STAT3-Dependent Gene Induced by IFN Identification of CXCL11 As A

Identification of CXCL11 as a STAT3-Dependent Gene Induced by IFN Chuan He Yang, Lai Wei, Susan R. Pfeffer, Ziyun Du, Aruna Murti, William J. Valentine, Yi Zheng and Lawrence This information is current as M. Pfeffer of September 29, 2021. J Immunol 2007; 178:986-992; ; doi: 10.4049/jimmunol.178.2.986 http://www.jimmunol.org/content/178/2/986 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Identiﬁcation of CXCL11 as a STAT3-Dependent Gene Induced by IFN1

Chuan He Yang,2* Lai Wei,2* Susan R. Pfeffer,* Ziyun Du,* Aruna Murti,* William J. Valentine,* Yi Zheng,† and Lawrence M. Pfeffer3*

IFNs selectively regulate gene expression through several signaling pathways. The present study explored the involvement of STAT3 in the IFN-induced expression of the gene encoding the CXCL11 chemokine. The CXCL11 gene was induced in IFN- sensitive Daudi cells, but not in an IFN-resistant DRST3 subline with a defective STAT3 signaling pathway. Although the IFN- stimulated gene ISG15 was induced to a similar extent in Daudi and DRST3 cells, expression of wild-type STAT3 in DRST3 cells restored the IFN inducibility of CXCL11. Reconstitution of STAT3 knockout mouse embryonic ﬁbroblasts with wild-type STAT3, or STAT3 with the canonical STAT3 dimerization site at Y705 mutated, restored IFN inducibility of the CXCL11 gene. These data indicate that CXCL11 gene induction by IFN is STAT3 dependent, but that phosphorylation of Y705 of STAT3 is not required. Downloaded from Chromatin immunoprecipitation assays demonstrated that IFN treatment of Daudi and DRST3 cells induced STAT3 binding to the CXCL11 promoter. Chromatin immunoprecipitation assays also revealed that NF-␬B family member p65 and IFN regulatory factor (IRF)1 were bound to CXCL11 promoter upon IFN treatment of Daudi cells. In contrast, IFN induced the binding of p50 and IRF2 to the CXCL11 promoter in DRST3 cells. The proﬁle of promoter binding was indistinguishable in IFN-sensitive Daudi cells and DRST3 cells reconstituted with wild-type STAT3. Thus, STAT3 also plays a role in the recruitment of the transcriptional activators p65 and IRF1, and the displacement of the transcriptional repressors p50 and IRF2 from the CXCL11 promoter also http://www.jimmunol.org/ appears to regulate the induction of CXCL11 gene transcription. The Journal of Immunology, 2007, 178: 986–992.

