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Inflammatory Diseases Research, The DuPont Pharmaceuticals Company, Wilmington, DE 19880
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and arachidonic acid
metabolites. This leads to the recruitment and activation of other
immune cells into sites of injury and infection. One of the most potent activators of monocytes is LPS, a component of the outer membrane of Gram-negative bacteria. At physiologic concentrations, LPS binds to the serum protein, LPS-binding protein, and this complex then interacts with the glycosyl phosphatidylinositol-linked cell surface glycoprotein, CD14, which is present on monocytes, macrophages, and neutrophils (1, 2). Because CD14 lacks both transmembrane and cytoplasmic domains, the mechanism by which CD14 transmits a stimulatory response is still unclear. It has been shown that a number of src family tyrosine kinases, such as lyn, hck, and fgr, become activated in LPS-stimulated macrophages (3, 4, 5) and that inhibitors of tyrosine phosphorylation block the secretion of cytokines (6). Activation of protein kinase C and phosphatidylinositol (PI)3-kinase has also been shown to occur (7, 8, 9).
Among the most prominently tyrosine phosphorylated proteins in LPS-activated macrophage cell lines are the p42 (extracellular signal-regulated kinase (ERK) 2)2 and p44 (ERK1) isoforms of the MAP kinase family of serine/threonine kinases (10). MAP kinases have been implicated in a number of signaling events that are potentially important in the inflammatory response. ERKs have been shown to phosphorylate and activate the transcription factors elk1 and NF-IL6 (11, 12). Elk1 has been shown to be required for c-fos up-regulation, which, as a component of the AP-1 transcription factor, plays an important role in the up-regulation of cytokines and metalloproteases (13) whereas NF-IL6 promotes the up-regulation of granulocyte (G)-CSF, IL-6, IL-8, IL-1, TNF, and the inducible form of nitric oxide synthase (14, 15, 16, 17). ERKs can also phosphorylate cytoplasmic phospholipase A2 (PLA2), which catalyzes the release of arachidonic acid (18).
There are additional MAPK family members, JNKs and p38s, whose activity has been shown to be up-regulated in response to LPS in fibroblast and macrophage cell lines (19, 20, 21, 22). Recent experiments with a small molecule inhibitor of p38 kinases show that blocking p38 prevents IL-1 and TNF production in LPS-stimulated monocytes at the level of translation (23). It has also been shown that dexamethasone inhibits TNF production at least in part by blocking induction of JNKs (22). These results suggest that JNKs and p38 kinases are also important in the regulation of cytokines in LPS-stimulated monocytes.
The ERK pathway is activated through the stimulation of Ras, which in turn activates the serine/threonine kinase Raf-1 (24). Raf-1 phosphorylates and activates the dual-specificity MAP kinase kinases, MEK1 and MEK2, which phosphorylate ERKs on two critical tyrosine and threonine residues. This event leads to an increase in ERK activity. In this report, we studied the role of the ERK pathway in the regulation of cytokines and PGs in LPS-stimulated monocytes by using a novel inhibitor of MEK, the kinase upstream of ERK in the signaling cascade. We show that treatment of monocytes with the selective MEK inhibitor U0126 blocks production of IL-1, IL-8, TNF, and PGE2 in response to LPS. These results demonstrate that, although multiple pathways are activated in response to LPS, activation of MEK/ERK is critical for cytokine and PG release.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 medium and DMEM were obtained from Life Technologies (Gaithersburg, MD). FCS was obtained from Hyclone (Logan, UT). LPS from Salmonella typhimurium was purchased from Calbiochem (San Diego, CA). All LPS samples were reconstituted to 10 mg/ml solutions in pyrogen-free water containing 0.5% triethylamine or in PBS. Abs to ERK2 and p38 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The Ab to ERK2 has been shown to cross-react with ERK1. Phosphospecific Abs were obtained from New England Biolabs (Beverly, MA). Compounds used in this study were prepared by the DuPont Pharmaceuticals Chemistry department and dissolved in 100% DMSO.
