Dampening of IFN-γ-Inducible Gene Expression in Human Choriocarcinoma Cells Is Due to Phosphatase-Mediated Inhibition of the JAK/STAT-1 Pathway This information is current as of October 10, 2021. Jason C. Choi, Renae Holtz, Margaret G. Petroff, Nadia Alfaidy and Shawn P. Murphy J Immunol 2007; 178:1598-1607; ; doi: 10.4049/jimmunol.178.3.1598

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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

Dampening of IFN-␥-Inducible Gene Expression in Human Choriocarcinoma Cells Is Due to Phosphatase-Mediated Inhibition of the JAK/STAT-1 Pathway1

Jason C. Choi,* Renae Holtz,* Margaret G. Petroff,† Nadia Alfaidy,‡ and Shawn P. Murphy2§

Trophoblast cells (TBCs) form the blastocyst-derived component of the placenta and play essential roles in fetal maintenance. The proinflammatory cytokine IFN-␥ plays a central role in activating cellular immunity, controlling cell proliferation, and inducing apoptosis. IFN-␥ is secreted by uterine NK cells in the placenta during pregnancy and in mice is required for proper formation of the decidual layer and remodeling of the uterine vasculature. Despite the presence of IFN-␥ in the placenta, TBCs do not express either MHC class Ia or class II Ags, and are resistant to IFN-␥-mediated apoptosis. In this study, we demonstrate that IFN-␥- induced expression of multiple genes is significantly reduced in human trophoblast-derived choriocarcinoma cells relative to HeLa epithelial or fibroblast cells. These results prompted us to investigate the integrity of the JAK/STAT-1 pathway in these cells. Downloaded from Choriocarcinoma cells and HeLa cells express comparable levels of the IFN-␥ receptor. However, tyrosine phosphorylation of JAK-2 is compromised in IFN-␥-treated choriocarcinoma cells. Moreover, phosphorylation of STAT-1 at tyrosine 701 is sub- stantially reduced in both IFN-␥-treated human choriocarcinoma and primary TBCs compared with HeLa cells or primary foreskin fibroblasts. A corresponding reduction of both IFN regulatory factor 1 mRNA and protein expression was observed in IFN-␥-treated TBCs. Treatment of choriocarcinoma cells with the tyrosine phosphatase inhibitor pervanadate significantly en- hanced IFN-␥-inducible JAK and STAT-1 tyrosine phosphorylation and select IFN-␥-inducible gene expression. We propose that http://www.jimmunol.org/ phosphatase-mediated suppression of IFN-␥ signaling in TBCs contributes to fetal maintenance by inhibiting expression of genes that could be detrimental to successful pregnancy. The Journal of Immunology, 2007, 178: 1598–1607.

rophoblast cells (TBCs)3 form the blastocyst-derived producing a variety of soluble and membrane-bound immunosup- component of the placenta and perform multiple functions pressive molecules (2, 3). Human TBCs also express a unique T that are critical for successful pregnancy. In the placentas repertoire of MHC Ags on their cell surface (4). Extravillous TBCs of humans and rodents, the trophoblast layer forms a protective express the MHC class Ia Ag HLA-C, and the nonpolymorphic

barrier surrounding the developing embryo/fetus and containing MHC class Ib Ags HLA-E and HLA-G on their cell surface, by guest on October 10, 2021 the only cells derived from the blastocyst that are directly exposed whereas syncytiotrophoblast cells express only soluble HLA-G (1, to maternal blood (1). Thus, TBCs play a significant role in pre- 4). These MHC Ags are believed to regulate the activities of uter- venting immune rejection reactions against the developing semi- ine NK cells (1, 4). Lastly, the expression of MHC class Ia allogeneic conceptus by the maternal immune system, in part by (HLA-A and HLA-B) (5) and class II Ags (5, 6) is silenced in all human TBCs, both constitutively and in response to IFN-␥. The lack of these classical MHC Ags on human TBCs may be critical *Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263; †Department of Anatomy and Cell Biology, University of Kansas Medical Center, for preventing transplant rejection reactions against the semiallo- Kansas City, KS 66160; ‡Institut National de la Sante´et de la Recherche Me´di- geneic conceptus by the maternal immune system (3, 5). cale, Equipe Mixte Institut National de la Sante´ et de la Recherche Me´dicale The proinflammatory cytokine IFN-␥ plays important roles in 01-05, De´partement Re´ponse et Dyamique Cellulaires, Laboratoire d’Angiogene´se, Commissariat a‘ l’Energie Atomique et UniversiteЈ Joseph Fou- diverse cellular processes, including activating innate and adaptive rier, Grenoble, France; and §Departments of Obstetrics and Gynecology, Micro- immune responses against pathogens and tumors, inhibiting cell biology and Immunology, University of Rochester, Rochester, NY 14642 proliferation, and inducing apoptosis (7, 8). Activation of adaptive Received for publication August 17, 2006. Accepted for publication November 11, 2006. immune responses by IFN-␥ is in part due to transcriptional in- The costs of publication of this article were defrayed in part by the payment of page duction of genes encoding MHC class I and class II Ags, invariant charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. chain, HLA-DM/H2-DM, transporters associated with Ag process- 1 This work was supported by grants from the National Institutes of Health (R01 ing (TAPs) and the immunoproteasome subunits LMP-2, LMP-7, HD37464), the Roswell Park Cancer Institute Alliance, and the Roswell Park Cancer and LMP-10 (7). Induction of apoptosis and cell-cycle arrest oc- Center Support Grant (P30 CA 16056). J.C.C. was supported by National Cancer curs through activation of caspase and p21 gene expression, re- Institute Predoctoral Training Grant 55640201. spectively (7, 8). Interestingly, IFN-␥ has been detected in the 2 Address correspondence and reprint requests to Dr. Shawn P. Murphy, Departments of Obstetrics and Gynecology, and Microbiology and Immunology, University of placentas of several mammals, including humans and mice (9–16). Rochester, 601 Elmwood Avenue, Box 668, Rochester, NY 14642. E-mail address: In mice, placental IFN-␥ is secreted primarily by uterine NK cells [email protected] (17), and studies using mice deficient for IFN-␥, IFN-␥R1, or NK 3 Abbreviations used in this paper: TBC, trophoblast cell; GBP, guanylate-binding cells demonstrated that this cytokine is essential for the pregnancy- protein; IRF-1, IFN regulatory factor 1; USF-1, upstream stimulatory factor 1; cTBC, cytotrophoblast cell; WCE, whole cell extract; PTP, protein tyrosine phosphatase; PV, induced remodeling of the uterine vasculature and proper forma- pervanadate; PIAS, protein inhibitor of activated STAT; SOCS-1, suppressors of tion and maintenance of the decidual layer of the placenta during cytokine signaling 1; ROS, reactive oxygen species; TcPTP, T cell protein tyrosine pregnancy (17). Although IFN-␥ is present in the placenta, neither phosphatase; SHP, Src homology region 2 domain-containing phosphatase. human nor rodent TBCs express MHC class II genes, due to si- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 lencing of expression of the CIITA (18–20), the master regulator www.jimmunol.org The Journal of Immunology 1599

