human primary monocytes/macrophages
Primary monocytes/macrophages were collected from heparinized venous
blood samples donated by healthy adult volunteers according to the Institutional Review Board protocol approved by Wake Forest University
(35). RBCs, platelets, and polymorphonuclear neutrophils were removed
through Isolymph (Gallard-Schlesinger Industries) centrifugation of whole
blood. Monocytes were then enriched through a 2-h adherence step, after
which nonadherent cells were removed. Cells were then cultured overnight
in fresh RPMI 1640 containing 10% FBS and either glucose or galactose,
with or without 100 ng/ml LPS to induce ex vivo endotoxin tolerance.
Brightfield analysis of morphology showed resulting cultures had .90%
monocytes and macrophages.
Metabolic assays
Assessment of oxygen consumption rate and extracellular acidification
rates (ECAR) were made using the Seahorse XF24 extracellular flux analyzer
(Seahorse Bioscience) (36). Plates were coated with Cell-Tak (BD Biosciences) (37) and dried overnight before addition of 25 3 104 cells/well in
unbuffered DMEM (10% FBS, 2 g/l glucose or galactose) and 1 h incubation
in a CO2-free 37˚C incubator. Plates were assayed according to the manufacturer’s instructions.
Lactate assays were performed using an L-Lactate assay kit (Eton
Bioscience) according to the manufacturer’s instructions (38). Cells
were kept in phenol red–free DMEM with 2 g/l glucose or galactose
during the assay.
FIGURE 1. Tolerance and galactose both affect TNF expression. (A) RT-qPCR assay comparing TNF mRNA expression in responsive, tolerant, and
galactose-fed cultures with or without a 1-h stimulation of 1 mg/ml LPS. Bars show average of five independent experiments 6 SEM. **p , 0.01 compared
with responsive counterpart, calculated by unpaired t test. (B) ELISA assay comparing TNF cytokine expression in responsive, tolerant, and galactose-fed
cultures with or without a 4-h stimulation with 1 mg/ml LPS. Bars show mean of n = 3 6 SEM. *p , 0.05, **p , 0.01 compared with responsive
counterpart. (C) RT-qPCR assay comparing rates of TNF mRNA decay in responsive, tolerant, and galactose-fed cultures following a 1-h stimulation with
1 mg/ml LPS and incubation with 5 mg/ml actinomycin D for indicated time. Points represent average of three independent experiments, shown as percentage
of (2)actinomycin D (0 h).
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study, we propose a mechanism where glycolysis directly affects
TNF expression through posttranscriptional regulation.
With our previous work in the background in regard to posttranscriptional repression of TNF mRNA and immunometabolic
shifts in monocytes during the endotoxin response, we speculated
that GAPDH–ARE binding might contribute to regulation of TNF
expression in monocytes. We hypothesized that if glycolysis was
limited, GAPDH would bind the ARE of TNF mRNA, thereby
limiting its translation. To test this, we first cultured our THP-1
cells in media where glucose was replaced by galactose. Because
galactose is metabolized more slowly than glucose (33), these
cells adopted a less glycolytic, more oxidative metabolism. We not
only found GAPDH binding to TNF mRNA in galactose-fed
monocytic cells, but that this binding also occurs in endotoxintolerant cells following the natural downregulation of glycolysis
that monocytes exhibit during tolerance. Furthermore, we found
that GAPDH–TNF mRNA binding is affected by pharmacological
manipulation of glycolysis. Our results indicate that this mechanism allows leukocyte cell metabolism to fine-tune TNF gene
expression. These findings have potential implications for any
number of disease states involving inflammation and metabolism,
such as immunoparalysis during septic shock.
GAPDH BINDING REPRESSES TNF TRANSLATION
The Journal of Immunology
2543
FIGURE 2. Tolerance and galactose both affect metabolism. (A) Lactate assay of responsive, tolerant, and galactose-fed cultures after addition of LPS
(n = 3 6 SEM). (B) Seahorse XF assay of ECAR of responsive, tolerant, and galactose-fed cultures before and after injection of 1 mg/ml LPS. Representative graph, n = 3.