Monocyte purification
Human monocytes were isolated by a combination of Ficoll-Hypaque gradient centrifugation, with elutriation as previously described (25). This procedure resulted in populations of monocytes of greater than 90% purity as determined by Wright-Giemsa staining.
Cell culture
Cells were plated at 2 x 106 cells per ml in DMEM or RPMI +10% FCS media (Life Technologies). Cells were plated in Costar (Cambridge, MA) 12-well culture dishes with 1 ml cell suspension per well. Cells were stimulated with 1 µg/ml LPS and incubated at 37°C for specific lengths of time (4 h, TNF; 16–18 h, IL-1, IL-8, and PGE2). In experiments to determine the effects of MEK inhibitors, compound was added at various concentrations immediately before addition of LPS. At the end of the incubation, supernatants were removed and assayed for cytokines or PGE2.
Cytokine and PGE2 assays
The quantitative analyses of TNF- and IL-1-ß were performed
by ELISA as previously described (26, 27). PGE2 levels were
determined by ELISA (PerSeptive Diagnostics, Cambridge, MA), as were
levels of IL-8 (R&D Systems, Minneapolis, MN), according to
manufacturer’s instructions.
Kinase assays
Cellular extracts were prepared from monocytes stimulated for various lengths of time with 1 µg/ml LPS in the presence or absence of U0126 as previously described (28). ERK, JNK, and p38 activities were measured by immunoprecipitation kinase assays as described (28, 29). Determination of phospho-ERK, JNK, and p38 levels was done by Western analysis using phosphospecific Abs (New England Biolabs) as previously described (29).
Northern analysis
The human monocytic cell line, THP-1, was stimulated with 10
µg/ml LPS in the presence or absence of compound for 2 h. Total
RNA was isolated using RNA-ZolB (Tel-Test, Friendswood,
TX), and Northern blot analyses were performed as described (30) using
10 µg of RNA/lane. The blots were probed with digoxygenin-labeled
cDNA probes for full-length human IL-1 ß, TNF-, and PGHS-2
according to the manufacturer (Boehringer Mannheim, Indianapolis, IN).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although it has previously been shown that ERKs, JNKs, and p38
kinases can be activated by LPS in fibroblasts and macrophage cell
lines (7, 10, 19, 20, 21, 22), we performed immune complex kinase assays on
extracts from LPS-stimulated human monocytes to determine the extent
and time course of activation of all three MAPK pathways in our system.
As shown in Fig. 1
, ERK, JNK, and p38 are
activated to a similar extent (5- to 10-fold) in human monocytes.
Maximal kinase activity is observed at 30 min for all of the pathways.
This response is somewhat delayed relative to other activators, such as
TPA, where kinase activity peaks after 5 min, but is in agreement with
previous studies on LPS signaling performed in macrophage cell lines
(10, 20, 21, 22). Although the reagents used in this experiment cannot
discriminate among the various ERK, JNK, and p38 family members, the
results discussed below suggest that specific family members are
preferentially used in this response.
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U0126 was initially identified as an inhibitor of AP-1-driven gene
transcription and was later shown to directly block ERK phosphorylation
by its upstream kinases, MEK1 and MEK2 (31). As reported previously,
U0126 inhibits MEK1 and MEK2 catalytic activity in a direct enzyme
assay but does not significantly inhibit the related MAP kinase family
members MKK3, MKK4, MKK6, ERK2, JNK1, or p38. We have also shown that
U0126 is a noncompetitive, reversible inhibitor of MEK and does not
inhibit unrelated kinases such as protein kinase C (PKC), abl, S6
kinase, or cdk2 (31). Since these studies were done in vitro, we wanted
to determine whether MEK1/2 was also selectively inhibited in cells.
