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Independent of Signal Transducer and Activator of Transcription 1
Center for Animal Biotechnology and Genomics and Department of Animal Science, Texas A&M University, College Station, Texas 77843
Address all correspondence and requests for reprints to: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, Texas 77843-2471. E-mail: tspencer{at}tamu.edu.
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Abstract |
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(IFN), a type I IFN produced by the conceptus trophectoderm, increases many type I IFN-stimulated genes (ISGs) in the ovine uterine endometrial stroma and glandular epithelium (GE) using signal transducer and activator of transcription 1 (STAT1)-dependent pathways. Most ISGs are not induced or increased by IFN in the STAT1-negative endometrial luminal epithelium (LE). The objective was to identify genes regulated by IFN in a STAT1-independent manner using DNA microarray and human cell lines. The RNA from human 2fTGH and U3A (STAT1 null 2fTGH) cell lines, stimulated for 24 h with nothing or recombinant ovine IFN, was profiled using an Affymetrix human genome U95Av2 microarray. In 2fTGH cells, IFN increased the expression of 101 genes at least 2-fold, including IFN-inducible 56-kDa protein (IFI56), ISG12 or p27, and guanylate binding protein isoform I (GBP-2). In U3A cells, IFN increased expression of 66 genes at least 2-fold, including Wnt7a. Steady-state levels of IFI56, ISG12, GBP-2, and Wnt7a mRNAs increased in the ovine uterine endometrium between d 10 and 16 of pregnancy but not during the estrous cycle. GBP-2 and IFI56 mRNAs were expressed only in endometrial stroma, ISG12 in both LE and GE, and Wnt7a only in LE of the ovine uterus. Intrauterine infusion of ovine IFN increased expression of all four genes in the endometrium of cyclic ewes. Therefore, IFN does regulate genes independent of STAT1 in the endometrial LE and U3A cells and dependent on STAT1 in the endometrial stroma and 2fTGH cells. These IFN -stimulated genes may be important in establishment of uterine receptivity to the embryo and conceptus implantation given their stage-specificity in endometrium across diverse species. |
Introduction |
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(IFN), a member of the type I IFN family, between d 11 and 21 of gestation with peak production on d 15–16 (1, 2). In the ovine uterus, IFN acts in a paracrine manner on the endometrial luminal epithelium (LE) and sGE [superficial ductal glandular epithelium (GE)] to suppress transcription of the estrogen receptor 
(ER) gene, thus preventing increases in oxytocin receptor (OTR) gene expression (3, 4). These antiluteolytic actions of IFN prevent development of the endometrial luteolytic mechanism by abrogating oxytocin-induced luteolytic pulses of prostaglandin F2, thereby maintaining a functional corpus luteum and progesterone secretion necessary for pregnancy (1, 2).
In addition to suppressing or silencing transcription of ER and OTR genes in the endometrial LE and sGE, IFN induces or increases transcription of a number of type I IFN-stimulated genes (ISGs) in the endometrium of the ovine uterus. These ISGs include signal transducer and activator of transcription (STAT) 1 and 2 (5), IFN regulatory factor-1 (IRF-1) and IRF-9 (5), ISG15/17 (6, 7, 8), Mx protein (9, 10), 2',5'-oligoadenylate synthetase (OAS) (11), and major histocompatibility complex (MHC) class I and ß-2-microglobulin (ß2MG) (12). During pregnancy recognition and establishment, expression of many ISGs, including STAT1, STAT2, IRF-9, ISG17, OAS, MHC Class I and ß2MG, are induced or increased only in the endometrial stroma and GE (5, 6, 7, 10, 11, 12). Indeed, expression of these ISGs is not detected in the endometrial LE and sGE in pregnant ewes between d 14 and 18 of pregnancy. In the ovine uterus, IRF-2, a known transcriptional repressor of type I ISGs as well as the type I IFN genes themselves (13, 14), is constitutively expressed in the endometrial LE and sGE and increases during early pregnancy, consequently preventing induction or increases in their transcription by IFN (5).
