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J. Biol. Chem., Vol. 275, Issue 23, 17728-17739, June 9, 2000
From the
Received for publication, October 4, 1999, and in revised form, February 8, 2000
Increased oxidative stress has been
reported in vivo in the diabetic state via the production
of reactive oxygen species (ROS). Such stress is bound to play a key
role on activation of circulating monocytes, leading to the accelerated
atherosclerosis observed in diabetics. However the exact molecular
mechanisms of monocyte activation by high glucose is currently unclear.
Here, we demonstrate that chronic high glucose (CHG) causes a dramatic
increase in the release of the inflammatory cytokine tumor necrosis
factor Cellular redox state has been shown to play an important role in
the pathogenesis of cardiovascular disease including atherosclerosis, the rate of which is higher in diabetics (1-3). Hyperglycemia in the
blood stream could generate free radicals and peroxide species by slow
"autooxidation" of glucose, causing oxidative stress to circulating
monocytes (4, 5). Furthermore, glycosylation of low density lipoprotein
increases its susceptibility to oxidation, generating byproducts in
circulation that preferentially accumulate in foam cell-generating
monocytes/macrophages (6, 7). Additionally, soluble advanced glycated
end products (AGEs)1 present
in the blood stream could also generate reactive oxygen species (ROS)
(8-10). AGEs deposited in the arterial walls generate free radicals
capable of oxidizing vascular lipids and accelerating atherogenesis in
hyperglycemia (9, 11).
As peripheral blood glucose levels increase in hyperglycemia, there is
simultaneous rise in intracellular glucose levels, utilizing the
sorbitol pathway and altering the redox balance inside the cells.
Hyperglycemia also leads to increased NADH/NAD+ ratio,
thereby decreasing the availability of NAD+ as a co-factor
for other metabolic events (12-14). The redox changes induced by
hyperglycemia, AGEs, and lipid peroxidation have been shown to alter
cellular functions via activation of key signal transduction pathways
involving MAPKs such as ERK 1/2, JNKs, and p38 (15-18). High glucose
and diabetes have been shown to specifically activate p38 MAPK via ROS
intermediates in smooth muscle cells (19-20), and oxidant stress has
been shown to incite macrophage spreading via the p38 MAPK pathway
(21). In addition, production of inflammatory cytokines such as TNF The inflammatory cytokine human tumor necrosis factor The role of monocytes in increased foam cell formation in diabetic
patients is well established (25). Hyperglycemia-induced oxidative
stress, further accentuated by the inactivation of superoxide dismutase
(12), along with soluble AGEs and products of lipid peroxidation
possibly serve as key activators of circulating monocytes via the
activation of upstream kinases, leading to induction of inflammatory
gene expression. However the signaling kinases or transcription factors
specifically involved in high glucose-induced monocyte activation
leading to the production of the inflammatory cytokine TNF Activation of genes in response to inflammatory stimuli has been shown
to involve coordinated participation of transcription factors NF- Since the exact mechanism for the activation of monocytes leading to
inflammatory cytokine production by hyperglycemia is currently unclear,
we evaluated some of the key molecular and cellular events leading to
TNF Cell Culture--
U937 (monoblastoid) cells and THP-1
(histiocytic) cells were obtained from ATCC and maintained in RPMI 1640 medium containing 7% heat-inactivated fetal calf serum,
Preparation of Human Monocytes--
Fresh human monocytes were
obtained from healthy donors using an approved Institutional Review
Board protocol and isolated as described previously (41). Autologous
serum was used for the attachment purification and culture of the monocytes.
Lucigenin Chemiluminescence Assay (LCA)--
This assay was
performed as described earlier (6) to measure superoxide anion
(O2 Inhibitors and
Reagents--
N-Acetyl-L-cysteine (NAC; 100 µM) was purchased from Calbiochem. It was dissolved in
phosphate-buffered saline, and the pH was adjusted to 7.2. Pyrrolidine
dithiocarbamate (PDTC; Sigma; 50-100 µM) was dissolved
in water. Me2SO was used to resuspend the rest of the
inhibitors and was added to the control plates. p38 MAPK inhibitor
SB202190 (SB, 10 µM) was purchased from Upstate Biotechnology. Mannitol, 3-O-methyl glucose, and
2-deoxyglucose were purchased from Sigma.
Detection of Secreted TNF Detection of TNF Plasmids and Luciferase Reporter Gene Assays--
The 295TNF Mutational Analysis of the NF- Preparation of Nuclear and Cytosolic Extract for Gel-shift
(Electromobility Shift Assay (EMSA)) and Western Analyses--
Cells
were cultured in NG or CHG, depleted of serum in depletion media, and
treated with TNF Binding Reaction and EMSA--
Oligonucleotide probes for EMSA
were synthesized in the City of Hope National Medical Center DNA
synthesis facility. The proximal NF- Western Analysis--
Nuclear extracts (5 µg) were mixed with
equal volume of 2× sample buffer (4% SDS, 10% glycerol, 0.006%
bromphenol blue, and 2% Statistical Analysis--
Results are expressed as the mean ± S.E. of the average responses in multiple experiments. Data were
analyzed by analysis of variance followed by Tukey's test or by
Student's t tests for paired components.
