U.S. patent application number 10/193657 was filed with the patent office on 2003-07-10 for regulation of cytokine production in a hematopoietic cell.
This patent application is currently assigned to National Jewish Center for Immunology and Respiratory Medicine. Invention is credited to Gelfand, Erwin W., Johnson, Gary L..
Application Number | 20030129752 10/193657 |
Document ID | / |
Family ID | 24633586 |
Filed Date | 2003-07-10 |
United States Patent
Application |
20030129752 |
Kind Code |
A1 |
Gelfand, Erwin W. ; et
al. |
July 10, 2003 |
Regulation of cytokine production in a hematopoietic cell
Abstract
A method useful for regulating cytokine production by a
hematopoietic cell by regulating an MEKK/JNKK-contingent signal
transduction pathway in such a cell is disclosed. Methods of
identifying compounds capable of specifically regulating an
MEKK/JNKK-contingent signal transduction pathway in hematopoietic
cells, a kit for identifying cytokine regulators, methods to treat
diseases involving cytokine production, and cells useful in such
methods are also set forth.
Inventors: |
Gelfand, Erwin W.;
(Englewood, CO) ; Johnson, Gary L.; (Boulder,
CO) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
National Jewish Center for
Immunology and Respiratory Medicine
|
Family ID: |
24633586 |
Appl. No.: |
10/193657 |
Filed: |
July 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10193657 |
Jul 10, 2002 |
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09305720 |
May 5, 1999 |
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6495331 |
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09305720 |
May 5, 1999 |
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08656563 |
May 31, 1996 |
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5910417 |
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Current U.S.
Class: |
435/455 ;
424/85.2; 435/372 |
Current CPC
Class: |
C12N 15/85 20130101;
C07K 14/4702 20130101; C12N 2830/00 20130101; C12N 5/0642 20130101;
G01N 33/6863 20130101; C12N 2830/85 20130101; C07K 16/40 20130101;
C12N 2501/70 20130101; A61K 31/352 20130101; C12N 2830/001
20130101 |
Class at
Publication: |
435/455 ;
435/372; 424/85.2 |
International
Class: |
C12N 005/08; C12N
015/85; A61K 038/20 |
Goverment Interests
[0001] This invention was made in part with government support
under: AI HL-36577 and DK-37871, each awarded by the National
Institutes of Health. The government has certain rights to this
invention.
Claims
What is claimed is:
1. A method to regulate cytokine production, comprising regulating
an MEKK/JNKK-contingent signal transduction pathway in a
hematopoietic cell to effect regulation of cytokine production in
said cell.
2. The method of claim 1, wherein said cytokine is selected from
the group consisting of TNF-a, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,
G-CSF, GM-CSF, TNF-b, TGF-b, IFN-.gamma., and
IFN-.alpha./.beta..
3. The method of claim 1, wherein said hematopoietic cell is
selected from the group consisting of a mast cell, a basophil, an
eosinophil, a neutrophil, a T cell, a B cell, a macrophage, a
dendritic cell, and a natural killer cell.
4. The method of claim 1, wherein said hematopoietic cell expresses
FceRI.
5. The method of claim 1, wherein said step of regulating said
MEKK/JNKK-contingent signal transduction pathway comprises
regulating a signal transduction molecule selected from the group
consisting of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1, and JNK2.
6. The method of claim 5, wherein said step of regulating a signal
transduction molecule comprises a method selected from the group
consisting of degrading said molecule, binding a regulatory
compound to said molecule, inhibiting transcription of said
molecule, inhibiting translation of said molecule, inhibiting
activation of said molecule, and inhibiting the interaction of said
molecule with another signal transduction molecule.
7. The method of claim 5, wherein said step of regulating a signal
transduction molecule results in modulation of the interaction of a
transcription factor selected from the group consisting of NF-AT,
AP-1, Jun, Fos, ATF-2, NF.kappa.B, and CBP, with a cytokine
promoter.
8. The method of claim 5, wherein said MEKK/JNKK-contingent signal
transduction pathway is regulated by contacting said cell with an
effective amount of a compound that interacts with said
molecule.
9. The method of claim 1, wherein said cytokine production is
inhibited.
10. The method of claim 1, wherein said MEKK/JNKK-contingent signal
transduction pathway is activated by a PI3-K signal transduction
pathway.
11. The method of claim 10, wherein said step of regulating said
MEKK/JNKK-contingent signal transduction pathway comprises
regulating PI3-K.
12. The method of claim 1, wherein said MEKK/JNKK-contingent signal
transduction pathway is activated by aggregation of FceRI on said
cell.
13. The method of claim 12, wherein said method further comprises
regulating a signal transduction pathway selected from the group
consisting of c-kit signal transduction pathway and p38 signal
transduction pathway.
14. The method of claim 12, wherein said regulation of cytokine
production does not entail regulation of the ERK signal
transduction pathway.
15. A method to regulate cytokine production in a hematopoietic
cell expressing FceRI, comprising regulating an
MEKK/JNKK-contingent signal transduction pathway in said cell to
effect regulation of cytokine production in said cell, said
MEKK/JNKK-contingent signal transduction pathway being activated by
the PI3-K signal transduction pathway of said cell.
16. The method of claim 15, wherein said cytokine is selected from
the group consisting of TNF-.alpha., IL-1, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,
IL-16, G-CSF, GM-CSF, TNF-b, TGF-b, IFN-.gamma., and
IFN-.alpha./.beta..
17. The method of claim 15, wherein said hematopoietic cell is
selected from the group consisting of a mast cell, a basophil and
an eosinophil.
18. The method of claim 15, wherein said cytokine production is
inhibited.
19. The method of claim 15, wherein said step of regulating said
MEKK/JNKK-contingent signal transduction pathway comprises
administering to said cell an effective amount of a compound that
interacts with a signal transduction molecule selected from the
group consisting of PI3-K, MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1,
and JNK2, wherein said molecule modulates the interaction of a
transcription factor selected from the group consisting of NF-AT,
AP-1, Jun, Fos, ATF-2, NF.kappa.B, and CBP, with a cytokine
promoter.
20. A method to regulate signal transduction pathways involved in
cytokine production in a hematopoietic cell, comprising: (a)
providing a hematopoietic cell having an MEKK/JNKK-contingent
signal transduction pathway; (b) regulating signal transduction in
said pathway by inhibiting the interactions between molecules in
said pathway, said molecules selected from the group consisting of
MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1, and JNK2, wherein said step
of regulating inhibits the production of cytokines by said
cell.
21. The method of claim 20, further comprising regulating other
signal transduction pathways that regulate an MEKK/JNKK-contingent
signal transduction pathway.
22. The method of claim 20, further comprising regulating c-kit and
p38 pathways to affect the regulation of cytokine production.
23. The method of claim 20, wherein said step of inhibiting
comprises administering to said cell an effective amount of a
compound which modulates interactions between said molecules.
24. The method of claim 23, wherein said compound modulates
interactions between said molecules by a method selected from the
group consisting of degrading said molecule, binding to said
molecule such that the function of said molecule is inhibited,
inhibiting transcription of said molecule, inhibiting translation
of said molecule, and inhibiting the interaction of said molecule
with another signal transduction molecule.
25. The method of claim 20, wherein said hematopoietic cell is
selected from the group consisting of a mast cell, a basophil, an
eosinophil, a neutrophil, a T cell, a B cell, a macrophage, a
dendritic cell, and a natural killer cell.
26. The method of claim 20, wherein said cytokine is selected from
the group consisting of TNF-a, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,
G-CSF, GM-CSF, TNF-b, TGF-b, IFN-.gamma., and
IFN-.alpha./.beta..
27. The method of claim 20, wherein said cytokine production is
inhibited.
28. The method of claim 20, wherein said MEKK/JNKK-contingent
signal transduction pathway is activated by a PI3-K signal
transduction pathway in said cell.
29. The method of claim 20, wherein said step of regulating signal
transduction comprises inhibiting the interactions between said
molecules and PI3-K.
30. The method of claim 20, wherein said MEKK/JNKK-contingent
signal transduction pathway is activated by aggregation of FceRI on
said cell.
31. A method to identify compounds capable of regulating cytokine
production in a hematopoietic cell, comprising: (a) providing a
hematopoietic cell having an MEKK/JNKK-contingent signal
transduction pathway; (b) contacting a putative regulatory compound
with said cell; and (c) determining whether said putative
regulatory compound is capable of regulating said
MEKK/JNKK-contingent signal transduction pathway to affect cytokine
production by said cell. (d)
32. The method of claim 31, wherein said hematopoietic cell is
selected from the group consisting of a mast cell, a basophil, an
eosinophil, a neutrophil, a T cell, a B cell, a macrophage, a
dendritic cell, and a natural killer cell.
33. The method of claim 31, wherein said cytokine is selected from
the group consisting of TNF-a, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,
G-CSF, GM-CSF, TNF-b, TGF-b, IFN-.gamma., and
IFN-.alpha./.beta..
34. The method of claim 31, wherein said cytokine production is
inhibited.
35. The method of claim 31, wherein said regulatory compound
regulates said MEKK/JNKK-contingent signal transduction pathway by
modulating the interaction between signal transduction molecules
selected from the group consisting of MEKK1, MEKK2, MEKK3, MEKK4,
JNKK, JNK1, and JNK2.
36. The method of claim 35, wherein step of modulating the
interaction between signal transduction molecules regulates the
interaction of a transcription factor selected from the group
consisting of NF-AT, AP-1, Jun, Fos, ATF-2, NF.kappa.B, and CBP,
with a cytokine promoter.
37. The method of claim 35, wherein said regulatory compound
modulates the interaction of said molecules with PI3-K.
38. A kit to identify compounds capable of regulating cytokine
production in a hematopoietic cell comprising: (a) a hematopoietic
cell having an MEKK/JNKK-contingent signal transduction pathway,
said cell producing an amount of at least one cytokine, wherein
production of said cytokine by said cell is dependent upon said
NEKK/JNKK-contingent signal transduction pathway; and (b) a means
for determining a change in the amount of cytokine produced by said
cell after said cell is contacted with a putative regulatory
compound.
39. The kit of claim 38, wherein said cell expresses FceRI and said
MEKK/JNKK-contingent signal transduction pathway is activated by
aggregation of said FceRI.
40. The kit of claim 38, wherein said hematopoietic cell is
selected from the group consisting of a mast cell, a basophil, an
eosinophil, a neutrophil, a T cell, a B cell, a macrophage, a
dendritic cell, and a natural killer cell.
41. The kit of claim 38, wherein said means for determining a
change in cytokine production is an assay selected from the group
consisting of an immunoassay, a transcription assay and a
biological assay which detects levels of cytokine production by
said cell.
42. The kit of claim 41, wherein said immunoassay is selected from
the group consisting of an enzyme-linked immunoassay, a
radioimmunoassay, a fluorescence immunoassay, and an immunoblot
assay.
43. The kit of claim 41, wherein said immunoassay is an antibody
capture enzyme-linked immunoassay.
44. The kit of claim 41, wherein said transcription assay is a
PCR-based assay for cytokine mRNA production.
45. The kit of claim 41, wherein said biological assay comprises
the use of a cell line which is responsive to a cytokine selected
from the group consisting of TNF-a, IL-1, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,
IL-16, G-CSF, GM-CSF, TNF-b, TGF-b, IFN-.gamma., and
IFN-.alpha./.beta..
46. The kit of claim 38, wherein said cell comprises heterologous
mammalian nucleic acid sequences encoding proteins involved in
MEKK/JNKK-contingent signal transduction.
47. The kit of claim 38, wherein said cell comprises heterologous
mammalian nucleic acid sequences encoding proteins involved in
signal transduction through FceRI.
48. A method to treat a disease involving cytokine production in an
animal, comprising regulating an MEKK/JNKK-contingent signal
transduction pathway to affect cytokine production by a
hematopoietic cell.
49. The method of claim 48, wherein said disease is selected from
the group consisting of allergic diseases, anaphylaxis, diseases
involving defects in hematopoietic cells, inflammation, mast cell
disorders, sepsis and cancer.
50. The method of claim 48, wherein said disease is allergic
inflammation.
51. The method of claim 48, wherein said step of regulating
comprises administering to said animal an effective amount of a
compound which interacts with a signal transduction molecule in
said NEKK/JNKK-contingent signal transduction pathway selected from
the group consisting of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 and
JNK2, said interaction resulting in the regulation of cytokine
production by a hematopoietic cell in said animal.
52. The method of claim 51, wherein said compound is selected from
the group consisting of an MEKK inhibitor, a JNKK inhibitor, and a
JNK inhibitor.
53. The method of claim 48, wherein said step of regulating
comprises administering to said animal an effective amount of a
compound which inhibits PI3-K.
54. A method to treat allergic inflammation in an animal,
comprising regulating cytokine production in a hematopoietic cell
in said animal, said cell expressing FceRI, wherein said step of
regulating comprises regulating an MEKK/JNKK-contingent signal
transduction pathway.
55. The method of claim 54, wherein said cell is selected from the
group consisting of a mast cell, a basophil and an eosinophil.
56. The method of claim 54, wherein said cytokine production is
inhibited.
57. The method of claim 54, wherein said step of regulating
MEKK/JNKK-contingent signal transduction pathway comprises
administering to said animal an effective amount of a compound
which interacts with a signal transduction molecule in said
MEKK/JNKK-contingent signal transduction pathway, said interaction
resulting in the regulation of cytokine production by said
hematopoietic cell in said animal.
58. The method of claim 54, wherein said animal is a mammal.
59. The method of claim 54, wherein said animal is a human.
60. A compound for regulating cytokine production in a
hematopoietic cell, wherein said compound interacts with a signal
transduction molecule in an MEKK/JNKK-contingent signal
transduction pathway, said molecule selected from the group
consisting of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2.
