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Gene Expression by a STAT6 and CCAAT/Enhancer-Binding Protein-Dependent Mechanism
* Department of Allergic Diseases, Novartis Forschungsinstitut, Vienna, Austria; and
Novartis Respiratory Research Center, Horsham, United Kingdom
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Introduction |
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55 million people affected worldwide. The current prevalence for asthma in the United States is estimated at 14.8% and is growing at 2.3% per annum (1). Animal models for asthma, despite their limitations, have been instrumental to dissect pathways contributing to the main disease phenotypes of airway hyperresponsiveness, eosinophilia, and increased mucus hyperplasia (2, 3). One of the critical molecules involved in the processes leading to the disease phenotype is the transcription factor STAT6. STAT6 is critically involved in the differentiation of naive T cell precursors into Th2 cells (4, 5, 6) and for class switching to the IgE isotype (7). STAT6 is activated by IL-4 or IL-13 (8) and thus represents an integral member of one of the IL-4 and IL-13 signal transduction pathways. Consequently, a number of important genes in an allergic immune response like the IL-4R (9), CD23 (10), the IgE germline promoter (GLP)2 (7, 11, 12), and eotaxin-1 and -3 (13, 14, 15) are directly regulated by STAT6. Not surprisingly, STAT6-deficient mice are unable to mount a pulmonary inflammation after allergen challenge (5, 16, 17).
Recently, a new gene family, called independently either found in inflammatory zone (FIZZ) (18) or resistin-like molecule (RELM) (19), was reported. Throughout this paper, we will refer to this gene family as FIZZ because this nomenclature was used in an experimental allergic inflammation system in the lung (18) similar to ours. Until now, three family members were identified in the mouse and two in humans, with supposedly human FIZZ1/RELM
still missing. The secreted form of a consensus FIZZ protein is 85–94 aa residues in length, with a motif consisting of 10 cysteine residues with a unique spacing of C-X11-C-X8-C-X-C-X3-C-X10-C-X-C-X-C-X9-CC at the C terminus. Murine (m) FIZZ2/RELM
and mFIZZ3/resistin/adipocyte-secreted factor (20) contain an additional cysteine residue in the variable N-terminal region, which is responsible for disulfide-linked homodimeric proteins, whereas FIZZ1 lacks this eleventh cysteine and therefore remains a monomeric protein (21).
The FIZZ1 protein was first described to be present in the bronchoalveolar lavage fluid in a murine allergic pulmonary inflammation model (18). mFIZZ1 mRNA was detected also in other tissues than lungs with its highest constitutive expression in white adipose, followed by mammary, tongue, and a weak expression in the heart. The other two FIZZ members have been reported to have a more restricted expression pattern. FIZZ2 was found exclusively in the epithelium of the colon and small bowel (18, 19). FIZZ3 is specifically expressed in adipose tissue but recently has also been detected in human circulating mononuclear cells (22).
Although there are also conflicting reports (23, 24), FIZZ3 is believed to play a role in insulin resistance and to serve as a link between obesity and type 2 diabetes (25, 26, 27). FIZZ1 was shown to inhibit nerve growth factor-mediated survival of neuronal cells and also blocked the increase of calcitonin gene-related peptide expression (18). Recently, it was shown that mFIZZ1 was expressed by alternatively activated macrophages in an IL-4-dependent manner (28). The biological function of FIZZ2 is completely unknown.
The present study extends our understanding of the regulation of FIZZ gene expression during the onset of allergic inflammation. All three FIZZ family members could be detected in Ag-challenged lungs albeit at very different expression levels. It is shown that FIZZ1 gene induction in allergically challenged lungs is an IL-4- or IL-13-driven process in which STAT6 and C/EBP are critical mediators. The FIZZ2 gene is also up-regulated in lung tissue in a STAT6-dependent fashion, however, presumably without direct involvement of IL-4 and IL-13. FIZZ3 gene expression, although constitutively present in lung tissue, did not change significantly upon allergic stimulation.
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Materials and Methods |
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The BMnot cell line was isolated from bone marrow of temperature-sensitive SV40 T-Ag transgenic mice which had been cultivated with GM-CSF for several weeks. Phenotypically, this cell line expresses myeloid markers CD11b, F4/80, and the dendritic cell marker CD11c, but no T cell marker and only CD45R/B220 among B cell markers. They show only low expression of MHC class II and costimulatory molecules CD40, CD80, and CD86.
BMnot was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated Ultra-Low IgG FBS (Life Technologies, Grand Island, NY), 25 mM HEPES, pH 7.3, 1 mM sodium pyruvate (Amimed, Allschwil, Switzerland), 50 µM 2-ME (Life Technologies), 200 ng/ml murine GM-CSF (Novartis, Basel, Switzerland), 2 mM glutamin, 100 U/ml penicillin, and 100 µg/ml streptomycin. Murine recombinant IL-4 and IL-13 were purchased from R&D Systems (Minneapolis, MN).