CHG Induces Oxidant Stress in Monocytes
CHG Increased Reactive Oxygen Species in Monocytic Cells Detected
by LCA--
To investigate whether CHG increased ROS in monocytic cell
lines (U937 and THP-1), we examined the effect of CHG culturing alone
on superoxide (O2 Elevated Phosphorylation of Stress-responsive MAPKs by CHG,
Confirming Oxidant Stress--
An important measure of oxidative
stress is the activation of upstream stress-responsive MAPKs. We
therefore examined the effects of CHG on phosphorylation of p38 (pp38),
JNK-1 (pJNK-1), and ERK 1/2 MAPKs using Western blot analysis. Fig.
1B, top panel 1, shows a
representative immunoblot probed with antibodies to pp38. In the
middle panel, the same blot was stripped and
re-probed with pJNK-1 antibody, and in the bottom
panel, the blot was stripped further and probed with
nonphosphorylated p38 antibody serving as loading control. THP-1 cells
cultured in CHG showed a striking increase in basal levels of pp38
(Fig. 1C, 2.6-fold) and pJNK-1 (Fig. 1D,
2.3-fold) over NG controls. Treatment of cells with TNF Elevated Levels of TNF TNF TNF To evaluate if CHG-induced TNF
Molecular Mechanisms of Tumor Necrosis Factor
Gene
Expression in Monocytic Cells via Hyperglycemia-induced Oxidant
Stress-dependent and -independent Pathways*
§,
, and
Department of Diabetes and Endocrinology and
Graduate School of Biological Sciences, City of Hope National
Medical Center, Duarte, California 91010, ¶ Genetics Institute,
Pasadena, California 91105, and Department of Internal
Medicine, University of Virginia, Charlottesville, Virginia 22908
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(TNF), at least in part through enhanced TNF mRNA
transcription, mediated by ROS via activation of transcription factors
nuclear factor 
B (NF-B) and activating protein-1 (AP-1). TNF
accumulation in the conditioned media was increased 10-fold and
mRNA levels were increased 11.5-fold by CHG. The following
observations supported that both NF-B and AP-1 mediated enhanced
TNF transcription by CHG: 1) A 295-base pair fragment of the
proximal TNF promoter containing NF-B and AP-1 sites reproduced
the effects of CHG on TNF transcription in a luciferase reporter
assay, 2) mutational analyses of both NF-B and the AP-1 sites
abrogated 90% of the luciferase activity, 3) gel-shift analysis using
the binding sites showed activation of NF-B and AP-1 in CHG nuclear
extracts, and 4) Western blot analyses demonstrated elevated nuclear
levels of p65 and p50 and decreased cytosolic levels of IB in
CHG-treated monocytes. That ROS acted as a key intermediate in the CHG
pathway was supported by the following evidence: 1) increased
superoxide levels similar to those observed with PMA or TNF, 2)
increased phosphorylation of stress-responsive mitogen-activated
protein kinases p38 and JNK-1, 3) counteraction of the effects of CHG on TNF production, the 295TNFluc reporter activity, activation of
NF-B, and repression of IB by antioxidants and p38
mitogen-activated protein kinase inhibitors. The study suggests that
ROS function as key components in the regulatory pathway progressing
from elevated glucose to monocyte activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and interleukin-6 by activated rat smooth muscle cells was regulated by
the p38 MAPK pathway (22). Activation of the p38 MAPK has been observed
in a number of physiological responses such as apoptosis of myocardial
cells (25) and adipogenesis in 3T3-L1 cells (23). Altered
NADH/NAD+ ratio caused by hyperglycemia results in de
novo synthesis of diacylglycerol and activation of various protein
kinase C (PKC) isoforms in cell/tissue-type and stimulus-specific
manner (7, 25, 44). That hyperglycemia induced ROS may function as a key intermediate leading to the activation of PKC has been shown in
many cell types of human and porcine origin (22, 26).
PKC-dependent and -independent activation of p38 MAPK
pathway was observed in smooth muscle cells (19) and mesangial cells,
respectively (24).
(hTNF) is
produced by activated monocytes in response to a variety of signals
including stress response, phorbol esters, cytokines, endotoxin, and
substrate adherence (27-31). TNF gene expression is regulated both
at the levels of transcription and post-transcription. Elevated levels
of TNF and other inflammatory cytokines have been detected in
atherosclerotic plaques of diabetic and nondiabetic patients (32).
are still
unclear and are the focus of the present study.
B
and AP-1. Regulation of many inflammatory cyokines, tissue factor, and
matrix metalloproteinases involve dual transcriptional regulation by
NF-B and AP-1 (33-36). NF-B/Rel proteins are heterodimeric transcription factors retained in the cytoplasm of unstimulated cells
by the inhibitory subunit IB, the NF-B/IB forming an inactive
ternary complex (37). Stimulation with stress-inducing agents or other
proinflammatory mediators causes rapid phosphorylation, ubiquitination,
and degradation of IB-subunit, allowing translocation of NF-B to
the nucleus (37). NF-B then induces transcription of several genes,
including that of its inhibitor IB (38). Another transcription
factor regulated by cellular stress is AP-1, a transacting molecule
consisting mainly of homodimers of Jun or heterodimers of Fos and Jun
(39). The hTNF gene has in its promoter region canonical binding
sites for transcription factors NF-B and AP-1 (40).
secretion by high glucose. Our study suggests that CHG-induced
monocyte activation, as evidenced by increased TNF expression, was
regulated at least in part through increased TNF mRNA
transcription. The process involved ROS-dependent and
-independent pathways, requiring coordinate activation of both p38 MAPK
and PKC as upstream kinases and NF-B and AP-1 as downstream
transcription factors.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol (50 µM), HEPES (10 mM),
glutamine (2 mM), streptomycin (50 µg/ml), penicillin (50 units/ml), and 5.5 mM glucose (NG). For chronic high
glucose (CHG) conditions, cells were cultured in 12.5, 15,
or 25 mM glucose for 2 days before being depleted of serum
and treated with various agents as indicated under "Results." High
glucose (HG) culturing was done in 15 mM glucose for
18 h. This HG condition was used to culture peripheral human
monocytes for TNF production and for 295TNFluc luciferase activity
studies. CHG- or HG-treated cells were washed and resuspended in
depletion medium containing 0.5% bovine serum albumin for 18 h or
for other periods as indicated prior to stimulation.
