Not Monocytes/Macrophages STAT1-PIAS1 Interactions In

TGF-β Suppresses IFN-γ-STAT1-Dependent Gene Transcription by Enhancing STAT1-PIAS1 Interactions in Epithelia but Not Monocytes/Macrophages This information is current as of September 28, 2021. Colin Reardon and Derek M. McKay J Immunol 2007; 178:4284-4295; ; doi: 10.4049/jimmunol.178.7.4284 http://www.jimmunol.org/content/178/7/4284 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

TGF-␤ Suppresses IFN-␥-STAT1-Dependent Gene Transcription by Enhancing STAT1-PIAS1 Interactions in Epithelia but Not Monocytes/Macrophages1

Colin Reardon and Derek M. McKay2

IFN-␥ and TGF-␤ are important regulators of mucosal immunity, typically functioning in opposition to each other. In this study, we assessed whether TGF-␤ could modulate IFN-␥-induced STAT1 signaling. Model epithelial cell lines (HEp-2, HT-29, and T84) or monocytes/macrophages (THP-1 cell line, human blood mononuclear cells) were pretreated with TGF-␤ (1 ng/ml; 5–60 min), followed by IFN-␥ exposure (20 ng/ml; 30 min), and then STAT1 transcriptional activity, DNA-binding activity, phosphorylation, and methylation were assessed. Some epithelia were transfected with an expression plasmid encoding SMAD7 to block TGF-␤- SMAD signaling. Epithelia, but not macrophages, pretreated with TGF-␤ were hyporesponsive to IFN-␥ stimulation as indicated Downloaded from by reduced expression of four STAT1-regulated genes and reduced STAT1 DNA binding on EMSA. However, STAT1 Tyr701-, Ser727 phosphorylation, and nuclear recruitment of STAT1 were not signiﬁcantly different in IFN-␥ with or without TGF-␤- treated cells, indicating that the effects of TGF-␤ are downstream of IFN-␥R-JAK-STAT1 interaction. The TGF-␤ effect was not dependent on ERK1/2, p38, or JNK activation but was prevented by overexpression of the inhibitory SMAD7 protein. Additional studies suggest that TGF-␤ blockade of IFN-␥ activity in epithelia is via enhanced sequestering of STAT1 by pre-existing protein inhibitor of ␤ ␥ activated STAT1. These results demonstrate that TGF- rapidly suppresses IFN- -driven STAT1 signaling by reducing DNA binding http://www.jimmunol.org/ via promotion of STAT1-protein inhibitor of activated STAT1 interactions and not inhibition of STAT1 activation; an event that may be speciﬁc to epithelia and represent a novel mode of action of TGF-␤. The Journal of Immunology, 2007, 178: 4284–4295.

he intestine is continuously exposed to potentially nox- synthesis (5–8). In contrast, TGF-␤ is an immunoregulatory cy- ious substances derived from the diet and the transient tokine produced by many cell types and, despite its ability to cause T and resident bacterial flora. Thus, not surprisingly, the fibrosis, is generally considered beneficial by virtue of its anti- intestine is the largest reservoir of immune cells in the body and inflammatory and immunosuppressive properties (9). For instance, may be considered as being in a continuous state of controlled loss of TGF-␤ signaling by expression of a dominant-negative re- inflammation. As such, the balance between normal physiology ceptor exacerbates colitis, and conversely enhancement of TGF-␤ by guest on September 28, 2021 and pathological processes is delicate and is dictated by the inter- production significantly ameliorates murine colitis (10, 11). In actions between the host and commensal and pathogenic bacteria. terms of direct action on the epithelium, TGF-␤ enhances enteric As a consequence of a perturbation of this homeostatic balance, epithelial barrier function, and the in vivo significance of this inappropriate, exaggerated, or otherwise dysregulated immune re- would be to control the movement of potentially antigenic material sponses can develop and result in immunopathology (1, 2). from the lumen into the mucosa/submucosa and access to the im- IFN-␥ and TGF-␤1 are key cytokines that often function in op- mune cells therein (7). Moreover, TGF-␤ blocks the loss of barrier position to modulate mucosal immunity and inflammation (3). function observed in monolayers of human colon-derived epithe- IFN-␥ is a proinflammatory cytokine that is produced primarily by ϩ lial cell lines caused by exposure to bacterial pathogens and im- CD4 T cells (particularly Th 1 cells) and NK cells. Exposure to mune mediators, such as IFN-␥ (12, 13). IFN-␥ can result in the activation of up to 500 genes (4) and, in the Under normal conditions and during active immune responses case of the enteric epithelium, causes reduced epithelial barrier function, reduced active ion transport, and increased chemokine or disease, multiple cytokines will be present in the interstitial milieu. Understanding the interplay between these signals on the various target cell types is crucial to fully appreciate cytokine reg- Gastrointestinal Research Group, University of Calgary, Calgary, Alberta, Canada ulation of mucosal immunity and gut function. IFN-␥ antagonism Received for publication June 1, 2006. Accepted for publication January 18, 2007. of TGF-␤-induced intracellular signaling is established and, in The costs of publication of this article were defrayed in part by the payment of page general, appears to be an IFN-␥-STAT1-dependent event (14): sig- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. nificantly less is known of how TGF-␤ might ablate IFN-␥ sig- 1 This work was supported by Canadian Institutes for Health Research Grant MT- naling. For example, TGF-␤ inhibition of IFN-␥-induced increases 13421 (to D.M.M.). D.M.M. is supported by an Alberta Heritage Foundation for in epithelial paracellular permeability (12) could be due to separate Medical Research Scientist Award and a Canada Research Chair (Tier 1). C.R. is a recipient of Natural Sciences and Engineering Research Council of Canada student- and opposing affects on the expression of tight junction proteins ship and had former support from a Premiers Research Excellence Award (to that gate the paracellular permeation pathway between adjacent D.M.M.), Canadian Institutes for Health Research, and Ontario Graduate epithelial cells or via direct cross-regulation of intracellular sig- studentships. naling pathways. In support of the latter hypothesis, TGF-␤ treat- 2 Address correspondence and reprint requests to Dr. Derek M. McKay, Gastrointes- tinal Research Group, Department of Physiology and Biophysics, HS-1877, Univer- ment reduces NF-␬〉-dependent IL-8 expression in enteric epithe- sity of Calgary, 3330 Hospital Drive Northwest, Calgary, Alberta T2N 4N1, Canada. lium (15) and prevents IL-6-induced JAK/STAT3 phosphorylation E-mail address: [email protected] through increased suppressor of cytokine signaling 1/3 expression Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 in airway epithelium (16). www.jimmunol.org The Journal of Immunology 4285