61. The compound of claim 60, wherein said compound does not
regulate an ERK-dependent signal transduction pathway.
62. A compound for regulating cytokine production in a
hematopoietic cell identified by the method comprising: (a)
providing a hematopoietic cell having an MEKK/JNKK-contingent
signal transduction pathway, wherein production of cytokines by
said cell is dependent solely on said pathway; (b) contacting said
compound with said cell; and (c) identifying said compound by its
ability to regulate said MEKK/JNKK-contingent signal transduction
pathway.
63. The compound of claim 62, wherein said method further comprises
determining the ability of said compound to inhibit cytokine
production in said cell.
64. A cell used in a method to identify compounds capable of
regulating cytokine production, comprising a cell having at least
one heterologous mammalian nucleic acid sequence encoding at least
one protein involved in an MEKK/JNKK-contingent signal transduction
pathway.
65. The cell of claim 64, wherein said cell is capable of producing
cytokines and production of said cytokines is regulated by said
MEKK/JNKK-contingent signal transduction pathway.
66. A method of using a hematopoietic cell to screen putative
regulatory compounds for their ability to regulate cytokine
production in said cell comprising: (a) providing a hematopoietic
cell having an MEKK/JNKK-contingent signal transduction pathway;
(b) contacting said cell with a putative regulatory compound; and
(c) determining whether said putative regulatory compound is
capable of modulating the amount of cytokine produced by said
cell.
67. The method of claim 66, wherein said cell is selected from the
group consisting of a mast cell, a basophil, an eosinophil, a
neutrophil, a T cell, a B cell, a macrophage, a dendritic cell, and
a natural killer cell.
68. The method of claim 66, said cell having at least one
heterologous mammalian nucleic acid sequence encoding at least one
protein involved in an MEKK/JNKK-contingent signal transduction
pathway.
69. The method of claim 66, wherein said cell lacks an
ERK-dependent signal transduction pathway.
Description
FIELD OF THE INVENTION
[0002] The present invention relates to a method for regulating an
MEKK/JNKK-contingent signal transduction pathway in a hematopoietic
cell in order to regulate cytokine production by such cell.
BACKGROUND OF THE INVENTION
[0003] Aggregation of the high-affinity Fc receptors for
immunoglobulin E (IgE) (Fc.epsilon.RI) on the surface of mast cells
initiates intracellular signal transduction pathways, involving the
tyrosine phosphorylation of cellular proteins, activation of
phospholipase C.gamma., hydrolysis of phosphoinositide, increase in
intracellular calcium, activation of protein kinase C and the
stimulation of phosphatidylinositol 3-kinase. These signal
transduction pathways are believed to be involved in the exocytic
release of inflammatory mediators such as vasoactive amines,
cytokines, and lipid metabolites. The production of cytokines by
mast cells is a critical event that influences the pathogenesis of
allergic inflammation in asthma and other allergic disorders.
[0004] In addition to the activation of phospholipase C.gamma. and
protein kinase C, which appears to be essential for the
Fc.epsilon.RI-mediated release of preformed mediators, the
aggregation of Fc.epsilon.RI on rat basophilic leukemia 2H3
(RBL-2H3) cells has been shown to induce histamine and leukotriene
release. Except for the activation of the extracellular
signal-regulated kinases/mitogen activated protein kinases
(ERKs/MAPKs), however, the downstream consequences of early
activation events in a signal transduction pathway leading to
cytokine production are not well defined.
[0005] The extracellular signal-regulated kinases (ERKs), ERK1 and
ERK2, are serine/threonine protein kinases that are activated
through concomitant phosphorylation of tyrosine and threonine
residues. Prior to the current invention, it was thought that ERKs
were one of the intermediates in the signal transduction pathway
leading to increases in gene transcription and proliferation,
including cytokine gene transcription. ERKs phosphorylate specific
transcription factors including members of the Ets family, such as
Elk-1, and it has been reported that ERKs are activated via
Fc.epsilon.RI on mast cells.
[0006] Despite the current understanding of early signal
transduction events in hematopoietic cells, there remains a need to
elucidate signal transduction pathways that specifically regulate
cytokine production in such cells and to determine what molecules
and/or functional elements of such molecules are responsible for
regulating such cellular pathways. There is also a need for
products and processes that permit the effective regulation of
specific steps in such a signal transduction pathway. Regulation of
specific steps of a signal transduction pathway which regulate
cytokine production permits the implementation of predictable
controls of such signal transduction in cells, thereby allowing
modulation of the effects of cytokine production in diseases
wherein such modulation can ameliorate disease pathogenesis.
SUMMARY OF THE INVENTION
[0007] The present invention generally relates to a method to
regulate a novel signal transduction pathway to modulate the
production of cytokines by a hematopoietic cell. The present
inventors have identified an MEKK/JNKK-contingent signal
transduction pathway which regulates the production of cytokines by
a hematopoietic cell. Prior to the present invention, it was
thought that signal transduction through the ERK pathway lead to
increases in gene transcription and proliferation, including
cytokine gene transcription. The ERK pathway is known to be
distinct from the pathway of the present invention; therefore, the
discovery that an ERK-independent signal transduction pathway
regulates cytokine production is unexpected. The present inventors
were the first to appreciate that an MEKK/JNKK-contingent signal
transduction pathway, and not an ERK-dependent pathway, regulates
cytokine production. Furthermore, the present inventors were the
first to appreciate that such an MEKK/JNKK-contingent pathway is
activated in mast cells through aggregation FceRI and activation of
PI3-kinase (PI3-K). The present inventors were also the first to
appreciate the method of regulation of such a signal transduction
pathway in order to regulate production of cytokines such as,
TNF-a, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, G-CSF, GM-CSF, TNF-b,
TGF-b, IFN-.gamma., and IFN-.alpha./.beta., in a hematopoietic cell
such as a mast cell, a basophil, an eosinophil, a neutrophil, a T
cell, a B cell, a macrophage, a dendritic cell, and a natural
killer cell.
[0008] One embodiment of the present invention relates to a method
to regulate cytokine production by regulating an
MEKK/JNKK-contingent signal transduction pathway in a hematopoietic
cell. Preferably, such a method comprises regulating one or more of
the signal transduction molecule selected from the group of MEKK1,
MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2.
[0009] In one embodiment, an MEKK/JNKK-contingent signal
transduction pathway can be regulated by administration of a
compound which regulates a signal transduction molecule selected
from the group of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2,
such that cytokine production is regulated. Preferably, such a
compound regulates such a signal transduction molecule by a method
such as degrading the molecule, binding an inhibitory compound to
the molecule, inhibiting transcription of the molecule, inhibiting
translation of the molecule, and inhibiting the interaction of the
molecule with another signal transduction molecule.
[0010] A preferred embodiment of the present invention relates to a
method to regulate cytokine production in a hematopoietic cell
expressing FceRI by regulating an MEKK/JNKK-contingent signal
transduction pathway in such cell. Regulation of an
MEKK/JNKK-contingent signal transduction pathway can further
comprise regulating other signal transduction pathways that affect
the MEKK/JNKK-contingent signal transduction pathway.
[0011] Another embodiment of the present invention relates to a
method to identify compounds which regulate cytokine production in
a hematopoietic cell. Such a method comprises contacting a cell
with a putative regulatory compound and determining whether such a
compound is capable of regulating cytokine production in a cell by
regulating an MEKK/JNKK-contingent signal transduction pathway in
the cell.
[0012] Yet another embodiment of the present invention relates to a
kit for identifying compounds which regulate cytokine production by
regulating an MEKK/JNKK-contingent signal transduction pathway.
[0013] Another embodiment of the present invention relates to a
method to treat a disease involving cytokine production in an
animal by regulating an MEKK/JNKK-contingent signal transduction
pathway. In one embodiment, such a treatment involves administering
to an animal an effective amount of a compound which interacts with
a signal transduction molecule in an MEKK/JNKK-contingent signal
transduction pathway such that cytokine production is
regulated.
[0014] A preferred embodiment of the present invention relates to a
method to treat allergic inflammation by regulating cytokine
production. Such regulation of cytokine production is effected by
regulation of an MEKK/JNKK-contingent signal transduction
pathway.
[0015] Yet another embodiment of the present invention relates to a
compound for regulating cytokine production. Such a compound
interacts with a signal transduction molecule in an
MEKK/JNKK-contingent signal transduction pathway in a manner
effective to regulate cytokine production.
[0016] Another embodiment of the present invention relates to a
cell used in a method to identify compounds capable of regulating
cytokine production, comprising a cell having at least one
heterologous mammalian nucleic acid sequence encoding at least one
protein involved in an MEKK/JNKK-contingent signal transduction
pathway. A preferred embodiment relates to a method of using such a
cell to screen putative regulatory compounds for their ability to
regulate cytokine production in said cell.
DESCRIPTION OF THE FIGURES
[0017] FIG. 1 schematically illustrates an MEKK/JNKK-contingent
signal transduction pathway of the present invention.
[0018] FIG. 2A demonstrates activation of JNK by OVA and OVA-IgE in
passively sensitized mast cells.
[0019] FIG. 2B shows an immunoblot demonstrating JNK activity
measured in passively sensitized mast cells over time.
[0020] FIG. 2C graphically shows fold-increases in JNK activity in
passively sensitized mast cells.
[0021] FIG. 2D shows an immunoblot demonstrating the
antigen-specificity of JNK activation in passively sensitized mast
cells.
[0022] FIG. 3A shows an immunoblot demonstrating the activation of
MEKK1 by antigen in passively sensitized mast cells.
[0023] FIG. 3B graphically shows fold-increases in MEKK1 activation
by antigen in passively sensitized mast cells.
[0024] FIG. 4A shows an immunoblot demonstrating the activation of
ERK2 by antigen in passively sensitized mast cells.
[0025] FIG. 4B shows an immunoblot demonstrating ERK2
phosphorylation measured in passively sensitized mast cells over
time.
[0026] FIG. 4C shows an immunoblot demonstrating ERK2 kinase
activity measured in passively sensitized mast cells for up to 90
minutes.
[0027] FIG. 5A shows an immunoblot demonstrating that wortmannin
inhibits JNK activation by antigen in mast cells.
[0028] FIG. 5B demonstrates that a decrease in JNK activity
correlates with increased wortmannin concentration.
[0029] FIG. 5C shows that wortmannin does not inhibit ERK2
activity.
[0030] FIG. 6 demonstrates that TNF-a is produced in response to
antigen by passively sensitized mast cells.
[0031] FIG. 7 shows the fold-increase in TNF-a production over time
in response to antigen activation of passively sensitized mast
cells.
[0032] FIG. 8 demonstrates that p38 MAP kinase is activated in
response to antigen activation of passively sensitized mast
cells.
[0033] FIG. 9 shows that wortmannin inhibits p38 MAP kinase
activation in activated MC/9 cells.
[0034] FIG. 10 demonstrates that wortmannin inhibits TNF-a
production by activated MC/9 mast cells.
[0035] FIG. 11 shows that an MEK inhibitor, PD 098059, inhibits
activation of ERK2 in activated MC/9 cells.
[0036] FIG. 12 shows that MEK inhibitor, PD 098059, does not
inhibit TNF-.alpha. production in MC/9 cells.
[0037] FIG. 13 demonstrates that the PI3-K inhibitor, wortmannin,
inhibits TNF-.alpha. promoter activity in MC/9 cells stimulated by
Fc.epsilon.RI aggregation.
[0038] FIG. 14 shows that MEK inhibitor, PD 098059, enhances
TNF-.alpha. promoter activity in MC/9 cells stimulated by
Fc.epsilon.RI aggregation.
[0039] FIG. 15 demonstrates that overexpression of MEKK1 greatly
enhances JNK activity in antigen-activated MC/9 cells.
[0040] FIG. 16 shows that overexpression of MEKK1 weakly enhances
p38 activity in antigen-activated MC/9 cells.
[0041] FIG. 17 demonstrates that overexpression of MEKK1 greatly
enhances TNF-.alpha. promoter activity in antigen-activated MC/9
cells. FIG. 18 demonstrates that cross-linking of c-kit on MC/9
cells synergizes with aggregation of FceRI to greatly enhance JNK
activation.
[0042] FIG. 19 demonstrates that cross-linking of c-kit on MC/9
cells synergizes with aggregation of FceRI to greatly enhance TNF-a
production.
[0043] FIG. 20 shows that aggregation of FceRI on MC/9 cells
activates the transcription factor, NFkB.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention relates to the elucidation of a novel
signal transduction pathway that regulates cytokine production in
hematopoietic cells and a method to target such a pathway to
regulate cytokine production by such cells. The regulation of
cytokine production in a hematopoietic cell is useful since
cytokines are known to play a critical role in the pathogenesis of
many diseases. In particular, regulation of cytokine production in
a cell expressing FceRI, such as a mast cell, is useful for
treating diseases involving allergic inflammation. Specifically,
the present invention relates to the regulation of an
MEKK/JNKK-contingent signal transduction pathway, in order to
regulate cytokine production in a hematopoietic cell.
[0045] Prior to the present invention, it was thought that signal
transduction through the ERK pathway lead to increases in gene
transcription and proliferation, including cytokine gene
transcription. Since the ERK pathway is known to be distinct from
the pathway of the present invention, the present discovery that an
ERK-independent signal transduction pathway regulates cytokine
production is unexpected and surprising.
[0046] As used herein, the phrase "signal transduction pathway"
refers to at least one biochemical reaction, but more commonly a
series of biochemical reactions, which result from interaction of a
cell with a stimulatory compound. Thus, the interaction of a
stipulatory compound with a cell generates a "signal" that is
transmitted through the signal transduction pathway, ultimately
resulting in a cellular response.
