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-Induced Fibroblasts
Department of Allergic Diseases, Novartis Research Institute, Vienna, Austria
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulation in a synergistic fashion. TNF- activated gene expression
at the transcriptional level in a STAT6-dependent manner, because: 1)
eotaxin-1 promoter luciferase constructs were TNF- inducible in
STAT6-defective HEK293 cells only on cotransfection of STAT6 expression
vector, an effect that was partially mediated by activation-induced
binding of NF-
B proteins to a composite STAT6/NF-B element; 2)
reporter constructs defective in STAT6 DNA binding did not respond to
TNF- stimulation; 3) eotaxin-1 protein secretion was detected only
in STAT6-transfected HEK293 cell supernatants on TNF- treatment; and
4) a trans-dominant negative STAT6 protein inhibited
TNF--induced eotaxin-1 secretion in primary fibroblasts. TNF-
inducibility of the IL-8 and monocyte chemoattractant protein-1 genes
was not dependent on STAT6 expression in the same experimental systems.
The inducing effect of IL-4 and IL-13 was also mediated by STAT6. The
synergistic effect of IL-4 and TNF- observed at the RNA and the
protein level was not seen at the promoter level. The data demonstrate
that both IL-4 and TNF- induce eotaxin-1 expression at the level of
transcription via a STAT6-mediated pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of eotaxin-1 itself is regulated by a number of cytokines.
It has been shown that the proinflammatory cytokines TNF- and IL-1
induce the production of eotaxin-1 (10, 11). More
recently, IL-4 and TNF- were reported to synergize in the secretion
of eotaxin-1 in human skin (12) and nasal fibroblasts
(13). Similarly, IL-13 which shares many biological
properties with IL-4 showed similar effects in human epithelial cells
(14, 15). The underlying mechanisms responsible for this
effect in epithelial cells have been characterized in more detail
(16). In that study, the transcription factor STAT6 was
shown to be responsible for the IL-4-mediated induction of eotaxin-1
promoter activity by binding to a specific DNA response element.
TNF- stimulation, in contrast, resulted in binding of NF-B
protein members to a site that overlapped with the STAT6-binding site,
and this interaction conferred promoter activation on TNF-
treatment. Incubation with the combination of the cytokines had an
additive effect.
The present study describes a similar analysis in human fibroblasts. We
confirm and extend the important role of STAT6 in induction of
eotaxin-1 promoter activity in response to IL-4 in human fibroblasts.
In contrast to the situation in epithelial cells (16),
TNF- stimulation was not additive or synergistic with IL-4.
Interestingly, the activating effect of TNF- on the promoter was
dependent on the presence of an intact STAT6-binding site and also on
the presence of functional STAT6 protein. In addition, TNF--induced
eotaxin-1 protein production was detected only in STAT6-expressing
cells and could be counteracted by a trans-dominant negative
STAT6 protein. Two other known TNF--inducible genes,
MCP-1 and IL-8, were not affected. The data show
that both IL-4 and TNF- require STAT6 as
mediator to activate eotaxin-1 gene expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal human adult dermal fibroblasts and neonatal fibroblasts
were cultured in FGM-2 medium (Clonetics, Walkersville, MD). HEK293
cells were carried at 37°C with 5% CO2 in DMEM
supplemented with 10% heat-inactivated FCS (Life Technologies, Grand
Island, NY), 100 U/ml penicillin, and 100 µg/ml streptomycin.
Purified human recombinant IL-4 was obtained from Novartis (Basel,
Switzerland) with a specific activity of 0.5 U/ng. Recombinant human
TNF- (Genzyme, Cambridge, MA) and recombinant human IL-13 (PeproTech
EC, London, U.K.) were used at a concentration of 100 U/ml. Human
eotaxin-1 and MCP-1 proteins were quantitated by commercially available
ELISA kits (R&D Systems, Minneapolis, MN).
