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Am J Physiol Lung Cell Mol Physiol 285: L137-L148, 2003; doi:10.1152/ajplung.00441.2002
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STAT-1 and c-Fos interaction in nitric oxide synthase-2 gene activation

Weiling Xu,1 Suzy A. A. Comhair,1 Shuo Zheng,1 Shan C. Chu,2 Joanna Marks-Konczalik,2 Joel Moss,2 S. Jaharul Haque,1 and Serpil C. Erzurum1

1Pulmonary and Critical Care Medicine and Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195; and 2Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

Submitted 30 December 2002 ; accepted in final form 12 March 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interferon-{gamma} (IFN-{gamma}) is required for induction of the human nitric oxide synthase-2 (NOS2) gene in lung epithelium. Although the human NOS2 promoter region contains many cytokine-responsive elements, the molecular basis of induction is only partially understood. Here, the major cis-regulatory elements that control IFN-{gamma}-inducible NOS2 gene transcription in human lung epithelial cells are identified as composite response elements that bind signal transducer and activator of transcription 1 (STAT-1) and activator protein 1 (AP-1), which is comprised of c-Fos, Fra-2, c-Jun, and JunD. Notably, IFN-{gamma} activation of the human NOS2 promoter is shown to require functional AP-1 regulatory region(s), suggesting a role for AP-1 activation/binding in the IFN-{gamma} induction of genes. We show that c-Fos interacts with STAT-1 after IFN-{gamma} activation and the c-Fos/STAT-1 complex binds to the {gamma}-activated site (GAS) element in close proximity to AP-1 sites located at 4.9 kb upstream of the transcription start site. Taken together, our findings support a model in which a physical interaction between c-Fos and STAT-1 participates in NOS2 gene transcriptional activation.

gene regulation; signal transduction

INTERFERON-{gamma} (IFN-{gamma}) induces gene expression through activation of specific members of the Janus kinase (JAK) family, which in turn phosphorylate signal transducer and activator of transcription 1 (STAT-1; see Refs. 14 and 15). The phosphorylated STAT-1 molecules form homodimers, translocate to the nucleus, and bind to {gamma}-activated sites (GAS) in the 5'-flanking regions of genes, such as the nitric oxide synthase-2 (NOS2) gene. The importance of STAT-1 for IFN signaling is clearly demonstrated by STAT-1-deficient mice, which fail to respond to IFNs and are consequently highly sensitive to microbial infection, which is the result of lack of induction of downstream target genes, such as NOS2 (5, 29, 32).

NOS2, which is inducible in diverse cell types by cytokines, converts L-arginine to L-citrulline, and nitric oxide (NO; see Refs. 30 and 36). NO, a short-lived, free radical gas, functions in essential biological processes, including regulation of vascular tone and host defense (30, 36). In addition to its beneficial effects, increased production of NO is associated with inflammatory tissue damage in human diseases, e.g., septic shock and asthma (6, 11, 25, 27, 44). The molecular basis for induction of the human NOS2 gene is only partially understood. The 8.3-kb human NOS2 promoter region contains clusters of cytokine-responsive elements, perfectly or partially matched to consensus sequences, including GAS and activator protein 1 (AP-1; see Refs. 3, 21, 35, 39). Previous studies have demonstrated that IFN-{gamma} is necessary for induction of the murine and human NOS2 promoter (3, 4, 8, 9, 24, 45, 47) and the GAS element in the NOS2 promoter is essential for IFN-{gamma} activation (8, 9, 45). However, prior work has also emphasized the importance of AP-1 binding sites in activation of the NOS2 promoter in response to cytokine combinations (3, 26). AP-1 is a complex composed of proteins of the Fos (c-Fos, FosB, Fra-1, and Fra-2) and Jun (c-Jun, JunB, and JunD) protooncogene families (2, 35, 43). In general, Fos and Jun family proteins function as dimeric transcription factors that bind to AP-1 regulatory elements in the promoter and enhancer regions of genes (2, 43).

Induction of NOS2 gene expression in the lung epithelial cell line A549 requires activation of AP-1, but the relevance of AP-1 to in vivo activation of the human NOS2 gene is not clear, since IFN-{gamma} alone is sufficient for induction of NOS2 in primary human airway epithelial cells (HAEC), the key cellular source of NO in the lung (12, 42). IFN regulatory factor (IRF)-1, another essential transcriptional activator of NOS2 gene expression (18, 41), is itself induced by IFN-{gamma} activation of STAT-1, which binds to GAS in the IRF-1 promoter (20, 38). IFN-{gamma} also activates c-fos gene expression through the sis-inducible element within the c-fos promoter, which binds sis-inducible factor complexes, comprised of homodimers of STAT-3, heterodimers of STAT-3 and STAT-1, and homodimers of STAT-1 (31, 46). STAT proteins demonstrate cooperative DNA binding not only with other STAT family members (e.g., STAT-1/STAT-2 and STAT-1/STAT-3; see Refs. 10, 14, and 16) but also with other proteins and transcription factors, including transcriptional activator specificity protein (SP)-1 and CCAAT enhancer binding protein (22, 23). Recently, physical association between STAT-3 and c-Jun on the {alpha}2-macroglobulin enhancer element has been shown to yield maximal enhancer functions (48).

Based upon this knowledge, we hypothesized that cooperative interaction between AP-1 and STAT-1 pathways may be important in the activation of IFN-{gamma}-activated genes, such as NOS2. Here, we show that c-Fos rapidly interacts with STAT-1 after IFN-{gamma} activation and the c-Fos/STAT-1 complex binds to the GAS element in close proximity to AP-1 sites located in a 665-bp region at 4.9 kb upstream of the transcription start site. Taken together, our findings support a physical interaction between c-Fos and STAT-1 and suggest a role for c-Fos and STAT-1 in transcriptional activation of NOS2 gene.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
NOS2 luciferase reporter constructs. A series of deletion constructs containing the NOS2 gene 5'-flanking region cloned into the pGL-3-basic luciferase reporter gene vector (Promega, Madison, WI), and named pGL3-8296, -7196, -6251, -5574, -4909, -4711, -4060, -3137, and -336, or the pGL3–8296 full-length NOS2 promoter that had undergone oligonucleotide-directed mutagenesis of AP-1-binding sites were used in experiments (3, 26). All constructs were subjected to digestion with restriction enzymes and sequence analysis to verify the 5'-end of the insert.

