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Inflammatory Diseases Research, DuPont Merck Pharmaceutical 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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The role of the MAP kinase pathways in inducing T cell tolerance is also of interest. Th1 cells can be rendered unresponsive (anergic) by TCR occupancy in the absence of costimulation. This results in suboptimal activation of the T cells, with no IL-2 production and no resultant proliferation (22). In addition, such cells are unresponsive to subsequent stimulation with normal APC plus Ag (22). In response to Ag plus APC, anergized T cells have been shown to undergo early events characteristic of T cell activation (23, 24), to have normal surface expression of TCR, CD4 (24), and CD28 (D. R. DeSilva and Marc K. Jenkins, unpublished data), but to fail to accumulate IL-2 mRNA.
Of the three MAP kinase pathways described in mammalian cells, the ERK pathway is the best defined. This pathway is up-regulated via Ras, which is transiently activated in response to TCR binding. Upon activation, Ras complexes with, and in turn activates the serine-threonine kinase Raf-1, which then phosphorylates the MAP kinase kinase, MEK (25). MEK, a dual-specific kinase, activates ERK by phosphorylating it on two critical residues, Tyr183 and Thr185. ERK then translocates into the nucleus and regulates the activities of several nuclear transcription factors.
In this study, we examined the effects of blocking the ERK pathway on T cell activation and anergy by using an inhibitor, U0126, which specifically inhibits the ability of MEK to phosphorylate ERK without affecting the JNK and p38 pathways. This compound blocked T cell proliferation in response to antigenic stimulation by decreasing IL-2 mRNA levels, but did not block the response of cells to exogenously added IL-2. Blocking the ERK pathway during T cell activation did not affect the response of the T cells to subsequent restimulation. Induction of anergy was also not affected by MEK inhibition, suggesting that immune suppressants that target the ERK pathway would not cause long-term perturbation of the immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.BR/SgSn mice were purchased from The Jackson Laboratory (Bar Harbor, ME). BALB/c mice were purchased from Charles River Laboratories (Raleigh, NC). Single-cell suspensions of whole spleen obtained from these mice were used as APCs for maintenance of Th1 clones and in proliferation assays. The compounds, U0126, U0124, PD098059, and SK&F86002, were synthesized by DuPont Merck Chemistry Department (Wilmington, DE).
T cell clones
A.E7.
This is a B10.A-derived CD4+8-,
CD28+ murine Th1 clone (26) that expresses a Vß3/V
11
TCR. A.E7 cells produce IL-2, IL-3, and IFN-
upon stimulation
with the C-terminal cyanogen bromide fragment (residues 81–104) of
pigeon cytochrome c or a synthetic peptide based on the moth
cytochrome c sequence called DASP (27) bound to
I-Ek molecules on the surface of APC. The DASP peptide was
synthesized using the Merrifield method at the University of Minnesota
(Minneapolis, MN) microchemical facility.
Th17.
This murine Th1 clone was derived from BALB/c mice immunized s.c. with
the influenza virus A/PR/8/34 (PR8), as described (28). It is
CD4+8-, CD28+, and produces
IL-2 and IFN- upon stimulation with the hemagglutinin molecule of
influenza virus PR8. The viral Ag was in the form of allantoic fluid
from virus-infected embryonated hen eggs. Virus titers were determined
by agglutination of chicken erythrocytes and are expressed as
hemagglutinating units (28).
Both A.E7 and Th17 cells were maintained in 24-well plates on a "rest-stimulation" protocol (26) using mitomycin C-treated APC, Ag, and human rIL-2 (Boehringer Mannheim, Indianapolis, IN). Cells used in experiments were rested at least 5 days after addition of IL-2 and were separated from debris and dead APC by Histopaque 1077 (Sigma, St. Louis, MO) density gradient centrifugation.
Northern blots
Total RNA was isolated using the RNA-ZolB method (Tel-Test, Friendswood, TX) from 20 to 50 x 106 Th17 T cells that were unstimulated or stimulated for 4.5 h with PMA plus ionomycin (100 ng/ml and 1 µM, respectively) in the absence or presence of 10 µM U0126, U0124 (an inactive analogue of U0126), the Parke-Davis (Ann Arbor, MI) MEK inhibitor (PD 098059) (29), or the SmithKline Beecham (Philadelphia, PA) p38 inhibitor (SK&F 86002) (8). Northern blots were performed as described (30) using 10 µg of total RNA/lane.
Cellular extracts
Total cellular extracts were made from A.E7 or Th17 T cells (10–50 x 106 cells/treatment) that were unstimulated, or stimulated for 15 min at 37°C with PMA plus ionomycin in the presence or absence of 10 µM U0126. Following the 15-min incubation, T cells were pelleted and washed twice with cold PBS. The cells were then lysed in 100 µl of cold lysis buffer (31). After a 10-min incubation on ice, the extracts were spun for 15 min at 14,000 rpm in Eppendorf tubes at 4°C to pellet cellular debris. The supernatant was removed and protein concentrations determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). These extracts were used in kinase assays and Western blots.
