Department of Nutrition, Case Western Reserve University, Cleveland, Ohio 44106-4906
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ABSTRACT |
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Lipopolysaccharide (LPS) is a potent
activator of tumor necrosis factor- (TNF-
) production by
macrophages. LPS stimulates the phosphorylation of extracellular
signal-regulated kinase (ERK) 1/2 and increases TNF-
mRNA and
protein accumulation in RAW 264.7 murine macrophages. However, the role
of ERK1/2 activation in mediating LPS-stimulated TNF-
production is
not well understood. Inhibition of ERK1/2 activation with PD-98059 or
overexpression of dominant negative ERK1/2 decreased LPS-induced
TNF-
mRNA quantity. LPS rapidly increased early growth response
factor (Egr)-1 binding to the TNF-
promoter; this response was
blunted in cells treated with PD-98059 or transfected with
dominant-negative ERK1/2. Using a chloramphenicol acetyltransferase
reporter gene linked to the Egr-1 promoter, we show that LPS increased
Egr-1 promoter activity via an ERK1/2-dependent mechanism. These
results delineate the role of ERK1/2 activation of Egr-1 activity in
mediating LPS-induced increases in TNF-
mRNA expression in macrophages.
monocytes/macrophages; lipopolysaccharide; protein
kinases/phosphatases; transcription factors; signal transduction; extracellular signal-regulated kinase 1/2; early growth response
factor-1; tumor necrosis factor-
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INTRODUCTION |
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MACROPHAGES PLAY AN
IMPORTANT role in regulating immune and inflammatory activities.
Upon activation by lipopolysaccharide (LPS), cell wall components of
gram-negative bacteria, macrophages secrete an array of proinflammatory
cytokines and oxidants, including tumor necrosis factor- (TNF-
).
TNF-
serves as an important mediator for activation of the host
immune response against infection and tumor formation, as well as in
tissue remodeling. However, in addition to its beneficial effects,
TNF-
also mediates septic shock during chronic infection, cachexia,
some autoimmune diseases, and activation of human immunodeficiency
virus (18).
LPS-induced TNF- production is controlled at transcriptional
(3, 19), posttranscriptional (8, 12), and
posttranslational levels (13). The following seven
transcription factor binding sites identified within the promoter
region of the TNF-
gene are required for full transcriptional
activation of the TNF-
gene after LPS treatment: an early growth
response factor (Egr)-1 site, three E26 transformation-specific (Ets)
sites, a cAMP response element (CRE)/activator protein (AP)-1 site, a
nuclear factor (NF)-
B site, and a promoter specificity protein 1 (Sp1) site (19, 23). LPS stimulates a complex
array of signal transduction pathways, leading to the activation of
the transcription factors regulating TNF-
expression.
Extracellular/signal-regulated kinase (ERK) 1/2 is a member of the
mitogen-activated protein kinase family, which are activated by protein
phosphorylation at tyrosine and threonine residues (20).
Although it is well documented that treatment of macrophages with LPS
activates ERK1/2 (5, 16, 21), the role of ERK1/2 in
regulation of LPS-induced TNF-
mRNA expression is controversial. One
report indicates that ERK1/2 activation is required for TNF-
mRNA
expression in murine J774 monocytes (19). In contrast,
another investigation found that inhibition of ERK1/2 activation had no
effect on TNF-
mRNA expression in RAW 264.7 macrophages
(11). Because of the potentially key role of LPS
activation of ERK1/2 in regulating TNF-
expression, here we have
made use of a specific inhibitor of mitogen/extracellular signal-regulated kinase kinase, PD-98059, and dominant-negative forms
of ERK1/2, to address the role of ERK1/2 in mediating LPS-stimulated TNF-
accumulation. We show that LPS-induced ERK1/2 activation increases TNF-
mRNA expression in RAW 264.7 cells via regulation of
Egr-1 production and binding to the TNF-
promoter.
