Title: Use of transcription factors for treating inflammation and other diseases
Abstract: The present invention provides a method of treating inflammation in a mammal comprising altering the activity of a transcription factor involved in the inflammatory response. The invention also relates to the use of transcription factors to screen compounds that are capable of reducing inflammation. The invention also relates to the use of transcription factors in methods of diagnosing the presence of an inflammatory disease in a tissue of a mammal and methods of monitoring the treatment of an inflammatory disease in a tissue of a mammal.
Patent Number: 6,869,929 Issued on 03/22/2005 to Oettgen, et al.
| Inventors:
|
Oettgen; Peter (Brookline, MA);
Libermann; Towia (Newton, MA);
Goldring; Mary (Auburndale, MA)
|
| Assignee:
|
Beth Israel Deaconess Medical Center (Boston, MA)
|
| Appl. No.:
|
393905 |
| Filed:
|
March 20, 2003 |
| Current U.S. Class: |
514/2; 530/350 |
| Intern'l Class: |
C07K 014//00 |
| Field of Search: |
514/2
530/350
|
References Cited [Referenced By]
U.S. Patent Documents
| 6024940 | Feb., 2000 | Ghio et al. | 424/45.
|
Other References
Rudders et al., "ESE-1 Is a Novel Transcriptional Mediator of Inflammation
That Interacts with NF-.kappa.B to Regulate the Inducible Nitric-oxide
Synthase Gene", The Journal of Biological Chemistry, 276(5):3302-3309
(2001).
|
Primary Examiner: Carlson; Karen Cochrane
Attorney, Agent or Firm: Conlin; David G., Rosenfield; Jennifer K.
Edwards & Angell, LLP
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
Funding for this invention was provided in part by the Government of the
United States of America National Institutes of Health Grant R01/CA76323,
by National Institutes of Health Grant K08/CA 71429; Grant No. RO1/AR45378
from NIH/NIAMS, Grant No. 1RO1/AI49527-01 from NIH/NIAID and Grant No.
1RO1/CA763230-02 from NIH/NCI. The Government has certain rights in this
invention.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation-in-part of PCT Application No.
PCT/US01/29340 and having an international filing date of Sep. 20, 2001
and published in English under PCT Article 21(2), which claims priority to
provisional application No. 60/234,379, filed Sep. 20, 2000, the entire
teachings of which are incorporated herein by reference.
Claims
We claim:
1. A method of screening compounds that are capable of reducing
inflammation comprising:
(a) providing cells which do not normally express a measurable amount of a
transcription factor except in the presence of a pro-inflammatory agent;
(b) providing two portions of said cells to form a control and exoerimental
group of cells;
c) providing the experimental group of cells with said compound;
(d) providing the pro-inflammatory agent to both the control and
experimental group of cells;
(e) measuring the expression of the transcription factor in both the
control and the experimental group of cells; and
(f) comparing the amount of expression of said transcription factor in the
experimental group of cells with the control group of cells,
wherein decreased expression of said transcription factor in the
experimental group of cells when compared to the control group of cells
indicates that the compound may be capable of reducing inflammation in
vivo.
2. The method according to claim 1, wherein the transcription factor
comprises a STAT transcription factor, C/EBPs, HMG protein, EGR-1 or AP-1.
3. The method according to claim 1, wherein the transcription factor
comprises an Ets transcription factor.
4. The method according to claim 3, wherein the Ets transcription factor
comprises ESE-1 or ESE-1 related factors, e.g., ESE-2 and ESE-3.
5. The method according to claim 3, wherein the Ets transcription factor
comprises Ets-1, Ets-2, ERG, SAP-1, ELK-1, Erp-1, TEL-1, TEL-2, PU.1 and
FLI-1.
6. The method according to claim 1, wherein the compound comprises a small
molecule, peptide, antisense RNA or viral DNA.
7. The method according to claim 1, wherein the inflammatory agent
comprises a pro-inflammatory cytokine, endotoxin or a virus.
8. The method according to claim 7, wherein the pro-inflammatory cytokine
comprises IL-1, IL-1.beta., TNF-.alpha., and IL-15, IL-17, IL-18,
oncostatin M, and leukemia inhibitory factor.
9. The method according to claim 1, wherein the cells comprises
fibroblasts, synoviocytes, chondrocytes, murine monocytes, glioma cells,
osteoblasts, smooth muscle cells, endothelial cells, or monocytic cells.
10. The method according to claim 9, wherein the smooth muscle cells
comprises vascular smooth muscle cells.
11. The method according to claim 7, wherein the endotoxin comprises LPS.
Description
FIELD OF THE INVENTION
This invention relates to methods of treating inflammation in a tissue
comprising altering the activity of a transcription factor, which is
involved in the inflammatory response. The transcription factor is
preferably expressed in said tissue in response to a pro-inflammatory
agent. The invention also relates to the use of transcription factors to
screen compounds that are capable of reducing inflammation. The invention
also relates to the use of transcription factors in methods of diagnosing
the presence of an inflammatory disease in a tissue of a mammal and
methods of monitoring the treatment of an inflammatory disease in a tissue
of a mammal.
BACKGROUND OF THE INVENTION
Inflammatory processes generally contribute to host defense against
infections and as a stress response to tissue injury. Conversely,
inflammation contributes to the chronic or acute pathological processes in
autoimmune and cardiovascular diseases and other conditions that lead to
tissue injury and destruction. Inflammation is a hallmark of several
vascular diseases including atherosclerosis, restenosis, and the
vasculopathy associated with transplantation. The most common vascular
disease, atherosclerosis, begins when lipoproteins, and in particular low
density lipoprotein (LDL) enter the subendothelium and become oxidized.
Oxidized LDL stimulates the production of interleukin-1 and other
inflammatory cytokines. These cytokines activate adhesion molecules,
including VCAM-1, ICAM-1, and E-selectin, on the endothelial surface,
which promote the attachment of, and transmigration of monocytes. The
expression of the inducible form of nitric oxide synthase (NOS2) has also
been shown to be upregulated by inflammatory cytokines and endotoxin in
cultured cells found in the atherosclerotic plaque including macrophages,
smooth muscle cells, T lymphocytes, and endothelial cells (Esaki, T., et
al. 1997. Atherosclerosis 128:39-46; Rikitake, Y., et al. 1998,
Atherosclerosis 136:51-7; Xu, R., et al. 1999, Life Sci 64:2451-62).
Furthermore, immunohistological studies have demonstrated the expression
of NOS2 in the atherosclerotic lesions in these cell types as well
(Buttery, L. D., et al. 1996, Lab Invest 75:77-85; Esaki, T., et al. 1997,
Atherosclerosis 128:39-46).
The induction of the NOS2 gene is also associated with more acute forms of
vascular inflammation such as endotoxemia. The generation of the potent
vasodilator, nitric oxide (NO) by NOS2, is at least in part responsible
for the hypotension seen in association with bacterial sepsis (Wei, 1995
#1021) (MacMicking, 1995 #1020). NOS2 gene expression is also induced in
other types of vascular inflammation including restenosis and in the
accelerated atherosclerosis associated with heart transplantation (Ikeda,
U., et al. 1998, Clin Cardiol 21:473-6; Lafond-Walker, A., et al. 1997, Am
J Pathol 151:919-25).