In this study, we assessed if, and how, TGF-␤ inhibits IFN-␥- CC-3Ј (3 pmol), and ␤-actin reverse, 5Ј-CTG TGG TGG TGA AGC TGT STAT1 gene transcription in model epithelia and monocytes/mac- AG-3Ј (3 pmol). Amplification by PCR was conducted using platinum Taq rophages. The data illustrate that a short duration, low-dose TGF-␤ (Invitrogen Life Technologies) and the following parameters: 95°C for 3 ␥ min, followed by 25 cycles of 1 min at 95°C, 60°C annealing for 1 min, exposure suppresses IFN- -driven STAT1-dependent gene expres- 72°C for 1 min, with a final elongation step conducted at 72°C for 10 min. sion and STAT1 DNA binding in epithelia but not in macrophages. Amplification products were subjected to electrophoresis in a 1% agarose TGF-␤ blockade of STAT1 activity in epithelia did not rely on gel in Tris-acetic acid EDTA buffer, and imaged on a Kodak EDAS 290 gel inhibition of Tyr710 or Ser727 phosphorylation or STAT1 methyl- documentation system (Kodak). ation; rather, it occurred through enhanced sequestering of STAT1 EMSA for STAT1 by existing protein inhibitor of activated STAT1 (PIAS1),3 which ␤ Following cytokine treatment, nuclear protein extracts were obtained, and may represent a novel mode of action for TGF- regulation of protein concentrations were determined using the Bradford assay. EMSA IFN-␥-STAT1 signaling in epithelial cells. were performed as previously described (22, 23): nuclear extracts (5–10 ␮g protein) in binding buffer were incubated for 30 min with [32P]dCTP (NEN Materials and Methods Life Science Products)-labeled oligonucleotide probe high-affinity sis-inducible element (hSIE) containing a high-affinity STAT1 binding site (5Ј- Cell culture GTCGACATTTCCCGTAAATC-3Ј and 5Ј-TCGACGATTTACGGGAA Ј Epithelial cells. Three human-derived epithelial cell lines that are widely ATG-3 (MOBIX; McMaster University) (24). Samples were electropho- used in the analysis of the effects of enteric pathogens and cytokines on resed through a nondenaturing 6% (40:1 bis/acrylamide) polyacrylamide epithelial function were used, namely HEp-2 (laryngeal origin), HT-29, gel for 2.5 h at 120 V, dried under vacuum at 80°C, and visualized by Ϫ and T84 (both of colonic origin) cells (American Type Culture Collection) autoradiography after overnight exposure ( 70°C) to Kodak BioMAX MS film. Specificity controls included use of a “cold” unlabeled hSIE dsDNA (7, 17–21). Each cell line was maintained at 37°C with 5% CO2 in a Downloaded from specific culture medium (Hep2 cells: MEM F11 supplemented with 2% oligonucleotide for competitive binding and inclusion of STAT1-specific sodium bicarbonate, 2.5% (v/v) penicillin-streptomycin, and 10% FBS; Abs (Santa Cruz Biotechnology) in the reaction mixture. HT-29 cells: DMEM plus 1% penicillin-streptomycin (v/v), 0.5% sodium Immunoblotting and immunoprecipitation bicarbonate (v/v), 0.1% L-glutamine, and 5% FBS; T84 cells: a 1/1 mixture of DMEM and Ham’s F-12 medium supplemented with 2% penicillin- Immunoblotting was used following a standard protocol (12, 22) to assay streptomycin, 1.5% (v/v) HEPES, and 10% FBS (all from Invitrogen Life for changes in whole-cell protein extracts after cytokine treatment using the Technologies) (18, 19)). following Abs: anti-TGF-␤ receptor I, anti-P-Tyr701-STAT1, anti-P-Ser727- Immune cells. The human THP-1 monocyte cell line (from American STAT1, anti-c-myc, anti-P-ERK1/2, anti-P-Ser63-JNK, anti-P-Ser73-JNK, http://www.jimmunol.org/ Type Culture Collection) was maintained as a suspension in RPMI 1640 anti-JNK, anti-p38, anti-phospho-p38 (Cell Signaling Technology), anti- culture medium supplemented with 2% (v/v) penicillin-streptomycin, 36 phosphoserine (Abcam), anti-SMAD2/3, anti-␤-actin, anti-STAT1, anti-IRF1, ␮M HEPES, and 10% FBS. Two million cells were seeded onto 3-cm petri anti-ERK1/2, anti-PIAS1 (Santa Cruz Biotechnology), and anti-FLAG dishes and induced into a macrophage phenotype by adding PMA (10 nM; (Sigma-Aldrich) at working dilutions of 1/1000. Secondary Abs were goat Sigma-Aldrich). Three days later, cells were serum starved for 16 h, fol- anti-rabbit-HRP IgG or goat anti-mouse-HRP IgG (Santa Cruz Biotechnology) lowed by cytokine treatment (22). PBMC from healthy volunteers were and were used at a working dilution of 1/5000. isolated as described previously (7). Briefly, venous blood was collected in Immunoprecipitations were conducted with EZview beads (Sigma- heparinized vaccu-tubes, diluted 1/2 with sterile PBS (37°C) in a sterile Aldrich), according to the manufacturer’s instructions. Briefly, cell lysates 50-ml tube, and underlain with 10 ml of Ficoll plaque (Amersham Bio- were precleared with 40 ␮l of the EZview bead slurry (1 h, 4°C). Cell