RT-PCR
Total RNA was isolated using the Trizol reagent (Life
Technologies) according to the instructions of the manufacturer. Total
RNA, 3 µg, was reverse transcribed using avian myeloblastosis virus
reverse transcriptase (Roche Molecular Biochemicals, Mannheim, Germany)
in a total volume of 50 µl. A 260-bp fragment of the eotaxin-1
transcript was amplified using the intron spanning PCR primers
5'-CATGAAGGTCTCCGCAGCACTTCT-3' and 5'-CCAGATACTTCATGGAATCCTGC-3'
from cDNA corresponding to 20 ng RNA. PCR was performed for 30 cycles
at 94°C and 30 s, 56°C and 30 s, and 68°C and 30
s). The PCR primer pair 5'-ATGGATGATGATATCGCCGCG-3' and
5'-AGTCCATCACGATGCCAGTGG-3' was used to amplify a 480-bp fragment
of the 
-actin mRNA using the same reaction conditions .
Cloning of eotaxin-1 reporter constructs
A 1.1-kb eotaxin-1 promoter fragment was amplified from genomic
DNA (Roche Molecular Biochemicals) using the PCR primers
5'-CTGACTCGAGCAGGTTTGCAGTACCTCCACACC-3' and 5'-
AGTCAAGCTTGTTGGAGGCTGAAGGTGTGAGC-3'. The PCR fragment was digested
with XhoI and HindIII and cloned into pGL3-Basic
(Promega, Madison, MA) to give pGL3-EO1. Another promoter fragment was
amplified between position -2250 relative to the transcriptional start
site (17) and a natural PstI site at position
-986 using the primer pair 5'-AGTCACGCGTTTCAGGCGTAGAGTAAATCC-3'
and 5'-AGTCACTGCAGCGGATTACAGC-3'. This fragment was digested with
MluI and PstI and inserted into pGL3-EO1
restricted with the same enzymes to give pGL3-EO2 with a total insert
size of 2.2 kb. Plasmid pGL3-EO3 was constructed by inserting a 1.4-kb
EcoRI/HindIII fragment in which the
EcoRI site was made blunt with Klenow polymerase (Roche
Molecular Biochemicals) into a
SmaI/HindIII-digested pGL3-Basic vector.
Site-directed mutations in the composite STAT6/NF-B site were
generated as reported earlier (18) using the following
oligonucleotides: M1,
5'-ATGGGCAAAGGCTATCCTGGAATCTCCCACACTGTCTGCT-3' and
5'-GGGAGCAGACAGTGTGGTCGATTCCAGGGAAGCCTTTGCC-3'; M2,
5'-ATGGGCAAAGGCTTCCCTGCTATCTCCCACACTGTCTGCT-3' and
5'-GGGAGCAGACAGTGTGGGAGATAGCAGGGAAGCCTTTGCC-3'; M3,
5'-ATGGGCAAAGGCTTCCCTGGAATCGACCACACTGTCTGCT-3' and
5'-GGGAGCAGACAGTGTGGTCGATTCCAGGGAAGCCTTTGCC-3'. The cloning
of the STAT6 expression vector (19) and the IL-8
promoter reporter construct IL-8p (20) has been described.
The STAT6-
TD expression vector was cloned by insertion of a
XhoI/SacI fragment containing the complete human
STAT6 cDNA except for the carboxy-terminal trans-activation
domain into the pcDNA3.1 vector. Plasmids were analyzed by digestion
with restriction endonucleases and DNA sequencing. Constructs
used for transient transfections were purified by cesium chloride
density gradients.
Transient transfection of HEK293 cells and primary fibroblasts
The day before transfection, 5 x 104
cells were seeded into 24-well culture plates in fresh medium.
Transient transfection of HEK293 cells was achieved using calcium
phosphate coprecipitation. Briefly, 1–2 µg plasmid DNA was diluted
in 42 µl H2O, mixed with 7 µl 2 M
CaCl2, and added dropwise to 50 µl 2x HEPES
buffered saline (280 mM NaCl, 1.5 mM
Na2HPO4, 50 mM HEPES (pH
7.05)). After a 2-min incubation period at room temperature, the
mixture was added to the cells. Primary fibroblasts were transfected
with DNA-containing liposomes using Effectene (Qiagen, Hilden, Germany)
according to the manufacturer’s protocol. After 24 h, cells were
washed and cultured for 12 h in the presence or absence of 50
ng/ml IL-4, 100 ng/ml IL-13, and/or 100 U/ml TNF- before luciferase
assays were conducted in triplicates according to the instructions of
the manufacturer using the Promega Luciferase Assay System (Promega
Biotech, Madison, WI). In some experiments, pRL-Tk (Promega) was
cotransfected as internal control for normalization of differences in
transfection efficiency. Lysates from these cells were quantitated for
luciferase using the Dual-Luciferase Reporter Assay System
(Promega).