[0047] A signal transduction pathway of the present invention can
include a variety of signal transduction molecules that play a role
in the transmission of a signal from one portion of a cell to
another portion of a cell. Signal transduction molecules of the
present invention include, for example, cell surface receptors and
intracellular signal transduction molecules. As used herein, the
phrase "cell surface receptor" includes molecules and complexes of
molecules capable of receiving a signal and the transmission of
such a signal across the plasma membrane of a cell. An example of a
"cell surface receptor" of the present invention is FceRI. As used
herein, the phrase "intracellular signal transduction molecule"
includes those molecules or complexes of molecules involved in
transmitting a signal from the plasma membrane of a cell through
the cytoplasm of the cell, and in some instances, into the cell's
nucleus. In the present invention, MEKK1, MEKK2, MEKK3, MEKK4,
JNKK, JNK1 and JNK2 are "intracellular signal transduction
molecules", but can also be referred to as "signal transduction
molecules".
[0048] A signal transduction pathway in a cell can be initiated by
interaction of a cell with a stimulator that is inside or outside
of the cell. If an exterior (i.e. outside of the cell) stimulator
interacts with a cell surface receptor, a signal transduction
pathway can transmit a signal across the cell's membrane, through
the cytoplasm of the cell, and in some instances into the nucleus.
If an interior (e.g. inside the cell) stimulator interacts with an
intracellular signal transduction molecule, a signal transduction
pathway can result in transmission of a signal through the cell's
cytoplasm, and in some instances into the cell's nucleus.
[0049] As used herein, the term "molecule" refers to a protein, a
lipid, a nucleic acid or an ion, and at times is used
interchangeably with such terms. In particular, a signal
transduction molecule refers to a protein, a lipid, a nucleotide,
or an ion involved in a signal transduction pathway.
[0050] A signal transduction molecule of the present invention can
regulate the activity of proteins involved in the transcription of
genes involved in cell growth within the nucleus of a cell, in
particular, cytokine genes, thereby altering the biological
function of a cell.
[0051] Signal transduction can occur through: the phosphorylation
of a molecule; non-covalent allosteric interactions; complexing of
molecules; the conformational change of a molecule; calcium
release; inositol phosphate production; proteolytic cleavage;
cyclic nucleotide production and diacylglyceride production.
Preferably, signal transduction occurs through phosphorylating a
signal transduction molecule including, MEKK1, MEKK2, MEKK3, MEKK4,
JNKK, JNK1 and JNK2. According to the present invention, all the
signal transduction molecules of the MEKK/JNKK-contingent signal
transduction pathway need not be known in order to successfully
utilize the methods of the present invention.
[0052] According to the present invention, an MEKK/JNKK-contingent
signal transduction pathway refers generally to a pathway in which
MEKK protein regulates a pathway that includes JNKK, molecules
which are active between MEKK and JNKK in the pathway, and
molecules downstream of JNKK, such as JNK1, JNK2, NF-AT, AP-1, Jun,
Fos, ATF-2, NF.kappa.B, and CBP. An MEKK/JNKK-contingent signal
transduction pathway is independent of an ERK-dependent signal
transduction pathway downstream from the effects of MEKK. In other
words, the regulation of the MEKK/JNKK-contingent signal
transduction pathway does not, of necessity, also affect signal
transduction downstream from the ERK protein. A suitable
MEKK/JNKK-contingent signal transduction pathway includes a pathway
involving an MEKK.backslash.JNKK-contingent signal transduction
molecule, including MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 and
JNK2, but not ERK molecules exclusively involved in an
ERK-dependent signal transduction pathway. More particularly, an
MEKK.backslash.JNKK-contingent molecule regulates a pathway that is
substantially independent of an ERK-dependent pathway if the
MEKK/JNKK-contingent protein induces phosphorylation of JNKK or a
member of the pathway downstream of JNKK (e.g., proteins including
JNK1, JNK2, NF-AT, AP-1, Jun, Fos, ATF-2, NF.kappa.B, and/or CBP),
and induces cytokine production in a cell having such a pathway. A
schematic representation of the proposed signal transduction
pathway of the present invention is shown in FIG. 1.
[0053] As a result of the elucidation by the present inventors of
the novel function of MEKK/JNKK-contingent signal transduction
pathway (i.e. regulation of cytokine production), one of skill in
the art can determine that regulation of such a pathway by an
MEKK/JNKK-contingent molecule is substantially independent of an
ERK-pathway. One can also determine how an MEKK/JNKK-contingent
molecule regulates the phosphorylation of a downstream member of an
MEKK/JNKK-contingent pathway or, alternatively, how to regulate
cytokine production in a cell through the inhibition and/or
stimulation of the MEKK/JNKK-contingent pathway.
[0054] An "ERK-dependent pathway" can refer to a signal
transduction pathway in which ERK protein regulates a signal
transduction pathway that is substantially independent of an
MEKK/JNKK-contingent pathway, or a pathway in which ERK protein
regulation converges with common members of a pathway involving
MEKK/JNKK-contingent protein. More particularly, an ERK-dependent
pathway includes components downstream of ERK proteins and
continues downstream in a series of signal transduction events. The
independence of regulation of a pathway by an ERK protein from the
regulation of a pathway by an MEKK/JNKK-contingent protein can be
determined using methods as described above in relation to the
MEKK/JNKK-contingent pathway. In particular, regulation of an
ERK-dependent pathway will not lead to the regulation of cytokine
production in a cell.
[0055] As referenced in the present invention, MEKK, or mitogen ERK
kinase kinase, is a signal transduction molecule that is capable of
phosphorylating mitogen ERK kinase or MAPK kinase (MEK) and/or
c-Jun amino-terminal kinase kinase (JNKK), thereby activating such
molecules. Several members of the MEKK family have been identified,
including MEKK1, MEKK2, MEKK3, and MEKK4. An MEKK molecule of the
MEKK/JNKK-contingent signal transduction pathway of the present
invention phosphorylates JNKK. In a preferred embodiment, MEKK1
phosphorylates JNKK in response to activation through an
FceRI-activated, PI3-kinase-dependent pathway in a mast cell. MEK
protein is not a component of the MEKK/JNKK-contingent signal
transduction pathway of the present invention.
[0056] JNK-activating protein kinase (JNKK), is a dual-specificity
threonine-tyrosine protein kinase that activates JNK and functions
downstream from MEKK. JNK is a distant member of the
mitogen-activated protein kinase superfamily, designated c-Jun
amino-terminal kinase (JNK). JNK is activated following dual
phosphorylation at a Thr-Pro-Tyr motif in response to diverse
stimuli including tumor necrosis factor-.alpha., heat shock, or
ultraviolet irradiation. Costimulation of T cells with antibodies
to the T cell receptor and CD28 or the stimulation of B cells with
anti-CD40 antibody also induces the activation of JNK. JNK
functions to phosphorylate c-Jun at the amino-terminal regulatory
sites, serine 63 and serine 73, mapping within its transactivation
domain. Phosphorylation of these sites in response to UV
irradiation also results in the transcriptional activation of
c-Jun. There are two members of the JNK family, designated JNK1 and
JNK2. It has been suggested that JNK may be involved in apoptosis,
but until the present invention, it was not appreciated that JNK
was involved in a signal transduction pathway that regulated
cytokine production in a cell.
[0057] One embodiment of the present invention is directed to a
method to regulate cytokine production in a hematopoietic cell,
comprising regulating an MEKK/JNKK-contingent signal transduction
pathway. In a preferred embodiment, regulation of such a pathway
results in inhibition of cytokine production.
[0058] As used herein, the term "regulate" can be used
interchangeably with the term "modulate". "To regulate" a molecule,
a pathway, or a function of such a molecule or pathway, in the
present invention refers to specifically controlling, or
influencing the activity of such a molecule, pathway, or function,
and can include regulation by activation, stimulation, inhibition,
alteration or modification of such molecule, pathway or
function.
[0059] In a preferred embodiment, regulating an
MEKK/JNKK-contingent signal transduction pathway comprises
regulating a signal transduction molecule selected from the group
consisting of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1, and JNK2.
Preferably, regulation of such a signal transduction molecule is
accomplished by a method including, but not limited to, degrading
said molecule, binding a regulatory compound to said molecule,
inhibiting transcription of said molecule, inhibiting translation
of said molecule, inhibiting activation of said molecule, and
inhibiting the interaction of said molecule with another signal
transduction molecule.
[0060] In a preferred embodiment of the present invention, an
MEKK/JNKK-contingent signal transduction pathway is regulated by
administration of an effective amount of a compound that interacts
with a signal transduction molecule of said pathway such that
cytokine production is regulated. Preferably, such a compound
regulates a signal transduction molecule selected from the group of
MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2. In another
embodiment, such a compound regulates PI3-K. A regulatory compound
of the present invention, however, does not regulate a molecule
specific to an ERK-dependent pathway. Thus, although in some cell
types, for example, PI3-K may regulate other signal transduction
pathways in addition to an MEKK/JNKK-contingent pathway, regulation
of PI3-K to effect regulation of an MEKK/JNKK-contingent pathway is
still within the scope of the present invention.
[0061] As used herein, an "effective amount" of a compound is at
least the minimum amount of a compound that is necessary to
minimally achieve, and more preferably, optimally achieve, the
desired effect (i.e. regulation of a signal transduction molecule).
An effective amount for use in a given method can be readily
determined by one skilled in the art without undue experimentation,
depending upon the particular circumstances encountered (e.g.
concentrations, cell type and number, etc.).
[0062] A regulatory compound of the present invention regulates
cytokine production in a hematopoietic cell, comprising a compound
that is capable of regulating an MEKK/JNKK-contingent signal
transduction pathway of the present invention. Such a regulatory
compound includes a compound that is capable of inhibiting an
MEKK/JNKK-contingent signal transduction pathway of the present
invention, a compound that is capable of stimulating an
MEKK/JNKK-contingent signal transduction pathway of the present
invention, or a compound that is capable of preventing both the
stimulation and the inhibition of the activity of an
MEKK/JNKK-contingent signal transduction pathway of the present
invention (i.e., maintaining the activity of a signal transduction
pathway). Such regulation by a compound can be effected by, but is
not limited to, any of the preferred methods of regulating a signal
transduction molecule as described above (i.e. degrading a signal
transduction molecule, etc.).
[0063] Acceptable protocols to contact a cell with a regulatory
compound in an effective manner can be accomplished by those
skilled in the art based on variables such as, the conditions under
which the compound is being administered, the type of cell being
regulated and the chemical composition of the regulatory compound
(i.e., size, charge etc.) being administered.
[0064] As used herein, "inhibiting the interaction of" one molecule
with another can be accomplished in a variety of ways including,
but not limited to, physically blocking the interaction between two
molecules (i.e. by a regulatory compound), moving one molecule
relative to the other such that interaction between the two can not
occur, dephosphorylating or preventing phosphorylation of one or
both molecules such that interaction can not occur, and
phosphorylating one or both molecules such that interaction can not
occur.
[0065] Inhibiting activation of a molecule can be accomplished by a
method including, but not limited to, preventing activation of said
molecule and deactivating a molecule that is activated. Such
methods include, but are not limited to, phosphorylating a
molecule, dephosphorylating a molecule, preventing phosphorylation
of a molecule, physically inhibiting activation of a molecule as
described above, and degrading a molecule.
[0066] In one embodiment of the present invention, signal
transduction pathways involved in cytokine production in a
hematopoietic cell are regulated by a method comprising inhibiting
the interactions between molecules selected from a group of MEKK1,
MEKK2, MEKK3, MEKK4, JNKK, JNK1 and JNK2, in a hematopoietic cell
having an MEKK/JNKK-contingent signal transduction pathway. Such
inhibition of interactions between such signal transduction
molecules can be effected by methods of regulation described
herein, including, but not limited to, contacting a cell with a
compound which modulates the interactions between such
molecules.
[0067] According to the present invention, a hematopoietic cell is
a cell which includes erythrocyte cells (i.e., a red blood cell),
certain leukocyte cells, including granular leukocytes
(eosinophils, basophils, neutrophils, and mast cells), non-granular
leukocytes (megakaryocytes, polymorphonuclear cells, lymphocytes
and monocytes), or thrombocyte cells (i.e., platelet cell). A
preferred hematopoietic cell of the present invention includes a
mast cell, a basophil, an eosinophil, a neutrophil, a T cell, a B
cell, a macrophage, a dendritic cell, and a natural killer
cell.
[0068] Cytokine production, as used in the present invention,
refers to the de novo synthesis of mRNA encoding such a cytokine
which results in the translation and exocytosis of such a cytokine.
A cytokine for which production can be regulated in the present
invention can be selected from the group consisting of TNF-a, IL-1,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IL-16, G-CSF, GM-CSF, TNF-b, TGF-b,
IFN-.gamma., and IFN-.alpha./.beta.. It is within the scope of the
present invention that cytokines that are unknown at the time of
the present invention, but are described in the art in the future,
may also be a cytokine that can be regulated by the
MEKK/JNKK-contingent pathway of the present invention. In a
preferred embodiment, production of TNF-a by a mast cell is
regulated. The de novo synthesis of cytokine production is
regulated by activation of an MEKK/JNKK-contingent signal
transduction pathway of the present invention. In a preferred
embodiment, activation of such a pathway results in modulation of
the interaction of transcription factors with cytokine promoters
which can thereby regulate cytokine production.
[0069] A cytokine promoter as described herein can include any DNA
sequence capable of being specifically bound by an RNA polymerase
in such a manner that the RNA polymerase can unwind the DNA strand
to initiate RNA synthesis of a cytokine gene.
[0070] A transcription factor of the present invention is capable
of mediating the rate of cytokine transcription in a cell. The rate
of transcription of a particular cytokine DNA molecule in a cell is
not necessarily fixed and can change according to the needs of the
cell in different conditions of growth. Such regulation of
transcription can be mediated by proteins that, by binding to DNA
near or within a promoter, can increase or decrease the rate at
which RNA polymerase initiates RNA synthesis. Transcription rates
can be mediated by proteins including transcription factors.