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EXPERIMENTAL PROCEDURES |
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Reagents. LPS from Escherichia coli serotype 026:B6 was purchased from Sigma (St. Louis, MO). PD-98059 was obtained from Calbiochem (La Jolla, CA). Antibodies were from the following sources: anti-active ERK1/2 polyclonal antibody (Promega, Madison, WI), anti-ERK1/2 (Upstate Biotechnology, Lake Placid, NY), anti-Egr-1, and anti-SP1 (Santa Cruz Biotechnology, Santa Cruz, CA). Oligonucleotide probes were synthesized by IDT Technologies (Coralville, IA). The ribonuclease protection assay system was from Ambion (Austin, TX). The template for transcribing the anti-sense chloramphenicol acetyltransferase (CAT) riboprobe was purchased from Promega. Kinase dead dominant-negative constructs for ERK1/2 were a gift from Dr. R. L. Eckert and have been described previously (14). An Egr-1 promoter linked to a CAT reporter construct (pEgr-1B950 CAT) and control vector (pCAT) were a gift from Dr. R. P. Huang (7).
Culture and transfection of RAW 264.7 macrophages. The mouse macrophage cell line RAW 264.7 was cultured as previously described (10). For transfections, RAW 264.7 macrophages were grown in 100-mm dishes to 60% confluency and were transiently transfected with either empty vectors or dominant-negative ERK1/2 and/or Egr-1 promoter-CAT reporter expression vectors using TransFast transfection reagent (Promega) following the manufacturer's instructions. A single plate of transfected cells was then used to set up the experimental cultures required for each assay, ensuring equal transfection efficiencies between different treatments, and cells were cultured for an additional 48 h before treatment with LPS.
ERK1/2 activation. After 48 h, the cells were washed one time with 2 ml of DMEM with 10% FBS and were treated with or without LPS in DMEM with 10% FBS for the indicated times. In some cases, cells were preincubated with PD-98059 for 2 h before addition of LPS. Activation of ERK1/2 was measured by Western blot analysis (9). Membranes were first probed with anti-active ERK1/2 antibody and then stripped and reprobed with antibody to total ERK1/2.
TNF- mRNA.
Cells were treated with or without LPS as described above and then were
washed one time with PBS and lysed in 1 ml of Trizol reagent (Life
Technologies, Gaithersburg, MD) for 5 min. Total RNA was isolated with
TRIzol reagent according to the manufacturer's instructions, and
TNF-
mRNA was quantified by Northern blot analysis or ribonuclease
protection assays.
Electrophoretic mobility shift assays.
RAW 264.7 cells were treated or not with LPS as described above, and
then nuclear extracts were prepared for electrophoretic mobility shift
assay as described (9). The binding of nuclear proteins to
an oligonucleotide corresponding to the Egr-1 binding site in the
promoter region of the murine TNF- gene (5'-AACCCTCTGCC CCCGCGATGGAG-3') was measured as described previously (9). In competition and antibody supershift assays, competing
oligonucleotides (50-fold excess) or 2 µg of antibodies were included
in the binding reaction mixtures 10 min before the addition of labeled oligonucleotides.
TNF- bioassay.
RAW 264.7 cells were treated or not with LPS for 2-4 h, and cell
culture media were removed. Accumulated TNF-
was measured by
bioassay (1).
Statistical analysis. All values are expressed as means ± SE. Student's t-test was used to compare between groups.
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RESULTS |
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ERK1/2 are involved in LPS-induced TNF- mRNA and protein
accumulation.
LPS treatment activated ERK1/2 in RAW 264.7 cells. Phosphorylation
peaked 40 min after LPS treatment (Fig.
1A) and returned to basal
levels by 3 h after LPS treatment (data not shown). LPS treatment
did not change the total amount of ERK1/2 protein (Fig. 1A).
Pretreatment of RAW 264.7 cells with PD-98059 for 2 h dose dependently decreased LPS-induced ERK1/2 phosphorylation (Fig. 1B). Phosphorylation of ERK1/2 was decreased by ~70% of
control in the presence of 50 µM PD-98059 (Fig. 1C). LPS
treatment also increased TNF-
mRNA accumulation (Fig. 1D)
and peptide secretion from RAW 264.7 cells (Fig. 1F).