All type I IFNs, including IFN, exert their action through a common receptor, which consists of two subunits, IFNAR1 and IFNAR2c (15). In the ovine uterus, both IFNAR1 and IFNAR2 are expressed in all endometrial cell types (16). Induction and increases of most ISGs in response to type I IFNs are mediated by an intracellular signal transduction system involving the transcription factor complexes IFN-stimulated gene factor 3 (ISGF3; heterotrimer of STAT1, STAT2, and IRF-9) and 
activation factor (GAF; homodimer of STAT1 or heterodimer of STAT1 and STAT2) (15). The human 2fTGH fibrosarcoma cells and derivative cell lines lacking specific IFN signaling components have been effectively used to determine the precise roles of STAT1, STAT2, and IRF-9 in the signal transduction pathway of IFN (17, 18). Results indicated that STAT1 is required for IFN effects on STAT2, ISGF3 or IRF-9, OAS (40/46, 69/71, and 100 kDa) and IRF-1 (17, 18). During early pregnancy, the endometrial LE and sGE do not express STAT1, STAT2, or IRF-9 genes (5), thereby precluding the ability of IFN to regulate expression of genes in those cell types using the classical JAK-STAT pathway involving ISGF3 and GAF transcription factors.
Our working hypothesis is that IFN regulates transcription of specific genes in the endometrial LE and sGE through an undefined signaling pathway in a STAT1-independent manner. Available studies indicate that the 2fTGH cells and U3A (STAT1-null 2fTGH) cells are good models for the endometrial stroma/GE and LE of the ovine uterus, respectively (17, 18) because these cells adequately recapitulate cell type-specific responses of the ovine uterus to IFN during pregnancy in terms of ISG expression (5, 6, 7, 10, 11, 12). Comprehensive genome-wide gene profiling studies to test hypotheses are possible in human tissues and cells, but not in domestic animals. Therefore, studies were conducted to: 1) identify genes increased by ovine IFN in human parental (2fTGH) and STAT1-null 2fTGH (U3A) cells by transcriptional profiling; and 2) determine effects of the estrous cycle, pregnancy, and intrauterine infusion of recombinant ovine IFN on expression of selected genes in the ovine uterus that were identified by transcriptional profiling.
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Materials and Methods |
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Experimental design
Study 1.
The 2fTGH (parental) and U3A (STAT1-deficient 2fTGH) cell lines have been described previously (19). The 2fTGH cells are derived from a human fibrosarcoma and respond to both type I and type II IFNs (19). The 2fTGH cells were maintained in basal medium containing DMEM with F-12 salts (DMEM-F12; Sigma-Aldrich Corp., St. Louis, MO) supplemented with 10% FBS and penicillin/streptomycin/amphotericin solution (Invitrogen, Carlsbad, CA) as described previously (17). The U3A cells were maintained in basal medium with hygromycin B (250 µg/ml; Invitrogen). Recombinant ovine IFN (roIFN) was prepared and assayed for biological activity as described previously (20). The activity of roIFN, determined in antiviral units (AVU), was 1 x 109 AVU per mg protein. Monolayer cultures of 2fTGH and U3A cells were grown in culture medium to 80–90% confluence on 100-mm tissue culture plates (n = 3 per treatment). Cells were either untreated, as a control, or treated with roIFN (105 AVU/ml) for 24 h. The chosen dose of roIFN is effectively inhibits transcriptional activity of the promoter/enhancer region of the ovine ER gene (4) and induces or increases expression of several ISGs in ovine uterine endometrial cells and human 2fTGH and U3A cell lines (8, 17, 18). The total RNA was isolated and used for transcriptional profiling or RT-PCR.
Study 2.
At estrus (d 0), ewes were mated to an intact or vasectomized ram as described previously (5). Ewes were then hysterectomized (n = 5 ewes/d) on d 10, 12, 14 or 16 of the estrous cycle or d 10, 12, 14, 16 or 18 of pregnancy. Pregnancy was confirmed on d 10–18 post mating by the presence of morphologically normal conceptus(es) in the uterus. The d 10–16 period of the estrous cycle encompasses development of the endometrial luteolytic mechanism (2), whereas the d 10–18 period of pregnancy encompasses the period of maternal recognition of pregnancy (d 12–14), peak production of IFN by the elongating conceptus (d 14–16), and the onset of conceptus implantation (d 16).