). Briefly, U937 or THP-1 cells
were cultured in NG versus CHG, depleted of serum using
depletion media, and prepared for the assay. Cells were treated with
TNF (5 ng/ml) or PMA (10 ng/ml; positive control) for 60 min, washed
in a balanced salt solution, and resuspended at 2 × 106 cells/ml in aerated balanced salt solution. 1 × 106 cells/ml was used for the assay.
O2 was measured in intact cells by
LCA. Cells from various treatments were added to a scintillation vial
containing lucigenin (500 µM) in the aerated balanced
salt solution. Photon emission was measured for 10 min using the
Beckman LS6500 Multipurpose Scintillation counter measuring single
photon emission. First, photon emission was measured using a buffer
blank and dark-adapted lucigenin, and the blank reading was subtracted
from the sample reading. A standard curve was generated using xanthine
and xanthine oxidase. Superoxide dismutase (100 units/ml) was used as
an inhibitor for superoxide production. H2O2
was added to the buffer blanks or to the NG control cells to determine
if the photon emission in the lucigenin assay was induced by
O2 or peroxide species.
in the Culture Supernatant by
Specific hTNF Enzyme-linked Immunosorbent Assay--
U937 or THP-1
cells were cultured in NG or CHG, depleted of serum in depletion media,
and cultured in NG, CHG, or NG + PMA for another 24 h.
Quantitative detection of hTNF in conditioned media was performed
using a specific antibody sandwich CytoscreenTM
enzyme-linked immunosorbent assay assay from
BIOSOURCE International using the manufacturer's
suggested directions. Known concentrations of hTNF were used to
generate the standard curves. Supernatant from NG-cultured cells
treated with PMA (10 ng/ml) for 24 h served as the positive
control. Streptavidin-horseradish peroxidase served as the detection
system. For each experiment, duplicate samples were measured. Data were
represented as the mean (pg/106 cells) ± S.E. The
assay was linear between 15.6 pg/ml and 1000 pg/ml.
Message by Competitive RT-PCR
Assay--
TNF message was measured in U937 cells using the
Quantitative RT-PCR Cytoexpress detection kit from
BIOSOURCE International. Cells were cultured and
depleted as in the preceding paragraph above, and RNA was extracted
from CHG or NG cells stimulated for 4 h with various
concentrations of TNF (0.25, 0.5, 1, or 5 ng/ml) or PMA (10 or 50 ng/ml). RNA was reverse-transcribed to cDNA using murine leukemia
virus reverse transcriptase. A known copy number of exogenously
synthesized DNA, known as the internal control standard (ICS) was mixed
with sample cDNA before PCR amplification. The ICS contained PCR
primer binding sites similar to the TNF cDNA and a unique
capture-binding site to distinguish the ICS amplicon from the TNF
amplicon. In samples containing ICS, two amplicon bands were visible
following PCR amplification, the 382-base pair TNF band and the
432-base pair ICS band. After amplification, the amplicons were
hybridized to the ICS or TNF-specific oligonucleotide-coated wells.
Biotinylation of an original primer allowed streptavidin coupled to
biotin to be used as the detection system. The signal generated in the
hybridization reaction was proportional to the number of amplicons
present in the starting cDNA. Since the ICS (known copy number) was
amplified at the same frequency as the TNF cDNA, it served to
determine the copy number of TNF cDNA in each sample (see
Equation 1).
(Eq. 1)
luciferase (295TNFluc) deletion construct from the human TNF
promoter was a kind gift from Dr. James S. Economou, UCLA (44). 5 × 106 U937 or THP-1 cells were depleted of serum 2 h
before transfection. The cells were transfected with 2 µg of the
295TNFluc deletion construct and co-transfected with 0.2 µg of
reporter plasmid (RSVGal) using the Effectene reagent (Qiagen Inc.)
in 2% serum-containing media overnight. The cells were washed and
depleted of serum for 4 h before stimulation with glucose (NG
versus HG) and/or TNF for 18 h. Alternatively, cells
were cultured in NG or CHG for 24 h, washed, depleted for 4 h, and transfected with 295TNFluc plasmid overnight. On day 3, cells
were washed, depleted of serum, and stimulated with NG or CHG
plus/minus TNF for 18 h, and stimulated luciferase activity was
measured. Treatment with PMA served as the positive control for the
stimulated luciferase activity. Inhibitors were added at the indicated
concentrations 1 h before CHG culturing or before treatment with
TNF or PMA. The inhibitors were present throughout the culturing and
the stimulation phase. The luciferase assay was performed using the
firefly luciferase kit (Promega) using a Turner TD-20e luminometer
measuring light intensity over a 5-log range. Results from the
luciferase assay were normalized to -galactosidase levels, and
relative luciferase units were determined. The results were reported as
fold stimulation over XP-1 control plasmids. Concentrations of
inhibitors used for the study are listed under "Inhibitors and
Reagents" of this section.