Monocytes were stimulated with LPS in the presence or absence of
various concentrations of U0126 for 15 min. Extracts were then prepared
and analyzed by Western blot for phosphorylated forms of ERK1/2,
JNK1/2, and p38 or by immune complex kinase assay for MAPK activity. As
shown in Fig. 2, ERK2 appears to be
preferentially phosphorylated in the LPS-stimulated monocytes, compared
with ERK1. The p54 isoform of JNK is also phosphorylated to a greater
extent than the p46 form. U0126 blocks ERK2 phosphorylation with an
IC50 value of 0.2 µM and does not inhibit p38 or JNK
phosphorylation by greater than 30% at concentrations as high as 100
µM. Further, U0126 blocks ERK activity >90% but not JNK or p38
activities (<20% at 10 µM) (Fig. 3).
The effect of U0126 on ERK phosphorylation vs p38 and jnk
phosphorylation is observed at all time points examined (not shown).
These data confirm the results of the direct enzyme assays and
demonstrate that U0126 is a selective inhibitor of MEK in cells.
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Although the MAPK pathways are activated by LPS, the role of MEK,
specifically, in LPS-mediated effects in monocytes is unclear. To
address this issue, we treated monocytes with LPS in the presence or
absence of various concentrations of U0126. The levels of IL-1, TNF,
and PGE2 produced were determined by ELISA. As shown in
Fig. 4, U0126 blocks IL-1 and TNF
production by LPS-stimulated monocytes, having IC50 values
of 1 and 0.3 µM, respectively. U0126 also inhibits the release of
PGE2, with an IC50 of 0.2 µM. In addition,
U0126 was shown to inhibit release of IL-6 and IL-8 in this system
(Fig. 5, and not shown). The
IC50 values for the inhibition of cytokine and
PGE2 release show a good correlation with the effects of
U0126 on ERK phosphorylation (IC50 = 0.2 µM), suggesting
that MEK inhibition is responsible for the effects observed.
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, PD98059 also blocked TNF and PGE2 release but was 10–30
times less potent (TNF IC50 = 10 µM; PGE2
IC50 = 2 µM). These results are in agreement with the
difference in potency between the two compounds in the direct enzyme
assay. U0126 blocks MEK activity with an IC50 of 70 nM
whereas PD98059 has an IC50 of 5 µM (data not shown).
There were no effects on cell viability or on total RNA and protein
levels at concentrations of U0126 as high as 20 µM, suggesting that
the cytokine inhibition observed was not due to toxicity (not shown).
However, to study this further, we examined the effects of U0126 on
cytokine production in response to different stimuli. As shown in Fig. 5, treatment of cells with 1 µM U0126 inhibited IL-8 production in
response to LPS or IL-1 but not in response to TNF. At higher
concentrations of U0126 (>10 µM), some inhibition of IL-8 secretion
in TNF-treated cells was observed. IL-1 and IL-6 levels were not
significantly increased in response to TNF in this system; therefore,
the effects of U0126 could not be examined. These results suggest that
the involvement of the ERK pathway in the regulation of cytokine
production is stimulus dependent and that the effects of U0126 are
not due to general toxicity.
U0126 blocks cytokine production at the level of transcription
It is known that ERK activation leads to the phosphorylation and
increased activity of several transcription factors such AP-1 and
NF-IL6, which have been shown to be involved in the transcriptional
up-regulation of cytokines. We therefore performed Northern analyses to
determine whether the effects of U0126 on cytokine release were due to
inhibition of transcription. We could demonstrate that U0126 blocks
IL-1 up-regulation of mRNA levels in the human monocytic cell line,
THP-1, in response to LPS stimulation (Fig. 6). U0126 has an IC50 value
of 0.2 µM in this system. U0126 also decreases the levels of TNF mRNA
(not shown). In both cases, the inactive analogue, U0124, had no
effect. The effects of the MEK inhibitor at the mRNA level are in
contrast to those observed with the p38 inhibitor SB203580, which has
been shown to prevent cytokine release at the level of translation
(23).