Animal model of allergic pulmonary inflammation, total RNA isolation, and cDNA synthesis
Female BALB/c mice were immunized on days 0 and 5 by i.p. injection of 20 µg of OVA (albumin, chicken egg; Sigma-Aldrich, St. Louis, MO) in 120 µl of NaCl adsorbed to 80 µl of 2% Al(OH)3 (Alu-Gel-S; Serva, Heidelberg, Germany) in a final volume of 0.2 ml. The mice were challenged once with either PBS aerosol or 5% OVA aerosol for 20 min on day 12. Six hours after challenge, lungs were excised and immediately put into liquid nitrogen. Frozen lungs (pooled or individually as mentioned in the text) were ground to a fine powder in the presence of liquid nitrogen and homogenized further with a Polytron homogenisator (Kinematika, Luzern, Switzerland). Total RNA isolation was performed with TRIzol (Invitrogen, Breda, The Netherlands) according to the manufacturer’s protocol. Contaminating DNA was removed from 15 µg of total RNA with RQ1 DNase digestion (Promega, Madison, WI). The RNA was reverse-transcribed with AMV reverse transcriptase (Roche Molecular Biochemicals, Mannheim, Germany).
C57BL/6 mice with targeted disruption of the gene encoding STAT6 (5) were provided by Dr. J. Ihle (St. Jude Children’s Research Hospital, Memphis, TN) and inbred by B&K Universal (North Humberside, U.K.). Age- and sex-matched C57/BL6 mice were purchased from Harlan (Oxon, U.K.). The animals were housed in plastic cages in an air-conditioned room at 24°C. Food and water were available ad libitum. The studies reported here were performed in compliance with the U.K. Animal (Scientific Procedures) Act 1986 and the guidelines issued by the Austrian Government (Tierversuchsgesetz 1988).
Quantitative PCR
The commercially available Sybr-Green PCR kit (Applied Biosystems, Warrington, U.K.) was used in an ABI7700 machine with the following PCR parameters: one cycle of 2 min at 50°C and 10 min at 95°C followed by 40 cycles of 15 sec at 95°C and 1 min at 62°C with continuous data measurement. All templates were analyzed in triplicates and the average was used for further calculation. The average cycle threshold (Ct) values of the FIZZ samples were normalized to the average Ct values of the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT) using the following equation: 2(Ct"HPRT"-Ct"FIZZ"). The average and SD of at least two independent Sybr-Green PCRs was used for the graph. The following primer pairs were used: mFIZZ1, 5'-CCAATCCAGCTAACTATCCCTCC-3' and 5'-AAGCCACAAGCACACCCAGT-3'; mFIZZ2, 5'-CTGGAGAGTGAATCTGCTC-3' and 5'-AAGGAGCATTGCGCGTTCC-3'; mFIZZ3, 5'-CTGTGTCCCATCGATGAAG-3' and 5'-GGAGCAGCTCAAGACTGCTG-3'.
As control, the expression levels of the HPRT gene was determined using the following primer pair: 5'-TTGCTCGAGATGTGATGAAGGA-3' and 5'-AAAGTTGAGAGATCATCTCCACCAA-3'.
Cloning of mFIZZ1 reporter constructs
A 409-bp mFIZZ1 promoter fragment was amplified from mouse genomic DNA (Clontech Laboratories, Palo Alto, CA) using the PCR primers 5'-CTGACTCGAGAGCAGGATCAGCTTGAATGG-3' and 5'-AGTCAAGCTTTTCCAGGACCTGGCCAGATG-3'. The PCR fragment was digested with XhoI/HindIII and cloned into pGL3basic (Promega) to give LUC-wild type (Wt). Site-directed mutations in the STAT6 site were generated as reported earlier (29) using the following oligonucleotides: mut2, 5'-GGCCTAGAATCATAATACACAAGAAATTGCTAA-3' and 5'-CTGTTTAGCAATTTCTTGTGTATTATGATTCTA-3'; mut3, 5'-GGCCTAGAATCATAATACACAAGCAATTGCTAA-3' and 5'-CTGTTTAGCAATTGCTTGTGTATTATGATTCTA-3'; mutA, 5'-GTGGGTTTACTGTCTCAAG-3'and 5'-GAGACTTGAGACAGTAAAC-3'; mutB, 5'-CACAAGAAACGGCTAAACA-3' and 5'-GTACTGTTTAGCCGTTTCT-3'; mutC, 5'-CTGAAAGTGCCGACTCCAC-3' and 5'-CCCAGTGGAGTCGGCACTT-3'; mutD, 5'-GTTAAGGGTACCTAGTCCAGC-3' and 5'-GATTGCTGGAC TAGGTACCCT-3'.
Plasmids were analyzed by digestion with restriction endonucleases and DNA sequencing.
Transient transfection
BMnot cells were harvested and washed twice in cold serum-free RPMI 1640 medium. Ten micrograms of supercoiled DNA was mixed with 5 x 106 cells in 250 µl of cold RPMI 1640 medium and electroporated at 950 µF and 230 V using a Bio-Rad Gene Pulser (Richmond, CA). Immediately after transfection, 700 µl of warm culture medium without GM-CSF were added and the cells were diluted with 3 ml of complete culture medium without GM-CSF. Aliquots were cultured for 24 h in the presence or absence of 5 ng/ml mIL-4 and/or 5 ng/ml mIL-13 before luciferase activity was measured.