Pretreatment of RAW 264.7 cells with 50 µM PD-98059 reduced
LPS-induced TNF-
mRNA accumulation by 65% at 30 min and 37% at 45 min after LPS treatment (Fig. 1, D and E).
Similarly, pretreatment with 50 µM PD-98059 decreased LPS-induced
secretion of TNF-
by 65% at 2-4 h after LPS treatment (Fig.
1F).
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ERK1/2 activation is required for LPS-induced Egr-1 binding to the
TNF- promoter.
Seven transcription factor binding sites have been identified in the
promoter region of the TNF-
gene; the sites contribute to
upregulation of the TNF-
gene after LPS treatment in macrophages and
T cells (3, 23). One of these transcription factors, Egr-1, is regulated by an ERK1/2- dependent mechanism in other cell
types (4, 22). Therefore, we asked whether
LPS-stimulated Egr-1 binding to the murine TNF-
was mediated via
ERK1/2. Although multiple proteins bound to the probe containing the
Egr-1 site, stimulation with LPS increased the binding activity of only
one protein (Fig. 3A).
Supershift assays utilizing antibodies against Sp1 and Egr-1, two
transcription factors reported to bind to Egr-1 consensus sites,
identified this protein as Egr-1 (Fig. 3A). Pretreatment of
RAW 264.7 cells with 50 µM PD-98059 for 2 h decreased
LPS-induced Egr-1 binding by 40 ± 11% (P < 0.05 compared with control, n = 3; Fig. 3A).
Increased Egr-1 DNA binding activity was associated with a greater
accumulation of Egr-1 protein in the nucleus after LPS treatment (Fig.
3B). Pretreatment of PD-98059 for 2 h decreased LPS-induced Egr-1 quantity in the nucleus by 42 ± 5%
(P < 0.05 compared with controls not treated with
PD-98059, n = 6; Fig. 3B). Overexpression of
dominant-negative ERK1/2 also decreased LPS stimulation of Egr-1
binding to the TNF-
promoter, with binding reduced to 21 ± 5%
(P < 0.05 compared with cells transfected with empty
vector, n = 3; Fig. 3C).
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LPS stimulates Egr-1 promoter activation.
Using a CAT reporter construct linked to the Egr-1 promoter, we tested
whether treatment of RAW 264.7 macrophages with LPS increased Egr-1
promoter activity. RAW 264.7 macrophages were transfected with pCAT
control vector or an Egr-1 promoter-CAT reporter construct and then
were stimulated or not with 100 ng/ml LPS for 60 min. LPS had
no effect on CAT mRNA expression in cells transfected with pCAT control
vector (Fig. 5) but increased CAT mRNA
expression by 2.2-fold in cells expressing the Egr-1 promoter-CAT construct (Fig. 5). Consistent with a role for ERK1/2 activation in
mediating LPS-induced increases in Egr-1 expression, cotransfection with dominant-negative ERK1/2 completely abrogated the ability of LPS
to increase Egr-1 promoter-driven CAT mRNA expression (Fig. 5).
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DISCUSSION |
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Activation of macrophages with LPS initiates a complex set of
signal transduction cascades, ultimately resulting in increased secretion of TNF-. Increased TNF-
expression in response to LPS
requires the activation of a distinct set of transcription factors
binding to the TNF-
promoter (19, 23). Although the exact array of transcription factors interacting with the TNF-
promoter is to some extent cell and species specific (11),
recruitment of NF-
B, Egr-1, and c-Jun appears to be required for
full activation of TNF-
expression in most types of macrophages
(19, 23). Despite advances in our understanding of the
transcriptional regulation of TNF-
expression, the signal
transduction cascades linking the binding of LPS to cell surface
receptors with increased TNF-
expression are only partially
understood. Here we report that LPS-induced activation of ERK1/2 is
required for LPS-stimulated TNF-
mRNA and peptide accumulation in
RAW 264.7 macrophages. In other cell types, activation of ERK1/2
activation leads to an increase in the activity of the transcription
factor Egr-1 (4, 22). Therefore, we asked whether
ERK1/2-dependent activation of Egr-1 contributed to LPS-stimulated
TNF-
production. We report that LPS-stimulated activation of ERK1/2
increased the binding of Egr-1 to the murine TNF-
promoter.