of constitutive and IFN-␥-inducible MHC class II gene transcrip- ranging from 100 to 1000 U/ml. Sodium orthovanadate (S6508 450243), tion (21, 22). Moreover, primary term human cytotrophoblast cells hydrogen peroxide (31642), and bovine liver catalase (C1345) were pur- (cTBCs) and human trophoblast-derived choriocarcinoma cells are chased from Sigma-Aldrich. resistant to apoptosis activated by IFN-␥ alone (23, 24). These Activation of orthovanadate and generation of PV properties of TBCs may be essential for the maintenance of suc- Activation of sodium orthovanadate was performed as described by Gordon cessful pregnancy. Despite these observations, several studies col- (36). PV solution (1 mM) was generated as described by Huyer et al. (37). ␥ ␮ lectively suggest that TBCs have the capacity to respond to IFN- . Excess H2O2 was removed by incubation for 5 min with 100 g/ml bovine Primary human TBCs and the choriocarcinoma cell lines Jar and liver catalase. The PV solution was used on cells at a final concentration of 100 ␮ JEG-3 express cell surface IFN-␥ receptors (IFN-␥R) (6, 15, 25). M within 5 min of generation. Furthermore, the expression of LMP-7, TAP-1, TAP-2, guanylate- Isolation and purification of human villous cTBCs binding protein (GBP), IFN regulatory factor 1 (IRF-1), and Cytotrophoblast cells were purified as previously described by Petroff et al. B7-H1 is up-regulated in both primary human TBCs and chorio- (27). In brief, ϳ40 g of term villous placental tissue were finely minced and carcinoma cell lines following exposure to IFN-␥ (19, 20, 26–29). subjected to three 30-min stages of digestion in a solution of trypsin and Taken together, these studies suggest that responses of TBCs to DNase. The resulting cell suspensions were layered over a discontinuous IFN-␥ may be selective. However, to date a comprehensive ex- 5–70% Percoll gradient (Sigma-Aldrich) and centrifuged. The cell layer ␥ located between the densities of 1.053 and 1.060 was collected and resus- amination of IFN- signal transduction has not been reported pended in culture medium (IMDM containing 10% FBS, 100 ␮g/ml strep- in TBCs. tomycin, and 100 U/ml penicillin), and further subjected to negative se- IFN-␥-mediated activation of Ag presentation gene expression, lection using anti-HLA class I Ab (clone W6/32; American Type Culture induction of apoptosis, and inhibition of cell proliferation are reg- Collection) coupled to magnetic microbeads (Miltenyi Biotec). The purity Downloaded from ulated by the JAK/STAT pathway (7, 8). Binding of IFN-␥ to its of the cTBCs used in these studies ranged from 96 to 99% based on im- munostaining with cytokeratin-7 Abs (clone OV-TL; DakoCytomation). cognate receptor results in activation of the receptor-associated kinases JAK-1 and JAK-2, which phosphorylate the intracellular RNA isolation and real-time RT-PCR ␥ domain of the IFN- R1 (7, 8). Monomers of the transcription fac- RNA was isolated using TRIzol (Invitrogen Life Technologies) as speci- tor STAT-1, which are localized in the cytoplasm, interact with the fied by the manufacturer. Reverse transcriptase reactions were performed phosphorylated IFN-␥R1 and are subsequently phosphorylated on on 2 ␮g of total RNA using Superscript II RT (Invitrogen Life Technol- tyrosine residue 701 by the JAKs (8, 30). Tyrosine phosphoryla- ogies) as described previously (19, 34). The primers and annealing tem- http://www.jimmunol.org/ peratures used for RT-PCR analysis were described previously and are as tion of STAT-1 leads to its homodimerization, translocation to the follows: CIITA (38), GBP (39), IRF-1 (18), LMP-2, LMP-7 (40), and nucleus, and transcriptional activation of multiple different genes GAPDH (26). Quantitative RT-PCR was performed in duplicate for that contain a IFN-␥-activating sequence in their promoters, such each sample on 1/100 of the total cDNA using an iCycler (Bio-Rad) as the gene encoding the transcription factor IRF-1 (7, 8). IRF-1 instrument and SYBR Green master mix (Bio-Rad) as directed by the can subsequently activate transcription of caspase genes involved manufacturer. Standard curves were generated using plasmids contain- ing the respective cDNAs for each gene examined. Expression of IFN- in initiating apoptosis, the p21 gene that inhibits cell growth, ␥-inducible genes was normalized to GAPDH gene expression, and data and/or the genes encoding MHC class Ia Ags, TAP-1, TAP-2, and are represented as the ratio of the relative cDNA copy number of each