Preparation of nuclear extracts and EMSA
Nuclear extracts from BMnot cells stimulated with 15 ng/ml mIL-4 for 30 min were prepared as described previously (30). C/EBP containing rat liver extracts were obtained from the commercially available human C/EBP Gelshift kit (Geneka Biotechnology, Québec, Canada). dsDNA was end-labeled using [-32P]dCTP (3000 Ci/Mol) (Amersham Life Sciences, Chalfont, U.K.) and Klenow polymerase (Bethesda Research Laboratories, Gaithersburg, MD). The labeled product was purified by size exclusion chromatography (G-25 Sephadex Microspin columns; Amersham Life Sciences) and polyacrylamide gel electrophoresis. The binding reaction with a 0.5-µg nuclear extract was performed as described previously (31). For competition experiments, if not mentioned otherwise, a 10-fold molar excess of unlabeled double-stranded oligonucleotide and for supershift experiments, 1 µg of specific Ab, was preincubated with nuclear extract on ice for 30 min before the addition of 0.5 ng of labeled dsDNA. After further incubation for 30 min on ice, nucleoprotein complexes were separated in a 4% native polyacrylamide gel at 4°C in 1x Tris-borate-EDTA at 200 V for 90 min. Gels were dried and exposed to x-ray films at -70°C (Kodak XOMAT MR; Rochester, NY). STAT6-M20 (sc-981), C/EBP
(sc-158X), C/EBP (sc-746X), and p50 (sc-114X) Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The following oligonucleotides were used in these experiments: mFIZZ1 Wt, 5'-GGCCTAGAATCATAATTCACAAGAAATTGCTAA-3' and 5'-CTGTTTAGCAATTTCTTGTGAATTATGATTCTA-3'; mFIZZ1 Mut2, 5'-GGCCTAGAATCATAATACACAAGAAATTGCTAA-3' and 5'-CTGTTTAGCAATTTCTTGTGTATTATGATTCTA-3'; mFIZZ1 Mut3, 5'-GGCCTAGAATCATAATACACAAGCAATTGCTAA-3' and 5'-CTGTTTAGCAATTGCTTGTGTATTATGATTCTA-3'; mFIZZ1-long Wt, 5'-TCATAATTCACAAGAAATTGCTAAACA-3' and 5'-GTACTGTTTAGCAATTTCTTGTGAATT-3'; mFIZZ1-long C/EBP mutB, 5'-TCATAATTCACAAGAAACGGCTAAACA-3' and 5'-GTACTGTTTAGCCGTTTCTTGTGAATT-3'.
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Results |
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To find new genes involved in early responses of allergic inflammation in the lung, a representational difference analysis (RDA) was performed. As templates, total RNA from whole lungs 6 h after a single challenge with either PBS or OVA was used. This early time point was chosen to focus on the first waves of gene expression initiated by resident cells, before an infiltration of effector cells had occurred. From 303 sequenced clones, enriched for induced genes in the OVA-challenged lungs, FIZZ1 was identified as the most abundant gene with 49 clones (16.17%). The same templates were also analyzed for differentially expressed genes using the Affymetrix chip technology. Also in this analysis, FIZZ1 (probe set 110838_at) was identified as being up-regulated in OVA-challenged lungs (data not shown).
To verify and quantitate these results, the same templates were tested in a quantitative RT-PCR (Fig. 1A). Indeed, FIZZ1 expression was strongly elevated by a factor of 33.6 in whole lungs 6 h after a single Ag challenge thus validating the RDA and chip data. In the same experiment, the expression levels of the two other FIZZ members were quantitated. FIZZ2 was not detectable in control animals consistent with the very restricted expression profile observed in previous studies (18, 19). The levels of FIZZ3 transcripts in PBS-challenged mice were 10-fold lower than that of FIZZ1. In OVA-challenged lungs, a clear up-regulation of 58.2-fold was detectable for FIZZ2. In contrast, FIZZ3 gene expression was not enhanced in Ag-challenged mice but was slightly reduced. The absolute levels of FIZZ2 transcripts were >1000-fold lower than those of FIZZ1 in allergen-challenged animals.
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IL-4 induces FIZZ1 gene expression in BMnot cells
The STAT6 protein is part of IL-4 and IL-13 signal transduction pathways. Therefore, a direct involvement of STAT6 in FIZZ gene regulation should be reflected by the ability of these cytokines to activate FIZZ. To test this, BMnot cells were cultured with IL-4 for different time periods and analyzed by RT-PCR for FIZZ transcripts. This myeloid-like cell line was found earlier to respond to IL-4 treatment (our unpublished observations). Induction of FIZZ1 gene expression was measured starting already 1 h after IL-4 was added and increased steadily up to the latest time point at 24 h (Fig. 2). At that time point, 10,000-fold more FIZZ1 transcripts were present in the cells compared with uninduced cultures. FIZZ2 expression was neither detectable at the onset of cytokine treatment nor at any time point during the experiment. FIZZ3 transcripts were readily observable in BMnot cells but their concentration did not change during the experiment. These results strongly suggested that STAT6 directly activated FIZZ1 gene expression. In contrast, FIZZ2 gene expression is likely not directly regulated by STAT6. This notion is supported by the fact that no STAT6 binding sites could be identified in the promoter region of the FIZZ2 gene (data not shown). Therefore, FIZZ2 induction in vivo is probably only indirectly regulated through a STAT6-dependent pathway.