Preparation of nuclear extracts and EMSA
Nuclear extracts from unstimulated adult dermal fibroblasts or
cells that had been stimulated for 30 min with IL-4 (50 ng/ml) or
TNF- (100 U/ml) were prepared according to the method described by
Andrews and Faller (21). A ds oligonucleotide probe
spanning the composite STAT6/NF-B site from position -82 and -46
was end-labeled using [32P]dCTP (Amersham,
Little Chalfont, U.K.) and Klenow polymerase (Roche Molecular
Biochemicals). The nucleoprotein binding reaction was done as described
(22) using 5 µg nuclear extracts. For competition and
supershift experiments, extracts were preincubated with a 50-fold
excess of competitor oligo or 2 µg Ab for 30 min before the
radiolabeled probe was added. All Abs used in supershift experiments
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the kinetics of eotaxin-1 induction in primary
fibroblasts, cells derived from two donors (donor 1, adult skin
fibroblasts; donor 2, neonatal fibroblasts) were cultured with IL-4,
TNF-, or the combination for different times. The supernatants were
analyzed for eotaxin-1 protein by ELISA (Fig. 1
A). Chemokine expression
became detectable at 8 h in IL-4 and IL-4/TNF--stimulated cells
and was maximal at 24 h. TNF- alone induced eotaxin-1
expression in donor 2 (23) but not in donor 1 cells,
whereas both samples were responsive to IL-4 alone. In accordance with
published data (12), the cytokine combination had a clear
synergistic effect. Essentially the same results were obtained when
IL-13 was used as stimulus alone or in combination with TNF- (Fig. 1B). TNF- was able to induce gene expression in donor 1
cells because MCP-1, a chemokine known to be induced by this factor,
could be easily detected in the same supernatants (Fig. 1A).
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C).
Eotaxin-1 specific RNA was detectable as early as 2 h in
IL-4-induced cells, was maximal at 8 h, and was much less abundant
at the later time points in donor 1 cells. In accordance with the
protein data, TNF- stimulation did not induce gene transcription.
Donor 2 cells responded more slowly to IL-4 stimulation and produced
significant amounts of chemokine transcripts on TNF- stimulation. In
IL-4/TNF--stimulated cells, there was significantly more mRNA
detectable at 2 h. At the 8 h time point, eotaxin message was
maximally induced and remained at that level throughout the observation
period. This result suggested that IL-4-induced eotaxin transcripts
follow the typical characteristics of an immediate early gene. This
also applies for the TNF- stimulation in donor 2 cells, whereas in
donor 1 TNF- acted on the steady state levels of IL-4-induced
transcripts either at the posttranscriptional or at the transcriptional
level.
STAT6 mediates eotaxin-1 promoter activation by IL-4 or
TNF-
The kinetics by which IL-4 induced gene expression suggested that
this effect may be caused by the transcription factor STAT6. A number
of STAT6-regulated genes, such as the IgE germline gene, CD23, or the
IL-4 receptor gene showed similar kinetics of induction (24, 25).