Suitable transcription factors include, but are not limited to, at
least a portion of a transcription factor. A transcription factor
as referred to herein has a transactivation domain capable of
regulating the activity of the transcription factor. Such a
transcriptional activation domain contains amino acid residues
capable of being phosphorylated, the phosphorylation of which
results in the regulation of the activity of the transcription
factor. Without being bound by theory, it is believed that
phosphorylation of residues contained in a transcriptional
activation domain alters the conformation of the transcription
factor such that the DNA binding domain of the transcription
complex can drive transcription. Preferred sites of phosphorylation
include serine residues and threonine residues spaced in such a
manner that the phosphorylation of such residues results in the
activation of the transcription factor. Preferred transcription
factors of the present invention include NF-AT, AP-1, Jun, Fos,
ATF-2, NF.kappa.B, and CBP.
[0071] In one embodiment of the present invention, a hematopoietic
cell, in which cytokine production is regulated, expresses FceRI.
Preferably, cytokine production in such a cell is inhibited by
regulation of an MEKK/JNKK-contingent signal transduction pathway.
In a preferred embodiment, activation of an MEKK/JNKK-contingent
signal transduction pathway is initiated through aggregation of
FceRI. FceRI is the high-affinity receptor for IgE. Hematopoietic
cells expressing FceRI in the present invention are preferably mast
cells, basophils and eosinophils, and most preferably mast cells.
The multivalent binding of an antigen to receptor-bound IgE and the
subsequent aggregation of the high-affinity Fc receptors for IgE
(Fc.epsilon.RI) provide the trigger for activation of mast cells.
The first demonstrable response to Fc.epsilon.RI aggregation is
tyrosine phosphorylation and activation of phospholipase C.gamma.,
which catalyzes the hydrolysis of phosphatidylinositol
4,5-bisphosphate resulting in the liberation of inositol
1,4,5-trisphosphate and diacylglycerol. The elevation of
diacylglycerol and the mobilization of Ca.sup.2+ from intracellular
and extracellular sources results in the activation of protein
kinase C.
[0072] The Fc.epsilon.RI is composed of three subunits, single
.alpha. and .beta. chains, and a homodimer of disulfide-linked
.gamma. chains. The intracellular tails of the .beta. and .gamma.
chains contain a motif that is important for signal transduction.
This motif has been called the antigen recognition activation motif
or tyrosine activation motif, which is thought to couple the
Fc.epsilon.RI to protein tyrosine kinases. Activation of protein
tyrosine kinases is one of the earliest signaling events induced by
aggregation of the Fc.epsilon.RI on mast cells. The aggregation of
Fc.epsilon.RI initiates diverse signal transduction pathways. As is
shown for the first time herein, one of these pathways is the
MEKK/JNKK-contingent signal transduction pathway which leads to
cytokine production.
[0073] In a preferred embodiment of the present invention, an
MEKK/JNKK-contingent signal transduction pathway is activated
through a phosphatidylinositol 3-kinase (PI3-K) signal transduction
pathway. More particularly, such a PI3-K signal transduction
pathway is activated through aggregation of FceRI on the surface of
a mast cell, a basophil, or an eosinophil. As used herein, a PI3-K
pathway is a signal transduction pathway that involves the signal
transduction molecule, PI3-K. PI3-K is a heterodimeric protein
composed of a non-catalytic p85 .alpha. subunit (85 kD) and a
catalytic p110 .beta. subunit (110 kD). PI3-K.gamma. is a 110 kD
enzyme specifically regulated by G proteins. PI3-K is capable of
phosphorylating inositol lipids on the D-3 hydroxyl position.
Contained within the p85 subunit are two proline-rich domains.
PI3-K.gamma. is reguled by G protein subunits, particularly
.beta..gamma. subunits.
[0074] The present inventors have unexpectedly found that the PI3-K
inhibitor, wortmannin, at concentrations that inhibit PI3-kinase
activity, also inhibited JNK activation, but not ERK activation.
This finding is the first demonstration of a role for PI3-kinase in
regulating a JNK pathway by an Src family tyrosine
kinase-associated receptor. Thus, in mast cells the regulation of
the MEKK1, JNKK, JNK pathway is dependent on the activation of
PI3-kinase, which in turn, is activated by aggregation of FceRI.
Mechanistically, there is a very early separation in the signal
pathways activated by the Fc.epsilon.RI to differentially regulate
JNK and ERK sequential protein kinase pathways. Without being bound
by theory, the present inventors believe that PI3-kinase activity
is involved in activating the MEKK/JNKK-contigent pathway in mast
cells downstream of tyrosine kinases and upstream of MEKK1.
[0075] In a preferred embodiment of the present invention,
regulation of cytokine production by regulating an
MEKK/JNKK-contingent signal transduction pathway further comprises
regulating PI3-kinase.
[0076] Such regulation can be effected by the methods described
herein for regulation of an MEKK/JNKK-contingent signal
transduction molecule, and/or by interfering with the capability of
PI3-K to trigger downstream signal transduction events. In yet
another preferred embodiment of the present invention, a method to
regulate cytokine production in a cell further comprises regulating
a signal transduction pathway selected from the group of a c-kit
signal transduction pathway and a p38 signal transduction pathway.
The c-kit and the p38 signal transduction pathways are distinct
from the MEKK/JNKK-contingent signal transduction pathway of the
present invention. It is appreciated for the first time in the
present invention, that both the c-kit and the p38 signal
transduction pathways can enhance the regulation of cytokine
production that is effected by the MEKK/JNKK-contingent signal
transduction pathway, c-kit is a cell-surface receptor that, when
bound by c-kit ligand, initiates a signal transduction pathway that
is incapable of regulating cytokine production itself, but that can
enhance the effects of the MEKK/JNKK-contingent pathway on cytokine
production. p38 is activated by dual phosphorylation at a
Thr-Gly-Tyr motif and is activated by cellular stress,
pro-inflammatory cytokines, and lipopolysaccharide (LPS). p38 can
be activated by a specific JNKK referred to as MKK3 or MKK6;
however, in the MEKK/JNKK-contingent signal transduction pathway of
the present invention, JNK is preferentially activated over p38.
Therefore, signal transduction through the MEKK/JNKK-contingent
signal transduction pathway is primarily responsible for regulation
of cytokine production in a cell, but can be enhanced by other
signal transduction pathways. Regulation of such other pathways can
be effected by the methods described herein for regulation of an
MEKK/JNKK-contingent signal transduction pathway.
[0077] One aspect of the present invention includes a method to
identify compounds that regulate an MEKK/JNKK-contingent signal
transduction pathway. Such compounds are referred to herein as
"putative regulatory compounds". As used herein, the term
"putative" refers to compounds having an unknown signal
transduction regulatory activity, at least with respect to the
ability of such compounds to regulate cytokine production via the
MEKK/JNKK-contingent pathway. Regulatory compounds, defined by
their identifying characteristics of being capable of regulating
signal transduction molecules of the present invention have been
previously described herein.
[0078] Putative regulatory compounds as referred to herein include,
for example, compounds that are products of rational drug design,
natural products and compounds having partially defined signal
transduction regulatory properties. A putative compound can be a
protein-based compound, a carbohydrate-based compound, a
lipid-based compound, a nucleic acid-based compound, a natural
organic compound, a synthetically derived organic compound, an
anti-idiotypic antibody and/or catalytic antibody, or fragments
thereof. A putative regulatory compound can be obtained, for
example, from libraries of natural or synthetic compounds, in
particular from chemical or combinatorial libraries (i.e.,
libraries of compounds that differ in sequence or size but that
have the same building blocks) or by rational drug design.
[0079] A method to identify a regulatory compound of the present
invention comprises the steps of providing a hematopoietic cell
having an MEKK/JNKK-contingent signal transduction pathway,
contacting such a cell with a putative regulatory compound, and
determining whether such a compound is capable of regulating said
MEKK/JNKK-contingent signal transduction pathway. In a preferred
embodiment, such a method is used to identify a regulatory compound
that inhibits cytokine production by said cell.
[0080] In particular, a preferred regulatory compound of the
present invention can be identified by determining the ability of
such a compound to modulate the interactions between signal
transduction molecules selected from the group of MEKK1, MEKK2,
NEKK3, MEKK4, JNKK, JNK1, and JNK2. More preferably, the regulation
of such a signal transduction molecule by a regulatory compound of
the present invention modulates the interaction of such a molecule
with a transcription factor selected from the group of NF-AT, AP-1,
Jun, Fos, ATF-2, NF.kappa.B and CBP. In another preferred
embodiment, a regulatory compound is identified by its ability to
modulate the interaction of PI3-K with signal transduction
molecules in the MEKK/JNKK-contingent pathway.
[0081] Another embodiment of the present invention is a kit for
identifying compounds capable of regulating cytokine production in
a hematopoietic cell. Such a kit comprises a hematopoietic cell
capable of producing an amount of at least one cytokine, such cell
having an MEKK/JNKK-contingent signal transduction pathway, and
production of such cytokine being dependent on such
MEKK/JNKK-contingent signal transduction pathway. Such a kit
further comprises a means for determining a change in said amount
of cytokine produced by such a cell after the cell is contacted
with a putative regulatory compound. In a preferred embodiment,
such a kit is useful for identifying compounds that inhibit
cytokine production by said cell.
[0082] As used herein, "at least one cytokine" means that a minimum
of one cytokine selected from the group of TNF-a, IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,
IL-14, IL-15, IL-16, G-CSF, GM-CSF, TNF-b, TGF-b, IFN-.gamma., and
IFN-.alpha./.beta.., can be produced by a cell of the kit of the
present invention. It is within the scope of the present invention
that more than one cytokine can be produced by such a cell. As used
herein, "an amount" of a cytokine refers to a quantity of cytokine
produced by a hematopoietic cell that is detectable by standard
methods of cytokine measurement in the art. "An amount" can refer
to a detectable protein concentration of cytokine in a sample, or
to units of activity of such a cytokine in a sample. "A change in
the amount" of cytokine produced by a hematopoietic cell after such
a cell is contacted by a putative regulatory compound, is any
detectable change, (i.e. increase or decrease) in the amount of
cytokine produced by such a cell after contact with such a
regulatory compound, as compared to the amount of cytokine produced
before such cell was contacted with such a compound.
[0083] Suitable cells for use with either the method to detect a
regulatory compound or with the kit useful for detecting a
regulatory compound of the present invention include any cell that
has an MEKK/JNKK-contingent signal transduction pathway. Such cells
can include normal cells or transformed derivatives thereof, that
express a receptor in a native physiological context (e.g.,
basophils, eosinophils, neutrophils, monocytes, macrophages, and
lymphoid cells). Alternatively, cells for use with the present
invention can include spontaneously occurring variants of normal
cells, or genetically engineered cells, that have altered signal
transduction activity, such as enhanced responses to particular
ligands. Signal transduction variants of normal cells can be
identified using methods known to those in the art. For example,
variants can be selected using fluorescence activated cell sorting
(FACS) based on the level of calcium mobilization by a cell in
response to a ligand. Genetically engineered cells can include
recombinant cells of the present invention (described in detail
below) that have been transformed with, for example, a recombinant
molecule encoding a signal transduction molecule and/or a
transcription indicator recombinant molecule of the present
invention.
[0084] Cells for use with the present invention include mammalian,
invertebrate, plant, insect, fungal, yeast and bacterial cells.
Preferred cells include mammalian, amphibian and yeast cells.
Preferred mammalian cells include primate, mouse and rat cells. In
a preferred embodiment, cells to be used in a method to identify
compounds which regulate an MEKK/JNKK-contingent signal
transduction pathway can be genetically manipulated to obtain cells
having an MEKK/JNKK-contingent signal transduction pathways wherein
production of cytokines by such cells is dependent on the
MEKK/JNKK-contingent signal transduction pathway and/or components
of such signal transduction pathway. In a preferred embodiment,
such cells are substantially devoid of any other signal
transduction pathways that result in significant production of
cytokines by such cell.
[0085] In yet another embodiment, a cell suitable for use in the
present invention further comprises a PI3-K signal transduction
pathway which activates said MEKK/JNKK-contingent pathway, and/or a
p38 signal transduction pathway or a c-kit signal transduction
pathway which enhances said MEKK/JNKK-contingent pathway.
[0086] In one embodiment, a cell suitable for use in the present
invention has at least one type of cell surface receptor. A cell
surface receptor as referred to herein includes those cell surface
receptors capable of binding to a ligand (as described in detail
below) and capable of initiating an MEKK/JNKK-contingent signal
transduction pathway in a cell upon ligand binding. A cell surface
receptor typically includes an external portion located on the
outer surface of a plasma membrane of a cell, a transmembrane
portion that spans the plasma membrane, and a cytoplasmic portion
located on the inner surface of the plasma membrane.
[0087] A cell surface receptor as described herein can be produced
by expression of a naturally occurring gene encoding a cell surface
receptor and/or a heterologous nucleic acid molecule transformed
into a cell. An example of a cell surface receptor of the present
invention could be, for example, CD40, CD28, or FceRI. A preferred
cell surface receptor of the present invention is FceRI.
[0088] An intracellular signal transduction molecule as described
herein can be produced in a cell by expression of a naturally
occurring gene and/or by expression of a heterologous nucleic acid
molecule transformed into the cell. Preferred intracellular signal
transduction molecules of the present invention are MEKK1, MEKK2,
MEKK3, MEKK4, JNKK, JNK1 and JNK2. MAPK/ERK molecules are not
intracellular signal molecules of the present invention.