Cell culture. HAEC were isolated from bronchoscopic brushing of the airway, or from surgical specimens of tracheas and main-stem bronchi, and cultured as previously described (11, 41). Primary cultures of passage 0–3 were used in experiments. The epithelial nature of primary and cultured cells was confirmed by immunocytochemical staining, as previously described (12). A549 cells, an epithelial cell line derived from lung adenocarcinoma, were cultured in MEM (Invitrogen, Carlsbad, CA) with 10% heat-inactivated FCS, or 24 h before cytokine stimulation with 1% FCS. A STAT-1-deficient fibrosarcoma cell line, U3A, was maintained in DMEM (Invitrogen) with 10% FCS (1, 7). 293T cells, a clone of 293 (human embryonic kidney fibroblast cells) that expresses the Simian virus 40 large-T antigen, were maintained in DMEM (Invitrogen) with 10% FCS. Human IFN-{gamma} was a gift from Genentech (South San Francisco, CA) or was purchased from R&D Systems (Minneapolis, MN). Recombinant human interleukin (IL)-1{beta} and tumor necrosis factor (TNF)-{alpha} were purchased from Biosource (Camarillo, CA).

Transient transfection and luciferase assay. With the use of Lipofectamine Reagent (Invitrogen), 40% confluent HAEC and 30% confluent A549 cells in six-well plates were transfected with various NOS2 luciferase reporter constructs. Transfections were performed using equal amounts of DNA, as previously described (3). After adding the DNA with Lipofectamine to each well and incubating for 4 h for HAEC and 10 h for A549, the medium was replaced with normal growth medium for HAEC and MEM with 1% FCS for A549 cells. After (24 h) transfection, cells were exposed to cytokine mixture (CK). Later (24 h), cells were washed in PBS, harvested after the addition of 250 µlof1x Passive Lysis Buffer, freeze-thawed two times, and centrifuged (12,000 g, 2 min). Supernatants were assayed for firefly luciferase activity using the Dual-Luciferase Reporter Assay (Promega) in which luciferase activities are normalized by dual (Renilla) luciferase assay (Promega). In separate experiments, pCMV-{beta}-Gal (Invitrogen) was used to determine the percentage of cells transfected. Relative luciferase activity is reported as means of values from more than three independent experiments, each performed in triplicate.

The antisense phosphorothioated oligodeoxynucleotide (5'-CCGAGAACATCATCGTGGCG-3') was directed against the translation initiation site of c-Fos mRNA (40). Corresponding sense oligodeoxynucleotide (5'-CGCCACGATGATGTTCTCGG-3') was used as a control. With the use of Lipofectamine reagent, A549 cells were cotransfected with a 8,296-bp full-length NOS2 promoter and the c-Fos antisense phosphorothioated oligodeoxynucleotide or sense oligodeoxynucleotide and then incubated in the presence or absence of CK for 6 or 12 h. Cells were harvested, and supernatants were assayed for luciferase activity.

With the use of Lipofectamine Reagent (Invitrogen), 293T cells or A549 cells were transfected with 2 µg DNA containing various expression vectors, HA-tagged STAT-1 (HA-STAT-1; see Ref. 34), HA-tagged TAK1 (HA-TAK1; see Ref. 37), or pRSV-c-Fos (33). The medium was replaced with 1% FCS after 10 h transfection. After transfection (24 h), cells were exposed to CK for 24 h, and then cells were washed in PBS and harvested.

EMSA. Whole cell extract (WCE) was prepared as previously described (11, 41). For nuclear extract, the cell suspensions were centrifuged and resuspended in 0.4 ml ice-cold buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM PMSF, and 1 mM DTT] by gentle pipetting in a yellow tip, and then cells were allowed to swell on ice for 15 min. Subsequently, 25 µl of 10% solution of Nonidet P-40 was added, vigorously vortexed for 10 s, and then spun for 30 s in a microfuge. The nuclear pellet was resuspended in 50 µl ice-cold buffer [20 mM HEPES (pH 7.9), 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, and 1 mM DTT].

The duplex oligonucleotides used in EMSA (Table 1) were synthesized by Operon (Alameda, CA) and then end-labeled with [{gamma}-32P]ATP by polynucleotide kinase (11, 41). For binding reactions, cell extract was incubated in 20 µl total reaction volume containing 20 mM HEPES (pH 7.9), 5% glycerol, 50 mM NaCl, 5 mM DTT, 0.1 mM EDTA, 100 µg/ml BSA, and 2 µg polydeoxyinosinic-polydeoxcytidylic acid (Amersham, Arlington Heights, IL) for 15 min at room temperature. The 32P-labeled oligonucleotide (2 x 105 counts/min) was added to the reaction mixture and incubated for 20 min at room temperature. The reaction mixture was analyzed by electrophoresis on a 4% polyacrylamide gel in 0.25x buffer containing 12.5 mM Tris, 12.5 mM borate, and 0.5 mM EDTA. The gels were dried and analyzed by autoradiography. To demonstrate specificity of binding, competition was performed by adding unlabeled wild-type and mutated oligonucleotide at a 100-fold molar excess of 32P-labeled oligonucleotide probe in the binding reaction. To specifically identify AP-1, GAS binding-factor, and NF-{kappa}B proteins in binding complexes, 4 µg rabbit anti-c-Fos, FosB, Fra-1, Fra-2, c-Jun and JunD, STAT-3, STAT-5, p50 or p65 polyclonal antibody (Ab; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-STAT-1 polyclonal Ab (11, 13), or nonimmune rabbit IgG (Biodesign, Saco, ME) was added to the binding reaction mix and incubated for 30 min at room temperature before adding the 32P-labeled oligonucleotide. Antibodies used in experiments include anti-c-Fos Ab (rabbit polyclonal Ab against domain of c-Fos p62 of human origin; Santa Cruz Biotechnology), and c-Fos(2) Ab (rabbit polyclonal Ab against the amino terminus of c-Fos p62 of human origin; Santa Cruz Biotechnology).