Pretreatment of RAW 264.7 macrophages with PD-98059 or overexpression
of dominant-negative ERK1/2 decreased LPS-stimulated Egr-1 binding to
the Egr-1 site from the TNF-
promoter.
Inhibition of ERK1/2 activation also decreased LPS-induced Egr-1
promoter activity and protein accumulation inside nuclei of RAW 264.7 macrophages (Figs. 3B and 5), suggesting that LPS-induced Egr-1 binding to the TNF- promoter is dependent on stimulation of
Egr-1 synthesis via ERK1/2 activation. Indeed, the time course for
activation of Egr-1 DNA binding activity is consistent with a maximal
increase in Egr-1 activity before maximal increases in TNF-
mRNA
accumulation (see Fig. 4). Egr-1 is an immediate early gene and a
member of the zinc finger transcription factor family. Its synthesis is
rapidly and transiently upregulated in response to a variety of
stimuli, including LPS in peritoneal macrophages (2) and
primary cultures of rat Kupffer cells (9). This response
is similar to the rapid increase in Egr-1 DNA binding activity in
response to LPS reported here (Fig. 4). Activation of Egr-1 promoter
activity, and induction of Egr-1 expression, was dependent on
activation of ERK1/2 in response to LPS in RAW 264.7 macrophages (Fig.
5). A recent report demonstrated a similar involvement of ERK1/2 and
Egr-1 in LPS-stimulated TNF-
production in the human macrophage cell
line THP-1 (6). Similarly, Egr-1 induction in a number of
other cell types, including astroglial cells (17),
endothelial cells (22), and hepatocytes (15), requires ERK1/2 activation.
The essential role of LPS-stimulated ERK1/2 activation in TNF- mRNA
accumulation reported here was based on data collected using two
different methods of inhibition of ERK1/2: pretreatment with PD-98059,
a specific inhibitor of ERK1/2 phosphorylation, and overexpression of
dominant-negative ERK1/2 construct. The use of these two distinct
methods of inhibiting ERK1/2 makes it unlikely that the results are
because of nonspecific effects of either inhibitor. Although
pretreatment of RAW 264.7 macrophages with 50 µM PD-98059 did not
completely inhibit ERK1/2 activation, significant reductions in nuclear
Egr-1 protein and DNA binding activity were observed (see Fig. 3).
Instead of using higher concentrations of PD-98059 to attempt to
completely inhibit ERK1/2 activation, which would have a higher risk
for potential nonspecific effects of the inhibitor, we made use of a
dominant-negative ERK1/2 construct. Overexpression of dominant-negative
ERK1/2 resulted in undetectable levels of ERK1/2 phosphorylation. With
complete inhibition of ERK1/2, Egr-1 binding activity was reduced to
baseline (Fig. 3C), and activation of the Egr-1 promoter was
completely blocked (Fig. 5). Taken together, the strong correlation
between the extent of inhibition of ERK1/2 with PD-98059 or
overexpression of dominant-negative ERK1/2 with decreased Egr-1 DNA
binding activity provides strong support for the role of ERK1/2 in
LPS-induced activation of Egr-1 DNA binding activity.
The data presented here demonstrating a role for ERK1/2 and Egr-1 in
mediating LPS-stimulated TNF- production differ from a previous
report indicating that LPS-induced TNF-
mRNA expression in RAW 264.7 cells was independent of ERK1/2 activation (11). Here we
have used a slightly higher concentration of the ERK1/2 inhibitor (50 µM) compared with 20 µM PD-98059 in the previous report
(14) and overexpression of a dominant-negative ERK1/2 construct, which may have resulted in a greater degree of inhibition of
ERK1/2 activation. Moreover, it is likely that inhibition of ERK1/2 has
a rapid effect on TNF-
mRNA. TNF-
mRNA accumulation peaked at
45-60 min after stimulation by LPS (Fig. 4). At 30-60 min
after LPS stimulation, activity of ERK1/2 contributed up to 50% of the
stimulus toward TNF-
mRNA accumulation (see Figs. 1, D
and E, and 2B). In the study by Means et al.