sciences). Following centrifugation, mononuclear cells were retrieved from lysates were diluted with radioimmunoprecipitation assay (RIPA) buffer to by guest on September 28, 2021 the Ficoll-PBS interface, rinsed twice in PBS, and resuspended in RPMI 1 mg/ml and were gently rocked with Ab (2 ␮g/ml, 2 h, 4°C), and then 1640 culture medium at 2 ϫ 106/ml. added to 40 ␮l of prewashed EZview beads while rocking (1.5 h, 4°C). The Cytokine treatment. Epithelia cells were seeded at 106 cells/ml into sterile beads were centrifuged and washed four times at 5 min to remove unbound petri dishes or 6-well culture plates and grown to ϳ70% confluence (de- proteins. Following the final wash, dissociation from the EZview beads termined by phase contrast microscopy), and the culture medium was re- was performed by adding SDS-loading buffer (laemmli) and boiling placed with serum-free medium for 16 h, followed by IFN-␥ (20 ng/ml) for 5 min. ␤ In additional experiments, we conducted in vitro binding assays. Protein with or without TGF- 1 (1 ng/ml, as a 5- to 60-min pretreatment) exposure ␤ (R&D Systems). We have previously shown that these doses of cytokine extracts (0.5 mg/ml) from HEp-2 epithelial cells with or without TGF- directly affect epithelial function (12, 22). treatments (1 ng/ml at 15, 30, and 60 min) were subjected to immunoprecipitation with anti-PIAS1 Ab or an isotype-matched irrelevant Ab (neg- RT-PCR ative control). The captured PIAS1 from control or TGF-␤-treated cells was incubated with 0.5 mg of protein obtained from STAT1-FLAG-trans- IFN-␥-STAT1-dependent gene expression was determined through semi- fected cells (see below) that had or had not (i.e., control) been exposed to quantitative measurements of IFN-␥-regulated factor-1 (IRF-1), major his- IFN-␥ (20 ng/ml; 30 min) overnight at 4°C. Following several washes in tocompatibility CIITA, guanylate-binding protein 1 (GBP-1), and induc- RIPA buffer, protein complexes were dissociated by boiling in SDS-load- ible NO synthase (iNOS) mRNA (22). Cells were pretreated with TGF-␤, ing buffer for 5 min, followed by immunoblotting with PIAS1- or exposed to IFN-␥ for 30 min, and rinsed in serum-free medium (three FLAG-specific Abs. times), and RNA was extracted 3.5 h later (i.e., 3 h following IFN-␥) using RNeasy columns (Qiagen). RNA yield and concentration were determined Construction of a SMAD7 expression vector ␮ spectrophotometrically, and 1 g of RNA was used to make cDNA by The viral genome was isolated from an adenovirus encoding mouse reverse transcription using an iSCRIPT kit (Bio-Rad). PCR was conducted SMAD7, provided by Dr. P. ten Dijke (Ludwig Institute for Cancer Re- on the resulting cDNA with primers designed using Primer3 software (www. search, Uppsala, Sweden) (25), through a modified small DNA isolation genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi/) from published Ј technique (Hirt protocol) as described previously (26). Briefly, subconflu- mRNA sequences (GenBank): IRF-1 forward, 5 -CGA TAC AAA GCA ent monolayers (60–80%) of HEK293 cells were infected at 5 multiplicity GGG GAA AA-3Ј (10 pmol), and IRF-1 reverse, 5Ј-TAG CTG CTG TGG Ј Ј of infection for 1 h with gentle agitation. Cytopathic effects were observed TCA TCA GG-3 (10 pmol); GBP-1 forward, 5 -TGG AAC GTG TGA 24 h postinfection, at which point cells were dislodged with a cell scrapper AAG CTG AG-3Ј (3 pmol), and GBP-1 reverse, 5Ј-TGA CAG GAA GGC Ј Ј and gentle tapping of the culture flask. Detached cells were incubated for TCT GGT CT-3 (3 pmol); CIITA forward, 5 -GGG AAA GCT TGT GCA 16 h on a plate rocker at 37°C in DNA extraction buffer (50 mM Tris, 2 GAC TC-3Ј (3 pmol), and CIITA reverse, 5Ј-CAC CCA GGT CAG TGA Ј Ј mM EDTA, 0.5% (w/v) polyoxyethylene sorbitan (Tween 20; Sigma- TGT TG-3 (3 pmol); iNOS forward, 5 -TGT GCT CTT TGC CTG TAT Aldrich), and 400 ␮g/ml proteinase K (Roche Diagnostics, Laval, PQ: GC-3Ј (3 pmol), and iNOS reverse, 5Ј-GGG GAT CTG AAT GTG CTG Ј ␤ Ј added immediately before use). DNA was isolated by a 25:24:1 phenol: TT-3 (3 pmol); and -actin forward, 5 -CCA CAG CAA GAG AGG TAT chloroform:isoamyl alcohol (v/v/v) extraction and precipitated with iso- propanol (Ϫ20°C overnight). Once precipitated, the DNA was collected by 3 Abbreviations used in this paper: PIAS1, protein inhibitor of activated STAT1; centrifugation (13,000 rpm, 30 min), washed once with 70% ethanol, air- GBP-1, guanylate binding protein 1; hSIE, high-affinity sis-inducible element; iNOS, dried, and resuspended in ultrapure water (Invitrogen Life Technologies). inducible NO synthase; IRF-1, IFN-␥-regulated factor-1; RIPA, radioimmunoprecipi- Endonuclease restriction sites for KpnI and BamHI were added to the 5Ј tation assay; SAPK, stress-activated protein kinase; siRNA, small interfering RNA. and 3Ј end of SMAD7, facilitating insertion into the pcDNA3.1 (Invitrogen 4286 TGF␤-IFN␥ INTERACTIONS AT THE EPITHELIUM Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Pretreatment of epithelial cells with TGF-␤ attenuates IFN-␥-induced STAT1-regulated gene expression. A, Representative image of RT-PCR product for IRF-1 mRNA expression in HEp-2 cells exposed to IFN-␥ (20 ng/ml, 30 min, followed by washout and RNA extraction 3 h later) with or without p Ͻ 0.05 ,ء) TGF-␤ (1 ng/ml) pretreatment (times indicated). ␤-Actin served as a housekeeper gene. B, Densitometric quantiﬁcation of the IRF-1:␤-actin ratio compared with control and other groups; n ϭ 4). C and D are representative images and subsequent densitometry (n ϭ 3), showing that IFN-␥-induced expression of CIITA, GBP-1, and iNOS mRNA is reduced in HEp-2 epithelial cells (left panel), but not THP-1 macrophages (right panels), pretreated with TGF-␤. E, IFN-␥-induced IRF-1 protein is reduced in HEp-2 but not THP1 cells by TGF-␤ pretreatment (␤-actin is included as a loading control; representative of n ϭ 3). The Journal of Immunology 4287

FIGURE 2. TGF-␤ pretreatment of epithelial cells inhibits IFN-␥-induced STAT1 DNA binding. TGF-␤ (1 ng/ml) pretreatment for 15–60 min significantly reduced IFN-␥-induced (20 ng/ml, 30 min) STAT1 DNA binding as determined by EMSA conducted on nuclear protein extracts from HEp-2 (n ϭ 5; A and B) and T84 (30 min TGF-␤ pretreatment; n ϭ 2; C) but not THP-1 cells (D) or human Downloaded from PBMC (n ϭ 3; E). TGF-␤ treatment alone fails to induce specific binding activity to the hSIE probe (lane 3 of A) (specificity of the STAT1 signal was confirmed by inclusion of an anti- STAT1 Ab (aS1-Ab.), which super-

shitfs a portion of the band (arrow, B) http://www.jimmunol.org/ and use of a cold competitor (cc, C) that obliterates DNA binding; NS, nonspeciﬁc band; fp, free probe; CON, extracts from control noncytokine-treated cells). by guest on September 28, 2021

Life Technologies) expression vector by PCR. Amplification was conducted using the SMAD7-specific primers: mSMAD7-KpnI, 5Ј-GGG GTA CCA TGT TCA GGA CCA AAC GAT CTG-3Ј, and mSMAD7-BamHI, 5Ј-CGG AAT TCC TAC CGG CTG TTG AAG ATG AC-3Ј, with hi- fidelity platinum Taq (Invitrogen Life Technologies) under the following conditions: 95°C 2 min, 30 cycles of 95°C 15 s, 64°C 30 s, 60°C 2 min, and a final 68°C extension for 10 min. Following PCR amplification, vector and SMAD7 insert were sequentially double digested with KpnI, followed by BamHI with 10-fold excess enzyme. Linearlized pcDNA3.1 was treated with shrimp alkaline phosphatase (Fermentas Life Sciences), purified by agarose gel electrophoresis (0.7%), and extracted using the QIAEX II kit (Qiagen). Recovered SMAD7 and lineralized pcDNA3.1 were then ligated overnight at a molar ratio of 3:1, respectively, with T4 DNA ligase (In- vitrogen Life Technologies). Competent DH5␣ Escherichia coli (Invitrogen Life Technologies) were transformed according to the manufacturer’s instructions and plated on Luria-Bertani-ampicillin (100 ␮g/ml) agar plates, and 24 h later, ampicillin resistant colonies were isolated for screening by restriction enzyme and PCR analysis (using primers directed against the T7 and BGH sequences flanking the multiple cloning site on the vector). Successfully transformed bacteria were further cultured, and plasmid DNA was purified with a QIA filter Maxiprep kit (Qiagen). FIGURE 3. TGF-␤ pretreatment does not affect IFN-␥-induced STAT1 Tyr701 phosphorylation. Whole-cell protein extracts from epithelial cells Transient transfection of epithelia (HEp-2 or T84) or THP-1 macrophages showed no significant differences in STAT1 Tyr701 phosphorylation (pSTAT1) levels induced by IFN-␥ (20 ng/ml, Subconfluent HEp-2 monolayers (ϳ60%) were transfected using GenePorter ␤ II (GPII) according to the manufacture’s recommendations (Gene Therapy 30 min) with or without a 5- to 60-min pretreatment with TGF- (1 ng/ml). Systems). Briefly, plasmid DNA was incubated in DNA dilution solution for Lower panel in each doublet is the upper membrane that was stripped and Ն5 min and added to the GPII transfection reagent diluted in serum and an- reprobed for total STAT1 to ensure equal protein loading. Images are repre- tibiotic free HEp-2 culture medium. The SMAD7 vector described above was sentative of three to five experiments. 4288 TGF␤-IFN␥ INTERACTIONS AT THE EPITHELIUM