B3 and AP-1 Sites in the TNF
Promoter--
Site-directed mutagenesis was performed at the NF-B3
and the AP-1 sites using the Quick-changeTM site-directed
mutagenesis kit from Stratagene using the manufacturer's suggested
directions. The NF-B3 (GGGTTTCTCC) and AP-1 (TGAATGA) sequences were
mutated to taGTTTCTCC (mNF-B3) and gtAATGA (mAP-1), respectively, using primers as suggested in published protocols (47).
or preincubated with the inhibitors at
concentrations for the time indicated under "Results." Nuclear and
cytosolic extracts were made by a modification of the protocol of Lin
et al. (42). The cytosolic extracts (Western blot analysis) and nuclear extracts (gel-shift assay and Western blot analysis) were
divided into aliquot and frozen at 
70 °C for future use. The
concentrations of inhibitors used for the study are listed above.
B (NF-B3) and AP-1 sites from
the TNF promoter were used for the EMSA. The sequence for the
NF-B3 site was: GCTCATGGGTTTCTCCACCAAG. The sequence for
the AP-1 site was: CCAGATGAGCTCATGGG. EMSA was performed
according to published protocol (43). The probes were labeled with
[
-32P]ATP using T4 kinase (Stratagene). Antibodies
used for supershifting experiments, anti-p50 (NLS), anti-p65 (c-20),
anti-C rel, anti-Rel B (C-19), and anti-p52 (K-27), were purchased from
Santa Cruz Biotechnology. The gels were dried and visualized using a
PhosphorImager screen (Molecular Dynamics, San Jose, CA) and quantified
using the Imagequant software (NIH, Bethesda, MD).
-mercaptoethanol), boiled for 5 min, and
run on SDS-polyacrylamide reducing gel (20). Antibodies for Western
analyses were purchased from Santa Cruz Biotechnology. Western blot
analysis was done using anti-p50, anti-p65, anti-IB,
anti-IB, and anti-Ib
antibodies at a dilution of 1:1000.
Anti-histone H1 (C-17), used at 1:10000 dilution, served as the loading
control for nuclear extracts. Cytosolic extracts were treated the same
way as the nuclear extracts, and Western blot analysis was done using
anti-IB and anti-IB antibodies. Anti -actin antibody
(1:20000 dilution), purchased from Sigma, served as the loading control
for the cytosolic extracts. Phosphorylated ERK 1/2, JNK-1, and p38 were
detected by Western blot analysis using phosphorylated ERK 1/2 (pERK), JNK (pJNK), and p38 (pp38) mitogen-activated protein kinase antibodies purchased from New England Biolabs. Nonphosphorylated ERK 1/2, JNK-1,
and p38 served as the loading controls. For these experiments, cells
lysates were made using lysis buffer described earlier (20). All
Western blots were developed using the ECL detection system (Amersham
Pharmacia Biotech), following the protocol suggested by the
manufacturer. Anti-rabbit horseradish peroxidase-linked secondary
antibody served as the detecting antibody. Stripping and reprobing of
the membranes for Western blot analysis was performed according to
stringent conditions suggested in the ECL handbook. Western blots were
quantified with the Alpha Imager documentation and analysis system
using the Alpha Imager 3.24 software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) production. CHG
increased O2 levels significantly
(p < 0.005) over NG-cultured U937 cells (2.3 ± 0.87-fold; Fig. 1A).
O2 levels generated by CHG were
comparable with that observed in NG cells following treatment with the
inflammatory cytokine TNF or phorbol ester PMA (positive control)
for 1 h. Treatment of cells with superoxide dismutase before
measuring O2 levels by LCA quenched
any detectable O2 produced in CHG-,
TNF-, or PMA-treated cells. Adding H2O2 to NG cells did not increase chemiluminescence counts, indicating that
O2 but not peroxide was the
contributor for the photon emission. THP-1 cells generated similar
trends but had higher levels of O2
than U937 cells.
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Fig. 1.
Increased oxidant stress induced in monocytic
cells by CHG. A, higher levels of superoxide production
by U937 cells cultured in CHG. Cells were cultured in NG or CHG for
48 h, then in serum-depleted medium with 0.5% bovine serum
albumin for 18 h (maintaining the appropriate glucose
concentration) and, finally, stimulated with TNF (5 ng/ml) or PMA
(10 ng/ml; positive control) for 1 h. LCA was performed as
explained under "Materials and Methods" ("Lucigenin
Chemiluminescence Assay"). Data are the means ± S.E. from 4 separate experiments and are represented as cpm/106 cells.
Data were analyzed using analysis of variance followed by Tukey's
test. Compared with NG control, superoxide production in CHG, NG/CHG
plus TNF, or NG plus PMA were significantly different
(p < 0.005). SOD, superoxide dismutase.