the immunoproteasome subunits LMP-2, LMP-7 and LMP-10, all of these genes per copy number of GAPDH. The results were statisti- by guest on October 10, 2021 of which are required for effective immune responses to pathogens cally analyzed using Student’s t test. and tumors (7, 31). Furthermore, STAT-1 and IRF-1 cooperate FACS analysis of IFN-␥ receptors with the ubiquitously expressed transacting factor upstream stim- ϳ 6 ␥ HeLa, Jar, and JEG-3 cells ( 10 ) were harvested by trypsinization and ulatory factor 1 (USF-1) to activate transcription from the IFN- - stained for 30 min at 4°C with PE-conjugated Abs to human IFN-␥R1 inducible CIITA promoter IV (32, 33). CIITA subsequently acti- (GIR-94; BioLegend) or PE-conjugated human IFN-␥R2 (2HUB-159; vates transcription of the MHC class II genes (21, 22). BioLegend) at concentrations suggested by the manufacturer. Background In this report, quantitative RT-PCR was used to demonstrate that staining was established using isotype control Abs sc-2868 for IFN-␥R1 ␥ the expression of several IFN-␥-responsive genes is reduced in the and sc-2875 for IFN- R2. The cells were subsequently washed with 2% BSA/0.1% sodium azide in PBS and fixed with 2% paraformaldehyde di- human choriocarcinoma lines Jar and JEG-3 relative to HeLa ep- luted in PBS. Cell surface fluorescence was analyzed with a FACS-440 ithelial cells. The dampening of IFN-␥-inducible gene expression instrument (BD Biosciences) using CellQuest and WinMDI software. The in human choriocarcinoma cells correlates with compromised ty- maximum fluorescence intensity values were obtained using WinList ver- rosine phosphorylation of JAK-2 and STAT-1, and reduced ex- sion 5.0 (Verity Software House). pression of IRF-1. Impaired phosphorylation of STAT-1 and re- Western blot analysis duced IRF-1 expression were also observed in primary human Whole cell and nuclear extracts were prepared as previously described (41) cTBCs. Furthermore, treatment of choriocarcinoma cells with the using radioimmunoprecipitation assay buffer (1ϫ PBS, 1% Nonidet P-40, tyrosine phosphatase inhibitor pervanadate (PV) enhanced IFN-␥- 0.5% sodium deoxycholate, 150 mM NaCl, and 0.1% SDS) or lysis buffer inducible JAK and STAT-1 phosphorylation and IFN-␥-inducible composed of: 20 mM HEPES (pH 7.9); 420 mM NaCl; 1.5 mM MgCl2; 0.2 gene expression. Taken together, these studies suggest that IFN-␥ mM EDTA; 25% glycerol (v/v); 10 mM sodium molybdate; 1.0 mM DTT, 50 ␮g/ml pepstatin, 25 ␮g/ml aprotinin, 25 ␮g/ml leupeptin, and 1.0 mM signaling is inhibited in human TBCs by tyrosine phosphatase(s) PMSF. Protease inhibitors and DTT were added to the lysis buffer just that target the JAKs. This phenomenon may have important im- before use. Radioimmunoprecipitation buffer was used in the analyses of plications for successful mammalian pregnancy, as well as cho- primary cytotrophoblast and fibroblast cells. Abs against IRF-1 (sc-497), riocarcinoma tumor survival. IRF-2 (sc-498), OCT-1 (sc-232), STAT-1 (sc-417), phosphotyrosine 701- STAT-1 (sc-7988), phosphoserine 727-STAT-1 (sc-16570-R), and USF-1 (sc-229) were purchased from Santa Cruz Biotechnology. Protein extracts Materials and Methods (15–30 ␮g) were fractionated on 7–8% polyacrylamide gels and trans- Cell culture and reagents ferred to nitrocellulose membranes (Schleicher & Schuell Microscience) using a semiwet transfer apparatus (Bio-Rad). For detection of STAT-1, Jar and JEG-3 choriocarcinoma, HeLa cervical carcinoma, 2fTGH fibro- IRF-1, IRF-2, USF-1, OCT-1, and heat shock cognate 70 (HSC70), mem- sarcoma cells, and Jar/2fTGH and JEG-3/2fTGH stable hybrids were cul- branes were blocked overnight in 5% milk/PBS-0.075% Tween 20 and tured as previously described (34). BG-9 primary human foreskin fibro- incubated with primary Ab for1hatroom temperature using the following blasts were cultured as described by Lin et al. (35). Human IFN-␥ was Ab concentrations: IRF-1 (100 ng/ml for cell lines, 250 ng/ml for cTBCs), purchased from PBL Biomedical Laboratories and used at concentrations IRF-2 (1 ␮g/ml), STAT-1 (200 ng/ml), USF-1 (333 ng/ml); OCT-1 (250 1600 DAMPENED JAK/STAT SIGNALING IN CHORIOCARCINOMA CELLS