FIGURE 4. Neither STAT1 nor SMAD2/3 nuclear localization is affected in HEp-2 epithelia by cotreat- ment with TGF-␤ and IFN-␥, respectively. Immunofluorescence images of STAT1 and SMAD2/3 cellular localization 30 min after exposure to IFN-␥ (20 ng/ml) with or without TGF-␤ (1 ng/ml; 30 min pretreatment) show that STAT1 and SMAD2/3 translocate to the nucleus of HEp-2 cells treated with IFN-␥, TGF-␤, or IFN-␥ ϩ TGF-␤ (STAT1 is in green (arrowheads), SMAD2/3 in red (small arrows), and colocalization is orange/ yellow; representative of five fields of view from three separate preparations; original magnification, ϫ63). Downloaded from

used at a final concentration 4 ␮g/ml, whereas the SMAD2-myc, SMAD3-myc Confocal microscopy http://www.jimmunol.org/ (provided by Dr. O. Eickelberg, University of Giessen, Giessen, Germany) (27), and STAT1-FLAG (provided by Dr. M. David, University of Cali- HEp-2 cells were grown in 8-well chamber slides (NUNC Labtek II; VWR fornia, San Diego, CA) (28) plasmids were used at a final concentration of Scientific) with or without cytokine treatment and fixed with 10% neutral- 2 ␮g/ml. Monolayers were rinsed in serum- and antibiotic-free medium buffered formalin. Cells were permeabilized with 0.1% Triton X-100/PBS (three times), the DNA transfection reagent added to the cells, and 24 h for 30 min at room temperature, followed by blocking in 5% BSA (Roche later, the solution was aspirated and replaced with fresh normal HEp-2 Diagnostics) and 5% normal goat serum (Sigma-Aldrich) for 1 h. After medium (transfection conditions were determined empirically by monitoring washing with 0.1% Triton X-100/PBS, preparations were incubated with protein expression of the plasmid encoded gene over a 48-h period). the specific primary Abs (i.e., those used in immunoblotting; 1/500) for 1 h at room temperature, rinsed extensively, and incubated with AlexaFluor Nucleofection of small interfering RNA (siRNA) 488- or AlexaFluor 594-conjugated secondary Abs (1/1000) (Molecular by guest on September 28, 2021 Nucleofection was performed according to manufacture’s instructions Probes) for1hatroom temperature. Slides were washed five times in 0.1% (Amaxa; EBSE Scientific). Briefly, cells were grown to 70% confluence Triton X-100/PBS, gel mount was applied, and coverslips were affixed. In and dissociated with trypsin EDTA to obtain a single-cell suspension. Cells experiments in which nuclei were counterstained, propidium iodide was (1 ϫ 106) were placed into a cuvette with nucleofactor solution V, with an added for 5 min and washed extensively before affixing the coverslip. Prep- experimentally determined amount of prevalidated “stealth” siRNA di- arations were examined on an inverted Zeiss laser-scanning microscope rected against PIAS1 (Invitrogen Life Technologies) and nucleofected us- (LSM 510, Axiovert 100 M; Zeiss) equipped with argon (450–514 nm) and ing program T-027. Verification of the nucleofection regime was ascer- helium-neon (543 and 633 nm) lasers. AlexaFluor 488 and AlexaFluor 594 tained in experiments using a dsRNA FITC-labeled oligo provided in the fluorescence were exited using the 488- and 543-nm laser with emissions siRNA kit and verified by confocal microscopy 48 h postnucleofection. collected using the standard FITC and rhodamine filter set, respectively.

FIGURE 5. Inhibition of IFN-␥-induced STAT1 DNA binding by TGF-␤ is SMAD dependent. Repre- sentative EMSA showing that IFN-␥ (20 ng/ml, 30 min)-induced STAT1 DNA binding is not affected by the transfection reagent, GenePorter II (GPII), or the plasmid encoding SMAD7 (pcSMAD7) only (lanes 3 and 4). TGF-␤ (1 ng/ml, 60 min pretreatment) inhibits the IFN-␥ mobilization of STAT1 (lane 8), and this is not observed in nuclear protein extracts from pcSMAD7-transfected HEp-2 cells, where STAT1 DNA binding in response to IFN-␥ is increased (lane 9)(n ϭ 3; fp, free probe). Inset,An immunoblot showing increased SMAD7 protein in HEp-2 cells treated 48 h previously with 4 ␮g/ml pcSMAD7 (membrane stripped and reprobed for ␤-actin (n ϭ 2); CON, protein extracts from non- transfected cells). The Journal of Immunology 4289

Propidium iodide-labeled nuclei were excited using the 633-nm laser and collected with a Cyto-red filter set (12). In vitro methylation assay Semiconfluent HEp-2 monolayers were transferred into serum-free medium lacking amino acids capable of donating methyl groups for 16 h. At this point, the protein synthesis inhibitors cycloheximide (40 ␮g/ml) and chloramphenicol (100 ␮g/ml; both from Sigma-Aldrich) were added 1 h before the tritiated methyl donor S-[methyl-3H]-adenosyl-L-methionine (10 FIGURE 6. Overexpression of SMAD7 prevents TGF-␤-induced ␮ Ci/ml; NEN Life Science Products), ensuring that radiolabeling was due ERK1/2 activation in HEp-2 cells. Basal expression of phosphorylated to methylation and not incorporation of the radiolabeled amino acid. Mono- (thus activated) ERK1/2 (pERK 1/2) MAPK is present in untreated control layers were stimulated with cytokines as indicated above, and whole-cell cells (CON) and those exposed to GenePorter II (GPII) only, and this is protein extracts were subjected to immunoprecipitation using anti-STAT1 ␤ Abs. Following SDS-PAGE, proteins were transferred onto polyvinylidene significantly increased 15 min after TGF- (1 ng/ml) stimulation. The ␤ difluoride membranes and exposed to Kodak BioMAX MS film in a Kodak TGF- -induced pERK 1/2 is not observed in protein extracts from cells LE autoradiography enhancement cassette. Exposure was allowed to pro- previously induced to overexpress SMAD7 by transfection with pcSMAD7 ceed for 1 wk at Ϫ70°C before development. (4 ␮g/ml) (upper panel). The lower panel is the upper membrane stripped and reprobed for total ERK1/2 to ensure equal protein loading (represen- Cell surface biotinylation tative of four separate experiments). Biotinylation of the HEp-2 surface proteins was conducted using EZ link Sulfo-NHS-LC biotin (Pierce) according to the manufacturer’s instruc-

tions. Brieﬂy, 48 h after transfection, monolayers were washed with ice- beled dsDNA probe (Fig. 2). Paralleling the reduced IRF-1 ex- Downloaded from cold PBS/Mg2ϩ/Ca2ϩ (1 mM MgCl and 0.1 mM CaCl in PBS) and 2 2 pression with TGF-␤ pretreatment, STAT1 DNA binding was re- subsequently incubated with biotin (0.75 mg/ml EZ link Sulfo-NHS-LC) for1honicewith gentle agitation. Following removal and quenching (100 duced in extracts from HEp-2, T84 (Fig. 2), or HT-29 cells (data mM glycine in PBS/Mg2ϩ/Ca2ϩ) of excess biotin, monolayers were not shown) pretreated for as little as 15 min with TGF-␤. Although ϩ ϩ washed three times with ice-cold PBS/Mg2 /Ca2 , and RIPA lysis buffer there was some variability in the degree of inhibition of STAT1 ␤ was added. Immunoprecipitation of the TGF RI was conducted as de- DNA binding, which likely reﬂects interpassage cell variability scribed above, followed by immunoblotting using an anti-biotin Ab (Pro-

␤ ␥ http://www.jimmunol.org/ mega). Membranes were stripped and reprobed with anti-TGF␤RI Ab to (e.g., degree of expression of TGF- or IFN- R), five separate ensure equal loading (29). experiments showed that a 15- to 60-min pretreatment with TGF-␤ consistently and significantly reduced IFN-␥-induced STAT1 Data presentation DNA binding. In stark contrast, inhibition of STAT1 DNA binding Numerical data are presented as mean Ϯ SEM and were compared by by TGF-␤ was not observed in either THP-1 cells or PBMC drawn ANOVA, followed by Newman-Keuls statistical comparisons, where p Յ from three healthy volunteers (Fig. 2, D and E). Reduced STAT1 0.05 was set as the level of statistically significant difference. Images shown are, in the main, representative of at least three separate experiments, with n values being stipulated in the figure legends.