lthough originally identified by their antiviral activity, tory factor (IRF)4 9, translocate into the nucleus, and bind to the IFNs are diverse multifunctional cytokines that also in- conserved IFN stimulation response element (ISRE) within the A hibit cell proliferation, regulate cell differentiation, reg- promoters of IFN-stimulated genes (ISGs). ulate apoptosis (programmed cell death), and modulate activities The importance of STAT3 in IFN-␣␤ action has been defined by of the immune system. Furthermore, IFNs have clinical utility in characterizing the IFN response pathway in Daudi lymphoblastoid diseases of diverse pathogenesis and manifestations including cells and in an IFN-resistant subclone (3–5). However, the role of by guest on September 29, 2021 hairy cell leukemia, Kaposi’s sarcoma, laryngeal and genital pap- STAT3 in IFN-induced gene expression is unknown. The general illomas, chronic viral hepatitis, and multiple sclerosis. The type I dogma is that a single tyrosine is phosphorylated in STATs upon IFNs, consisting of IFN-␣, IFN-␤, and IFN-␻ (IFN-␣␤), bind to a cytokine stimulation (Y705 in STAT3), which results in STAT multisubunit cell surface receptor complex that is distinct from the dimerization and subsequent binding to the promoter of STAT receptor for type II IFN, IFN-␥. IFN-␣␤ elicits diverse biological target genes (6). However, 21 of 22 potential tyrosine-based motifs effects by altering the pattern of gene expression through the ac- are perfectly conserved in human, murine, and rat STAT3 ho- tivation of the JAK1 and TYK2 nonreceptor protein tyrosine ki- mologs. Although Daudi cells are extremely sensitive to the anti- nases, which mediate the tyrosine phosphorylation of several proliferative and antiviral actions of IFN, IFN-resistant Daudi cells STAT proteins. STAT1 and STAT2 play crucial roles in the tran- are relatively nonresponsive and stably maintain their resistance in scriptional response to IFN-␣␤ and in the induction of antiviral the absence of IFN addition (7–14). IFN-sensitive and IFN- activity (1, 2). In the classical JAK-STAT signaling pathway, these resistant cell lines have similar numbers of IFN receptors, and phosphorylated STAT proteins form a complex with IFN regula- activate the JAK-STAT signaling pathway as defined by IFN- dependent tyrosine phosphorylation of JAK1, TYK2, STAT1, and STAT2 and the induction of ISG transcription. Although IFN-resistant Daudi cells have detectable levels of STAT3, *Department of Pathology and Laboratory Medicine, University of Tennessee Health these cells exhibit a defective STAT3-dependent IFN signaling Science Center and University of Tennessee Cancer Institute, Memphis, TN 38163; pathway, which can be rescued by stable expression of STAT3. and †Division of Experimental Hematology, Children’s Hospital Research Founda- tion, University of Cincinnati, Cincinnati, OH 45229 Hence Daudi cells are designated DRST3 (for Daudi cells that Received for publication May 19, 2006. Accepted for publication October 25, 2006. are IFN-resistant to the antiviral and antiproliferative effects and exhibit a defective STAT3-dependent signaling pathway). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance The present study explored the involvement of STAT3 in the with 18 U.S.C. Section 1734 solely to indicate this fact. expression of the IFN-induced gene CXCL11, which encodes the 1 This work was supported by grant CA73753 from the National Institutes of Health (to L.M.P.) and by funds from the Muirhead Chair Endowment at the University of Tennessee Health Science Center (L.M.P.). 4 2 Abbreviations used in this paper: IRF, IFN regulatory factor; ChIP, chromatin im- C.H.Y. and L.W. contributed equally to this work. munoprecipitation; ISRE, IFN stimulation response element; SIE, c-sis inducible el- 3 Address correspondence and reprint requests to Dr. Lawrence M. Pfeffer, Depart- ement; ISG, IFN-stimulated gene; KO, knockout; MEF, mouse embryonic fibroblast; ment of Pathology and Laboratory Medicine, University of Tennessee Health Science WT, wild type. Center, 930 Madison Avenue, Room 530, Memphis, TN 38163. E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 987

chemokine also called ␤-R1 and I-TAC. Our data indicate that 5Ј-TCACTGCTTTTACCCCAGGG-3Ј (reverse); human ISG15,5Ј-GGTCCA CXCL11 gene induction by IFN is STAT3 dependent, and that GTTGCTGAAAGAGC-3Ј (forward), 5Ј-TTTCGTCTGGAGATCCTGTA-3Ј ␤ Ј Ј phosphorylation of the Y705 (the canonical dimerization site) of (reverse); human -actin,5-AGAAGGAGATCACTGCCCTG-3 (forward), 5Ј-CACATCTGCTGGAAGGTGGA-3Ј (reverse); mouse CXCL11,5Ј-AGGA STAT3 is not required for the IFN inducibility of CXCL11 gene AGGTCACAGCCATAGC-3Ј (forward), 5Ј-CGATCTCTGCCATTTTGA expression. Other events such as the recruitment of the transcrip- CG-3Ј (reverse); and mouse ␤-actin,5Ј-AAGGAGATTACTGCTCTGGC-3Ј tional activators p65, which is a NF-␬B family member, and IRF1 (forward), 5Ј-ACATCTGCTGGAAGGTGGAC-3Ј (reverse). and the displacement of the transcriptional repressors p50 and Reverse transcription was performed at 48°C for 45 min and RT-PCR cycling parameters were as follows: denaturation at 95°C for 2 min, am- IRF2 from the CXCL11 promoter are also STAT3 dependent and plification at 94°C for 30 s, and 60°C for 30 s for 35 cycles. The product regulate the IFN induction of CXCL11 gene transcription. size was initially monitored by agarose gel electrophoresis, and melting curves were analyzed to control for specificity of PCR. The data on IFN- Materials and Methods induced genes were normalized to the expression of the housekeeping gene ␤ Biological reagents and cell culture -actin. The relative units were calculated from a standard curve, plotting three different concentrations against the PCR cycle number at which the InterMune and Biogen-Idec provided recombinant human IFN␣Con1 and measured intensity reaches a fixed value (with a 10-fold increment equiv- rat IFN-␤, respectively. IFN biological activity was expressed in interna- alent to ϳ3.1 cycles). tional reference units per milliliter as assayed by protection against the cytopathic effect of vesicular stomatitis virus on fibroblasts, using the appropriate National Institutes of Health reference standard. Abs directed Chromatin immunoprecipitation (ChIP) against the following proteins were used: STAT1, phospho-STAT1, ChIP experiments were performed using the ChIP-IT kit (Active Motif) STAT2, IRF1, IRF2, p65, and p50 from Santa Cruz Biotechnology; according to the manufacturer’s instructions with the average size of Downloaded from STAT3 from BD Biosciences; and Y705 phosphorylated of STAT3 from sheared fragments ϳ200 bp. The forward and reverse primers used were Upstate Biotechnology. IFN-sensitive Daudi cells and the IFN-resistant human CXCL11 5Ј-GGTTTTCACAGTGCTTTCAC-3Ј (forward), 5Ј-TT ϫ 5 Daudi (DRST3) cells were maintained at 2–10 10 cells/ml in RPMI TCCCTCTTTGAGTCATGC-3Ј (reverse). 1640 containing 10% defined calf serum (HyClone). STAT3-knockout (STAT3-KO) mouse embryonic fibroblasts (MEFs) and reconstituted MEFs were generated as previously described (15) and plated at 1 ϫ 104 cells/cm2 every 3 days in DMEM supplemented with 10% defined calf Results ␮ Induction of CXCL11 by IFN is dependent on STAT3