ELISA
Real-time quantitative PCR
RNA was isolated using STAT60 (Tel-Test) when isolation was required
outside the context of RNA immunoprecipitation (40). RNA quality was
measured on a NanoDrop 1000 (Thermo Scientific) before reverse tran-
RNA immunoprecipitation
RNA immunoprecipitation was performed using the Magna RIP kit
(Millipore) according to manufacturer’s instructions (42). Briefly, cultures
of 10 3 106 cells were prepared as described above, spun down, washed,
and lysed with 280˚C freezing. Lysates were then spun down and supernatants transferred to tubes with magnetic beads that were previously
FIGURE 3. GAPDH binds to TNF-a mRNA in galactose-fed cells. (A) TNF-a mRNA expression in glucose-fed or galactose-fed cells, relative to actin,
with or without addition of LPS (1 mg/ml) for 1 h. Both the table and the blackened portions of bars (GAPDH-IP) show percentage of TNF-a mRNA
captured by GAPDH Ab during RNA-IP, relative to total RNA as determined from input. Bars show mean of n = 5 6 SEM. The p values are compared with
glucose-fed counterpart, calculated by unpaired t test. Nonsignificant values (p . 0.05) are not shown. (B) GAPDH mRNA expression of same cells
previously described. Bars show mean of n = 5 6 SEM. Table and blackened portion of bars (GAPDH-IP) show percentage of GAPDH mRNA captured by
GAPDH Ab during RNA-IP. No significant change in GAPDH protein binding to its own RNA was observed, as expected. (C) Western blot of GAPDH,
actin in glucose- and galactose-fed cells. Blots are representative of three independent observations. No significant difference was observed with media or
LPS treatment.
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A Quantikine TNF ELISA kit (R&D Systems) was used according to the
manufacturer’s instructions for measuring TNF protein concentration (39).
Cells were washed twice with PBS and resuspended to a density of 80 3
104 cells/ml in appropriate media before incubation with or without LPS.
Supernatant of resulting cultures was collected when indicated and used
for assay.
scription using the qScript cDNA synthesis system (Quanta BioSciences)
(41). Quantitative PCR was done using TaqMan reagents and probe/primer
mixes (Applied Biosystems) on the ABI 7500 Fast system.
For RNA stability assay, cells were stimulated with LPS for 1 h and then
given 5 mg/ml actinomycin D for indicated time. Cells were then pelleted
and RNA isolated as described above (23).
2544
GAPDH BINDING REPRESSES TNF TRANSLATION
treated with 5 mg anti-GAPDH Ab (Sigma-Aldrich) or nonspecific IgG.
Lysates were rotated with beads overnight, washed the next day, eluted
(alongside input RNA), isolated with phenol-chloroform-isoamyl alcohol,
ethanol precipitated, and resuspended in RNase-free water. Quality of input RNA was assessed and all samples were measured through quantitative
real-time PCR (RT-qPCR) as described above.
mics) encoding Renilla luciferase with 39UTR regions indicated in the
figure legends. Transfections included Cypridina TK loading control
Western blotting
THP-1 cells were cultured and treated as indicated in text. Cells were
pelleted and lysed in RIPA buffer. Protein (50 mg) was loaded into each
well of a 4–20% Precise protein gel (Thermo Fisher). Blot was run and
transferred according to the gel manufacturer’s instructions (43).
RNA interference
THP-1 cells (5 3 106) were transfected with 1 mM small interfering RNA
(siRNA) targeting either GAPDH mRNA or not targeting any mRNA
(control). Transfection was done using the Amaxa Nucleofector II according
to the manufacturer’s instructions (23). Cells were cultured in appropriate
media for 3 d following transfection before use in Western blot or ELISA
experiments.