Results by guest on September 28, 2021 We have shown that enteric epithelia and THP-1 cells mobilize STAT1 in response to IFN-␥ (22). However, while direct effects of TGF-␤ on HEp-2, T84 and HT-29 have been shown, neither receptor expression nor SMAD protein mobilization have been presented. In initial experiments, immunoblotting for phosphorylated SMAD2 and immunolocalization of SMAD2 to the nucleus confirmed that HEp-2 cells stimulated with TGF-␤ (1 ng/ml, 15 min) mobilize SMAD2/3 (data not shown). TGF-␤ pretreatment prevents IFN-␥-induced STAT1-regulated gene expression To determine whether TGF-␤ would alter IFN-␥-driven STAT1- dependent gene expression, HEp-2 cells were pretreated with TGF-␤ (1 ng/ml: 15, 30, or 60 min) before IFN-␥ stimulation and assayed for IRF-1, CIITA, GBP-1, and iNOS mRNA expression (all STAT1-regulated genes). As shown in (Fig. 1, A–D), pretreatment with TGF-␤ attenuated the IFN-␥-induction of mRNA expression of all four genes in HEp-2 epithelia but not THP-1 macrophages (IFN-␥-induced IRF-1 mRNA expression in T84 epithelia was also reduced by TGF-␤ pretreatment (data not shown)). Corroborating these findings, IFN-␥-induced IRF-1 pro- FIGURE 7. Suppression of STAT1 DNA binding by TGF-␤ in HEp-2 tein expression was suppressed in TGF-␤-pretreated HEp-2 but not cells is independent of ERK1/2 MAPK. Representative EMSA demonstrat- THP-1 cells (Fig. 1E). ing that STAT1 DNA binding is not restored by inhibition of ERK MAPK activity by U0126 (5 ␮M) pretreatment of epithelia cotreated with TGF-␤ IFN-␥-induced STAT1 DNA binding is reduced by TGF-␤ (1 ng/ml, 15 or 30 min) ϩ IFN-␥ (20 ng/ml) (compare lanes 5 and 6 and pretreatment lanes 7 and 8 with lane 2)(n ϭ 3). Inset shows inhibition of TGF-␤- induced ERK1/2 phosphorylation by a 1-h pretreatment with 5–20 ␮M ␥ To determine whether the reduced IFN- -STAT1 driven expres- U0126 before cytokine stimulation (n ϭ 3; cc, cold competitor nonlabeled sion was due to decreased STAT1 DNA binding we used EMSA hSIE probe; NS, nonspecific band; fp, free probe; CON, extracts from analysis. Nuclear protein extracts from IFN-␥-treated (20 ng/ml, nontreated cells; DMSO is included with TGF-␤ only because it was used 30 min) HEp-2 cells showed specific binding to the hSIE radiola- to solubilize U0126). 4290 TGF␤-IFN␥ INTERACTIONS AT THE EPITHELIUM Downloaded from http://www.jimmunol.org/

FIGURE 8. Immunoprecitiations and reciprocal STAT1 and SMAD3 immunoblotting (IB) reveals no evidence of physical interaction between these transcription factors following TGF-␤ ϩ IFN-␥ treatment of HEp-2 epithelial cells. Cells were cotransfected with 2 ␮g/ml each of SMAD3-myc and STAT1-FLAG, 48 h before cytokine treatment, and subsequently, cells were pretreated with TGF-␤ (1 ng/ml) before IFN-␥ (20 ng/ml, 30 min) exposure and protein extraction. A, Protein extracts were immunoprecipitated (IP) using an anti-FLAG Ab followed by IB for either the myc or FLAG tag. The upper panel reveals that myc (i.e., the SMAD3 surrogate) was not detected in any of the cellular extracts that had undergone IP with the anti-FLAG Ab, whereas FLAG, as expected, was found in all the transfected cells (middle panel). Assessment of whole-cell extracts (WCE (i.e., no IP step)) revealed that both tagged proteins were expressed in the transfected cells (lower panel) (CON, nontreated cells; GPII, GenePorter II the transfection reagent; pd, plasmid only). B repeats this experiment but assesses endogenous STAT1 and SMAD2/3. Whole-cell protein extracts were prepared from HEp-2 epithelia treated with by guest on September 28, 2021 TGF-␤ (1 ng/ml, 10–60 min) before IFN-␥ (20 ng/ml, 30 min) stimulation. One milligram of protein was subjected to IP with anti-STAT1 (B), and another 1 mg was IP with anti-SMAD2/3 (C). IB was then conducted for SMAD2/3 or STAT1, respectively. SMAD2/3 or STAT1 was not observed in the STAT1 IP or SMAD2/3 IP, respectively (upper panels in B and C), indicating these proteins do not interact. Stripping and reprobing the membrane revealed STAT1 and SMAD2/3 in the extracts that had undergone IP with anti-STAT1 and anti-SMAD2/3 Abs, respectively (IgG, are protein samples from an IP with an irrelevant isotype matched Ab; ϩve CON, protein samples from IFN-␥- or TGF-␤-treated Hep-2 cells known to contain STAT1 and SMAD2/3 respectively, and so acts as positive controls for detecting authentic STAT1 and SMAD2/3 on the immunoblots; images are representative of two or three experiments). binding was not due to TGF-␤-elicited SMAD2/3 binding to the was increased ϳ2-fold by IFN-␥ (20 ng/ml, 30 min), this was not hSIE probe as indicated by the complete absence of a signal in altered by TGF-␤ pretreatment (n ϭ 3; data not shown). epithelial extracts from cells treated with TGF-␤ only. This sug- Confocal microscopy revealed that IFN-␥-induced STAT1 nu- gests that the effect TGF-␤ is not at the promoter but is affecting clear translocation (Fig. 4, IFN-␥, arrowheads) was unaffected by the ability of STAT1 to bind DNA directly. In addition, the data pretreatment with TGF-␤ and reciprocally that the SMAD2/3 nu- indicate that this TGF-␤ inhibition of STAT1 DNA binding is not clear localization induced by TGF-␤ (Fig. 4, TGF-␤, arrows) was a global response and may be speciﬁc and aligned with the func- not blocked by subsequent IFN-␥ exposure. tion of specialized cell types. Overexpression of the inhibitory SMAD7 protein restores IFN-␥-induced STAT1 phosphorylation and nuclear localization IFN-␥-induced STAT1 DNA binding ␤ are not altered by TGF- pretreatment Many TGF-␤ signal transduction events are mediated by the mobili- Inhibition of STAT1 DNA binding in nuclear protein extracts from zation of proteins unique to the TGF-family of cytokines, designated TGF-␤ plus IFN-␥-treated cells could be due to reduced STAT1 as SMAD proteins. To determine the contribution of this pathway to phosphorylation (a critical modiﬁcation to allow maximal nuclear the inhibition of STAT1 signaling, HEp-2 cells were transiently trans- import and DNA binding (30)) or impaired nuclear import in gen- fected with a plasmid (pcSMAD7) encoding the inhibitory SMAD7 eral. Immunoblot analyses revealed that the levels of total STAT1 protein. Transfection of HEp-2 epithelial cells with 4 ␮g/ml and IFN-␥-induced STAT1 Tyr701 phosphorylation in epithelia pcSMAD7 for 48 h resulted in negligible cytotoxicity and increased and THP1 cells were unaffected by a 5- to 60-min pretreatment SMAD7 protein expression (Fig. 5, inset). HEp-2 cells in which with TGF-␤ (Fig. 3). SMAD7 was overexpressed were insensitive to TGF-␤ inhibition of STAT1 phosphorylation of Ser727 may be critical for optimal IFN-␥-induced STAT1 DNA binding; indeed, SMAD7 overexpres- transcriptional activity (31). Consistent with other studies, we ob- sion restored IFN-␥-induced STAT1 DNA binding in TGF-␤-pre- served constitutive Ser727 phosphorylation of HEp-2 STAT1, even treated cells (rightmost lane in Fig. 5; n ϭ 3). Increased SMAD7 after an 18-h serum starvation, and while Ser727 phosphorylation expression can evoke TGF␤RI internalization and degradation (32). The Journal of Immunology 4291

