From the Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
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ABSTRACT |
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The inflammatory cytokines interleukin-1
(IL-1
) and tumor necrosis factor-
(TNF), elevated in
inflammatory, malignant, and infectious diseases, induce low density
lipoprotein (LDL) receptor transcription in HepG2 cells, and such an
induction can account for hypocholesterolemia associated with these
states. However, the signaling mechanisms of cytokine-mediated LDL
receptor induction are largely unexplored. In the present studies, we
examined the potential involvement of different mitogen-activated
protein kinase (MAPK) pathways. Northern analysis demonstrated that
IL-1
or TNF significantly increased LDL receptor transcript in HepG2 cells, whereas expression of another tightly regulated
sterol-responsive squalene synthase gene was unaffected. IL-1
treatment resulted in transient activation of three MAPK cascades,
namely p46/54JNK, p38MAPK, and ERK-1/2,
with maximal activation of 20-, 25-, and 3-fold, respectively,
occurring 15-30 min after cytokine addition. PD98059, a specific
inhibitor of MAPK kinase activity, inhibited IL-1
-induced LDL
receptor expression. In contrast, SB202190, a specific inhibitor of
p38MAPK, enhanced IL-1
-induced LDL receptor expression,
with a concomitant increase in ERK-1/2 activity. Similarly, TNF induced
LDL receptor expression also required ERK-1/2 activation. Finally,
sterols repressed IL-1
induced receptor expression, without
affecting ERK-1/2 activation. These results show that IL-1
- or
TNF-induced LDL receptor expression requires ERK-1/2 activation, that
the p38MAPK pathway negatively regulates LDL receptor
expression, and that sterols inhibit induction at a point downstream of
ERK-1/2 in HepG2 cells.
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INTRODUCTION |
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Cytokines interleukin-1
(IL-1)1 and tumor necrosis
factor- (TNF) are potent immunoregulatory and proinflammatory
cytokines secreted by a variety of cells in response to infection,
activated lymphocyte products, microbial toxins, and other stimuli (1, 2). These cytokines induce a variety of biochemical and functional responses in hepatic cells (3-6). IL-1, TNF, and other cytokines have
been demonstrated to act as the principal mediators that influence
liver metabolism and induction of a variety of hepatic genes during
liver cell infection and injury. Elevated plasma levels of cytokines,
including IL-1
and TNF found in various inflammatory, infectious,
and malignant diseases, are often associated with hypocholesterolemia
(7-11). Systemic infusion of cytokines has been shown to lower serum
cholesterol levels in animals and humans (12-14). The concentrations
of IL-1
and TNF similar to those found following infection induce
low density lipoprotein (LDL) receptor expression in human hepatoma
(HepG2) cells, and this effect is not part of its mitogenic response,
as IL-1
did not increase DNA synthesis (15-18). Interestingly, a
1.56-kilobase pair region of the 5'-flanking region of the human LDL
receptor promoter has been shown to confer
IL-1
-dependent induction to an heterologous gene,
suggesting that increased receptor expression results from activation
of LDL receptor transcription and is not due to an alteration in LDL
receptor mRNA stability (18). These studies have also established
that, unlike TNF, IL-1
-induced LDL receptor transcription does not
require protein synthesis and, therefore, likely depends on activation
of a preexisting component(s). The initial step in their action is the
association of agonist with its cell surface receptors, followed by
intracellular protein phosphorylation/dephosphorylation (19-21). The
downstream effectors linking receptor activation with the different
cellular responses are still largely to be defined, although several
signaling pathways have been proposed (20, 21). The signal transduction mechanisms by which these cytokines stimulates LDL receptor expression are poorly characterized.