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  		Table 1. Oligonucleotides used in EMSA

 

Immunoprecipitation and Western blot analysis. Extracts were prepared by lysing the cells in ice-cold buffer containing 50 mM Tris (pH 7.9), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% Nonidet P-40, 10% glycerol, 1 mM PMSF, 5 µg/ml leupeptin, 10 µg/ml pepstatin A, 200 µM NaOV, and 20 µg/ml aprotinin on ice for 30 min and centrifuging at 13,000 g for 30 min at 4°C. The cleared supernatant containing 400–600 µg proteins was incubated with 4–6 µg antibodies (anti-c-Fos, c-Jun, STAT-1, STAT-3, STAT-5 Ab, and anti-HA Ab; Upstate Biotechnology, Lake Placid, NY) or nonimmune rabbit IgG for 2 h at 4°C, and then 100 µl protein G-Sepharose (Amersham) was added and incubated for 2 h at 4°C. The captured beads were washed and boiled in denaturing buffer, and the released proteins were analyzed by Western blot. Protein was separated by electrophoresis on an 8 or 12.5% SDS-polyacrylamide gel and then electrophoretically transferred to nitrocellulose (Osmonics, Minnetonka, MN). Signal detection was accomplished with primary rabbit polyclonal Abs, followed by a secondary anti-rabbit Ab (Amersham).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of CK and IFN-{gamma} on activity of human NOS2 promoter constructs in HAEC and A549 cells. NOS2 mRNA is induced by IFN-{gamma} in HAEC (12, 42). Here NOS2 protein expression in HAEC is also induced by IFN-{gamma} alone (Fig. 1). In contrast, NOS2 expression in most cell lines, including A549, requires exposure to IFN-{gamma} in combination with a mixture of cytokines, such as IL-1{beta} and/or TNF-{alpha} (3). The time course of NOS2 protein induction in HAEC by IFN-{gamma} is slower than CK, with equivalent protein present at 48 h. The time course of NOS2 mRNA induction by IFN-{gamma} is also delayed compared with CK (42). The lack of NOS2 induction in U3A, a cell line lacking STAT-1, may be because of the fact that STAT-1 activation and binding to GAS elements is required for NOS2 expression in cells (11, 29).



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  		Fig. 1. Nitric oxide synthase-2 (NOS2) protein expression by interferon-{gamma} (IFN-{gamma}) or cytokine mixture [CK: IFN-{gamma}, interleukin (IL)-1{beta}, and tumor necrosis factor (TNF)-{alpha}] in human airway epithelial cells (HAEC) and A549 cells. Western analyses of NOS2 in extracts from nonstimulated HAEC (lane 1) and HAEC induced by IFN-{gamma} (lanes 2, 4, and 6)orCK(lanes 3, 5, and 7), and from A549 cells induced by CK (lane 9) for the times indicated. U3A cells lacking signal transducer and activator of transcription (STAT)-1 do not express NOS2 with CK (lane 8). Similar results were obtained in 3 independent experiments.

 

To identify IFN-{gamma}-responsive transcriptional elements in the 5'-flanking region of the NOS2 gene, HAEC and A549 cells were transiently transfected with constructs containing various segments of the proximal 5'-flanking region of the human NOS2 gene driving luciferase expression. After transfection, cells were exposed for 24 h to 10,000 U/ml IFN-{gamma} or CK containing 10,000 U/ml IFN-{gamma}, 0.5 ng/ml IL-1{beta}, and 10 ng/ml TNF-{alpha} or were left untreated. Transfection of pGL-3-basic reporter constructs lacking promoter served as a negative control. Efficiency of Lipofectamine transfection in HAEC determined by {beta}-galactosidase expression plasmid (pCMV-{beta}-Gal) was 5 ± 2% cells/high-power field, whereas 15 ± 5% A549 cells were transfected. As previously shown (3, 26), the NOS2 promoter (regions containing 5,574 bp or greater up to 8,296 bp) was activated by CK in A549 cells, whereas CK inducibility of constructs in A549 cells was lost with the 4,909-bp or shorter regions of the 5'-flanking promoter region construct (Fig. 2A). In contrast, full-length or deletion constructs of the NOS2 promoter were not activated by IFN-{gamma} alone in A549 cells (degree of induction compared with unstimulated A549 cells of the 5,574-bp promoter construct: CK induced 7 ± 1, IFN-{gamma} induced 1.4 ± 0.2; n >= 3).



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  		Fig. 2. Effect of CK or IFN-{gamma} on activity of human NOS2 promoter constructs and effect of mutation of the activator protein (AP)-1-binding sites in the NOS2 gene in A549 cells and HAEC. Serial-deletion luciferase constructs of the 5'-flanking region of human NOS2 gene and 8,296-bp full-length promoter with mutation of the AP-1 sites (mAP-1) fused to luciferase were transfected in A549 (A) or HAEC (B) and then incubated in the presence and absence of CK or IFN-{gamma} alone. Data are expressed as the degree of induction compared with unstimulated cells (n >= 3 experiments).

 

Similar to A549 cells, induction of the NOS2 promoter by CK in HAEC also occurred with 5,574-bp or longer constructs (Fig. 2B). However, IFN-{gamma} alone activated the NOS2 promoter (regions containing 5,574 bp or greater up to 8,296 bp) in HAEC (degree of induction compared with unstimulated HAEC of the 5,574-bp promoter construct: CK induced 2.4 ± 0.3, IFN-{gamma} induced 2.5 ± 0.2; n >= 3). CK or IFN-{gamma} inducibility of constructs in HAEC was lost with the 4,909-bp or shorter regions of the 5'-flanking promoter region construct, indicating that the 665-bp region upstream of the 4,909-bp fragment contained important transcription regulatory elements (degree of induction compared with unstimulated HAEC of the 4,909-bp promoter construct: CK induced 1.2 ± 0.3, IFN-{gamma} induced 1.4 ± 0.3; n >= 3). In HAEC incubated with CK or IFN-{gamma} alone, the luciferase activities increased at 24 h and were highest at 48 h (Fig. 3). Thus IFN-{gamma} or CK similarly induced the NOS2 promoter in HAEC but not in A549 cells. Yet the important cytokine-responsive elements for IFN-{gamma} or CK activation of the promoter in both A549 cells and HAEC were located within the 665-bp region between -5,574 and -4,909 bp.



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  		Fig. 3. Time course of effect of CK or IFN-{gamma} on activity of human NOS2 promoter construct (5,574 bp) in HAEC. Cells were transfected with 5,574-bp-length promoter and then incubated in the presence and absence of CK or IFN-{gamma} alone for times indicated. Effect of CK or IFN-{gamma} inducible activity in HAEC is expressed as the degree of induction compared with unstimulated cells (n >= 2 for each time point).