B, Western blot analyses showing increased phosphorylation
of p38 (pp38) and JNK-1 (pJNK-1) stress-responsive kinases by CHG or
TNF in THP-1 cells. Cells were cultured in NG versus CHG
for 48 h, then in serum-depleted medium as in A and,
finally, stimulated with TNF for 5 or 10 min. Western blots were
probed with anti-pp38 antibody (top panel), stripped and
reprobed with anti-pJNK-1 antibody (middle panel), and
finally, stripped and reprobed with nonphosphorylated p38 antibody
(bottom panel) to show equal loading. C, the
bar graph represents density of pp38 from B. D, the bar graph represents density of pJNK-1
band from B.
for 5 or 10 min (Fig. 1C) showed a stronger increase in pp38 levels in
NG (2.1-fold) compared with CHG (0.6-fold). On the contrary, the pJNK-1
levels (Fig. 1D) following TNF treatment showed a much
stronger and faster increase (2.5-fold) in CHG compared with NG
(1.7-fold). The activation profile of the two stress-responsive kinases
in CHG showed positive correlation with
O2 data. TNF-induced elevated pp38
and pJNK-1 levels in CHG were additive. In contrast, the third member
of the MAPK family, ERK 1/2, which generally is responsive to stress
and mitogenic stimuli, showed no activation by either high glucose or
TNF in these monocytic cells. CHG or TNF showed similar trends in
U937 cells as THP-1. Increased superoxide levels and elevated
phosphorylation of stress-responsive MAPKs, p38, and JNK-1 by CHG
demonstrate that ROS induced by high glucose could potentially
contribute to downstream signaling via activation of MAPKs.
Accumulate in Conditioned Media of U937,
THP-1, or Normal Human Monocytes Cultured in CHG
is a potent cytokine involved in inflammation, and elevated
levels of TNF are seen in atherosclerotic plaques of diabetics. We
therefore evaluated if culturing monocytic cells in CHG could lead to
increased TNF accumulation in conditioned medium. CHG alone induced
a dramatic increase in TNF accumulation in conditioned media of U937
(Fig. 2A, 10.7-fold), THP-1
(Fig. 2B, 8.17-fold), and normal isolated human monocytes
(Fig. 2C, 10.2-fold) compared with their NG counterparts.
PMA was used as a positive control. To determine whether the effects of
CHG (15 mM)-induced TNF accumulation was due to
increased osmolality of CHG, mannitol (9.5 mM) in NG was
used as a control. To evaluate if glucose metabolism was required for
the increased TNF secretion by CHG, 3-O-methyl glucose
(9.5 mM) in NG was tested. Culturing cells in
mannitol, or 3-O-methyl glucose showed comparable levels of
TNF in the conditioned media as NG controls. These results confirmed
that the effect of CHG on elevated TNF accumulation was not due to
hyperosmolality of CHG and that glucose metabolism was essential for
the elevated levels of TNF.
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Fig. 2.
TNF accumulation in
conditioned media was elevated by CHG in U937, THP-1, or normal human
monocytes. A and B, U937
(A) or THP-1 (B) cells were cultured in NG or CHG
for 24 h, then in serum-depleted medium as in Fig. 1A.
Cells were washed, and 1 × 106 cells were cultured in
NG, CHG (15 mM), NG + 9.5 mM mannitol, NG + 9.5 mM 3-O-methyl glucose (3-O-MG), or NG + 10 ng/ml PMA in depletion medium for an additional 24 h.
Conditioned medium was collected as described under "Materials and
Methods" ("Detection of Secreted TNF in the Culture Supernatant
By Specific hTNF Enzyme-linked Immunosorbent Assay"), and TNF
was quantified using enzyme-linked immunosorbent assay assay. Data
represent the mean ± S.E. of four separate experiments, each
sample run in triplicate. C, primary human monocytes
(C) were isolated and cultured as outlined under
"Materials and Methods" ("Preparation of Human Monocytes"), and
TNF levels were quantified in the conditioned media. The data
represent the average ± range of two separate donor samples, each
sample run in triplicate. TNF secretion by normal human monocytes in
HG was significantly higher compared with NG.
Is Transcriptionally Regulated in U937 Cells Cultured in
CHG
is regulated transcriptionally
in monocytic cells, we used competitive RT-PCR to monitor the levels of
TNF message. Since TNF is regulated both by autocrine and
paracrine pathways, we compared the levels of TNF message induced by
the cytokine to that induced by CHG. Ethidium bromide staining of
products from RT-PCR analysis (Fig.
3A) demonstrated that low
levels of TNF (0.25, 0.5, or 1.0 ng/ml), similar to that secreted in
conditioned media by CHG culturing, could stimulate TNF message in
an autocrine fashion (Fig. 3A, top
panel, lanes 5-7). The peak of this stimulation
was at 1 ng/ml. The addition of higher levels of TNF (5 ng/ml) did
not further increase the levels of the cytokine message (Fig.
3A, top panel, lane 8). Interestingly, CHG induced TNF message at levels similar to that induced by 0.25-0.5 ng/ml TNF (Fig. 3A, top panel,
lanes 11 and 12). PMA at 10 ng/ml induced about
2.4-fold higher message compared with CHG (Fig. 3A,
top panel, lane 4) and served as the positive
control for TNF message induction. Glyceraldehyde-3-phosphate
dehydrogenase was used to check the integrity of RNA (Fig.
3A, bottom panel). TNF cDNA was also
amplified using competitive PCR in tubes containing known copy numbers
of ICS and hybridized to wells containing either the ICS
oligonucleotide or the TNF-specific oligonucleotide to determine
copy number of TNF message in the different cDNA samples (Fig.
3B). At low levels (0.25-1 ng/ml), TNF showed a
dose-dependent increase in the copy number of specific
TNF message. Copy number of TNF message induced by CHG was
comparable with that induced by TNF (0.5 ng/ml) (Fig.
3B). The competitive RT-PCR data in U937 cells suggest that
secreted TNF, at levels induced by CHG, could act through an
autocrine loop to transcriptionally regulate further TNF message in
monocytic cell lines. THP-1 cells showed a similar trend as U937 cells
but gave a higher copy number of TNF message following different
stimulations (data not shown). Data from RT-PCR studies indicate that
the regulation of TNF by CHG in U937 and THP-1 cells is controlled,
at least in part, transcriptionally, although post-transcriptional
control may also play a significant role in TNF regulation.