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The fast onset of FIZZ1 expression in response to the cytokines is reminiscent of other IL-4-regulated genes, such as the IgE germline gene or the chemokines eotaxin-1 and -3 (11, 13, 14, 15). In all these genes, STAT6 appears to be responsible for the fast kinetic by interacting with a cis-acting regulatory element in the promoter region of the responding gene within minutes. To evaluate whether similar mechanisms are operative for STAT6-driven FIZZ1 gene induction, the promoter region of FIZZ1 was analyzed for STAT6 binding sites.
Inspection of the sequence upstream of the most 5'-located exon revealed a possible STAT6 site 5'-TTCACAAGAA-3' which corresponded well with the STAT6 consensus TTC(N4)GAA (Fig. 3). The sequence motif differed from the STAT6 site in the human IgE germline promoter by only 1 bp in the variable part (11). Four additional binding sites for transcription factors could be identified using the "findpatterns" command in the GCG package. Upstream of the putative STAT6 binding site, a putative Ets family member binding site could be detected, whereas downstream, two potential C/EBP binding sites and one peroxisome proliferator-activated receptor (PPAR) binding site were recognized (Fig. 3).
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B/PU.1 site of the IgE-GLP (32). Competition with the FIZZ1 STAT6 sequence containing a single mutation in the STAT6 core region (mut2), was only very weak. Furthermore, no competition at all could be detected with the mut3 double-stranded oligonucleotide (Fig. 3), containing two point mutations in the STAT6 core region. These data demonstrated that STAT6 core region nucleotides were responsible for binding of the IL-4-induced protein to the Wt double-stranded oligonucleotide. Moreover, the IL-4-induced band could be completely supershifted with a specific anti-STAT6 Ab, whereas the negative control, an anti-NF-B p50 Ab, had no effect. Finally, incubation of the same extracts with the labeled mut3 double-stranded oligonucleotide did not lead to any shifted band (Fig. 4A). Therefore, the IL-4-induced band was identified as STAT6. Functional STAT6 binding was also observed when IL-13-induced nuclear extracts were used, resulting in exactly the same binding pattern (data not shown).
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To show that the STAT6 site was involved in the cytokine-mediated induction of FIZZ1 gene expression, a 409p DNA fragment encompassing the STAT6 site (Fig. 3) was amplified from mouse genomic DNA and cloned into the pGL3basic vector. The luciferase gene was fused to exon1 of the FIZZ1 gene. In addition to the Wt promoter, reporter constructs were created carrying the same mutations in the STAT6 site used for the EMSA study in Fig. 4A (mut2, mut3). The reporter gene plasmids were transiently transfected into BMnot cells and stimulated with IL-4, IL-13, or the cytokine combination. Transient transfection of the control vector without insert (LUC vector) into BMnot cells resulted in very low constitutive activity barely above background. This activity could not be increased after induction with either IL-4, IL-13, or the combination of both (Fig. 4B). A completely different picture was obtained in cells transfected with the Wt FIZZ1 promoter plasmid LUC-Wt. The constitutive activity of this construct was similar to the control vector, but cultivation with IL-4 and/or IL-13 for 24 h led to a strong induction of promoter activity. The IL-4 treatment increased the promoter activity by 30.1-fold, the IL-13-mediated induction factor was 69.7-fold and the combination of both cytokines led to a 73.8-fold increase in FIZZ1 promoter activity.
Importantly, the LUC-Mut2 reporter construct, in which the STAT6 site is severely compromised to interact with the factor (Fig. 4A), was strongly impaired to respond to the cytokine stimulus with an increased promoter activity. The LUC-Mut3 reporter construct, whose STAT6 site is unable to bind STAT6, was completely unable to react to IL-4 and/or IL-13. Thus, a correlation of STAT6 binding and cytokine-mediated inducibility of the reporter constructs could be established. Together, these experiments demonstrated that STAT6 binding is absolutely necessary and of functional consequence for IL-4- and IL-13-mediated induction of the FIZZ1 promoter.
A C/EBP site is critically involved in IL-4-mediated inducibility of the mFIZZ1 promoter
Besides STAT6, other cis-acting elements were predicted in the FIZZ1 promoter sequence (Fig. 3). To test a potential regulatory role of these sites, reporter constructs were generated which carried point mutations in the predicted core motifs. The IL-4/IL-13-mediated inducibility of these plasmids was tested in transiently transfected BMnot cells in comparison to the Wt construct. The empty vector, the mFIZZ1 Wt and the STAT6 mut3 construct gave results as described before. Plasmids harboring mutations in the predicted Ets site (MutA), the proximal C/EBP site (MutC), and the PPAR site (MutD) responded to the cytokine stimulus as well as the Wt plasmid (Fig. 5A). The induction pattern of the construct containing the mutations in the distal C/EBP site (MutB), adjacent to the STAT6 site, differed markedly from the other three plasmids. In this case, IL-4 induced only a 3.3-fold increase in promoter activity compared with a 21-fold increase of the Wt plasmid. A similar reduction of up-regulation in response to IL-13 was observed where the Wt induction factor of 94-fold was reduced to 17-fold. The combination of both IL-4 and IL-13 gave very similar results with an 83% reduction in promoter activity compared with the Wt construct. These data showed that this putative C/EBP binding site was involved in the IL-4-mediated inducibility of the FIZZ1 promoter.