Rheumatoid arthritis (RA) is a prototypical immune-mediated disease
characterized by chronic inflammation in the synovium and the destruction
of joints, in which, similar to other inflammatory disorders, a central
role for interleukin (IL)-1 and tumor necrosis factor (TNF)-.alpha. has
been established. These cytokines and bacterial endotoxins have major
roles in inflammatory responses via the activation of a variety of
transcription factors.
Upon binding of cytokines or other inflammatory mediators to their
corresponding receptors, several classes of transcription factors function
as mediators of these stimuli. For example, within minutes of
interleukin-1 beta (IL-1.beta. treatment, the expression of the immediate
early genes cFos and c-Jun are induced. These transcription factors are
the constituent proteins for AP-1 (Conca, W., et al. 1991, J Biol Chem
266:16265-8; Conca, W., et al. 1989, J Clin Invest 83:1753-7). One of the
target genes of IL-1.beta., the collagenase gene, can be activated by AP-1
alone (Angel, P., I. et al. 1987, Mol Cell Biol 7:2256-66). Multiple
signaling pathways have been implicated in the activation of these
immediate early genes by IL-1.beta. including the Janus kinases (JAKs),
MAP kinases, and protein kinase A (Hill, C. S., and R. Treisman. 1995,
Cell 80:199-211; Karin, M. 1995, J Biol Chem 270:16483-6; Sadowski, H. B.,
et al. 1993, Science 261:1739-44; Treisman, R. 1996, Curr Opin Cell Biol
8:205-15; Wagner, B. J., et al. 1990, Embo J 9:4477-84).
The propagation of inflammation is dependent on several other transcription
factors for the activation of multiple genes. The nuclear factor kappa B
(NF-.kappa.B) transcription factors are dimeric proteins involved in the
activation of a large number of genes in response to inflammatory stimuli.
Although originally described to have been important in lymphoid cells and
lymphoid specific genes, NF-.kappa.B has clearly been shown to play an
important role in a whole host of other cell types and target genes. The
p50 and p65 subunits of NF-.kappa.B have also been shown to bind to other
transcription factors through protein interactions often resulting in
synergistic transactivation of the target genes of NF-.kappa.B (Baeuerle,
P. A., and D. Baltimore. 1996, Cell 87:13-20; DiDonato, J. A., et al.
1997, Nature 388:548-54).
One of the major transcriptional circuits implicated in inflammation is the
NF-.kappa.B/I.kappa.B pathway. NF-.kappa.B is rapidly activated by
proinflammatory cytokines and endotoxins and is involved in the regulation
of a large set of inflammatory response genes including various cytokines
and chemokines, acute phase proteins, cell adhesion proteins,
immunoglobulins, and viral genes. Most of these genes are directly
regulated by NF-.kappa.B via high affinity binding sites within their
respective promoter regions. However, the regulation of a significant
number of inflammatory response genes by cytokines cannot be attributed
exclusively to direct interaction of NF-.kappa.B with binding sites within
their regulatory regions, thereby suggesting that additional pathways play
critical roles in the transcriptional regulation of these genes.
Osteoarthritis (OA) is another example of a disease having an inflammatory
response. OA is a slowly progressive disease with multiple etiologies
involving biomechanical, biochemical, and genetic factors, all of which
may contribute to the OA lesion in cartilage by disrupting
chondrocyte-matrix associations and altering metabolic responses in the
chondrocyte. The central role of cytokines, particularly interleukin
(IL)-1 and tumor necrosis factor (TNF)-.alpha., in causing the destruction
of articular cartilage is well established. It is generally accepted that
the chondrocyte is the target of cytoline action, although the sources
responsible for generating the cytokines are less clear in the context of
OA pathogenesis. Even in the absence of classical inflammation
characterized by infiltration of neutrophils and macrophages into joint
tissues, elevated levels of proinflammatory cytokines have been measured
in OA synovial fluids. Nevertheless, symptoms of local inflammation and
synovitis are present in many patients and in animal models of OA. Thus,
the fibroblast- and macrophage-like synovial cells, as well as the
chondrocytes themselves, are potential sources of cytokines that could
induce chondrocytes to synthesize and secrete cartilage-degrading
proteases and other cytokines and proinflammatory mediators.
The complexity of the cytokine network that may be involved in OA has
increased with the recent discoveries of additional proinflammatory and
destructive, as well as inhibitory, cytokines that may amplify or modify
the effects of the primary catabolic cytokines. Changes in the patterns of
the production of growth factors or their receptors may also contribute to
the course of the disease. Aspects of the role of the chondrocyte in OA
and lessons from animal models have been reviewed recently. (Goldring M B,
Connec Tiss Res 1999, 40:1-11; Goldring M B. Arthritis Rheum 2000, in
press.).
Cytokines and growth factors are produced in joint tissues and released
into the synovial fluid, and they act on the resident cells in an
autocrine-paracrine manner. Many of these factors are necessary at low
levels for normal homeostasis, but in OA their balance may be disturbed.
The major proinflammatory cytokines, which are generally also catabolic,
include IL-1.alpha. and .beta., TNF-.alpha., IL-6, leukemia inhibitory
factor (LIF), oncostatin-M (OSM), IL-8, IL-17, and IL-18. The
anti-inflammatory cytokines, IL-4, IL-10, IL-11, IL-13, IL-1 receptor
antagonist (IL-1ra) and IFN-.gamma., are classified as inhibitory
cytokines, since they may block the actions of catabolic cytokines.
Members of the transforming growth factor (TGF)-.beta./bone morphogenetic
protein (BMP) family, insulin-like growth factor-I, and fibroblast growth
factors (FGFs) are considered to be major anabolic factors for cartilage,
since they may oppose cartilage destruction by promoting synthesis of
matrix proteins. Some of these factors, such as IL-6 and TGF-.beta., may
have dual roles. The role of cytokines in osteoarthritis is described in
further detail in Goldring, M. B., Current Rheumatology Reports, Jul. 1,
2000, incorporated by reference in its entirety.
The Ets genes are a family of at least thirty members that function as
transcription factors (Wasylyk B., H. S. L., Giovane A. 1993, Eur. J.
Biochem. 211:7-18). All Ets factors share a highly conserved 80-90 amino
acid long DNA binding domain, the ETS domain. Ets factors play a central
role in regulating genes involved in development, cellular differentiation
and proliferation. Many macrophage, B and T cell specific genes are
regulated by Ets factors. The role of Ets factors in the immune system has
been substantiated by experiments in mice where the genes encoding several
Ets factors have been disrupted by homologous recombination. The PU.1
knockout is characterized by a lack of immune system development (Scott,
E. W., et al. 1994, Science 265:1573-7). The Ets-1 knockout mice are
characterized by T cell apoptosis and increased terminal B cell
differentiation (Muthusamy, N., K. Barton, and J. M. Leiden. 1995, Nature
377:639-42).