FIGURE 9. Pretreatment with TGF-␤ (1 ng/ml) before IFN-␥ (20 ng/ml, 30 min) increases PIAS1-STAT1 interaction in Hep-2 epithelia (A) but not THP-1 macrophage-like cells (B). Cells were treated as indicated Downloaded from above the blots, and protein extracts were immunoprecipitated (IP) with anti-PIAS1 or anti-STAT1 Abs, followed by immunoblotting (IB) for STAT1 (upper panels) or PIAS1 (middle panels), respectively. Critically, cytokine treatment during this time did not alter the expression levels of either total STAT1 or PIAS1 in

whole-cell extracts (WCE) (lower panels) (IgG, repre- http://www.jimmunol.org/ sents samples that were IP with an irrelevant isotype- matched Ab that did not capture either STAT1 or PIAS1, thus conﬁrming the speciﬁcity of the data). Im- ages are representative of three experiments. by guest on September 28, 2021

TGF␤RI internalization induced by pcSMAD7 was not observed by the ability of TGF-␤ to inhibit IFN-␥-induced STAT1 DNA binding neither confocal microscopy (analysis of five randomly selected fields was assessed in the presence of U0126, a pharmacological inhibitor of of view on three separate epithelial preparations/condition) nor by MEK1/2, the upstream activating kinase of ERK1/2. Although U0126 cell surface biotinylation followed by immunoprecipitation of the (1 h pretreatment; 5 ␮M (dose determined empirically; Fig. 7 inset)) TGF␤RI and antibiotin immunoblotting (n ϭ 3, data not shown). prevented TGF-␤-induced ERK1/2 activation, this ERK inhibition In addition to SMAD signaling, stimulation with TGF-␤ acti- failed to restore STAT1 DNA binding in TGF-␤ plus IFN-␥-treated vates the MAPK ERK1/2, and the stress activated kinases p38 and cells (Fig. 7). This demonstrates that TGF-␤ inhibition of STAT1- JNK. Overexpression of SMAD7 was not a specific antagonist of mediated signaling is independent of TGF-␤-mobilized ERK1/2. SMAD-mediated signaling as evidenced by the prevention of Attenuation of STAT1 signaling mediated by TGF-␤ was not TGF-␤-elicited ERK1/2 phosphorylation (Fig. 6, n ϭ 4). dependent on SAPK activity as stimulation of HEp-2 epithelial cells with TGF-␤ (1 ng/ml; 5–90 min) failed to activate, as gauged ␤ Inhibition of STAT1 signal transduction by TGF- is by phosphorylation on immunoblots, either p38 or JNK (data not independent of MAPK/stress-activated protein kinase (SAPK) shown; n ϭ 6 and n ϭ 3, respectively). activity ␤ Phosphorylation of ERK1/2 MAPK was apparent after TGF-␤ expo- TGF- mobilized SMAD2/3 does not physically interact with ␥ sure (Figs. 6 and 7 inset), allowing for the possibility that ERK1/2 IFN- -induced STAT1 signaling could participate in the TGF-␤-induced inhibition of IFN- The colocalization of SMAD2/3 and STAT1 in the nucleus of ␥-STAT1 gene transcription in epithelial cells. To test this hypothesis, TGF-␤ plus IFN-␥-treated cells (Fig. 4) suggested the possibility 4292 TGF␤-IFN␥ INTERACTIONS AT THE EPITHELIUM

FIGURE 10. TGF-␤ treatment (1 ng/ml) promotes the ability of HEp-2 cell-derived PIAS to bind FLAG-tagged STAT1 in vitro. A shows that PIAS1 immunoprecipitated from control (CON, noncytokine-treated cells) or IFN-␥-treated (20 ng/ml, 30 min) cells has a limited ability to bind to FLAG-tagged STAT1 in protein extracts from transfected cells and that this is signiﬁcantly enhanced in cells pretreated with TGF-␤. Lower panel in A indicates approximately equal loading of PIAS1 in each lane as determined by stripping the upper blot and reprobing with PIAS1 Ab. B, Immunoblots of whole-cell Downloaded from extracts (WCE) conﬁrming that only the transfected cells express FLAG-STAT1 and that total PIAS1 is not increased by exposure to IFN-␥ (IgG, extracts obtained by IP with an irrelevant isotype-matched Ab; blots are representative of three separate experiments).

that the proximity of the transcription factors could allow for a data to support STAT1 methylation in IFN-␥-treated HEp-2 epi-