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Ab generated a supershifted complex (Fig. 5B). These data showed that BMnot cells indeed contain C/EBP protein albeit at low abundance. Incubation of the labeled Wt double-stranded oligonucleotide (mFIZZ1-longWT) with BMnot nuclear extracts yielded reproducibly three closely spaced complexes. However, large amounts of extract and long x-ray film exposure times were necessary to visualize these bands making a reliable interpretation of the results impossible (data not shown). To circumvent this problem, the same probe was incubated with a C/EBP containing rat liver nuclear extract provided in a commercially available EMSA detection kit for C/EBP. This extract contains much higher concentrations of C/EBP family members. The binding pattern obtained consisted of three complexes with similar sizes (Fig. 5C). Their migration properties matched closely the ones of the BMnot extract (data not shown). Preincubation with an anti-C/EBP Ab which recognizes a common epitope present in all C/EBP family members resulted in a supershift together with a weaker binding of one of the original three bands. Use of a specific C/EBP Ab recognizing only this isoform also gave rise to a supershift and an almost complete loss of the uppermost band. Use of an anti-NF-B p50 Ab did not change the binding pattern. These data suggested that indeed C/EBP family members could interact with the sequence motif next to the STAT6 site. Importantly, none of these large C/EBP-containing complexes were observed when a labeled double-stranded oligonucleotide containing the C/EBP MutB mutation was used as probe in the EMSA experiments (Fig. 5C). This showed that loss of C/EBP protein binding in the MutB reporter gene construct was responsible for the inability of this plasmid to respond to IL-4 or IL-13 stimulation. STAT6 binding is not influenced by the C/EBP mutation
The MutB C/EBP mutation is separated by only one nucleotide from the STAT6 core motif. Thus, the defect in IL-4-mediated promoter inducibility of this construct may be due to impaired STAT6 binding in addition to defective C/EBP binding. To rule out this possibility, STAT6 binding to the C/EBP mutant oligonucleotide was tested in an EMSA experiment using IL-4-induced BMnot extracts (Fig. 6). Compared with the Wt probe, no difference in the amount of STAT6 binding was observed. However, it was still possible that the C/EBP mutation led to diminished STAT6 binding affinity. To test for such a mechanism, competition with increasing molar excess of Wt or C/EBP mutant double-stranded oligonucleotides were performed. The potency of both double-stranded oligonucleotides to compete with the radiolabeled probes was indistinguishable from each other. These data support the conclusion that the affinity of STAT6 for its binding site was not changed by introduction of the C/EBP MutB mutation. This leads to the conclusion that the presence or absence of C/EBP itself is responsible for the observed phenotype of the MutB reporter construct.
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Discussion |
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IL-4 and IL-13 activate STAT6 by tyrosine phosphorylation leading to homodimerization and translocation of the protein into the nucleus. There, STAT6 homodimers can interact with specific DNA elements in promoters of IL-4 responsive genes (34). Indeed, a functional STAT6 binding site was identified in the FIZZ1 promoter. FIZZ1 promoter luciferase plasmids encompassing the STAT6 site could be activated by IL-4 and IL-13 in transient transfection experiments. Abrogation of STAT6 binding correlated with loss of IL-4-mediated transcriptional activation of FIZZ1 promoter reporter constructs, demonstrating that interaction of STAT6 with the DNA was essential for promoter activation. The STAT6 site is positioned within 200 bp of the transcriptional start site. Such a location appears to be typical for STAT6-regulated genes because the STAT6 sites in the IgE germline gene or the eotaxin-1 and -3 promoters are similarly spaced relative to the transcriptional start sites (9, 10, 11, 13, 14). The 409bp mFIZZ1 promoter piece used likely contains most binding sites necessary for efficient transcription in response to the cytokine stimuli. In fact, a 1.2-kb promoter construct was only twice as efficient after IL-4 treatment, and promoter reporter constructs in which the luciferase gene was fused to exon 2 instead of exon 1 responded equally well to IL-4 (data not shown).
The response of the reporter gene plasmids to IL-13 was always higher than to IL-4. Titration of the cytokines revealed that the response to IL-13 leveled off at a higher plateau than the response to IL-4 (data not shown). The same phenomenon was observed with a minimalistic STAT6-driven reporter construct (33) ruling out the possibility of an IL-13-mediated autocrine mechanism acting specifically on the FIZZ1 promoter (data not shown). The most likely explanation for the differences of cytokine responsiveness may be a higher number of IL-13Rs on the BMnot cell surface.