The inspection of the published eotaxin-1 promoter sequence
(17) revealed the presence of two potential high affinity
STAT6-binding sites as defined by the
5'-TTC(N)4GAA-3' consensus sequence
(26). The proximal site between position -74 and position
-60 relative to the transcriptional start site overlapped with a
putative binding sequence for NF-B proteins. The distal site is
located between positions -2204 and -2195. Reporter constructs were
generated in which the firefly luciferase reporter gene is driven by
the human eotaxin-1 promoter. The sequence of the 2.2-kB insert in
pGL3-EO2 corresponded well with the published sequence. Of interest,
one difference was noted in the putative distal STAT6-binding sequence
where the cytidine at position 3 in the consensus sequence
5'-TTC(N)4GAA-3' was changed to a thymidine (Fig. 2). The presence of this substitution was
verified by using another PCR primer pair amplifying a small DNA
fragment encompassing this site (data not shown). A
5'-TTT(N)4GAA-3' sequence is generally not
recognized by STAT6 with high affinity (26), making the
proximal sequence a more likely candidate for a potential regulatory
role. Three 5' deletion promoter constructs (pGL3-EO1, -EO2, and -EO3)
(Fig. 2) were transiently transfected into the STAT6-defective HEK293
cell line (27) and tested for cytokine inducibility. In
the absence of STAT6 expression, none of the constructs was inducible
with IL-4, TNF- or the combination thereof (Fig. 3, left). The constitutive
promoter activity of the shortest EO-1 plasmid was lower than that of
the other two constructs, suggesting a positive regulatory element
between positions -1036 and -1359. In the presence of cotransfected
STAT6 expression vector, all three constructs were inducible on IL-4
stimulation. IL-13 stimulation was as effective as IL-4 and was also
dependent on the presence of cotransfected STAT6 (Fig. 3, right). The IL-4 induction index of the plasmids pGL3-EO2
and -EO3 was consistently higher than that of the EO1 construct,
suggesting that the sequence between positions -1036 and -1359
contributed to IL-4 inducibility as well as constitutive promoter
activity. Because the EO3 plasmid was as IL-4 responsive as the EO2
construct, a major regulatory role of the distal STAT6-like binding
site can be ruled out. All three constructs responded to TNF-
treatment albeit at a somewhat lower rate than IL-4. Interestingly,
TNF- responsiveness, like IL-4, was dependent on cotransfected
STAT6. The shortest pGL3-EO1 plasmid was less well inducible with
TNF-, suggesting that the same region that contributed to IL-4
up-regulation also was involved in TNF- regulation. The cytokine
combination activated promoter activity to the same degree as IL-4
alone. This demonstrated that the synergistic effect of the stimuli
seen at the protein level was not due to synergistic activation of the
eotaxin-1 promoter. Overall, these results suggested that the
stimulatory potential of these reporter constructs to both IL-4 and
TNF- was dependent on STAT6 coexpression. Because they all contain
the proximal STAT6/NF-B element, an involvement of this sequence
motif appeared likely.
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The interaction of STAT6 with the proximal STAT6/NF-B binding
sequence was assessed by EMSAs. Nuclear extracts prepared from human
dermal fibroblasts were incubated with a ds oligonucleotide probe
(-82/46), and the nucleoprotein complexes were resolved in native
polyacrylamide gels. In uninduced cells, two specific bands were
detected (Fig. 4A). Extracts
from IL-4-treated cells produced an additional band which migrated more
slowly than the two original complexes. Addition of anti-STAT6 Abs
before addition of the labeled probe specifically reduced the large
IL-4-induced complex and led to the formation of a supershifted
complex. Although an anti-PU.1-specific as well as an Ab directed
against the NF-B family member p50 did not change the banding
pattern, preincubation with an anti-p65 Ab led to disappearance of
the original two complexes present in uninduced extracts but not the
IL-4-induced band. This suggested that small amounts of NF-B p65
were present in uninduced cells and interacted with the -82/46 ds
probe. The addition of an 50-fold excess of unlabeled ds
oligonucleotide containing the functional STAT6-binding site of the
human IgE germline promoter (IgE104/83) specifically competed with the
formation of the IL-4-induced nucleoprotein complex to the radiolabeled
probe. These data demonstrated that the IL-4-induced band contains
STAT6.