Study 3.
Ten cyclic ewes were ovariectomized and fitted with intrauterine catheters on d 5 post estrus as described previously (12). Ewes (n = 5 ewes/treatment) received im injections of 50 mg progesterone daily from d 5–16 and intrauterine infusions of either 200 mg control serum proteins (CX; ovine serum proteins) or roIFN (2 x 107 AVU) from d 11–16. When infused into the uterus of cyclic ewes on d 11, the chosen dose of roIFN will suppress development of the endometrial luteolytic mechanism and prevents luteal regression (3) and adequately mimics effects of the conceptus in terms of endometrial ISG expression (5, 7, 11, 12, 21). Proteins were prepared for intrauterine injection as described previously (21). All ewes were hysterectomized on d 17.
For both studies 2 and 3, portions (
1 cm) of the from the middle region of the uterine horn were fixed at hysterectomy in fresh 4% paraformaldehyde in PBS (pH 7.2) for 24 h, washed in 70% (vol/vol) ethanol for 24 h, and then embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO). The remaining endometrial tissue was physically dissected from myometrium, frozen in liquid nitrogen, and then stored at -80 C for RNA extraction.
RNA isolation
Total cellular RNA was isolated from cultured cells and frozen endometrial tissue using Trizol reagent (Life Technologies, Inc.-BRL, Bethesda, MD) according to manufacturer’s recommendations. The quantity and quality of total RNA was determined by spectrometry and denaturing agarose gel electrophoresis, respectively.
Microarray analysis
Experimental procedures, including synthesis of double stranded cDNA and biotin-labeled cRNA target, were performed by the Texas A&M University Microarray Core Facility in the Laboratory for Functional Genomics according to the recommendations in the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Inc., Santa Clara, CA). Briefly, double stranded cDNA was synthesized from three independent samples of total RNA (15–20 µg) isolated from monolayer cultures of 2fTGH or U3A cells treated with either nothing or roIFN for 24 h. Purified double stranded cDNA was used to synthesize biotin-labeled cRNA targets for hybridization of a GeneChip Array using an Affymetrix BioArray HighYield RNA Transcript labeling Kit (catalog no. 900182). Purified labeled cRNA (20–30 µg) then was fragmented to the size of 35–200 bp. Before hybridization, the quality of labeling and fragmentation was determined using agarose gel electrophoresis. The fragmented biotinylated cRNA (15 µg), generated from each RNA sample (n = 12 total), was hybridized to a human U95Av2 Affymetrix GeneChip Array (n = 12 total). After washing, the chips were scanned using an Affymetrix GeneArray Scanner at the excitation wavelength of 570 nm. After scanning, each image was checked for major chip defects or abnormalities in hybridization signal as a quality control. Based on a proprietary algorithm developed by Affymetrix, analysis of hybridization signal intensity over the results for each gene determined if genes were present, marginal, or absent in the data set. Genes that were deemed absent at all time points in the experiment were eliminated from further analysis.
Data were first analyzed using Affymetrix Microarray Suit Software 5.0 version for absolute and pair-wise comparison analyses. Normalized expression values for the mean and SD of three replicate average difference scores were calculated for each gene. Comparisons between sample types were performed using the Student’s t test (P < 0.05 was considered significant). The raw data were further analyzed and interpreted using GeneSpring software (version 5.0, Silicon Genetics, Redwood, CA).