(11), which reported no effect of ERK1/2 inhibition on
LPS-dependent responses, TNF-
mRNA was not measured until 4 h
after LPS stimulation, well past the peak of LPS-stimulated TNF-
mRNA observed here (Fig. 4B). Despite this potentially
transient inhibition of mRNA accumulation, inhibition of ERK1/2
activation, either with PD-98059 or overexpression of dominant-negative
ERK1/2, inhibited TNF-
protein production at 2 and 4 h after
LPS stimulation (Figs. 1F and 2C). Thus LPS stimulation of ERK1/2 contributes to maximal TNF-
production in RAW
264.7 macrophages by increasing Egr-1 quantity and DNA binding activity
to the TNF-
promoter.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Alcoholism and Alcohol Abuse Grant AA-11975 (to L. E. Nagy).
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FOOTNOTES |
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3 Address for reprint requests and other correspondence: L. E. Nagy, Dept. of Nutrition, Case Western Reserve Univ., 2123 Abington Rd., Rm. 201, Cleveland, OH 44106-4906 (E-mail: len2{at}po.cwru.edu).
Present address for L. Shi: Dept. of Pharmacology, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106.
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.
First published February 6, 2002;10.1152/ajpcell.00511.2001
Received 24 October 2001; accepted in final form 30 December 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aldred, A,
and
Nagy LE.
Ethanol dissociates hormone-stimulated cAMP production from inhibition of TNF- production in rat Kupffer cells.
Am J Physiol Gastrointest Liver Physiol
276:
G98-G106,
1999
2.
Coleman, DL,
Bartiss AH,
Sukhatme VP,
Liu J,
and
Rupprecht HD.
Lipopolysaccharide induces Egr-1 mRNA and protein in murine peritoneal macrophages.
J Immunol
149:
3045-3051,
1992
3.
Delgado, M,
Munoz-Elias EJ,
Kan YQ,
Gozes I,
Fridkin M,
Brenneman DE,
Gomariz RP,
and
Ganea D.
Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit tumor necrosis factor a transcriptional activation by regulating nuclear factor-kB and cAMP response element-binding protein/c-Jun.
J Biol Chem
273:
31427-31436,
1998
4.
Dieckgraefe, BK,
and
Weems DM.
Epithelial injury induces Egr-1 and fos expression by a pathway involving protein C and ERK.
Am J Physiol Gastrointest Liver Physiol
276:
G322-G330,
1999
5.
Dong, Z,
Qi X,
and
Fidler IJ.
Tyrosine phosphorylation of mitogen-activated protein kinases is necessary for activation of murine macrophages by natural and synthetic bacterial products.
J Exp Med
177:
1071-1077,
1993[Abstract].
6.
Guha, M,
O'Connell MA,
Pawlinski R,
Hollis A,
McGovern P,
Yan SF,
Stern D,
and
Mackman N.
Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor expression by inducing Elk-1 phosphorylation and Egr-1 expression.
Blood
98:
1429-1439,
2001
7.
Huang, RP,
Ngo L,
Okamura D,
Tucker M,
and
Adamson ED.
V-sis induces Egr-1 expression by a pathway mediated by c-Ha-Ras.
J Cell Biochem
56:
469-479,
1994[ISI][Medline].
8.
Jarrous, N,
Osman F,
and
Kaempfer R.
2-Aminopurine selectively inhibits splicing of tumor necrosis factor alpha mRNA.
Mol Cell Biol
16:
2814-2822,
1996[Abstract].
9.
Kishore, R,
Hill JR,
McMullen MR,
Frenkel J,
and
Nagy LE.
ERK1/2 and Egr-1 contribute to increased TNF- production in rat Kupffer cells after chronic ethanol feeding.