FIGURE 1. IFN-␥-inducible gene expression in hu- man choriocarcinoma cells. RNA was isolated from HeLa, Jar, and JEG-3 cells exposed for 24 h to 0, 500, or 1000 U/ml IFN-␥ and subjected to SYBR Green- based quantitative RT-PCR using primers specific for IRF-1, GBP, LMP-2, LMP-7, and GAPDH. Relative copy number from 20 ng RNA of each cell type was determined by generating a standard curve using known amounts of plasmids containing the respective cDNAs of the genes examined. The data are the average of three independent experiments and are represented as the ra- tio of the relative mRNA expression of each gene (i.e., Downloaded from IRF-1)vsGAPDH. Student’s t test was used to compare the relative levels of IFN-␥-inducible gene expression in HeLa vs Jar and JEG-3 cells that were exposed to the same concentrations of IFN-␥. http://www.jimmunol.org/ by guest on October 10, 2021 ng/ml), and HSC70 (1/20,000 monoclonal 3a3 ascites) (42). The mem- Results branes were washed four times with PBS-0.075% Tween 20 at room tem- IFN-␥-induced gene expression is reduced in Jar and JEG-3 perature, incubated with HRP-conjugated secondary Ab (100 ng/ml; Pro- mega) for 45 min, and washed five times with PBS-0.075% Tween 20. For choriocarcinoma cells compared with HeLa cells examination of phosphorylated proteins, membranes were blocked with 3% Previous studies demonstrated that the expression of a subset of BSA/TBS/0.1% Tween 20, incubated with primary phospho-specific Abs ␥ ␥ (phosphotyrosine 701-STAT-1 (200 ng/ml) or phosphoserine 727-STAT-1 IFN- -inducible genes is enhanced in TBCs in response to IFN- , (200 ng/ml)), washed with TBS/0.075% TBS, and incubated with second- but these studies did not precisely distinguish whether there were ary Ab as described above. Signals were detected using the SuperSignal quantitative differences in IFN-␥ induction of these genes in TBCs West Pico Chemiluminescent Substrate (Pierce) as described by the man- compared with other cell types, such as fibroblasts and epithelial ufacturer and subsequent exposure to Kodak Scientific Imaging film (Kodak). Relative levels of transcription factors were determined by dilu- cells. To address this possibility, RNA was isolated from Jar and tion analysis of the cell extracts, followed by Western blot analysis. Signals JEG-3 choriocarcinoma cells and HeLa epithelial cells cultured for were quantitated using a Molecular Dynamics Computing Densitometer 24 h in 0, 500, or 1000 U/ml IFN-␥ and subjected to quantitative model 300S. RT-PCR using primers for IRF-1, GBP, LMP-2 and LMP-7. Rel- ative levels of expression of these genes were normalized to JAK-1/2 immunoprecipitation GAPDH mRNA expression. Fig. 1 demonstrates that although ϳ ϫ 7 basal expression of IRF-1 and GBP mRNAs is comparable in HeLa, Jar, and JEG-3 cells ( 1.2 10 ) were treated with 1000 U/ml ␥ IFN-␥ for various times as indicated. Whole cell extracts (WCE) were HeLa, Jar, and JEG-3 cells, IFN- -induced expression of these isolated using lysis buffer composed of 20 mM Tris (pH 8.0), 150 mM genes is clearly significantly reduced in Jar and JEG-3 cells NaCl, 1% Nonidet P-40 (v/v), 50 mM NaF, 1 mM sodium orthovanadate, when compared with HeLa cells. Specifically, IRF-1 mRNA 50 ␮g/ml pepstatin, 50 ␮g/ml aprotinin, 50 ␮g/ml leupeptin, and 1 mM ␥ ␮ expression is 4.8-fold lower (500 U/ml IFN- ) to 6.7-fold lower PMSF. Aliquots (50 l) were removed from each sample before immuno- ␥ precipitation to use in Western blot analysis for assessment of STAT-1 (1000 U/ml IFN- ) in Jar vs HeLa cells and 4-fold lower in tyrosine phosphorylation. The extracts were precleared by overnight incu- JEG-3 cells. Similarly, GBP mRNA expression is reduced ϳ33- bation at 4°C with 30 ␮l of protein A-agarose beads (Upstate Biotechnol- fold and 8-fold in IFN-␥-treated Jar and JEG-3 cells vs HeLa cells, ogy), followed by immunoprecipitation with an isotype-matched Ab for respectively. Moreover, LMP-2 mRNA was expressed constitu- 1 h. Immunoprecipitations were performed by incubating extracts for 4–16 ␥ hat4oC with 1 ␮g of Abs to JAK-1 or JAK-2 followed by3hat4oC with tively in all three cell lines, but following exposure to IFN- was 30 ␮l of protein A-agarose beads. The immunoprecipitates were washed also lower in Jar (ϳ30-fold reduction) and JEG-3 (ϳ6-fold reduc- three times with lysis buffer, eluted from the beads, and subjected to West- tion) cells compared with HeLa cells (Fig. 1). Lastly, basal ex- ern blot analysis using Abs to JAK-1 (sc-277), JAK-2 (sc-294), phospho- pression of LMP-7 mRNA was substantially higher in HeLa cells tyrosine 4G10 (100 ng/ml; Upstate Biotechnology), or phospho-JAK-2- Y1007/Y1008 (sc-16566-R; Santa Cruz Biotechnology and 44-426G, 200 compared with Jar and JEG-3, and was enhanced following expo- ng/ml; BioSource International) as described above. sure to IFN-␥. LMP-7 mRNA expression was also up-regulated by The Journal of Immunology 1601 Downloaded from

FIGURE 2. IFN-␥ receptor expression on human choriocarcinoma cells. FACS analysis was performed on HeLa, Jar, and JEG-3 cells using PE-conjugated Abs to the IFN-␥R1 chain (A) and IFN-␥R2 chain (B). The open histograms represent staining with the IFN-␥R Abs and the filled http://www.jimmunol.org/ histograms represent staining with isotype-control Abs, respectively. The horizontal axes represent the maximum fluorescence intensity. The FACS analysis was repeated three times with similar results.

IFN-␥ in Jar and JEG-3 cells, but the absolute levels remained substantially lower than those in HeLa cells (Fig. 1). GAPDH mRNA was expressed at comparable levels in all three cell lines in by guest on October 10, 2021 the absence or presence of IFN-␥, demonstrating that the differ- ences in gene expression were not due to differences in RNA quan- FIGURE 3. JAK-1 and JAK-2 expression and phosphorylation in hu- titation or integrity (Fig. 1). Similar trends of gene expression were man choriocarcinoma cells. A, WCE were prepared from equal numbers of observed in two experiments using 100 U/ml IFN-␥ (data not HeLa, Jar, and JEG-3 cells cultured in 1000 U/ml IFN-␥ for 0, 0.25, 1, and shown). Taken together, these results indicate that although cho- 3 h and subjected to immunoprecipitation (IP) using Abs to JAK-1 or riocarcinoma cells clearly respond to IFN-␥ by up-regulating tran- JAK-2. Western blot (WB) analysis was subsequently performed on the scription of a number of genes, both the absolute levels of expres- immunoprecipitated products using Ab 4G10 to phosphotyrosine or Abs to JAK-1 and JAK-2. These experiments were repeated four times with sim- sion and the fold induction are significantly lower compared with ilar results. B, Densitometric analysis of the Western blots was performed HeLa epithelial cells. to quantify the differences in JAK phosphorylation among HeLa, Jar, and JEG-3 cells. The data are represented as the fold induction of JAK-1 and The IFN-␥R is expressed in human choriocarcinoma cells JAK-2 phosphorylation by IFN-␥ relative to untreated cells. Student’s t test One explanation for the relative dampening of IFN-␥-inducible was used to compare the fold induction of JAK-1 and JAK-2 phosphory- gene expression in Jar and JEG-3 cells compared with HeLa cells lation in HeLa vs Jar and JEG-3 cells at the corresponding time points. is that choriocarcinoma cells express reduced numbers of IFN-␥R. To address this possibility, Jar, JEG-3, and HeLa cells were sub- jected to flow cytometric analysis using Abs specific to the IFN- JAKs were assessed. WCE were prepared from equal numbers of ␥R1 and IFN-␥R2 chains, respectively. As shown in Fig. 2, cell HeLa, Jar, and JEG-3 cells cultured for 0, 0.25, 1, and3hin1000 surface expression of both the IFN-␥R1 and IFN-␥R2 chains was U/ml IFN-␥ and subjected to immunoprecipitation using Abs to detected on Jar and JEG-3 cells, at levels comparable to HeLa either JAK-1, JAK-2, or isotype-matched Abs. Immunoprecipi- cells. Therefore, reduced numbers of cell surface IFN-␥R on cho- tated products were subsequently subjected to Western blot anal- riocarcinoma cells do not account for the dampening of IFN-␥- ysis with Abs to JAK-1, JAK-2, P-Y1007/Y1008-JAK-2, or phos- inducible gene expression. photyrosine. As shown in Fig. 3A, JAK-1 is expressed in Jar and JEG-3 cells, but expression is 2.4-fold lower relative to HeLa cells, JAK phosphorylation is compromised in human whereas JAK-2 expression is comparable among the three lines. ␥ choriocarcinoma cells exposed to IFN- Tyrosine phosphorylation of JAK-1 was detected within 0.25 h and Binding of IFN-␥ to its receptor results in activation of JAK-1 and sustained for up to 3 h after exposure to IFN-␥ in all three cell lines JAK-2 through the phosphorylation of tyrosine 1022/1023 on (Fig. 3A). However, although we observed an average 10.6-fold JAK-1 and tyrosine 1007/1008 on JAK-2, respectively (43, 44). To increase of phosphorylated JAK-1 in HeLa cells at all time points determine whether this step in the JAK/STAT-1 pathway is intact following IFN-␥ treatment, only an average 2.5-fold increase was in choriocarcinoma cells, expression and phosphorylation of the detected in both Jar and JEG-3 cells exposed to IFN-␥ at the same 1602 DAMPENED JAK/STAT SIGNALING IN CHORIOCARCINOMA CELLS