Besides STAT6, a C/EBP site adjacent to the STAT6 binding motif was identified to be critically involved in cytokine-induced FIZZ1 promoter activation. Our results show that C/EBP family members, in particular C/EBP, are able to interact with this site. However, these data were generated with nuclear extracts derived from rat liver cells and therefore the identity of the family members involved in FIZZ1 promoter activation remain to be determined. Unfortunately, the levels of C/EBP in BMnot extracts were too low to allow such an analysis. Nevertheless, the results support the conclusion that STAT6 and C/EBP members functionally cooperate in the induction of FIZZ1 promoter activity. The present data do not allow a statement if such a cooperation requires physical contact of the proteins. However, direct interaction of STAT6 with C/EBP family members has been demonstrated recently in other promoter systems, such as the IgE germline promoter (12, 35) and the CD23 promoter (36).
Interestingly, C/EBP was recently shown to bind to the FIZZ3 promoter and was sufficient for activity of the promoter (37). Furthermore, it was reported that C/EBP binding occurred only in adipocytes whereas in pre-adipocytes no binding, and therefore no FIZZ3 promoter activity, was detected. Therefore, C/EBP binding is thought to be responsible for the restricted cell type and differentiation-dependent gene expression of FIZZ3. By comparison to mFIZZ3 gene regulation, C/EBP proteins may not only cooperate with STAT6 for FIZZ1 gene induction but may also be involved in the cell type-specific expression of this gene. Such a scenario is supported by data demonstrating that IL-4 could not activate FIZZ1 promoter constructs in a number of IL-4-inducible cell lines derived from different tissues (data not shown). In summary, a model of mFIZZ1 activation is proposed in which the driving force behind mFIZZ1 activation in response to IL-4 or IL-13 is STAT6 which rapidly translocates into the nucleus after cytokine stimulation. Activated STAT6 is able to transactivate mFIZZ1 expression upon binding to its site. Transactivation requires cooperation with C/EBP family members which are constitutively bound on the adjacent C/EBP binding site. Thus, activation of FIZZ1 expression may be limited to certain cell types in which the correct C/EBP family members are present.
The function of FIZZ1 so far is not well understood. In the study of Moore et al. (26) it was shown that FIZZ1 expression was strongly diminished in diabetic mice compared with nondiabetic littermates, suggesting that normal FIZZ1 levels may be involved in adipocyte homeostasis. If this was true, then elevated FIZZ1 levels may have an antiadipogenic effect. Support for such a hypothesis comes from recent data describing a strong reduction of fat tissue in the skin of mice overexpressing IL-4 (38). It would be interesting to measure the levels of FIZZ1 expression in these animals.
A negative regulatory role of FIZZ1 as suggested above is consistent with its potential function in pulmonary inflammation. It has been demonstrated that FIZZ1 antagonizes the effects of nerve growth factor (18), a protein thought to be amplifying Th2 effector functions (39). However, the very fast time kinetic of FIZZ1 clearly precedes the influx of Th2 cells in the lung. In addition, all genes which are currently known to be directly regulated by STAT6 promote the allergic phenotype in some way or another. Therefore, it cannot be excluded that FIZZ1 positively contributes to an allergic inflammation. Further studies are required to clarify the exact function of this molecule.
Besides FIZZ1, FIZZ2 gene expression was also up-regulated in allergen-challenged lung tissue. The absolute levels of FIZZ2 transcripts were >1000-fold lower compared with FIZZ1. The low abundance may explain our inability to identify FIZZ2 clones in the RDA (data not shown). In control lungs, no FIZZ2 mRNA was measured confirming prior results that this gene was constitutively expressed only in certain parts of the colon (18, 19). Similar to FIZZ1, induction of FIZZ2 expression was significantly impaired in STAT6-deficient mice. It cannot be ruled out that this effect was due to differences in the kinetics of FIZZ2 induction in STAT6 knock-out mice compared with Wt animals. However, in the literature, no precedence for kinetic differences in STAT6-deficient vs Wt animals is reported. It is more likely that FIZZ2 gene regulation may also be dependent on STAT6. However, unlike FIZZ1, IL-4-treated BMnot cells did not respond with induction of FIZZ2 expression and inspection of the putative promoter region of the FIZZ2 gene did not reveal STAT6 binding sites (data not shown). From that, it is conceivable that the involvement of STAT6 on FIZZ2 gene induction in vivo is more indirect, for instance by STAT6-driven activation of an as yet unknown factor which then turns on FIZZ2 transcription in a STAT6-independent fashion.
In summary, the data in this study identify FIZZ1 and FIZZ2 as early induced gene products during the initial stages of allergen-triggered pulmonary inflammation. FIZZ1 represents another member of an IL-4- or IL-13-inducible gene in which STAT6 and C/EBP play pivotal roles in mediating the cytokine effect at the level of transcription. Elucidation of the functional roles of the FIZZ proteins in allergic conditions both in rodent model systems as well as in humans will determine their importance as therapeutic targets.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: GLP, germline promoter; FIZZ, found in inflammatory zone; RELM, resistin-like molecule; m, murine; HPRT, hypoxanthine phosphoribosyl transferase; Ct, cycle threshold; RDA, representational difference analysis; PPAR, peroxisome proliferator-activated receptor; Wt, wild type.