Western blots
Cellular extracts from A.E7 or Th17 T cells were used in Western analysis, as described earlier (31). Briefly, 10 to 20 µg of protein/sample were analyzed by SDS-PAGE on 10% Tris-Tricine gels (Novex, San Diego, CA). Protein was electrotransferred to polyvinylidene difluoride membrane, blocked with a solution of PBS containing 5% milk and 0.1% Tween-20, and probed with a rabbit polyclonal phosphospecific Ab against ERK or p38 (New England Biolabs, Beverly, MA), followed by a peroxidase-conjugated goat anti-rabbit secondary Ab. Washes following incubation with Abs were done in PBS + 0.1% Tween-20. Bands were detected using the Luminol chemiluminescent detection reagents (New England Biolabs). Blots were exposed to autoradiographic film (DuPont) for 1 to 2 min for detection.
Kinase assay
JNK assays were performed using cellular extracts of Th1 cells that were unstimulated or stimulated in the presence or absence of the drug U0126, as described previously (31).
T cell proliferation assays
A.E7 or Th17 cells (2 x 104/well) were incubated with 5 x 105 mitomycin C-treated B10.BR or BALB/c splenocytes plus varying concentrations of pigeon cytochrome c or PR8 Ag (28), or with 5 U/ml human rIL-2. In addition, some assays contained U0126 or an inactive analogue, U0124, to determine direct effects of MEK inhibition on T cell proliferation. Two days after culture initiation, each well was pulsed with 1 µCi of [3H]TdR (DuPont NEN, Boston, MA) and harvested the following day. The incorporation of [3H]TdR into DNA was quantitated on a Packard Matrix 96 direct beta counter without the use of liquid scintillation mixtures.
Effects of U0126 on T cell proliferation and response to restimulation
During the preculture, A.E7 or Th17 cells (5 x 105/well) were cultured in 24-well plates for 3 days with 5 x 106 mitomycin C-treated B10.A or BALB/c splenocytes with or without 0.1 µM Ag. Some of the cultures also contained 10 µM U0126 as an inhibitor of proliferation. Controls included Th1 cells incubated with APC alone in the presence or absence of the drug and the positive control for anergy that consisted of Th1 cells incubated on immobilized anti-CD3 alone, as described below. Following the preincubation step, viable cells were enumerated by trypan blue dye exclusion to determine the extent of proliferation. The T cells were then isolated by density-gradient centrifugation on Histopaque 1077 and rested in medium alone for an additional 4 days. Following the rest period, A.E7 cells (1–2 x 104) were washed and restimulated with normal APC plus Ag or with exogenous IL-2 alone in 96-well plates, as described below under restimulation.
Anergy induction
During the preculture, A.E7 or Th17 T cells (1–3 x 106/plate) were incubated in 10-cm Falcon 3003 petri plates coated with 10 µg/ml purified anti-CD3 mAb (clone 145-2C11; Boehringer Mannheim) (32) to induce anergy, as previously described (33). Some of the induction cultures also contained 10 µM U0126 or the inactive analogue, U0124. T cells were removed from the immobilized anti-CD3 after 1 to 2 days, washed, and transferred into fresh wells, where they were allowed to rest in medium alone for 2 to 3 days before restimulation.
Restimulation of T cells
Following the rest period, Th1 cells were restimulated with APC plus Ag (for proliferation assays) or PMA plus ionomycin (100 ng/ml and 1 µM, respectively, for cellular extracts used in kinase assays) as a stimulus to detect differences between control resting T cells and anergic T cells.
Mixed leukocyte reaction
PBMC were isolated from normal human donors by centrifugation through Vacutainer cell preparation tubes with sodium heparin gel (Becton Dickinson, Franklin Lakes, NJ). Cells from two unrelated donors were cultured together at a final concentration of 2 x 105 cells/well in 96-well plates at 37°C in 5% CO2. Various concentrations of compound were added at the initiation of culture. After 5 days, the plates were then pulsed with 1 µCi/well [3H]TdR and harvested after 16 to 18 h.
Cell cycle analysis
PBMC were stimulated for 72 h with 1 µg/ml Con A in the presence or absence of 10 µM U0126 or 1 µM cyclosporin A (CsA). In some cultures, exogenous IL-2 was added at 10 U/ml. The cells were then fixed with ethanol, stained with propidium iodide, and analyzed by flow cytometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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U0126 (Fig. 1
) was initially
identified as an inhibitor of AP-1 activity, and was shown to
specifically inhibit MEK in a direct enzyme assay (manuscript in
preparation). To determine whether U0126 showed selective inhibition of
MEK in T cells, cellular extracts from murine Th1 clones that were
stimulated for 15 min with PMA plus ionomycin in the presence or
absence of U0126 were examined for activation of ERK, JNK, and p38. As
shown in Figure 2A, ERK
phosphorylation during PMA/ionomycin stimulation was blocked 96% by
the presence of 10 µM U0126. Under these conditions, U0126 was shown
to inhibit ERK phosphorylation with an IC50 value of 0.5
µM (data not shown). In contrast, U0126 had only a slight effect on
p38 phosphorylation (Fig. 2B) and no effect on JNK
activity (Fig. 2C). Results from immune complex
kinase assays showed that the slight effect of U0126 on p38
phosphorylation did not result in a decrease in p38 kinase activity
(not shown). These data indicate that U0126 selectively blocks the ERK
pathway in T cells.