serum, 100 U/ml penicillin G, and 100 g/ml streptomycin. http://www.jimmunol.org/ Transfection conditions and constructs To determine the effect of STAT3 on IFN-induced CXCL11 expression, quantitative real-time PCR assays were performed for To determine the role of STAT3 in ISG expression, cells were transfected CXCL11 and ISG15 using RNA from Daudi cells and DRST3 cells with expression plasmids. Transient and stable transfection of cells (107) was accomplished by electroporation (capacitance 300 ␮F, 250 V) with 50 that were treated with IFN for 6 h. The expression of ISG15 was ␮g of salmon sperm DNA and 20 ␮g of plasmid DNA for each sample. determined because the IFN induction of this gene is through the STAT3 was cloned in the pcEF expression vector, which provides a c-myc classical JAK-STAT pathway involving the binding of STAT1- epitope tag at the COOH terminus of the protein (5). STAT2-IRF9 complexes to the ISRE. As shown in Fig. 1, IFN Immunoprecipitations and immunoblot analysis treatment induced a dose-dependent increased expression of CXCL11 (Fig. 1A) and ISG15 (Fig. 1B) in IFN-sensitive Daudi by guest on September 29, 2021 For immunoprecipitation studies, transiently transfected cells were treated cells. In contrast, IFN treatment induced expression of ISG15, but with IFN (1000 U/ml) at 37°C for the indicated time periods and then washed with ice-cold PBS and lysed for 20 min in lysis buffer (50 mM Tris not CXCL11 in IFN-resistant DRST3 cells. CXCL11 gene expres- (pH 7.4), 150 mM NaCl, 1 mM EDTA 0.5% Nonidet P-40, and 15% sion was not observed in DRST3 cells up to 24 h after IFN addition ␮ glycerol) containing 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 5 g/ml and could not be induced at IFN concentrations as high as 10,000 ␮ ␮ soybean trypsin inhibitor, 5 g/ml leupeptin, and 1.75 g/ml benzamidine. U/ml (data not shown). Samples were clarified by centrifugation at 12,000 ϫ g for 15 min at 4°C and were immunoprecipitated with anti-myc (Santa Cruz Biotechnology) DRST3 cells have a defective STAT3-dependent signaling path- overnight at 4°C. Immune complexes were collected using protein way, which can be rescued by expression of wild-type (WT) A-Sepharose beads (Pharmacia) and eluted in sample buffer. Samples were STAT3 in these cells (4). To determine whether STAT3 can rescue separated on SDS-7.5% PAGE, transferred to polyvinylidene difluoride the IFN-induced expression of the CXCL11, RNA was isolated membranes (Millipore) and probed with anti-STAT1 or anti-STAT3 (di- from DRST3 cells stably expressing WT STAT3 (STAT3-DRST3 lution 1/1000), followed by anti-mouse IgG coupled with HRP (Santa Cruz Biotechnology). Blots were developed using ECL (Pierce). cells) after IFN treatment and subjected to quantitative real-time PCR assays for CXCL11 and ISG15. As shown in Fig. 1A, STAT3 Nuclear extracts and DNA binding activity assays expression was required for the induction of CXCL11 gene expres- Nuclei isolated from control and IFN-treated cells were extracted with sion by IFN. In contrast, expression of STAT3 in these IFN-resis- buffer (20 mM Tris-HCl (pH 7.85), 250 mM sucrose, 0.4 M KCl, 1.1 mM tant cells had no effect on the induction of ISG15 by IFN. Taken ␮ MgCl2, 5 mM 2-ME, 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 5 g/ml together these results suggested that IFN-induced expression of soybean trypsin inhibitor, 5 ␮g/ml leupeptin, and 1.75 ␮g/ml benzamidine), and extracts were frozen and stored at Ϫ80°C (5). The nuclear ex- CXCL11 is STAT3 dependent, but expression of ISG15 is STAT3 tracts were incubated with a 32P-labeled probe for the high affinity c-sis independent. inducible element (SIE) in the c-fos gene (5Ј-AGCTTCATTTCCCGTA To further characterize the induction of CXCL11 expression by ATCCCTAAAGCT-3Ј) and subjected to EMSA (4). To define the pres- IFN, we examined the effect of inhibitors of RNA synthesis and ence of specific STAT proteins in DNA-protein complexes, nuclear histone deacetylase on IFN-induced gene expression. Inhibition of extracts were preincubated with a 1/50 dilution of anti-STAT Abs at 25°C for 20 min before EMSA. Gels were quantitated by PhosphorIm- RNA synthesis by treatment with actinomycin D blocked the in- age autoradiography. duction of ISG15 and CXCL11 by IFN (data not shown), demon- strating that the induction of ISG15 and CXCL11 by IFN reflects Quantitative real-time PCR transcription activation rather than an effect on RNA stability. Total RNA was isolated from untreated and IFN-treated cells using TRIzol Moreover, as shown in Fig. 1C treatment of Daudi cells with the reagent (Invitrogen Life Technologies). Quantitative RT-PCR was per- histone deacetylase inhibitor trichostatin A did not increase the formed on an iCycler (Bio-Rad) using the AccessQuick RT-PCR system (Promega), and SYBR Green I (Molecular Probes) according to the man- basal expression of ISG15 or CXCL11, but did block the IFN- ufacturer’s instructions. The following forward and reverse primers used induced expression of these genes. Our results are consistent with were: human CXCL11,5Ј-ATGAGTGTGAAGGGCATGGC-3Ј (forward), previous finding that the inhibition of histone deacetylase function 988 IFN-INDUCED CXCL11 GENE EXPRESSION IS STAT3 DEPENDENT Downloaded from