Many extracellular signals elicit specific biological responses through
activation of MAPK cascades (22-26). The three major subfamilies of
MAPKs in higher eukaryotes include ERK-1/2, 46/54JNK, and
p38MAPK, all of which are activated by phosphorylation of a
tyrosine and a threonine residue catalyzed by a dual specificity MAPK
kinase. ERKs are most strongly activated by mitogenic signals such as growth factors or 12-O-tetradecanoylphorbol-13-acetate
(TPA), whereas p46/54JNK, and p38MAPK are
activated by stressful stimuli such as the inflammatory cytokines, IL-1, and TNF, thermal shock, and osmotic shock (23, 27). Activation
of MAPKs leads to distinct cellular responses mediated by
phosphorylation of specific target substrates (25). Recently, we have
shown involvement of ERK-1/2 signaling cascade in TPA-induced LDL
receptor expression in HepG2 cells (28).
In this study, we have investigated the early in vivo
signaling events triggered by IL-1 that elicit LDL receptor
activation in HepG2 cells. We show that, although IL-1
at
physiological concentrations strongly activates the stress-activated
p46/54JNK and p38MAPK in these cells, induction
of LDL receptor expression actually depends on the mild induction of
the ERK-1/2 signaling cascade by this cytokine. We also demonstrate
that the stress-activated p38MAPK cascade negatively
regulates LDL receptor expression via ERK-1/2. These results provide
new insight into the mechanisms of IL-1
action and suggest that the
MAPKs may be the critical components that control LDL receptor
expression.
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EXPERIMENTAL PROCEDURES |
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Materials--
IL-1 was purchased from R & D Systems Inc.
(Minneapolis, MN). TNF was obtained from Collaborative Biomedical
(Bedford, MA). The mitogen/extracellular-regulated protein kinase
kinase-1 and -2 (MEK-1/2) inhibitor PD98059 and p38MAPK
inhibitor SB202190 were purchased from Calbiochem. PD98059 from Research Biochemicals Inc. (Natick, MA) was also used in some experiments. Phospho-specific antibodies to the activated forms of
ERK-1/2 (Thr-202/Tyr-204), p46/54JNK (Thr-183/Tyr-188),
p38MAPK (Thr-180/Tyr-182), and MEK-1/2 (Ser-217/221) were
from New England Biolabs (Beverly, MA). Antibodies to ERK-1/2 and MAPK
phosphatase-1 (MKP-1) were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). TRIzol and all tissue culture supplies were from Life
Technologies Inc. A Zeta probe blotting membrane and the protein assay
reagent were purchased from Bio-Rad. [
-32P]dCTP (3000 Ci/mmol) was obtained from Dupont (Boston, MA), and the enhanced
chemiluminescence (ECL) detection kit was obtained from Amersham
International. Cholesterol, 25-hydroxycholesterol, and other chemicals
were obtained from Sigma.
Cell Culture-- HepG2 cells were maintained as monolayer cultures in a humidified 5% CO2 atmosphere at 37 °C in Eagle's minimum essential medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (Life Technologies Inc.), 2 mM L-glutamine, 20 units/ml penicillin, and 20 µg/ml streptomycin sulfate.
Immunoblot Analysis-- Proteins were fractionated by SDS-PAGE with an 10% acrylamide separation gel. Proteins were transferred to nitrocellulose in 25 mM Tris-HCl, 192 mM glycine, and 10% methanol at 4 °C for 12-16 h at a constant current of 50 mA or for 2 h at 300 mA with similar results. Nitrocellulose membranes were processed as described previously (28, 29). Briefly, membranes were incubated in 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.2% v/v Tween 20 (Tris/NaCl/Tween 20) with 5% w/v non-fat dried milk for 1 h, washed in Tris/NaCl/Tween 20 (3 × 5 min), and incubated for 1 h with primary antibody in Tris/NaCl/Tween 20 containing 1% milk at room temperature for non-phospho-antibodies and overnight at 4 °C for phospho-specific antibodies. The following dilutions were used for individual antibodies against different proteins: ERK-1/2 (1:1600), phospho-ERK-1/2 (1:1000), phospho-p46/54JNK (1:1000), phospho-p38MAPK (1:1600), phospho-MEK-1/2 (1:1000), and MKP-1 (1:1500). After further washing in Tris/NaCl/Tween 20, membranes were incubated for 1 h with horseradish peroxidase-linked anti-IgG secondary antibody (Bio-Rad, diluted 1:5000), and immunoreactive proteins were detected by ECL as described by the supplier. Quantitative analyses of protein levels were performed by scanning of the autoradiograms and are representative of three or more independent experiments.