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As reported previously (10, 11) and as shown in Figure 2
, all
three MAP kinase pathways are up-regulated in T cells upon activation
with PMA/ionomycin or anti-CD3/anti-CD28 cross-linking. We were
therefore interested in determining what effect, if any, blocking only
the ERK pathway would have on T cell proliferation and IL-2 production.
Ag-specific T cell clones were stimulated with APC plus Ag or
anti-CD3 plus anti-CD28 in the presence or absence of 10 µM
U0126, and proliferation was measured by [3H]TdR
incorporation after 3 days. As shown in Figure 3A, T cell proliferation was
blocked significantly in the presence of U0126, whereas the inactive
analogue, U0124, had no effect. Up-regulation of IL-2R during
stimulation of this Th1 clone with APC plus Ag was not inhibited by
U0126 (data not shown) and, as shown in Figure 3B, the T
cell clones can proliferate if IL-2 is added exogenously to the
culture. Therefore, blocking the ERK pathway inhibits T cell
proliferation in response to TCR cross-linking, but is not inhibitory
to cell cycle progression in general if mitosis is initiated by a
method that bypasses TCR signaling. This experiment also establishes
that U0126 is not inherently toxic to T cells.
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The experiments described above were conducted in murine T cell
clones. Since there may be differences in the signaling events in
freshly isolated cells, we also tested the effects of U0126 in an MLR
using PBLs isolated from normal human donors. As shown in Figure 4, U0126 effectively suppressed an
allogeneic response, having an IC50 value of 2 µM.
U0126 also blocked the proliferative response of PBLs to Con A and
anti-CD3 cross-linking (not shown).
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).
However, in agreement with the studies in the murine Th1 clones, the
PBLs can enter into cell cycle if IL-2 is exogenously provided. This
result is similar to the effects observed with the known
immunosuppressant, CsA.
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The results shown above suggest that T cell proliferation is
blocked by U0126 because the cells fail to produce IL-2 since
proliferation occurs if IL-2 is added exogenously. To determine whether
T cell proliferation is blocked by U0126 at the level of IL-2
transcription, or at a later stage affecting IL-2 synthesis and/or
secretion, we performed Northern analysis on cloned T cells activated
by PMA plus ionomycin in the presence or absence of the drug. As shown
in Figure 5, U0126 prevented the
PMA/ionomycin-induced up-regulation of IL-2 mRNA levels by 87% at 10
µM (lane 3), indicating that U0126 blocks T
cell proliferation by interfering with IL-2 production, as would be
expected of an AP-1 suppressor. In contrast, the inactive analogue,
U0124, the Parke-Davis MEK inhibitor PD 098059 (lane
5), or the p38 inhibitor SK&F 86002 (lane
6) did not affect IL-2 mRNA levels (lane
4) appreciably at 10 µM. However, treatment with the
Parke-Davis MEK inhibitor at a 20 µM concentration did result in a
50% reduction of IL-2 levels in Jurkat cells (data not shown),
indicating that it is less potent than U0126. This is consistent with
the differences in their potencies against the MEK enzyme. U0126 has an
IC50 of 70 nM, whereas PD098059 has an IC50 of
5 µM (data not shown). It should be noted that although U0126
treatment results in decreased IL-2 mRNA levels, these experiments do
not distinguish between an effect on IL-2 transcription or mRNA
stability.
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Previous studies have indicated that TCR occupancy in the absence
of proliferation induces anergy in murine Th1 clones (34). In these
early studies, signaling through the TCR was allowed to proceed to
completion, resulting in IL-2 production. Proliferation was then
blocked by interfering with IL-2R binding/signaling or by methods that
blocked entry into the cell cycle. It was therefore interesting to
determine whether inhibiting proliferation by blocking ERK activation
would also result in anergy, since we would in effect be preventing
completion of TCR signaling into the nucleus. To study this, T cells
were preincubated with APC plus Ag in the presence or absence of U0126
for 3 days, after which the drug was washed off and the T cells rested
in medium alone for 4 days before antigenic restimulation. To rule out
cytotoxicity, T cells were also incubated with APC plus drug in the
absence of Ag. T cells exposed to immobilized anti-CD3 in the
absence of costimulation were used as a positive control for anergy.
