|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1249-1257
GENE THERAPY
Targeted disruption of Stat6 DNA binding activity by an
oligonucleotide decoy blocks IL-4-driven TH2 cell response
Li Hua Wang,
Xiao Yi Yang,
Robert A. Kirken,
James H. Resau, and
William L. Farrar
From the Cytokine Molecular Mechanisms Section, Laboratory of
Molecular Immunoregulation, Division of Basic Sciences; the Intramural
Research Support Program, SAIC Frederick, National Cancer
Institute-Frederick Cancer Research and Development Center, Frederick,
MD; the Department of Integrative Biology, Pharmacology, and
Physiology, University of Texas at Houston, TX; and the ABL-Basic
Research Program, National Cancer Institute, Frederick, MD.
 |
Abstract |
The transcription factor, signal transducer and activator of
transcription (Stat) 6, regulates TH2-lymphocyte activity
by controlling the expression and responsiveness to interleukin
(IL)-4, which plays a key role in numerous allergic maladies.
Therefore, we sought to use a phosphorothiolate cis-element decoy to
target disruption of Stat6 transcriptional activity. Here we showed
that the Stat6 decoy potently ablated the messenger RNA expression and
production of IL-4, but not of several other cytokines.
The Stat6 decoy functionally disrupted IL-4-inducible
cell proliferation of murine TH2 cells and primary human
CD4+ T lymphocytes. Specificity of the decoy was
demonstrated by its ability to directly block Stat6 binding to a
cis-element probe and transactivation, but not affect Stat6 tyrosine
phosphorylation or expression of the IL-4 receptor chains. Moreover,
the decoy failed to inhibit non-Stat6-dependent signaling pathways
since IL-2 was competent to induce cell proliferation and activation of
Stats 1, 3, and 5a/b. With the use of laser scanning confocal microscopy, fluorescently tagged Stat6 decoy was detectable in the
cytoplasm and nucleus; however, greater levels of oligonucleotide were
present in the latter following IL-4 treatment. Taken together, these
data suggest that IL-4-driven TH2 cell activity can be
preferentially restricted via targeted disruption of Stat6 by a novel
and specific decoy strategy that may possess gene therapeutic potential.
(Blood. 2000;95:1249-1257)
© 2000 by The American Society of Hematology.
| |
Introduction |
Functionally distinct T-helper cell subsets, known as
TH1 and TH2 cells, are characterized by the
patterns of cytokines they secrete.1-3 Different
pathological states can be mediated by selective activation of either
TH subset.4,5 For example, release of
TH1 cytokines can promote disease based on cell-mediated immunity, including multiple sclerosis, insulin-dependent diabetes mellitus, and allograft rejection. In contrast, overproduction of
TH2 cells and their products is characteristic of allergic maladies (eg, asthma, allergic rhinitis, atopic dermatitis) and is also
reported to promote susceptibility to infectious agents, such as
Leishmania major, leprosy, and human immunodeficiency virus.6,7 Therefore, blocking the production of
Th-subset cytokines and its consequent cellular activities
(ie, growth, differentiation, or cytokine production) by antigene
strategies or pharmaceuticals would be expected to have therapeutic
potential for lymphoid- and myeloid-derived pathologies.
The development of TH1/TH2 functional subsets
appears to be tightly regulated by specific transcription
factors.8-11 The signal transducer and activator of
transcription (Stat) proteins are a family of cytokine-activated,
tyrosine-phosphorylated transcription factors. Stat6 is one member
reported to play a significant role in the coordinated transcription of
various cytokine genes.12 Stat6 has been postulated to
mediate TH2-specific expression of interleukin (IL)-4
through an autocrine mechanism by binding and transactivating the IL-4
promoter.13,14 As IL-4 is a potent inducer of
TH2 development, transcription factors activated by IL-4
are potential candidates for regulating IL-4 gene expression and
TH2 phenotype development.15 A critical role
for Stat6 in TH2 development was first demonstrated by the
targeted disruption of the Stat6 gene in mice. Similar to
IL-4 / mice,16 Stat6-deficient
(Stat6/) mice also lack IL-4-mediated
activities, including TH2-cell differentiation, expression
of cell surface markers, and immunoglobulin class switching to
immunoglobulin (Ig) E.17-19
While in vivo targeted disruption of transcription factors such as
Stat6 has provided a unique pattern of phenotypic deficiencies, some
difficulties can arise and have done so. Unconditional deletion of some
specific transcription functions can lead to lethal
embryogenesis.20-22 These consequences can make the study
of certain transcription factors in fully developed cells or animals
difficult to assess. Moreover, targeted disruption of transcription
factors by genetic deletion has not been as successful in vivo as in
human cells. Here, we present a novel strategy for in vitro targeted
disruption of Stat6 with several distinct advantages and therapeutic potential.
Transfection of cis-element double-stranded oligodeoxynucleotides
(ODNs), referred to as "decoy" ODNs, has been
reported to be a powerful tool that provides a new class
of antigene strategies for gene therapy and for the study
of gene transcription.23-28 Synthetic ODNs act
as decoy cis-elements to block the binding of nuclear factors to
promoter regions of targeted genes, resulting in the inhibition of gene
transactivation in vitro and in vivo. Therefore, the decoy approach may
permit treatment of human diseases by modulation of
endogenous transcription gene.29-33 For example, an NF- B
decoy has been shown to reduce myocardial reperfusion injury by
inhibiting the protein expression of cytokines (IL-6 and IL-8) and
adhesion molecules in aortic endothelial cells.26,34 Treatment of hypertension by means of decoy ODNs for
angiotensinogen gene-activating elements was also
reported.35 It has been
shown that intrarenal arterial perfusion of E2F decoy ODNs
inhibited mesangial cell proliferation.36,37 However,
little is known about the effect of transcription factor decoys on
TH cells, which have a significantly shorter half-life than
the above-mentioned cells.
In order to investigate the efficacy of cis-element decoy ODN against
Stat6 binding site (Stat6 decoy ODN) for TH2 cell activity, we provide novel evidence that fluorescent dye N, N, N',
N'-tetramethyl-6-carboxyrhodamine (TAMRA)-labeled
double-stranded Stat6 decoys were successfully transfected into
TH2 cells with the use of a cationic liposome-mediated method of gene transfer. We found that the decoy ODN could effectively block Stat6 binding to its specific cis-element by electrophoretic mobility shift assay (EMSA) and inhibit IL-4-induced Stat6 promoter transactivation detected by a luciferase reporter construct containing the Stat6 binding element. Furthermore, our results revealed that the
Stat6 decoy ODN could potently and effectively ablate messenger RNA
(mRNA) expression and production of IL-4. Stat6 decoy ODN was also
found to inhibit IL-4-mediated cell proliferation in TH2
cells and primary human T cells, but not affect IL-2-mediated T-cell
activity. Taken together, these data suggest that Stat6 decoy ODN
provides rapid and efficient means to assess the roles of specific
transcription factors in T-cell development and has genetic therapeutic
potential that could be applied to human cells.
| |
Materials and methods |
First, ODN was synthesized and sequence targets were selected. The
Stat6 decoy ODN is a double-stranded phosphorothioate 28mer that
exhibits a high sequence-specific binding affinity to the transcription factor Stat6. Sequences utilized were as follows: Stat6
decoy ODN, 5'-GAT CAA GAC CTT TTC CCA AGA AAT CTA T-3' and 3'-CAT GTT CTG GAA AAG GGT TCT TTA GAT A-5'; scrambled
decoy ODN, 5'-CGA AAA TTC GTT AAA TCA CTA GCT TAC C-3' and
3'-GCT TTT AAG CAA TTT AGT GAT CGA ATG G-5'. Synthetic ODNs
were dissolved in sterile TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 8.0), purified by high-performance liquid
chromatography and quantitated by
spectrophotometry (Operon Tec, Alameda, CA). Each pair of
single-stranded ODN was annealed for 3 hours, during which
time the temperature was reduced from 90°C to 25°C.