Epithelial cell-specific members of the Ets transcription factor family,
i.e., ESE-2, ESE-3, and PDEF have been isolated. Recently, a novel member
of the Ets factor, called ESE-1, was discovered. Under normal
physiological conditions ESE-1 expression is restricted to many epithelial
cell types in a variety of tissues with highest expression in the
gastrointestinal tract.
ESE-1 is the prototype member of a new subclass of Ets factors and has
several interesting features when compared to other Ets family members.
First, unlike other Ets factors which are either ubiquitously expressed or
primarily expressed in lymphoid cells, ESE-1 appears to have an
epithelial-specific expression pattern under basal conditions. Second,
unlike all other Ets factors, ESE-1 has two DNA binding domains, a
classical Ets domain and in addition an A/T hook domain also found in high
mobility group (HMG) proteins. (See Oettgen, P., et al. 1996, Mol Cell
Biol 16:5091-106; Oettgen, P., et al. 1999, Genomics 55:358-62; Oettgen,
P., et al. 1997, Genomics 445:456-7; and Oettgen, et al., 1997, Mol. Cell
Biol. 17(8):4419-33. These references are incorporated herein in their
entirety).
One important medical need is the effective treatment of cardiovascular
disease, inflammation, and autoimmune diseases. At the moment these
diseases are treated with drugs that have inadequate safety profiles and
limited efficacy. Currently, the therapeutic alternatives available to
treat inflammation consist of the use of corticosteroids or non-steroidal
anti-inflammatory agents (NSAIDS). Unfortunately, all of these
anti-inflammatory agents are associated with significant side effects,
including gastrointestinal irritation and bleeding, bone loss, and fluid
retention, some of which can be life-threatening. These anti-inflammatory
drugs are therefore not ideal therapeutic agents. Other drugs that target
only a single gene involved in inflammatory processes are not effective
enough, since only a single component of inflammation is targeted leaving
all the other components untouched.
For example, experimental approaches for OA therapy have targeted
production or activities of catabolic cytokines. In addition to
anticytokine therapy, selective MMP inhibitors targeting enzymes that
degrade cartilage-specific collagens and aggrecan also offer the potential
to halt cartilage damage. Protein kinases that regulate signal
transduction pathways induced by catabolic cytokines have also been
proposed as therapeutic targets (Lewis A J, et al. Curr Opin Chem Biol
1999, 3:489-494; Badger A M, et al. Arthritis Rheum 2000, 43:175-183).
These include the stress-activated protein kinases (SAPKs), c-Jun
N-terminal kinases (JNKs) and p38 MAP kinase, and the IKK1 and IKK2
kinases that release nuclear factor (NF)-.kappa.B from its inhibitor
I.kappa.B, which are known to be activated in chondrocytes by catabolic
cytokines. The loss of cartilage in OA is a consequence not only of the
disturbed production of and responsiveness to catabolic factors, but also
the failure of cartilage repair once it begins to breakdown. Thus, therapy
should begin early and target pivotal catabolic pathways without affecting
normal homeostasis. Current procedures for repairing or transplanting
articular cartilage that is more severely damaged do not provide long-term
restoration of the normal cartilage surface (Buckwalter J A, et al.
Arthritis Rheum 1998, 42:1331-1342.). Autologous chondrocyte
transplantation has been used somewhat successfully in traumatic defects
of knee cartilage in young adults. However, the repair of more extensive
defects in OA patients will require the development of approaches for
genetically engineering chondrocytes prior to transplantation to not only
promote cartilage-specific matrix synthesis, but also to counteract the
effects of inflammatory and catabolic cytokines (Evans C H, et al.,
Arthritis Rheum 1999, 42:1-16.).
Pharmacological interventions for OA have focused primarily on improving
symptoms, although new agents may offer the possibility of preserving
normal homeostasis. Since the increased synthesis of MMPs, prostaglandins,
and other inflammatory and catabolic factors in OA tissues appears to be
related to elevated levels of IL-1 and TNF-.alpha., therapies that
interfere with the expression or actions of these cytokines are most
promising.
It would be useful to have effective methods of treating different types of
inflammation, such as vascular inflammatory disorders, rheumatologic
disorders, dermatologic inflammatory diseases, gastrointestinal
inflammatory diseases and kidney disorders, to name a few. It would be
especially useful to have methods of treating inflammation that modulate
the transcription factors that mediate inflammation. It would also be
useful to have methods of screening compounds that are capable of reducing
an inflammatory response, especially methods that modulate the expression
of the transcription factors involved in the response.
SUMMARY OF THE INVENTION
The present invention provides a method of treating inflammation in a
mammal comprising altering the activity of a transcription factor involved
in the inflammatory response. In preferred methods, the transcription
factor is expressed in said mammal in response to a pro-inflammatory agent
and the transcription factor is not normally expressed, or is expressed at
a low level, in the absence of the pro-inflammatory agent. The methods of
the present invention are useful for treating inflammation located in a
tissue, organ or synovial fluid of the mammal. In certain methods of the
present invention, altering the activity comprises decreasing the activity
of the transcription factor. In certain of these embodiments, the step of
decreasing the activity of a transcription factor further comprises either
decreasing the function of the transcription factor or blocking the
expression of the transcription factor.
In yet other methods, altering the activity comprises increasing the
activity of the transcription factor. In certain of these methods, the
step of increasing the activity of a transcription factor further
comprises either increasing the function of the transcription factor or
increasing the expression of the transcription factor.
As aforesaid, the transcription factor is one that is involved in an
inflammatory response. Examples of transcription factors useful in the
methods of the present invention include, but are not limited to an Ets
transcription factor, a STAT transcription factor, C/EBPs, HMG protein,
e.g., SOX protein, and other proteins having A/T hook domains, EGR-1 or
AP-1. One of ordinary skill in the art can readily select useful
transcription factors for use in the present methods, based on the
teachings disclosed herein.
In preferred embodiments, the transcription factor is selected from the
family of Ets transcription factors, including, ESE-1, and ESE-1 related
factors, ESE-2, and ESE-3. In especially preferred methods of the present
invention, the transcription factor comprises ESE-1. The methods and
products of the present invention will be described with reference to ETS
transcription factors, and ESE-1 in particular. However, it is to be
understood that the invention is not limited thereto. Other transcription
factors may also be useful in the present invention. Furthermore, it is to
be understood that such reference to the ESE-1 polypeptide refers to
naturally occurring and non-naturally occurring peptides and variants
thereto. One of ordinary skill in the art can readily determine useful
variants of the polypeptides.
The inflammation treated by the present methods, includes inflammation
associated with an inflammatory disease, e.g., vascular inflammatory
disorders, rheumatologic disorders, dermatologic inflammatory diseases,
gastrointestinal inflammatory diseases and kidney disorders. Examples of
the rheumatologic disorders include, but are not limited to, rheumatoid
arthritis, osteoarthritis, vasculitis, sclereoderma, systemic lupus
erthymotosus and collagen vascular disorder. Examples of vascular
inflammatory disorders include, but are not limited to bacterial sepsis.