FIGURE 4. Kinetic analysis of STAT-1 phosphorylation and IRF-1 ex- pression in human choriocarcinoma cells exposed to IFN-␥. WCE were isolated from HeLa, Jar, and JEG-3 cells cultured in the presence of 1000 U/ml IFN-␥ for 0, 1, 3, 6, 12, 16, and 24 h and subjected to Western blot analysis using Abs to STAT-1 (91 kDa), P-Y701-STAT-1, IRF-1 (50 kDa), IRF-2 (48 kDa), and USF-1 (43 kDa). The two bands detected by the P-Y701-STAT-1 Ab correspond to STAT-1␣ (p91) and STAT-1␤ (p84), respectively. Blots were stripped and reprobed with Abs to HSC70 as a control for protein extract integrity and quantity. Time-course analyses Downloaded from were performed a minimum of four times for each cell line, and the figure is representative of the data from all experiments.

FIGURE 5. Expression of STAT-1 and IRF-1 in primary term human time points (Fig. 3B). Phosphorylated JAK-1 was also observed in cTBCs and primary fibroblast cells. A, WCE were prepared from HeLa all three lines after 6-h IFN-␥ treatments (data not shown). Impor- cells and purified primary human cTBCs cultured in the presence or ab- sence of 1000 U/ml IFN-␥ for 0, 3, and 24 h and subjected to Western blot http://www.jimmunol.org/ tantly, the induction of JAK-2 phosphorylation was significantly 701 lower in Jar and JEG-3 cells (average, 1.3- and 1.5-fold increases, analysis for STAT-1, P-Y -STAT-1, and IRF-1 as described for Fig. 4. Human cTBCs isolated from a total of six different term placentas were respectively) vs HeLa cells (average, 10.9-fold increase) at all time ␥ used in the analyses. Representative data are shown. B, WCE were pre- points following exposure to IFN- (Fig. 3B), whether Abs to pared from 2fTGH, HeLa, and primary human foreskin fibroblast BG-9 1007 1008 phosphotyrosine (Fig. 3A)orP-Y /Y JAK-2 (data not cells cultured in the presence or absence of 1000 U/ml IFN-␥ for 0, 1, 3, shown) were used in Western blot analysis. Immunoprecipitations and/or 24 h and subjected to Western blot analysis for P-Y701-STAT-1 and with isotype-matched Abs demonstrated the specificity of the IRF-1 as described for Fig. 4. The analysis was performed on at least three JAK-1 and JAK-2 Abs (data not shown). These studies suggest independent preparations of WCE from each cell type, and the figure is representative of the data from all experiments. that phosphorylation and therefore activation of the JAKs, partic- by guest on October 10, 2021 ularly JAK-2, is compromised in human choriocarcinoma cells.

Tyrosine phosphorylation of STAT-1 is reduced in magnitude ␥ and duration in IFN- -treated human choriocarcinoma cells with HeLa cells), and they decreased further by 3 h and remained Transcriptional activation of genes regulated by the JAK/STAT-1 low thereafter. P-Ser727-STAT-1 was comparable, albeit slightly pathway requires phosphorylation of tyrosine residue 701 on reduced, in IFN-␥-treated Jar and JEG-3 cells relative to HeLa STAT-1 by the JAKs (8, 30). In addition, phosphorylation of cells (data not shown). serine 727 on STAT-1␣ plays a role in maximizing the transcrip- Accumulation of IRF-1 protein was detectable within1hin tional capacity of STAT-1 through interactions with histone HeLa cells, peaked at3h(ϳ10-fold induction relative to untreated acetyltransferases (45). The reduced levels of JAK phosphoryla- HeLa), and remained at maximal levels through 24 h (Fig. 4). tion in Jar and JEG-3 cells exposed to IFN-␥ prompted us to ex- Conversely, IRF-1 protein reached peak levels by3hinIFN-␥- amine the expression and phosphorylation status of STAT-1 in treated Jar and JEG-3 cells, but was markedly decreased within 6 h these cells. Because activation of STAT-1 in response to IFN-␥ (ϳ7-fold lower than HeLa cells), and remained low throughout the occurs very rapidly, and in many cell types is transient, particularly remainder of the time course. The maximal levels of IRF-1 protein at low concentrations of IFN-␥ (30), a kinetic analysis of STAT-1 detected in Jar and JEG-3 cells at 3 h after initiation of IFN-␥ tyrosine phosphorylation and IRF-1 protein expression was per- treatment were still ϳ2- to 4-fold lower compared with HeLa cells formed in IFN-␥-treated Jar and JEG-3 cells. WCE were isolated at the same time point. These results are consistent with the re- from HeLa, Jar, and JEG-3 cells treated with IFN-␥ for various duced levels of IRF-1 mRNA detected in IFN-␥-treated Jar and times from 1 to 24 h and subjected to Western blot analyses using JEG-3 cells vs HeLa cells (Fig. 1). Lastly, both the basal and Abs to STAT-1, tyrosine-phosphorylated STAT-1 (P-Y701-STAT- IFN-␥-induced expression of IRF-2 was dramatically lower in Jar 1), and IRF-1. Basal STAT-1 expression was comparable among or JEG-3 cells compared with HeLa cells at all time points (Fig. 4). the three cell lines (Fig. 4). Over the 24-h time course, total The levels of USF-1 and HSC70 were comparable at all time STAT-1 levels increased ϳ4–5-fold in HeLa cells treated with points in Jar and JEG-3 cells vs HeLa cells, indicating that the IFN-␥ as previously described (46). However, only a ϳ1.5- to differences in P-Y701-STAT-1, STAT-1, IRF-1, and IRF-2 expres- 2-fold increase of total STAT-1 was observed in IFN-␥-treated Jar sion were specific (Fig. 4). Similar results for P-Y701-STAT-1, and JEG-3 cells. Importantly, high levels of P-Y701-STAT-1 were IRF-1, and IRF-2 were obtained when cells were treated with 100 detected within1hinIFN-␥-treated HeLa cells, and the levels U/ml IFN-␥ (data not shown). Collectively, these studies indicate were sustained through 24 h (Fig. 4). In contrast, substantially that the magnitude and duration of STAT-1 Y701 phosphorylation reduced levels of P-Y701-STAT-1 were detected in Jar and JEG-3 and IRF-1 expression are significantly reduced in IFN-␥-treated cells treated with IFN-␥ for 1 h (at least ϳ7-fold lower compared Jar and JEG-3 cells compared with HeLa cells. The Journal of Immunology 1603