Northern Analysis--
HepG2 cells were grown and treated as
described in the figure legends. Total RNA was isolated using TRIzol,
and Northern blotting was done essentially as described earlier (28,
30). Briefly, 20 µg of total cellular RNA were fractionated on 1%
formaldehyde-agarose gel and transferred to a Zeta probe membrane by
capillary blotting. RNA blots were hybridized with LDL receptor and
squalene synthase-specific single-stranded M13 probes labeled with
[-32P]dCTP. Hybridized filters were washed and exposed
to Kodak x-ray film. The relative intensities of specific bands were
determined densitometrically within the linear range of film on a model
300A laser densitometer (Molecular Dyanamics, Sunnyvale, CA) and Image Quant software. LDL receptor mRNA was normalized to squalene
synthase (included in this study) or to
-actin (data not shown)
mRNA level, and data for each points were plotted as the percentage
of LDL receptor mRNA as compared with controls.
Transfection Studies--
Human LDL receptor promoter-reporter
constructs were transfected into HepG2 cells using LipofectAMINE (31),
and the effect of PD98059 on IL-1-induced expression of the
luciferase gene was determined.
Measurement of ERK-1/2 Activity--
Cells were washed in
ice-cold phosphate-buffered saline and removed from the flask by gentle
scraping into 0.25 ml of lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM sodium vanadate, 50 mM sodium fluoride, 20 mM -glycerophosphate, 0.1 µM okadaic acid,
1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin,
50 µg/ml leupeptin, and 10 µM pepstatin). After 15 min
on ice, insoluble material was removed by sedimentation for 20 min at
100,000 × g, and ERK-1/2 activity was determined by an
immune complex kinase assay with myelin basic protein as the substrate
as described previously (32). Briefly, each cell extract (400 µg) was
mixed with 10 µl of anti-ERK-1/2 antibody for 1 h, and then 30 µl of 50% protein A-Sepharose in lysis buffer was added for an
additional 1 h. The immune complex was recovered by sedimentation
for 5 min in a microcentrifuge, washed three times with 0.5 ml of
phosphate-buffered saline containing 1% Nonidet P-40 and 2 mM sodium vanadate and once with ERKs reaction buffer (25 mM Hepes, pH 7.5, 10 mM MgCl2, 20 mM
-glycerophosphate, 0.5 mM sodium
vanadate, 0.5 mM EDTA, 10 mM dithiothreitol, 10 µg/ml leupeptin, 6 µM pepstatin). The immunoprecipitate
was resuspended in 30 µl of reaction buffer containing 6 µg of
myelin basic protein and 5 µl of 0.3 mM
[
-32P]ATP (30,000 cpm/pmol). After incubation for 20 min at 30 °C, the reaction was terminated by 8 µl of 5× SDS
sample buffer and heating to 95 °C for 5 min. Samples were analyzed
by SDS-PAGE (15% acrylamide), and gels were stained with Coomassie
Blue, dried, and subjected to autoradiography.
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RESULTS |
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Kinetics of IL-1-induced LDL Receptor Expression--
Northern
blot analysis of HepG2 cells that had been incubated in the absence and
presence of 5 ng/ml IL-1
for various periods of time showed that LDL
receptor induction reached a peak at 4 h after IL-1
stimulation
and remained elevated thereafter (Fig. 1). Furthermore, we tested whether
IL-1
would similarly stimulate another tightly regulated gene
involved in cholesterol homeostasis, squalene synthase. Our results
show that, in contrast to LDL receptor gene, IL-1
did not affect
expression of the squalene synthase gene (Fig. 1).
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The ERK-1/2 Cascade Positively Regulates LDL Receptor Expression in
HepG2 Cells--
The activation of MAPKs requires specific
phosphorylation of the threonine and tyrosine residues within the
TXY motif (33, 34). We first evaluated the temporal
characteristics of MAPKs activation in response to 5 ng/ml IL-1
stimulation in HepG2 cells by using antibodies specific for
phosphorylated (activated) forms of ERK-1/2, p46/54JNK, and
p38MAPK. These antibodies do not recognize inactive,
unphosphorylated enzymes. As shown in Fig.