To create a cell culture, the TH2 cell line, D10, was
maintained in RPMI-1640 medium containing 10% fetal calf
serum (Sigma, St Louis, MO), 2 mmol/L L-glutamine and
penicillin-streptomycin (50 IU/mL and 50 µg/mL, respectively), IL-1
(2 U/mL; PeproTech, Rock Hill, NJ), recombinant human IL-2 (25 U/mL;
Hoffmann-LaRoche, Nutley, NJ), Concanavalin A (2 µg/mL; Sigma), 35 µmol/L  -ME and 6 mmol/L
HEPES. The TH2 cell clone was
a generous gift from Dr Minute Li-Weber. Fresh human T lymphocytes were
obtained from normal donors and purified by isocentrifugation and
activated for 72 hours with phytohemagglutinin (PHA) in
RPMI-1640 medium containing 10% fetal calf serum, 2 mmol/L
L-glutamine and penicillin-streptomycin (50 IU/mL and 50 µg/mL, respectively), and then made quiescent by washing and
incubating for 24 hours in RPMI-1640 medium containing 1% fetal calf
serum. Human CD4+ T cells were purified from the above
quiescent T cells by the use of human T-cell CD4 subset column kit (R&D
Systems, Minneapolis, MN) according to the manufacturer's instructions
before transfection. More than 95% of the cells were CD4+
as assessed by fluorescence-activated cell sorter.
Cationic liposomes (Boehringer Mannheim, Indianapolis, IN) were used to
transfect double-stranded decoy ODN into D10 cells. Liposome-ODN complexes were formed by mixing 30 µg of lipid
in HEPES buffer, 20 mmol/L, pH 7.4, to a final
volume of 100 µL while 5 µg ODN were diluted to a concentration of
0.1 µg/µL in HEPES buffer, 20 mmol/L, pH
7.4. The solution was gently mixed and incubated
at room temperature for 15 minutes to allow liposomes to form. D10 cells (1.5 × 106) in 5 to 6 mL
OPTI-MEM (Life Technologies, Gaithersburg, MD) were mixed gently with
liposome-ODN mixture or control liposome without ODN and then
incubated for 6 hours at 37°C. The medium was then
changed to fresh D10 cell-culture medium. We confirmed that this
quantity of DNA and cationic liposomes had no toxic effect
on cell viability, which was judged to be
more than 90% viable on the basis of trypan blue dye exclusion.
To assess ODN uptake, cells were transfected with double-stranded ODN
tagged with TAMRA at their 3' end, then at different time points
fixed with an equal volume of 5% paraformaldehyde, neutralized by
one-tenth volume of 1 mol/L Tris-HCl (pH 7.2), and
centrifuged onto a glass slide by means of a cytospin
apparatus.36 The plates were allowed to air
dry. Dried plates were then stained with 2 µg/mL
4,6-diamidino-2-phenylindole (DAPI) and examined
with the use of a laser scanning confocal microscope (Model 310, Carl Zeiss, Thornwood, NY). TAMRA (565 nm) and DAPI (UV 364 nm) images (red and blue, respectively) were prepared for each
specimen. Blue and red images were superimposed onto the
Nomarski image. Photographs were taken with the
use of a Sony color video printer, UP5200 MD Mavigraph. Quantitative
measurement of nuclear/cytoplasmic fluorescence was also performed.
After transfection with the Stat6 or scrambled decoy
ODN, D10 cells (108 cells/mL) were
solubilized in lysis buffer.38 Cell lysates were rotated
end over end at 4°C for 60 minutes, and insoluble material was
pelleted at 12 000g for 20 minutes. The supernatants were
incubated with 5 µg/mL rabbit polyclonal  -Stat6
(R&D Systems) for 2 hours at 4°C. Antibodies were captured by
incubating for 30 minutes with protein A-Sepharose beads (Pharmacia,
Piscataway, NJ). Precipitated material was eluted by boiling in sodium
dodecyl sulfate (SDS) sample buffer for 4 minutes and subjected to
7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. All proteins were transferred to Immobilion-P (PVDF) membrane as previously described.38 Analysis was done by
Western blotting with monoclonal antiphosphotyrosine or
-Stat6 antibodies that were diluted 1:1000 in
blocking buffer as described.38
D10 cells were washed with the hypotonic buffer (10 mmol/L
HEPES, pH 7.9, 10 mmol/L KCl, 1.5 mmol/L
MgCl2, 0.5 mmol/L DTT), then lysed in the
same buffer supplemented with 1% NP-40 and incubated for
20 minutes on ice. The nuclei-containing pellet was resuspended in
equal volumes of low-salt buffer (10 mmol/L HEPES, 25%
glycerol, 1.5 mmol/L MgCl2, 20 mmol/L KCl, 0.5 mmol/L DTT,
0.2 mmol/L EDTA) and high-salt buffer (low-salt buffer containing 800 mmol/L KCl). The nuclear extract was centrifuged at 4°C for 15 minutes. Supernatants were saved as nuclear protein extract and stored
at  70°C. For the EMSA, end-labeled [32P]-Stat6
oligonucleotide probes corresponding to the C element sequence
5'-AGT CAA GAC CTT TTC CCA AGA AAT CTA
TC-3'),39 Stat5 probes corresponding to the
-casein gene sequence
5'-AGATTTCTAGGAATTCAATCC-3'),40 and Stat1/3
probes corresponding to the m67 SIE gene sequence 5'-AGCTTGTCGACATTTCCCGTAAATCGTCGAG-3')41,42
were used to detect DNA binding activities of the above transcriptional
factors. The probe was then incubated with 5 µg of nuclear extracted
proteins in 15 µL of binding cocktail (50 mmol/L Tris-Cl, pH 7.4, 25 mmol/L MgCl2, 0.5 mmol/L DTT, 50% glycerol) at 4°C for
15 minutes. For supershift assay, the nuclear extracts were
preincubated with 1 µg of either normal rabbit serum or antisera
specific to Stat6 at 4°C for 30 minutes. The DNA-protein complexes
were resolved on a 5% polyacrylamide gel. The dried gels were exposed
to x-ray film.
An oligonucleotide consisting of 3 copies of IL-4 nuclear-activated
factor Stat6 binding promoters of the murine C (119 to
104)39 or Stat1/3 binding promoter of
the SIE from c-fos gene in a direct repeat was synthesized with SacI
and XhoI overhangs and ligated into pGL3 luciferase reporter vector
(Promega, Madison, WI). The correct reporter construct sequence was
confirmed by DNA sequencing. Plasmid DNA was prepared with the use of
Wizard (Promega) maxipreps DNA purfication system vector.
Cationic liposomes were used to co-transfect the Stat6 or SIE
reporter plasmid and decoy ODN into D10 cells with the use
of 3 µg of plasmid with 20 µg of lipid according to the
manufacter's instructions. After 6 hours of
transfection, cells were resuspended and cultured in fresh medium for
24 hours. Cells were then stimulated in the presence or absence of IL-4
for another 24 hours at 37 °C. Cell extracts were
prepared with the use of the reporter lysis buffer in the
Luciferase Assay System (Promega) and centrifuged at
12 000g for 2 minutes at 4°C. Supernatants were
transferred to new tubes. 20 µL of lysate was mixed with 100 µL
luciferase assay reagent in cuvettes and measured by a
luminometer (Monolight 3010; PharMingen, San Diego, CA)
according to the manufacturer's instructions. To correct for
variations in transfection efficiencies, the luciferase values
were normalized against protein concentration.