Polysome fractionation profiling
Polysome fractionation analysis was performed as previously described
(44). Briefly, 10 3 106 THP-1 cells were incubated with 100 mg/ml
cyclohexamide before lysis in hypotonic buffer. Lysates were pelleted,
and the supernatant was placed on top of a 10–45% continuous sucrose
gradient. Samples were then centrifuged at 222,228 3 g for 2 h at 4˚C.
After ultracentrifugation, tubes were pierced at the bottom and fractions
were collected. UV absorbance was measured for each fraction using a
NanoDrop 1000 (Thermo Scientific). RNA was extracted from each fraction using STAT50 (Tel-Test). RT-qPCR analysis was then performed as
previously described.
Luciferase reporter
THP-1 cells were plated in white 96-well plates in phenol red–free DMEM
(5% FBS, 2 g/l glucose or galactose). Cells were then transfected with
FuGENE transfection reagent and GoClone plasmids (SwitchGear Geno-
FIGURE 5. TNF cytokine expression increases in galactose-fed
GAPDH knockdown cells. (A) Western blot of GAPDH and actin in glucose or galactose-fed cultures, following transfection with either GAPDH
or control (ctrl) siRNA. Blots are representative of three independent assays. (B) ELISA assay comparing TNF cytokine expression in glucoseand galactose-fed cultures following transfection with GAPDH or control
(ctrl) siRNA after 4 h stimulation with 1 mg/ml LPS. Bars show mean of
n = 3 6 SEM. *p , 0.05 compared with control knockdown counterpart.
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FIGURE 4. GAPDH binds to TNF-a mRNA in endotoxin-tolerant cells. (A) TNF-a mRNA expression in responsive or tolerant cultures, relative to actin, with
or without addition of LPS (1 mg/ml) for 1 h. Both the table and the blackened portions of bars (GAPDH-IP) show percentage of TNF-a mRNA captured by
GAPDH Ab during RNA-IP, relative to total RNA as determined from input. Bars show mean of n = 4 6 SEM. The p values are compared with the responsive
counterpart, calculated by an unpaired t test. Nonsignificant values (p . 0.05) are not shown. (B) GAPDH mRNA expression of same cells previously described.
Bars show mean of n = 4 6 SEM. Table and blackened portion of bars (GAPDH-IP) show percentage of GAPDH mRNA captured by GAPDH Ab during RNAIP. No significant change in GAPDH protein binding to its own RNA was observed, as expected. (C) Western blot of GAPDH and actin in responsive and tolerant
cell cultures. Blots are representative of three independent observations. No significant difference in GAPDH density was observed.
The Journal of Immunology
plasmid. Transfection procedure followed the manufacturer’s instructions.
Assay of luciferase activity was done 24 h after transfection using
LightSwitch dual assay reagents (Active Motif) and the MicroLumat Plus
LB96V (Berthold Technologies) plate luminometer. Relative luciferase
units were calculated by subtracting background signal and normalizing
Renilla signal to loading plasmid.
Statistical analysis
Statistical analysis and graphical presentations were performed using
Microsoft Excel 2010. Significance was calculated using an unpaired
Student t test. All data shown represent results from three or more independent observations, expressed as mean 6 SEM.
Results
Tolerance and galactose both affect metabolism and TNF-a
expression
cells use more mitochondrial oxidation and less glycolysis (15, 45,
46). Thus, this model allowed us to separate the metabolic impact
of tolerance from its other effects on gene expression.
We first measured expression of TNF in three different culturing
conditions: responsive (glucose-based media), tolerant (glucosebased media, prior overnight exposure to 1 mg/ml LPS), and galactose fed (galactose-based, glucose-free media). At the RNA
level, we observed no significant difference between responsive
versus galactose-fed cultures, with or without addition of LPS
(Fig. 1A). TNF mRNA levels were significantly different in tolerant cultures, in line with previous reports (17). Despite showing
no difference in TNF mRNA, however, galactose-fed cultures did
show a significant reduction in TNF protein expression, as measured by ELISA (Fig. 1B). Culturing conditions did not appear to
significantly impact stability of TNF transcript (Fig. 1C).