FIGURE 2. IFN induces Y705 phosphorylation in DRST3 cells and the formation of STAT1/STAT3 dimers. A, Lysates prepared from Daudi and ␣

DRST3 cells at the indicated time after IFN- addition (1000 U/ml) were http://www.jimmunol.org/ immunoprecipitated with anti-STAT3. The proteins were resolved by SDS- PAGE, blotted onto polyvinylidene diﬂuoride membranes and probed with anti-STAT3, anti-Y705-STAT3, and anti-phosphotyrosine (clone 4G10; Upstate Biotechnology). B, Lysates prepared from control and IFN-␣- treated (1000 U/ml, 15 min) DRST3 cells transfected with either WT STAT3, F705 mutant STAT3, or empty vector (EV) were immunoprecipitated with anti-myc. The proteins were resolved by SDS-PAGE, blotted onto polyvinylidene diﬂuoride membranes, and probed with either anti- STAT1 or anti-STAT3. Blots were visualized by ECL (Pierce). C, Nuclear

extracts from control or IFN-␣-treated (1000 U/ml, 15 min) DRST3 cells by guest on September 29, 2021 transfected with WT STAT3, F705 mutant STAT3, or empty vector (EV) were incubated with a 32P-labeled SIE probe. To deﬁne speciﬁc STAT proteins, extracts were preincubated with anti-STAT1 or anti-STAT3 Abs before EMSA. Results from one of two experiments are shown.