[0089] In certain embodiments, a cell of the present invention is
transformed with at least one heterologous nucleic acid sequence. A
nucleic acid sequence, or molecule, as described herein can be DNA,
RNA, or hybrids or derivatives of either DNA or RNA. Nucleic acid
molecules as referred to herein can include regulatory regions that
control expression of the nucleic acid molecule (e.g.,
transcription or translation control regions), full-length or
partial coding regions, and combinations thereof. It is to be
understood that any portion of a nucleic acid molecule can be
produced by: (1) isolating the molecule from its natural milieu;
(2) using recombinant DNA technology (e.g., PCR amplification,
cloning); or (3) using chemical synthesis methods. A gene includes
all nucleic acid sequences related to a natural cell surface
receptor gene such as regulatory regions that control production of
a cell surface receptor encoded by that gene (such as, but not
limited to, transcription, translation or post-translation control
regions) as well as the coding region itself.
[0090] A nucleic acid molecule can include functional equivalents
of natural nucleic acid molecules encoding a protein or functional
equivalents of natural nucleic acid sequences capable of being
bound by proteins. Functional equivalents of natural nucleic acid
molecules can include, but are not limited to, natural allelic
variants and modified nucleic acid molecules in which nucleotides
have been inserted, deleted, substituted, and/or inverted in such
molecules without adversely affecting the function of products
encoded by such sequences.
[0091] As guidance in determining what particular modifications can
be made to any particular nucleic acid molecule, one of skill in
the art should consider several factors that, without the need for
undue experimentation, permit a skilled artisan to appreciate
workable embodiments of the present invention. For example, such
factors include modifications to nucleic acid molecules done in a
manner so as to maintain particular functional regions of the
encoded proteins including, a ligand binding site, a target binding
site, a catalytic domain, etc. Functional tests for these various
characteristics (e.g., ligand binding studies and signal
transduction assays such as kinase assays, transcription assays,
and other assays described in detail herein) allows one of skill in
the art to determine what modifications to nucleic acid sequences
would be appropriate and which would not.
[0092] Transformation of a heterologous nucleic acid molecule
(e.g., a heterologous cell surface receptor encoding nucleic acid
molecule) into a cell suitable for use in the present invention can
be accomplished by any method by which a gene is inserted into a
cell. Transformation techniques include, but are not limited to,
transfection, retroviral infection, electroporation, lipofection,
bacterial transfer and spheroplast fusion. Nucleic acid molecules
transformed into cells suitable for use in the present invention
can either remain on extra-chromosomal vectors or can be integrated
into the cell genome.
[0093] Expression of a nucleic acid molecule of the present
invention in a cell can be accomplished using techniques known to
those skilled in the art. Briefly, the nucleic acid molecule is
inserted into an expression vector in such a manner that the
nucleic acid molecule is operatively joined to a transcription
control sequence in order to be capable of affecting either
constitutive or regulated expression of the gene when the gene is
transformed into a host cell. The phrase "recombinant molecule", as
used herein refers to a gene operatively linked to at least one
transcription control sequence on an expression vector. The phrase
"expression vector", as used herein refers to a DNA or RNA vector
that is capable of transforming a host cell, of replicating within
the host cell, and of affecting expression of the operatively
linked gene. Expression vectors are capable of replicating to
either a high or low copy number depending on their inherent
characteristics. Transcription control sequences, which can control
the amount of protein produced, include sequences that control the
initiation, elongation, and termination of transcription.
Particularly important transcription control sequences are those
which control transcription initiation, such as promoter and
upstream activation sequences.
[0094] Construction of desired expression vectors can be performed
by methods known to those skilled in the art and expression can be
in eukaryotic or prokaryotic systems. Procaryotic systems typically
used are bacterial strains including, but not limited to various
strains of E. coli, various strains of bacilli or various species
of Pseudomonas. In prokaryotic systems, plasmids are used that
contain replication sites and control sequences derived from a
species compatible with a host cell. Control sequences can include,
but are not limited to promoters, operators, enhancers, ribosome
binding sites, and Shine-Dalgarno sequences. Expression systems
useful in eukaryotic host cells comprise promoters derived from
appropriate eukaryotic genes. Useful mammalian promoters include
early and late promoters from SV40; other viral promoters such as
those derived from baculovirus, polyoma virus, adenovirus, bovine
papilloma virus, avian sarcoma virus or cytomegalovirus; or
collagenase promoters. Expression vectors include any vectors that
function (i.e., direct gene expression) in recombinant cells of the
present invention including bacterial, yeast, other fungal, insect,
and mammalian cells. Particularly preferred expression vectors
include promoters useful for expressing recombinant molecules in
human cells.
[0095] An expression system can be constructed from any of the
foregoing control elements operatively linked to nucleic acid
sequences using methods known to those of skill in the art. (see,
for example, Sambrook et al., ibid.).
[0096] The conditions under which the cell of the present invention
is contacted with, such as by mixing, a putative regulatory
compound are conditions in which the cell can transduce a normal
signal if essentially no regulatory compound is present. Such
methods are within the skill in the art, and include an effective
medium in which the cell can be cultured such that the cell can
exhibit signal transduction activity. A preferred number of cells
to use in the method or test kit of the present invention includes
a number of cells that enables one to detect a change in activity
of a signal transduction molecule using a detection method of the
present invention (described in detail below).
[0097] In another embodiment of the present invention, cells
suitable for use in the present invention are stimulated with
ligands capable of binding to cell surface receptors of the present
invention to initiate an MEKK/JNKK-contingent signal transduction
pathway and thereby regulate cytokine production. Suitable ligands
can include, for example, hormones, growth factors, antigens,
peptides, ions, other differentiation agents and other cell type
specific mitogens. Preferred ligands include IgE, anti-FceRI, and
c-kit ligand.
[0098] In another embodiment of the present invention, cells
suitable for use in the present invention are stimulated with
intracellular initiator molecules capable of initiating a signal
transduction pathway from inside a cell. Examples of intracellular
initiator molecules as referred to herein include, but are not
limited to, phorbol esters, calcium ionophores, ALF4, phenyloxide,
mastoparans, sodium orthovanadate, arachidonic acid and
ceramides.
[0099] A suitable amount of putative regulatory compound(s)
suspended in culture medium is added to the cells that is
sufficient to regulate the activity of a signal transduction
molecule inside the cell such that the regulation is detectable
using a detection method of the present invention. A preferred
amount of putative regulatory compound(s) comprises between about 1
nM to about 10 mM of putative regulatory compound(s) per well of a
96-well plate. The cells are allowed to incubate for a suitable
length of time to allow the putative regulatory compound to enter a
cell and interact with a signal transduction molecule. A preferred
incubation time is between about 1 minute to about 12 hours.
[0100] In one embodiment, the method and kit of the present
invention include determining if a putative regulatory compound is
capable of regulating an MEKK/JNKK-contingent signal transduction
pathway by regulating cytokine production in a cell. Such methods
of determining a change in an amount of a cytokine after contact of
a putative regulatory compound with a cell include: immunoassays
for cytokine production, such as by enzyme-linked immunoassay
(e.g., ELISA), radioimmunoassay analysis, fluorescence immunoassay
or immunoblot assay (as generally described in Sambrook et al.,
ibid.); transcription assays to detect the activation of cytokine
transcription, such as measuring the increase or decrease in mRNA
transcription of a cytokine gene by PCR-based technology; and
biological assays in which a cytokine-indicator cell is used to
determine a change in an amount of cytokine (such cells are known
in the art for a large number of cytokines). Particularly useful
assays are antibody-based capture assays that comprise: (1)
attaching a capture antibody having specificity for a specific
cytokine to a support, such as an ELISA plate; (2) contacting a
cell supernate with the substrate-bound antibody to form an immune
complex; (3) contacting the substrate-bound immune complex with a
detection antibody specific for an epitope on the cytokine; and (4)
detecting the association of the detection antibody to the immune
complex.
[0101] It is within the scope of the present invention to determine
regulation of an MEKK/JNKK-contingent signal transduction pathway
by measuring the activation of signal transduction molecules
selected from the group of MEKK1, MEKK2, MEKK3, MEKK4, JNKK, JNK1
and JNK2, by methods such as: kinase assays to detect the
phosphorylation of such signal transduction molecules, calcium
mobilization assays to detect increases in calcium levels in a
cell's cytoplasm, and immunoassays such as those listed above. Such
methods are known in the art. The methods of the present invention
can further include the step of performing a toxicity test to
determine the toxicity of a putative regulatory compound.
[0102] One embodiment of the present invention relates to a method
to treat a disease involving cytokine production in an animal,
comprising regulating an MEKK/JNKK-contingent signal transduction
pathway to affect cytokine production by a hematopoietic cell. Such
diseases include medical disorders and diseases in which the
pathogenesis of the disease and/or the physiological effects of the
disease might be ameliorated by regulation of cytokine production.
Such diseases include, but are not limited to, allergic diseases,
anaphylaxis, diseases involving defects in hematopoietic cells,
inflammation, mast cell disorders, sepsis and cancer. According to
the present invention, the term treatment can refer to the
regulation of the progression of a disease or the complete removal
of a disease (e.g., cure).
[0103] The present invention preferably relates to a method to
treat allergic inflammation, comprising regulating cytokine
production in a hematopoietic cell in an animal by regulating an
MEKK/JNKK-contingent signal transduction pathway. In one
embodiment, such a hematopoietic cell is selected from a group of a
mast cell, a basophil and an eosinophil, such a cell expressing
FceRI.
[0104] In a preferred embodiment, a disease involving cytokine
production can be treated by administering to an animal an
effective amount of a compound which interacts with a signal
transduction molecule in an MEKK/JNKK-contingent signal
transduction pathway in a hematopoietic cell of said animal, such
that cytokine production by such a cell is regulated. In a
preferred embodiment, cytokine production is inhibited.
[0105] Signal transduction molecules of an MEKK/JNKK-contingent
signal transduction pathway that can be regulated by administration
of a regulatory compound include MEKK1, MEKK2, MEKK3, NEKK4, JNKK,
JNK1 and JNK2. A preferred compound to administer to an animal to
regulate cytokine production by a hematopoietic cell is a compound
selected from the group of an MEKK inhibitor, a JNKK inhibitor, and
a JNK inhibitor. In a preferred embodiment, a disease involving
cytokine production by a hematopoietic cell of an animal can be
regulated by administering an effective amount of a compound which
inhibits PI3-K.
[0106] As used herein, an "inhibitor" of a particular signal
transduction molecule inhibits, prevents, decreases, or impedes,
the normal activity of such a molecule. An inhibitor can inhibit a
specific signal transduction molecule by a means including, but not
limited to: causing such a molecule to be degraded, binding to such
a molecule such that the molecule is incapable of being activated,
binding to such a molecule such that the molecule is unable to
interact with other signal transduction molecules, inhibiting
transcription of such a molecule, and inhibiting translation of
such a molecule.
[0107] As used herein, "an effective amount" of such a compound is
an amount, or dose, of a regulatory compound, that when
administered to an animal, is capable of regulating cytokine
production by a hematopoietic cell in said animal.
[0108] Effective doses to administer to an animal include doses
administered over time that are capable of regulating cytokine
production by a hematopoietic cell in the animal. For example, a
first effective dose can comprise an amount of a regulatory
compound of the present invention that causes a minimal change in
cytokine production by a hematopoietic cell when administered to an
animal. A second effective dose can comprise a greater amount of
the same compound than the first dose. Effective doses can comprise
increasing concentrations of the compound necessary to regulate
cytokine production and ameliorate a disease involving such
cytokine production in an animal such that the animal does not have
an immune response to subsequent exposure to the compound. A
suitable single dose of a regulatory compound of the present
invention is a dose that is capable of substantially regulating
cytokine production by a hematopoietic cell when administered one
or more times over a suitable time period. A preferred single dose
of a regulatory compound ranges from about 0.01 .mu.g to about
1,000 milligrams (mg) of such a compound per subject, more
preferred ranges being from about 0.1 .mu.g to about 100 mg of a
compound per subject, and even more preferred ranges being from
about 1 .mu.g to about 10 mg of a compound per subject.
[0109] A regulatory compound of the present invention can be
administered to any animal, preferably to mammals, and even more
preferably to humans. Acceptable protocols to administer a
regulatory compound of the present invention in an effective manner
include individual dose size, number of doses, frequency of dose
administration, and mode of administration. Determination of such
protocols can be accomplished by those skilled in the art depending
upon a variety of variables, including the animal to be treated and
the stage of disease. Modes of delivery can include any method
compatible with prophylactic or treatment of a disease. Modes of
delivery include, but are not limited to, parenteral, oral,
intravenous, topical administration, local administration, and ex
vivo administration to isolated hematopoietic cells.
[0110] The following experimental results are provided for purposes
of illustration and are not intended to limit the scope of the
invention.
EXAMPLES
[0111] For the following Examples 1-12, the materials and methods
used herein are the same throughout the examples and are therefore
described in detail only upon the first appearance of such material
or method.
Example 1
[0112] The following example demonstrates that JNK is activated
through aggregation of FceRI by antigen or anti-IgE in MC/9
cells.
[0113] MC/9, a mouse mast cell line, was originally derived from
fetal liver cells cultured in concanavalin A-conditioned medium
followed by culture with irradiated syngeneic bone marrow cells.
The MC/9 mast cell line was identified as a source of mast cells by
light microscopy and the appearance of metachromatic granules.
Further characterization includes the findings on electron
microscopy and the ability to release histamine upon stimulation
with A23187 or with antigen following passive sensitization with
IgE. MC/9 cells express Fc.epsilon.RI on the cell surface.