2A, stimulation by IL-1
preferentially activated p46/54MAPK (20-fold) and
p38MAPK (25-fold) as compared with ERK-1/2 (3-fold). The
increased activation of p46/54JNK and p38MAPK
were detected within 5 min of exposure to IL-1
, reached a peak in
15-30 min, and returned to the basal level at 90 min. Consistent with
the immunoblot analyses, an increase in ERK-1/2 activity after 30 min
of IL-1
treatment, which declined to the basal level at 90 min, was
observed in immune complex kinase assays using myelin basic protein as
a substrate (Fig. 2B). As a positive control for ERK-1/2
activation in the kinase assay, extracts from cells treated with 100 nM TPA for 15 min were included because we have observed
earlier TPA-dependent ERK-1/2 activation in HepG2 cells (28). The kinetics and fold activation of ERK-1/2 by immune complex
kinase assay were similar to those obtained on immunoblotting with
phospho-specific antibody. Therefore, we have used the phospho-specific immunoblot as an index of kinase activity in our subsequent
experiments. Taken together, these results establish that the different
MAPKs are rapidly and transiently activated by IL-1
in HepG2
cells.
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The p38MAPK Cascade Antagonizes IL-1-induced LDL
Receptor Expression in HepG2 Cells--
As many of the effects of
IL-1
have been shown to be mediated through p38MAPK
pathway (2), we tested the effect of SB202190, a potent
p38MAPK inhibitor (47, 48), on IL-1
-mediated LDL
receptor induction. SB202190 is known to inhibit p38MAPK
with an IC50 of 0.6 µM and even at 100 µM had no effect on activities of 12 other protein
kinases tested, including ERK-1/2 or p46/54JNK (48). We
also confirmed that 5 µM SB202190 did not inhibit the
phosphorylation levels of p38MAPK and p46/54JNK
(Fig. 4). Pretreatment of HepG2 cells for 30 min with or without 0.5 to
5 µm SB202190, followed by incubation with IL-1
for 4 h
reproducibly enhanced LDL receptor expression (Fig. 5). Interestingly, this induction was accompanied by a concomitant increase in ERK-1/2 activity without affecting phosphorylation levels of the
p38MAPK and P46/54JNK (Fig. 4). Furthermore,
enhanced LDL receptor expression and increased ERK-1/2 activity was not
observed with SB202190 in the presence of PD98059 (data not shown).
These results strongly suggests that the p38MAPK negatively
regulates IL-1
-induced ERK-1/2 activity and LDL receptor induction.
Sterols Block IL-1-induced LDL Receptor Expression at a Point
Downstream of ERK-1/2--
Treatment of HepG2 cells with IL-1
in
the presence of sterols (25-hydroxycholesterol and cholesterol)
resulted in loss of IL-1
-induced LDL receptor expression (Fig.
6A), an observation consistent
with an earlier study (18). The reduced expression of squalene synthase
in the presence of sterols (lane 3) is consistent with its
well established negative regulation by sterols in HepG2 cells (49). To
test whether the induction is lost due to lack of activation of
ERK-1/2, we evaluated the effects of sterols on
IL-1
-dependent activation of this kinase in HepG2 cells.
IL-1
was added to the cells pretreated with sterols, and ERK-1/2
activation was measured. As shown in Fig. 6B, irrespective
of sterol concentrations used, IL-1
induced ERK-1/2 activity,
suggesting that the loss of an IL-1
-dependent increase
in LDL receptor induction is not due to lack of activation of ERK-1/2
in the presence of sterols in HepG2 cells.
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TNF-induced LDL Receptor Expression Is Mediated by ERK-1/2
Cascade--
Earlier studies had proposed that IL-1 and TNF
possibly enhance LDL receptor expression by different mechanisms in
HepG2 cells (18). Therefore, we tested the involvement of ERK-1/2 in
TNF-dependent activation of LDL receptor expression in this cell line. TNF (100 ng/ml) treatment for 4 h increased LDL
receptor expression in HepG2 cells (Fig.