We next compared the differences in glycolysis between cells
grown in responsive, tolerant, or galactose-fed culturing conditions.
This was done in two ways. Lactate concentration following addition of LPS was measured using a commercial biochemical
lactate assay (Fig. 2A). Responsive cells showed the highest
concentration of lactate, followed by tolerant and galactosefed cells, respectively. We also measured the ECAR of responsive, tolerant, and galactose-fed cells using the Seahorse XF24
(Fig. 2B). As a measurement of the rate of proton output by live
FIGURE 6. TNF mRNA is not present in polysomes in galactose-fed cells. (A) Absorbance of glucose and galactose cultures following separation by
density on sucrose gradient. Gradients were fractionated and measured for absorbance at 254 nm to identify 40S, 60S, 80S, and polysome fractions. Graph
shows representative of three independent experiments. (B) Distribution of TNF mRNA in fractions. Total RNA was extracted from each fraction, and then
TNF mRNA was measured by quantitative PCR. Graph shows representative of three independent experiments. (C) Relative portion of TNF mRNA from
(B) found in translated polysome fractions versus nontranslated fractions. (D) Distribution of actin mRNA in fractions. Total RNA was extracted and
measured by quantitative PCR, as in (B). Graph shows representative of three independent experiments. (E) Relative portion of actin mRNA from (D) found
in translated polysome fractions versus nontranslated fractions.
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As our laboratory has previously reported (17, 22), endotoxin
tolerance includes two distinct phenotypic characteristics in THP1 monocytic cells. One characteristic of tolerance is an inability to
produce TNF-a mRNA or protein in response to LPS restimulation. The other characteristic is a preference for fatty acid
oxidation over glycolysis (8). To test our hypothesis that the latter
influences the former, we compared responsive and tolerant cells
to those grown in galactose-based media. The literature suggests
that when glucose is replaced by galactose in cell culture media,
2545
2546
GAPDH BINDING REPRESSES TNF TRANSLATION
FIGURE 7. Glycolysis is subject to artificial manipulation in tolerant THP-1 cells. (A) Table of drugs used to block or increase glycolysis, with brief
description of mechanism. (B) ECAR of tolerant cell cultures with or without drug treatments as indicated. Changes in ECAR were consistent with expected
effects on glycolysis. Data are representative of n = 3. **p , 0.01 compared with tolerant cells.
GAPDH binds to TNF mRNA in THP-1 cells with low
glycolysis
Our observation that TNF protein but not mRNA was reduced in
galactose-fed cells (Fig. 1A, 1B) suggests a mechanism of posttranscriptional repression. These data are consistent with our hypothesis that low glycolysis causes GAPDH to bind the ARE of
TNF mRNA. To determine whether this was the case, we used RNA
immunoprecipitation (RNA-IP) with an anti-GAPDH Ab to probe
for an interaction between GAPDH protein and TNF-a mRNA.
FIGURE 8. GAPDH binding to TNF mRNA is sensitive to changes in glycolysis. (A) TNF mRNA expression in tolerant cells, relative to actin, with or
without addition of drugs as indicated. Table and shaded portions of bars (GAPDH-IP) show percentage of TNF-a mRNA captured by GAPDH Ab during
RNA-IP, relative to total RNA as determined from input. Bars show mean of n = 3 6 SEM. The p values are compared with tolerant cells, calculated by an
unpaired t test. (B) GAPDH mRNA expression of same cells previously described. Bars show mean of n = 3 6 SEM. Table and shaded portion of bars
(GAPDH-IP) show percentage of GAPDH mRNA captured by GAPDH Ab during RNA-IP. (C) Western blot of GAPDH and actin in tolerant cell cultures
with or without indicated treatments. Blots are representative of three independent assays. No significant difference in GAPDH density was observed.