Semiquantitative RT-PCR analysis
The cDNA was synthesized from total cellular RNA (5 µg) isolated from cells using random primers (Invitrogen), oligo(deoxythymidine) primers, and SuperScript II Reverse Transcriptase (Invitrogen) as described previously (22). Newly synthesized cDNA was acid-ethanol precipitated, resuspended in 20 µl of water, and stored at -20 C. PCR was performed using an ABI PRISM 7700 (Applied Biosystems, Foster City, CA) with SYBR Green PCR Master Mix (Applied Biosystems) as the detector according to manufacturer’s recommendations. Primers were designed to amplify cDNAs of less than 200 bp to maximize efficiency (Table 1
). PCR cycle parameters were 95 C for 15 sec and 60 C for 1 min for 40 cycles. Data were analyzed by using GeneAmp 5700 SDS software (version 1.4). The threshold line was set in the linear region of the plots above the baseline noise, and threshold cycle (CT) values were determined as the cycle number at which the threshold line crosses the amplification curve. The results are expressed in terms of change in CT values (
CT), which refer to the cycle number during exponential amplification at which the PCR product (measured in real-time by SYBR green fluorescence) crosses a set threshold. To adjust for variations in the amount of input RNA/cDNA, the average CT values for each gene were normalized against average CT values for the housekeeping gene (cyclophilin C) as follows: CT = average CT specific gene - average CT CypC. This is subtraction of an arbitrary constant, so the SD of CT is the same as the SD of the CT value. Finally, the relative value of each mRNA was calculated by the formula: 2- CT. PCR without template or template substituted with total RNA was used as a negative control to verify experimental results.
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(Table 1
). A partial Wnt7a cDNA was amplified by RT-PCR of total RNA from U3A cells treated with roIFN using specific primers (Table 1). RT of cellular total RNA into cDNA was performed as described previously (22). PCR amplification was conducted as follows for GBP-2, ISG12, IFI56, and Wnt7a using PCR Optimized Buffer (Invitrogen): 1) 95 C for 2 min; 2) 95 C for 30 sec, 54.5 C for 40 sec (for GBP-2), 53 C for 40 sec (for ISG12), 54 C for 40 sec (for IFI56), 53 C for 40 sec (for Wnt7a), and 72 C for 1 min for 35 cycles; and 3) 72 C for 10 min. The partial cDNAs for human GBP-2, ISG12, IFI56, and Wnt7a PCR products were cloned into pCRII using a T/A Cloning Kit (Invitrogen) and sequenced verified using an ABI PRISM Dye Terminator Cycle Sequencing Kit and ABI PRISM automated DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA).
Slot blot hybridization and statistical analyses
Steady-state levels of mRNA in ovine endometrium were assessed by slot blot hybridization as described previously (22). Radiolabeled antisense cRNA probes were generated from linearized GBP-2, IFI56, ISG12, or Wnt7a partial cDNAs by in vitro transcription with [-32P]-UTP. Denatured total endometrial RNA (20 µg) from each ewe was hybridized with radiolabeled cRNA probes. To correct for variation in total RNA loading, a duplicate RNA slot membrane was hybridized with radiolabeled antisense 18S cRNA (pT718S; Ambion, Austin, TX). Following washing, the blots were digested with ribonuclease A and radioactivity associated with slots quantified using a Typhoon 8600 MultiImager (Molecular Dynamics, Piscataway, NJ). Data are expressed as relative units (RU x 103).
In situ hybridization analyses
Location of mRNA expression in sections (5 µm) of the ovine uterus was determined by radioactive in situ hybridization analysis as described previously (23). Briefly, deparaffinized, rehydrated, and deproteinated uterine tissue sections were hybridized with radiolabeled antisense or sense cRNA probes generated from linearized GBP-2, ISG12, Wnt7a, or IFI56 partial cDNAs using in vitro transcription with [-35S]-uridine triphosphate. After hybridization, washing and ribonuclease A digestion, slides were then dipped in NTB-2 liquid photographic emulsion (Kodak, Rochester, NY), and exposed at 4 C for 1–2 wk. Slides were developed in Kodak D-19 developer, counterstained with Gill’s hematoxylin (Fisher Scientific, Fairlawn, NJ), and then dehydrated through a graded series of alcohol to xylene. Coverslips were then affixed with Permount (Fisher). Images of representative fields were recorded under bright-field or dark-field illumination using a Nikon Eclipse 1000 photomicroscope (Nikon Instruments Inc., Lewisville, TX) fitted with a Nikon DXM1200 digital camera.