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The enzyme PG H synthase (PGHS) has been shown to be essential for
PG release by catalyzing the conversion of arachidonic acid to PG
endoperoxide (PGH2), the precursor to various PGs,
including PGE2. (33, 34, 35). Two isoforms of PGHS-2 have been
identified, a constitutive form (PGHS-1) and an inducible form
(PGHS-2). The inducible PGHS-2 has previously been shown to be
selectively increased in LPS-stimulated macrophages (36). Since U0126
was shown to block the up-regulation of IL-1 and TNF mRNA, it was
possible that U0126 might exert its effects on PGE2 release
by regulating the level of the inducible PGHS-2 enzyme. We therefore
examined PGHS-2 mRNA levels in the presence or absence of U0126 by
Northern analysis (Fig. 7). We could show
that U0126 blocks the up-regulation of PGHS-2 at the mRNA level and
therefore demonstrate at least one mechanism whereby MEK inhibition can
affect PGE2 release.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It had previously been shown that overexpression of a Raf1:estrogen
receptor (Raf1:ER) chimeric protein in the murine macrophage
cell line RAW 264.7 resulted in the rapid and prolonged activation of
ERK1 and ERK2 upon treatment with estradiol (38). When the levels of
TNF protein and mRNA were measured, however, it was found that they
were induced only weakly by Raf1:estrogen receptor. This suggested that
other signaling events, such as NF-
B activation, might be required
to achieve the maximum response observed with LPS. Although this may be
the case in some systems, our data clearly demonstrate that blocking
only the ERK pathway is sufficient to inhibit cytokine production in
freshly isolated monocytes. These results suggest that, although
environmental stimuli may require multiple signaling events to elicit
a cellular response, blocking any one of them is adequate for
inhibition.
Our results are in agreement with a recent study showing that PD98059 partially inhibits monocyte production of IL-1 and TNF in response to LPS stimulation (39). In this study, they also demonstrated that, in contrast to TNF and IL-1, neither IL-10 secretion nor IL-10 mediated responses are affected, demonstrating that not all LPS-mediated effects will be ERK dependent. Our data show that, similar to IL-1 and TNF, monocyte production of IL-6, IL-8, and PGE2 in response to LPS are dependent on activation of the MEK, further confirming that proinflammatory events will be suppressed by blocking the ERK pathway. Although these conclusions are based on results with small molecule inhibitors and one can never absolutely exclude the possibility of a second target other than MEK, the finding that two structurally distinct inhibitors give similar results strongly implicates the importance of MEK in LPS-stimulated cytokine production.
The role of the ERK pathway in cytokine production appears to be
stimulus dependent since U0126 blocks IL-8 secretion in response to LPS
and IL-1 but not TNF. Although we and others have shown that TNF can
up-regulate the ERK pathway in monocytes (40), the response is weaker
and more transient (not shown), suggesting that other pathways may be
more important. In support of this, we have shown that the p38
inhibitor SB203580 does block IL-8 production in response to TNF as
well as LPS (not shown). How MEK inhibition results in decreased IL-8
production in response to LPS is under study. IL-8 up-regulation has
been shown previously to be dependent on NF-B sites in its promoter.
The role of MEK in regulating NF-B activity is unclear, but recent
studies demonstrating that PD98059 can reduce NF-B transactivational
activity (41) suggest that there is some interaction.