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Fig. 3.
TNF , at levels
secreted during CHG culturing, can further stimulate
TNF message. A, U937 cells
were cultured in NG or CHG for 48 h and then in serum-depleted
medium as in Fig. 1A. TNF message was determined as
described under "Materials and Methods" ("Detection of TNF
Message by Competitive RT-PCR Assay"). The top panel shows
representative ethidium bromide-stained agarose gel. Lanes are as
follows: 1, molecular weight markers; 2, NG CTRL;
3, NG PMA, 50 ng/ml; 4, PMA, 10 ng/ml,
5-8, 0.25, 0.5, 1.0, 5 ng/ml TNF; 9, water CTRL;
10, RT CTRL; 11-12, CHG. Bottom panel,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) analysis of
the same samples shown in the top panel. B,
mRNA copy number for data shown in A. Bars 1-7 are in NG. Bar 8 is CHG. Calculations are described
under "Materials and Methods" ("Detection of TNF Message by
Competitive RT-PCR Assay"). C, representation of the
295TNFluc construct with important transcription factor binding
sites.
Identification of Cis-elements Involved in CHG Mediated Increased
Transcriptional Regulation of TNF
TNF is regulated both transcriptionally and
post-transcriptionally in response to various stimuli. In our study,
the competitive RT-PCR data demonstrated that TNF expression in
monocytic cells has a transcriptional component in response to CHG, and
therefore, some of the cis-elements in the TNF promoter involved in
the induction of this inflammatory cytokine by CHG was evaluated. The
295TNFluc promoter construct selected for our study has been used to
study the cis-elements involved in TNF promoter regulation following
induction by various stimuli such as TNF, PMA, LPS, and cyclosporin
A (29, 30, 39). U937 cells cultured in NG or CHG were transfected with
the 295TNFluc plasmid and cotransfected with the -galactosidase as
internal control, and normalized luciferase activity was measured
following stimulation with or without TNF for 18 h. CHG
stimulated similar luciferase activity as TNF-treated NG cells
(Table I). However, the effect of CHG
plus TNF on luciferase activity was additive over CHG alone (Table
I), possibly suggesting the involvement of more than one pathway in
transcriptional regulation of TNF by CHG and TNF. Specificity for
CHG-stimulated luciferase activity was determined by using mannitol
(control for osmolality), 3-O-methyl glucose and
2-deoxyglucose (control for glucose metabolism). All of the controls
demonstrated near NG levels of luciferase activity (Table I). In some
experiments luciferase activity was measured in transfected NG cells
stimulated with HG (15 mM) for 18 h. HG in the
stimulation phase showed a similar increase in luciferase activity as
CHG. These results demonstrate that 295 base pairs of proximal promoter
region, adjacent to the transcriptional start site, in the TNF
promoter could reproduce the effects observed on the TNF message by
competitive RT-PCR following stimulation by CHG or TNF in the
luciferase reporter assay.
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Mutational Analysis of the NF-B and AP-1 Sites in the Proximal
TNF Promoter
The 295TNFluc plasmid has, in its proximal promoter, consensus
binding sites for NF-B and AP-1 transcription factors (Fig. 3C). Site-directed mutagenesis was used to introduce two
point mutations each, at the NF-B3 (GGGTTTCTCC mutated to
taGTTTCTCC) and AP-1 sites (TGAATGA mutated to
gtAATGA). Mutation of these sites completely abrogated
binding of NF-B or AP-1 to their respective sites using CHG- or
TNF-stimulated nuclear extracts in EMSA (data not shown). Mutations
at the NF-B3 and the AP-1 sites reduced CHG-stimulated 295TNFluc
activity by 71.8 ± 3.73 and 31.5 ± 4.7%, respectively
(Table II). Interestingly, double mutations of both the sites abrogated CHG-stimulated luciferase activity by 89.3 ± 4.31%, confirming the critical contributions of NF-B (major) and AP-1 (minor) in CHG-mediated induction of the
TNF promoter.
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EMSA to Study the Effect of CHG Culturing on NF-B
Activation
The pleiotropic transcription factor NF-B has been shown to be
responsive to oxidant stress in endothelial cells, smooth muscle cells,
and mesangial cells, all of which play a critical role in
atherosclerosis (1, 4, 11). Mutational analyses demonstrated NF-B as
a major transcription factor in CHG-stimulated luciferase activity.
Therefore CHG- and/or TNF-mediated activation of NF-B in
monocytic cell lines was confirmed using EMSA, performed with a 22-base
pair oligonucleotide containing the NF-B3 site from the hTNF
promoter. Nuclear extracts from U937, THP-1, and fresh human monocytes
(from healthy donors) were used for EMSA (Fig.
4, A-D). Culturing U937cells
in 5.5 mM (NG), 15 mM (CHG), or 25 mM (VHG) glucose alone for 3 days showed a
dose-dependent increase in NF-B binding (Fig.
4A, first, third, and fifth lanes). PhosphorImager quantitation of data from several experiments showed that NF-B binding was significantly increased (p < 0.05) in 15 mM (2.1 ± 0.83-fold) and 25 mM glucose (3.2 ± 0.68-fold) over NG (Fig.
4C). Representative gels showing the effect of CHG on NF-B binding are shown in Fig. 4, A and B.