Statistical analyses
All quantitative data were subjected to least-squares regression analyses (ANOVA) using the General Linear Models (GLM) procedures of the Statistical Analysis System (SAS Institute, Cary, NC) (24). Slot blot hybridization data were corrected for differences in sample loading using the 18S rRNA data as a covariate. Data from study 2 were analyzed for effects of day, pregnancy status (cyclic or pregnant), and their interaction. Within pregnancy status, least squares regression analyses were used to determine effects of day on endometrial mRNA levels. Data from Study Three were subjected to one-way least-square-ANOVA. All tests of significance were performed using the appropriate error terms according to the expectation of the mean squares for error. A P value of 0.10 or less was considered significant. Data are presented as least-square means with SE.
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Results |
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differentially regulates gene expression in 2fTGH and U3A (STAT1-deficient 2fTGH) cells (study 1) for 24 h and profiled using an Affymetrix human genome U95Av2 GeneChip array. This array contains probes for approximately 12,000 genes of which about 2,400 are known genes with the remainder being expressed sequence tags. In 2fTGH cells, roIFN increased expression of 101 genes at least 2-fold, and genes increased at least 5-fold are presented in Table 2. The majority of genes increased by IFN in 2fTGH cells were known type I ISGs, including Mx, ISG15/17, OAS, IFI56, ISG12, IFP35, IFI6–16, and ISGF3. In U3A cells, roIFN increased expression of 66 genes at least 2-fold, and genes increased at least a 5-fold are presented in Table 3. None of the genes increased by IFN in U3A cells overlapped with genes in the 2fTGH cells.
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on selected genes in 2fTGH cells, including IFN-inducible 56 kDa protein or IFI56, ISG12 or p27, peroxisome proliferating-activated receptor 
, and guanylate binding protein isoform I (GBP-2). Similarly, RT-PCR analyses were performed on selected genes in U3A cells, including glucokinase, c-myb, Wnt7a, flt3 and glucocorticoid receptor . Analyses verified that roIFN increased (P < 0.001) levels of GBP-2, ISG12, and IFI56 mRNAs in 2fTGH cells (Table 4). However, roIFN increased (P < 0.001) only Wnt7a mRNA in U3A cells. Analyses failed to verify effects of IFN on expression of peroxisome proliferating-activated receptor in 2fTGH cells and c-myb, glucokinase, glucocorticoid receptor , and flt3 in U3A cells. Many of these genes were expressed at low abundance in both 2fTGH and U3A cells based on their normalized expression levels relative to the verified genes (data not shown). Partial cDNAs for IFI56, ISG12, GBP-2, and Wnt7a were amplified from either 2fTGH or U3A cells by RT-PCR using specific primers (Table 1), cloned, and sequence verified (data not shown).
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). Expression of GBP-2, IFI56, and ISG12 mRNAs in the endometrium was not affected (P > 0.10) by day of the estrous cycle, whereas Wnt7a mRNA levels decreased (P < 0.001) after d 10 of the estrous cycle. Expression of GBP-2, IFI56, ISG12, and Wnt7a mRNAs in the endometrium increased (P < 0.001, quadratic) between d 10 and 16 of pregnancy. On d 16 of pregnancy, levels of GBP-2, IFI56, ISG12 and Wnt7a mRNA were maximum in the endometrium and higher than on d 16 of the estrous cycle (P < 0.001, day x pregnancy status).
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and 3). On d 10 and 12, GBP-2 mRNA was low, confined to the endometrial stroma, and not different in uteri from cyclic or pregnant ewes (Fig. 2A). However, by d 14 of pregnancy, GBP-2 mRNA increased in abundance predominantly in the stratum compactum stroma underneath the LE. Expression of GBP-2 mRNA in uteri of d 16 pregnant ewes was abundant in the endometrial stroma and lower in GE. Expression of GBP-2 mRNA, but not IFI56 mRNA, was detected in the conceptus trophectoderm on d 18 of pregnancy. During early pregnancy, expression of IFI56 mRNA was not different in uteri from d 10 cyclic compared with d 10 pregnant ewes, but increased in the endometrial stratum compactum stroma on d 12 of pregnancy (Fig. 2A). Between d 12 and 16 of pregnancy, IFI56 mRNA abundance increased only in the stroma and GE, but was not detected in LE or sGE (Fig. 2B).