[0114] The MC/9 murine mast cell clone was obtained from the
American Type Culture Collection (Rockville, Md.) and maintained in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 5.times.10.sup.-5 M 2-mercaptoethanol (Life
Technologies, Inc.), 10% fetal bovine serum (Summit Biotechnology,
Ft. Collins, Colo.), and 5% conditioned medium (rat growth factor
obtained from Collaborative Biomedical (Bedford, Mass.)). Purified
rat anti-mouse IgE monoclonal antibody (R35-72) was purchased from
Pharmingen (San Diego, Calif.). Ovalbumin (OVA, grade V) was
obtained from Sigma. Recombinant protein G-Sepharose 4B was
purchased from Zymed Laboratories (San Francisco, Calif.). An
anti-OVA IgE antibody-secreting hybridoma cell line was generated
as described. A hybridoma cell line producing monoclonal mouse
IgE-specific for 2,4,6-trinitrophenol (TNP), IGEL b4 was purchased
from ATCC.
[0115] MC/9 cells (5.times.10.sup.6/ml) were cultured with 500
ng/ml anti-OVA IgE for 2 h. The cells were washed with medium three
times and cultured with fresh medium for an additional 2 h. OVA
dissolved in PBS or anti-IgE was added for the stimulation, and PBS
was used as a control vehicle.
[0116] The activity of JNK was measured by monitoring the activity
of a Glutathione S-transferase-c-Jun-(1-79) fusion protein. Cells
(3.times.10.sup.6) were lysed in a buffer (20 mM Tris-HCl, pH 7.6,
250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5% NP-40, 2 mM
Na.sub.3VO.sub.4, 1 mM dithiothreitol (DTT), 1 mM
phenylmethylsulfonyl fluoride (PMSF), 20 .mu.g/ml aprotinin, 5
.mu.g/ml leupeptin). The lysates were mixed with
GST-c-Jun-Sepharose beads and rotated at 4.degree. C. for 3 h. The
beads were washed twice in lysis buffer and once in kinase assay
buffer (20 mM Hepes, pH 7.5, 20 mM .beta.-glycerophosphate, 10 mM
MgCl.sub.2, 1 mM DTT, 50 mM Na.sub.3VO.sub.4, 10 mM p-nitrophenyl
phosphate). After the final wash, 40 .mu.l of a kinase assay buffer
containing 10 .mu.Ci of .gamma.-.sup.32P-ATP (ICN Pharmaceuticals,
Irvine, Calif.) were added per sample. The sample was incubated for
20 min at 30.degree. C. and the reaction was stopped by addition of
13 .mu.l of 4.times. protein loading buffer (188 mM Tris (pH 6.8),
30% glycerol, 6% sodium dodecyl sulfate (SDS), 15%
2-mercaptoethanol, 0.4% bromophenol blue). The samples were boiled
for 3 min, and GST-c-Jun was separated by SDS-12% polyacrylamide
gel. The gel was stained with Coomassie brilliant blue,
exhaustively destained, dried, and subjected to autoradiography.
The bands corresponding to GST-c-Jun were cut out of the gel, and
radioactivity was determined by liquid scintillation counting.
[0117] MC/9 cells were incubated with 500 ng/ml mouse monoclonal
IgE specific for OVA (OVA-IgE) for 2 h. After washing, sensitized
MC/9 cells were incubated in the presence of 10 ng/ml to 100
.mu.g/ml OVA for 10 min. Following addition of OVA to MC/9 cells
sensitized with OVA-specific IgE (OVA-IgE), JNK was significantly
activated in a dose-dependent manner. 10 .mu.g/ml OVA induced
maximal activation of JNK (FIG. 2A).
[0118] MC/9 cells sensitized with OVA-IgE were incubated in the
presence of PBS (0 min) or 10 .mu.g/ml OVA for 1, 3, 5, 10, 15, 20,
30, 40, or 60 min. FIGS. 2B and 2C show representative
autoradiography from four independent experiments (2B) and fold
increases in JNK activity (mean.+-.S.D., n=4) (2C). JNK was
significantly activated within 5 min, and its activation was
maximal at 15-20 min after the addition of OVA (FIGS. 2B and
2C).
[0119] MC/9 cells were incubated with 500 ng/ml mouse monoclonal
IgE specific for TNP (TNP-IgE) or 500 ng/ml OVA-IgE for 2 h. FIG.
2D shows MC/9 cells sensitized with TNP-IgE were incubated together
with PBS (control), 10 .mu.g/ml OVA (OVA), 1 .mu.g/ml rat
anti-mouse IgE monoclonal antibody (anti-IgE (1)), or 10 .mu.g/ml
anti-mouse IgE (anti-IgE (10)) for 10 min. MC/9 cells sensitized
with OVA-IgE were incubated with PBS (control), 10 .mu.g/ml OVA
(OVA), 10 .mu.g/ml BSA (BSA), or 1 .mu.g/ml anti-IgE (anti-IgE (1))
for 10 min. GST, glutathione S-transferase. JNK activation by OVA
was not induced in MC/9 cells sensitized with TNP-specific IgE
(TNP-IgE) and BSA did not activate JNK in MC/9 cells sensitized
with OVA-IgE. Anti-mouse IgE antibody activated JNK in both TNP-IgE
and OVA-IgE-sensitized cells (FIG. 2D). Student's t test, Welch's t
test, or a paired t test was used for the statistical analysis.
Example 2
[0120] The following example demonstrates that MEKK1 is activated
by antigen in MC/9 cells.
[0121] Affinity-purified rabbit polyclonal anti-mouse MEK kinase 1
(MEKK1) antibody was prepared by immunizing rabbits with a
recombinant fragment of the amino-terminal domain of MEKK1.
[0122] Addition of OVA (10 .mu.g/ml) induced MEKK1 activation in
MC/9 cells sensitized with OVA-IgE. As a positive control in the
kinase assay for MEKK1, cell lysates from Cos cells that
transiently overexpressed full-length MEKK1 were used.
[0123] To assay for MEKK1, MEKK1 was first immunoprecipitated by
lysing 5.times.10.sup.6 cells by vigorous mixing in 0.4 ml of
extraction buffer (1% Triton X-100, 10 mM Tris-HCl (pH 7.4), 5 mM
EDTA, 50 mM NaCl, 50 mM NaF, 0.1% bovine serum albumin (BSA), 20
.mu.g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 2 mM
Na.sub.3VO.sub.4). The lysate was incubated with the
affinity-purified rabbit anti-MEKK1 antibody (1:100 dilution) for 2
h at 4.degree. C. Recombinant protein G-Sepharose 4B was added to
the lysate and incubated for an additional 30 min at 4.degree. C.
The immune complexes were washed twice with
radioimmunoprecipitation assay buffer (10 mM sodium phosphate (pH
7.0), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40 (Nonidet P-40), 0.1%
SDS, 10 .mu.g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride,
0.1% 2-mercaptoethanol, 50 mM NaF, and 200 .mu.M Na.sub.3VO.sub.4),
twice with PAN buffer (10 mM PIPES pH 7.0), 100 mM NaCl, 20
.mu.g/ml aprotinin) containing 0.5% Nonidet P-40 and once with PAN.
For the in vitro kinase assay, the PAN immune complex suspension
was incubated with catalytically inactive JNKK (JNKK-KR) and 30
.mu.Ci of [.gamma.-.sup.32P]ATP in 1.times.universal kinase buffer
(20 mM PIPES (pH 7.0), 10 mM MnCl.sub.2, and 20 .mu.g/ml aprotinin)
in a final volume of 40 .mu.l for 30 min at 30.degree. C. MEKK1 was
transiently expressed in Cos cells by using lipofectamine (Life
Technologies, Inc.), and the cell lysate was used as a positive
control in the MEKK1 kinase assay. The kinase reaction was
terminated by the addition of 4.times.protein loading buffer, and
the mixture was boiled for 5 min, separated by SDS-10%
polyacrylamide gel, and transferred to nitrocellulose for
autoradiography and immunoblotting. The kinase activity was
quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale,
Calif.). The membranes were probed using the same anti-MEKK1
antibody with an alkaline phosphatase visualization system (Promega
protoblot alkaline phosphatase system, Madison, Wis.).
[0124] MC/9 cells sensitized with OVA-IgE were incubated together
with PBS (0 min) or 10 .mu.g/ml OVA for 0.5, 1, 3, 5, 10, or 20
min. The cell lysate from Cos cells which expressed full-length
MEKK1 (Cos (MEKK1)) was used as a positive control in the kinase
assay. MEKK1 activation in MC/9 cells was observed 30 s after the
addition of OVA to IgE-sensitized cells. MEKK1 activity reached
maximal levels 3 min after OVA addition. MEKK1 activity was
increased to 2.5-3-fold over basal activity. FIGS. 3A and 3B show a
representative autoradiograph from three independent experiments
(FIG. 3A) and fold increases in MEKK1 activity (mean.+-.S.E., n=3)
(FIG. 3B). Kinase activities decreased gradually by 10 min after
addition of OVA. The same membrane in the kinase assay was probed
with the anti-MEKK1 antibody used for immunoprecipitation, and
reactivity was visualized by the alkaline phosphatase system to
ensure that the same amounts of MEKK1 were present in each sample.
Immunoblotting showed a 98-kDa bank of MEKK1, and the density in
each sample was comparable (data not shown).
Example 3
[0125] The following example demonstrates that ERK2 is
phosphorylated and activated by antigen ligation in MC/9 cells.
[0126] The mouse monoclonal anti-mouse ERK2 antibody and bovine
myelin basic protein were obtained from Upstate Biotechnology (Lake
Placid, N.Y.). Goat affinity-purified polyclonal anti-ERK2 (C-14,
amino acids 345-358) antibody was purchased from Santa Cruz
Biotechnology (Santa Cruz, Calif.).
[0127] ERK2 activation induced by tyrosine-threonine
phosphorylation was observed in immunoblots using anti-ERK2
antibody. After different treatments, 1.times.10.sup.6 cells were
lysed in buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic
acid sodium salt, 0.1% SDS, 50 mM Tris (pH 7.6), 10 .mu.g/ml
aprotinin, 5 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride).
Samples were electrophoresed on SDS-10% polyacrylamide gels and
proteins were transferred to nitrocellulose membranes. Membranes
were incubated overnight in blocking buffer containing 1% BSA at
4.degree. C. The monoclonal anti-ERK2 antibody (200 .mu.g/69 .mu.l,
Upstate Biotechnology) was added to the blocking buffer (1:1000),
and blots were incubated for an additional 1 h at room temperature.
The blots were washed in TBST (25 mM Tris (pH 8.0), 125 mM NaCl,
0.025% Tween 20), and specific reactive proteins were detected by
an enhanced chemiluminescence method, employing a sheep anti-mouse
Ig antibody linked to horseradish peroxidase (Amersham Corp.).
[0128] MC/9 cells sensitized with OVA-IgE were incubated with PBS
(0) or 100 pg/ml to 100 .mu.g/ml OVA for 1 min. Cell lysates were
analyzed by SDS-10% polyacrylamide gel and immunoblotting using an
anti-ERK2 antibody. ERK2 was phosphorylated in the presence of
1-100 .mu.g/ml OVA (FIG. 4A). MC/9 cells sensitized with OVA-IgE
were incubated with PBS (0 min) or 10 .mu.g/ml OVA for 0.5, 1, 5,
10, 20, 30, or 40 min. ERK2 phosphorylation was elicited within 30
s, and a clear shift in mobility was observed at 1-20 min after 10
.mu.g/ml OVA stimulation. Phosphorylated ERK2 protein was decreased
30-40 min after OVA addition (FIG. 4B). MC/9 cells sensitized with
OVA-IgE were incubated with PBS or 10 .mu.g/ml OVA for 1, 5, 20,
40, 60, or 90 min. Kinase activity of ERK2 was measured as .sup.32P
incorporation into myelin basic protein. ERK2 was significantly
activated at 1 min after the addition of OVA, and its activation
was maximal at 5-20 min. Significant activation was still observed
at 90 min after OVA stimulation (FIG. 4C).
Example 4
[0129] The following example demonstrates that wortmannin inhibits
JNK activation but not ERK2 activation.
[0130] Wortmannin was obtained from Calbiochem and stored as a 10
mM stock in dimethyl sulfoxide (Me.sub.2SO).
[0131] Wortmannin has been shown to inhibit PI3-kinase when used at
concentrations below 1 .mu.M. The effect of wortmannin (3 nM to 1
.mu.M) on JNK and ERK2 activation induced by 10 .mu.g/ml OVA
stimulation in OVA-IgE-sensitized MC/9 cells was examined. MC/9
cells sensitized with OVA-IgE were incubated with 0.01% Me.sub.2SO
(control and 0 nM) or 3-1000 nM wortmannin for 15 min. The cells
were then incubated with 10 .mu.g/ml OVA or PBS (control) for 10
min. The data are expressed as the percentage of JNK activity
detected in the presence of 10 .mu.g/ml OVA and 0.01% Me.sub.2SO
(FIGS. 5A and 5B) or as the percentage of ERK2 activity stimulated
by 10 .mu.g/ml OVA in the presence of 0.01% Me.sub.2SO (FIG. 5C).
(*, p<0.05; **, p<0.01.)
[0132] Wortmannin inhibited JNK activity in a dose-dependent
manner. The kinase activity in cells treated with 100 nM wortmannin
was decreased to 8% of that observed in the absence of treatment
(FIGS. 5A and 5B). In contrast, 100-300 nM wortmannin did not
significantly inhibit ERK2 activation induced by OVA (FIG. 5C).
[0133] The aggregation of Fc.epsilon.RI initiates diverse signal
transduction pathways. In addition to the release of mast cell
granule contents, these pathways lead to late responses such as the
increase in c-fos and c-jun expression and modulation of cytokine
gene expression. Electrophoretic migration and activation of ERK2
was observed in antigen-stimulated MC/9 cells. Based on the present
invention, it is believed that a role for ERKs in mast cells is the
activation of cytosolic phospholipase A2, which would result in the
production of arachidonic acid derivatives such as LTC4 and PGD2.