scription factors (2). For STAT3, phosphorylation at Y705 has been shown previously to be required for its dimerization and function. To determine the phosphorylation of STAT3 at this critical tyrosine residue, we immunoprecipitated STAT3 from cell lysates prepared from Daudi cells and DRST3 cells after IFN treatment, and then performed Western blot analysis with Y705 phospho-specific Ab. As shown in Fig. 2A, STAT3 underwent phosphorylation at Y705 in both IFN-sensitive Daudi cells and FIGURE 1. IFN induces CXCL11 expression in IFN-sensitive Daudi IFN-resistant DRST3 cells within 10 min of IFN addition with cells and STAT3-expressing DRST3 cells but not in DRST3 cells. Real- significant levels of phosphorylation observed at 30 min after IFN time PCR for CXCL11 (A) and ISG15 (B) gene expression was performed addition. Consistent with previous studies relatively low levels of on cDNAs prepared from Daudi cells, DRST3 cells, and WT-STAT3 expressing DRST3 cells before and after IFN addition (6 h, 1000 U/ml). STAT3 tyrosine phosphorylation were detected in IFN-treated C, Daudi cells were pretreated with the histone deacetylase (HDAC) inhibitor DRST3 cells as compared with Daudi cells with a general anti- trichostatin A (400 ng/ml for 30 min) before IFN addition. Gene expression phosphotyrosine Ab (4). Blotting with anti-STAT3 verified that was normalized to actin expression. Data are shown as fold induction rel- equivalent amounts of STAT3 were expressed in Daudi and ative to untreated cells and are the mean Ϯ SEM (n ϭ 3 experiments). DSRT3 cells. Thus, STAT3 undergoes phosphorylation of Y705 in response to IFN in IFN-resistant DRST3 cells. led to impairment of IFN-induced ISG expression, with little effect To assess for the role of Y705 in the formation of STAT3 com- on basal ISG expression (16). plexes, DRST3 cells were stably transfected with either WT- STAT3 or a mutant form of STAT3 (F705-STAT3), in which ty- Tyrosine phosphorylation, dimerization, and binding of STAT3 rosine residue 705 was mutated to a phenylalanine, and STAT3 to the CXCL11 promoter immunoprecipitates were probed with anti-STAT1. As shown in Phosphorylation of STAT proteins at a critical tyrosine residue is Fig. 2B, IFN treatment of IFN-resistant DRST3 cells did not in- necessary for their appropriate transactivation function as tran- duce formation of STAT1-STAT3 complexes. However, IFN The Journal of Immunology 989

FIGURE 3. Phosphorylation of Y705 of STAT3 is not required for IFN-induced CXCL11 expression and the effect of protein kinase inhibitors on IFN-induced CXCL11 expression. A, Real-time PCR for CXCL11 gene expression was performed on cDNAs prepared from WT MEFs, STAT3-KO cells,

or STAT3-KO MEFs reconstituted with WT STAT3 or the F705-STAT3 mutant at varying times after IFN addition (1000 U/ml). B, Real-time PCR for Downloaded from CXCL11 gene expression was performed on cDNAs prepared from STAT3-KO MEFs reconstituted with the F705-STAT3 mutant, which were pretreated with DMSO as vehicle, the tyrosine kinase inhibitor genistein (100 ␮M), the PI3K inhibitor LY294002 (10 ␮M), or the general protein kinase inhibitor staurosporin (50 nM) for 1 h before IFN addition (1000 IU/ml, 5 h). Gene expression was normalized to actin expression. Data are shown as fold induction relative to untreated cells and are mean Ϯ SEM (n ϭ 3 experiments).