FIGURE 6. IFN-␥-inducible gene expression in sta- ble hybrids between human choriocarcinomas and fi- brosarcoma cells. Stable hybrids between Jar choriocar- cinoma cells and 2fTGH fibrosarcoma cells were cultured in the absence (Ϫ) or presence (ϩ) of 1000

U/ml IFN-␥ for 24 h and harvested for isolation of WCE Downloaded from and RNA. A, WCE were subjected to Western blot anal- ysis using Abs to STAT-1, P-Y701-STAT-1, IRF-1, IRF-2, OCT-1, and HSC70 as described for Fig. 4. B, RNA was subjected to quantitative RT-PCR using prim- ers for IRF-1, GBP, LMP-7, and GAPDH as described for Fig. 1. Western blot and RT-PCR analyses were per- formed on three independent preparations of WCE and http://www.jimmunol.org/ RNA, respectively, with similar results. Cl., Clone; MC, mixed clone. by guest on October 10, 2021

Tyrosine phosphorylation of STAT-1 in response to IFN-␥ is tained STAT-1 Y701 phosphorylation observed in HeLa cells is transient in purified primary human TBCs unique to this cell line and not reflective of a normal cell type. The transient phosphorylation of STAT-1 Y701 and relatively low However, high level, sustained STAT-1 phosphorylation was ob- levels of IRF-1 expression in Jar and JEG-3 cells exposed to IFN-␥ served in BG-9 primary human foreskin fibroblasts exposed to ␥ could be a reflection of: 1) the normal trophoblastic phenotype, 2) IFN- , and this correlated with high levels of IRF-1 expression the fact that the cells are transformed, or 3) adaptation to tissue (Fig. 5B). Similar results were observed with 2fTGH fibrosarcoma culture. To distinguish these possibilities, Western blot analysis was cells (Fig. 5B and see Fig. 6). Taken together, these studies used to examine STAT-1 phosphorylation and IRF-1 expression in strongly suggest that transient phosphorylation of STAT-1 and ␥ human villous cTBCs freshly isolated from term placentas. As ob- dampening of IRF-1 expression in response to IFN- are normal served in Jar and JEG-3 cells, basal STAT-1 expression was trophoblast phenotypes and not due to transformation or adaptation clearly detectable in human cTBCs, but was only very weakly to tissue culture. up-regulated following a 24-h IFN-␥ treatment (Fig. 5A). Impor- ␥ tantly, STAT-1 Y701 phosphorylation was very low in primary Dampening of IFN- -inducible gene expression in cTBCs treated for 3 h with IFN-␥ and decreased to basal levels by choriocarcinoma cells is recessive 24 h (Fig. 5A). Moreover, the levels of IRF-1 protein detected in We previously generated stable hybrids between 2fTGH fibrosar- IFN-␥-treated human villous cTBCs were significantly lower than coma cells (which express CIITA in response to IFN-␥) and Jar or in HeLa cells (Fig. 5A). IRF-2 expression was not detected in JEG-3 cells and demonstrated that silencing of IFN-␥-inducible WCE from two preparations of primary cTBCs (data not shown). CIITA gene transcription in human choriocarcinoma cells is ge- Comparable results were observed using 100 U/ml IFN-␥ (data not netically recessive (34). To determine whether the ability of the shown). An alternative explanation for our results is that the sus- stable hybrids to express CIITA in response to IFN-␥ correlates 1604 DAMPENED JAK/STAT SIGNALING IN CHORIOCARCINOMA CELLS

FIGURE 7. Effects of tyrosine phosphatase inhibi- tors on JAK and STAT-1 phosphorylation and IFN-␥- inducible gene expression in Jar cells. A, WCE were prepared from Jar cells cultured for 0, 0.25, 1, and 3 h with 1000 U/ml IFN-␥, 100 ␮M PV, or the combination and subjected to immunoprecipitation and Western blot analyses as described for Fig. 3. B, WCE were prepared from Jar choriocarcinoma cells cultured for 0, 3, and 16 h with 1000 U/ml IFN-␥, 100 ␮M PV, or the com- bination and subjected to Western blot analysis using Abs to STAT-1, P-Y701-STAT-1, IRF-1, USF-1, and

HSC70 as described for Fig. 4. C, RNA was isolated Downloaded from from Jar cells treated as in B and subjected to SYBR Green-based quantitative RT-PCR using primers for IRF-1, GBP, LMP-7, and GAPDH as described for Fig. 1. The data are the average of three independent exper- iments and are represented as the ratio of the mRNA expression of each gene (i.e., IRF-1, GBP)vsGAPDH.