 

Effect of mutation in AP-1-binding sequence of NOS2 gene in HAEC and A549 cells. Previous studies have demonstrated that the GAS element in NOS2 promoter is essential for IFN-{gamma} activation (8, 9, 45). However, prior work has also emphasized the importance of AP-1-binding sites in activation of NOS2 promoter in response to cytokine combinations (3, 26). Multiple cytokine binding sites are present in the nucleotide sequence from -5,574 to -4,909 bp of the NOS2 promoter (Fig. 4). Based on the consensus sequence TTN5AA (14), this region contains two putative GAS, two consensus-binding sites (TGANTCA) for AP-1 (3, 35), and two consensus-binding sites (GGGRNWYYCC) for NF-{kappa}B (3, 39).



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  		Fig. 4. Nucleotide sequence of the 5'-flanking region of human NOS2 gene from -5,574 to -4,909 bp (GenBank accession no. AF017634 [GenBank] ). This 665-bp region contains putative sequences for {gamma}-activated sites (GAS; boxed), AP-1 (straight underline), and NF-{kappa}B (wavy underline).

 

To determine whether AP-1 sites are important in induction of NOS2 in HAEC by IFN-{gamma} alone, 8,296-bp full-length NOS2 promoter bearing three-base mutations in the AP-1-binding sites termed mAP-1 (26) was studied. HAEC and A549 cells transfected with mutated NOS2 promoter (mAP-1) were exposed to CK or IFN-{gamma} for 24 h. As previously shown, A549 cells transfected with the AP-1 mutated construct had a marked decrease in luciferase activity with CK (Fig. 2A; see Ref. 26). Compared with the wild-type promoter construct, HAEC transfected with mutant AP-1 constructs had decreased induction by CK and, unexpectedly, by IFN-{gamma} (Fig. 2B). These results suggested that CK or IFN-{gamma} induction of the human NOS2 promoter requires AP-1 regulatory region(s).

STAT-1 and c-Fos interaction in IFN-{gamma} induced NOS2 GAS activation. To determine which transcription factors bind to the GAS in the region from -5,574 to -4,909 bp of the NOS2 promoter, DNA-protein interactions were investigated by EMSA using extracts from HAEC or A549 cells after exposure to IFN-{gamma} for 30 min. With the use of oligonucleotides bearing both the sequences of upstream AP-1 (AP-1u) and GAS elements in the NOS2 promoter (Table 1), WCE from HAEC had binding activity at baseline that increased by exposure of cells to IFN-{gamma} (Fig. 5). Antibodies to c-Fos or c-Jun produced supershift of the complex, indicating that these proteins are present in the DNA-protein complexes. With the use of an oligonucleotide only bearing the sequence of the GAS element in the NOS2 promoter (Table 1), DNA binding activity was present in HAEC WCE (Fig. 6A) or A549 cell nuclear extract (data not shown) exposed to IFN-{gamma} but not in nonstimulated cells. Antibodies against c-Fos or STAT-1 supershifted DNA-protein bands, indicating that both proteins are present in the binding complex. On the other hand, antibodies against STAT-3, STAT-5, c-Jun (Fig. 6), FosB, Fra-1, Fra-2, or JunD (data not shown) did not produce a supershift of the binding complex, indicating that no other members of the AP-1 complex (FosB, Fra-1, Fra-2, c-Jun, or JunD) interact with STAT-1. NF-{kappa}B binding sites overlap GAS in the NOS2 promoter. To determine whether NF-{kappa}B binding occurs in this region, EMSA was performed on WCE of TNF-{alpha}-stimulated A549 cells using an oligonucleotide containing the NOS2 NF-{kappa}B binding sequence that overlaps GAS (Table 1). WCE from CK- or IFN-{gamma}-exposed cells contained binding activity to GAS that was the result of STAT-1 and AP-1 binding but not the result of NF-{kappa}B (Fig. 6B). Notably, competitive binding of STAT-1 and NF-{kappa}B has been demonstrated in the region of the GAS site such that STAT-1 precludes NF-{kappa}B binding (8).



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  		Fig. 5. Identification of the NOS2 AP-1- and {gamma}-activated site (GAS)-binding protein in HAEC by EMSA. Whole cell extract (WCE) from nonstimulated (NS) HAEC (lanes 1 and 8) or HAEC stimulated with IFN-{gamma} for 30 min (lanes 2–7 and 9–14) was analyzed for DNA-binding activity by EMSA using radiolabeled oligonucleotide AP-1 upstream (u) ~ GAS or NOS2 GAS. The specificity of the binding complex (arrow a) was assessed by the addition of a 100-fold molar excess of unlabeled wild-type (lane 4) or mutant GAS oligonucleotides (lane 3) before incubation with the labeled probe. Supershift of the complex with anti-c-Fos (lanes 5 and 12; arrow c) or anti-STAT-1 (lanes 7 and 14; arrow b) polyclonal antibody (Ab) added to binding reaction revealed that STAT-1 and c-Fos were present in the binding complexes. Supershift of the binding complex with anti-c-Jun was noted in EMSA using NOS2 AP-1u ~ GAS oligonucleotide (lane 6). The autoradiograph is representative of 3 independent experiments.

 


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  		Fig. 6. NOS2 GAS binding activity in HAEC and A549 cells treated with IFN-{gamma}. A: WCE from nonstimulated HAEC (lanes 1) or HAEC stimulated with IFN-{gamma} for 30 min (lanes 2–10) was analyzed by EMSA using oligonucleotide bearing the sequence of the NOS2 GAS sequence motif. The specificity of the binding complex (arrow a) was assessed by the addition of a 100-fold molar excess of unlabeled wild-type (lane 4) or mutant GAS oligonucleotides (lane 3) before incubation with the labeled probe. Anti-c-Fos, c-Jun, STAT-1, STAT-3, STAT-5, or NF-{kappa}B p65 polyclonal Ab was added to binding reactions to identify proteins in the binding complex. IFN-{gamma} led to binding activity (arrow a) that was supershifted by anti-STAT-1 (lane 7, arrow b) or anti-c-Fos Ab (lane 5, arrow c). Similar results were obtained in 3 separate experiments. B: WCE from HAEC stimulated with CK for 30 min (lanes 1–3), IFN-{gamma} for 30 min (lanes 4–6), or TNF-{alpha} for 30 min (lanes 7–9) was analyzed by EMSA using the NOS2 GAS. CK or IFN-{gamma} led to a prominent binding complex (arrow). STAT-1 and c-Fos were both present in the complex, as shown by supershift with anti-STAT-1 (lanes 3 and 6) and anti-c-Fos Ab (lanes 2 and 5). Similar results were obtained in a minimum of 3 separate experiments.