Received for publication August 22, 2002. Accepted for publication December 6, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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promoter: regulation by NF-IL-4, a C/EBP family member and NFB/p50. J. Exp. Med. 181:181. induced fibroblasts. J. Immunol. 166:4507.B and STAT6 in human airway epithelial cells. J. Immunol. 163:6876.
action in humans. Diabetes 50:2199. agonists. J. Biol. Chem. 276:25651.B or PU.1 to mediate IL-4-induced activation of IgE germline gene transcription. J. Immunol. 163:4383.B/rel in the IL-4-induced up-regulation of the human IgE germline promoter. J. Immunol. 159:3330.[Abstract]
promoter mediated by Stat6 and C/EBP. J. Immunol. 161:1822.B and C/EBP in CD23 expression and IgE production. Int. Immunol. 10:1529.This article has been cited by other articles:
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  T. Liu, B. Hu, Y. Y. Choi, M. Chung, M. Ullenbruch, H. Yu, J. B. Lowe, and S. H. Phan Notch1 Signaling in FIZZ1 Induction of Myofibroblast Differentiation Am. J. Pathol., May 1, 2009; 174(5): 1745 - 1755. [Abstract] [Full Text] [PDF]
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W. Stenzel, U. Muller, G. Kohler, F. L. Heppner, M. Blessing, A. N.J. McKenzie, F. Brombacher, and G. Alber IL-4/IL-13-Dependent Alternative Activation of Macrophages but Not Microglial Cells Is Associated with Uncontrolled Cerebral Cryptococcosis Am. J. Pathol., February 1, 2009; 174(2): 486 - 496. [Abstract] [Full Text] [PDF]
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 N. M. Heller, X. Qi, I. S. Junttila, K. A. Shirey, S. N. Vogel, W. E. Paul, and A. D. Keegan Type I IL-4Rs Selectively Activate IRS-2 to Induce Target Gene Expression in Macrophages Sci. Signal., December 23, 2008; 1(51): ra17 - ra17. [Abstract] [Full Text] [PDF]
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 M.-L. Wang, S. A. Keilbaugh, T. Cash-Mason, X. C. He, L. Li, and G. D. Wu Immune-mediated signaling in intestinal goblet cells via PI3-kinase- and AKT-dependent pathways Am J Physiol Gastrointest Liver Physiol, November 1, 2008; 295(5): G1122 - G1130. [Abstract] [Full Text] [PDF]
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 J. J. Reece, M. C. Siracusa, T. L. Southard, C. F. Brayton, J. F. Urban Jr., and A. L. Scott Hookworm-Induced Persistent Changes to the Immunological Environment of the Lung Infect. Immun., August 1, 2008; 76(8): 3511 - 3524. [Abstract] [Full Text] [PDF]
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 C. T. Migliaccio, M. C. Buford, F. Jessop, and A. Holian The IL-4R{alpha} pathway in macrophages and its potential role in silica-induced pulmonary fibrosis J. Leukoc. Biol., March 1, 2008; 83(3): 630 - 639. [Abstract] [Full Text] [PDF]
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 E. Daley, C. Emson, C. Guignabert, R. de Waal Malefyt, J. Louten, V. P. Kurup, C. Hogaboam, L. Taraseviciene-Stewart, N. F. Voelkel, M. Rabinovitch, et al. Pulmonary arterial remodeling induced by a Th2 immune response J. Exp. Med., February 18, 2008; 205(2): 361 - 372. [Abstract] [Full Text] [PDF]
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 S. L. Barnes, A. Vidrich, M.-L. Wang, G. D. Wu, F. Cominelli, J. Rivera-Nieves, G. Bamias, and S. M. Cohn Resistin-Like Molecule beta (RELMbeta/FIZZ2) Is Highly Expressed in the Ileum of SAMP1/YitFc Mice and Is Associated with Initiation of Ileitis J. Immunol., November 15, 2007; 179(10): 7012 - 7020. [Abstract] [Full Text] [PDF]
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S. D. Swain, S. Han, A. Harmsen, K. Shampeny, and A. G. Harmsen Pulmonary Hypertension Can Be a Sequela of Prior Pneumocystis Pneumonia Am. J. Pathol., September 1, 2007; 171(3): 790 - 799. [Abstract] [Full Text] [PDF]
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 A. Mishra, M. Wang, J. Schlotman, N. M. Nikolaidis, C. W. DeBrosse, M. L. Karow, and M. E. Rothenberg Resistin-like molecule-beta is an allergen-induced cytokine with inflammatory and remodeling activity in the murine lung Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293(2): L305 - L313. [Abstract] [Full Text] [PDF]
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R. J. Homer Airway remodeling and RELM-beta Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293(2): L303 - L304. [Full Text] [PDF]
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H. Novak, A. Muller, N. Harrer, C. Gunther, J. M. Carballido, and M. Woisetschlager CCL23 Expression Is Induced by IL-4 in a STAT6-Dependent Fashion J. Immunol., April 1, 2007; 178(7): 4335 - 4341. [Abstract] [Full Text] [PDF]