Am J Physiol Gastrointest Liver Physiol
282:
G6-G15,
2002
10.
Kishore, R,
McMullen MR,
and
Nagy LE.
Stabilization of TNF mRNA by chronic ethanol: role of A+U rich elements and p38 mitogen activated protein kinase signaling pathway.
J Biol Chem
276:
41930-41937,
2001
11.
Means, TK,
Pavlovich RP,
Roca D,
Vermeulen MW,
and
Fenton MJ.
Activation of TNF-alpha transcription utilizes distinct MAP kinase pathways in different macrophage populations.
J Leukoc Biol
67:
885-893,
2000[Abstract].
12.
Osman, R,
Jarrous N,
Ben-Asouli Y,
and
Kaempfer R.
A cis-acting element in the 3'-untranslated region of human TNF-alpha mRNA renders splicing dependent on the activation of protein kinase PKR.
Genes Dev
13:
3280-3293,
1999
13.
Ribeiro, SP,
Villar J,
Downery GP,
Edelson JD,
and
Slutsky AS.
Effects of the stress response in septic rats and LPS-stimulated alveolar macrophages: evidence for TNF alpha posttranslational regulation.
Am J Respir Crit Care Med
154:
1843-1850,
1996[Abstract].
14.
Robbins, DJ,
Zhen E,
Owaki H,
Vanderbilt CA,
Ebert D,
Geppert TD,
and
Cobb MH.
Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro.
J Biol Chem
268:
5097-5106,
1993
15.
Rokos, CL,
and
Ledwith BJ.
Peroxisome proliferators activate extracellular signal-regulated kinases in immortalized mouse liver cells.
J Biol Chem
272:
13452-13457,
1997
16.
Sanghera, JS,
Weinstein SL,
Lem L,
and
DeFranco AL.
Activation of multiple proline-directed kinases by bacterial lipopolysaccharide in murine macrophages.
J Immunol
156:
4457-4465,
1996[Abstract].
17.
Sato, K,
Ishikawa K,
and
Okajima F.
Spingosine 1-phosphate induces expression of early growth response-1 and fibroblast growth factor-2 through mechanism involving extracellular signal-regulated kinase in astroglial cells.
Brain Res Mol Brain Res
74:
182-189,
1999[ISI][Medline].
18.
Tracey, KJ,
and
Cerami A.
Tumor necrosis factor: a pleiotropic cytokine and therapeutic target.
Annu Rev Med
45:
491-503,
1994[ISI][Medline].
19.
Tsai, EY,
Falvo JV,
Tsytsykova AV,
Barczak AK,
Reimold AM,
Glimcher LH,
Fenton MJ,
Gordon DC,
Dunn IF,
and
Goldfeld AE.
A lipopolysaccharide-specific enhancer complex involving Ets, Elk-1, Sp1 and CREB binding protein and p300 is recruited to the tumor necrosis factor alpha promoter in vivo.
Mol Cell Biol
20:
6084-6094,
2000
20.
Waskiewicz, AJ,
and
Cooper JA.
Mitogen and stress response pathways: MAP kinase cascades and phosphatase regulation in mammals and yeast.
Curr Opin Cell Biol
7:
798-805,
1995[ISI][Medline].
21.
Weinstein, SL,
Sanghera JS,
Lemke K,
DeFranco AL,
and
Pelech SL.
Bacterial lipopolysaccharide induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in macrophages.
J Biol Chem
267:
14955-14962,
1992
22.
Wung, BS,
Cheng JJ,
Chao YJ,
Hsieh HJ,
and
Wang DL.
Modulation of ras/raf/ERK pathway by reactive oxygen species is involved in cyclic strain-induced early growth response-1 gene expression in endothelial cells.
Circ Res
84:
804-812,
1999
23.
Yao, J,
Mackman N,
Edgington TS,
and
Fan ST.
Lipopolysaccharide induction of the tumor necrosis factor a promoter in human monocytic cells. Regulation by egr-1, c-jun and NFkB transcription factors.
J Biol Chem
272:
17795-17801,
1997