Functionally, antigen-stimulated JNK activity in mast cells
functions in the regulation of AP-1 activity and cytokine gene
expression. The present invention provides the first evidence for
the regulation of the MEKK/JNKK-contingent pathway, and not the ERK
pathway, in mast cell cytokine production, thus permitting genetic
analysis of the role of this pathway in mast cell biology.
Example 5
[0134] The following example demonstrates that TNF-.alpha. is
generated by antigen in passively sensitized MC/9 cells.
[0135] Recombinant mouse TNF-.alpha., purified rat anti-mouse
TNF-.alpha. monoclonal antibody (ELISA Capture), and biotinylated
rabbit anti-mouse TNF-.alpha. polyclonal antibody (ELISA Detection)
were purchased from Pharmingen (San Diego, Calif.).
[0136] An ELISA for TNF-.alpha. production was performed as
follows. Purified rat anti-mouse TNF-.alpha. monoclonal antibody
was diluted to 2 .mu.g/ml in coating solution (0.1 M NaHCO.sub.3,
pH 8.2) and 50 .mu.l was added to wells of an ELISA plate (Dynatech
Laboratories). After overnight incubation at 4.degree. C., wells
were washed twice with washing solution (0.05% Tween-20/PBS) and
blocked with PBS containing 10% FCS (10% FCS/PBS ) at room
temperature for 2 hrs. After washing twice, standards (16 pg/ml-4
ng/ml recombinant mouse TNF-.alpha.) and samples were added at 100
.mu.l per well and incubated overnight at 4.degree. C. After
washing four times, biotinylated rabbit anti-mouse TNF-.alpha.
polyclonal antibody (1 .mu.g/ml) was added to wells and incubated
at room temperature for 45 min and wells were washed six times. 2
.mu.g/ml avidin-peroxidase was added to wells, incubated at room
temperature for 30 min, and wells were washed eight times. ABTS
(2,2'-Azino-bis(3-ethylbe- nzthiazoline-6-sulfonic acid) substrate
(30 mg/ml 0.1 M citric acid, pH 4.35) containing 0.03%
H.sub.20.sub.2 was added at 100 .mu.l per well and color reaction
was allowed to develop at room temperature for 30 min. A plate was
read at OD 410 nm and analyzed by Mycroplate Manager (Bio Rad,
Hercules, Calif.).
[0137] MC/9 cells (1.times.10.sup.6/ml) were incubated with 500
ng/ml mouse monoclonal IgE specific for OVA (OVA-IgE) for 2 h.
After washing, 1.times.10.sup.6 sensitized MC/9 cells were
incubated in the presence of 10 ng/ml to 100 .mu.g/ml OVA for 3 h.
After the incubation, the cell supernatant was harvested and
TNF-.alpha. production was measured by ELISA. TNF-.alpha.
production reached maximal by addition of 10 .mu.g/ml OVA
(mean.+-.S.D., n=4). 1-100 .mu.g/ml OVA induced TNF-.alpha.
production at 3 h after addition of OVA (FIG. 6). 3.6 ng
TNF-.alpha. was produced from 1.times.10.sup.6 cells in the
presence of 10 .mu.g/ml OVA.
[0138] MC/9 cells sensitized with OVA-IgE (1.times.10.sup.6/ml)
were incubated in the presence of PBS (0 h) or 10 .mu.g/ml OVA for
0.5, 1.0, 1.5. 2.0, 2.5, 3.0, or 4.0 h. TNF-.alpha. was detected 1
h after addition of OVA and reached maximal at 2.5-3.0 h
(mean.+-.S.D., n=4). TNF-.alpha. was not detected in the
supernatant at 30 min after the addition of 10 .mu.g/ml OVA.
TNF-.alpha. production leveled at 2.5-3 hrs after the addition of
OVA (FIG. 7).
[0139] The effects of a protein synthesis inhibitor, cycloheximide,
and an RNA transcription inhibitor, actinomycin D, on TNF-.alpha.
production were examined. 1 .mu.g/ml cycloheximide or 1 .mu.g/ml
actinomycin D was incubated with cells 15 min before the addition
of OVA. Both cycloheximide and actinomycin D completely blocked
TNF-.alpha. production 3 h after stimulation (less than 30 pg per
million cells) (data not shown).
[0140] The aggregation of Fc.epsilon.RI on mast cells is essential
for the induction of allergic inflammation. Following aggregation,
mast cells secrete a variety of preformed chemical mediators, such
as histamine, and newly synthesized arachidonic acid derivatives.
In addition to these biologically active substances, aggregation of
Fc.epsilon.RI on mast cells leads to the production of cytokines
and chemokines such as IL-3, IL-5, IL-6, TNF-.alpha., GM-CSF, and
MIP-1.alpha.. Among these cytokines, TNF-.alpha., is produced in
large amounts in mast cell lines. Both mouse bone marrow-derived
mast cells and human cultured mast cells also produce TNF-.alpha..
Therefore, TNF-.alpha. is likely to be involved in allergic
inflammation initiated by mast cell activation. TNF-.alpha. is a
multifunctional cytokine which has effects in inflammation. Like
other cytokines, TNF-.alpha. is newly synthesized following the
aggregation of Fc.epsilon.RI on mast cells. Stimulation via the
Fc.epsilon.RI markedly increases the levels of TNF-.alpha. mRNA in
BMMC and some mast cell lines. The TNF-.alpha. gene shows the
characteristics of an immediate-early gene in activated mast cells.
It is strongly induced within 30 min in antigen-stimulated mast
cells. MC/9, which does not have preformed TNF-.alpha., produces
TNF-.alpha. following the aggregation of Fc.epsilon.RI. The present
inventors have shown herein that cycloheximide and actinomycin D
completely inhibited TNF-.alpha. production induced by
Fc.epsilon.RI aggregation, demonstrating that TNF-.alpha. is
synthesized de novo following activation in MC/9 cells.
Example 6
[0141] The following example illustrates that p38 is activated by
antigen in MC/9 cells.
[0142] MC/9 cells sensitized with OVA-IgE were incubated in the
presence of PBS (0 min) or 10 .mu.g/ml OVA for 1, 5, 15, 30, or 60
min. To immunoprecipitate p38 kinase, 3.times.10.sup.6 cells were
lysed by vigorous mixing in 0.4 ml of extraction buffer (1% Triton
X-100, 10 mM Tris-HCl (pH 7.4), 5 mM EDTA, 50 mM NaCl, 50 mM MaF,
0.1% bovine serum albumin (BSA), 20 .mu.g/ml aprotinin, 1 mM PMSF,
and 2 mM Na.sub.3VO.sub.4). The lysate was incubated with the
rabbit anti-serum raised against the COOH-terminal peptide sequence
of p38 (1:400 dilution) for 2 h at 4.degree. C. Recombinant protein
G sepharose 4B was added to the lysate and incubated for an
additional 1 h at 4.degree. C. The immunoprecipitates were washed
once with extraction buffer, twice with PAN buffer (10 mM
piperazine-N,N'-bis (2-ethanesulfonic acid) (PIPES, pH 7.0), 100 mM
NaCl, 20 .mu.g/ml aprotinin). For the in vitro kinase assay, the
immunoprecipitates were suspended in 25 .mu.l of assay buffer (25
mM Hepes, pH 7.4, 25 mM .beta.-glycerophosphate, 25 mM NaCl.sub.2,
2 mM dithiothreitol, 0.1 mM Na.sub.3VO.sub.4) containing a
recombinant NH.sub.2-terminal fragment of ATF-2 (20-50 ng) as a
substrate and 5 .mu.Ci [.gamma..sup.32P] ATP. The kinase reaction
was terminated by the addition of 4.times.protein loading buffer,
and the mixture was boiled for 5 min and separated by SDS-12%
polyacrylamide gel. The gel was fixed with 5% acetic acid and 10%
methanol solution, dried, and subjected to autoradiography. The
kinase activity was quantified with a PhosphorImager (Molecular
Dynamics, Sunnyvale, Calif.).
[0143] Following addition of OVA to MC/9 cells sensitized with
OVA-IgE, p38 was significantly activated. OVA induced p38
activation in a dose-dependent manner (data not shown). p38 was
significantly activated at 1 min and its activation was maximal
(about 12-fold increase) at 5 min after the addition of OVA. p38
activities decreased gradually after 15-60 min (FIG. 8).
[0144] Example 1 demonstrates that JNK is strongly activated in
mast cells following aggregation of Fc.epsilon.RI. This example
demonstrates that another member of MAP kinase family, p38, the
osmotic imbalance responsive kinase similar to the yeast Hogl
enzyme (#Lin) is also activated in mast cells by aggregation of
Fc.epsilon.RI. p38, like JNK, is also activated by treatment of
cells with pro-inflammatory cytokines and environmental stress such
as extracellular changes in osmolarity. Its activation depends on
dual phosphorylation on threonine-180 and tyrosine-182.
Example 7
[0145] The following example demonstrates that wortmannin inhibits
both p38 MAP kinase activation and TNF-.alpha. production.
[0146] MC/9 cells sensitized with OVA-IgE(3.times.10.sup.6) were
preincubated with either 1-1000 nM wortmannin or a control (0.01%
DMSO) for 15 min and stimulated for 5 min by addition of 10
.mu.g/ml OVA. Wortmannin inhibited p38 MAPK activity in a
dose-dependent manner. 100 nM-1 .mu.M wortmannin significantly
inhibited p38 MAPK activation (50-60% decrease in kinase
activities) (FIG. 9). The data are expressed as the percentage of
p38 activity detected in the presence of 10 .mu.g/ml OVA and 0.01%
DMSO (*, p<0.05). However, the inhibitory effects of wortmannin
on p38 MAP kinase activation in stimulated MC/9 cells were not as
strong as the inhibitory effects of wortmannin on JNK activation as
shown in Example 4.
[0147] The effect of wortmannin on TNF-.alpha. production in MC/9
cells was also examined. MC/9 cells sensitized with OVA-IgE
(1.times.10.sup.6/ml) were incubated with 0.01% DMSO (control and 0
nM) or 1-1000 nM wortmannin for 15 min. The cells were then
incubated with 10 .mu.g/ml OVA for 3 h. TNF-.alpha. in the medium
was measured by ELISA. 1 nM-1 .mu.M wortmannin inhibited
TNF-.alpha. production in a dose-dependent manner (mean.+-.S.D.,
n=4, *, p<0.05; **, p<0.01). TNF-.alpha. production was
decreased by 77% in the presence of 100 nM wortmannin (FIG.
10).
[0148] This example shows that p38 activation, like JNK activation,
is significantly inhibited by the PI3-kinase inhibitor, wortmannin,
treatment. PI3-kinase is an enzyme important in intracellular
trafficking, actin polymerization, and growth factor signaling.
PI3-kinase is activated following aggregation of Fc.epsilon.RI in a
rat basophilic leukemia cell line, RBL-2H3. The inhibitory effects
of wortmannin on JNK and p38 activation were observed in
antigen-stimulated mouse bone marrow derived mast cells as well as
MC/9 cells (data not shown). Wortmannin also inhibited TNF-.alpha.
production of antigen-stimulated MC/9 cells in a dose-dependent
manner. The concentrations of wortmannin which inhibited
TNF-.alpha. production were similar to concentrations at which
wortmannin inhibits PI3-kinase. These results indicate that
inhibition of PI3-kinase by wortmannin decreases JNK and p38
activation following FceRI aggregation and that activation of p38
can enhance the effects of the MEKK/JNKK-contingent pathway of the
present invention on TNF-.alpha. production in antigen-stimulated
mast cells.
Example 8
[0149] The following example illustrates that the MEK inhibitor, PD
098059, inhibits ERK2 activation, but does not inhibit TNF-.alpha.
production, JNK activation, or p38 activation in MC/9 cells.
[0150] MEK inhibitor, PD#098059, was kindly provided by Dr. David
Dudley (Warner Lambert Company, Ann Arbor, Mich.) and stocked 100
mM in DMSO. PD 098059 is a noncompetitive inhibitor of MAP kinase
kinase (MEK). It exerts its effect by binding to the inactive form
of MEK1.
[0151] MC/9 cells sensitized with OVA-IgE (1.times.10.sup.6/ml)
were incubated with 0.1% DMSO (control and 0 nM) or 3-30 .mu.M PD
098059 for 1 h and the reaction was stopped by centrifugation at 5
min after the stimulation. The cells were then incubated with 10
.mu.g/ml OVA or PBS (control) for 5 min. Kinase activity of ERK2
was measured as .sup.32P incorporation into myelin basic protein
(MBP).
[0152] 1.times.10.sup.6 cells were lysed in buffer (150 mM NaCl, 1%
NP40, 0.5% deoxycholic acid sodium salt, 0.1% SDS, 50 mM Tris (pH
7.6), 10 .mu.g/ml aprotinin, 5 mg/ml leupeptin, 1 mM PMSF). Samples
were electrophoresed on SDS-10% polyacrylamide gels and proteins
were transferred to nitrocellulose membranes. Membranes were
incubated overnight in blocking buffer containing 1% BSA at
4.degree. C. The monoclonal anti-ERK 2 antibody (200 .mu.g/69
.mu.l, Upstate Biotechnology) was added to the blocking buffer
(1:1000) and blots were incubated for an additional 1 h at room
temperature. The blots were washed in TBST (25 mM Tris, pH 8.0, 125
mM NaCl, 0.025% Tween 20) and specific reactive proteins were
detected by an enhanced chemiluminescence method, employing a sheep
anti-mouse Ig antibody linked to horseradish peroxidase (Amersham,
Arlington Heights, Ill.).