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-induced extracts
(Fig. 4B). TNF- induced the formation of two
nucleoprotein complexes that migrated at the positions of the weak
constitutive complexes and were different from the one induced by IL-4,
suggesting that NF-B p65 was induced. Incubation with Abs directed
against STAT6, bcl-6, or NF-B p50 had no effect on the formation of
the complexes. In contrast, NF-B p65 Abs led to almost complete
disappearance of the TNF--induced (and constitutive; compare with
Fig. 4A) bands and formation of a supershifted complex. In
addition, successful competition with an authentic NF-B ds
oligonucleotide from the murine Ig C enhancer for both
TNF--induced complexes was observed (data not shown). These data
suggested that this cytokine stimulated the interaction of p65
containing B proteins but not STAT6 to the composite STAT6/NF-B
element. Involvement of the STAT6 response element in promoter activation
To determine whether the STAT6/NF-B-binding site was involved
in STAT6-mediated cytokine induction, three different point mutations
were introduced into the pGL3-EO2 construct. One mutation (M1)
specifically altered the palindromic TTC of the STAT6 site into TAT
(Fig. 2). This change has been shown earlier to ablate IL-4-induced
STAT6 binding and trans activation in the human IgE germline
promoter (28). Mutation M3 affected the polypyrimidine
half site of the NF-B element and mapped outside of the STAT6 core
sequence. The M2 mutant affected the overlapping portion of this
putative regulatory unit. The effect of these changes on transcription
factor interaction was monitored by EMSA. A ds oligonucleotide probe
containing the M1 mutation (-82/46 m1) was unable to bind STAT6 in
IL-4-induced extracts but still retained the ability to form NF-B
nucleoprotein complexes on TNF- stimulation (Fig. 5). Conversely, a M3 mutation containing
probe was able to bind STAT6 but not NF-B proteins. STAT6 binding
appeared to be much stronger than with the wild-type probe. This may be
explained either by a higher specific activity of the M3 mutant ds
oligonucleotide or by an increased affinity for the protein. It is
known that neighboring nucleotides are involved in fine tuning of the
affinity of DNA-binding proteins to its core recognition sequence. The
ds oligonucleotide M2 probe did not form any nucleoprotein complexes
consistent with the mutation located in the overlapping portion of the
composite STAT6/NF-B element.
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A, left). This
suggested that the STAT6/NF-B site acted as a positive regulatory
cis element. In STAT6-cotransfected cells, the IL-4 response
of the M1 and M2 mutant plasmids was abrogated, demonstrating that
cytokine inducibility by STAT6 was mediated via the proximal
STAT6-binding site. In contrast, the NF-B-specific M3 mutation
responded to IL-4 in a manner comparable to that of the wild-type
plasmid, suggesting that the NF-B binding site was not involved in
IL-4-mediated promoter trans activation. The phenotype of
the three mutant reporter constructs was identical when the
transfectants were incubated with IL-13 (data not shown).
Interestingly, TNF- inducibility of the STAT6-specific M1 mutation
was also completely abrogated. The double defective M2 mutant plasmid
showed the same phenotype, whereas in the NF-B-specific M3 plasmid
TNF- inducibility was partially reduced. These data showed that
eotaxin-1 promoter activation by both cytokines required the presence
of STAT6 and a functional STAT6-binding site. The overlapping NF-B
element was partially involved in the TNF- but not in the IL-4
response. To assess the specificity of STAT6 in eotaxin-1 promoter
activation after TNF- stimulation, an IL-8 promoter luciferase
reporter construct which has been shown to respond to TNF- treatment
(20) was tested under identical experimental conditions.
The inducible phenotype of this plasmid was observed in the absence or
presence of cotransfected STAT6 expression vector (Fig. 6A,
right). These results demonstrated that the dependence of
eotaxin-1 promoter activation on STAT6 in response to TNF- was
specific.
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B). Similar to the situation in HEK293
cells, the wild-type EO2 construct was inducible with IL-4 or TNF-
alone, whereas the cytokine combination did not lead to a further
increase in luciferase expression. Also the phenotype of the three
mutation plasmids was essentially identical with the one in HEK293
cells, with the exception that the constitutive promoter activity of
the M3 construct was significantly lower compared with the wild-type
plasmid. These data demonstrated that in primary human fibroblasts an
intact STAT6 site was required for mediating inducibility of the
eotaxin-1 promoter against both IL-4 and TNF-. Eotaxin-1 protein production is STAT6 dependent
Further confirmation for the involvement of STAT6 in eotaxin-1
regulation was obtained at the protein level. Eotaxin-1 was produced in
IL-4-stimulated HEK293 cells that had been transiently transfected with
a STAT6 expression vector but not in mock transfected cells (Fig. 7). Interestingly, TNF- stimulation
also led to eotaxin-1 production in STAT6-expressing cells only.