Student’s t test was used to compare the levels of http://www.jimmunol.org/ mRNA expression in Jar cells treated with the combi- nation of IFN-␥/PV vs IFN-␥ alone. Tx, Treatment. by guest on October 10, 2021

with sustained tyrosine phosphorylation of STAT-1, Western blot Treatment of choriocarcinoma cells with the tyrosine analysis was performed on WCE prepared from 2fTGH clone 3, phosphatase inhibitor PV enhances tyrosine phosphorylation Jar clone 3, and Jar/2fTGH stable hybrids grown for 24 h in the of the JAKs and STAT-1 absence or presence of IFN-␥. As expected, tyrosine 701 phos- Our collective results suggest that dampening of IFN-␥-inducible phorylation of STAT-1 was observed in the IFN-␥-treated 2fTGH gene expression in human choriocarcinoma cells is due, at least in clone 3, but not Jar clone 3 (Fig. 6A). Sustained STAT-1 Y701 part, to compromised JAK tyrosine phosphorylation and the con- phosphorylation was also observed in all of the stable Jar/2fTGH sequential reduction in the level of activated STAT-1. Previous hybrids (Fig. 6A). In addition, IFN-␥-inducible IRF-1 expression studies show that JAK activity is negatively regulated in various in the stable hybrids was similar to that observed in 2fTGH clone cell types by at least two distinct mechanisms: 1) dephosphoryla- 3 and elevated relative to Jar clone 3 (Fig. 6A). Similar results were observed for both basal and IFN-␥-induced expression of IRF-2 in tion by protein tyrosine phosphatases (PTPs) (47, 48) or 2) inhi- the Jar/2fTGH hybrids (Fig. 6A). The differences in STAT-1 ty- bition of JAK phosphorylation and catalytic activity by suppres- rosine phosphorylation and IRF-1/IRF-2 expression between the sors of cytokine signaling 1 (SOCS-1) (48). To determine whether ␥ stable hybrids and Jar clone 3 were not due to variations in extract PTPs play a role in inhibiting JAK phosphorylation in IFN- - integrity or quantitation, because OCT-1, HSC70 (Fig. 6A), and treated choriocarcinoma cells, Jar cells were treated for 0, 0.25, 1, ␥ USF-1 (data not shown) expressions were all comparable. Lastly, and 3 h with IFN- alone and in combination with the tyrosine quantitative RT-PCR analyses demonstrated that, following expo- phosphatase inhibitor PV. Tyrosine phosphorylation of JAK-1 and sure to IFN-␥, expression of the IRF-1, GBP, and LMP-7 mRNAs JAK-2 was assessed by immunoprecipitation/Western blot analy- in the stable hybrids was similar to 2fTGH clone 3 and signifi- ses as described for Fig. 3. In IFN-␥-treated HeLa cells, we ob- cantly higher than Jar clone 3 (Fig. 6B). Identical results were served significant JAK-1 and JAK-2 phosphorylation at all time obtained in the analyses of stable hybrids between JEG-3 and points examined (Fig. 7A). As previously observed, phosphoryla- 2fTGH cells (data not shown). Collectively, these studies suggest tion of JAK-1 and JAK-2 was very low to undetectable at all time that transient phosphorylation of STAT-1 and the general damp- points in Jar cells exposed to IFN-␥ alone (Fig. 7A). In contrast to ening of IFN-␥-inducible gene expression is recessive in human previous studies on HeLa cells (49), treatment of Jar cells with PV choriocarcinoma cells. alone had only minor effects on JAK-1 phosphorylation, but no The Journal of Immunology 1605 apparent effect on JAK-2 (Fig. 7A). However, simultaneous treat- compromised in IFN-␥-treated Jar and JEG-3 cells, even at times ment of Jar cells with IFN-␥ and PV resulted in robust phosphor- as early as 0.25 h; 2) there is a substantial dampening of IFN-␥- ylation of JAK-1 and JAK-2 (average, 7.2- and 8.7-fold increases, inducible gene expression in the choriocarcinoma cell lines rela- respectively, relative to treatment with IFN-␥ alone) at all time tive to epithelial or fibroblast cells, even at high doses of IFN-␥; points examined (Fig. 7A). The enhancement of phosphorylated and 3) simultaneous treatment of Jar and JEG-3 cells with IFN-␥ JAKs in Jar cells exposed to IFN-␥/PV was not due to increases in and the PTP inhibitor PV results in sustained, high-level phos- total JAK-1 or JAK-2 protein expression (Fig. 7A). Jar cells were phorylation of the JAKs and STAT-1 and a significant enhance- subsequently treated with IFN-␥ and/or PV for 0, 3, and 16 h and ment of expression of multiple IFN-␥-inducible genes. harvested for isolation of WCE and RNA. Western blot analysis Although our data strongly support the concept that PTPs play demonstrated that the observed increase in phosphorylated JAKs a central role in inhibiting JAK phosphorylation in IFN-␥-treated 701 correlated with a significant enhancement of P-Y -STAT-1 and human choriocarcinoma cells, the precise molecular basis for the increased IRF-1 expression in Jar cells (Fig. 7B). IFN-␥/PV treat- PTP-mediated dampening of IFN-␥ responses in Jar and JEG-3 ment had little effect on either USF-1 or HSC70 expression, dem- cells vs HeLa and 2fTGH cells is currently unclear. The observa- onstrating that the effect of the treatment was selective (Fig. 7B). tion that JAK-1 and JAK-2 are only weakly phosphorylated even Consistent with the enhancement of IRF-1 protein, quantitative at early time points (0.25 h) of IFN-␥ treatment in choriocarcinoma ␥ RT-PCR demonstrated that the combined IFN- /PV treatment of cells suggests that the PTP(s) is constitutively active. Furthermore, Jar cells significantly increased the expression of IRF-1 mRNA the fact that high levels of STAT-1 phosphorylation and IFN-␥- (2.8- and 7.3-fold enhancements, respectively, at 3 and 16 h com- inducible gene expression are observed in stable hybrids of 2fTGH pared with IFN-␥ treatment alone; Fig. 7C). Similar enhancements fibrosarcoma and choriocarcinoma cells suggests that PTP-medi- Downloaded from of GBP (25.6-fold) and LMP-7 (11.8-fold) mRNA expression ated dampening of IFN-␥ responses is recessive in TBCs. One ␥ were observed in Jar cells exposed for 16 h to IFN- /PV relative mechanism to explain these results is that TBCs express a novel to IFN-␥ alone (Fig. 7C). Prolonged exposure of Jar cells to PV 701 and/or trophoblast-specific tyrosine phosphatase(s), the expression alone had little to no effect on phosphorylation of STAT-1 Y of which is extinguished in the stable