7A). Like IL-1
, TNF
strongly activated p46/54JNK and p38MAPK (data
not shown) with a slight activation of the ERK-1/2 (Fig. 7B). Pretreatment with 50 µM PD98059 for 30 min blocked TNF induced LDL receptor expression (Fig. 7A),
thus supporting the involvement of the ERK-1/2 signaling in the
induction process. Furthermore, similar to IL-1
, treatment with 5 µM SB202190 enhanced TNF induced LDL receptor
expression (Fig. 7A), with a parallel increase in ERK-1/2 activation (Fig. 7B).
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DISCUSSION |
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The results presented in this study establish a role for the
ERK-1/2 activation in IL-1-induced LDL receptor expression in HepG2
cells. First, IL-1
treatment leads to an activation of ERK-1/2 in a
time-dependent manner (Fig. 2). The kinetics of ERK-1/2 activation and LDL receptor induction are closely related, thereby providing the first in vivo evidence linking them
kinetically. Second, PD98059, a specific inhibitor of MEK activity,
inhibits both ERK-1/2 activation and LDL receptor expression induced by IL-1
, without affecting phosphorylation levels of
p46/54JNK and p38MAPK. Although IL-1
treatment resulted in activation of other MAPKs, the complete loss of
IL-1
induced LDL receptor expression on treatment with PD98059 rules
out involvement of other MAPKs in LDL receptor induction by IL-1
.
Furthermore, IL-1
-induced phosphorylation of MEK-1/2 correlates with
the ERKs activation, which is consistent with the notion that ERK-1/2
induction is mediated through MEK activation. These results show that
IL-1
signal acts upstream of ERK-1/2 phosphorylation, at least at
the level of MEK-1/2 activation. The ERK cascade is normally required
for the essential function as a positive regulator of cell
proliferation in response to growth signals, and strong correlations
between ERKs activation and DNA synthesis have been shown in earlier
studies (27, 50, 51). In view of an earlier demonstration that
IL-1
-induced activation of LDL receptor expression in HepG2 cells is
not a consequence of increased DNA synthesis, involvement of ERK-1/2 in
IL-1
-dependent LDL receptor induction is surprising, and
suggests that these isoforms can be coordinately activated in response
to a wide range of mitogenic as well as nonmitogenic stimuli.
Furthermore, although physiological concentrations of IL-1
strongly
activated p46/54JNK and p38MAPK, the lessor
induction of ERK-1/2 proved to be the crucial mediator of the
cytokine's action in LDL receptor induction. These results emphasize a
general point of significance, namely, that although a specific
signaling pathway may appear to dominate a response, other mildly
activated pathways can be critical, and thus should not be overlooked
or ignored.
The present study has focused on the role of MAPKs in IL-1-induced
LDL receptor expression. Results presented here along with an earlier
report (18) are consistent with the notion that IL-1
induces LDL
receptor expression at the transcriptional level. In most cell types,
the mitogenic signal is relayed from the cytoplasm into the nucleus by
the nuclear translocation of the phosphorylated ERK-1/2, resulting in
phosphorylation and activation of a range of transcription factors
(52). The precise role of phosphorylation in transcriptional activation
of LDL receptor expression in response to IL-1
is unclear but may
involve phosphorylation of transcription factors critical for LDL
receptor expression (53-55), or modification of co-activators such as
the cAMP-response element binding (CREB) protein (56). Given that
ERK-1/2 activation occurs rapidly and transiently (Fig. 2), and LDL
receptor induction follows relatively slow kinetics (Fig. 1), the
mechanism likely involves this protein kinase cascade in the initial
step and does not require it for sustained activation of LDL receptor
expression by IL-1
in HepG2 cells. Our demonstration that IL-1
can regulate gene expression through selective activation of ERK-1/2
may explain the lack of effect of p38MAPK inhibitor on
IL-1
induction of E-selectin expression on vascular endothelial
cells, or IL-1-induced IL-6 production in gingival fibroblasts (57).