Similar to primary fibroblasts, a synergistic effect was measured in
the presence of both cytokines. In mock transfected cells, no eotaxin-1
secretion was measured. Importantly, the secretion of the known
TNF--inducible chemokine MCP-1 measured in the same supernatants was
very similar irrespective of the presence of cotransfected STAT6,
demonstrating that HEK293 cells are not generally defective in TNF-
signal transduction and that the effect seen with eotaxin-1 was
specific. Further evidence for the involvement of STAT6 in eotaxin-1
regulation was obtained in normal fibroblasts transfected with
increasing amounts of STAT6-TD DNA. The plasmid expresses a mutant
STAT6 protein that lacks the carboxy-terminal trans
activation domain. This polypeptide has been shown earlier to act in a
trans-dominant negative fashion (29). The
transfected cells were induced with cytokines for 48 h before
eotaxin-1 protein was monitored in the cell supernatants. Expression of
this STAT6 mutant in transiently transfected primary fibroblasts led to
strong inhibition of eotaxin-1 protein expression in either IL-4- or
TNF--induced cells (Fig. 8). Higher
amounts of DNA were not well tolerated by the cells. The effect,
however, was specific because transfection of empty vector was
significantly less inhibitory. In addition, no difference between
vector and STAT6-TD plasmid was observed when the same supernatants
were assayed for MCP-1 (data not shown). These results supported the
conclusion that STAT6 is a mediator of both TNF-- and IL-4-driven
eotaxin-1 secretion in fibroblasts but not of TNF--driven MCP-1
synthesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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during their synergistic induction
of eotaxin-1 expression in fibroblasts. A similar study was recently
conducted in epithelial cells (16). The results obtained
in the present effort extend the role of STAT6 as a mediator of
IL-4-induced eotaxin-1 production to fibroblasts. In addition, we
demonstrate that TNF- stimulation also is dependent on STAT6;
TNF--inducible reporter gene expression was dependent on an intact
STAT6-binding site both in HEK293 cells and in primary fibroblasts. In
addition, eotaxin-1 protein synthesis driven by TNF- was observed
only in STAT6-expressing HEK293 cells and could be inhibited by a
trans-dominant negative form of STAT6 in primary
fibroblasts. The data demonstrated that HEK293 cells displayed the same
phenotype as primary fibroblasts and therefore represent a valid
cellular system with which to study these aspects of eotaxin-1 gene
regulation. A number of possible scenarios can be envisioned to explain
this result. The most straightforward explanation would be the direct
activation of STAT6 by TNF-. Support for this comes from a recent
article describing STAT6 phosphorylation in adipocytes upon TNF-
treatment (30). Our efforts including EMSA analysis and
immunoblotting with phosphotyrosine-specific anti-STAT6 mAb have
thus far failed to detect a similar phenomenon in HEK293 cells or in
primary adult fibroblasts (Figs. 4B and 5 and data not
shown). Even in HEK293 cells that ectopically expressed wild-type
STAT6, no activation of this factor in response to TNF- could be
measured (data not shown). A more indirect possibility may be that
TNF- induces the expression of IL-4 or IL-13 (31) which
then, in an autocrine manner, activates eotaxin-1 promoter activity via
the STAT6 pathway. This possibility was addressed by measuring IL-4 and
IL-13 protein in supernatants of TNF--induced fibroblasts. In
addition, we attempted to block a possible autocrine action of IL-4 by
adding neutralizing anti-IL-4 Abs to TNF--stimulated cultures
and to measure IL-4 mRNA (data not shown). All efforts failed to
indicate the presence of IL-4, suggesting that a TNF--induced IL-4
autocrine mechanism was unlikely. A related possibility may be that
TNF- stimulation induced the expression of other factors able to
activate STAT6, such as platelet-derived growth factor
(32). Another candidate may be bradykinin, which has been
shown recently to induce eotaxin synthesis in fibroblasts
(33). Experiments are currently ongoing to address these
various possibilities.