 

To investigate AP-1 activation and which transcription factors bind to the AP-1 site, EMSA was also performed using oligonucleotide containing only the AP-1u binding sequence (Table 1). Basal AP-1 binding activity in nonstimulated A549 was low and significantly increased by exposure of cells to CK for 3 h (Fig. 7A). Anti-c-Fos, Fra-2, c-Jun, or JunD led to significant supershift of the complex. No supershift was detected with FosB or Fra-1 (Fig. 7A). WCE from HAEC had basal binding activity that was not appreciably increased by exposure to CK (Fig. 7B) or IFN-{gamma} (data not shown). Antibodies to c-Fos or c-Jun supershifted the complex, indicating that c-Fos and c-Jun are activated and bind to the AP-1 element even in the absence of cytokine stimulation in HAEC. This basal AP-1 activation may explain, in part, why IFN-{gamma} alone is sufficient to activate NOS2 in HAEC, whereas multiple cytokines are required in A549 cells. With the use of an oligonucleotide containing only the downstream AP-1 (AP-1d) sequence, binding activity determined by EMSA was much weaker with AP-1d than with AP-1u (Fig. 7C). This indicated that the AP-1u is more relevant to promoter activation.



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  		Fig. 7. Identification of the NOS2 AP-1-binding proteins in A549 and HAEC by EMSA. A: EMSA of WCE from nonstimulated A549 (lane 1) or A549 stimulated with CK for 3 h (lanes 2–8) was analyzed using the radiolabeled NOS2 AP-1u oligonucleotide. Antibodies were added to reactions to identify binding proteins as indicated. Anti-c-Fos (lane 3), Fra-2 (lane 6), c-Jun (lane 7), or JunD (lane 8) led to significant supershift of the complex. No supershift was detected with FosB or Fra-1. The autoradiograph is representative of 2 independent experiments. B: EMSA of WCE from nonstimulated HAEC (lane 1) or HAEC stimulated with CK for 3 h (lanes 2–7) using radiolabeled AP-1u sequence. The specificity of the binding complex (arrow) was assessed by the addition of a 100-fold molar excess of unlabeled wild-type (lane 4) or mutant AP-1u (lane 3) oligonucleotide before the labeled probe. Anti-c-Fos, c-Jun, or STAT-1 polyclonal Ab was added to reactions to identify binding proteins. Anti-c-Fos (lane 5), anti-c-Jun (lane 6), and perhaps anti-STAT-1 (lane 7) led to supershift of the complex. The autoradiographs are representative of 3 experiments. C: NOS2 AP-1u and AP-1d binding activation in A549 cells by EMSA. WCE from nonstimulated A549 (lanes 1 and 4) or A549 stimulated with CK for 3 h (lanes 2–3 and 5–6) was analyzed by EMSA using radiolabeled oligonucleotide AP-1u or downstream (d) AP-1. Anti-c-Fos Ab was added to reactions to identify binding protein.

 

Effect of c-Fos antisense phosphorothioated oligodeoxynucleotide on CK induction of human NOS2 promoter. To further test whether c-Fos is important in induction of NOS2, A549 cells were cotransfected with the 8,296-bp full-length NOS2 promoter and the c-fos antisense phosphorothioated oligodeoxynucleotide. Compared with the sense oligodeoxynucleotide and no oligo, CK induction of the NOS2 promoter was decreased significantly in A549 cells exposed to CK for 6 or 12 h (P < 0.05; Fig. 8), which indicates the essential role of c-Fos in NOS2 induction. However, STAT-1 and c-Fos overexpression did not produce any significant increase in NOS2 expression in A549 and 293T cells (data not shown).



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  		Fig. 8. Effect of c-fos antisense phosphorothioated oligodeoxynucleotide on CK induction of human NOS2 promoter transfected with 8,296-bp full-length NOS2 promoter. A549 cells were cotransfected the c-Fos antisense phosphorothioated oligodeoxynucleotide (AS), sense oligodeoxynucleotide (S), or no oligo (N) and then cultured in the presence or absence of CK for 6 or 12 h. Data are expressed as the degree of induction compared with unstimulated A549 cells transfected with full-length NOS2 promoter (n = 2). P < 0.05 vs. N and S.

 

Interaction between c-Fos and STAT-1 proteins. In the context that STAT-1 and c-Fos are present in the binding complexes with the GAS sites, we investigated the interactive binding of c-Fos and STAT-1, and whether c-Fos interaction with STAT-1 requires DNA-binding, using coimmunoprecipitation and Western blot analysis. The cell lysate of HAEC exposed to IFN-{gamma} (400 µg total protein) was imunoprecipitated with an anti-c-Fos polyclonal Ab. The immunocomplex was resolved on an 8% SDS-polyacrylamide gel, and the immunoblot was probed with an anti-STAT-1 polyclonal Ab. Cell lysates of HAEC and A549 cells immunoprecipitated with anti-STAT-1 polyclonal Ab and probed with anti-STAT-1 polyclonal Ab served as a positive control, and the STAT-1-deficient fibrosarcoma cell line, U3A, served as a negative control. The time course (0–24 h) and the dose response to IFN-{gamma} were evaluated. STAT-1 coimmunoprecipitated with c-Fos by 30 min after IFN-{gamma} exposure (Fig. 9A). An IFN-{gamma} dose of 100 U/ml was necessary to detect STAT-1 coimmunoprecipitation with c-Fos (Fig. 9B). Lack of coimmunoprecipitation at baseline suggests that STAT-1 activation/phosphorylation is required for c-Fos/STAT-1 interaction.