Furthermore, the lack of activation of the squalene synthase gene by
IL-1
suggests that the members of the MAPK family selectively
regulate expression of specific genes involved in cholesterol
homeostasis.
Although p38MAPK has been found to be activated by several
forms of environmental stress and cytokines, the physiological and pathophysiological function of this kinase in mammalian cells is still
unclear. Recently, roles for p38MAPK in the regulation of
cell quiescence and in the promotion of programmed cell death in
differentiated cellular systems have been reported (58-63). Our
demonstration that treatment with SB202190 resulted in superinduction
of the LDL receptor by IL-1 suggests that the p38MAPK
signaling cascade exerted a negative effect on LDL receptor expression through negative regulation of the ERK-1/2. Although the precise role
for this level of regulation is not yet clear, such a mechanism may
provide cells a means to counteract the actions of cytokines and growth
factors on LDL receptor expression.
It has been demonstrated earlier that, unlike IL-1, TNF requires
protein synthesis for induction of LDL receptor transcription in HepG2
cells (18). Based on this observation, it was suggested that
biologically redundant cytokines, TNF and IL-1
, may act via
different mechanisms to increase LDL receptor expression in HepG2
cells. In the present study, we have shown that both cytokines require
ERK-1/2 for induction of LDL receptor expression. PD98059 was found to
block the IL-1
- and TNF-dependent induction of LDL receptor expression at a concentration shown to completely inhibit ERKs
in HepG2 cells. It is safe to conclude from the above studies that
whatever differences exist in their upstream signal transduction pathways, both cytokines activate ERK-1/2 and this step is critically required for LDL receptor induction in response to them. Furthermore, an observation that IL-1
-dependent induction of LDL
receptor expression is sensitive to cellular cholesterol levels allowed us to test whether sterols exert their effect upstream or downstream of
ERKs activation. The lack of effect of sterols on ERK-1/2 activation under conditions shown to inhibit IL-1
induced LDL receptor
expression suggests that sterols acts at a step downstream of this
protein kinase pathway. On the other hand, lack of activation of the
sterol-sensitive squalene synthase gene by IL-1
(Fig. 1), which is
known to contain a functional SRE-1 (44), suggests that SREBP alone may
not be solely responsible for LDL receptor induction by these
cytokines.
In conclusion, we have provided evidence that MAPK signal transduction
pathways play essential and differential roles in the induction of LDL
receptor transcription by IL-1, thus establishing a link between
MAPK-mediated intracellular signaling and regulation of LDL receptor
gene expression. LDL receptor induction by IL-1
occurs via a complex
chain of events initiated after ligand binding that leads to the
activation of certain kinases that promote MAPKs phosphorylation. Our
results support the possibility that p38MAPK negatively
regulates ERK-1/2 activation and some of the responses mediated by this
kinase in the presence of IL-1
. The cross-talk between various
signaling pathways involving protein phosphorylation is an emerging
theme in intracellular communication. Based on the above results, we
propose that interplay between these pathways probably has a central
role in the processing circuits that direct the transcription of LDL
receptor gene. Results presented here will help form a framework for
further investigation of the signaling pathways responsible for LDL
receptor transcriptional regulation by mitogenic and nonmitogenic
stimuli.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Research Grant HL-51592-04), American Heart Association Grant 94012580, and the University of Arkansas for Medical Sciences Hornick Endowment Award (to K. D. M.).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.
Present address: Molecular and Cellular Endocrinology Branch,
NIDDK, Bethesda, MD 20892.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205. Tel.: 501-686-8053; Fax: 501-686-8169; E-mail: mehtakamald{at}exchange.uams.edu.
1
The abbreviations used are: IL, interleukin;
TNF-, tumor necrosis factor; LDL, low density lipoprotein; MAPK,
mitogen-activated protein kinase; ERK, extracellular signal-regulated
kinase; MEK, mitogen/extracellular-regulated protein kinase kinase;
MKP, MAPK phosphatase; TPA,
12-O-tetradecanoylphorbol-13-acetate; PAGE, polyacrylamide
gel electrophoresis.
2 A. Kumar and K. D. Mehta, unpublished results.
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REFERENCES |
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