D10 cells were transfected as described above with Stat6 or scrambled
decoy ODN, or treated with 200 ng/mL of antimouse IL-4 or antimouse
IL-3 (Biosource, Camarillo, CA). Total RNA was isolated from treated or
control cells with the use of TRIzol (Life Technologies, Gaithersburg,
MD). Cytokine RNA-message was examined by ribonuclease (Rnase) protection assay with the use of 20 µg of total RNA
hybridized to 2 × 106 cpm of
[33P]-labeled probe corresponding to mCK-1 or mCR-1
(PharMingen) overnight at 56°C. Unhybridized RNA was digested with
RNase T1 and RNase A for 45 minutes at 30°C, then digested with
proteinase K for 15 minutes at 37°C. After phenol/chloroform
extraction and sodium acetate/ethanol precipitation, hybridized RNA
probes were denatured at 90°C for 3 minutes and electrophoresed on
a 5% polyacrylamide gel. The dried gels were exposed to x-ray film.
After transfection with the Stat6 or scrambled decoy
ODN, D10 cells were grown to approximately
2.5 × 106 cell/mL and transferred to
25-mL flasks. Supernatants were collected after 24 hours and assayed
for murine cytokines (IL-4, IL-5, IL-6, IL-10, IL-13) by means of
enzyme-linked immunosorbent assay (ELISA) Endogen kits (Wolburn, MA),
according to the manufacturer's instructions, by the Clinical Services
Department, Frederick Cancer Research and Development Center,
Frederick, MD.43
After transfection with the Stat6 decoy or scrambled
ODN, quiescent cells (50 × 103/well)
were plated in flat bottom 96-well microtiter plates in 200 µL of
growth media (described above), supplemented with 5% fetal calf serum
and stimulated with 1 nmol/L of IL-4, or IL-2, or media alone for 16 hours, then pulsed for the remaining 4 hours of the assay with
[3H]-thymidine (0.5 µCi/200 µL) and
harvested onto glass-fiber filters. [3H]-thymidine incorporation was analyzed
by liquid scintillation counting as previously described.38
| |
Results |
In order to study the efficacy of transcription factors decoy on
T-lymphocyte function, we chose the D10 cell line as a model system.
This cell line represents the prototypic TH2 cell, which secretes and responds to IL-4.43 Using cationic liposome
gene transfer, we first verified that double-stranded ODN tagged with TAMRA at the 3' end could be efficiently introduced into D10
cells. TAMRA-labeled ODNs produce red fluorescence when excited at 565 nm, whereas DAPI, a nuclear fluorescence marker, produces blue fluorescence when UV excited. With the use of laser scanning confocal microscopy combined with DAPI staining, it was seen that transfection of TAMRA-labeled ODNs for 6 hours resulted in nearly 60% of the D10
cells exhibiting intense red cytoplasmic fluorescence, but a weak nuclear signal (Figure 1A). After
IL-4 stimulation (Figure 1B), red fluorescence staining was increased
in both the nuclei and the cytoplasm of cells. This effect was further
confirmed by quantification of nuclear/cytoplasmic fluorescence
intensity. The ratio of nuclear to cytoplasmic fluorescence in
IL-4-stimulated cells (31.62 ± 2.96%) was compared with
non-IL-4-stimulated cells (1.20 ± 0.18%) from 3 sample sets
(n = 100). In the absence of cationic
liposomes, D10 cells did not significantly take up TAMRA-labeled ODN
(data not shown). These data suggest that double-stranded ODN can be
successfully transfected into TH2 cells via cationic liposomes. Moreover, detectable levels of TAMRA-labeled ODN were elevated in nucleus following IL-4 stimulation.
View larger version (91K):
[in this window]
[in a new window]
| Fig 1.
TAMRA-labeled (ODN) uptake in D10 cells.
Phase-contrast (left panel) and fluorescence (right panel) confocal
photomicrographs of D10 cells that were transfected with
TAMRA-labeled double-stranded ODN (2 µmol/L) for 6 hours with the use
of cationic liposomes. Cells were then stimulated without (A) or with
(B) 100 nmol/L IL-4 at 37°C for 10 minutes. Cells
were fixed with 5% paraformaldehyde and co-loaded with DAPI and
examined by laser scanning confocal microscope.
|
|
Stat6 decoy markedly reduces mRNA expression of IL-4 but not other
cytokines in D10 cells. Because Stat6 transactivation is necessary for
driving IL-4 transcription in TH2 cells, we next investigated whether the Stat6 decoy could block IL-4 mRNA expression. For this experiment, total RNA was isolated from cells treated with the
corresponding Stat6 decoy for 24 hours and probed for cytokine message
with the use of RPA (Figure
2). The IL-4 transcript was found to be
greatly reduced in Stat6 decoy-treated cells (lane c) as compared with
control cells, which were IL-4-stimulated (lane b), unstimulated (lane
a), or scrambled decoy-ODN-treated (lane d). In
contrast, pretreatment of D10 cells with antibodies to the
Stat6-activating cytokine IL-4 (lane f) displayed no significant inhibitory or stimulatory effects as compared with anti-IL-3 antibody (lane e) or nontreated control (lane g) samples. Densitometric analysis
of IL-4 mRNA message was standardized against L32/GAPDH messages. The IL-4 transcript reduced by the Stat6 decoy was greater than 90% as compared with controls, as measured by densitometric analysis. This evidence suggests that Stat6 decoy can specifically block transcriptional regulation of IL-4, since no detectable loss in
mRNA expression of IL-5, IL-6, IL-9, IL-10, IL-13, or IFN was observed.
View larger version (63K):
[in this window]
[in a new window]
| Fig 2.
Stat6 decoy inhibits mRNA expression of IL-4 but not
other cytokines.
RNA for RPA analysis was obtained from untreated control samples (lanes
a, b, g), Stat6 decoy (lane c), scrambled (lane d) ODN-transfected D10
cells, or cells pretreated with anti-IL-3 or anti-IL-4 antibodies
(lanes e and f, respectively). Cells were stimulated with IL-4 (lanes b
through d). RNA was isolated and hybridized with
[33P]-labeled RNA probes corresponding to transcripts for
murine cytokines (mCK1). The RNase-protected fragments were separated
on 5% PAGE, dried, and exposed to x-ray. Densitometric analysis of
IL-4 RNA message compared with L32/GAPDH indicated a greater than 90%
reduction in Stat6 decoy-treated samples.
|
|
Stat6 decoy decreases IL-4 protein production in TH2
cells. To determine if the Stat6 decoy could inhibit
TH2 cell function, ELISA was used to measure the production
of TH2 cytokines in D10 cells after transfection with the
Stat6 or scrambled decoy ODN. Following transfection, D10 cells were
cultured in fresh medium for 24 hours, and supernatants were collected
and assayed for TH2 cytokines (Figure
3). IL-4 production was dramatically
reduced (approximately 70%), while little effect was observed on the
remaining TH2 cytokines (ie, IL-5, IL-6, IL-10, IL-13).
These data indicated that the Stat6 decoy selectively blocks IL-4
production over other TH2 cytokines.
View larger version (27K):
[in this window]
[in a new window]
| Fig 3.
Stat6 decoy treatment of TH2 clone D10 cells
preferentially inhibits IL-4 production.