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). In pregnant ewes, ISG12 mRNA was also very low in the endometrial LE and GE on d 10 and 12 and then increased in abundance between d 14 and 16 of pregnancy. Expression of ISG12 mRNA was not observed in the endometrial stroma or myometrium. In cyclic ewes, Wnt7a mRNA was detected only in endometrial LE on d 10, but not thereafter (Fig. 3B). In uteri of pregnant ewes, Wnt7a mRNA was expressed at relatively low abundance specifically in the endometrial LE on d 10 and 12. However, expression of Wnt7a mRNA was particularly abundant in the endometrial LE on d 16 and 18 of pregnancy. Neither ISG12 nor Wnt7a mRNA was detected in the conceptus on d 18 of pregnancy.
Intrauterine administration of ovine IFN increases GBP-2, IFI56, ISG12, and Wnt7a mRNA in the endometrium (study 3)
To determine if the differences in expression of the selected genes in uteri of pregnant compared with cyclic ewes was due to IFN from the conceptus, ewes were ovariectomized on d 5 post estrus, treated daily with progesterone to d 16, received intrauterine infusions of control proteins or roIFN from d 11 to 16, and hysterectomized on d 17. Intrauterine administration of roIFN increased (P < 0.001) steady-state levels of GBP-2, IFI56, ISG12, and Wnt7a mRNA in the endometrium (Fig. 4, A, C, E, and G). In situ hybridization analyses verified that roIFN increased GBP-2, IFI56, ISG12, and Wnt7a mRNA expression in a cell-type specific manner consistent with their expression in the uterus of d 16 and 18 pregnant ewes in study 2. Infusion of roIFN increased GBP-2 and IFI56 mRNA only in the endometrial stroma and GE (Fig. 4, B and D). The roIFN increased ISG12 mRNA predominantly in the endometrial GE (Fig. 4F), whereas Wnt7a mRNA was increased only in the endometrial LE (Fig. 4H).
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Discussion |
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in human 2fTGH and U3A fibrosarcoma cells is an effective heterologous approach to identifying genes regulated by IFN in the ovine endometrium during pregnancy recognition and implantation. In 2fTGH cells, roIFN increased expression of many ISGs previously shown to be induced or increased by IFN in the ovine uterus. The present study focused on GBP-2, IFI56, and ISG12, because they have not been investigated in the ovine uterus. In the present study, in situ hybridization analyses revealed that expression of GBP-2 and IFI56 was low in uteri of cyclic ewes and increased in the endometrial stroma and GE between d 12 and 16 of pregnancy, but not of the estrous cycle. The temporal nature of the increases in these ISGs in the endometrium correlate directly with the amount of IFN, produced by the rapidly elongating conceptus, which is maximum on d 14–16 (25). In the present study, intrauterine infusion of recombinant ovine IFN into uteri of cyclic ewes mimicked the effects of the conceptus during pregnancy, indicating that both GBP-2 and IFI56 are IFN-stimulated genes in the ovine uterus. IFN appears to have access to the endometrial stroma and glands due to transepithelial endocytotic activity (vesicles) and passage through intercellular spaces around tight junctions (26). Interestingly, expression of GBP-2 and IFI56 were not detected in the endometrial LE or sGE in uteri of pregnant ewes. This pattern of expression is comparable to that of other ISGs, including STAT1, STAT2, IRF-9, IRF-1, ISG15/17, OAS, MHC class I, and ß2MG (5, 6, 7, 10, 11, 12). The expression of all of these ISGs is induced or increased only in the endometrial stroma and GE while being simultaneously repressed or silenced in the endometrial LE and sGE in response to pregnancy or IFN during the periimplantation period of pregnancy.