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M. G. Nair, K. J. Guild, and D. Artis Novel Effector Molecules in Type 2 Inflammation: Lessons Drawn from Helminth Infection and Allergy J. Immunol., August 1, 2006; 177(3): 1393 - 1399. [Full Text] [PDF]
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 A. L. Greene, M. S. Rutherford, R. R. Regal, G. H. Flickinger, J. A. Hendrickson, C. Giulivi, M. E. Mohrman, D. G. Fraser, and J. F. Regal Arginase Activity Differs with Allergen in the Effector Phase of Ovalbumin- versus Trimellitic Anhydride-Induced Asthma Toxicol. Sci., December 1, 2005; 88(2): 420 - 433. [Abstract] [Full Text] [PDF]
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M.-L. Wang, M. E. Shin, P. A. Knight, D. Artis, D. G. Silberg, E. Suh, and G. D. Wu Regulation of RELM/FIZZ isoform expression by Cdx2 in response to innate and adaptive immune stimulation in the intestine Am J Physiol Gastrointest Liver Physiol, May 1, 2005; 288(5): G1074 - G1083. [Abstract] [Full Text] [PDF]
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 M. P. Reilly, M. Lehrke, M. L. Wolfe, A. Rohatgi, M. A. Lazar, and D. J. Rader Resistin Is an Inflammatory Marker of Atherosclerosis in Humans Circulation, February 22, 2005; 111(7): 932 - 939. [Abstract] [Full Text] [PDF]
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M. G. Nair, I. J. Gallagher, M. D. Taylor, P. Loke, P. S. Coulson, R. A. Wilson, R. M. Maizels, and J. E. Allen Chitinase and Fizz Family Members Are a Generalized Feature of Nematode Infection with Selective Upregulation of Ym1 and Fizz1 by Antigen-Presenting Cells Infect. Immun., January 1, 2005; 73(1): 385 - 394. [Abstract] [Full Text] [PDF]
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 X. Liang, Y. C. Shin, R. E. Means, and J. U. Jung Inhibition of Interferon-Mediated Antiviral Activity by Murine Gammaherpesvirus 68 Latency-Associated M2 Protein J. Virol., November 15, 2004; 78(22): 12416 - 12427. [Abstract] [Full Text] [PDF]
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O. Norkina, T. G. Burnett, and R. C. De Lisle Bacterial Overgrowth in the Cystic Fibrosis Transmembrane Conductance Regulator Null Mouse Small Intestine Infect. Immun., October 1, 2004; 72(10): 6040 - 6049. [Abstract] [Full Text] [PDF]
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P. A. Knight, A. D. Pemberton, K. A. Robertson, D. J. Roy, S. H. Wright, and H. R. P. Miller Expression Profiling Reveals Novel Innate and Inflammatory Responses in the Jejunal Epithelial Compartment during Infection with Trichinella spiralis Infect. Immun., October 1, 2004; 72(10): 6076 - 6086. [Abstract] [Full Text] [PDF]
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 D. Artis, M. L. Wang, S. A. Keilbaugh, W. He, M. Brenes, G. P. Swain, P. A. Knight, D. D. Donaldson, M. A. Lazar, H. R. P. Miller, et al. RELM{beta}/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal tract PNAS, September 14, 2004; 101(37): 13596 - 13600. [Abstract] [Full Text] [PDF]
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 K. F. Wagner, A.-K. Hellberg, S. Balenger, R. Depping, J. Dodd-O, R. A. Johns, and D. Li Hypoxia-Induced Mitogenic Factor Has Antiapoptotic Action and Is Upregulated in the Developing Lung: Coexpression with Hypoxia-Inducible Factor-2{alpha} Am. J. Respir. Cell Mol. Biol., September 1, 2004; 31(3): 276 - 282. [Abstract] [Full Text] [PDF]
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T. Liu, H. Jin, M. Ullenbruch, B. Hu, N. Hashimoto, B. Moore, A. McKenzie, N. W. Lukacs, and S. H. Phan Regulation of Found in Inflammatory Zone 1 Expression in Bleomycin-Induced Lung Fibrosis: Role of IL-4/IL-13 and Mediation via STAT-6 J. Immunol., September 1, 2004; 173(5): 3425 - 3431. [Abstract] [Full Text] [PDF]
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O. Norkina, S. Kaur, D. Ziemer, and R. C. De Lisle Inflammation of the cystic fibrosis mouse small intestine Am J Physiol Gastrointest Liver Physiol, June 1, 2004; 286(6): G1032 - G1041. [Abstract] [Full Text] [PDF]
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D. Hebenstreit, P. Luft, A. Schmiedlechner, G. Regl, A.-M. Frischauf, F. Aberger, A. Duschl, and J. Horejs-Hoeck IL-4 and IL-13 Induce SOCS-1 Gene Expression in A549 Cells by Three Functional STAT6-Binding Motifs Located Upstream of the Transcription Initiation Site J. Immunol., December 1, 2003; 171(11): 5901 - 5907. [Abstract] [Full Text] [PDF]
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 A. Schaffler, A. Ehling, E. Neumann, H. Herfarth, I. Tarner, J. Scholmerich, U. Muller-Ladner, and S. Gay Adipocytokines in Synovial Fluid JAMA, October 1, 2003; 290(13): 1709 - 1710. [Full Text] [PDF]
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