D10 cells were transfected with Stat6 decoy or scrambled ODN at
37°C for 6 hours, then resuspended and cultured in normal D10
medium for 24 hours. ELISA was performed for each cytokine indicated
(n = 3) and plotted as the mean ± the standard error (abscissa)
based on ELISA values (pg/mL) (ordinate). Actual values obtained were
as follows: For control cells, IL-4 (4980 ± 518), IL-5
(7646 ± 511), IL-6 (5904 ± 518), IL-10 (8860 ± 240),
and IL-13, (9926 ± 516). For Stat6 decoy ODN-transfected D10
cells, IL-4 (1461 ± 264), IL-5 (7448 ± 760), IL-6
(4906 ± 423), IL-10 (8479 ± 808), and IL-13
(8233 ± 431). For scrambled decoy ODN-transfected D10 cells,
IL-4 (4308 ± 339), IL-5 (7260 ± 609), IL-6
(5276 ± 366), IL-10 (7972 ± 448), and IL-13
(10 996 ± 947).
|
|
Stat6 decoy inhibits IL-4-mediated proliferation of D10 cells and
primary human CD4+ T cells. Since IL-4 plays a key role in
the differentiation and autocrine expansion of TH2 cells,
we asked whether the Stat6 decoy could block IL-4-mediated
TH2 cell growth. For this assay, Stat6 or scrambled decoy
ODN was used to transfect D10 cells. Cells were then cultured in growth
medium with or without 1 nmol/L IL-4. As shown in Figure
4 (upper panel), the Stat6 decoy markedly
suppressed IL-4-inducible [3H]-thymidine incorporation
(61%) compared with the unstimulated control cells, whereas scrambled
ODN alone did not affect cell proliferation. By contrast, the Stat6
decoy did not block IL-2-inducible cell proliferation of D10 cells
(Figure 4, middle panel). Interestingly, the same inhibitory effect of
the Stat6 decoy was also observed on IL-4-inducible proliferation in
PHA-activated primary human CD4+ T cells (Figure 4, lower
panel). The D10 and human T cells used for the
proliferation assay were judged to be more than 90% viable on the
basis of trypan blue dye exclusion (data not shown). These findings
suggest the Stat6 decoy effectively inhibits IL-4 growth-promoting signals in TH2 cells lines and primary human
CD4+ T cells.
View larger version (18K):
[in this window]
[in a new window]
| Fig 4.
Stat6 decoy inhibits IL-4-inducible cell proliferation
on a TH2 cell line and human CD4+ T cells.
Quiescent D10 cells (upper and middle panel) or human CD4+
T cells (lower panel) (50 × 103/well) were
transfected with Stat6 decoy or scrambled decoy ODN for
6 hours and cultured in 200 µL of growth media in the presence of
IL-4 ( , upper and lower panel) or IL-2 (, middle panel) or their
absence ( ) for 16 hours at 37°C. Cells were then pulsed with
[3H]-thymidine (0.5 µCi/200 µL) for an additional 4 hours and incorporation of radiolabeled probe plotted on the abscissa
(expressed as total cpm for 6 samples).
|
|
Treatment of D10 cells with Stat6 decoy does not alter mRNA expression
of IL-4 receptor chains. To investigate whether the Stat6 decoy
mediated loss of IL-4-regulated cell proliferate owing to reduced
expression of IL-4 receptor chains, we performed RNase protection
assays. For this analysis, total mRNA was isolated from control, Stat6
decoy-treated, or scrambled ODN-treated cells (as described in
"Materials and methods") and hybridized against [33P]-labeled receptor probes. RNase-protected samples
were then electrophoretically separated by PAGE, dried, and
autoradiographically shown (Figure 5). The
IL-4-receptor mRNA message from non-IL-4-stimulated control cells
(lanes a, e through g) and IL-4-stimulated (lanes b through d), Stat6
decoy (lane c), and scrambled ODN (lane d) failed to show a loss in
cytokine receptor expression. Similarly, pretreatment of D10 cells with
anti-IL-4 antibody (lane f) or anti-IL-3 antibody (lane e) and
untreated control samples also did not display a significant change in
expression of IL-4 receptor or c chains compared with the
control housekeeping genes, L32/GAPDH. From this data we conclude that
the loss of IL-4-mediated cell growth and IL-4 message following Stat6
decoy treatment is not due to a significant reduction in IL-4R or
c expression but likely occurs at a site distal to the receptor.
View larger version (60K):
[in this window]
[in a new window]
| Fig 5.
Stat6 decoy does not alter mRNA expression of IL-4
receptors.
Freshly isolated mRNA was obtained from control (lanes a, b, g), Stat6
decoy (lane c), scramble ODN-transfected D10 cells (lane d), or
anti-IL-4 antibody treated (lane f) or anti-IL-3 antibody treated
(lane e) cells in the absence (lanes a, e, f) or presence of IL-4
(lanes b through d). RNA was then hybridized with
[33P]-labeled RNA probes corresponding to transcripts for
individual murine c-receptors (mCR-1) according to PharMingen
protocol (see "Materials and methods"). The Rnase-protected
fragments were separated on 5% PAGE, dried, and exposed to x-ray
film.
|
|
Stat6 decoy does not affect IL-4-dependent Stat6 tyrosine
phosphorylation in vivo. It is well established from Stat6 knockout mice studies that this transcription factor provides a key role in
T-cell development, IL-4 responsiveness, and TH2
differentiation.19,44 To clarify whether the Stat6 decoy
affects IL-4-dependent Stat6 tyrosine phosphorylation, cells were
transiently transfected with decoy and stimulated with or without IL-4
for 10 minutes and assayed by Western analysis for tyrosine
phosphorylated Stat6. Tyrosine-phosphorylated Stat6 was observed in
exogenous IL-4-stimulated cells (Figure 6,
lanes b, d, f), but not in lysates from unstimulated cells transfected
with Stat6 (lane d) or scrambled decoy ODN (lane f). Immunoblotting of
Stat6 (indicated beneath phosphorylation blots) verified equivalent
loading and no loss of protein expression. These data suggest that the
Stat6 decoy does not affect IL-4-dependent Stat6 tyrosine
phosphorylation and directly competes for its ability to bind DNA.
View larger version (37K):
[in this window]
[in a new window]
| Fig 6.
Stat6 decoy does not affect Stat6 tyrosine
phosphorylation.
D10 cells were treated with Stat6 decoy (lanes c, d) or scrambled ODN
(lanes e, f) for 6 hours and then stimulated with or without 100 nmol/L
IL-4 at 37°C for 10 minutes. Cells were lysed, immunoprecipitated
with anti-Stat6 (-Stat6), and then Western
blotted with PY (upper panel) or anti-Stat6 (lower
panel) to verify equivalent loading. Arrows indicate location of
Stat6.
|
|
Stat6 decoy specifically inhibits IL-4-induced Stat6 DNA binding. To
provide evidence that the Stat6 decoy ODN prevented the binding of
endogenous Stat6 to its target sites, we performed an
electrophoretic mobility-shift assay in the presence of the Stat6 decoy or the scrambled control ODN, both in vitro and in vivo.
First, we examined whether the Stat6 decoy can compete for binding the
sequence-specific DNA binding proteins. As shown in Figure
7A, IL-4-induced Stat6 DNA binding was
abolished by preincubating nuclear extracts with an excess
of unlabeled C oligonucleotide probe and Stat6 decoy ODN (lanes e,
c). However, no effect was observed when this was performed with the
unlabeled scrambled ODN (lane d). By contrast, the Stat6 decoy failed
to interfere with IL-2-induced Stat1, Stat3, and Stat5 DNA binding
activities (Figure 7B, 7C, lane d).
View larger version (3643K):
[in this window]
[in a new window]
| Fig 7.
Binding site specificity of Stat6 decoy.