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cells, ECAR serves as an indicator of lactic acid production and
glycolysis (36). Basal ECAR was the highest in responsive cells,
followed respectively by tolerant and galactose-fed cells. Interestingly, responsive cells showed a sharp increase in ECAR after
an injection of LPS into the assay wells, whereas neither tolerant
nor galactose cells showed any significant change in ECAR in
response to LPS. These differences in lactate (Fig. 2A) and ECAR
(Fig. 2B) both indicate that galactose-fed THP-1 cells have a
lower rate of glycolysis than do their glucose-fed counterparts.
The Journal of Immunology
2547
FIGURE 9. Changes in GAPDH binding TNF mRNA correlate with changes in TNF protein levels in tolerant cells. (A) RT-qPCR of TNF mRNA with or
without second dose of LPS for 1 h, expressed as relative fold of tolerant (2)LPS (0 h). No significant differences observed. Bars show mean of n = 3 6
SEM. (B) ELISA of TNF-a cytokine with or without second dose of LPS for 22 h. *p , 0.05 compared with respective tolerant cultures without drug
treatment, calculated by an unpaired t test.
during endotoxin tolerance. Tolerant THP-1 cells show reduced
glycolysis (Fig. 2) and serve as a model for septic shock (47–49).
To determine whether this mechanism participated in tolerance,
we again used RNA-IP to probe for interactions between GAPDH
protein and TNF-a mRNA. Tolerant cultures were stimulated with
LPS for 24 h prior to assay, whereas responsive cultures were not
exposed to any LPS prior to assay.
Real-time PCR analysis of the RNA pulled down by the GAPDH
Ab shows that GAPDH binds to TNF mRNA in tolerant cells
(Fig. 4A). The amount of TNF mRNA bound by GAPDH was
significantly greater in tolerant cells than responsive cells, despite
the repression of TNF mRNA in tolerant cells. As in the glucose versus galactose model, no significant off-target binding to
GAPDH mRNA is observed (Fig. 4B). We also observed no significant change in total GAPDH protein level (Fig. 4C).
GAPDH is responsible for posttranscriptional repression
Our observation that GAPDH binds to TNF mRNA when TNF
protein, but not mRNA, is reduced immediately suggests a mechanism of posttranscriptional repression. To test this potential
mechanism, we used siRNA to knock down GAPDH expression in
both glucose and galactose-fed cells (Fig. 5A). We observed that
whereas the GAPDH knockdown had no significant effect on
glucose-fed cells, the knockdown increased production of TNF
FIGURE 10. Transcripts with the 39UTR of TNF-a mRNA are repressed in a metabolism-sensitive manner. (A) Schematic of luciferase reporter plasmids
used. Plasmids encoded Renilla luciferase, which does not require ATP for luminescence. Transcripts contained either the TNF-a 39UTR or had no 39UTR
(control). Reporter plasmid transcription was controlled by a constitutive promoter (RPL10). Cells were also transfected with a Cypridina loading control
plasmid, which uses a different substrate. (B) Relative luciferase activity of reporter plasmids, normalized to loading plasmid. Data shown in log scale. Bars
show mean of n = 3 6 SEM. *p , 0.05, **p , 0.01 compared with respective tolerant wells without drug treatment, calculated by an unpaired t test.
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Our initial RNA-IP experiments compared responsive, glucosefed cells with responsive, galactose-fed cells. As shown in Figs. 1
and 2, these cultures differed in metabolism, but not TNF mRNA.
After stimulation with LPS for 1 h, significantly more TNF mRNA
was pulled down by the GAPDH Ab in galactose-fed cultures than
in glucose-fed cultures (Fig. 3A). This indicates greater GAPDH
protein–TNF mRNA binding occurs in galactose-fed cells.
Additionally, GAPDH showed no off-target binding to its own
mRNA (Fig 3B). GAPDH mRNA is constitutively expressed and
lacks an ARE, making it an unlikely target for GAPDH protein to
bind. This made GAPDH mRNA a suitable negative indicator of
nonspecific RNAs isolated by the RNA-IP. As shown in Fig. 3B,
minimal GAPDH mRNA was pulled down during the RNA-IP.