Other examples of diseases that can be treated by the present methods
include, but are not limited to, atherosclerosis, restenosis,
transplantation associated arteriopathy, psoriasis, transplant rejection,
multiple sclerosis, diabetes, and Alzheimer's disease and fever.
The step of blocking the expression of the transcription factor can be
accomplished in many ways that are known to one of ordinary skill in the
art, e.g., inhibiting the activation of the promoter for the gene encoding
the transcription factor. In certain embodiments, the step of inhibiting
activation further comprises providing a substance that blocks the
function or expression of the transcription factor. The substance can be
selected by one of ordinary skill in the art but include, e.g., small
molecules, peptides, dominant negative mutants, antisense RNAs, and DNA
viruses. Examples of a substance that inhibits activation of the ESE-1
transcription factor include ESE-1 dominant negative proteins, e.g., ESE-1
DN1 or ESE-1 DN2.
Similarly, in the methods in which the activity of the transcription factor
is increased, this can be accomplished in many ways that are known to one
of ordinary skill in the art, e.g., activating the promoter for the gene
encoding the transcription factor. In certain embodiments, the step of
increasing activation further comprises providing a substance that
increases the function or expression of the transcription factor. The
substance can be selected by one of ordinary skill in the art but include,
small molecules, peptides, dominant positive mutants, antisense RNAs, and
DNA viruses. Examples of such substances include, e.g., I.kappa.B kinase
and p38 kinase. Preferred substances mimic or enhance the activity of the
transcription factor.
In certain methods, the transcription factor comprises ESE-1 and the step
of inhibiting activation further comprises preventing the binding of
binding proteins, e.g., p50 and p65 subunits of NF-.kappa.B, to the ESE-1
promoter NF-.kappa.B site. In certain embodiments, the step of preventing
binding comprises the step of mutating the ESE-1 promoter NF-.kappa.B site
or otherwise blocking the binding site.
In other methods, the step of inhibiting ESE-1 function comprises
preventing or enhancing ESE-1 phosphorylation or acetylation or preventing
nuclear translocation.
The substance that alters the activity of the transcription factor can be
provided in vivo systemically, or alternatively, the substance is provided
to the site of inflammation, depending on the result desired. For example,
the substance, e.g., small molecule drugs, peptides, dominant negative
mutants by gene delivery mechanisms, antisense RNA, can be used to block
the function or expression of the transcription factor, e.g., ESE-1,
systemically to treat a disease such as atherosclerosis or rheumatoid
arthritis. Alternatively, local delivery of an ESE-1 blocking agent can be
used to treat localized inflammation as is seen in restenosis after
balloon angioplasty for the treatment of coronary artery disease, or in
the joints of rheumatoid arthritis patients or to treat transplant
associated arteriopathy. Methods of in vivo administration, e.g., gene
therapy are know to one of ordinary skill in the art.
The invention provides methods of screening compounds that are capable of
reducing inflammation. One such method comprises: (a) providing cells
which do not normally express a measurable transcription factor but do
express the transcription factor in the presence of a pro-inflammatory
agent; (b) providing to a portion of the cells a compound to be screened;
(c) providing a portion of the cells as a control without the compound;
(d) providing the pro-inflammatory agent to the cells; (e) measuring the
expression of the transcription factor in the cells, and (f) comparing the
amount of expression of the transcription factor in the cells containing
the compound with the control portion of cells. In preferred methods, the
transcription factor is an Ets transcription factor. In especially
preferred methods, the transcription factor is ESE-1.
The invention further provides screening methods where the compound of
interest is a small molecule, peptide, antisense RNA or viral DNA. The
inflammatory agent can be selected by one of ordinary skill in the art. In
preferred methods the inflammatory agent comprises a pro-inflammatory
cytokine, endotoxin, IL-17, IL-18, oncostatin M, and leukemia inhibitory
factor. Examples of pro-inflammatory cytokines include IL-1.beta.,
TNF-.alpha., and IL-15, and examples of endotoxin include LPS. Another
pro-inflammatory agent comprises a virus.
Examples of cells, which are useful in screening methods of the present
invention include, but are not limited to, fibroblasts, synoviocytes,
chondrocytes, murine monocytes, glioma cells, osteoblasts, smooth muscle
cells, endothelial cells, monocytic cells, and keratinocytes. Examples of
smooth muscle cells include vascular smooth muscle cells.
In accordance with another aspect of the present invention, there are
provided ESE-1 agonists. Among preferred agonists are molecules that mimic
ESE-1, that bind to ESE-1-binding molecules or receptor molecules, and
that elicit or augment ESE-1-induced responses. Also among preferred
agonists are molecules that interact with ESE-1 or ESE-1 polypeptides, or
with other modulators of ESE-1 activities, and thereby potentiate or
augment an effect of ESE-1 or more than one effect of ESE-1.
In accordance with yet another aspect of the present invention, there are
provided ESE-1 antagonists. Among preferred antagonists are those which
mimic ESE-1 so as to bind to an ESE-1 receptor or binding molecules, but
not elicit an ESE-1-induced response or more than one ESE-1-induced
response. Also among preferred antagonists are molecules that bind to or
interact with ESE-1 so as to inhibit an effect of ESE-1 or more than one
effect of ESE-1 or which prevent expression of ESE-1.
The invention also provides methods of diagnosing the presence of an
inflammatory disease in a mammal comprising: (a) removing a sample from
the mammal and (b) measuring the presence and/or amount of a transcription
factor wherein the transcription factor is not present in detectable
amounts in the sample in the absence of the inflammatory disease. In the
methods of the present invention, the sample comprises tissue, synovial
fluid, cerebrospinal fluid (CSF), urine or blood. Preferably, the
transcription factor is an Ets transcription factor. In certain
embodiments, the transcription factor is ESE-1. The inflammatory diseases
diagnosed by the methods of the present invention include rheumatological
or autoimmune diseases, atherosclerosis, restenosis, transplantation
associated arteriopathy, psoriasis, for example. In certain methods, the
rheumatological or autoimmune disease comprise rheumatoid arthritis,
osteoarthritis, vasculitis, sclereoderma, systemic lupus erthymotosus. The
presence or amount of the transcription factor can be measured by methods
known in the art and also by methods described herein.