Culturing primary human monocytes overnight in 15 mM
glucose (HG) also caused a marked increase in NF-B binding over NG
controls (Fig. 4D, first and second lanes). Since
CHG leads to accumulation of TNF in the conditioned media, we
evaluated the NF-B binding response to TNF in CHG
versus NG cells. After 1 h of continuous treatment with
TNF, NF-B binding in NG and CHG (15 mM) cells were
not significantly different (representative gels shown in Fig. 4, A and B, second and fourth lanes). CHG
at 25 mM, however, showed an additive increase in NF-B
binding with TNF (13.8 ± 1.77-fold) compared with NG + TNF
(Fig. 4A, second lane versus sixth
lane) in U937 cells, possibly due to significantly higher
osmolality of glucose at such high concentration. The time course of
NF-B activation following continuous treatment with TNF (5-60
min) showed early activation (5-30 min) in CHG versus NG
(Fig. 5, A and B).
PhosphorImager quantitation of NF-B binding using nuclear extracts
from NG- or CHG-cultured U937 cells treated with TNF for 30 min was
performed, and the results are presented graphically in Fig.
4C. Stimulation with TNF for 30 min in CHG showed a
significant increase in NF-B binding (Fig. 4C; 2.01 ± 0.87, p < 0.05) over CHG alone, suggesting an
autocrine role of this cytokine in CHG-stimulated TNF gene
regulation. The stimulatory effect of CHG and TNF were specific to
NF-B, because the ubiquitous transcription factor Oct-1 was not
affected (panels 2 of Figs. 4, A and
B).
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Specificity for CHG-stimulated NF-B binding was determined by
culturing U937 cells in NG plus 9.5 mM mannitol, as a
control for osmolality (Fig. 4E, third lane). To
determine if glucose metabolism was essential for NF-B activation,
nuclear extracts were made from U937 cells cultured in NG plus 9.5 mM 3-O-methyl glucose (Fig. 4E,
panel 1, second lane). These results confirm that
effects of CHG on NF-B activation were not due to increased osmolality of CHG and that metabolism of glucose was necessary for
NF-B activation by CHG.
The subunits of NF-B stimulated by CHG were compared with that
stimulated by NG plus TNF in U937 by supershift analysis (EMSA).
Both TNF (Fig. 4F) and CHG (Fig. 4G)
demonstrated the same supershift profile of the stimulated NF-B
complex, showing p50 and p65 as the major subunits. THP-1 cells showed
similar shifts as U937 cells (data not shown). Primary human monocytes cultured in HG (15 mM) overnight also demonstrated p50 and
p65 as the major components of the activated NF-B complex (Fig.
4D, fourth and fifth lanes). The basal NF-B
complex in NG showed weak supershifting only with the anti-p50 antibody
but not with the anti-p65 antibody (data not shown). The supershift
data confirm that p50/p65 heterodimers and p65 homodimers are the major
transcriptionally active NF-B complexes stimulated by CHG, important
in regulating TNF gene expression.
Elements of NF-B Complex Affected by CHG Culturing and/or TNF
Treatment
To identify the elements of NF-B complex affected by CHG or
TNF stimulation, a time course study was performed in U937 cells cultured in NG or CHG following TNF treatment. The regulation of the
NF-B complex over time was monitored using EMSA and Western blot
analysis. Western blots of nuclear extracts were sequentially probed
with anti-p65, anti-p50, anti-IB, and anti-Histone H1 (loading
control) antibodies, and those of cytosolic extracts were sequentially
probed with anti-IB, anti-IB, and anti-actin (loading
control) antibodies with stripping of the blots following each
detection. Representative gels from the time course study are shown in
Fig. 5, A, C, and E (NG) and
B, D, and F (CHG), and some of the key
data from the same study are graphically summarized in Fig. 5,
G, I, and K (NG) and H,
J, and L (CHG).
The time course of NF-B activation in NG showed a gradual increase
in NF-B binding, with peak binding at 60 min after TNF treatment.
The increased binding persisted for 2 h post-TNF stimulation (Fig. 5A, panel 1, fifth and sixth
lanes) and fell to basal NG levels by 4 h (data not
shown). In CHG, NF-B activation following TNF treatment was
strongly detectable as early as 5 min (Fig. 5B, panel
1, second lane). There was a gradual increase in
NF-B binding, which peaked around 15-30 min following TNF
treatment (Fig. 5B, panel 1, third and fourth
lanes) and remained sustained even at 18 h (data not
shown). Oct-1 (Fig. 5, A and B, panels 2) served as a control for nuclear extract preparation and was not
affected by CHG or TNF at any time points. Under CHG conditions, basal levels of NF-B binding were about 2-fold higher compared with
basal NG levels (Fig. 5, A versus B,
panel 1, first lane). The NF-B binding data
suggest that CHG-cultured U937 cells are primed for a faster
responsiveness to TNF.
To explore the mechanism for this observed increase in NF-B binding
in CHG, we examined the levels of the p50 and p65 subunits in nuclear
extracts, since they were the only subunits identified by
supershifting. The Western blots from nuclear extracts demonstrated higher p65 levels in CHG (2.67 ± 0.69-fold) compared with the barely detectable levels in NG (Fig. 5, C and D,
panel 1, first lane). Basal p50 levels in CHG
were also higher (3.25 ± 0.73-fold) compared with NG (Fig. 5,
C and D, panel 2, first
lane). Mimicking the EMSA (Fig. 5, A
versus B), the graphical results of the Western analysis (Fig. 5, I versus J) showed
significantly higher levels of p65 in CHG compared with NG
(p < 0.05) at the early time points (5-15 min) of
TNF treatment. However at the later time points (60-120 min) of
continuous TNF treatment, p65 levels in NG and CHG were equal,
drawing similarity to the EMSA binding data. These results confirm that
CHG-cultured U937 cells are primed to respond faster to the
inflammatory cytokine TNF compared with NG cultured cells.