We have shown that U0126 inhibition of PGE2 release may be due in part to its effects on PGHS-2 since U0126 was shown to block the up-regulation of mRNA levels of this enzyme. This result supports a previous study that demonstrated that overexpression of dominant negative mutants of Raf-1 and ERK blocked induction of PGHS-2 in 3T3 cells stimulated with platelet-derived growth factor (PDGF) or serum (42). Our results are also in agreement with a recent study demonstrating that the Parke-Davis MEK inhibitor, PD98059, blocks PGHS-2 transcription in the RAW 264.7 murine macrophage cell line (43). There is an NF-IL6 site in the PGHS-2 promoter that has been shown to be important for PGHS-2 up-regulation by LPS in vascular endothelial cells (44). Since ERK has been shown to phosphorylate NF-IL6, this may be how the Ras/Raf/MEK/ERK pathway mediates its effects on PGHS-2 transcription. In addition, it is known that the rate-limiting step in PG biosynthesis is the liberation of arachidonic acid and that the release of arachidonic acid requires PLA2 activation (45). Since ERK has been shown to phosphorylate and activate PLA2 in vitro (18), it is possible that U0126 blocks PGE2 release in LPS-stimulated monocytes at this step as well.
Previous studies with inhibitors of the p38 MAP kinase pathway have demonstrated that these molecules have antiinflammatory properties in vivo (46). Our results showing that U0126 inhibits cytokine and PGE2 production by LPS-stimulated monocytes in vitro suggest that MEK inhibitors may also prevent inflammation in vivo. Based on these results, we have tested U0126 in several animal models of inflammation such as the carrageenan paw and TPA ear edema models and have demonstrated that the compound is efficacious (our unpublished data). Our data therefore demonstrate that MEK is another potential target for the design of therapeutic strategies in the treatment of inflammatory diseases.
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Footnotes |
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2 Abbreviations used in this paper: ERK, extracellular signal-regulated kinase; MAP kinase, mitogen-activated protein kinase; JNK, Jun NH2-terminal kinase; AP-1, activator protein-1; MEK, MAP/ERK kinase; PLA2, phospholipase A2; PGHS-2, PG H synthase 2; GAPDH, glyceraldehyde phosphate dehydrogenase; MKK, MAP kinase kinase.
Received for publication January 14, 1998. Accepted for publication July 15, 1998.
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H.-Y. Hsu and M.-H. Wen Lipopolysaccharide-mediated Reactive Oxygen Species and Signal Transduction in the Regulation of Interleukin-1 Gene Expression J. Biol. Chem., June 14, 2002; 277(25): 22131 - 22139. [Abstract] [Full Text] [PDF]
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 M. Takeda, A. Takamiya, A. Yoshida, and H. Kiyama Extracellular Signal-Regulated Kinase Activation Predominantly in Muller Cells of Retina with Endotoxin-Induced Uveitis Invest. Ophthalmol. Vis. Sci., April 1, 2002; 43(4): 907 - 911. [Abstract] [Full Text] [PDF]
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J. Cuschieri, D. Gourlay, I. Garcia, S. Jelacic, and R. V. Maier Hypertonic Preconditioning Inhibits Macrophage Responsiveness to Endotoxin J. Immunol., February 1, 2002; 168(3): 1389 - 1396. [Abstract] [Full Text] [PDF]
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 T. L. Mynott, B. Crossett, and S. R. Prathalingam Proteolytic Inhibition of Salmonella enterica Serovar Typhimurium-Induced Activation of the Mitogen-Activated Protein Kinases ERK and JNK in Cultured Human Intestinal Cells Infect. Immun., January 1, 2002; 70(1): 86 - 95. [Abstract] [Full Text] [PDF]