Figure 6A shows that
proliferation in response to APC plus Ag was blocked effectively by
U0126 during the preincubation step. As shown in Figure 6B,
Th1 cells preincubated with APC plus Ag in the presence of the MEK
inhibitor showed a good restimulation response, equal to resting T
cells. This finding suggests that to induce anergy, TCR signaling has
to be completed, and that blocking the ERK pathway does not interfere
with the ability of the T cells to respond to subsequent
restimulation.
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To study the effects of a MEK inhibitor on anergy induction, Th1
cells were exposed to immobilized anti-CD3 with no costimulation in
the presence or absence of U0126. After 2 days, the cells were removed
from the anti-CD3, washed, and rested for 3 days before
restimulation with APC plus Ag. As shown in Figure 7A, blocking the ERK pathway
did not interfere with the induction of anergy. Th1 cells anergized in
the presence or absence of U0126 showed much lower levels of
proliferation during restimulation compared with resting T cells. The
cells were still viable, though, because they proliferated well in
response to exogenous IL-2. In fact, they responded better to exogenous
IL-2 than resting T cells (Fig. 7B) perhaps due to
the fact that IL-2R had been up-regulated during incubation on
anti-CD3. These results indicate that signaling through the ERK
pathway is not a prerequisite for the induction of anergy.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clonal anergy is a state of long-lasting unresponsiveness to antigenic stimulation induced in Th1 cells as a result of TCR occupancy in the absence of proliferation. It is induced half maximally by 5 h and maximally after 16 h of TCR occupancy in the absence of costimulation (23). The anergic state involves Ca2+ increases within the cell since anergy can be induced by increasing intracellular Ca2+ (23) and is also blocked by CsA (33, 37). Anergy induction requires new protein synthesis (38) and appears to be a result of an IL-2 production defect since anergic cells can proliferate in response to exogenously added IL-2.
Because these features suggest that anergy results from an active signaling process, we were interested in examining the effects of a MEK inhibitor on anergy induction. We could show that blocking T cell proliferation via inhibition of ERK activation did not induce anergy. Cells stimulated with Ag in the presence of U0126 show a restimulation response identical to resting T cells or T cells activated in the absence of U0126, indicating that blocking the ERK pathway in and of itself does not result in anergy. In addition, blocking the ERK pathway did not prevent anergy induction following TCR occupancy in the absence of costimulation. These results suggest that in contrast to the critical role of the ERK signaling pathway in the events leading to IL-2 production, this pathway does not regulate the induction of anergy when the TCR is cross-linked in the absence of costimulation. The reason for this may be that the anergy program is set in motion at a point upstream or parallel to MEK, possibly at the level of Ca2+ increases within the cell following receptor cross-linking. In support of this, it is known that CsA blocks the induction of anergy in Th1 cells (33). This is thought to be due to the fact that this drug abrogates Ca2+ signaling by blocking translocation of the NF-ATp subunit into the nucleus following TCR ligation. NF-ATp then cannot complex with AP-1, bind to NF-AT sites, and up-regulate transcription of NF-AT-responsive genes. However, we have shown in this study that U0126, which blocks the PKC-induced arm of TCR signaling leading to the up-regulation of AP-1, does not affect the outcome of anergy induction. This finding is interesting because it suggests that anergy is not induced by a gene product that is regulated by binding of the NF-AT heterotrimer composed of the NF-ATp plus AP-1 subunits. If this were the case, then one would expect both CsA and a MEK inhibitor (by blocking NF-ATp and AP-1, respectively) to prevent anergy. Because only CsA blocked induction of anergy, it suggests instead that anergy is induced by either a Ca2+-responsive gene product or by a gene with an enhancer that is bound by the NF-ATp subunit alone. Characterization of the genes induced by TCR occupancy in the presence of a MEK inhibitor vs CsA will be of interest.
In these respects, U0126 belongs to a novel class of immunosuppressants that effectively block IL-2 synthesis and T cell proliferation without affecting the long-term outcomes of either T cell activation or tolerance. This may be of importance clinically. It has been reported that T cell-mediated autoimmunity results when the widely used immunosuppressive drug CsA is withdrawn following prolonged treatment (39), perhaps due to the fact that tolerance via clonal anergy cannot take place in the presence of CsA. In addition to inhibiting anergy induction in vitro, CsA has been shown to interfere with the deletion of thymocytes bearing self-reactive TCRs in vivo (40). U0126 is also distinct from immunosuppressants such as rapamycin that inhibit T cell proliferation by interfering with IL-2R signaling and induce T cell anergy (34). Although the simultaneous induction of anergy in the subset of T cells whose activation one is trying to block by the use of immunosuppressants is advantageous, other immune reactions are constantly ongoing. Agents that induce anergy nonspecifically may also lead to tolerance induction in useful immune responses that could become disruptive to the functioning of the immune system as a whole. Efforts to determine the viability of using a MEK inhibitor as an immune suppressant in vivo will therefore be important.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peggy A. Scherle, The DuPont Merck Pharmaceutical Co., Experimental Station, E-400 Room 5438, Route 141, Wilmington, DE 19880-0400. E-mail address:
3 Abbreviations used in this paper: ERK, extracellular signal-regulated kinase; AP-1, activator protein-1; CsA, cyclosporin A; JNK, Jun NH2-terminal kinase; MAP, mitogen-activated protein; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; NF-AT, nuclear factor of activated T cells.
Received for publication October 6, 1997. Accepted for publication December 29, 1997.
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References |
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/CD3-CD4 and CD28 signaling in T cells: SH2/SH3 domains, protein-tyrosine and lipid kinases. Immunol. Today 15:225.[Medline]
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 G. Bandyopadhyay, T. Biswas, K. C. Roy, S. Mandal, C. Mandal, B. C. Pal, S. Bhattacharya, S. Rakshit, D. K. Bhattacharya, U. Chaudhuri, et al. Chlorogenic acid inhibits Bcr-Abl tyrosine kinase and triggers p38 mitogen-activated protein kinase-dependent apoptosis in chronic myelogenous leukemic cells Blood, October 15, 2004; 104(8): 2514 - 2522. [Abstract] [Full Text] [PDF]
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K. Ueda, H. Kosako, Y. Fukui, and S. Hattori Proteomic Identification of Bcl2-associated Athanogene 2 as a Novel MAPK-activated Protein Kinase 2 Substrate J. Biol. Chem., October 1, 2004; 279(40): 41815 - 41821. [Abstract] [Full Text] [PDF]
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 D. E. Spaner Amplifying cancer vaccine responses by modifying pathogenic gene programs in tumor cells J. Leukoc. Biol., August 1, 2004; 76(2): 338 - 351. [Abstract] [Full Text] [PDF]
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C. L. Adams, A. M. Grierson, A. M. Mowat, M. M. Harnett, and P. Garside Differences in the Kinetics, Amplitude, and Localization of ERK Activation in Anergy and Priming Revealed at the Level of Individual Primary T Cells by Laser Scanning Cytometry J. Immunol., August 1, 2004; 173(3): 1579 - 1586. [Abstract] [Full Text] [PDF]
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 R. K. Upmacis, R. S. Deeb, M. J. Resnick, R. Lindenbaum, C. Gamss, D. Mittar, and D. P. Hajjar Involvement of the mitogen-activated protein kinase cascade in peroxynitrite-mediated arachidonic acid release in vascular smooth muscle cells Am J Physiol Cell Physiol, June 1, 2004; 286(6): C1271 - C1280. [Abstract] [Full Text] [PDF]
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H.-O. Pae, G.-S. Oh, B.-M. Choi, S.-C. Chae, Y.-M. Kim, K.-R. Chung, and H.-T. Chung Carbon Monoxide Produced by Heme Oxygenase-1 Suppresses T Cell Proliferation via Inhibition of IL-2 Production J. Immunol., April 15, 2004; 172(8): 4744 - 4751. [Abstract] [Full Text] [PDF]
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 D.-G. Cho, M. R. Mulloy, P. A. Chang, M. D. Johnson, A. S. Aharon, T. A. Robison, T. L. Buckles, D. W. Byrne, and D. C. Drinkwater Jr Blockade of the extracellular signal-regulated kinase pathway by U0126 attenuates neuronal damage following circulatory arrest J. Thorac. Cardiovasc. Surg., April 1, 2004; 127(4): 1033 - 1040. [Abstract] [Full Text] [PDF]
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E. Rottinger, L. Besnardeau, and T. Lepage A Raf/MEK/ERK signaling pathway is required for development of the sea urchin embryo micromere lineage through phosphorylation of the transcription factor Ets Development, March 1, 2004; 131(5): 1075 - 1087. [Abstract] [Full Text] [PDF]
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 H. Guo, R. M. Ray, and L. R. Johnson RhoA stimulates IEC-6 cell proliferation by increasing polyamine-dependent Cdk2 activity Am J Physiol Gastrointest Liver Physiol, October 1, 2003; 285(4): G704 - G713. [Abstract] [Full Text] [PDF]
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T. J. Dillon, V. Karpitski, S. A. Wetzel, D. C. Parker, A. S. Shaw, and P. J. S. Stork Ectopic B-Raf Expression Enhances Extracellular Signal-regulated Kinase (ERK) Signaling in T Cells and Prevents Antigen-presenting Cell-induced Anergy J. Biol. Chem., September 19, 2003; 278(38): 35940 - 35949. [Abstract] [Full Text] [PDF]
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M. J. Smit, P. Verdijk, E. M. H. van der Raaij-Helmer, M. Navis, P. J. Hensbergen, R. Leurs, and C. P. Tensen CXCR3-mediated chemotaxis of human T cells is regulated by a Gi- and phospholipase C-dependent pathway and not via activation of MEK/p44/p42 MAPK nor Akt/PI-3 kinase Blood, September 15, 2003; 102(6): 1959 - 1965. [Abstract] [Full Text] [PDF]
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L. L. Molinero, M. B. Fuertes, L. Fainboim, G. A. Rabinovich, and N. W. Zwirner Up-regulated expression of MICA on activated T lymphocytes involves Lck and Fyn kinases and signaling through MEK1/ERK, p38 MAP kinase, and calcineurin J. Leukoc. Biol., June 1, 2003; 73(6): 815 - 822. [Abstract] [Full Text] [PDF]
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 S. K. Sharma, C. M. Sherff, J. Shobe, M. W. Bagnall, M. A. Sutton, and T. J. Carew Differential Role of Mitogen-Activated Protein Kinase in Three Distinct Phases of Memory for Sensitization in Aplysia J. Neurosci., May 1, 2003; 23(9): 3899 - 3907. [Abstract] [Full Text] [PDF]
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S. K. Sharma, M. W. Bagnall, M. A. Sutton, and T. J. Carew Inhibition of calcineurin facilitates the induction of memory for sensitization in Aplysia: Requirement of mitogen-activated protein kinase PNAS, April 15, 2003; 100(8): 4861 - 4866. [Abstract] [Full Text] [PDF]
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P. Gupta and R. Prywes ATF1 Phosphorylation by the ERK MAPK Pathway Is Required for Epidermal Growth Factor-induced c-jun Expression J. Biol. Chem., December 20, 2002; 277(52): 50550 - 50556. [Abstract] [Full Text] [PDF]
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 E. M Fitzgerald The presence of ca2+ channel {beta} subunit is required for mitogen-activated protein kinase (mapk)-dependent modulation of {alpha}1b ca2+ channels in cos-7 cells J. Physiol., September 1, 2002; 543(2): 425 - 437. [Abstract] [Full Text] [PDF]
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 W.-H. Zhu, A. MacIntyre, and R. F. Nicosia Regulation of Angiogenesis by Vascular Endothelial Growth Factor and Angiopoietin-1 in the Rat Aorta Model : Distinct Temporal Patterns of Intracellular Signaling Correlate with Induction of Angiogenic Sprouting Am. J. Pathol., September 1, 2002; 161(3): 823 - 830. [Abstract] [Full Text] [PDF]
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 W. Wang, J. X. Chen, R. Liao, Q. Deng, J. J. Zhou, S. Huang, and P. Sun Sequential Activation of the MEK-Extracellular Signal-Regulated Kinase and MKK3/6-p38 Mitogen-Activated Protein Kinase Pathways Mediates Oncogenic ras-Induced Premature Senescence Mol. Cell. Biol., May 15, 2002; 22(10): 3389 - 3403. [Abstract] [Full Text] [PDF]
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 J. C. Selcher, E. J. Weeber, A. W. Varga, J. D. Sweatt, and M. Swank Book Review: Protein Kinase Signal Transduction Cascades in Mammalian Associative Conditioning Neuroscientist, April 1, 2002; 8(2): 122 - 131. [Abstract] [PDF]
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L. J. Appleman, A. A. F. L. van Puijenbroek, K. M. Shu, L. M. Nadler, and V. A. Boussiotis CD28 Costimulation Mediates Down-Regulation of p27kip1 and Cell Cycle Progression by Activation of the PI3K/PKB Signaling Pathway in Primary Human T Cells J. Immunol., March 15, 2002; 168(6): 2729 - 2736. [Abstract] [Full Text] [PDF]
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D. E. Kling, H. K. Lorenzo, A. M. Trbovich, T. B. Kinane, P. K. Donahoe, and J. J. Schnitzer Pre- and Postnatal Lung Development, Maturation, and Plasticity: MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs Am J Physiol Lung Cell Mol Physiol, March 1, 2002; 282(3): L370 - L378. [Abstract] [Full Text] [PDF]
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J. Adnane, E. Seijo, Z. Chen, F. Bizouarn, M. Leal, S. M. Sebti, and T. Munoz-Antonia RhoB, Not RhoA, Represses the Transcription of the Transforming Growth Factor beta Type II Receptor by a Mechanism Involving Activator Protein 1 J. Biol. Chem., March 1, 2002; 277(10): 8500 - 8507. [Abstract] [Full Text] [PDF]
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J. Dierov, Q. Xu, R. Dierova, and M. Carroll TEL/platelet-derived growth factor receptor beta activates phosphatidylinositol 3 (PI3) kinase and requires PI3 kinase to regulate the cell cycle Blood, March 1, 2002; 99(5): 1758 - 1765. [Abstract] [Full Text] [PDF]
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 M. Li, C. Torres, C. Acuna-Castillo, R. Walter, E. M. Gardner, D. M. Murasko, and F. Sierra Defect in ERK2 and p54JNK Activation in Aging Mouse Splenocytes J. Gerontol. A Biol. Sci. Med. Sci., February 1, 2002; 57(2): B41 - 47. [Abstract] [Full Text]