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  		Fig. 9. Interaction of c-Fos and STAT-1. A: STAT-1 coimmunoprecipitated with c-Fos from HAEC lysate after IFN-{gamma} exposure. Lysates from nonstimulated HAEC (lane 1) or HAEC stimulated with IFN-{gamma} (lanes 2–5) were immunoprecipitated using anti-c-Fos Ab, run on 8% gel, and immunoblotted with anti-STAT-1 antibody. Immunoprecipitation with anti-STAT-1 and immunoblot with anti-STAT-1 of HAEC lysate (lane 6) or A549 lysate (lane 7) served as a positive control, whereas U3A cells that lack STAT-1 expression were a negative control (lane 8). STAT-1 (arrow) coimmunoprecipitated with c-Fos from HAEC lysate after IFN-{gamma} exposure (nonspecific band noted above STAT-1 band). B: STAT-1 coimmunoprecipitated with c-Fos from lysate of HAEC exposed to a minimum of 100 U/ml IFN-{gamma}. Lysates of HAEC exposed to increasing amounts of IFN-{gamma} were immunoprecipitated using anti-c-Fos and immunoblotted with anti-STAT-1 antibody (lanes 2–5). Immunoprecipitation with anti-STAT-1 and immunoblot with anti-STAT-1 in A549 lysate derived from CK-stimulated cells served as a positive control (lane 7), whereas U3A cells, which lack STAT-1 expression, were a negative control (lane 8). Arrow: STAT-1 coimmunoprecipitated with c-Fos from lysate of HAEC exposed to a minimum of 100 U/ml IFN-{gamma}. C: HA-STAT-1 coimmunoprecipitated with c-Fos from 293T cells transfected with HA-STAT-1. Immunoprecipitation with anti-STAT-1 or anti-HA Ab in 293T cells transfected with HA-STAT-1 served as positive controls (lanes 3 and 4), whereas immunoprecipitation with nonimmune rabbit IgG was a negative control (lanes 5 and 8). Arrow: STAT-1 coimmunoprecipitated with c-Fos from 293T cell lysate nontransfected (lane 6) or transfected (lane 2) with HA-STAT-1. D: c-Fos coimmunoprecipitated with STAT-1. Lysates from A549 stimulated with CK (lanes 1–3) were immunoprecipitated using nonimmune rabbit IgG, anti-c-Fos, and STAT-1 Ab, run on 12.5% gel, and immunoblotted with anti-STAT-1 or anti-c-Fos(2) Ab. Immunoprecipitation with nonimmune rabbit IgG was a negative control (lane 1). Arrows: STAT-1 coimmunoprecipitated with c-Fos and c-Fos coimmunoprecipitated with STAT-1.

 

To further confirm the interactive binding of c-Fos and STAT-1, 293T cells were transfected with an expression construct for HA-STAT-1 (34) or as a control with the expression construct or HA-TAK1, a protein involved in the IL-1 signaling pathway (37). Lysates from 293T cells stimulated with CK were immunoprecipitated using anti-c-Jun, c-Fos, HA or STAT-1 Ab or nonimmune rabbit IgG, electrophoresed on 8% gel, and immunoblotted with anti-HA or STAT-1 Ab. Immunoprecipitation with anti-STAT-1 or anti-HA Ab of lysates from 293T cells transfected with HA-STAT-1 served as a positive control, whereas immunoprecipitation with nonimmune rabbit IgG was a negative control. HA-STAT-1 was coimmunoprecipitated with c-Fos in 293T transfected with HA-STAT-1 (Fig. 9C) but not in lysates from 293T cells nontransfected (Fig. 9C) or transfected with HA-TAK1 (data not shown). Endogenous STAT-1 coimmunoprecipitated with c-Fos in 293T cells nontransfected, transfected with HA-STAT-1 (Fig. 9C), or transfected with HA-TAK1 (data not shown). Thus either endogenous STAT-1 or HA-STAT-1 interacts with c-Fos, but not c-Jun. Coimmunoprecipitation of c-Fos and STAT-1 or HA-STAT-1 from 293T cells transfected with HA-STAT-1 was also confirmed using different c-Fos antibodies (data not shown). In addition, c-Fos coimmunoprecipitated with STAT-1 in lysates from A549 cells (Fig. 9D).

These results confirm that c-Fos interacts with STAT-1, indicating that activation/phosphorylation of STAT-1 is necessary for c-Fos interaction.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrate the physical interaction between c-Fos and STAT-1, which participate in NOS2 gene transcriptional activation after IFN-{gamma} activation. Fos and Jun family proteins usually function as dimeric transcription factors that bind to AP-1 regulatory elements [TGA(C/G)TCA] in the promoter of numerous genes, including NOS2 (2, 35, 43). Jun proteins can form stable homodimers or heterodimers with Fos proteins, but Fos proteins do not form stable homodimers. Fos-Jun heterodimers bind DNA more stably than Jun homodimers (2, 28, 35, 43). Thus c-Fos heterodimerization with Jun family members enhances association of Jun proteins to DNA. Although other Fos family members may be capable of substituting functionally for c-Fos in c-Fos-deficient mice, our studies show that only c-Fos physically associates with STAT-1 after IFN-{gamma} activation (17). Notably, AP-1 is activated in unstimulated HAEC in culture, with no appreciable increase in activation after stimulation with CK or IFN-{gamma}. This suggests that basal AP-1 activation is sufficient to allow subsequent activation of NOS2 gene expression by IFN-{gamma} alone in the HAEC, a unique feature of these cells (12, 42).

Previous studies indicate that genistein, a tyrosine kinase inhibitor of the JAK-STAT-1 pathway, abolishes induction of NOS2 by IFN-{gamma} in airway epithelial cells (11), and tyrophostin A25, a pharmacological inhibitor of Jak 2 kinase, inhibits cytokine-induced NOS2 expression in a dose-dependent manner in A549 cells (8). These data suggest that the JAK-STAT pathway is involved in regulating cytokine or IFN-{gamma}-induced NOS2 expression in lung epithelial cells. Furthermore, cotransfection with the dominant-negative STAT-1 expression vector significantly inhibits cytokine-induced NOS2 reporter expression (8), implicating STAT-1 as a positive regulator of NOS gene transcription in these cells.

Cooperative DNA binding of proteins usually involves regions in close proximity, which functionally represent a composite regulatory element (2, 48). In this study, the 100-bp region encompassing the GAS and AP-1 site of NOS2 promoter may serve as composite binding elements. These closely located sites support that heterodimeric c-Fos interaction with STAT-1 in binding complexes on DNA elements is important for maximal gene activation. Experimental support of this is provided by the decreased IFN-{gamma} inducibility of the NOS2 promoter containing mutated AP-1 sites. Definitive evidence of physical association between c-Fos and STAT-1 is provided by coimmunoprecipitation of endogenously expressed or exogenously expressed factors from cells after exposure to IFN-{gamma}. We speculate that STAT-1 binding may be facilitated on the GAS element through interaction with c-Fos, i.e., STAT-1 may more firmly associate with GAS, in part through interaction with c-Fos. In support of this concept, previous studies have shown that low-affinity and low-specificity Smad-family DNA binding proteins rely on interactions with other DNA-binding proteins, including Jun, to target them to specific regulatory DNA elements (2). Similarly, a previous study of IFN-{gamma} induction of intercellular adhesion molecule-1 in primary human airway cells has shown that STAT-1 activation and DNA binding require the transcriptional activator SP-1 (23). Furthermore, transcription factor TFII-I can form protein-protein complexes with STAT-1, STAT-3, and serum response factor, which enhances the response of the c-fos promoter (19). Taken together, pairing of STAT-1 and c-Fos to the promoter provides maximal activation of NOS2 expression in cells.