The invention further provides a method of monitoring the treatment of an
inflammatory disease in a mammal comprising: removing a sample from the
mammal subsequent to said treatment and measuring the presence or amount
of a transcription factor wherein the transcription factor is not present
in detectable amounts in the mammal in the absence of the inflammatory
disease. In the methods of the present invention, the sample comprises
tissue, synovial fluid, urine, CSF or blood. Preferably, the transcription
factor is an Ets transcription factor. In certain embodiments, the
transcription factor is ESE-1. The inflammatory diseases diagnosed by the
methods of the present invention include rheumatological or autoimmune
diseases, atherosclerosis, restenosis, transplantation associated
arteriopathy, psoriasis, for example. Other examples of diseases include
rheumatological or autoimmune disease such as rheumatoid arthritis,
osteoarthritis, vasculitis, sclereoderma, systemic lupus erthymotosus. In
certain embodiments of these methods, the method further comprises
repeating the removing and measuring steps at subsequent intervals and
comparing the amounts of the transcription factor to determine if the
treatment is effective.
The present invention also relates to a pharmaceutical composition for the
treatment of inflammation comprising a compound that alters the expression
of a transcription factor, e.g., Ets, and a pharmaceutically acceptable
carrier. Preferred compositions comprise compounds that alter the
expression of ESE-1. Examples of compounds that are useful in such
compositions include small molecules, peptides, or antisense RNA. In
certain embodiments, the composition further comprises an agent that
stimulates cartilage repair, e.g., a cartilage inducing growth and
differentiation factor in a collagen scaffold or synthetic polymer.
The present invention further relates to altering the expression of an
inflammatory response gene comprising modulating the expression of a
transcription factor which affects the expression of the gene. In some
embodiments, the step of altering the expression of an inflammatory
response gene comprises decreasing the expression or the activity of the
transcription factor. The step of decreasing the activity of the
transcription factor further comprises either decreasing the function of
the transcription factor or blocking the expression of the transcription
factor. In other embodiments, altering the expression of an inflammatory
response gene comprises increasing the activity of the transcription
factor. In certain of these embodiments, the step of increasing the
activity of a transcription factor further comprises either increasing the
function of the transcription factor or increasing the expression of the
transcription factor.
Preferably, the transcription factor is an epithelium specific, e.g., Ets,
transcription factor. In certain embodiments, the transcription factor is
ESE-1. Examples of inflammatory response genes comprise genes for
metalloproteinases, genes associated with apoptosis, genes for nuclear
orphan receptor (MINOR), inducible nitric oxide synthase (NOS-2),
cyclooxygenase (COX-2), phospholipase A2, angiogenesis genes (VEGF and FGF
and their receptors). Examples of metalloproteinases include, e.g., MMP-1,
MMP-3, MMP-8, MMP-9, MMP-13, MMP-14, aggrecanases, e.g., ADAM-TS4,
ADAM-TS5. Genes associated with apoptosis include FAS and DR5, for
example.
A further approach would be to use the methods of the present invention to
prevent the detrimental effects of proinflammatory cytokines on cartilage
specific matrix protein synthesis by inhibiting expression or action of
ESE-1. For example, the methods can be used to reverse the inhibitory
effect of ESE-1 on Type II collagen gene expression.
The present invention also relates to a method of treating a disease
comprising increasing the activity of a transcription factor, wherein the
transcription factor is either not expressed in diseased tissue or
expressed in low amounts. The transcription factor increases the
expression of a product that is useful for treating the disease. For
example, it would be useful to increase nitric oxide synthase (NOS-2),
cyclooxygenase (COX-2) or Prostaglandin E2 (PGE2) in certain instances.
For increasing PGE2 may actually counter act some of the IL-1 induced
effects (e.g., inhibition of type II collagen) in osteoarthritis. Thus, by
using the present methods to increase PGE2 activity, instead of other
treatments, e.g., NSAIDS, which lose the protective effects of
prostaglandins, treatments may be more effective.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows ESE-1 induction to inflammatory stimuli. FIG. 1(a) shows
RT-PCR analysis of ESE-1 expression in human aortic smooth muscle cells
(HASMCs), human umbilical vein endothelial cells(HUVECs), and the THP-1
monocytic cell line at different time points after stimulation with
IL-1.beta., TNF-.alpha., or LPS (See methods for details of PCR). FIG.
1(b) shows Northern blot analysis of ESE-1 induction in primary human
aortic smooth muscle cells. 10 ug of total RNA were used per lane. (See
methods for details of Northern blot analysis).
FIG. 2 shows a schematic diagram of the human ESE-1 promoter demonstrating
conserved transcription factor binding sites (Ets, NF-.kappa.B, Ap-1, and
CRE) and the TATA box.
FIG. 3 shows ESE-1 promoter induction by LPS. FIG. 3(A) shows induction of
the ESE-1 promoter in RAW 264.7 cells in response to LPS. FIG. 3(B) Effect
of mutation in NF-.kappa.B site(m ESE-1 promoter) upon activation by LPS.
FIG. 4 shows electrophoretic mobility shift assays (EMSA) with ESE-1
promoter NF-.kappa.B site. FIG. 4(A) shows EMSA using an oligonucleotide
probe encoding the ESE-1 promoter NF-.kappa.B site, with whole cell
extracts derived from human aortic smooth muscle cells at different time
points(0,1,2,4,6,24 hours). FIG. 4(B) shows EMSA using ESE-1 NF-.kappa.B
oligonucleotide probe and whole cell extracts derived from IL-1 stimulated
cell at 0 hours(control) or 1 hour, in the presence or absence of
antibodies to the different Rel family members. An antibody to the
unrelated Bcl-3 protein is used as a negative control.
FIG. 5 shows ESE-1 transactivation of the promoters of potential target
genes. FIG. 5(A) shows cotransfection of the mammalian expression plasmid
with luciferase reporter constructs of different potential target genes,
including CD44, interleukin-6, NOS2, E-selectin, and ICAM-2. FIG. 5(B)
shows cotransfection of ESE-1 with the long (-1485 to +31) and short (-233
to +31) forms of the murine NOS2 promoter.
FIG. 6 shows Electrophoretic Mobility Shift Assay (EMSA) of in vitro
translated ESE-1 with an oligonucleotide probe encoding either the PSP
promoter Ets site(lane 2), or the NOS2 promoter Ets site(lane 4) compared
with unprogrammed reticulocyte lysate control(lanes 1 and 3).
FIG. 7 shows Mutational analysis of NOS2 Ets binding site. FIG. 7(A) shows
cotransfection of wild type NOS2 promoter or the mutated NOS2 promoter
with pCI or pCI-ESE-1 mammalian expression vectors. FIG. 7(B) shows
evaluation of effect of Ets site mutation on LPS induction.
FIG. 8 shows synergistic effect of NF-.kappa.B with ESE-1 upon NOS2
transactivation. Cotransfection experiments of different combinations of
the pCI mammalian expression plasmid containing cDNAs encoding either
ESE-1, p50, or p65, were performed in RAW 264.7 or RASMCs, with the NOS2
promoter luciferase reporter construct(short).
FIG. 9 shows ESE-1 interaction with p50. FIG. 9(A) shows .sup.35
S-methionine in vitro translated rabbit reticulocyte lysates of p50, p65,
and unprogrammed lysate(control) separated by SDS gel electrophoresis.
