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1598.Full.Pdf 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 http://www.jimmunol.org/content/178/3/1598 Downloaded from References This article cites 60 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/178/3/1598.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 10, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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.
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