hybrids. Alternatively, a ␥ (Fig. 7B) or IFN- -inducible gene expression (Fig. 7C). Lastly, ubiquitously expressed tyrosine phosphatase(s) may control JAK combined IFN-␥/PV treatment had no apparent effect on CIITA or phosphorylation in TBCs, but the activity of the PTP(s) may be http://www.jimmunol.org/ MHC class I mRNA expression (data not shown). Taken together, differentially regulated in TBCs vs HeLa and 2fTGH cells. Several these results strongly suggest that IFN-␥ signaling is inhibited in PTPs have previously been shown to attenuate IFN-␥ signaling by human choriocarcinoma cells by protein tyrosine phosphatase(s) dephosphorylating JAK-1 and/or JAK-2, and these include CD45, that prevent productive phosphorylation and, thus, activation of T cell protein tyrosine phosphatase (TcPTP), Src homology region the JAKs. 2 domain-containing phosphatase (SHP) 1, SHP-2, and PTP-1B Discussion (48). CD45 and SHP-1 are primarily expressed in cells of hemo- poietic origin and are therefore unlikely to inhibit IFN-␥ responses Despite the fact that the proinflammatory cytokine IFN-␥, which in human TBCs. In contrast, TcPTP, SHP-2, and PTP-1B may plays critical roles in regulating immune responses, apoptosis and by guest on October 10, 2021 potentially function in this capacity in TBCs, for they are all ubiq- cell proliferation, is produced in the human placenta during normal uitously expressed, and have all been detected in the placenta (51). pregnancy, a comprehensive and quantitative analysis of the IFN-␥ Interestingly, glucose levels, growth factors, and environmental signaling pathway in human TBCs has not been reported. In this study, we demonstrate that the expression of multiple IFN-␥-in- stress modulate the intracellular localization of SHP-2 and TcPTP ducible genes is significantly reduced in human choriocarcinoma (52–54), and therefore the specific target proteins subject to control cells relative to epithelial or fibroblast cells. These results suggest by these PTPs. Thus, differences in the intracellular localization of that there is a general dampening of IFN-␥ signal transduction in a ubiquitously expressed PTP in Jar and JEG-3 cells vs HeLa cells, choriocarcinoma cells. Consistent with this proposal, the IFN-␥R, 2fTGH cells, and Jar/2fTGH stable hybrids may account for the JAK-1, and JAK-2 are present in Jar and JEG-3 cells, but JAK differential effects of PTP activity on JAK phosphorylation in these phosphorylation, particularly JAK-2, is inhibited due to the activ- cells. Alternatively, the enzymatic activity of a ubiquitous PTP ity of PTPs. Furthermore, a corresponding inhibition of STAT-1 may be differentially regulated in TBCs vs epithelial or fibroblast Y701 phosphorylation and reduced IRF-1 expression are also ob- cells. Signaling through the BCR and in response to multiple cy- served in Jar and JEG-3 cells exposed to IFN-␥, as well as primary tokines and growth factors is attenuated by PTPs that are consti- human cTBCs isolated from term placentas. tutively active (55–57). In these cases, binding of ligand to recep- The regulation of cellular responses to IFN-␥ is very complex tor results in the generation of the reactive oxygen species (ROS) and is mediated by the equilibrium between the activities of the hydrogen peroxide, which transiently inactivates PTP activity by JAKs and STAT-1, and a number of negative regulatory mole- reversible oxidation of an invariant cysteine residue in the PTP cules, which include SOCS-1, protein inhibitor of activated STAT catalytic site (58). This results in amplification of the initial kinase (PIAS), and PTPs (48). SOCS-1 down-regulates IFN-␥ responses activation and generation of a robust and sustained signal (55–57). by interacting with JAK-2 and either inhibiting kinase activity Importantly, the concentration of the hydrogen peroxide produced and/or promoting JAK-2 degradation (48). PIAS1 directly blocks appears to regulate the magnitude and duration of the correspond- IFN-␥-inducible transcription by inhibiting STAT-1 DNA-binding ing signal (57). Furthermore, the receptors, kinases, and PTPs are activity, while PIASy inhibits STAT-1 transactivation by a mech- in close physical proximity, and this appears to control the spec- anism that has not yet been elucidated (48). PTPs fine tune or ificity of the cellular response to the receptor signal (57). Although attenuate responses to a wide array of cytokines and growth fac- a role for ROS-mediated inhibition of PTP activity has not been tors, including IFN-␥, by dephosphorylating JAK and/or STAT directly demonstrated in IFN-␥ signaling, two pieces of evidence family members (48, 50). However, rather than merely fine tuning, strongly support this possibility: 1) the catalytic sites of PTPs in- PTPs appear to play a key role in suppressing the initiation of volved in regulating cytokine signaling are highly conserved (58) IFN-␥ signaling in human choriocarcinoma cells, based on our and 2) PTPs previously shown to down-regulate IFN-␥ responses observations that: 1) phosphorylation of JAK-2 and STAT-1 is in some cell types are inactivated by ROS in response to other 1606 DAMPENED JAK/STAT SIGNALING IN CHORIOCARCINOMA CELLS stimuli (56, 57). Thus, in response to IFN-␥, ubiquitously ex- riocarcinoma cells may contribute to tumor survival for the same pressed PTP(s) may be inhibited by a ROS-mediated mechanism reasons. Future studies will address these hypotheses. in cells that display robust and sustained phosphorylation of JAKs and STAT-1, such as HeLa, 2fTGH, and Jar/2fTGH stable hybrids. Acknowledgments In contrast, robust activation of the JAKs may not occur in IFN- We thank Dr. Kailash Chadha for providing BG-9 primary human foreskin ␥-treated human TBCs due to insufficient inactivation of the neg- fibroblast cells. We are grateful to Drs. Willis Li, Edith Lord, and Richard ative regulatory PTP(s) by ROS. Future studies will address the K. Miller, and Kelly Cycon for critical reading of this manuscript. mechanisms involved in regulating PTP activity in Jar and JEG-3 cells. Disclosures 701 The demonstration that STAT-1 Y phosphorylation and The authors have no financial conflict of interest. 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