This indicates that there is specificity to the GAPDH protein–
TNF-a mRNA interaction. To test whether the increase in GAPDH–
TNF mRNA binding reflected an increase in total GAPDH protein,
we measured GAPDH protein levels by Western blotting (Fig. 3C).
We observed no significant change in GAPDH protein concentration in response to galactose-based media, or in response to
stimulation with LPS.
Comparison of glucose-fed and galactose-fed cultures indicated
that our hypothesized mechanism of metabolism-sensitive RNA
binding took place in monocytes, but under idealized and artificial
conditions. We next sought to investigate whether it also took place
2548
GAPDH BINDING REPRESSES TNF TRANSLATION
protein in galactose-fed cells (Fig. 5B). This supports our hypothesis that GAPDH protein limits production of TNF protein in
cells with reduced glycolysis.
We next sought to verify that posttranscriptional repression was,
in fact, responsible for the loss of TNF protein. To test this, we
used polysome fractioning to determine whether TNF mRNA
ceased associating with polyribosomes when glycolysis was limited. Lysates from glucose and galactose-fed cells were separated
over a 10–45% sucrose gradient and fractionated by density. Polysome fractions were determined by UV absorbance at 254 nm
(Fig. 6A). We observed that in glucose-fed cells, a greater portion
of the TNF mRNA was present in the polysome fractions (Fig. 6B,
6C). In galactose-fed cells, TNF mRNA was found in less dense
fractions, indicating fewer associated ribosomes. Neither media
affected the density of actin mRNA (Fig. 6D, 6E).
GAPDH binding to TNF mRNA is sensitive to changes in
glycolysis
After demonstrating GAPDH binding to TNF mRNA in two
conditions with low glycolysis, we sought to further establish that
glycolysis regulated the level of this binding. We also sought to
determine whether this binding was reversible. To test this, we
treated tolerant THP-1 cells with different substances that alter
glycolysis. We then used RNA-IP to study corresponding changes
in GAPDH–TNF mRNA binding.
Based on the literature and our past experience, we selected four
substances, each with a distinct mechanism of affecting glycolysis
(Fig. 7A). To block glycolysis, we used 2-DG, an inhibitor of
hexokinase and phosphoglucose isomerase (50). To promote glycolysis, we used EX527, a sirtuin 1 inhibitor that limits the ability
of cells to transition from glycolysis to fatty acid oxidation (8);
human insulin, which increases glucose uptake and phosphorylation (51, 52); and oligomycin, an ATP synthase inhibitor that
blocks mitochondrial ATP production (53) and causes an acute
increase in glycolysis.
The effects of these four substances on glycolysis were verified
by Seahorse XF analysis (Fig. 7B). Tolerant cell cultures were
treated with 2-DG (5 mM, 1 h before assay), EX527 (5 mM, 18 h
before assay), human insulin (100 nM, 18 h before assay), or
oligomycin (10 mM, 15 min before assay) as indicated. Cultures
were then lysed and analyzed by RNA-IP. Inhibition of glycolysis
using 2-DG resulted in a greater level of TNF-a mRNA in the
resulting GAPDH RNA-IP (Fig. 8A). Similarly, promotion of
glycolysis with any of the other three treatments decreased the
level of TNF mRNA isolated by RNA-IP. This indicates that
lowering glycolysis increases GAPDH–TNF mRNA binding,
whereas increasing glycolysis reduces that binding. This reciprocal relationship is predicted by our hypothesis. No significant
binding to GAPDH mRNA was observed (Fig. 8B), again indicating that the GAPDH–TNF mRNA interaction is specific. Additionally, we saw no significant change in total GAPDH protein
in response to the treatments (Fig. 8C).