(A) D10 cells were incubated with medium () or 100 nmol/L IL-4
(+) for 10 minutes at 37°C. Nuclear extracts were incubated with a
[32P]-labeled Stat6 oligonucleotide probe corresponding
to the C gene promoter. Competition was performed with 100-fold
molar excess of unlabeled Stat6 decoy ODN (lane c), scrambled ODN (lane
d), or cold C element ODN (lane e). Arrow indicates migrational
location of Stat6-DNA complex or free probe. (B) D10 cells were
incubated with medium () or 100 nmol/L IL-2 (+) for 10 minutes
at 37°C. Nuclear extracts were incubated with a
[32P]-labeled Stat5 oligonucleotide probe corresponding
to the prolactin response element of the -casein gene
promoter. Competition was performed with 100-fold molar excess of
unlabeled cold Stat5 ODN (lane c), Stat6 decoy ODN (lane d), or
scrambled ODN (lane e). Arrow indicates migrational location of
Stat5-DNA complex or free probe. (C) D10 cells were incubated with
medium () or 100 nmol/L IL-2 (+) for 10 minutes at 37°C.
Nuclear extracts were incubated with a [32P]-labeled
Stat1/3 oligonucleotide probe corresponding to the SIE gene promoter.
Competition was performed with 100-fold molar excess of unlabeled cold
Stat1/3 ODN (lane c), Stat6 decoy ODN (lane d), or scrambled ODN (lane
e). Arrow indicates migrational location of each Stat1- or Stat3-DNA
complex or free probe.
|
|
We next determined the ability of the Stat6 decoy to penetrate cells
and compete with the native Stat6 transcription activity in vivo.
Nuclear extracts prepared from D10 cells transfected with either Stat6
decoy ODN or scrambled decoy ODN or prepared from control cells were
tested for their ability to bind a radiolabeled C oligonucleotide
probe (Figure 8). Control extracts readily displayed IL-4-modulated Stat6 DNA binding (lanes b through d); however, equivalent protein from nuclear extracts of Stat6 decoy ODN-treated cells showed greatly diminished DNA binding capacity (lanes e, f). Control scrambled decoy ODN had no effect (lanes g, h).
To confirm that the identity of Stat6 DNA binding activity was IL-4
dependent, complexes were incubated with anti-Stat6 antibody or normal
rabbit serum. Only the anti-Stat6 antibody (lane c) could supershift
the IL-4-inducible protein-DNA complex, unlike normal rabbit serum
(lane d), thus verifying the identify of the radiolabeled band. These
findings suggest that the Stat6 decoy is a highly specific
competitor for activated Stat6.
View larger version (71K):
[in this window]
[in a new window]
| Fig 8.
Stat6 decoy specifically blocked Stat6 DNA binding
activity.
Control (lanes a, b, c, d) and Stat6 decoy ODN-treated (lanes e, f) or
scrambled decoy ODN-treated (lanes g, h) D10 cells were
incubated with medium () or 100 nmol/L IL-4 (+) for 10 minutes
at 37°C. Nuclear extracts were obtained, and 5 µg of protein were
incubated in the absence of antibody (lanes a, b, e through h),
-Stat6 (lanes c), or normal rabbit serum (nrs; lane d) and then with
a [32P]-labeled Stat6 oligonucleotide probe corresponding
to the C gene promoter. Arrows indicate migrational location of each
nonsupershifted Stat6-DNA complex or free probe.
|
|
Stat6 decoy inhibits IL-4-stimulated Stat6 transactivation. We used
the Stat6-luciferase reporter gene construct to quantitatively assess
the effect of the Stat6 decoy on IL-4-stimulated transcriptional
activation. As shown in Figure 9
(upper panel), Stat6 luciferase activity of IL-4-stimulated cells was
substantially reduced in Stat6 decoy-treated samples as compared with
the control and the scrambled ODN-treated cells. Interestingly, basal
levels of luciferase activity were higher for cells transfected with
the Stat6 binding element construct than for cells transfected with
control plasmid, but was attenuated in the presence of the Stat6 decoy.
Specificity of the decoy was further assessed by measuring
IL-2-inducible transactivation of Stat1/3 to an SIE reporter. IL-2
activation of the SIE-reporter construct was not blocked by the decoy
(Figure 9, lower panel). These observations suggest that the
ODN decoy can specifically inhibit constitutive Stat6
promoter transactivation potential and subsequent production and
response to IL-4.
View larger version (25K):
[in this window]
[in a new window]
| Fig 9.
Effects of Stat6 decoy ODN on transactivation of Stat6
compared with Stat1/3 in cultured D10 cells.
(A) D10 cells treated with Stat6 decoy ODN or scrambled decoy ODN were
co-transfected with a 3 × Stat6 binding element pGL3
promoter-luciferase construct or with pGL3 promoter-luciferase
construct alone. After transfection, cells were stimulated with or
without IL-4 (100 nmol/L). Cells were harvested after an additional 24 hours, and luciferase activity was measured and normalized against
protein concentration. (B) D10 cells treated with Stat6 decoy ODN, or
scrambled decoy ODN were co-transfected with a 3 × SIE binding
element pGL3 promoter-luciferase construct or with pGL3
promoter-luciferase construct alone. After transfection, cells were
stimulated with or without IL-2 (100 nmol/L). Cells were harvested
after an additional 24 hours, and luciferase activity was measured and
normalized against protein concentration.
|
|
| |
Discussion |
IL-4 is a pleiotropic cytokine that plays a prominent role in
driving inflammatory and cell-mediated responses in
numerous types of immune cells.44-48 IL-4 is the
hallmark cytokine produced by TH2
cells49 and is critical for TH cell
differentiation. While Stat6 plays an essential role in TH2
differentiation, its role in IL-4-mediated transcription remains unclear.12,50 We propose that IL-4 is a target gene for the Stat6 decoy, which in turn ultimately blocks TH2-cell
activity. The results of the present study clearly demonstrated that
TH2 cells transfected with the Stat6 decoy ODN showed
significantly lower Stat6 transcriptional activity and significantly
lower production of IL-4 than TH2 cells
transfected with the scrambled ODN. Thus, blocking Stat6 may be useful
for inhibiting IL-4-derived TH2 cell activity and proliferation.
The Stat6 decoy is a double-stranded phosphorothioate 28mer
ODN. As emphasized by Bielinska et
al,24,51-53 oligonucleotides with modified
phosphodiester bonds, such as phosphorothioate, methyl phosphate,
phosphoramidite, or methyl phosphonate derivatives, are relatively
resistant to nucleases. Recently, Park et al54 reported
that phosphorothioate ODNs can be stable up to 48 hours in cell
systems, on the basis of evidence that a 24mer decoy phosphorothioate ODN accumulated in cells at a size consistent with the duplex/hairpin forms. On the basis of these findings, we hypothesized that the Stat6
decoy could be used to transfect cells. To determine the site of action
for the decoy, we employed an ODN tagged with fluorescent group TAMRA
that could be efficiently introduced and monitored in D10 cells. Using
a cationic liposome delivery and laser scanning confocal microscopy, we
combined TAMRA (red) with DAPI (blue) and found that the
Stat6 decoy ODN could be introduced into TH2 cells without
causing cellular toxicity. As shown in Figure 1A, under these
transfection conditions, resting levels of TAMRA-labeled ODN were
significantly higher in the cytoplasm than in the nucleus. However,
following addition of IL-4, we observed a dramatic increase in the
ratio of nuclear to cytoplasmic fluorescence intensity, from 1:100 to
1:3 (Figure 1B). These findings suggest that Stat6 dimer may bind the
decoy and be translocated into the nucleus as a complex. Another
possibility is that the increase in fluorescence intensity following
IL-4 stimulation could be due to increased stability of the decoy since
dimers of activated Stat6 would be expected to bind and shield the
Stat6 ODN core sequence from nucleases. Indeed, using crystallographic
evidence, Chen et al55 recently demonstrated that a complex
of Stat1 homodimers form a C-shaped complex that surrounds a 15-bp
region of the DNA. From either scenario, we conclude that the decoy
binds Stat6 with sufficient affinity to compete for
IL-4-induced Stat6 DNA binding and disrupt Stat6 activity.