The dependence of TNF- on STAT6 for eotaxin-1 promoter activation
appeared to be specific. In the same experimental system, an IL-8
promoter construct was responsive to the cytokine in the absence of
functional STAT6, and ectopic expression of the transcription factor
did not lead to further activation. Similar results were obtained at
the protein level where the TNF--induced production of MCP-1 was not
dependent on STAT6, whereas eotaxin-1 could be secreted only in
STAT6-transfected cells. These results demonstrated that the three
different TNF--inducible genes could be distinguished by
their requirement for functional STAT6 protein to respond to the
cytokine stimulus.
The composition of NF-B proteins induced by TNF- in fibroblasts
contained NF-B p65 but not p50, whereas both subunits were detected
in epithelial cells (16). Perhaps related to this
difference was the finding that mutations in the NF-B-specific
pyrimidine half site of the composite STAT6/NF-B sequence abrogated
TNF- inducibility in an epithelial cell line (16) in
contrast to only a partial effect in HEK293 cells.
Our current model of the individual functions of IL-4 and TNF- to
stimulate eotaxin-1 gene promoter in HEK293 and fibroblasts can be
summarized as follows. IL-4 activates STAT6 which is able to bind DNA
and acts as the predominant player at the level of transcriptional
activation of the eotaxin-1 promoter. TNF--induced NF-B may act
as transcriptional costimulus in a STAT6-dependent manner using an as
yet unknown mechanism.
The synergistic effect of the stimuli on eotaxin-1 protein production
is another point worth discussing. The data demonstrate that TNF-
did not synergize with IL-4 in the activation of eotaxin-1 promoter
reporter gene constructs both in HEK293 cells and in donor 2
fibroblasts. It is possible, however, that the reporter constructs used
did not contain important cis-acting regulatory DNA elements
involved in a possible synergism at the level of transcription
initiation. Alternatively, synergy may be achieved by a possible role
of IL-4 or TNF- in chromatin opening and gene accessibility in
combination with transcription initiation. Such effects would not be
seen in transient transfection experiments.
Another explanation for the synergistic effect is suggested by the time
course experiments. TNF- alone was not able to induce eotaxin-1
expression in donor 1 fibroblast cells, yet it synergized with IL-4 to
increase the levels of eotaxin-1 mRNA and also secreted chemokine. Its
effect on eotaxin-1 transcripts was clearly seen as early as 2 h.
This suggested a direct mode of action. Collectively, it is therefore
conceivable that TNF- is the predominant cytokine driving the
synergy and that it acts mainly at the posttranscriptional level. This
role of TNF- has been described for a number of other genes,
including bradykinin receptor (34), syndecan-4
(35), and Fc
RIIb (36).Two AUUUA sequence
motifs are located in the 3'-untranslated region of the eotaxin-1 mRNA.
These sequences have been implicated in the stabilization of mRNA
(37). Therefore, the possibility existed that the
synergistic function of TNF- may be related to stabilization of
eotaxin-1 transcripts. We have compared the half-lives of IL-4-induced
eotaxin-1 mRNA with the one of IL-4/TNF--induced transcripts in
cells treated with actinomycin D. The results demonstrated that
eotaxin-1 transcripts under both conditions are very stable with a
half-life of >18 h (data not shown). The long stability of eotaxin-1
mRNA is in good agreement with previous measurements (38).
These data suggested that TNF--induced stabilization of eotaxin-1
transcripts is unlikely to be the mode of action of the cytokine to
synergize with IL-4.
The architecture of the eotaxin-1 promoter is reminiscent of the
regulatory region of the human IgE germline gene (39, 40).
In both promoters a STAT6 binding site is flanked by a NF-B-binding
motif. Although binding of B-factors appear to be dispensable for
IL-4-mediated eotaxin-1 promoter activation, NF-B proteins, together
with the transcription factor PU.1, are critically involved in
cytokine-driven IgE germline promoter activity (19). This
functional difference may be related to the different stimuli that
induce DNA binding of NF-B to their respective binding site.
Agonistic anti-CD40 Abs did not synergize with IL-4 to express
eotaxin-1 (data not shown), whereas they act as costimulus for
IL-4-induced IgE germline gene expression (28).