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 M. Guha, M. A. O'Connell, R. Pawlinski, A. Hollis, P. McGovern, S.-F. Yan, D. Stern, and N. Mackman Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor {alpha} expression by inducing Elk-1 phosphorylation and Egr-1 expression Blood, September 1, 2001; 98(5): 1429 - 1439. [Abstract] [Full Text] [PDF]
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 G. Dong, Z. Chen, Z.-Y. Li, N. T. Yeh, C. C. Bancroft, and C. Van Waes Hepatocyte Growth Factor/Scatter Factor-induced Activation of MEK and PI3K Signal Pathways Contributes to Expression of Proangiogenic Cytokines Interleukin-8 and Vascular Endothelial Growth Factor in Head and Neck Squamous Cell Carcinoma Cancer Res., August 1, 2001; 61(15): 5911 - 5918. [Abstract] [Full Text] [PDF]
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L. Rabehi, T. Irinopoulou, B. Cholley, N. Haeffner-Cavaillon, and M.-P. Carreno Gram-Positive and Gram-Negative Bacteria Do Not Trigger Monocytic Cytokine Production through Similar Intracellular Pathways Infect. Immun., July 1, 2001; 69(7): 4590 - 4599. [Abstract] [Full Text] [PDF]
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S. Ibata-Ombetta, T. Jouault, P.-A. Trinel, and D. Poulain Role of extracellular signal-regulated protein kinase cascade in macrophage killing of Candida albicans J. Leukoc. Biol., July 1, 2001; 70(1): 149 - 154. [Abstract] [Full Text] [PDF]
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 D. Meerzaman, P. S. Shapiro, and K. C. Kim Involvement of the MAP kinase ERK2 in MUC1 mucin signaling Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L86 - L91. [Abstract] [Full Text] [PDF]
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X. Zhu, H. Sano, K. P. Kim, A. Sano, E. Boetticher, N. M. Munoz, W. Cho, and A. R. Leff Role of Mitogen-Activated Protein Kinase-Mediated Cytosolic Phospholipase A2 Activation in Arachidonic Acid Metabolism in Human Eosinophils J. Immunol., July 1, 2001; 167(1): 461 - 468. [Abstract] [Full Text] [PDF]
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Y.-W. Hsu, K.-H. Chi, W.-C. Huang, and W.-W. Lin Ceramide Inhibits Lipopolysaccharide-Mediated Nitric Oxide Synthase and Cyclooxygenase-2 Induction in Macrophages: Effects on Protein Kinases and Transcription Factors J. Immunol., May 1, 2001; 166(9): 5388 - 5397. [Abstract] [Full Text] [PDF]
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D. Chakravortty, Y. Kato, T. Sugiyama, N. Koide, M. M. Mu, T. Yoshida, and T. Yokochi The Inhibitory Action of Sodium Arsenite on Lipopolysaccharide-Induced Nitric Oxide Production in RAW 267.4 Macrophage Cells: A Role of Raf-1 in Lipopolysaccharide Signaling J. Immunol., February 1, 2001; 166(3): 2011 - 2017. [Abstract] [Full Text] [PDF]
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K. M. Krishna Rao MAP kinase activation in macrophages J. Leukoc. Biol., January 1, 2001; 69(1): 3 - 10. [Abstract] [Full Text]
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P.-Y. Perera, T. N. Mayadas, O. Takeuchi, S. Akira, M. Zaks-Zilberman, S. M. Goyert, and S. N. Vogel CD11b/CD18 Acts in Concert with CD14 and Toll-Like Receptor (TLR) 4 to Elicit Full Lipopolysaccharide and Taxol-Inducible Gene Expression J. Immunol., January 1, 2001; 166(1): 574 - 581. [Abstract] [Full Text] [PDF]
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 A. K. Ho and C. L. Chik Adrenergic Regulation of Mitogen-Activated Protein Kinase in Rat Pinealocytes: Opposing Effects of Protein Kinase A and Protein Kinase G Endocrinology, December 1, 2000; 141(12): 4496 - 4502. [Abstract] [Full Text] [PDF]