Molecular weight markers are shown on the left. FIG. 9(B) shows binding of
p50 to ESE-1, another Ets factor NERF-2, and several deletion constructs,
and the GST-fusion protein alone(control). FIG. 9(C) shows a schematic of
ESE-1 constructs used in GST-fusion experiments. (See methods for details
of the GST-pull down experiment).
FIG. 10 shows expression of ESE-1 in the rat aorta during acute
inflammation. Immunohistological evaluation of ESE-1 protein expression in
the rat aorta before and 24 hours after systemic administration of
endotoxin(see methods for details). HP (high power) LP(low power).
FIG. 11 shows induction of ESE-1 mRNA in patients with rheumatoid arthritis
and PVNS and in non-epithelial cells by inflammatory stimuli.
FIGS. 11(a) and 1(b) show induction of ESE-1 mRNA in human chondrocytes by
proinflammatory cytokines and endotoxin.
FIG. 11(c) shows induction of ESE-1 mRNA in human synovial fibroblasts by
proinflammatory cytokines and endotoxin.
FIG. 11(d) shows induction of ESE-1 mRNA in human LB-12 osteoblasts by
proinflammatory cytokines and endotoxin.
FIG. 11(e) shows induction of ESE-1 mRNA in THP-1 human monocytes by
proinflammatory cytokines and endotoxin.
FIG. 11(f) shows induction of ESE-1 mRNA in U-138 MG and U-373 MG human
glioma cells by proinflammatory cytokines and endotoxin.
Subconfluent cultures of the human costal chondrocyte cell lines, T/C28 a2,
C28/I2, C20A4 were incubated in the absence or presence of IL-1.beta.,
TNF-.alpha., or IFN-.gamma. for 24 h (a) or for 0.5, 2, 6, 12, or 24 hours
(b) and ESE-1 mRNA was analyzed by RT-PCR. Human synovial fibroblasts (c)
at passage 4 were incubated in the absence or presence of IL-1.beta.,
indomethacin, or a combination of both for 6 hours, 24 hours, and 5 days.
RT-PCR analysis was performed using ESE-1 and GAPDH specific primers.
Northern blot analysis of ESE-1 mRNA expression in human osteoblasts (d)
upon stimulation by IL-1.beta.. Cultures of the human LB-12 osteoblast
cell line were incubated in the absence or presence of cycloheximide,
IL-1.beta., hydrocortisone, or combinations thereof for 1, 2, 6, and 24
hours. Stimulation of ESE-1 mRNA by LPS in human monocytes (e). THP-1
cells were grown in the absence or presence of LPS for 1, 2, 6, or 24
hours and ESE-1 mRNA was analyzed by RT-PCR. Kinetics of ESE-1 mRNA
induction by IL-1.beta. in human glioma cell lines (f). U-138 MG and U-373
MG glioma cells were grown in the absence or presence of IL-1.beta. for 1,
2, 6, or 22 hours.
FIG. 12 shows transcriptional activation of the ESE-1 promoter by
IL-1.beta. and LPS involves NF-.kappa.B.
FIG. 12(a) shows U-138 MG cells which were transfected with either the
parental pXP2 luciferase plasmid or the pXP2 luciferase construct
containing the ESE-1 promoter (pXP2/ESE-1) and incubated in the absence or
presence of IL-1.beta. for 16 h.
FIG. 12(b) shows RAW cells which were transfected with the pXP2/ESE-1
construct and incubated in the absence or presence of LPS for 16 h. Data
shown are means of duplicate measurements from one representative
transfection. Experiments were repeated three times with different plasmid
preparations with comparable results.
FIG. 12(c) shows interaction of NF-.kappa.B with the NF-.kappa.B binding
site in the ESE-1 promoter. Whole cell extracts isolated from U-138 MG
cells stimulated with IL-1.beta. for 0, 3, and 8 hours were analyzed by
EMSA using the labeled human ESE-1/NF-.kappa.B site oligonucleotide or the
human IL6/NF-.kappa.B site oligonucleotide as probes. Competitions were
carried out with either no competitor, or 1, 10, and 100 ng of unlabeled
wild type or mutant ESE-1/NF-.kappa.B oligonucleotides.
FIG. 12(d) shows that the NF-.kappa.B/rel family members p50 and p65
interact with the NF-.kappa.B binding site in the ESE-1 promoter.
Supershift-EMSAs using whole cell extracts from U-138 MG cells stimulated
with IL-1.beta. for 8 hours and the ESE-1/NF-.kappa.B probe were carried
out with either no antibody, or antibodies against p50, p65, relB, p52,
c-rel, and bcl-3. The arrow indicates the NF-.kappa.B DNA-protein complex.
FIG. 12(e) shows sequences of the wild type ESE-1/NF-.kappa.B site (SEQ ID
NO: 24) and the mutation (SEQ ID NO: 25) introduced within the ESE-1
promoter.
FIG. 12(f) shows that mutation of the NF-.kappa.B site within the ESE-1
promoter abolishes induction by IL-1.beta.. U-138 MG cells were
transfected with the ESE-1 promoter/pXP2 luciferase construct containing
either the wild type or a mutant NF-.kappa.B site and incubated in the
absence or presence of IL-1.beta.. Luciferase activity in the lysates was
determined 16 h later. Data shown are means of duplicate measurements from
one representative transfection. The experiment was repeated four times
with different plasmid preparations with comparable results.
FIG. 12(g) shows that adenovirus-mediated overexpression of I.kappa.B
inhibits ESE-1 induction by IL-1.beta.. U-138 MG cells were infected with
an adenovirus expressing I.theta.B or as a control an adenovirus
expressing .beta.-galactosidase prior to stimulation by IL-1.beta. for 8
hours. Total RNA was extracted and analyzed by RT-PCR using ESE-1 and
GAPDH specific primers.
FIG. 13(a) shows RAW cells which were co-transfected with the pXP2
luciferase construct containing the COX-2 promoter (pXP2/COX-2) and the
pCI/ESE-1 expression vector and incubated in the absence or presence of
LPS. Luciferase activity in the lysates was determined 16 h later.
FIG. 13(b) shows the sequence of the COX-2 promoter (SEQ ID NO: 26). Five
putative Ets binding sites are highlighted within the COX-2 promoter
sequence.
FIG. 13(c) shows that mutation of multiple Ets binding sites within the
COX-2 promoter inhibits induction by ESE-1. RAW cells were co-transfected
with the pCI/ESE-1 expression vector and the COX-2 promoter luciferase
constructs containing either wild type (WT) or combinations of multiple
mutant Ets binding sites (Ets MUT). Luciferase activity in the lysates was
determined 16 h later.
FIG. 13(d) shows that mutation of the Ets binding sites reduces LPS-induced
transactivation of the COX-2 promoter. RAW cells were transfected with the
wild type COX-2 promoter luciferase construct or the COX-2 promoter
luciferase containing mutations in the Ets binding sites 1, 2, 3, and 5.
Luciferase activity in the lysates was determined 16 h later.