The dependence of eotaxin-1 synthesis on STAT6 on cytokine stimulation may also explain some aspects of the phenotype observed in STAT6-deficient mice. In mouse models of allergic lung inflammation (41, 42, 43), contact hypersensitivity (44), and allergic diarrhea (45), STAT6 deficiency was accompanied by a lack of eosinophil influx into the sites of allergic inflammation. This effect may be at least partially due to defective eotaxin-1 production in these animals.
In summary, the data strengthen the importance of STAT6 as key player in situations of allergic inflammation and therefore as target for therapeutic intervention.
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Acknowledgments |
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Footnotes |
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2 Abbreviation used in this paper: MCP, monocyte chemoattractant protein.
Received for publication September 14, 2000. Accepted for publication January 17, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synergistically induce eotaxin production in human nasal fibroblasts. Clin. Exp. Allergy 30:348.[Medline]
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D. K. Fritz, C. Kerr, L. Tong, D. Smyth, and C. D. Richards Oncostatin-M Up-Regulates VCAM-1 and Synergizes with IL-4 in Eotaxin Expression: Involvement of STAT6 J. Immunol., April 1, 2006; 176(7): 4352 - 4360. [Abstract] [Full Text] [PDF]
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M. Nie, A. J. Knox, and L. Pang {beta}2-Adrenoceptor Agonists, Like Glucocorticoids, Repress Eotaxin Gene Transcription by Selective Inhibition of Histone H4 Acetylation J. Immunol., July 1, 2005; 175(1): 478 - 486. [Abstract] [Full Text] [PDF]
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H. S. Chang, J. S. Kim, J. H. Lee, J. I. Cho, T. Y. Rhim, S.-T. Uh, B. L. Park, I. Y. Chung, C.-S. Park, and H. D. Shin A Single Nucleotide Polymorphism on the Promoter of eotaxin1 Associates with Its mRNA Expression and Asthma Phenotypes J. Immunol., February 1, 2005; 174(3): 1525 - 1531. [Abstract] [Full Text] [PDF]
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N. Torpey, S. E. Maher, A. L. M. Bothwell, and J. S. Pober Interferon {alpha} but Not Interleukin 12 Activates STAT4 Signaling in Human Vascular Endothelial Cells J. Biol. Chem., June 18, 2004; 279(25): 26789 - 26796. [Abstract] [Full Text] [PDF]
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Q. Peng, T. Matsuda, and S. J. Hirst Signaling Pathways Regulating Interleukin-13-stimulated Chemokine Release from Airway Smooth Muscle Am. J. Respir. Crit. Care Med., March 1, 2004; 169(5): 596 - 603. [Abstract] [Full Text] [PDF]
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V. Hwa, B. Little, E. M. Kofoed, and R. G. Rosenfeld Transcriptional Regulation of Insulin-like Growth Factor-I by Interferon-{gamma} Requires STAT-5b J. Biol. Chem., January 23, 2004; 279(4): 2728 - 2736. [Abstract] [Full Text] [PDF]
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K. Chibana, Y. Ishii, T. Asakura, and T. Fukuda Up-Regulation of Cysteinyl Leukotriene 1 Receptor by IL-13 Enables Human Lung Fibroblasts to Respond to Leukotriene C4 and Produce Eotaxin J. Immunol., April 15, 2003; 170(8): 4290 - 4295. [Abstract] [Full Text] [PDF]
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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]
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S. J. Hirst, M. P. Hallsworth, Q. Peng, and T. H. Lee Selective Induction of Eotaxin Release by Interleukin-13 or Interleukin-4 in Human Airway Smooth Muscle Cells Is Synergistic with Interleukin-1beta and Is Mediated by the Interleukin-4 Receptor alpha -Chain Am. J. Respir. Crit. Care Med., April 15, 2002; 165(8): 1161 - 1171. [Abstract] [Full Text] [PDF]
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J. Hoeck and M. Woisetschlager Activation of Eotaxin-3/CCL26 Gene Expression in Human Dermal Fibroblasts Is Mediated by STAT6 J. Immunol., September 15, 2001; 167(6): 3216 - 3222. [Abstract] [Full Text] [PDF]
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