We next explored whether these changes in glycolysis produced measurable changes in TNF protein. If GAPDH–TNF mRNA
binding truly represents a mechanism of posttranscriptional repression, we would expect that treatments that increase glycolysis
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FIGURE 11. GAPDH binds to TNF-a mRNA in primary cells. (A) Lactate assays of primary cells kept in responsive, tolerant, and galactose-fed
culturing conditions, before and after addition of LPS (100 ng/ml). Points show mean of n = 4 6 SEM. (B) ELISA assay comparing TNF cytokine expression of primary cells kept in responsive, tolerant, and galactose-fed culturing conditions with or without 5 h stimulation with 100 ng/ml LPS. Bars show
mean of n = 3 6 SEM. *p , 0.05 compared with responsive cells. (C) TNF-a mRNA expression in responsive, tolerant, or galactose-fed primary cultures,
relative to actin, with or without addition of LPS (100 ng/ml) for 1 h. Both the table and the blackened portions of bars (GAPDH-IP) show percentage of
TNF-a mRNA captured by GAPDH Ab during RNA-IP, relative to total RNA as determined from input. Bars show mean of n = 5 6 SEM. The p values are
compared with responsive culture counterpart, calculated by an unpaired t test. Nonsignificant values (p . 0.05) are not shown.
The Journal of Immunology
2549
mRNA binding results in posttranscriptional repression, altering
glycolysis should alter luciferase signal in a consistent manner.
We observed a significant reduction in luciferase signal in tolerant cells transfected with the TNF 39UTR reporter, compared
with those with the control 39UTR (Fig. 10B). This immediately
demonstrated the importance of posttranscriptional repression of
TNF, which has been previously shown (25, 26). When cells
transfected with the TNF 39UTR reporter were treated with substances that affected both glycolysis and GAPDH–TNF mRNA
binding (Figs. 7B, 8A), luciferase signal was also affected (Fig. 10B).
Addition of 2-DG caused a decrease in luciferase signal, whereas
addition of insulin or oligomycin resulted in increased signal.
These results match the RNA-IP data (Fig. 8A), which indicated
the treatments respectively increased or decreased posttranscriptional
repression of TNF mRNA.
GAPDH binds to TNF-a mRNA in primary cells
and decrease GAPDH–TNF binding would increase TNF protein
production. To test this, we measured expression of TNF mRNA
and protein in tolerant THP-1 cells treated with either EX527 or
insulin versus untreated. We were unable to use 2-DG or oligomycin
in this study due to higher toxicity and the longer incubation period
required for ELISA.
TNF mRNA levels were not increased by addition of insulin or
EX527 to tolerant cultures (Fig. 9A); however, we observed small
but statistically significant increases in TNF protein levels following treatment with either substance (Fig. 9B). Because the
increase in cytokine production cannot be explained by an increase in RNA, it follows that a greater amount of the transcript is
translated. This supports our hypothesis that GAPDH binding
represses translation of TNF mRNA.
Transcripts with the 39UTR of TNF mRNA are repressed in a
metabolism-sensitive manner
Our data indicate that GAPDH–TNF mRNA binding correlates with
a decrease in TNF-a protein expression. To further demonstrate that
this decrease in cytokine production is due to posttranscriptional
repression, we used a luciferase reporter system (Fig. 10A). We used
plasmids encoding a Renilla luciferase transcript, with or without
the TNF 39UTR present. Because the plasmids contained the same
constitutive promoter, and because Renilla luciferase is not affected
by ATP, changes in luminescence should be directly attributable
to posttranscriptional regulation. We reasoned that if GAPDH–TNF
After characterizing this mechanism of posttranscriptional repression in THP-1 cells, we tested whether this mechanism was also
present in primary human monocytes. Primary monocytes were
isolated from whole blood samples collected from healthy donors.
Donor monocytes were either cultured overnight in glucose-based
media, tolerized ex vivo, or cultured overnight in galactose-based
media. Examination of cell morphology the following day by
brightfield staining showed .90% of isolated cells were monocyte/
macrophage cell types (data not shown).