FIG. 13(e) shows that dominant-negative mutant ESE-1 inhibits LPS mediated
transactivation of the COX-2 promoter. RAW cells were co-transfected with
the pCI expression vector containing a dominant-negative mutant of ESE-1
(ESE-1/DN-MUT) and the COX-2 promoter luciferase constructs. Cells were
grown in the absence or presence of LPS. Luciferase activity in the
lysates was determined 16 h later. Data shown are means of duplicate
measurements from one representative transfection.
FIG. 14 shows stimulation of DR5 in the presence of ESE-1, EGFP.
FIG. 15 shows the increase in MMP1 in the presence of ESE1, EGFP.
FIG. 16 shows the structure of the COL2A1 promoter. The ETS sites that are
potential ESE-1 binding sites are previously identified Sp1, Egr-1/Sp1,
and TATA-box sites are represented schematically. The oligonucleotides
(SEQ ID NOS 27-38. respectively in order of appearance), containing
wild-type and mutant COL2A1 sequences, which were use for EMSAs and
site-directed mutagenesis, are listed below. The ESE-1 binding site is
marked with a circle and mutations are underlined.
FIG. 17 shows the induction of ESE-1 mRNA by IL-1.beta. in human
chondrocytes.
FIG. 17 shows C-28/I2ells plated in serum-containing culture medium and
cultured for 5 days, and confluent cultures were changed to serum-free
medium containing 1% Nutridoma 24 h before treatment with IL-1.beta. for
the times indicated. Cycloheximide (10 .mu.g/ml) was also added in the
absence or presence of IL-1.beta..
FIG. 18 shows the effects of ESE-1, ELF-1 and NERF-2 overexpression on
COL2A1 promoter activity in cotransfection assays. The T/C-28a2 cells were
cotransfected with pGL2-COL2/4.0 and the empty vector, pCI, or (A) with
increasing amounts of the pCI-ESE1 expression vector, (B) with an
expression vector, pCI-ESE1, pCI-ELF1, or pCI-NERF2, or with increasing
amounts of pCI-ESE1 or pCI-ESE3 (not shown). Cotransfections with the
pGL2-basic vector gave values of <1.0 and did not respond to any of the
expression vectors (not shown). After lipofection, the cells were
incubated for 24 h prior to the addition of IL-1.beta. for a further 18 h,
cell harvest, and luciferase assay.
FIG. 19 is a deletion analysis of COL2A1 promoter activity and response to
IL-1.beta. and pCI-ESE1 in transient transfections. The C-28/I2 cells were
transfected with pGL2 constructs containing COL2A1 sequences, -577/+127
bp, -530/+127 bp, -403/+127 bp, -131 /+127 bp, and -83/+127 bp.
Cotransfection with 200 ng of pCI (empty vector) or 50, 100, or 200 ng of
pCI-ESE1 was also performed. The cultures were then incubated for 24 h to
permit expression of recombinant proteins. The cultures cotransfected with
pCI were then treated without or with IL-1.beta. and the incubations
continued for a further 18 h. All cultures were harvested at the same time
for luciferase assay of reporter gene expression.
FIG. 20A is an EMSA analysis of ESE-1 binding to the ESE-1 consensus and
COL2A1 promoter sequences. End-labeled double-stranded oligonucleotides
containing the ESE-1 consensus or wild-type COL2A1 oligonucleotides (see
FIG. 1) were incubated in the absence (-) or presence (+) of recombinant
ESE-1.
FIG. 20B is an EMSA analysis of nuclear protein binding to COL2A1 promoter
sequences. End-labeled double-stranded oligonucleotides containing COL2A1
sequences (see FIG. 1) were incubated with nuclear extracts from untreated
(-) or IL-1.beta.-treated (+) C-28/I2 cells.
FIG. 21A shows the structure of an ESE-1 dominant negative protein (ESE-1
DN 1).
FIG. 21B shows the ability of ESE-1 DN1 to block ESE-1 in vitro.
FIG. 21C shows the effect of ESE-1 DN1 on LPS induction of the NOS2
promoter.
FIG. 22A shows the amino acid sequence of ESE-1 DN1 (SEQ ID NO: 39), a
dominant negative protein of ESE-1.
FIG. 22B shows the amino acid sequence ESE-1 DN2 (SEQ ID NO: 40), a
dominant negative protein of ESE-1.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
"Isolated" means altered "by the hand of man" from its natural state; i.e.,
that, if it occurs in nature, it has been changed or removed from its
original environment, or both.
For example, a naturally occurring polynucleotide or a polypeptide
naturally present in a living animal in its natural state is not
"isolated," but the same polynucleotide or polypeptide separated from the
coexisting materials of its natural state is "isolated", as the term is
employed herein. For example, with respect to polynucleotides, the term
isolated means that it is separated from the chromosome and cell in which
it naturally occurs.
As part of or following isolation, such polynucleotides can be joined to
other polynucleotides, such as DNAs, for mutagenesis, to form fusion
proteins, and for propagation or expression in a host, for instance. The
isolated polynucleotides, alone or joined to other polynucleotides such as
vectors, can be introduced into host cells, in culture or in whole
organisms. Introduced into host cells in culture or in whole organisms,
such DNAs still would be isolated, as the term is used herein, because
they would not be in their naturally occurring form or environment.
Similarly, the polynucleotides and polypeptides may occur in a
composition, such as a media, formulations, solutions for introduction of
polynucleotides or polypeptides, for example, into cells, compositions or
solutions for chemical or enzymatic reactions, for instance, which are not
naturally occurring compositions, and, therein remain isolated
polynucleotides or polypeptides within the meaning of that term as it is
employed herein.
This invention relates to the use of a transcription factor, particularly
an Ets transcription factor, for treating and/or diagnosing inflammatory
diseases. It was previously believed that Ets transcription factor ESE-1
was implicated in the regulation of epithelial-specific genes and under
normal physiological conditions ESE-1 expression is restricted to cells of
the epithelial cell lineage. It was discovered that ESE-1 is a master
switch of inflammation.
This invention pertains to the discovery that this transcription factor is
inducible in non-epithelial cells and that stimulation by pro-inflammatory
agents, e.g., IL-1.beta. (interleukin-1.beta.) and TNF-.alpha. (-.alpha.,
tumor necrosis factor-.alpha.), and endotoxins, e.g., LPS, results in
rapid induction of ESE-1 in various non-epithelial cell types. Examples of
non-epithelial cell types include, but are not limited to, synovial
fibroblasts, chondrocytes, osteoblasts, monocyte/macrophages, and glial
cells, as well as endothelial cells and vascular smooth muscle cells.