   ACKNOWLEDGMENTS
 
We are indebted to Andrew Larner for the gift of the HA-STAT-1 expression construct, Xiaoxia Li for the HA-TAK1 expression construct, F. Dong for pRSV-c-Fos expression construct, and to Xiaoxia Li and Donna Driscoll for helpful suggestions to the manuscript.

This work was supported by National Heart, Lung, and Blood Institute Grant HL-60917.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. C. Erzurum, Cleveland Clinic Foundation, Lerner Research Institute, 9500 Euclid Ave./NB40, Cleveland, OH 44195 (E-mail: erzurus{at}ccf.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Chin YE, Kitagawa M, Su WC, You ZH, Iwamoto Y, and Fu XY. Cell growth arrest and induction of cyclin-dependent kinase inhibitor p21 WAF1/CIP1 mediated by STAT1. Science 272: 719-722, 1996.[Abstract]
  2. Chinenov Y and Kerppola TK. Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory specificity. Oncogene 20: 2438-2452, 2001.[ISI][Medline]
  3. Chu SC, Marks-Konczalik J, Wu HP, Banks TC, and Moss J. Analysis of the cytokine-stimulated human inducible nitric oxide synthase (iNOS) gene: characterization of differences between human and mouse iNOS promoters. Biochem Biophys Res Commun 248: 871-878, 1998.[ISI][Medline]
  4. de Vera ME, Shapiro RA, Nussler AK, Mudgett JS, Simmons RL, Morris SM Jr, Billiar TR, and Geller DA. Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter. Proc Natl Acad Sci USA 93: 1054-1059, 1996.[Abstract/Free Full Text]
  5. Durbin JE, Hackenmiller R, Simon MC, and Levy DE. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 84: 443-450, 1996.[ISI][Medline]
  6. Dweik RA, Comhair SA, Gaston B, Thunnissen FB, Farver C, Thomassen MJ, Kavuru M, Hammel J, Abu-Soud HM, and Erzurum SC. NO chemical events in the human airway during the immediate and late antigen-induced asthmatic response. Proc Natl Acad Sci USA 98: 2622-2627, 2001.[Abstract/Free Full Text]
  7. Farrar JD, Smith JD, Murphy TL, and Murphy KM. Recruitment of Stat4 to the human interferon-alpha/beta receptor requires activated Stat2. J Biol Chem 275: 2693-2697, 2000.[Abstract/Free Full Text]
  8. Ganster RW, Taylor BS, Shao L, and Geller DA. Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-kappa B. Proc Natl Acad Sci USA 98: 8638-8643, 2001.[Abstract/Free Full Text]
  9. Gao J, Morrison DC, Parmely TJ, Russell SW, and Murphy WJ. An interferon-gamma-activated site (GAS) is necessary for full expression of the mouse iNOS gene in response to interferon-gamma and lipopolysaccharide. J Biol Chem 272: 1226-1230, 1997.[Abstract/Free Full Text]
  10. Ghislain JJ, Wong T, Nguyen M, and Fish EN. The interferon-inducible Stat2: Stat1 heterodimer preferentially binds in vitro to a consensus element found in the promoters of a subset of interferon-stimulated genes. J Interferon Cytokine Res 21: 379-388, 2001.[ISI][Medline]
  11. Guo FH, Comhair SA, Zheng S, Dweik RA, Eissa NT, Thomassen MJ, Calhoun W, and Erzurum SC. Molecular mechanisms of increased nitric oxide (NO) in asthma: evidence for transcriptional and post-translational regulation of NO synthesis. J Immunol 164: 5970-5980, 2000.[Abstract/Free Full Text]
  12. Guo FH, Uetani K, Haque SJ, Williams BR, Dweik RA, Thunnissen FB, Calhoun W, and Erzurum SC. Interferon gamma and interleukin 4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators. J Clin Invest 100: 829-838, 1997.[ISI][Medline]
  13. Haque SJ, Flati V, Deb A, and Williams BR. Roles of protein-tyrosine phosphatases in Stat1 alpha-mediated cell signaling. J Biol Chem 270: 25709-25714, 1995.[Abstract/Free Full Text]
  14. Haque SJ and Williams BR. Signal transduction in the interferon system. Semin Oncol 25: 14-22, 1998.[ISI][Medline]
  15. Haspel RL and Darnell JE Jr. A nuclear protein tyrosine phosphatase is required for the inactivation of Stat1. Proc Natl Acad Sci USA 96: 10188-10193, 1999.[Abstract/Free Full Text]
  16. Ihle JN. STATs: signal transducers and activators of transcription. Cell 84: 331-334, 1996.[ISI][Medline]
  17. Jain J, Nalefski EA, McCaffrey PG, Johnson RS, Spiegelman BM, Papaioannou V, and Rao A. Normal peripheral T-cell function in c-Fos-deficient mice. Mol Cell Biol 14: 1566-1574, 1994.[Abstract/Free Full Text]
  18. Kamijo R, Harada H, Matsuyama T, Bosland M, Gerecitano J, Shapiro D, Le J, Koh SI, Kimura T, Green SJ, Mak TW, Taniguchi T, and Vilcek J. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science 263: 1612-1615, 1994.[Abstract/Free Full Text]
  19. Kim DW, Cheriyath V, Roy AL, and Cochran BH. TFII-I enhances activation of the c-fos promoter through interactions with upstream elements. Mol Cell Biol 18: 3310-3320, 1998.[Abstract/Free Full Text]
  20. Kroger A, Koster M, Schroeder K, Hauser H, and Mueller PP. Activities of IRF-1. J Interferon Cytokine Res 22: 5-14, 2002.[ISI][Medline]
  21. Laubach VE, Zhang CX, Russell SW, Murphy WJ, and Sherman PA. Analysis of expression and promoter function of the human inducible nitric oxide synthase gene in DLD-1 cells and monkey hepatocytes. Biochim Biophys Acta 1351: 287-295, 1997.[Medline]
  22. Lee PJ, Camhi SL, Chin BY, Alam J, and Choi AM. AP-1 and STAT mediate hyperoxia-induced gene transcription of heme oxygenase-1. Am J Physiol Lung Cell Mol Physiol 279: L175-L182, 2000.[Abstract/Free Full Text]