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A. K. De, K. M. Kodys, B. S. Yeh, and C. Miller-Graziano Exaggerated Human Monocyte IL-10 Concomitant to Minimal TNF-{alpha} Induction by Heat-Shock Protein 27 (Hsp27) Suggests Hsp27 Is Primarily an Antiinflammatory Stimulus J. Immunol., October 1, 2000; 165(7): 3951 - 3958. [Abstract] [Full Text] [PDF]
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E. Y. Tsai, J. V. Falvo, A. V. Tsytsykova, A. K. Barczak, A. M. Reimold, L. H. Glimcher, M. J. Fenton, D. C. Gordon, I. F. Dunn, and A. E. Goldfeld 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., August 15, 2000; 20(16): 6084 - 6094. [Abstract] [Full Text]
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R. Rezzonico, R. Chicheportiche, V. Imbert, and J.-M. Dayer Engagement of CD11b and CD11c beta 2 integrin by antibodies or soluble CD23 induces IL-1beta production on primary human monocytes through mitogen-activated protein kinase-dependent pathways Blood, June 15, 2000; 95(12): 3868 - 3877. [Abstract] [Full Text] [PDF]
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H. Liu, P. Sidiropoulos, G. Song, L. J. Pagliari, M. J. Birrer, B. Stein, J. Anrather, and R. M. Pope TNF-{alpha} Gene Expression in Macrophages: Regulation by NF-{kappa}B Is Independent of c-Jun or C/EBP{beta} J. Immunol., April 15, 2000; 164(8): 4277 - 4285. [Abstract] [Full Text] [PDF]
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M. E. Bates, V. L. Green, and P. J. Bertics ERK1 and ERK2 Activation by Chemotactic Factors in Human Eosinophils Is Interleukin 5-dependent and Contributes to Leukotriene C4 Biosynthesis J. Biol. Chem., April 6, 2000; 275(15): 10968 - 10975. [Abstract] [Full Text] [PDF]
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A. F. Valledor, M. Comalada, J. Xaus, and A. Celada The Differential Time-course of Extracellular-regulated Kinase Activity Correlates with the Macrophage Response toward Proliferation or Activation J. Biol. Chem., March 15, 2000; 275(10): 7403 - 7409. [Abstract] [Full Text] [PDF]
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J. A. Nick, S. K. Young, K. K. Brown, N. J. Avdi, P. G. Arndt, B. T. Suratt, M. S. Janes, P. M. Henson, and G. S. Worthen Role of p38 Mitogen-Activated Protein Kinase in a Murine Model of Pulmonary Inflammation J. Immunol., February 15, 2000; 164(4): 2151 - 2159. [Abstract] [Full Text] [PDF]
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A. Aicher, G. L. Shu, D. Magaletti, T. Mulvania, A. Pezzutto, A. Craxton, and E. A. Clark Differential Role for p38 Mitogen-Activated Protein Kinase in Regulating CD40-Induced Gene Expression in Dendritic Cells and B Cells J. Immunol., December 1, 1999; 163(11): 5786 - 5795. [Abstract] [Full Text] [PDF]
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H. Yang, D. W. Young, F. Gusovsky, and J. C. Chow Cellular Events Mediated by Lipopolysaccharide-stimulated Toll-like Receptor 4. MD-2 IS REQUIRED FOR ACTIVATION OF MITOGEN-ACTIVATED PROTEIN KINASES AND Elk-1 J. Biol. Chem., June 30, 2000; 275(27): 20861 - 20866. [Abstract] [Full Text] [PDF]
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K. Rutault, C. A. Hazzalin, and L. C. Mahadevan Combinations of ERK and p38 MAPK Inhibitors Ablate Tumor Necrosis Factor-alpha (TNF-alpha ) mRNA Induction. EVIDENCE FOR SELECTIVE DESTABILIZATION OF TNF-alpha TRANSCRIPTS J. Biol. Chem., February 23, 2001; 276(9): 6666 - 6674. [Abstract] [Full Text] [PDF]
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 R. Kishore, J. R. Hill, M. R. McMullen, J. Frenkel, and L. E. Nagy ERK1/2 and Egr-1 contribute to increased TNF-alpha production in rat Kupffer cells after chronic ethanol feeding Am J Physiol Gastrointest Liver Physiol, January 1, 2002; 282(1): G6 - G15. [Abstract] [Full Text] [PDF]
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