treatment of DRST3 cells expressing WT-STAT3 induced the for- cate that although STAT3 is required for IFN-induced expression http://www.jimmunol.org/ mation STAT1/STAT3 dimers. In DRST3 cells expressing the of the CXCL11 gene, tyrosine phosphorylation on Y705 of STAT3 F705-STAT3 mutant, although similar levels of STAT3 were ex- is not required for IFN-induced CXCL11 gene expression. This pressed, STAT3 did not form a complex with STAT1. These data result is surprising in light of the critical role Y705 of STAT3 indicate that IFN-induced phosphorylation of Y705 is necessary apparently plays in tumorigenesis and JAK-STAT signal transduc- for the formation of STAT1-STAT3 complexes. tion by cytokines. The activation of STAT3 can also be demonstrated by its pres- IFN-mediated signal transduction involves not only JAK-medi- ence in DNA-protein complexes using an oligonucleotide SIE ated tyrosine phosphorylation, but also activation of a PI3K-Akt probe (3). This finding led us next to investigate the IFN-depen- serine phosphorylation pathway (3, 17, 18). To characterize the dent activation of STAT3-containing SIE binding complexes. As role of protein kinases in IFN induction of CXCL11 gene expres- by guest on September 29, 2021 illustrated in Fig. 2C and consistent with our previous findings (5), sion, MEFs stably expressing the F705-STAT3 mutant were IFN only induces the formation of STAT1/STAT1 homodimers in treated with genistein (tyrosine kinase inhibitor), LY294002 (PI3K IFN-resistant DRST3 cells, which are supershifted by anti-STAT1 inhibitor), or staurosporin, which blocks IFN-induced biological but not by anti-STAT3. However, expression of WT-STAT3 in the events mediated by protein kinase C and by tyrosine kinases (8, 19, IFN-treated DRST3 cells results in the induction by IFN of two 20). As shown in Fig. 3B treatment with genistein, staurosporin, or additional STAT3-dependent SIE binding complexes (STAT1/ LY294002 blocked induction of CXCL11 expression by IFN. The STAT3 heterodimers and STAT3/STAT3 homodimers) as dem- data indicate that both JAK-mediated tyrosine phosphorylation, onstrated by supershift assays with anti-STAT1 and anti-STAT3. and activation of a PI3K-Akt serine phosphorylation pathway are All three SIE binding complexes are also formed in IFN-sensitive involved in IFN-induced CXCL11 gene expression. Daudi cells (5). In DRST3 cells expressing the F705-STAT3 mutant, IFN did not induce either of the STAT3-dependent DNA binding complexes, although similar levels of STAT3 were ex- The binding of transcription factors to the CXCL11 promoter pressed. These data indicate that IFN-induced phosphorylation of To investigate whether STAT3 directly bound to the CXCL11 pro- Y705 is necessary for the formation of STAT3-dependent DNA moter, ChIP assays were performed. As shown in Fig. 4, IFN treat- binding complexes. ment induced the binding of STAT3 to the CXCL11 promoter in Daudi cells as early as1hofIFNtreatment and STAT3 remained Phosphorylation of Y705 of STAT3 is not required bound to the CXCL11 promoter up to3hofIFNtreatment. Sur- for IFN-induced CXCL11 expression prisingly, STAT3 was also recruited to CXCL11 promoter after To further characterize the role of STAT3 as well as the tyrosine IFN treatment in DRST3 cells. Moreover, ChIP assays performed phosphorylation of Y705 of STAT3 in CXCL11 induction by IFN, with Y705 phospho-specific Ab showed that upon IFN treatment MEFs with a germline deletion of STAT3 were examined (15). Y705 phosphorylated STAT3 was recruited onto CXCL11 pro- These STAT3-KO MEFs were reconstituted with WT-STAT3 or moter in both Daudi and DRST3 cells. These results indicated that the F705-STAT3 mutant, and quantitative real-time PCR assays recruitment of STAT3 to the CXCL11 promoter may be important were performed for CXCL11. As shown in Fig. 3A, CXCL11 for IFN-induced CXCL11 expression, but this event alone may not reached an induction level of ϳ150-fold in STAT3-KO MEFs sta- be sufficient for gene expression. bly expressing either WT-STAT3 or F705-STAT3 after6hofIFN Because Y705 of STAT3 is not required for IFN-induced treatment, but was only slightly induced in STAT3-KO MEFs. CXCL11 expression, we performed ChIP assays to profile binding Similar expression levels of IFN-induced CXCL11 were observed of other potential transcription factors to the CXCL11 promoter. with WT MEFs that have STAT3 protein levels comparable to Previous studies showed that IFN treatment of Daudi cells induced STAT3-KO MEFs reconstituted with STAT3. These results indi- the formation of multiple STAT dimers, which include various 990 IFN-INDUCED CXCL11 GENE EXPRESSION IS STAT3 DEPENDENT

FIGURE 4. The effects of IFN on the binding of STAT, NF-␬B, and IRF proteins to the CXCL11 promoter. ChIP assays were performed on extracts from control and IFN-treated Daudi cells, DRST3 cells, and DRST3 cells expressing WT STAT3 using the indicated Abs for precipitation and primers that targeted the CXCL11 promoter as described in Materials and Meth- ods. Similar results were obtained in at least three independent experiments. Downloaded from

complexes of STAT1, STAT2, and STAT3 (4). These STAT pro- the pattern of transcription factor binding was indistinguishable