physical interaction and thus reduced STAT1 DNA binding: it has thelial cells. http://www.jimmunol.org/ been reported recently that SMAD2/3 and STAT1 may physically interact (33). We assessed whether SMAD2/3 activation would Sequestering of STAT1 by PIAS1 is enhanced by pretreatment ␤ result in sequestering of STAT1, directly preventing DNA binding. with TGF- To determine whether sequestering was occurring, coimmuno- Sequestering of phosphorylated STAT1 in the nucleus, thereby precipitation of epitope-tagged STAT1 and SMAD2 or SMAD3 preventing DNA binding and resultant gene transcription, can oc- was used. In this manner, cells were transfected with 2 ␮g/ml cur through the PIAS1 protein. To assess whether sequestering of full-length STAT1-FLAG and either SMAD2-cmyc or SMAD3- STAT1 by PIAS1 was enhanced by TGF-␤, coimmunoprecipita- cmyc. Whole-cell protein lysates from IFN-␥ plus TGF-␤-treated tion of endogenous proteins were conducted for epithelia (HEp-2 cells were divided in two, followed by anti-FLAG or anti-c-myc cells) and macrophages (THP-1) treated with TGF-␤ with or with- by guest on September 28, 2021 immunoprecipitation, electrophoresis, and immunoblotting for the out IFN-␥. Whole-cell protein lysates from IFN-␥ with or without reciprocal epitope tag. With this approach, we observed no evi- TGF-␤-treated cells were divided in two, followed by anti-STAT1 dence of a physical interaction between STAT1-FLAG and or anti-PIAS1 immunoprecipitation and immunoblotting for the SMAD2-myc (data not shown) or SMAD3-myc (Fig. 8A). It is reciprocal protein. Using this approach, and in accordance with possible that the epitope tags may have prevented the STAT1 and other reports (36), we observed a low level of interaction of SMAD2 or SMAD3 interaction. However, immunoprecipitation of STAT1-PIAS in IFN-␥-treated epithelial cells and constitutive in- endogenous STAT1, followed by immunoblotting for SMAD2 or teraction in THP-1 cells. Moreover, enhancement of STAT1-PIAS SMAD3, or the reciprocal approach (i.e., immunoprecipitation for interaction by TGF-␤ alone was restricted to HEp-2 cells (Fig. 9A, SMAD 2/3 and immunoblotting for STAT1), revealed no evidence upper panel), and this association was even more evident when of physical interaction between the endogenous proteins (Fig. 8B). protein extracts from TGF-␤ plus IFN-␥-treated HEp-2 cells were assessed (Fig. 9A, n ϭ 3). TGF-␤ promotion of STAT1-PIAS1 ␤ TGF- does not affect STAT1 methylation interaction was not apparent in THP-1 cells (Fig. 9, n ϭ 3). Crit- Methylation of arginine residue 31 of STAT1 may inhibit DNA ically, increased STAT1-PIAS1 binding was not due to TGF-␤- binding and gene transcription events by enhancing PIAS1-medi- induced production of PIAS1 because immunoblotting conducted ated sequestering of STAT1 (28). However, recent publications on whole-cell extracts from the immunoprecipitated experiments have questioned this postulate, demonstrating that STAT1 is not revealed equivalent PIAS1 expression in TGF-␤ with or without methylated in response to IFN-␥ (34, 35). Nevertheless, assessing IFN-␥-treated cells (Fig. 9, bottom panels). the possibility that STAT1 methylation could contribute to gene In addition, we performed complementary in vitro-binding ex- regulation in epithelia, HEp-2 cells were treated with IFN-␥ with periments, in which PIAS1 was captured from protein extracts or without TGF-␤ in the presence of a 3H-radiolabeled methyl from HEp-2 cells treated with or without TGF-␤. Captured PIAS1 donor. Methylation was determined by immunoprecipitation of was mixed with equal amounts of protein extracts from STAT1- STAT1 followed by SDS-PAGE, transfer to polyvinylidene diﬂu- FLAG-expressing cells with or without IFN-␥ treatment (20 ng/ml, oride membranes, and autoradiography. Although an abundance of 30 min), followed by immunoblotting for FLAG. As shown in Fig. methylated proteins was detected in whole-cell extracts and in the 10A, PIAS1 retrieved from TGF-␤-treated epithelia showed an en- STAT1 immunoprecipitates, they were also observed in the control hanced interaction with FLAG (i.e., the tagged STAT1). This fur- precipitates obtained with an irrelevant isotype-matched Ab (data ther supports our contention that TGF-␤ promotes PIAS1-STAT1 not shown; n ϭ 3). This lack of methylated STAT1 was not a interaction. consequence of reduced STAT1 in the extracts because total To conclusively demonstrate that increased PIAS1 sequestering STAT1 was readily detected by immunoblotting the membranes of STAT1 induced by TGF-␤ was the mechanism through which used for the autoradiography (data not shown). Thus, we have no STAT1 DNA binding was reduced, a validated siRNA targeted The Journal of Immunology 4293