We next investigated the molecular mechanism by which
transcription factor Stat6 decoy ODN inhibited the gene expression of cytokine IL-4, resulting in the suppression of TH2 cells.
Stat6 is recognized to perform a key role in mitogenic and pleiotropic functional response induced by cytokines such as IL-4.19,44 Selective activation of Stat6 through phosphorylation and dimerization results in its translocation to the nucleus, where it activates gene
transcription.56 As shown in Figure 5, the Stat6 decoy failed to inhibit IL-4 receptor -chain expression. While a Stat6 binding element resides in the promoter of this receptor, a role for
Stat6 in maintaining or promoting IL-4 receptor expression in committed
TH2 cell lines is not readily known. Indeed, convincing results have failed to demonstrate a direct role for the IL-4 receptor
pathway in IL-4 gene expression in fully committed TH2 cells.57-59 Using neutralizing anti-IL-4 antibodies, we
failed to observe a significant reduction in IL-4 and IL-4 receptor
expression (Figures 2 and 5). This suggests that additional effector
proteins and/or transcriptional elements activated by IL-4, other than Stat6, are required to maintain IL-4 receptor -chain expression in
committed TH2 cells. On the other hand, Stat6 tyrosine
phosphorylation following IL-4 stimulation was not markedly affected by
the Stat6 decoy, suggesting that the decoy does not block IL-4 receptor activation of Stat6 and that activated Stat6 is sequestered by the
decoy. These results tend to support the imaging results observed from
laser scanning confocal microscope in that the Stat6 decoy does not
block Stat6 translocation to the nucleus but does compete for its
ability to regulate gene transcription such as IL-4 (Figures 2 and 3)
or proliferative growth signals (Figure 4).
In order to characterize the specificity of Stat6 decoy on Stat6 DNA
binding and transactivation, we employed gel shift analysis and
reporter gene assay. First, IL-4-inducible Stat6 DNA binding was
abolished by Stat6 decoy ODNs. The binding-site specificity of Stat6
decoy was confirmed since a 100-fold excess of unlabeled double-stranded Stat6 ODN competed away the Stat6 complex, while similar concentration of scrambled ODN did not (Figure 7A). Also, we
demonstrate that the decoy did not interfere with IL-2-induced Stat1/3
DNA binding to the SIE-probe (Figure 7C) or with Stat5a/b DNA binding
to -casein probe (Figure 7B). Second, the highly specific effects of
the Stat6 decoy on transactivation were found by reporter gene assay.
Stat6-luciferase activity was significantly reduced (fivefold) in D10
cells co-transfected with the Stat6 decoy ODN as compared with
Stat6-luciferase activity in the scrambled ODN-co-transfected cells
(Figure 9), whereas no statistical loss in the transcriptional
activation of Stat1/3 (Figures 7 and 9) was detectable. Moreover,
specificity of the decoy was supported by its functional inability to
affect expression of other TH2 cytokines (Figures 2 and 3)
or IL-2-inducible TH2-cell growth (Figure 4B). Therefore,
we believe that transfecting a Stat6 decoy corresponding to the
cis-sequence may specifically result in the attenuation of an authentic
cis/trans interaction, leading to the removal of trans-factors from the
endogenous cis-element. This event subsequently inhibits IL-4 gene expression.
While transcription factors play a critical role in the development of
mature cell function, functional characterization of these proteins
in fully differentiated mammalian cells is not possible if it is based
entirely on gene knockout studies.
Transcription factor decoys provide a novel strategy for disrupting
gene expression and have several substantive advantages over the use of
knockout animals. This strategy is a rapid, specific, and
cost-effective means to analyze the function of a specific
transcription factor in the fully differentiated cells, which suggests
that it has therapeutic applications in treating human disease.
Sullenger et al31 reported that
overexpression of the sequences containing HIV trans-activation
response element (TAR decoy) rendered CD4+ human T-lymphoid
cells resistant to human immunodeficiency virus replication. Using a
similar approach, we have successfully introduced a transcription
factor decoy that blocked IL-4-inducible proliferation of
PHA-activated primary human CD4+ T cells (Figure 4).
In conclusion, we have identified a cis-element decoy that potently
blocks Stat6 signaling pathway in murine and human T cells. Our results
indicate that Stat6 decoy ODNs can bind Stat6 with sufficient affinity
and scavenge activated Stat6 dimers and inhibit their ability to
recognize native DNA binding sites, which can ultimately suppress
TH2 cell activity. This approach represents a
rapid and specific strategy to investigate transcription factor functions and their roles in gene expression and T cell biology.
| |
Acknowledgments |
We thank Dr Minute Li-Weber for generously providing the
TH2 cell line, D10. We also acknowledge Dr Joost Oppenheim
for critical review of the manuscript.
| |
Footnotes |
Submitted March 23, 1999; accepted October 19, 1999.
Funded in whole or in part with federal funds from the National Cancer
Institute, National Institutes of Health, under contract NO1-CO-56000 and sponsored in part by the National Cancer Institute, US
Department of Health and Human Services, under contract with ABL.
The content of this article does not necessarily reflect the views or
policies of the Department of Health and Human Services, nor does
mention of trade names, commercial products, or organizations imply
endorsement by the US government.
Reprints: William L. Farrar, National Cancer Institute,
PO Box B, Bldg 560, Room 31-68, Frederick, MD 21702; e-mail: farrar{at}mail.ncifcrf.gov.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Mosmann TR, Sad S.
The expanding universe of T-cell subsets: Th1, Th2 and more.
Immunol Today.
1996;17:138[Medline]
[Order article via Infotrieve].
2.
Carter LL, Dutton RW.
Type 1 and type 2: a fundamental dichotomy for all T-cell subsets.
Curr Opin Immunol.
1996;8:336[Medline]
[Order article via Infotrieve].
3.
Dutton RW.
The regulation of the development of CD8 effector T cells.
J Immunol.
1996;157:4287[Medline]
[Order article via Infotrieve].
4.
Umetsu DT, DeKruyff R.
Th1 and Th2 CD4+ cells in the pathogenesis of allergic diseases.
Proc Soc Exp Biol Med.
1997;215:11[Medline]
[Order article via Infotrieve].
5.
D'Elios M, Del Prete G.
Th1/Th2 balance in human disease.
Transplant Proc.
1998;30:2373[Medline]
[Order article via Infotrieve].
6.
Romagnani S.
Regulation of the development of type 2 T-helper cells in allergy.
Curr Opin Immunol.
1994;6:838[Medline]
[Order article via Infotrieve].
7.
Lafaille JJ.
The role of helper T cell subsets in autoimmune diseases.
Cytokine Growth Factor Rev.
1998;9:139[Medline]
[Order article via Infotrieve].
8.
Agarwal S, Rao A.
Long-range transcriptional regulation of cytokine gene expression.
Curr Opin Immunol.
1998;10:345[Medline]
[Order article via Infotrieve].
9.
Mikita T, Campbell D, Wu P, Williamson K, Schindler U.
Requirements for interleukin-4-induced gene expression and functional characterization of Stat6.
Mol Cell Biol.
1996;16:5811[Abstract].
10.
Lederer JA, Perez VL, DesRoches L, Kim SM, Abbas AK, Lichtman AH.
Cytokine transcriptional events during helper T cell subset differentiation.
J Exp Med.
1996;184:397[Abstract/Free Full Text].
11.
Zheng W, Flavell RA.
The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells.
Cell.
1997;89:587[Medline]
[Order article via Infotrieve].
12.
Szabo SJ, Glimcher LH, Ho IC.
Genes that regulate interleukin-4 expression in T cells.
Curr Opin Immunol.