  23. Look DC, Pelletier MR, Tidwell RM, Roswit WT, and Holtzman MJ. Stat1 depends on transcriptional synergy with Sp1. J Biol Chem 270: 30264-30267, 1995.[Abstract/Free Full Text]
  24. Lowenstein CJ, Alley EW, Raval P, Snowman AM, Snyder SH, Russell SW, and Murphy WJ. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide. Proc Natl Acad Sci USA 90: 9730-9734, 1993.[Abstract/Free Full Text]
  25. MacMicking JD, Nathan C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, Stevens K, Xie QW, Sokol K, Hutchinson N, Chen H, and Mudgett JS. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81: 641-650, 1995.[ISI][Medline]
  26. Marks-Konczalik J, Chu SC, and Moss J. Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor kappaB-binding sites. J Biol Chem 273: 22201-22208, 1998.[Abstract/Free Full Text]
  27. Mashimo H and Goyal RK. Lessons from genetically engineered animal models. IV. Nitric oxide synthase gene knockout mice. Am J Physiol Gastrointest Liver Physiol 277: G745-G750, 1999.[Abstract/Free Full Text]
  28. McBride K and Nemer M. The C-terminal domain of c-fos is required for activation of an AP-1 site specific for jun-fos heterodimers. Mol Cell Biol 18: 5073-5081, 1998.[Abstract/Free Full Text]
  29. Meraz MA, White JM, Sheehan KC, Bach EA, Rodig SJ, Dighe AS, Kaplan DH, Riley JK, Greenlund AC, Campbell D, Carver-Moore K, DuBois RN, Clark R, Aguet M, and Schreiber RD. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 84: 431-442, 1996.[ISI][Medline]
  30. Nathan C and Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell 78: 915-918, 1994.[ISI][Medline]
  31. Sadowski HB, Shuai K, Darnell JE Jr, and Gilman MZ. A common nuclear signal transduction pathway activated by growth factor and cytokine receptors. Science 261: 1739-1744, 1993.[Abstract/Free Full Text]
  32. Samardzic T, Jankovic V, Stosic-Grujicic S, and Trajkovic V. STAT1 is required for iNOS activation, but not IL-6 production in murine fibroblasts. Cytokine 13: 179-182, 2001.[ISI][Medline]
  33. Schuringa JJ, Timmer H, Luttickhuizen D, Vellenga E, and Kruijer W. c-Jun and c-Fos cooperate with STAT3 in IL-6-induced transactivation of the IL-6 respone element (IRE). Cytokine 14: 78-87, 2001.[ISI][Medline]
  34. Sekimoto T, Nakajima K, Tachibana T, Hirano T, and Yoneda Y. Interferon-gamma-dependent nuclear import of Stat1 is mediated by the GTPase activity of Ran/TC4. J Biol Chem 271: 31017-31020, 1996.[Abstract/Free Full Text]
  35. Shaulian E and Karin M. AP-1 in cell proliferation and survival. Oncogene 20: 2390-2400, 2001.[ISI][Medline]
  36. Stuehr DJ. Mammalian nitric oxide synthases. Biochim Biophys Acta 1411: 217-230, 1999.[Medline]
  37. Takaesu G, Ninomiya-Tsuji J, Kishida S, Li X, Stark GR, and Matsumoto K. Interleukin-1 (IL-1) receptor-associated kinase leads to activation of TAK1 by inducing TAB2 translocation in the IL-1 signaling pathway. Mol Cell Biol 21: 2475-2484, 2001.[Abstract/Free Full Text]
  38. Taniguchi T, Lamphier MS, and Tanaka N. IRF-1: the transcription factor linking the interferon response and oncogenesis. Biochim Biophys Acta 1333: M9-M17, 1997.[Medline]
  39. Taylor BS, de Vera ME, Ganster RW, Wang Q, Shapiro RA, Morris SM Jr, Billiar TR, and Geller DA. Multiple NF-kappaB enhancer elements regulate cytokine induction of the human inducible nitric oxide synthase gene. J Biol Chem 273: 15148-15156, 1998.[Abstract/Free Full Text]
  40. Touyz RM, He G, Wu XH, Park JB, Mabrouk ME, and Schiffrin EL. Src is an important mediator of extracellular signal-regulated kinase 1/2-dependent growth signaling by angiotensin II in smooth muscle cells from resistance arteries of hypertensive patients. Hypertension 38: 56-64, 2001.[Abstract/Free Full Text]
  41. Uetani K, Der SD, Zamanian-Daryoush M, de La Motte C, Lieberman BY, Williams BR, and Erzurum SC. Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase. J Immunol 165: 988-996, 2000.[Abstract/Free Full Text]
  42. Uetani K, Thomassen MJ, and Erzurum SC. Nitric oxide synthase 2 through an autocrine loop via respiratory epithelial cell-derived mediator. Am J Physiol Lung Cell Mol Physiol 280: L1179-L1188, 2001.[Abstract/Free Full Text]
  43. Van Dam H and Castellazzi M. Distinct roles of Jun: Fos and Jun: ATF dimers in oncogenesis. Oncogene 20: 2453-2464, 2001.[ISI][Medline]
  44. Wei XQ, Charles IG, Smith A, Ure J, Feng GJ, Huang FP, Xu D, Muller W, Moncada S, and Liew FY. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature 375: 408-411, 1995.[Medline]
  45. Xie QW, Whisnant R, and Nathan C. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide. J Exp Med 177: 1779-1784, 1993.[Abstract/Free Full Text]
  46. Yu CL, Meyer DJ, Campbell GS, Larner AC, Carter-Su C, Schwartz J, and Jove R. Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. Science 269: 81-83, 1995.[Abstract/Free Full Text]
  47. Zhang X, Laubach VE, Alley EW, Edwards KA, Sherman PA, Russell SW, and Murphy WJ. Transcriptional basis for hyporesponsiveness of the human inducible nitric oxide synthase gene to lipopolysaccharide/interferon-gamma. J Leukoc Biol 59: 575-585, 1996.[Abstract]
  48. Zhang X, Wrzeszczynska MH, Horvath CM, and Darnell JE Jr. Interacting regions in Stat3 and c-Jun that participate in cooperative transcriptional activation. Mol Cell Biol 19: 7138-7146, 1999.[Abstract/Free Full Text]



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