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Y. Zaffran, O. Destaing, A. Roux, S. Ory, T. Nheu, P. Jurdic, C. Rabourdin-Combe, and A. L. Astier CD46/CD3 Costimulation Induces Morphological Changes of Human T Cells and Activation of Vav, Rac, and Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase J. Immunol., December 15, 2001; 167(12): 6780 - 6785. [Abstract] [Full Text] [PDF]
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N. Kinkl, J. Sahel, and D. Hicks Alternate FGF2-ERK1/2 Signaling Pathways in Retinal Photoreceptor and Glial Cells in Vitro J. Biol. Chem., November 16, 2001; 276(47): 43871 - 43878. [Abstract] [Full Text] [PDF]
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S. Mariathasan, A. Zakarian, D. Bouchard, A. M. Michie, J. C. Zuniga-Pflucker, and P. S. Ohashi Duration and Strength of Extracellular Signal-Regulated Kinase Signals Are Altered During Positive Versus Negative Thymocyte Selection J. Immunol., November 1, 2001; 167(9): 4966 - 4973. [Abstract] [Full Text] [PDF]
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 J.-I. Park, H.-S. Choi, J.-S. Jeong, J.-Y. Han, and I.-H. Kim Involvement of p38 Kinase in Hydroxyurea-induced Differentiation of K562 Cells Cell Growth Differ., September 1, 2001; 12(9): 481 - 486. [Abstract] [Full Text] [PDF]
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N. Iida, K. Namikawa, H. Kiyama, H. Ueno, S. Nakamura, and S. Hattori Requirement of Ras for the Activation of Mitogen-Activated Protein Kinase by Calcium Influx, cAMP, and Neurotrophin in Hippocampal Neurons J. Neurosci., September 1, 2001; 21(17): 6459 - 6466. [Abstract] [Full Text] [PDF]
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S. Namura, K. Iihara, S. Takami, I. Nagata, H. Kikuchi, K. Matsushita, M. A. Moskowitz, J. V. Bonventre, and A. Alessandrini Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia PNAS, August 10, 2001; (2001) 181213498. [Abstract] [Full Text] [PDF]
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W. Li, C. D. Whaley, J. L. Bonnevier, A. Mondino, M. E. Martin, K. M. Aagaard-Tillery, and D. L. Mueller CD28 Signaling Augments Elk-1-Dependent Transcription at the c-fos Gene During Antigen Stimulation J. Immunol., July 15, 2001; 167(2): 827 - 835. [Abstract] [Full Text] [PDF]
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O. Planz, S. Pleschka, and S. Ludwig MEK-Specific Inhibitor U0126 Blocks Spread of Borna Disease Virus in Cultured Cells J. Virol., May 15, 2001; 75(10): 4871 - 4877. [Abstract] [Full Text]
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J. D. Richards, S. H. Dave, C.-H. G. Chou, A. A. Mamchak, and A. L. DeFranco Inhibition of the MEK/ERK Signaling Pathway Blocks a Subset of B Cell Responses to Antigen J. Immunol., March 15, 2001; 166(6): 3855 - 3864. [Abstract] [Full Text] [PDF]
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H. Sano, X. Zhu, A. Sano, E. E. Boetticher, T. Shioya, B. Jacobs, N. M. Munoz, and A. R. Leff Extracellular Signal-Regulated Kinase 1/2-Mediated Phosphorylation of Cytosolic Phospholipase A2 Is Essential for Human Eosinophil Adhesion to Fibronectin J. Immunol., March 1, 2001; 166(5): 3515 - 3521. [Abstract] [Full Text] [PDF]
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 C. C. Bancroft, Z. Chen, G. Dong, J. B. Sunwoo, N. Yeh, C. Park, and C. Van Waes Coexpression of Proangiogenic Factors IL-8 and VEGF by Human Head and Neck Squamous Cell Carcinoma Involves Coactivation by MEK-MAPK and IKK-NF-{{kappa}}B Signal Pathways Clin. Cancer Res., February 1, 2001; 7(2): 435 - 442. [Abstract] [Full Text]
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P. Bostik, A. E. Mayne, F. Villinger, K. P. Greenberg, J. D. Powell, and A. A. Ansari Relative Resistance in the Development of T Cell Anergy in CD4+ T Cells from Simian Immunodeficiency Virus Disease-Resistant Sooty Mangabeys J. Immunol., January 1, 2001; 166(1): 506 - 516. [Abstract] [Full Text] [PDF]
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Y. Yi, M. McNerney, and S. K. Datta Regulatory Defects in Cbl and Mitogen-Activated Protein Kinase (Extracellular Signal-Related Kinase) Pathways Cause Persistent Hyperexpression of CD40 Ligand in Human Lupus T Cells J. Immunol., December 1, 2000; 165(11): 6627 - 6634. [Abstract] [Full Text] [PDF]
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E M Fitzgerald Regulation of voltage-dependent calcium channels in rat sensory neurones involves a Ras-mitogen-activated protein kinase pathway J. Physiol., September 15, 2000; 527(3): 433 - 444. [Abstract] [Full Text] [PDF]
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C. W. M. Reuter, M. A. Morgan, and L. Bergmann Targeting the Ras signaling pathway: a rational, mechanism-based treatment for hematologic malignancies? Blood, September 1, 2000; 96(5): 1655 - 1669. [Abstract] [Full Text] [PDF]
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T.-K. Yu, E. G. Caudell, C. Smid, and E. A. Grimm IL-2 Activation of NK Cells: Involvement of MKK1/2/ERK But Not p38 Kinase Pathway J. Immunol., June 15, 2000; 164(12): 6244 - 6251. [Abstract] [Full Text] [PDF]
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O. Witt, K. Sand, and A. Pekrun Butyrate-induced erythroid differentiation of human K562 leukemia cells involves inhibition of ERK and activation of p38 MAP kinase pathways Blood, April 1, 2000; 95(7): 2391 - 2396. [Abstract] [Full Text] [PDF]
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D. Zella, F. Romerio, S. Curreli, P. Secchiero, C. Cicala, D. Zagury, and R. C. Gallo IFN-{alpha}2b Reduces IL-2 Production and IL-2 Receptor Function in Primary CD4+ T Cells J. Immunol., March 1, 2000; 164(5): 2296 - 2302. [Abstract] [Full Text] [PDF]
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S. Murakami, M. Kan, W. L. McKeehan, and B. de Crombrugghe Up-regulation of the chondrogenic Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway PNAS, February 1, 2000; 97(3): 1113 - 1118. [Abstract] [Full Text] [PDF]
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 I.-H. Lee, W. P. Li, K. B. Hisert, and L. B. Ivashkiv Inhibition of Interleukin 2 Signaling and Signal Transducer and Activator of Transcription (STAT)5 Activation during T Cell Receptor-mediated Feedback Inhibition of T Cell Expansion J. Exp. Med., November 1, 1999; 190(9): 1263 - 1274. [Abstract] [Full Text] [PDF]
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I. P. Whitehead, Q. T. Lambert, J. A. Glaven, K. Abe, K. L. Rossman, G. M. Mahon, J. M. Trzaskos, R. Kay, S. L. Campbell, and C. J. Der Dependence of Dbl and Dbs Transformation on MEK and NF-kappa B Activation Mol. Cell. Biol., November 1, 1999; 19(11): 7759 - 7770. [Abstract] [Full Text] [PDF]
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H. Shao, B. Wilkinson, B. Lee, P.-C. Han, and J. Kaye Slow Accumulation of Active Mitogen-Activated Protein Kinase During Thymocyte Differentiation Regulates the Temporal Pattern of Transcription Factor Gene Expression J. Immunol., July 15, 1999; 163(2): 603 - 610. [Abstract] [Full Text] [PDF]
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M. Saxena, S. Williams, J. Brockdorff, J. Gilman, and T. Mustelin Inhibition of T Cell Signaling by Mitogen-activated Protein Kinase-targeted Hematopoietic Tyrosine Phosphatase (HePTP) J. Biol. Chem., April 23, 1999; 274(17): 11693 - 11700. [Abstract] [Full Text] [PDF]
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S. J. Kempiak, T. S. Hiura, and A. E. Nel The Jun Kinase Cascade Is Responsible for Activating the CD28 Response Element of the IL-2 Promoter: Proof of Cross-Talk with the I{kappa}B Kinase Cascade J. Immunol., March 15, 1999; 162(6): 3176 - 3187. [Abstract] [Full Text] [PDF]
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A. M. Domina, J. H. Smith, and R. W. Craig Myeloid Cell Leukemia 1 Is Phosphorylated through Two Distinct Pathways, One Associated with Extracellular Signal-regulated Kinase Activation and the Other with G2/M Accumulation or Protein Phosphatase 1/2A Inhibition J. Biol. Chem., July 7, 2000; 275(28): 21688 - 21694. [Abstract] [Full Text] [PDF]
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K. M. F. Khan, D. J. Falcone, and R. Kraemer Nerve Growth Factor Activation of Erk-1 and Erk-2 Induces Matrix Metalloproteinase-9 Expression in Vascular Smooth Muscle Cells J. Biol. Chem., January 11, 2002; 277(3): 2353 - 2359. [Abstract] [Full Text] [PDF]
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S. Namura, K. Iihara, S. Takami, I. Nagata, H. Kikuchi, K. Matsushita, M. A. Moskowitz, J. V. Bonventre, and A. Alessandrini Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia PNAS, September 25, 2001; 98(20): 11569 - 11574. [Abstract] [Full Text] [PDF]
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