1997;9:776[Medline]
[Order article via Infotrieve].
13.
Takeda K, Kishimoto T, Akira S.
STAT6: its role in interleukin 4-mediated biological functions.
J Mol Med.
1997;75:317[Medline]
[Order article via Infotrieve].
14.
Kaplan MH.
Stat6 is required for mediating responses to IL-4 and for development of Th2 cells.
Immunity.
1996;4:313[Medline]
[Order article via Infotrieve].
15.
Nakamura T, Lee RK, Nam SY, Podack ER, Bottomly K, Flavell RA.
Roles of IL-4 and IFN-gamma in stabilizing the T helper cell type 1 and 2 phenotype.
J Immunol.
1997;158:2648[Abstract].
16.
Kuhn R, Rajewsky K, Muller W.
Generation and analysis of interleukin-4 deficient mice.
Science.
1991;254:707[Abstract/Free Full Text].
17.
Linehan LA, Warren WD, Thompson PA, Grusby MJ, Berton MT.
STAT6 is required for IL-4-induced germline Ig gene transcription and switch recombination.
J Immunol.
1998;161:302[Abstract/Free Full Text].
18.
Akimoto T, Numata F, Tamura M, et al.
Abrogation of bronchial eosinophilic inflammation and airway hyperreactivity in signal transducers and activators of transcription (STAT)6-deficient mice.
J Exp Med.
1998;187:1537[Abstract/Free Full Text].
19.
Shimoda K, Deursent JV, Sangster MY, et al.
Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene.
Nature.
1996;380:630[Medline]
[Order article via Infotrieve].
20.
Liu KD, Gaffen SL, Goldsmith MA.
JAK/STAT signaling by cytokine receptors.
Curr Opin Immunol.
1998;10:271[Medline]
[Order article via Infotrieve].
21.
Takeda K, Noguchi K, Shi W, et al.
Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality.
Proc Natl Acad Sci U S A.
1997;94:3801[Abstract/Free Full Text].
22.
Morriss-Kay GM, Sokolova N.
Embryonic development and pattern formation.
FASEB J.
1996;10:961[Abstract].
23.
Isner JM.
Oligonucleotide therapeutics: novel cardiovascular targets.
Nat Med.
1997;3:834[Medline]
[Order article via Infotrieve].
24.
Bielinska A, Shivdasani RA, Zhang L, Nabel GJ.
Regulation of gene expression with double-stranded phosphorothioate oligonucleotides.
Science.
1990;250:997[Abstract/Free Full Text].
25.
Morishita R, Gibbons GH, Horiuchi M, et al.
A novel molecular strategy using cis element "decoy" of E2F binding site inhibits smooth muscle proliferation in vivo.
Proc Natl Acad Sci U S A.
1995;92:5855[Abstract/Free Full Text].
26.
Morishita R, Sugimoto T, Aoki M, et al.
In vivo transfection of cis element "decoy" against NFB binding site prevents myocardial infarction as gene therapy.
Nat Med.
1997;3:894[Medline]
[Order article via Infotrieve].
27.
Sawa Y, Morishita R, Suzuki K, et al.
A novel strategy for myocardial protection using in vivo transfection of cis element "decoy" against NFB binding site: evidence for a role of NFB in ischemic-reperfusion injury.
Circulation.
1997;965(suppl II):II-280.
28.
Dzau VJ, Horiuchi M.
In vivo gene transfer and gene modulation in hypertension research.
Hypertension.
1996;28:1132[Abstract/Free Full Text].
29.
Yamashita J, Yoshimasa T, Arai H, et al.
Identification of cis-elements of the human endothelin-A receptor gene and inhibition of the gene expression by the decoy strategy.
J Biol Chem.
1998;273:15,993[Abstract/Free Full Text].
30.
Morishita R, Higaki J, Tomita N, Ogihara T.
Application of transcription factor "decoy" strategy as means of gene therapy and study of gene expression in cardiovascular disease.
Circ Res.
1998;82:1023[Abstract/Free Full Text].
31.
Sullenger BA, Gallardo HF, Ungers GE, Gilboa E.
Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication.
Cell.
1990;63:601[Medline]
[Order article via Infotrieve].
32.
Tomita N, Morishita R, Higaki J, et al.
Transient decrease in high blood pressure by in vivo transfer of antisense oligodeoxynucleotides against rat angiotensinogen.
Hypertension.
1995;26:131[Abstract/Free Full Text].
33.
Sharma HW, Perez JR, Higgins-Sochaski K, Hsiao R, Narayanan R.
Transcription factor decoy approach to decipher the role of NF-kappa B in oncogenesis.
Anticancer Res.
1996;16:61[Medline]
[Order article via Infotrieve].
34.
Sharma HW, Narayanan R.
The NF-kappaB transcription factor in oncogenesis.
Anticancer Res.
1996;16:589[Medline]
[Order article via Infotrieve].
35.
Morishita R, Higaki J, Tomita N, et al.
Role of transcriptional cis-elements, angiotensinogen gene-activating elements, of angiotensinogen gene in blood pressure regulation.
Hypertension.
1996;27:502[Abstract/Free Full Text].
36.
Maeshima Y, Kashihara N, Yasuda T, et al.
Inhibition of mesangial cell proliferation by E2F decoy oligodeoxynucleotide in vitro and in vivo.
J Clin Invest.
1998;101:2589[Medline]
[Order article via Infotrieve].
37.
Tomita N, Horiuchi M, Tomita S, et al.
An oligonucleotide decoy for transcription factor E2F inhibits mesangial cell proliferation in vitro.
Am J Physiol.
1998;275:F278[Abstract/Free Full Text].
38.
Kirken RA, Rui H, Malabarba MG, et al.
Activation of JAK3, but not JAK1, is critical for IL-2-induced proliferation and Stat5 recruitment by a COOH-terminal region of the IL-2 receptor beta-chain.
Cytokine.
1995;7:689[Medline]
[Order article via Infotrieve].
39.
Kotanides H, Nancy CR.
Requirement of tyrosine phosphorylation for rapid activation of a DNA binding factor by IL-4.
Science.
1993;262:1265[Abstract/Free Full Text].
40.
Wakao H, Gouilleux F, Groner B.
Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response.
EMBO J.
1994;13:2182[Medline]
[Order article via Infotrieve].
41.
Wagner BJ, Hayes TE, Hoban CJ, Cochran BH.
The SIF binding element confers sis/PDGF inducibility onto the c-fos promoter.
EMBO J.
1990;9:4477[Medline]
[Order article via Infotrieve].
42.
Sadowski HB, Shuai K, Darnell JE Jr, Gilman MZ.
A common nuclear signal transduction pathway activated by growth factor and cytokine receptors.
Science.
1993;261:1739[Abstract/Free Full Text].
43.
Schmidt J, Hatzelmann A, Fleissner S, Heimann-Weitschat I, Lindstaedt R, Szelenyi I.
Effect of phosphodiesterase inhibition on IL-4 and IL-5 production of the murine Th2-type T cell clone D10.G4.1.
Immunopharmacology.
1995;30:191[Medline]
[Order article via Infotrieve].
44.
Takeda KT, Tanaka W, Shi M, et al.
Essential role of Stat6 in IL-4 signaling.
Nature.
1996;380:627[Medline]
[Order article via Infotrieve].
45.
Brusselle G, Kips J, Joos G, Bluethmann H, Pauwels R.
Allergen-induced airway inflammation and bronchial responsiveness in wild-type and interleukin-4-deficient mice.
Am J Respir Cell Mol Biol.
1995;12:254[Abstract].
46.
Gavett SH, O'Hearn DJ, Karp CL, et al.
Interleukin-4 receptor blockade prevents airway responses induced by antigen challenge in mice.