teins undergo IFN-induced tyrosine phosphorylation in Daudi from that in IFN-sensitive Daudi cells, i.e., the IFN-induced loss of http://www.jimmunol.org/ cells. In contrast only STAT1/STAT2 and STAT1/STAT1 dimers p50 and IRF2 promoter binding, and the IFN-induced recruitment are formed upon IFN treatment of DRST3 cells, as determined by of p65 and IRF1 binding to the CXCL11 promoter. gel shift assays. As shown in Fig. 4 the CXCL11 promoter contains a potential ISRE site, which would be expected to bind STAT1/ Discussion STAT2 dimers. Therefore, we examined the binding of STAT1 STAT3, the transcription factor for acute phase response genes, is and STAT2 to the CXCL11 promoter. As shown in Fig. 4, STAT1 activated by a wide variety of cytokines suggesting that it may and phospho-STAT1 were recruited to the CXCL11 promoter in integrate diverse signals into common transcriptional responses (5, Daudi and in DRST3 cells. However, although STAT2 is activated 22, 23). A critical role of STAT3 in cellular physiology is impli- in both Daudi cells and DRST3 cells upon IFN addition (4), cated by the finding that the KO of the STAT3 gene in mice leads by guest on September 29, 2021 STAT2 was not bound to the CXCL11 promoter. These results to early embryonic lethality, and STAT3-deficient cell lines could indicate that although the CXCL11 promoter contains a potential not be isolated (24). Moreover, STAT3 apparently plays an im- ISRE site, the formation of STAT1-STAT2-IRF9 complexes is not portant role in tumorigenesis by regulating cell cycle progression, involved in IFN-induced regulation of CXCL11 expression. More- apoptosis, angiogenesis, invasion and metastasis, and evasion of over, because STAT1 and phospho-STAT1 are bound to the immune surveillance (25–28). A tyrosine-based YXXQ motif in CXCL11 promoter in Daudi and DRST3 cells, the difference in the cytosolic tails of type I IFN receptor 1 (IFNAR1) ␣ chain of the IFN-induced gene expression does not reflect alterations at the IFN-␣␤ receptor and in the shared signal-transducing gp130 chain level of STAT1. of the IL-6R family is required for cytokine-dependent STAT3 We have recently shown that NF-␬B proteins play a key regu- tyrosine phosphorylation and dimerization (12, 29–31). Although latory role in IFN-induced gene expression (21). In Daudi cells, the the role that STAT1/STAT2 dimers play in ISG activation is well p50 NF-␬B subunit was found basally bound to the CXCL11 pro- understood, it is unknown how STAT3-containing dimers form moter and by1hofIFNtreatment the p50 subunit of NF-␬B was and what is their role in IFN-responsive gene expression. no longer found associated with the CXCL11 promoter. In contrast, We examined the role of STAT3 in the regulation of IFN-re- the p65 subunit of NF-␬B was not basally promoter-bound but its sponsive gene expression. STATs, like many cytoplasmic effec- binding was induced by 1 h after IFN addition. In DRST3 cells, tors, contain tyrosine-based motifs as well as Src homology 2 do- although p50 was not basally bound to the CXCL11 promoter, IFN mains (32) that bind tyrosine-based motifs. The general dogma in induced p50 binding within 1 h after addition, and p65 was neither the cytokine field is that a single tyrosine is phosphorylated in basally promoter-bound nor was its binding IFN induced. More- STATs upon cytokine stimulation (Y705 in STAT3), followed by over, IFN induced the binding of the transcriptional activator IRF1 STAT dimerization and the transcriptional activation of selective to the CXCL11 promoter in Daudi cells. In contrast, in DRST3 gene expression (6). We found that STAT3 is required for IFN- cells IFN induced the binding of another IRF family member, induced expression of CXCL11 expression in both STAT3-KO IRF2, which is a transcriptional repressor. These results indicate MEFs and in STAT3-defective Daudi human lymphoblastoid cells. that the defective STAT3 signaling pathway in DRST3 cells affects However, although phosphorylation of the Y705 residue of STAT3 the binding of NF-␬B and IRF proteins, which cooperate in the is critical for the formation of STAT1/STAT3 heterodimers as de- transcription regulation of CXCL11. To rescue the defective tected by coimmunoprecipitation or EMSA, this event is not re- STAT3 signaling pathway in the DRST3 cells, we examined the quired for IFN-induced CXCL11 gene expression. Thus, in future effect of expression of WT-STAT3 on the binding of NF-␬B and studies it will be important to identify and characterize other IRF proteins to the CXCL11 promoter. As shown in Fig. 4, ex- STAT3-dependent genes induced by IFN that do not require Y705 pression of WT-STAT3 in DRST3 cells rescued the recruitment of phosphorylation of STAT3. The finding that Y705 phosphoryla- NF-␬B and IRF proteins to the CXCL11 promoter by IFN so that tion is not required for IFN regulation of CXCL11 gene expression The Journal of Immunology 991 is somewhat surprising in light of the findings that constitutive DRST3 cells IFN induces the binding of IRF1 to the CXCL11 Y705 phosphorylation of STAT3 occurs in a number of human promoter, IFN induces the binding of IRF2 to the CXCL11 pro- cancers and is considered a marker of activated STAT3. We hy- moter in IFN-resistant DRST3 cells. IRF1 and IRF2 are members pothesize that phosphorylation of different tyrosine and/or serine of the IRF family of transcriptional regulators that exert distinct residues of STAT3 may result in differential regulation of STAT3 roles in biological processes such as pathogen responses, cytokine target genes. For example, the IFN-induced phosphorylation of a signaling, cell growth regulation, and hemopoietic development Y657 KIM motif of STAT3 appears to act as a docking site for the (37). IRF1 and IRF2 have antagonistic roles in gene regulation: lipid/serine kinase PI3K (3). In the present study, we show that they bind to the same DNA elements, but IRF1 acts as a stimulator pharmacological inhibitors of PI3K and tyrosine kinases inhibit of transcription whereas IRF2 acts as a repressor (38). Apparently, IFN induction of CXCL11 gene expression, which is in agreement alterations of the IRF1 to IRF2 ratio can have significant conse- with previous finding for a role of JAK-STAT and PI3K signaling in quences for cell growth; restrained cell growth depends on a bal- the activation of a CXCL11 promoter-reporter construct by IFN (33). ance between these two mutually antagonistic transcription factors The CXCL11 gene has been shown to be selectively induced by (39). Our results are consistent with the hypothesis that upon IFN IFN-␤ relative to IFN-␣ in human cells (34). Consistent with these treatment IRF1 activates CXCL11 gene transcription by directly results we show that in murine fibroblasts IFN-␤ results in a higher binding to the ISRE site, whereas IRF2 binding to the CXCL11 CXCL11 gene induction than does IFN-␣ in human lymphoblas- promoter represses its transcription. toid cells. Moreover, previous studies established that the activa- In summary, we show that CXCL11 is a gene target of STAT3 tion of the JAK-STAT signaling pathway as well as the NF-␬B during IFN signaling. The IFN-induced expression of CXCL11 pathway is necessary for CXCL11 gene induction (35). Site-di- involves STAT3 but not the canonical Y705 dimerization site. A Downloaded from rected mutagenesis of the ISRE or the NF-␬B binding site in a number of other factors are involved in IFN-induced CXCL11 ex- promoter-reporter construct of the CXCL11 gene abrogated IFN pression including the binding of NF-␬B proteins (p50 and p65) induction of the reporter construct. Our studies show that the “clas- and IRF proteins (IRF1 and IRF2). Moreover, p50 and p65, as well sical” ISRE binding protein complex composed of STAT1-STAT2 as IRF1 and IRF2, have antagonizing roles in CXCL11 expression. does not bind to the CXCL11 promoter, although both STAT1 and