[0153] In vitro kinase assay of ERK2 was carried out as described
above with some minor modifications. 3.times.10.sup.6 cells were
lysed by vigorous mixing in 0.4 ml of lysis buffer (20 mM Tris-HCl
(pH 8.0), 1% Triton X-100, 10% glycerol, 137 mM NaCl, 1.5 mM
MgCl.sub.2, 1 mM EGTA, 50 mM NaF, 1 mM Na.sub.3VO.sub.4, 1 mM PMSF,
5 .mu.g/ml leupeptin, and 10 .mu.g/ml aprotinin). The lysate was
incubated with anti-ERK2 antibody (2 .mu.g/ml) for 2 hrs at
4.degree. C. Recombinant protein G sepharose 4B was added to the
lysate and incubated for an additional 1 hr at 4.degree. C. The
immune complexes were washed three times with lysis buffer, and
once with kinase buffer (30 mM Tris-HCl (pH 8.0), 20 mM MgCl.sub.2,
2 mM MnCl.sub.2). For the in vitro kinase assay, the immune complex
suspension was incubated with 9 .mu.g myelin basic protein (MBP)
and 10 .mu.Ci of [.gamma.-.sup.32P] ATP in kinase buffer in a final
volume of 30 .mu.l for 30 min at 30.degree. C. The reaction was
stopped by an additional 10 .mu.l of 4.times. protein loading
buffer. After the samples were boiled for 3 min, they were
separated by SDS-12% polyacrylamide gel and stained with Coomassie
brilliant blue, exhaustively destained, dried, and subjected to
autoradiography. The bands corresponding to MBP were cut out of the
gel, and radioactivity was determined by liquid scintillation
counting.
[0154] 10-30 .mu.M PD 098059 significantly inhibited ERK2
activities in MC/9 cells. OVA-induced ERK2 activity was decreased
65% in the presence of 30 .mu.M PD 098059 (FIG. 11). The data are
expressed as the percentage of ERK2 activity detected in the
presence of 10 .mu.g/ml OVA and 0.1% DMSO (*, p<0.05; **,
p<0.01). However, PD 098059 did not inhibit JNK or p38
activation (data not shown).
[0155] MC/9 cells sensitized with OVA-IgE (1.times.10.sup.6/ml)
were incubated with 0.1% DMSO or 30 .mu.M PD 098059 for 1 h. The
cells were then incubated with 10 .mu.g/ml OVA for 3 h. TNF-.alpha.
in the medium was measured by ELISA. 30 .mu.M PD 098059 did not
affect TNF-.alpha. production (mean.+-.S.D., n=4, NS, no
significance). In contrast to the effect seen with wortmannin in
example 7, PD 098059 did not affect on TNF-.alpha. production at 3
h after addition of OVA (FIG. 12).
[0156] Cytokine production in mast cells via Fc.epsilon.RI is
mediated by the phosphoinositide hydrolysis, an increase in
intracellular calcium, and protein kinase C (PKC) activation
because such production can be induced by calcium ionophore or the
protein kinase C activator, PMA. MC/9 cells also produce cytokines
including IL-2, IL-3, IL-4, and GM-CSF, after stimulation with PMA
(phorbol myristate acetate) plus the calcium ionophore A23187.
Prior to the present invention, it was thought that the
Ras-dependent ERK signal transduction pathway was the intermediate
in the transduction pathway leading to increases in gene
transcription and proliferation in mast cells. ERKs phosphorylate
specific transcription factors including members of the Ets family
such as Elk-1. MEK inhibitor, PD 098059, selectively blocks the
activation of MEK1 by Raf or MEKK in vitro. It exerts its effect by
binding to the inactive form of MEK1, inhibiting both the
phosphorylation and activation of ERK.
[0157] The present inventors have shown herein that PD 098059
strongly inhibited both ERK2 activity and phosphorylation induced
by the aggregation of Fc.epsilon.RI in MC/9 cells. However, it did
not inhibit TNF-.alpha. production, JNK activation or p38
activation. On the contrary, PD 098059 enhanced OVA-induced
promoter activity of TNF-.alpha. although it did not enhance the
TNF-.alpha. level in the medium. This demonstrates that ERK2
activation is not required for TNF-.alpha. production in mast
cells. Without being bound by theory, the present inventors believe
that ERK2 might regulate be a negative regulator of transcription
of TNF-.alpha. since inhibition of ERK2 activation enhanced
activity of the TNF-.alpha. promoter in antigen-stimulated MC/9
cells.
Example 9
[0158] The following example demonstrates that wortmannin, but not
PD 098059, inhibits TNF-.alpha. promoter activity in MC/9 cells
stimulated by Fc.epsilon.RI aggregation.
[0159] The pXP1 plasmid containing full length of the human
TNF-.alpha. promoter just upstream of the luciferase gene,
designated pTNF(-1311)Luc, was provided by Dr. James S. Economou
(Division of Surgical Oncology, UCLA School of Medicine, Los
Angels, Calif.). pTNF(-1311)Luc was transfected into MC/9 by the
DEAE-dextran method. 2.times.10.sup.6 cells were washed once with
1.times.TBS (25 mM Tris, 137 mM NaCl, 5 mM KCl, 0.5 mM
Na.sub.2HPO.sub.4, 0.49 mM MgCl.sub.2, 0.68 mM CaCl.sub.2, pH 7.5).
Cells were suspended with 0.4 ml of 500 .mu.g DEAE-dextran/4
.mu.gDNA mixture and incubated at room temperature for 30 min.
After washed with 1.times.TBS, cells were suspended with 10 ml of
culture medium and plated on culture dish. After 24 h of the
transfection, cells were passively sensitized with OVA-IgE and
incubated with 10 .mu.g/ml OVA for additional 15 h. In the
experiment for cotransfection, 10 .mu.g pCMV5MEKK.sub.COOH,
expression plasmid encoding MEKK.sub.COOH, a truncated activated
form of MEKK1 or 10 .mu.g pCMV5, control empty plasmid, was
transfected with 4 .mu.g pTNF(-1311)Luc and transfected cells were
harvested after 24 h of the cotransfection. For in vitro kinase
assay, 4 .mu.g or 10 .mu.g pCMV5MEKK.sub.COOH, or equivalent amount
of control empty plasmid was transfected similarly and cells were
harvested for in vitro kinase assay 24 h after the
transfection.
[0160] Luciferase activity was measured to measure TNF-.alpha.
promoter activity. Cell pellets were lysed in 200 .mu.l of a buffer
containing 25 mM Tris, pH 7.8, 2 mM EDTA, 2 mM dithiothreitol, 10%
glycerol, 1% Triton X-100. 30 .mu.l of the lysate was mixed with
the same volume of Luciferase Assay Substrate containing beetle
luciferin as a substrate (Promega, Madison Wis.), and
chemiluminescence was measured for 30 sec as relative light units
by a luminometer (Monolight 2010, Analytical Luminescence
Laboratory, San Diego, Calif.).
[0161] MC/9 cells transiently transfected by pTNF(-1311)Luc were
passively sensitized with OVA-IgE, and incubated for an additional
15 h with 10 .mu.g/ml OVA or PBS in the presence of 0.01% DMSO or
100 nM wortmannin. Similarly, cells were incubated with 10 .mu.g/ml
OVA or PBS in the presence of 0.1% DMSO or 30 .mu.M PD 098059.
Luciferase activity in cell lysates was measured as relative light
units (RLU) and standardized by control RLU (mean.+-.S.D., N=4,
p<0.01). OVA addition elicited a 5-6 fold induction of
luciferase activity in the cells. 100 nM wortmannin significantly
inhibited luciferase activity induced by addition of OVA (40%
decrease in luciferase activity) (FIG. 13). In contrast to
wortmannin, PD 098059 enhanced OVA-induced luciferase activity
(FIG. 14). Therefore, the PI3-K inhibitor, wortmannin, inhibited
TNF-a promoter activity in MC/9 cells, whereas the MEK inhibitor,
PD 098059, enhanced TNF-a promoter activity.
Example 10
[0162] The following example demonstrates that overexpression of
MEKK1 by an MC/9 cells greatly enhances the activity of JNK and
TNF-.alpha. promoter, weakly enhances p38 activity and ERK2
activity in antigen-activated MC/9 cells.
[0163] 4 or 10 .mu.g of pCMV5MEKK.sub.COOH (MEKK1(4) or MEKK1(10))
or equivalent amount of pCMV5 empty plasmid (pCMV5(4) or pCMV5(10))
was transfected into MC/9 cells. Cells treated with only
DEAE-dextran were used as a control. JNK activities (FIG. 15), p38
activities (FIG. 16), and ERK2 activities (data not shown) were
measured at 24 h after the transfection. FIGS. 15-16 graphically
show representative autoradiographs from each three of independent
experiments. Kinase activities were standarized by control activity
and expressed as fold-activation (mean.+-.S.D., n=3, *, p<0.05;
**, p<0.01).
[0164] JNK activity in the MC/9 cells transiently transfected by
pCMV5MEKK.sub.COOH was strongly increased compared with the cells
transfected by pCMV5 empty plasmid or treated with DEAE-dextran
(Pharmacia Biotech, Uppsala, Sweden) alone. More than 11-15 fold
increases of JNK activity was observed in
pCMV5MEKK.sub.COOH-transfected cells compared with pCMV5 empty
plasmid-transfected cells (FIG. 15). In contrast, p38 activity was
increased to a much lesser degree by the transfection of
pCMV5MEKK.sub.COOH (FIG. 16). ERK2 was also activated by
pCMV5MEKK.sub.COOH-transfection. However, the degree of ERK2
activation was less than that of JNK (data not shown).
[0165] 10 .mu.g of pCMV5MEKK.sub.COOH (MEKK1) or 10 .mu.g of pCMV5
empty plasmid (pCMV5) was transfected into MC/9 cells with 4 .mu.g
of pTNF(-1311)Luc. Luciferase activities were measured as relative
light units at 24 h after the transfection.
[0166] Cotransfection of pCMV5MEKK.sub.COOH and pTNF(-1311)Luc
elicited a 1000-fold induction of luciferase activities compared
with cotransfection of pCMV5 empty plasmid and pTNF(-1311)Luc,
demonstrating that overexpression of MEKK1 results in an increase
in the level of TNF-a promoter activity (FIG. 17).
[0167] A protein kinase cascade leading to activation of JNK is
dependent on MEK kinase 1 (MEKK1). MEKK1 was identified as
MEK-activating kinase unrelated to Raf-1. MEKK1 is activated in
both a Ras-dependent and -independent manner. Examples 3A and 3B
show, for the first time, that MEKK1 is also activated following
the aggregation of Fc.epsilon.RI in MC/9 cells. Furthermore, the
present inventors show herein that overexpression of MEKK1 strongly
induces JNK activation and only weakly induces ERK activation.
[0168] JNKK activates both JNK and p38. As shown herein, however,
JNK was strongly activated, but the activation of p38 was poor, in
pCMV5.sub.COOH-transfected MC/9 cells. Overexpression of MEKK1
induced by transfection of pCMV5MEKK.sub.COOH enhanced the activity
of TNF-.alpha. promoter strongly in MC/9 cells. Luciferase activity
in pCMV5MEKK.sub.COOH-transfected cells was 1000-fold higher than
that in the cells transfected by control vector. These results
demonstrate that activation of TNF-.alpha. promoter induced by
MEKK1 overexpression is caused by JNK activation. Thus, MEKK1 and
JNKK activation leading to JNK activation is directly involved in
the gene transcription of TNF-.alpha. in antigen-stimulated because
aggregation of Fc.epsilon.RI induces the activation of both MEKK1
and TNF-.alpha. promoter. Taken together, the above examples show
that the MEKK/JNKK-contingent pathway is the primary signal
transduction pathway leading to TNF-.alpha. production by mast
cells, but other wortmannin sensitive pathways such as p38 can
enhance such transcription of TNF-.alpha. in antigen-stimulated
mast cells.
Example 11
[0169] The following example illustrates that cross-linking of
c-kit on MC/9 cells synergizes with FceRI aggregation to greatly
enhance both JNK activation and TNF-a production. FIG. 18 shows
that JNK activity is greatly enhanced in MC/9 cells activated by
FceRI aggregation. Cross-linking of c-kit by c-kit ligand in the
absence of FceRI aggregation only weakly activates JNK. However,
when both c-kit and FceRI are cross-linked, JNK activation is
enhanced almost 4 fold above the level achieved with FceRI
aggregation alone.
[0170] FIG. 19 shows the results of a similar experiment in which
TNF-a production was measured. Like JNK, TNF-a production was
greatly enhanced when both c-kit and FceRI were cross-linked as
compared to cross-linking either receptor alone.
[0171] Together, these experiments further demonstrate that
activation of signal transduction by c-kit synergizes with FceRI
aggregation to activate the MEKK/JNKK-contingent signal
transduction pathway of the present invention.
Example 12
[0172] This example demonstrates that aggregation of FceRI on MC/9
cells activates the transcription factor NF.kappa.B. FIG. 20 shows
that cross-linking of FceRI on MC/9 cells activates the
transcription factor NF.kappa.B. Since NF.kappa.B is known to
interact with various cytokine promoters to induce cytokine
production, this example illustrates that FceRI aggregation, which
has been shown herein to induce the MEKK/JNKK-contingent pathway of
the present invention, can activate transcription factors involved
in cytokine production.
[0173] Another transcription factor, the nuclear factor of
activated T cells (NF-AT) is essential for transcription of the
IL-2 gene in activated T cells. Without being bound by theory, it
is believed that NF-AT may be one in a family of related
transcription factors that regulate the transcription of cytokine
genes in mast cells as well as T cells because aggregation of the
Fc.epsilon.RI induces NF-AT DNA binding activity in rat mast cells.
NF-AT binding motifs are present in the promoter region of
TNF-.alpha. genes as well as in the IL-2, IL-3, IL-4, and GM-CSF
promoters.
[0174] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. It is to be expressly understood, however, that such
modifications and adaptations are within the scope of the present
invention, as set forth in the following claims:
* * * * *