Am J Physiol.
1997;272:L253[Abstract/Free Full Text].
47.
Paul WE, Ohara J.
B-cell stimulatory factor-1/interleukin 4.
Annu Rev Immunol.
1987;5:429[Medline]
[Order article via Infotrieve].
48.
Yoshimoto T, Paul WE.
CD4pos, NK1.1pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3.
J Exp Med.
1994;179:1285[Abstract/Free Full Text].
49.
Sad S, Marcotte R, Mosmann TR.
Cytokine-induced differentiation of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or Th2 cytokines.
Immunity.
1995;2:271[Medline]
[Order article via Infotrieve].
50.
Georas SN, Cumberland JE, Burke TF, Chen R, Schindler U, Casolaro V.
Stat6 inhibits human interleukin-4 promoter activity in T cells.
Blood.
1998;92:4529[Abstract/Free Full Text].
51.
Buck HM, Koole LH, van Genderen MH, et al.
Phosphate-methylated DNA aimed at HIV-1 RNA loops and integrated DNA inhibits viral infectivity.
Science.
1990;248:208[Abstract/Free Full Text].
52.
Matsukura M, Shinozuka K, Zon G, et al.
Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus.
Proc Natl Acad Sci U S A.
1987;84:7706[Abstract/Free Full Text].
53.
Agrawal S, Goodchild J, Civeira MP, Thornton AH, Sarin PS, Zamecnik PC.
Oligodeoxynucleoside phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus.
Proc Natl Acad Sci U S A.
1988;85:7079[Abstract/Free Full Text].
54.
Park YG, Nesterova M, Agrawal S, Cho-Chung YS.
Dual blockade of cyclic AMP response element- (CRE) and AP-1-directed transcription by CRE-transcription factor decoy oligonucleotide. gene-specific inhibition of tumor growth.
J Biol Chem.
1999;274:1573[Abstract/Free Full Text].
55.
Chen X, Vinkemeier U, Zhao Y, Jeruzalmi D, Darnell JE Jr, Kuriyan J.
Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA.
Cell.
1998;93:827[Medline]
[Order article via Infotrieve].
56.
Hou J, Schindler U, Henzel W, Ho T, Brasseur M, McKnight S.
An interleukin-4-induced transcription factor: IL-4 Stat.
Science.
1994;265:701.
57.
Moriggl R, Kristofic C, Kinzel B, Volarevic S, Groner B, Brinkmann V.
Activation of STAT proteins and cytokine genes in human Th1 and Th2 cells generated in the absence of IL-12 and IL-4.
J Immunol.
1998;60:3385.
58.
Morris SC, Coffman RL, Finkelman FD.
In vivo IL-4 responses to anti-IgD antibody are MHC class II dependent and beta 2-microglobulin independent and develop normally in the absence of IL-4 priming of T cells.
J Immunol.
1998;160:3299[Abstract/Free Full Text].
59.
Huang H, Hu-Li J, Chen H, Ben-Sasson SZ, Paul WE.
IL-4 and IL-13 production in differentiated T helper type 2 cells is not IL-4 dependent.
J Immunol.
1997;159:3731[Abstract].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
P. R. Hoffmann, C. Jourdan-Le Saux, F. W. Hoffmann, P. S. Chang, O. Bollt, Q. He, E. K. Tam, and M. J. Berry
A Role for Dietary Selenium and Selenoproteins in Allergic Airway Inflammation
J. Immunol.,
September 1, 2007;
179(5):
3258 - 3267.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Miyazaki, T. Satoh, K. Nishioka, and H. Yokozeki
STAT-6-Mediated Control of P-Selectin by Substance P and Interleukin-4 in Human Dermal Endothelial Cells
Am. J. Pathol.,
August 1, 2006;
169(2):
697 - 707.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Taha, Q. Hamid, L. Cameron, and R. Olivenstein
T Helper Type 2 Cytokine Receptors and Associated Transcription Factors GATA-3, c-MAF, and Signal Transducer and Activator of Transcription Factor-6 in Induced Sputum of Atopic Asthmatic Patients
Chest,
June 1, 2003;
123(6):
2074 - 2082.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. H. Wang, X. Y. Yang, X. Zhang, K. Mihalic, W. Xiao, and W. L. Farrar
The cis Decoy against the Estrogen Response Element Suppresses Breast Cancer Cells via Target Disrupting c-fos not Mitogen-activated Protein Kinase Activity
Cancer Res.,
May 1, 2003;
63(9):
2046 - 2051.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Benekli, M. R. Baer, H. Baumann, and M. Wetzler
Signal transducer and activator of transcription proteins in leukemias
Blood,
April 15, 2003;
101(8):
2940 - 2954.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. L. Leong, G. A. Andrews, D. E. Johnson, K. F. Dyer, S. Xi, J. C. Mai, P. D. Robbins, S. Gadiparthi, N. A. Burke, S. F. Watkins, et al.
Targeted inhibition of Stat3 with a decoy oligonucleotide abrogates head and neck cancer cell growth
PNAS,
April 1, 2003;
100(7):
4138 - 4143.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. H. Von der Thusen, J. Kuiper, T. J. C. Van Berkel, and E. A. L. Biessen
Interleukins in Atherosclerosis: Molecular Pathways and Therapeutic Potential
Pharmacol. Rev.,
March 1, 2003;
55(1):
133 - 166.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. M. Litterst and E. Pfitzner
An LXXLL Motif in the Transactivation Domain of STAT6 Mediates Recruitment of NCoA-1/SRC-1
J. Biol. Chem.,
September 20, 2002;
277(39):
36052 - 36060.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. von Knethen and B. Brune
Activation of Peroxisome Proliferator-Activated Receptor {gamma} by Nitric Oxide in Monocytes/Macrophages Down-Regulates p47phox and Attenuates the Respiratory Burst
J. Immunol.,
September 1, 2002;
169(5):
2619 - 2626.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. F. Skinnider, A. J. Elia, R. D. Gascoyne, B. Patterson, L. Trumper, U. Kapp, and T. W. Mak
Signal transducer and activator of transcription 6 is frequently activated in Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma
Blood,
January 15, 2002;
99(2):
618 - 626.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.C. Kips, K.G. Tournoy, and R.A. Pauwels
New anti-asthma therapies: suppression of the effect of interleukin (IL)-4 and IL-5
Eur. Respir. J.,
March 1, 2001;
17(3):
499 - 506.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. VON KNETHEN and B. BRUNE
Delayed activation of PPAR{gamma} by LPS and IFN-{gamma} attenuates the oxidative burst in macrophages
FASEB J,
February 1, 2001;
15(2):
535 - 544.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. M. McKay, F. Botelho, P. J. M. Ceponis, and C. D. Richards
Superantigen immune stimulation activates epithelial STAT-1 and PI 3-K: PI 3-K regulation of permeability
Am J Physiol Gastrointest Liver Physiol,
November 1, 2000;
279(5):
G1094 - G1103.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. J. M. Ceponis, F. Botelho, C. D. Richards, and D. M. McKay
Interleukins 4 and 13 Increase Intestinal Epithelial Permeability by a Phosphatidylinositol 3-Kinase Pathway. LACK OF EVIDENCE FOR STAT 6 INVOLVEMENT
J. Biol. Chem.,
September 8, 2000;
275(37):
29132 - 29137.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. H. Wang, X. Y. Yang, K. Mihalic, W. Xiao, D. Li, and W. L. Farrar
Activation of Estrogen Receptor Blocks Interleukin-6-inducible Cell Growth of Human Multiple Myeloma Involving Molecular Cross-talk between Estrogen Receptor and STAT3 Mediated by Co-regulator PIAS3
J. Biol. Chem.,
August 17, 2001;
276(34):
31839 - 31844.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction