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Biology of Reproduction 60, 312-321 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Defective Induction of the Transcription Factor Interferon-StimulatedGene Factor-3 and Interferon {alpha} Insensitivity in Human Trophoblast Cells1

James C. Cross2,a,b, Shirley Lama, Simcha Yagel3,b, and Zena Werbb

a Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and the Departments of Molecular and Medical Genetics, and Obstetrics and Gynaecology, University of Toronto, Toronto, Ontario, Canada M5G 1X5 b Department of Anatomy, University of California, San Francisco, California 94143


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During pregnancy, trophoblast cells of the placenta contact maternal immune cells and yet are protected from attack. One mechanism that may account for this is that trophoblasts show altered expression of major histocompatibility complex (MHC) antigens. The gene for human leukocyte antigen G (HLA-G), a nonclassical gene, is expressed at high levels in trophoblast. Unlike other MHC class I genes, the HLA-G gene lacks an interferon (IFN) response element. Moreover, we demonstrate here that IFN, which regulates classical MHC class I genes in other cell types, does not affect these genes in trophoblast, owing to inactivation of an IFN{alpha} signaling pathway. Trophoblast cells (JEG-3 and JAR) were found to be selectively refractory to IFN. Specifically, although IFN{alpha} induced the transcription factors STAT1, STAT2, and IFN regulatory factor-1, and a protective response against encephalomyocarditis virus, it failed to protect the cells from vesicular stomatitis virus, activate a transfected MHC class I gene promoter, and induce the transcription factor IFN-stimulated gene factor (ISGF)-3. The lack of ISGF3 DNA-binding activity apparently was due to diminished p48/ISGF3{gamma} subunit activity since ISGF3 DNA-binding activity and IFN{alpha} induction of MHC class I promoter activity were reconstituted by p48/ISGF3{gamma} supplementation. These data indicate that a specific IFN signaling pathway is inactive in JEG-3 trophoblast cells because of altered activity of p48/ISGF3{gamma}, and they suggest IFN insensitivity as a mechanism that may help promote feto-placental survival.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Viruses and other pathogens often suppress the immune system in order to promote their own survival [1]. Similarly, normal immune functions are subverted during pregnancy in order to protect the conceptus from maternal immune attack [2]. The trophoblast component of the placenta lies in intimate contact with maternal blood and other bone marrow-derived cells that populate the uterus. Although it is of fetal origin and could potentially express paternal antigens, this "fetal allograft" is tolerated by the maternal immune system. The mechanisms that ensure survival of the graft remain mysterious, although several studies suggest that major histocompatibility complex (MHC) antigen expression in the placenta is atypical. Noninvasive trophoblast cells of the human placenta, which cover the floating chorionic villi (syncytiotrophoblast) and are bathed in maternal blood, express neither MHC class I nor class II antigens [3, 4]. However, the extravillous cytotrophoblast cells that deeply invade the uterine wall express class I antigens with unusual antigenic properties [5]. These antigens react with antibodies that recognize conserved framework determinants but not polymorphic regions of the molecule. This paradox was resolved with the discovery that extravillous cytotrophoblast cells express the nonclassical MHC class Ib molecule human leukocyte antigen G (HLA-G) [6, 7]. Like MHC class Ia molecules, the gene for HLA-G is found on chromosome 6 [8], but unlike the classical transplantation antigens HLA-A, B, and C, it is minimally polymorphic, and its expression is restricted to specific sites such as extravillous cytotrophoblast [6, 7, 9] and the eye [10], another immunologically protected site. HLA-G is also produced by BeWo [6, 11] and JEG-3 cells [12–14], which were derived from human trophoblast tumors (choriocarcinomas).

The fact that HLA-G is the predominant MHC class I molecule detectable on extravillous cytotrophoblast cells [7] implies that specific cellular mechanisms not only allow transcription of HLA-G but prevent transcription of HLA-A and -B. HLA-C may be expressed at low levels [15, 16]. The HLA-G promoter has not been characterized, but the regulation of the classical MHC class I promoters is well described. The promoters for these genes contain an enhancer [17] that binds proteins of the nuclear factor (NF)-{kappa}B/rel family [18] and an adjacent interferon (IFN)-stimulated response element (ISRE) [17, 19–21] that is similar to the ISREs found in other IFN-responsive genes [22]. IFN is expressed in the placenta of several mammalian species [23–26] and might, therefore, be expected to control MHC expression. However, experiments designed to test the effect of IFN on MHC class I expression in human trophoblast cells have yielded conflicting results [27–30].

Transcription of IFN-stimulated genes (ISGs) is controlled by transcription factors whose activities are regulated by IFN. IFN{alpha} binding to its receptor induces the rapid formation of three transcription factors called IRF-2 (also called ISGF1), IRF-1 (also called ISGF2), and ISGF3 [31,32]. Formation of IRF-1 and IRF-2 is inducible by both IFN{gamma} and IFN{alpha}, whereas activation of ISGF3 is dependent on IFN{alpha} [31, 32]. Thus, the induction of ISGF3 is a specific early marker of IFN{alpha} action. ISGF3 is composed of three proteins—p91/84 (STAT1), p113 (STAT2), and p48 (ISGF3{gamma})—which are thought to reside in the cytoplasm as latent transcription factors and are activated by the members of the JAK tyrosine kinase family after cytokine treatment [33]. Upon activation, the individual protein components are translocated to the nucleus, resulting in formation of ISGF3. In most cells, acute IFN treatment induces immediate transcriptional responses through activation of STATs. This is followed by synthesis of the factor IRF-1 and subsequent transcriptional activation. STAT-STAT dimers can activate transcription by binding to gamma-interferon activation site (GAS)-like elements such as those present in the IRF-1 promoter [34], whereas the STAT1-STAT2-ISGF3{gamma}-containing ISGF3 complex binds to a distinct element, called the ISRE [31, 35], present in the promoter of target genes such as MHC class I, ISG-15, ISG-54, and oligoadenylate synthetase (OAS). The importance of IFN-responsive transcription has been proven through analysis of mutant mice lacking elements of IFN signaling pathways. For example, IRF-1-deficient mice show reduced basal, as well as IFN-induced, transcription of MHC class I [36]. p48/ISGF3{gamma}-deficient cells showed diminished responses to type I and type II IFNs and an associated decrease in ISRE-binding activity [37].

JEG-3 choriocarcinoma cells share several features with primary cytotrophoblast cells [38], including expression of HLA-G. We demonstrate here that JEG-3 cells, like cytotrophoblasts in vivo [7], fail to express the classical MHC class I antigens and, therefore, are an appropriate model in which to study MHC regulation in cytotrophoblast cells. The objectives of our studies were to compare the transcriptional regulation of the classical MHC class I genes with that of the HLA-G gene and to address the role that IFNs play in controlling MHC class I expression in trophoblast cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Immunoprecipitations

The monoclonal antibody W6/32 reacts with all human class I HLAs and has been well described elsewhere [7,13, 29]. The HLA-G-specific mouse monoclonal antibody, recognizing a portion of the {alpha}1 domain, was previously described [39]. This antibody detects a surface antigen present on invasive, extravillous cytotrophoblast cells in the placenta and does not cross-react with other MHC class I antigens. Immunoprecipitations were performed on lysates made from [35S]methionine-labeled cells, as described previously [7].

Northern Blot Analysis

Total cell RNA extraction and Northern analyses using random primed cDNA probes were performed as previously described [40]. The human cDNA fragments used as probes were as follows: HLA-G-specific cDNA probe [39] was from Mike McMaster and Susan Fisher (University of California, San Francisco, CA); ß2-microglobulin [41] from John Chamberlain (Hospital for Sick Children, Toronto, ON, Canada); IRF-1 [42] from Tadatsugu Taniguchi (Osaka University, Osaka, Japan); p48/ISGF3{gamma} [43], ISG15, and ISG54 [44] from David Levy (New York University, New York City, NY); guanylate binding protein (GBP)-1 [45,46] from Thomas Decker (Vienna Biocenter, Vienna, Austria); and 2'-5' oligoadenylate synthetase (OAS) [47] from Bryan Williams (The Cleveland Clinic, Cleveland, OH).

Cells and Transfections

HeLa, JAR, and JEG-3 cells were grown in Dulbecco's Modified Eagle's medium (DMEM) supplemented with glutamine and 10% fetal bovine serum. Human IFN{alpha} (lymphoblastoid IFN), purchased from Sigma (St. Louis, MO), and human IFN{gamma}, a gift from Biogen (Cambridge, MA), were used as indicated in the figure legends. HeLa and JEG-3 cells were transfected by using the calcium phosphate precipitation method [48]. For transient transfections, cells were harvested 48 h after recovery from the transfection, and efficiencies were assessed by cotransfecting a Rous sarcoma virus-ß-galactosidase vector. The murine H-2Kb reporter constructs and their activity in human cells have been described [20, 21] and were provided by Michael Blanar (Bristol-Myers Squibb, Princeton, NJ). The p48/ISGF3{gamma} expression vector [43, 49] was from David Levy (New York University).

For biochemical analysis, cells were lysed in 250 mM Tris-HCl buffer (pH 8.0) by three freeze-thaw cycles. Luciferase activity was measured by using a kit purchased from Promega (Madison, WI) and a luminometer from Analytical Luminescence (San Diego, CA). ß-Galactosidase activity was measured by using a luminescence-based assay kit from Tropix (Bedford, MA). Chloramphenicol acetyltransferase (CAT) activity was measured by using the ethyl acetate extraction method [50]. All experiments were repeated at least three times, and raw data from a single representative experiment are presented. Differences among treatment means were analyzed statistically using the paired t-test or ANOVA.

Electrophoretic Mobility Shift Analysis (EMSA)

Whole-cell extracts [51], and nuclear and cytoplasmic extracts [52] were prepared as described previously. Binding assays were performed as described for the ISRE from ISG-15 [31, 32] and the GAS element from Ly-6A [53]. EMSA probes were synthesized as oligonucleotides, labeled with T4 polynucleotide kinase, and annealed. Recombinant p48/ISGF3{gamma} [43] and IRF-1 [42] were prepared by in vitro transcription and translation using the TnT kit from Promega.

Antiviral Assays

HeLa, JAR, and JEG-3 cells were seeded in wells of a 96-well plate (approximately 50 000 cells per well) containing serial dilutions of IFN{alpha}, incubated for 18–24 h, and then challenged with either vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV). Plates were incubated for an additional 24 h and washed, and viable cells were fixed with formaldehyde and stained with Giemsa stain.

Western Blot and Immunofluorescence Analysis

Protein nuclear extracts, separated by SDS-PAGE and blotted to nitrocellulose, were probed using antisera against STAT1 (p91/84) (Signal Transduction Laboratories), STAT2 (p113) (Santa Cruz Biotech), and p48/ISGF3{gamma} (Santa Cruz Biotech), and developed using the ECL kit from Amersham. On Western blots, the antisera detected single bands of the expected molecular weight. For some extracts, however, the p48/ISGF3{gamma} antiserum detected bands of other molecular weights on Western blots, although this was not consistent. It was otherwise specific because it detected a 48-kDa protein in cells that expressed the p48/ISGF3{gamma} mRNA; in the absence of primary antibody, or using unrelated primary antibody, the 48-kDa immunoreactive band was not observed; IFN induction that increased p48 mRNA levels also led to an increase in the abundance of the 48-kDa immunoreactive protein. For STAT2 and p48/ISGF3{gamma} immunostaining, JEG-3 cells were fixed with 4% paraformaldehyde for 15 min at 4°C. The primary antibody was diluted 1:200 in PBS/1% BSA and incubated for 1 h at room temperature. The secondary antibody was tetramethylrhodamine isothiocyanate (TRITC)-conjugated, anti-rabbit IgG (Sigma) used at a dilution of 1:100. Cell nuclei were visualized by staining with bisbenzamide (Sigma).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MHC Class I Expression in JEG-3 Cells Was Limited to HLA-G and Was Not Regulated by IFN

Immunoprecipitation experiments have suggested that HLA-G is the predominant MHC class I antigen expressed by primary cytotrophoblast cells in culture [7]. Similar experiments were performed to determine whether this was also true in the JEG-3 trophoblast cell line, and whether IFN affected MHC class I expression. [35S]Methionine-labeled HeLa and JEG-3 cells were left untreated or were primed for 14 h with IFN{gamma} and then treated for 48 h with IFN{alpha}. Proteins were immunoprecipitated with an HLA-G-specific antibody or an antibody (W6/32) that reacts with all MHC class I proteins, including HLA-G. Proteins of 40–43 kDa that reacted with W6/32, but not with the anti-HLA-G antibody (consistent with their being HLA-A, -B, or -C), were expressed constitutively in HeLa cells and were up-regulated by IFN treatment (Fig. 1). In contrast, these proteins were not detected in JEG-3 cells, even after IFN treatment. Two HLA-G-immunoreactive proteins of 29 and 36 kDa (Fig. 1), which may represent products of alternatively spliced mRNAs [10] and/or glycosylation variants, were expressed constitutively in JEG-3 cells but were not increased by IFN. These data indicate that HLA-G is the predominant, and perhaps only, MHC class I antigen produced by JEG-3 cells, but that its level is unaffected by IFN, unlike the classical HLA genes in other cell types. The failure of IFN to induce HLA-G protein expression was also associated with lack of mRNA induction. By Northern blot analysis, treatment of JEG-3 cells with either IFN{alpha} or IFN{gamma} failed to alter HLA-G mRNA levels (Fig. 2).



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  		FIG. 1. Immunoprecipitation of MHC class I antigens from untreated or IFN-treated cells. Cells were incubated as controls (-) or primed with 200 U/ml of IFN{gamma} for 14 h and then treated with 1000 U/ml of IFN{alpha} for 48 h (+). Cell lysates were incubated with control IgG (Con), W6/32 (Anti-Class I), or anti-HLA-G antibody, and immunoprecipitates were analyzed by SDS-PAGE and autoradiography. The bracket shows 40- to 43-kDa proteins induced by IFN in HeLa cells. The two arrows indicate HLA-G-specific bands present in JEG-3 but not HeLa cells.



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  		FIG. 2. RNA blot analysis of ISG expression in JEG-3 cells after IFN{alpha} or IFN{gamma} treatment. Total RNA was isolated from JEG-3 cells after IFN treatment (2000 U/ml) for 2 or 15 h. Replicate Northern blots containing 10 µg of RNA were probed for expression of HLA-G, ß2-microglobulin, IRF-1, p48/ISGF3{gamma}, GBP-1, ISG-15, ISG-54, and OAS, and were exposed to film for 24 h. Note that ISG-54 signals were undetectable even after prolonged exposure for several days.

Specific IFN{alpha}-Induced Signaling Pathways WereInactive in JEG-3 Cells

One reason for the failure of IFN to alter HLA-G expression may be that the HLA-G promoter differs from classical HLA gene promoters in lacking a conserved ISRE sequence [54]. However, the expression of ß2-microglobulin, a well known ISG, was also unaffected by IFN{alpha} treatment of JEG-3 cells (Fig. 2), suggesting a generally diminished IFN responsiveness. To test whether other ISGs were regulated by IFN in JEG-3 cells, we analyzed Northern blots with several other probes (Fig. 2). IRF-1 mRNA was rapidly induced by both IFN{alpha} and IFN{gamma} in JEG-3 cells. Exposure to IFN for several hours was associated with an increase in IRF-1 DNA binding activity (see Fig. 4; data not shown). In contrast, other ISGs showed unexpected responses in JEG-3 cells, compared with other cell types. IFN{alpha} was unable to induce ISG-54 expression, and ISG-15 and OAS mRNA expression levels were unaffected at 2 h after stimulation, although they were ultimately induced by IFN{alpha}, but only much later in the time course (Fig. 2). In contrast to these unusual blunted responses to IFN{alpha}, IFN{gamma} responses were apparently normal. IFN{gamma} acutely induced the expression of IRF-1, p48/ISGF3{gamma}, and GBP-1 (Fig. 2).



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  		FIG. 3. Choriocarcinoma cells were selectively resistant to the antiviral activity of IFN{alpha}. A) HeLa, JEG-3, and JAR cells were incubated as control cells (C), plated in the absence of IFN and then infected with VSV (-), or incubated in 5-fold dilutions of IFN{alpha} before VSV infection (one virus pfu per cell). B) HeLa and JEG-3 cells were treated with dilutions of IFN{alpha} and then challenged with EMCV at different concentrations as indicated. At the end of the assay, viable cells were stained with Giemsa. Details of the antiviral assay are described in the Materials and Methods.



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  		FIG. 4. IFN{alpha}-induced transcription is defective in JEG-3 cells. A) MHC class I transcription is not activated by IFN{alpha} in JEG-3 cells. HeLa and JEG-3 cells were transfected with the H-2KCAT reporter plasmid and left untreated (control, CON), treated with IFN{alpha} alone (2000 U/ml) or with IFN{gamma} alone (200 U/ml), or primed with IFN{gamma} for the first 14 h after which IFN{alpha} was added. Cells were harvested and assayed for CAT activity 62 h after the initial addition of IFN. Data represent mean relative CAT activity ± SE. Superscripts indicate significant differences (p < 0.05). B) EMSA analysis of ISGF3 binding activity. Nuclear extracts were prepared from untreated HeLa or JEG-3 cells (-) or cells treated with IFN{gamma} (200 U/ml) for 14 h and then with IFN{alpha} (10 000 U/ml) for 2 h (+). *ISGF3 complex; arrowhead, faster-migrating complex that comigrates with recombinant IRF-1, IRF-2, and p48/ISGF3{gamma}.

IFN{alpha} Induced Antiviral Response Against EMCV, but Not VSV, in JEG-3 and JAR Cells

To determine whether the atypical activation of ISGs had functional consequences, we tested cells for their ability to respond to IFN{alpha} in antiviral assays. HeLa and JEG-3 cells were treated for 18 h with IFN{alpha} and then challenged with VSV (1 plaque-forming unit [pfu] per cell). Whereas HeLa cells were protected from the cytopathic effects of the virus by a concentration of IFN{alpha} as low as 0.3 U/ml, JEG-3 cells were not protected by even 10 000 U/ml (Fig. 3A; Table 1). To determine whether this was a property only of JEG-3 cells, we tested the response of another human trophoblast cell line called JAR. These cells were also refractory to the protective effects of IFN{alpha} with respect to VSV cytopathic effects (Fig. 3A). We next tested whether the defect was virus-specific, since protection against different viruses can be dependent on separate IFN-stimulated transcription factors [55], by using EMCV as the challenge virus. IFN{alpha} protected both HeLa and JEG-3 cells against EMCV infection in a dose-dependent manner, although JEG-3 cells were approximately 10-fold less sensitive than HeLa cells (Fig. 3B; Table 1). These experiments clearly indicated that JEG-3 cells had functional receptors for IFN{alpha} and that certain IFN responses were induced but that a specific subset of IFN{alpha}-induced responses were defective in JEG-3 cells.


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  		TABLE 1. Antiviral activity of IFN-{alpha} against VSV and EMCV infection in HeLa and JEG-3 cells reported as the concentration of IFN-{alpha} (in U/ml) required to protect 50% of cells in a monolayer from the cytopathic effects of the virus.

IFN Could Not Induce Transcription of the Classical MHC Class I Genes in Choriocarcinoma Cells

IFN treatment of JEG-3 cells failed to induce the expression of genes for either HLA-G or the classical MHC class I antigens. This latter finding was surprising since these genes have ISREs in their promoters that mediate IFN responsiveness in other cell types. To determine whether IFN-induced transcription was interrupted in these cells, we tested the activity of a transfected MHC class I promoter from the murine H-2Kb gene, whose activity has been previously studied in human cells [20, 21]. In HeLa cells, priming with IFN{gamma} for 14 h followed by stimulation with IFN{alpha} increased the activity of the H-2K promoter to 3- to 4-fold that in controls (Fig. 4; p < 0.05); this was similar to the effects observed previously [20, 21]. In contrast, the H-2K promoter was not significantly stimulated by IFN treatment in JEG-3 cells. In some experiments, a statistically significant increase in H-2K promoter activity was detected in JEG-3 cells after treatment with IFN{gamma} alone, although this response was not observed when a point mutation that interrupts the binding of an IFN{gamma}-specific transcription factor [21] was introduced into the promoter (data not shown). These data indicate that IFN{alpha}-induced transcription was defective in JEG-3 cells.

IFN{alpha} Activated STAT1 and STAT2, but Not ISGF3 Complex Formation

We next sought to define the nature of the IFN signaling defect in JEG-3 cells. Because IFN{alpha} was able to induce IRF-1, we focused on the activation of ISGF3. Extracts prepared from IFN-treated cells were tested by EMSA analysis with an ISRE probe to determine whether IFN{alpha} induced ISGF3 complex formation in JEG-3 cells. Treatment of cells with IFN induces several ISRE-binding complexes [31, 32]. We found that treatment of HeLa cells with IFN induced a slower-migrating complex as well as a broad, faster-migrating complex (Fig. 4B). The relative migration of these bands was similar to that observed previously by others, who have characterized the faster-migrating bands as IRF-1, IRF-2, and/or p48/ISGF3{gamma}, whereas the characteristically slower-migrating (IFN{alpha}-specific) complex is ISGF3 [31, 32]. IFN treatment of JEG-3 cells increased the rapidly migrating binding activity (Fig. 4B, see arrowhead). In contrast, the slower-migrating band (ISGF3-like) activity was barely detectable in both JEG-3 cell cytoplasmic and nuclear extracts compared to HeLa cells. ISGF3-like complexes were apparent only after overexposure of films (Fig. 4B, see asterisk).

To investigate which specific components of the ISGF3 complex failed to be expressed or activated in JEG-3 cells, we first performed Western blot analysis to assess the expression and nuclear translocation of STAT1 and STAT2. Both STAT1 and STAT2 were readily detectable in JEG-3 cells and were exclusively detected in cytoplasmic extracts of untreated cells (data not shown). IFN{alpha} treatment resulted in the appearance of STAT1 and STAT2 immunoreactivity in nuclear extracts of JEG-3 cells (Fig. 5A) similarly to that in HeLa cells (data not shown). To confirm these results, cells were fixed and stained for STAT2 in order to observe intracellular localization. STAT2 immunoreactivity was apparent in the cytoplasm but was undetectable in the nuclei of untreated cells. After IFN{alpha} treatment, however, all cells showed nuclear accumulation of STAT2 (Fig. 5B). While STAT2 activation is specific to IFN{alpha}, STAT1 becomes tyrosine-phosphorylated in response to several cytokines, presumably due to the JAK1 kinase [33]. We found that activation of STAT1 was also normal in response to IFN{gamma} (Fig. 5A) and epidermal growth factor (data not shown) in JEG-3 cells. These data indicate that IFN{alpha} induces apparently normal nuclear translocation of the STAT components of ISGF3.



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  		FIG. 5. IFN{alpha} induced STAT1 and STAT2 nuclear translocation. A) Western blot analysis of STAT1 and STAT2 in nuclear extracts. JEG-3 cells were left untreated (-), or treated with IFN{alpha} (1000 U/ml) or IFN{gamma} (2000 U/ml) for 15 min and then harvested for preparation of nuclear extracts. Extracts were analyzed for nuclear content of STAT1 (p91) and STAT2 (p113) by Western blot. The arrows indicate the migration of the respective proteins. B) Immunofluorescent detection of STAT2 in untreated JEG-3 cells and cells treated with IFN{alpha} (2000 U/ml) for 10 min. Staining was performed as described in Materials and Methods. The "antibody control" frame indicates untreated cells stained with control normal rabbit serum. Cell nuclei were visualized by DNA staining (left) whereas STAT2 staining was visualized by immunofluorescence (right).

Low Levels of p48/ISGF3{gamma} Were Detectable in JEG-3 Nuclear Extracts

Because STAT1 and STAT2 appeared to be properly activated by IFN{alpha} in JEG-3 cells, one possible mechanism for the failure to induce ISGF3 would be reduced expression of the p48/ISGF3{gamma} component. However, JEG-3 cells contained readily detectable amounts of the p48/ISGF3{gamma} protein, although somewhat lower than in HeLa cells, as shown by Western blot analysis of whole cell extracts (Fig. 6A). In fractionated JEG-3 cells, compared to HeLa cells, the levels of p48/ISGF3{gamma} were lower in both cytoplasmic and nuclear extracts (Fig. 6B). On Western blots of fractionated HeLa cells, we noted that multiple immunoreactive bands were detectable: a faster-migrating band was consistently present in cytoplasmic extracts compared to nuclear extracts (Fig. 6B). The slower-migrating band could represent a phosphorylated form; although not reported for p48/ISGF3{gamma}, related proteins of the IRF family have been shown to be tyrosine-phosphorylated [56]. p48/ISGF3{gamma} expression is inducible by IFN [31, 32, 43], and therefore we tested whether IFN treatment could increase p48/ISGF3{gamma} content in JEG-3 cells. p48/ISGF3{gamma} mRNA was inducible by IFN{alpha} with slow kinetics but more rapidly by IFN{gamma} (Fig. 2). IFN{gamma} treatment for 16 h was associated with a significant increase in total cellular content of p48/ISGF3{gamma} protein in JEG-3 cells, similar to that in HeLa cells (Fig. 6B). Immunostaining of JEG-3 cells demonstrated that p48/ISGF3{gamma} was up-regulated after IFN{gamma} treatment, but only in nuclei of a small fraction of cells (Fig. 6C). Together these data indicate that while pretreatment of JEG-3 cells with IFN{gamma} can increase total cell levels of p48/ISGF3{gamma}, it clearly fails to render them IFN{alpha}-responsive (Fig. 4).



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  		FIG. 6. Expression of p48/ISGF3{gamma} protein. A) Western blot detection of p48/ISGF3{gamma} in whole cell extracts of HeLa and JEG-3 cells. The arrow indicates the 48-kDa immunoreactive band. The higher and faint lower-molecular weight cross-reacting bands were not consistently observed. B) Detection of p48/ISGF3{gamma} protein in fractionated cytoplasmic and nuclear extracts. Western blots were performed using 50 µg each of cytoplasmic (Cy) and nuclear (Nu) extract from untreated HeLa and JEG-3 cells, or those treated with IFN{gamma} for 16 h to increase expression levels. Panel represents results from two different experiments. C) Immunofluorescent detection of p48/ISGF3{gamma} in untreated JEG-3 cells and cells primed with IFN{gamma} (200 U/ml) for 16 h. Staining was performed as described in the Materials and Methods. The antibody control frame indicates untreated cells stained with control normal rabbit serum. Cell nuclei were visualized by DNA staining (left) whereas p48/ISGF3{gamma} staining was visualized by immunofluorescence (right). Note that after IFN{gamma} treatment, only a limited number of cells showed increased p48/ISGF3{gamma} nuclear staining (denoted by asterisks) whereas most cells showed no increase in nuclear expression (arrowheads).

We further assessed p48/ISGF3{gamma} expression by virtue of the fact that it is able to bind to ISRE sequences on its own. The complex migrates faster than the ISGF3 complex during EMSA [43]. An IFN{gamma}-inducible complex of this faster mobility was detected in both cytoplasmic and nuclear extracts from HeLa cells (Fig. 7A), and this activity comigrated with recombinant p48/ISGF3{gamma} (Fig. 7B). A comigrating complex was also induced by IFN{gamma} treatment in the cytoplasm of JEG-3 cells (Fig. 7A). However, it was undetectable in nuclear extracts (Fig. 7A). Because a complex of the appropriate mobility was not detected, we concluded that p48/ISGF3{gamma}-like binding activity was absent or at low levels in the nucleus of JEG-3 cells, consistent with the Western blotting results. To test whether JEG-3 cells contained an activity that prevented p48/ISGF3{gamma} binding activity, as has been reported in other cell types [57–59], we mixed recombinant p48/ISGF3{gamma} with JEG-3 nuclear extracts. Binding of both p48/ISGF3{gamma} and the related protein, IRF-1, were unaffected by preincubation with JEG-3 extracts (Fig. 7C).



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  		FIG. 7. p48/ISGF3{gamma} binding activity. A) ISRE binding activity present in nuclear (Nucl) and cytoplasmic (Cyto) extracts from untreated and IFN{gamma}-treated cells. Arrows indicate the migration of the p48/ISGF3{gamma} containing complex. B) ISRE-binding activity of recombinant p48/ISGF3{gamma} and IRF-1. Control reticulocyte lysate (-) or lysates programmed with p48/ISGF3{gamma} or IRF-1 mRNA were analyzed by EMSA with an ISRE probe. Note that the upper, nonspecific band is present in all lanes. C) Binding of in vitro-translated p48/ISGF3{gamma} and IRF-1 in the presence of HeLa and JEG-3 nuclear extracts. The in vitro-translated proteins were analyzed alone or after preincubation with nuclear extracts from HeLa or JEG-3 cells. In A and C, the origin of the lower band is unknown; it was not consistently observed.

ISGF3{gamma} Was Limiting to ISGF3 Complex Formation

Low levels of p48/ISGF3{gamma} protein and binding activity in the nucleus of control and IFN-treated JEG-3 cells suggested that it alone may limit formation of ISGF3. To test this idea, we took advantage of the fact that ISGF3 binding activity can be reconstituted in vitro by adding p48/ISGF3{gamma} to extracts containing activated STAT proteins [31, 32, 43]. We therefore mixed both cytoplasmic and nuclear extracts from IFN{alpha}-treated cells from JEG-3 cells, which contain activated STAT1 and STAT2 proteins, with recombinant p48/ISGF3{gamma}. Whereas the ISGF3 complex was not observed in control extracts derived from either the cytoplasm or nucleus, addition of p48/ISGF3{gamma} generated an ISGF3 binding complex in both (Fig. 8A). To determine whether supplementation with p48/ISGF3{gamma} could restore ISGF3 activity in vivo, and therefore restore ISGF3-mediated transcriptional activation, JEG-3 cells were transfected with an expression vector for p48/ISGF3{gamma} and tested for IFN{alpha}-inducibility of the H-2Kb promoter. As before, IFN{alpha} failed to induce promoter activity in control cells (Fig. 8B; fold induction = 1.17 ± 0.04, p > 0.1). Transfection with p48/ISGF3{gamma} alone did not increase promoter activity in untreated cells. However, together with increasing amounts of cotransfected p48/ISGF3{gamma}, IFN{alpha} was able to induce H-2K promoter activity 2- to 3-fold (Fig. 8B).



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  		FIG. 8. Addition of p48/ISGF3{gamma} restored ISGF3 activity. A) Addition of p48/ISGF3{gamma} to nuclear extracts from IFN{alpha}-treated JEG-3 cells generated an ISGF3 binding complex. Nuclear and cytoplasmic extracts from JEG-3 cells were analyzed for ISRE binding activity by EMSA. Cells were left untreated or were treated with IFN{alpha} for 20 min. Before analysis, extracts were mixed with control reticulocyte lysate or in vitro-translated p48/ISGF3{gamma}. Note that a p48/ISGF3{gamma} binding complex appears with addition of recombinant protein to all extracts. However, the ISGF3 complex was apparent only with addition of protein to extracts from IFN{alpha}-treated cells. B) Transfection of cells with p48/ISGF3{gamma} reconstituted the ability of IFN{alpha} to activate the H-2K promoter. JEG-3 cells were transfected with the H-2Kb-CAT reporter gene with or without a p48/ISGF3{gamma} expression vector. After 16 h, cells were treated with 4000 U/ml IFN{alpha} and harvested for CAT assay 24 h later. Data represent mean relative CAT activity ± SE. Different superscripts indicate significant differences (p < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we found that human trophoblast cells show diminished responses to IFN{alpha} including the inability of IFN to induce MHC class I expression. One surprising explanation for this finding is that IFN{alpha} signal transduction, which normally culminates via one signaling pathway in activation of the ISGF3 transcription factor, is impaired in these cells. In dissecting this pathway in detail, we found that while all components of the ISGF3 complex are expressed in JEG-3 trophoblast cells, their appearance and activation in the nucleus is abnormal. In particular, STAT1 and STAT2 are translocated to the nucleus after IFN treatment. However, the p48/ISGF3{gamma} component of ISGF3 was found at low levels in the nucleus, and transfection of cells with p48/ISGF3{gamma} restored IFN{alpha} responsiveness. These data suggest that signal transduction is interrupted because of specific steps in ISGF3 complex activation or formation. Consistent with the specificity of the effect, transcription of ISGF3-responsive genes was affected: ß2m and ISG-54 were not induced at all, and ISG-15 and OAS induction was delayed (inconsistent with the normal kinetics of ISGF3-stimulated responses) after IFN{alpha} treatment of JEG-3 cells. The overall significance of changes in gene expression for trophoblast cell function is unclear at present. However, we suggest that this defect in IFN signal transduction may be one mechanism, perhaps among others, for preventing the expression of the classical MHC class I genes in trophoblast cells. IFNs are constitutively expressed by several tissues, and notably in the placenta of several species including humans [23, 24]. The present data provide a plausible mechanism for excluding trophoblast cells as potential target cells for some of the effects of these IFNs. The importance of IFN signaling in regulating MHC class I expression in nonlymphoid cell types under normal physiological conditions has been demonstrated by the reduction in MHC class I expression in mice that lack STAT1 [60] and IRF-1 [36]. In U2A cells that lack p48/ISGF3{gamma}, IFN induction of MHC class I transcription is abolished [61], indicating that p48/ISGF3{gamma} is also essential.

Although JEG-3 cells lack one subset of responses to IFN{alpha}, our experiments indicated that they retained a response to IFN{gamma}. For example, H-2K promoter activity was increased and the transcription factors IRF-1 and p48/ISGF3{gamma} were induced by IFN{gamma} in JEG-3 cells. IFN{gamma} uses receptors and signal transduction pathways that are distinct from those used by type I IFN [22]. Receptors for IFN{gamma} are present on JEG-3 cells [62] as well as in normal placenta [63]. Nonetheless, in our experiments, overall MHC class I expression was unaffected by IFN (including IFN{alpha} and IFN{gamma}), indicating that the response of JEG-3 cells to IFN{gamma} is insufficient to induce HLA-A, -B, and -C genes. This conclusion is supported by others [13], although another report indicated that IFN{gamma} could induce MHC class I expression in JEG-3 cells [29]. Several reports have described the use of IFN{gamma} with human cytotrophoblast cells [27–30], but with conflicting results on MHC expression. These data suffer from the fact that cytotrophoblast preparations are often contaminated by nontrophoblast cells. The suggestion that MHC expression in trophoblast cells is insensitive to IFN is supported by in vivo experiments in mice that showed that IFN{gamma} is unable to enhance MHC class I or class II antigen expression in trophoblast cells [64].

Several IFN{alpha}-induced responses were suppressed in JEG-3 cells, including the induction of the classical transplantation antigens and induction of the antiviral state against VSV. These observations reflect a general property of cytotrophoblast cells and are not an effect in these trophoblast cell lines only, because IFN{alpha} fails to induce MHC class I expression in primary cultured cytotrophoblast cells [65]. The HCCM-5 and BeWo trophoblast cell lines were also reported to be refractory to the antiviral effect of IFNß towards VSV [66]. Type I IFNs ({alpha}, ß, {omega}) activate several independent signal transduction pathways downstream of a common receptor [22]. Activation of the transcription factor ISGF3 in response to IFN{alpha} occurs rapidly and results in translocation of ISGF3 into the nucleus [32]. Tyrosine phosphorylation is required for this activation [32, 67, 68], an event that is mediated by TYK2 and JAK1 [33]. The transcription factor IRF-1 appears to be a component of a second signal transduction pathway that is independent of ISGF3. Stimulation of IRF-1 activity by IFN is dependent on IRF-1 gene transcription and protein synthesis [35]. Transcriptional activation of IRF-1 expression in response to IFN is probably mediated by the activation of STAT1-containing transcription factors that bind GAS-like elements present in the IRF-1 gene promoter [34, 46, 69]. Gene-targeting experiments have shown that alternative antiviral pathways are required for protection against different viruses and specifically that IRF-1 is essential for mediating the antiviral effects of IFN against EMCV, but not VSV [70]. Our finding that JEG-3 cells could be protected by IFN against EMCV is similar to results in BeWo and HCCM-5 trophoblast cells [66] and suggests that IRF-1-dependent pathways are normally stimulated in trophoblast cells. In support of this, IFN{alpha} induction of IRF-1 mRNA and DNA-binding activity appeared normal in JEG-3 cells; because IFN-stimulated transcription of IRF-1 is mediated by STATs, it was therefore likely that STAT1/2 activation was normal. In contrast to stimulation of IRF-1, the pathway resulting in activation of ISGF3 was clearly nonfunctional in JEG-3 cells.

The major abnormality in IFN signaling observed in JEG-3 cells was the relative deficiency of p48/ISGF3{gamma} protein and/or activity, which correlated with the lack of ISGF3 DNA-binding activity. The p48/ISGF3{gamma} protein, while absolutely required for ISGF3 activity [37, 43, 71], has been previously described as a more passive participant in ISGF3 complex formation and nuclear translocation. Indeed, it is present in both cytoplasm and nuclear fractions in most cell types under resting conditions. However, in JEG-3 trophoblast cells, the relative absence of p48/ISGF3{gamma} activity is sufficient to explain the failure of ISGF3 formation. This hypothesis is supported by the findings that addition of recombinant p48/ISGF3{gamma} protein to extracts of IFN{alpha}-treated JEG-3 cells reconstituted ISGF3 binding activity, and also that transfection of cells with p48/ISGF3{gamma} reconstituted the ability of IFN{alpha} to activate an IFN-responsive promoter. Steady-state levels of p48/ISGF3{gamma} protein were indeed low in resting JEG-3 cells relative to the IFN-responsive HeLa cells used as controls. It was surprising, though, that while IFN{gamma} was able to increase p48/ISGF3{gamma} in JEG-3 cytoplasmic extracts to levels similar to HeLa cells, this still did not restore ISGF3-inducibility. This represents a significant difference between JEG-3 and HeLa cells. It seems likely that in JEG-3 cells, much higher levels of p48/ISGF3{gamma} protein, which could only achieved by transfection, are required to reconstitute signaling. We considered and tested two hypotheses to explain this difference in JEG-3 cells. First, it was apparent in some experiments that p48/ISGF3{gamma} was less abundant in nuclear compared to cytoplasmic extracts from JEG-3 cells, implying that nuclear accumulation may be regulated. However, this difference was apparent, albeit to a lesser extent, even in HeLa cells. The adenovirus E1A protein, which interferes with ISGF3 activation, significantly lowers total cellular levels of p48/ISGF3{gamma} protein [72, 73], an effect that appears different from the one observed here. Second, our ability to detect at least low levels of protein by Western blotting in JEG-3 nuclear extracts, compared to an apparent absence of its activity based on EMSA, suggested that binding activity rather than localization may be regulated. p48/ISGF3{gamma} is the target for inhibitors of DNA binding, such as ICSBP [58, 59] and a 19-kDa protein present in some cancer cell lines [57]. As described above, however, we have not found an activity with similar inhibitory properties in JEG-3 cells. Therefore, the precise mechanism interfering with p48/ISGF3{gamma} expression and/or activity remains elusive.

The screening of cell lines for mutations in IFN signal transduction pathways has significantly helped to unravel the complexity of cytokine signal transduction [33]. In addition, the study of several cell lines naturally resistant to IFN has implicated mechanisms by which pathogens or tumor cells may perturb normal signaling. For example, Epstein-Barr virus suppresses IFN{alpha} signal transduction at a step subsequent to ISGF3 activation [74]. In cells infected with hepatitis B virus [75] or adenovirus [76], ISGF3 is not activated in response to IFN{alpha}, a result similar to our findings in JEG-3 cells. These effects are mediated by the hepatitis B virus terminal protein [75] and the adenovirus E1a 12S protein [76], respectively. Recently, Leonard and Sen showed that E1A lowers both STAT1 and p48/ISGF3{gamma} levels [72, 73]. In addition, several cervical cancer cell lines show defective activation of ISGF3 due to the presence of an inhibitor of p48/ISGF3{gamma} DNA binding [57]. Although the general mechanisms leading to a signaling defect may be different, it is intriguing that interruption of the IFN pathway is common to several human diseases. The specificity of the signaling defect in trophoblast suggests that failure to express ISGF3-dependent genes during pregnancy may be an important mechanism by which human trophoblasts can avoid maternal immune surveillance. For example, it seems likely that resistance of trophoblast cells to IFN ensures that they do not express the classical, polymorphic MHC class I antigens that might otherwise provoke a maternal immune response [2].


   ACKNOWLEDGMENTS
 
We thank Drs. Mike McMaster, John Chamberlain, Tadatsugu Taniguchi, Bryan Williams, David Levy, and Michael Blanar for plasmids; Susan Kovats for advice on the immunoprecipitations; Susan Fisher and Mike McMaster for antisera; Dianna Smith for help with the antiviral assays; and Michael Roberts and Susan Fisher for comments on the manuscript.


   FOOTNOTES
 
1 This work was supported by grants from the Medical Research Council of Canada to J.C.C. (MT-12894) and the National Institutes of Health to Z.W. (HD 26732), and by the Office of Health and Environmental Research, U.S. Department of Energy (contract no. DE-AC03–76-SF01012). J.C.C. is a Scholar of the Medical Research Council of Canada. Back

2 Correspondence: James C. Cross, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Room 880, 600 University Ave., Toronto, ON, Canada M5G 1X5. FAX: 416 586 8588; cross{at}mshri.on.ca Back

3 Current address: Hadassah-Hebrew University Medical Center, Department of Obstetrics and Gynecology, Hadassah Mount Scopus, P.O.B. 24035, Jerusalem, 91240 Israel. Back

Accepted: September 11, 1998.

Received: April 6, 1998.


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