We first measured the effect of our responsive, tolerant, and
galactose-fed culturing conditions on glycolysis. As in our THP-1
model, responsive cultures showed the highest level of glycolysis
before and after the addition of LPS (Fig. 11A). Tolerant and
galactose-fed cell cultures both showed reduced concentration of
lactate, indicating a reduced rate of glycolysis.
We next determined whether culturing conditions affected
production of TNF cytokine. ELISA analysis of cell supernatant
revealed that cells in tolerant and galactose cultures produced less
cytokine in response to LPS than did their responsive counterparts (Fig. 11B). These results are consistent with THP-1 results
(Fig. 1B), supporting the hypothesis that a similar mechanism
was responsible. When analyzed by RNA immunoprecipitation,
GAPDH binding to TNF mRNA was confirmed (Fig. 11C). We
found significantly greater GAPDH–TNF mRNA binding in tolerant and galactose-cultured cells than in responsive-cultured
cells. This difference is particularly prominent when responsive
and galactose cultures are compared.
Discussion
In this study, we show that TNF mRNA is posttranscriptionally
repressed by GAPDH binding to the 39UTR. As summarized in
Fig. 12, this mechanism of repression is sensitive to changes in
cellular metabolism, specifically the rate of glycolysis. When the
rate of glycolysis is high, GAPDH binds TNF mRNA at a relatively low level. When glycolysis is downregulated due to limited
availability of glucose or endotoxin tolerance, GAPDH binds TNF
mRNA to a greater degree. This binding inhibits translation of the
transcript, thus limiting TNF cytokine production.
This study further demonstrates that GAPDH binding to TNF
mRNA can be reversed by increasing glycolysis. Others have
shown that GAPDH metabolic substrates G3P and NAD+ interfere with GAPDH binding to ARE (10, 14, 15). As neither G3P
nor NAD+ is membrane permeable, however, these data were
observed in ex vivo experiments or in saponin-permeabilized
cells. We think our approach of reversing binding by increasing glycolysis better illustrates the central role of metabolism in
regulating translation.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 12. Experimental model of posttranscriptional repression of
TNF-a by GAPDH. Schematic of experimental model. Left portion represents high glucose, high glycolysis conditions such as those found in
responsive, glucose-fed monocytes. Right portion represents conditions of
low glycolysis, such as those found in endotoxin tolerance or in galactosefed monocytes. When monocytes are stimulated by a molecule such
as LPS, they respond by upregulating transcription of inflammatory
genes such as TNF-a. The 39UTR of TNF-a mRNA contains an ARE.
Depending on the cellular environment, GAPDH can bind this ARE and
repress translation of the TNF-a mRNA. In our experimental model, the
rate of glycolysis determines whether TNF-a mRNA is posttranscriptionally repressed by GAPDH. In a high glycolysis environment, such as
the one depicted in Fig. 8A, the high concentration of GAPDH’s metabolic
substrates outcompetes the interaction between the enzymatic site of
GAPDH and the ARE of TNF-a mRNA. With GAPDH occupied with
glycolysis, TNF-a mRNA is free to be translated. In low glycolysis environments, as depicted in Fig. 8B, there is a relatively low concentration
of metabolic substrates for GAPDH. Without those substrates present,
GAPDH is better able to associate with the ARE of TNF-a mRNA. Once
bound, translation of the transcript is repressed. This mechanism is likely
meant to prevent the production of the TNF-a cytokine when monocytes
are not acting as effector cells.
2550
Acknowledgments
We acknowledge David Long and Dr. Michael Seeds for technical assistance during this project as well as Dr. Martha Alexander-Miller and
Dr. Anthony Molina for guidance during this project.
Disclosures
The authors have no financial conflicts of interest.
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We propose that this mechanism of posttranslational repression
through GAPDH–TNF mRNA binding represents a way of finetuning the inflammatory response. Our data indicate that glycolysis affects production of TNF cytokine, although only modestly
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