STAT2 are activated in Daudi cells, as demonstrated by the for- Acknowledgments http://www.jimmunol.org/ mation of an ISRE complex using a consensus ISRE oligonucle- We thank Lawrence Blatt and Darren Baker for generously providing re- otide probe as well as by their tyrosine phosphorylation upon IFN combinant human IFN␣Con1 and rat IFN-␤. We also thank Sandhya Rani treatment (4). In contrast, we show that STAT3 and STAT1 bind and Richard Ransohoff (Cleveland Clinic Foundation, Cleveland, OH) for to the CXCL11 promoter, as well as the ISRE binding proteins useful discussions during initial studies on CXCL11 gene expression in IRF1 and IRF2. Daudi and DRST3 cells. In addition, we show that NF-␬B proteins regulate the IFN- induced expression of CXCL11 gene. The NF-␬B proteins p50, Disclosures p52, p65 RelB, and c-Rel are transcription factors that regulate the The authors have no financial conflict of interest. expression of genes involved in the immune response, inflamma- by guest on September 29, 2021 tion, and cell survival. These proteins dimerize and bind to the References promoter elements of genes to regulate their expression. In a series 1. Meraz, M. A., J. M. White, K. C. F. Sheehan, E. A. Bach, S. J. Rodig, A. S. Dighe, D. H. Kaplan, J. K. Riley, A. C. Greenlund, D. Campbell, et al. 1996. of recent studies we show that different NF-␬B proteins bind to the Targeted disruption of the Stat1 gene in mice reveals unexpected physiological promoters of ISGs to regulate their expression (21). We previously specificity in the JAK-STAT signaling pathway. Cell 84: 431–442. 2. Darnell, J. E. J., I. M. Kerr, and G. R. Stark. 1994. 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USA 95: upon IFN treatment p65 displaces p50 binding to the promoter. 5568–5572. However, in STAT3-defective Daudi cells, IFN treatment induces 5. Yang, C. H., W. Shi, L. Basu, A. Murti, S. N. Constantinescu, L. Blatt, E. Croze, J. E. Mullersman, and L. M. Pfeffer. 1996. Direct association of STAT3 with the the binding of p50 to the CXCL11 promoter, whereas p50 protein IFNAR1 signal transducing chain of the type I IFN receptor. J. Biol. Chem. 271: is not basally bound. This result is in contrast to other NF-␬B- 8057–8061. regulated ISGs heretofore examined in which p50 was found 6. Kaptein, A., V. Paillard, and M. Saunders. 1996. Dominant negative stat3 mutant inhibits interleukin-6-induced Jak-STAT signal transduction. J. Biol. Chem. 271: basally bound to the promoters (21). Protein p50 differs from other 5961–5964. NF-␬B proteins in that it does not have a transactivation domain 7. Constantinescu, S. N., E. Croze, C. Wang, A. Murti, L. Basu, J. E. 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