The upregulation of ESE-1 by the pro-inflammatory agents, e.g.,
pro-inflammatory cytokines and endotoxin, indicates that certain
transcription factors, e.g., ESE-1, are novel mediators of the
inflammatory response. Further evidence of a role in inflammatory
disorders is provided by the fact that ESE-1 is expressed in the synovium
of rheumatoid arthritis patients and patients with Pigmented villonodular
synovitis (PVNS) (see examples below). ESE-1 is also expressed in synovial
tissues in osteoarthritis patients. Furthermore, ESE-1 is rapidly and
transiently induced in non-epithelial cell types, i.e., synovial
fibroblasts, chondrocytes, osteoblasts and monocytes/macrophages, by
IL-1.beta., TNF-.alpha. and endotoxin (e.g., lipopolysaccharide (LPS)) via
activation of NF-.kappa.B. See Grall, F., et al., "Responses to the
Proinflammatory Cytokines IL-1 and TNF-.alpha. in Cells Derived from
Rheumatoid Synovium and Other Joint Tissues Involves Nuclear
Factor-.kappa.B-Mediated Induction of the Ets Transciption Factor ESE-1,
Arthritis & Rheumatism, Vol. 48, No., 2003 (in press) (incorporated by
reference in its entirety).
This invention relates to the use of transcription factors in modulating
inflammation in response to many types of different stimuli. More
specifically, the invention relates to ESE-1, a novel member of the Ets
transcription factor, which is inducible in, e.g., vascular smooth muscle
cells, endothelial cells, and cells of the monocyte-macrophage lineage in
response to inflammatory stimuli. This induction appears to be mediated
via NF-.kappa.B. ESE-1 is able to directly bind to the p50 subunit of
NF-.kappa.B and can augment the NF-.kappa.B mediated activation of genes
that are induced during inflammation.
While the inventors do not intend to be bound by theory, the following is a
description of the basis for the invention. Proinflammatory cytokines and
endotoxin bind to their respective cell surface receptors and elicit their
pleiotropic responses. Activation of NF-.kappa.B is a common rapid
response, which in turn leads to translocation of NF-.kappa.B from the
cytoplasm to the nucleus and activation of a whole set of genes including
genes for transcription factors. ESE-1 is one of these target genes
related to the presence of a high affinity NF-.kappa.B binding site within
the ESE-1 promoter. Induction of ESE-1 expression by NF-.kappa.B leads to
the induction or repression of a set of genes that are regulated by ESE-1.
Some of these ESE-1 target genes might be direct targets for NF-.kappa.B
as well and, as shown below, ESE-1 directly interacts with and cooperates
with NF-.kappa.B and thereby enhances the response. ESE-1 target genes
represent only a subset of cytokine-responsive genes and may specify a
functionally related class of genes. Thus, ESE-1 is an alternative target
for anti-inflammatory drugs. See Grall, F., et al., J. Biol. Chem. (in
press) (incorporated herein by reference).
In the methods of the present invention, the transcription factor modulates
a variety of genes (target genes) that contribute to local inflammatory
processes in disorders such as rheumatoid arthritis and other inflammatory
diseases. Target genes include, but are not limited to, the COX-2 gene,
inducible nitric oxide synthase (iNOS), matrix metalloproteinases (MMPs),
adhesion molecules, other cytokines, and chemokines. Additional targets
are likely to be found among other cytokine-responsive genes. The COX-2
gene is one example of a target for ESE-1, since ESE-1 binds to several
sites within the COX-2 promoter and enhances COX-2 promoter activity. We
show here that the COX-2 promoter is indeed a target for ESE-1, since a
dominant-negative ESE-1 mutant as well as mutation in multiple ESE-1
binding sites efficiently inhibit LPS-mediated activation of the COX-2
promoter.
Unlike other Ets transcription factors, ESE-1 has two DNA binding domains,
a classical Ets domain and an A/T hook domain found in HMG proteins. Both
DNA binding domains are capable of binding to the p50 subunit of
NF-.kappa.B. The p50 and p65 subunits of NF-.kappa.B can act
synergistically with ESE-1 to enhance the transactivation of the NOS2
promoter by ESE-1. An ESE-1 binding site within the NOS2 promoter has been
identified, the site directed mutagenesis of which completely abolishes
the ability of ESE-1 to transactivate the NOS2 promoter, and leads to a
60% reduction in the inducibility of promoter by endotoxin. Finally in a
rat model of endotoxemia, associated with acute vascular inflammation,
ESE-1 is strongly expressed in vascular endothelium, and vascular smooth
muscle cells by immunohistological analysis.
The inducible form of nitric oxide synthase (NOS2) is also a preferred
target for ESE-1. NOS2 is induced in response to inflammatory cytokines in
vascular smooth muscle cells, endothelial cells, and monocytes. Ets
factors have not previously been shown to be important for the
inducibility of the NOS2 gene. Although NOS2 is generally considered an
inducible enzyme, that is not constituitively expressed, NOS2 has been
shown to be highly expressed in fetal and adult bronchial epithelium
(Sherman, T. S., et al. 1999, Am J Physiol 276:L383-90). Interestingly, we
also determined the highest level of ESE-1 expression in bronchial
epithelium. The functions of NO in the mature airways include smooth
muscle relaxation, neurotransmission, bacteriostasis, and modulation of
plasma exudation, mucin secretion, and ciliary motility. Constituitive
expression of NOS2 has also recently been demonstrated at lower levels in
both gastric and colonic epithelium, and is enhanced in association with
infection or neoplasia (Ambs, S., et al. 1998, Cancer Res 58:334-41; Fu,
S., et al. 1999, Gastroenterology, 116:1319-29).
Our results are the first demonstration that Ets factors may be able to
regulate NOS2 gene expression. Rudders, S., et al., J. Biol. Chem. (in
press) (incorporated herein by reference).
Other gene targets for ESE-1 include several keratinocyte terminal
differentiation markers such as transglutaminase 3, SPRR1, SPRR2A (Oettgen
et al., 1997), and profilaggrin, as well as the transforming growth factor
.beta. type II receptor (TGF-.beta.RII) (Choi et al., 1998),
endo-A/keratin-8, and HER-2 gene. Each of these genes contain a
functionally relevant ESE-1 binding site within their regulatory regions
that binds and responds to ESE-1.
Other genes, including urokinase-type plasminogen activator (U-PA), MMP-1,
MMP-3, TNF-.alpha., scavenger receptor, ICAM-1, ICAM-2, and IL-12 have
been shown to depend on Ets factors for their inducibility by cytokines
such as IL-1 or TNF-.alpha., and are also useful targets in the present
invention. Many additional cytokine-responsive genes contain putative Ets
binding sites within their regulatory regions, including COX-2, NOS-2, and
MMP-13.
Thus, any gene that contains a functionally relevant ESE-1 binding sites
within its regulatory region that bind and respond to ESE-1 is a useful
target in the methods of the present invention.
In addition to the role of ESE-1 as a transcriptional activator, ESE-1 also
acts as a transcriptional repressor of various genes including the
prostate specific antigen and keratin 4 gene. Several of these ESE-1
target genes have been associated with inflammatory processes. IL-1
induces SPRR1 expression in differentiating keratinocytes which directly
correlates with the upregulation of ESE-1 and the IL-1 receptor type I
during keratinocyte differentiation. ESE-1 is not expressed in
undifferentiated keratinocytes.
In osteoarthritis, there is a two pronged effect caused by inflammatory
cytokines, s