To further elucidate the mechanism of NF-B activation, the levels of
the inhibitory IB subunits (IB, and IB) were checked in
cytosolic and nuclear extracts under basal conditions and following TNF challenge in NG and CHG. TNF mediates degradation of
pre-existing IB, releasing the transacting NF-B complex,
allowing its translocation to the nucleus (37). In addition, the
re-synthesized IB translocates to the nucleus, binding to the
p50·p65 complex, thereby exposing the nuclear export signal for the
removal of the latter out of the nucleus (37). We therefore examined
re-synthesized levels of IB in the nuclear extracts of NG-
versus CHG-treated U937 cells following 60-120 min of
continuous TNF treatment. The re-synthesized levels of IB in
NG were significantly higher (2.87 ± 0.78-fold) compared with its
CHG counterpart (Fig. 5, C versus D,
panel 3, fifth and sixth lanes). The Western
blots of the cytosolic extracts from the time course study were also
sequentially probed with anti-IB and anti-IB antibodies
with intermediate stripping. We report here for the first time that the
level of IB in the cytosol of NG-cultured cells was significantly
higher (2.65 ± 0.53-fold) compared with that observed in CHG
(Fig. 5, E versus F, panel 1, first
lane). Re-synthesized levels of IB in the cytosol
following 60 or 120 min of TNF treatment were also higher in NG
versus CHG cells (1.85 ± 0.71-fold, Fig. 5,
E versus F, panel 1, sixth
lane), lending additional support to the higher basal p65
levels in CHG-cultured cells. In contrast to IB, levels of
IB (data not shown) and IB were not affected at any of the
time points evaluated (Fig. 5, E versus
F, panel 2).
Data from the time course study indicated that U937 cells cultured in
CHG are primed for faster response to TNF and show a stronger rapid
activation of NF-B compared with NG cells. The levels of IB
were higher in the cytosol of NG versus CHG cells, suggesting a possible mechanism for the higher basal levels of p65 in
CHG, which could further explain the increased DNA binding and
luciferase activity.
Effects of Antioxidants on CHG-induced NF-B Activation
U937 cells were incubated with antioxidants NAC (100 µM) and PDTC (50 µM) for 1 h before
culturing them in CHG for 24 h in the presence of the inhibitors,
following which nuclear extracts were made for EMSA. The antioxidants
showed striking inhibition of CHG-induced NF-B binding by blocking
the translocation of the p65 subunit to the nucleus (Fig.
6, A and B, third
lane versus eighth lane). In addition,
the antioxidants also partially blocked TNF-induced NF-B
activation in NG and CHG (Fig. 6, A and B, second
and fourth lanes versus fifth and sixth
lanes). PhosphorImager data from multiple EMSA are tabulated
in Table III.
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Activation of AP-1 by High Glucose but Not TNF
Mutational analysis of the TNF promoter confirmed a cooperative
role of AP-1 with NF-B in CHG-mediated 295TNFluc activity. The U937
nuclear extracts from the time course study (Fig. 5, A and
B) were used to perform EMSA with a 17-base pair
oligonucleotide encompassing the AP-1 site from the proximal TNF
promoter ("Materials and Methods"). AP-1 activation was
dramatically stimulated by CHG over NG (Fig.
7A, first lane
versus seventh lane). TNF treatment showed a
rapid increase in AP-1 activation in NG (Fig. 7A, first lane versus second through sixth
lanes). However, in CHG cells TNF down-regulated AP-1
activation compared with that observed in CHG alone (Fig.
7A, seventh lane versus ninth or tenth
lane). Activation of AP-1 was also confirmed in normal human
monocytes cultured overnight in HG (Fig. 7B). Supershift
analysis confirmed c-Fos and c-Jun to be a part of the CHG-activated
AP-1 complex (Fig. 7B, fourth and fifth lanes).
Specificity of the AP-1 complex was confirmed by competition of the
specific complex with cold AP-1 probe (Fig. 7B, third
lane), and the complex was supershifted with c-Fos and c-Jun
antibodies (Fig. 7B, fourth through sixth lanes).
The PKC inhibitor GF109203X blocked CHG-induced AP-1 binding by 68%
and reversed the down-regulation observed in CHG following TNF
greater than 75%, further suggesting involvement of similar PKC
isoforms in CHG and TNF-mediated activation of AP-1 (data not
shown).
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Mechanism of CHG-induced Monocyte Activation
Pharmacological inhibitors were used to further evaluate the
mechanism of CHG-induced monocyte activation. Antioxidants, a p38 MAPK
inhibitor, and PKC inhibitors were used to block each of the activated
pathways so far identified to be important in monocyte activation, and
the counter effects of each inhibitor were evaluated separately or in
combination. In our study, CHG induced oxidant stress (higher
O2) in monocytes and increased
phosphorylation of oxidant stress-sensitive MAPKs (p38 and JNK-1). The
antioxidants NAC and PDTC had similar counter effects on CHG- or
TNF-induced NF-B binding (Table III) and 295TNFluc activity
(Table IV) or CHG-induced TNF
secretion (Table V). The inhibitory
effects of PDTC, known to function as an antioxidant and a NF-B
inhibitor, were slightly greater than that seen with NAC. These results
suggest that CHG- and TNF-induce