FIGURE 11. Silencing PIAS1 using siRNA restores IFN-␥-induced STAT1 DNA binding in TGF-␤-treated HEp-2 epithelia. Nucleofection of HEp-2 cells with 100 nM PIAS1 siRNA prevented the ability of TGF-␤ to attenuate IFN-␥-induced STAT1 DNA binding on EMSA. Cells were nucleofected 48 h before pretreatment with TGF-␤ (1 ng/ml), followed by stimulation with IFN-␥ (20 ng/ml, n ϭ 2) (CON, noncytokine- treated cells). Inset demonstrates that silencing of PIAS1 is dose dependent, with maximal silencing achieved with 100 nM PIAS1 siRNA (n ϭ 2, CON, control nucleofection without siRNA; fp, free probe; ␤-actin included as a protein loading control). Downloaded from http://www.jimmunol.org/ against PIAS1 was used. Specific knockdown of PIAS1 was ac- with IFN-␥-STAT1-driven gene transcription. A short pretreat- complished by using an empirically determined dose of prevali- ment with TGF-␤ significantly reduced STAT1 DNA binding and dated siRNA directed against PIAS1 (Fig. 11, inset). Nucleofec- transcriptional activity of four target genes evoked by a 20-fold tion of epithelia with 100 nM PIAS1 siRNA restored the ability of higher dose of IFN-␥ in model epithelia. This rapid inhibition of STAT1 to bind DNA in TGF-␤ plus IFN-␥-treated cells (Fig. 11). STAT1 signaling by TGF-␤ was not observed in macrophages. These data are not due to a direct response to the siRNA as evi- Divergence in TGF-␤ regulation of IFN-␥-STAT1 signaling in ep- denced by lack of STAT1 DNA binding in nonstimulated nucleo- ithelia and macrophages could be due to differences in expression, by guest on September 28, 2021 fected controls (data not shown). or affinity, of the cytokine receptors or the efficiency of downstream signals that transduce receptor-ligand interaction into tran- Discussion scriptional activity, aligning with the function of these cells. Given Immunopathology is due to perturbations in the control of the im- the role of TGF-␤ in oral tolerance, intestinal cells may be con- mune system that allows proinflammatory events to outweigh pro- tinually exposed to low levels of this cytokine, and this could be tective or reparatory processes. In this context, IFN-␥ and TGF-␤ coupled to rapid and efficient degradation of SMAD7, as recently are key cytokines: the former mobilizing and perpetuating immune suggested by Monteleone et al. (41). Thus, TGF-␤ inhibition of the and inflammatory responses, and the latter exerting counterbalanc- effects of proinflammatory stimuli, such as IFN-␥, that lead to loss ing immunosuppressive and immunoregulatory functions (3). of epithelial integrity and active participation in mucosal immunity Strategies are being deployed to treat disease by restoring this (e.g., IFN-␥ can increase epithelial MHC II expression (39)) would balance through blocking proinflammatory cytokines or delivering be beneficial. Alternatively, the macrophages’ role is to respond to anti-inflammatory cytokines (37). Moreover, cytokine production microbial pathogens, and this function is enhanced by IFN-␥. The does not occur in isolation: multiple cytokines are present at any ability of low-dose TGF-␤ (found constitutively in the gut) to given time, and defining the constituents of the tissue cytokine override this could be deleterious, preventing local containment of milieu and how these stimuli interact are significant challenges. bacteria that could lead to systemic infection, sepsis, and toxic Knowledge of the intracellular signaling cascades evoked by cy- shock. tokines could identify targets to either inhibit or promote cytokine Focusing on the epithelium and using HEp-2 as a model cell responses in a cell-specific manner (38). Mechanistic insight on line, we sought to determine the mechanism responsible for inhi- how IFN-␥ suppresses TGF-␤ signal transduction has been pre- bition of STAT1-mediated events by TGF-␤. Signal transduction sented (14), but despite numerous examples of TGF-␤ inhibi- through STAT1 is initiated by phosphorylation of Tyr701 by JAK2 tion of IFN-␥-driven events (13, 39, 40), little is known of and is a prerequisite for nuclear localization and DNA binding TGF-␤ modulation of intracellular signal transduction pathways (42). IFN-␥-evoked STAT1 Tyr701 phosphorylation was not al- evoked by IFN-␥. tered by TGF-␤, indicating that the TGF-␤ effect is downstream of Mucosal tissues are unique environments, whose dynamic mul- IFN-␥R-JAK-STAT1 interaction and is not due to enhancement of tifunctional nature is the net effect of the integrated responses of phosphatase activity. Indeed, data in favor of (43), and refuting many cell types: epithelium, fibroblasts, immune cells, and nerves. (44), TGF-␤-inhibition of IL-12-induced JAK and STAT1 tyrosine Epithelia and macrophages are important components of the innate phosphorylation in T cells have been reported. In light of normal immune system, whose activity influence subsequent adaptive im- STAT1 tyrosine phosphorylation in the TGF-␤ plus IFN-␥-treated munity. As such, both cell types are important targets for IFN-␥ epithelia, TGF-␤ could prevent nuclear import or additional and TGF-␤. We assessed if, and then how, TGF-␤ could interfere STAT1 modifications that are believed to maximize transcriptional 4294 TGF␤-IFN␥ INTERACTIONS AT THE EPITHELIUM activity such as serine phosphorylation (45, 46). However, reduced STAT1-PIAS1-SMAD complex since this would have been de- nuclear translocation was not diminished in TGF-␤ plus IFN-␥- tected in the coimmunoprecipitation assessment of STAT1 and treated epithelia, and IFN-␥ induced STAT1 Ser727 phosphoryla- SMAD2/3 interactions. tion that was not altered by TGF-␤ pretreatment. In addition, we Three separate lines of evidence point to TGF-␤ promotion of found no evidence of STAT1 methylation in our model system, PIAS-STAT1 interaction as a mechanism for TGF-␤ inhibition of and SUMOlation, that could affect transcriptional, also seems un- IFN-␥-evoked transcription: 1) coimmunoprecipitations show en- likely because a higher m.w. STAT1 should have been observed hanced detection of STAT1-PIAS1 in TGF-␤ plus IFN-␥-treated on immunoblot and/or EMSA analysis. cells; 2) in vitro binding assays show that PIAS1 obtained from TGF-␤ activates two major intracellular signal cascades, the TGF-␤-treated cells has an enhanced ability to bind STAT1 (in this unique SMAD2 and 3 proteins that bind with the regulatory instance FLAG-tagged STAT1); and, 3) siRNA directed against SMAD4 protein and move to the nucleus and the ubiquitous PIAS1 restored the STAT1 DNA1 binding activity in nuclear ex- MAPK (47, 48). Overexpression of the inhibitory SMAD protein, tracts from TGF-␤ plus IFN-␥-treated cells. Furthermore, this SMAD7, reduced the capacity of TGF-␤ to inhibit IFN-␥-induced PIAS1-STAT1 interaction is not due to new protein synthesis (or STAT1 DNA binding. Importantly, despite the precedent for a technical protein loading issue) and, as discussed above, is un- SMAD7 causing internalization and degradation of the TGF␤RI likely to be a consequence of TGF-␤ modification of STAT1. Thus (49, 50), there were no differences in the amount of cell surface our data fit best with a model in which TGF-␤, via a SMAD- TGF␤RI between SMAD7 overexpressing and control epithelia as dependent pathway, results in alterations to PIAS1 that promotes determined by confocal microscopy and biotinylation of cell sur- its interaction with STAT1 and inhibition of STAT1-directed tran- face proteins: this is in accordance with the recent suggestion that scription; the modification to PIAS1 needs to be determined. Downloaded from distinct endocytotic pathways control TGF␤RI turnover (51). In summary, TGF-␤ (low dose, short duration exposure) is a Thus, restoration of STAT1 DNA binding in epithelia with en- potent inhibitor of STAT1-mediated signal transduction in epithe- hanced SMAD7 expression is due to disrupted signaling down- lia, but not monocytes/macrophages, that is dependent on en- stream of the TGF-␤ receptor as opposed to simply a lack of the hanced STAT1 sequestering in the nucleus by preexisting PIAS1. TGF␤RI. We suggest that defining the exact mechanism of TGF-␤ interfer-

Inhibition of TGF-␤ signal transduction was not restricted to ence with IFN-␥-STAT1 signaling in epithelia will be important in http://www.jimmunol.org/ SMAD-dependant signaling, as SMAD7 overexpression also pre- understanding the complexity of cytokine cross-talk in mucosal vented TGF-␤ activation of ERK1/2 MAPK. Thus, we sought to compartments and as such will be relevant to oral tolerance, main- ascertain the involvement of MAPK/SAPK involvement in TGF-␤ tenance of the epithelial barrier and mucosal immunity in general. inhibition of IFN-␥-STAT1 signaling. Stimulation with TGF-␤ (1 ng/ml) activated ERK1/2 MAPK but was insufficient to activate Acknowledgments p38 or JNK in HEp-2 cells, and subsequent use of a selective We thank Jun Lu, Arthur Wang, and Cindy James for assistance with MEK1/2 inhibitor, U0126, failed to restore STAT1 DNA binding various aspects of this study. in IFN-␥ plus TGF-␤-pretreated cells. Taken together, these results demonstrate that the TGF-␤-evoked reductions in STAT1 DNA Disclosures by guest on September 28, 2021 binding and STAT1-dependent gene transcription are SAPK- and The authors have no financial conflict of interest. MAPK-independent events. The colocalization of SMAD2/3 and STAT1 in the nuclei of References TGF-␤ plus IFN-␥-treated cells, and a precedent for physical in- 1. Hooper, L. V., and J. I. Gordon. 2001. Commensal host-bacterial relationships in the gut. Science 292: 1115–1118. teraction between STAT and SMAD proteins (52), pointed to the 2. MacPherson, A. J., and N. L. Harris. 2004. Interactions between commensal possibility of a direct STAT1-SMAD2/3 association blocking intestinal bacteria and the immune system. Nat. Rev. Immunol. 4: 478–485. STAT1 DNA binding and subsequent transcriptional activity. 3. Strober, W., B. L. Kelsall, I. Fuss, T. Marth, B. R. Ludviksson, R. O. Ehrhardt, and M. Neurath. 1997. Reciprocal IFN-␥ and TGF-␤ responses regulate the oc- Testing this hypothesis via coimmunoprecipitation of transfected currence of mucosal inflammation. Immunol. Today 18: 61–64. epitope-tagged STAT1 and SMAD2/3 or endogenous STAT1 and 4. Ramana, C. V., M. P. Gil, Y. Han, R. M. Ransohoff, R. D. Schreiber, and SMAD2/3 proteins, we found no evidence in support of a physical G. R. Stark. 2001. Stat1-independent regulation of gene expression in response to IFN-␥. Proc. Natl. Acad. Sci. USA 98: 6674–6679. association between STAT1 and SMAD2/3. Although a reduction 5. Adams, R. B., S. M. Planchon, and J. K. Roche. 1993. IFN-␥ modulation of in STAT1-driven gene transcription could be the result of epithelial barrier function. J. Immunol. 150: 2356–2363. 6. Madara, J. L., and J. Stafford. 1989. Interferon-␥ directly affects barrier function SMAD2/3 binding with cofactors important in STAT1-mediated of cultured intestinal epithelial monolayers. J. Clin. 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