The absence of ISGs, including GBP-2 and IFI56, in LE and sGE has been attributed to IRF-2, a potent transcriptional repressor in the IFN signaling system (13, 14). In the ovine uterus, IRF-2 is expressed only in LE and sGE of cyclic and pregnant ewes and increases during early pregnancy (5). Ovine IRF-2 binds to IFN consensus sequences (ICS), IFN-stimulated response elements (ISRE), and IRF elements (IRFE) and represses their basal and stimulated transcriptional activity driven by ISGF3 and IRF-1 (5). The promoter/enhancer region of the mouse GBP-2 gene contains conserved cis-acting elements that include activation sequences that bind GAF and IRFE that bind IRFs (27). Furthermore, the promoter region of the mouse IFI56 gene contains conserved cis-acting elements that include two ISRE that bind ISGF3 and IRFs (28). In the ovine uterus, the endometrial LE and sGE does not express ISGF3 or IRF-1 during d 10 to 20 of pregnancy, presumably due to a lack of STAT1, STAT2, and IRF-9 expression (5). Therefore, the inability of IFN to induce or increase GBP-2 and IFI56 in the endometrial LE is likely to be due to the lack of STAT1 and its derivate transcription factors ISGF3 and GAF. In the present study, IFN induction of GBP-2 and IFI56 gene transcription was dependent on STAT1, because these genes were unresponsive to IFN in U3A (STAT1-null 2fTGH) cells in the present study.
The function of GBP-2 and IFI56 in the ovine uterus is not known. GBP-2 is a member of GBP family of 65- to 67-kDa proteins whose induction by IFNs has been studied in a number of cell types (27, 29, 30). GBP-1, a related GBP family member, is an unconventional GTPase that converts GTP to GMP (31). Interestingly, the closely related GBP-1 is increased in endometrium from humans during the putative window of implantation (32). In that study, GBP-1 was increased only in the endometrial stroma and GE. Similarly, GBP-1 induction in human endometrium during implantation and GBP-2 induction in ovine endometrium during the periimplantation period of pregnancy suggests that GBPs may mediate endometrial responses to conceptus implantation. IFI56 was originally identified as a 56-kDa protein induced in human cells treated with IFN (33, 34). The functions of IFI56 and the closely related IFI54 are not known, but are part of an integrated gene family that responds to type I IFNs (28). Expression of IFI56 has not been reported in the uterus of any other species.
In addition to GBP-2 and IFI56, the ISG12 gene was also responsive to IFN in 2fTGH, but not U3A cells. In a manner different from GBP-2 and IFI56, ISG12 mRNA increased in the endometrial LE and GE during pregnancy and in response to intrauterine administration of IFN; however, ISG12 mRNA did not increase in the stroma. Based on available results, ISG12 is the first known ISG to be increased in ovine endometrial LE in response to IFN. Although Mx is expressed constitutively in the endometrial LE during early pregnancy, Mx mRNA expression is not increased in the LE and sGE by the conceptus or IFN (9, 10, 21). However, the conceptus and IFN increases Mx throughout the endometrial stroma and middle to deep glands. ISG12 was originally designated p27 and cloned as an estrogen-induced gene in the human breast MCF-7 epithelial cell line (35) and is highly inducible by IFN in a number of different human cell lines (36). The function of ISG12 is unknown at present. In the present study, IFN induced ISG12 expression in 2fTGH cells, but not U3A (STAT1-null) cells. The promoter region of the human ISG12 gene contains conserved cis-acting elements that include a putative ISRE, IRFE, and activation sequences. Although the endometrial stroma and GE of the ovine uterus are STAT1-positive during early pregnancy, the endometrial LE and superficial GE are STAT1-negative (5). Therefore, the ability of ISG12 to respond to IFN in the endometrial LE and GE must be through an unknown signaling pathway activated by IFN that is independent of STAT1-containing transcription factors and not active in the stroma. Recently, ISG12 mRNA was found to be markedly increased in the endometrial GE of the mid-secretory phase human uterus during the putative window of implantation (38). In the same study, IRG1, which displays high homology to ISG12 and ISG 6–16, was transiently increased primarily in the endometrial LE and GE and, at lower levels, in the stroma of the pregnant rat uterus on d 4 immediately preceding implantation (38). Further, treatment of female rats with IFN increased endometrial expression of IRG1 in the uterus. Available results from the present study and others indicate that ISGs are induced or increased in the endometrium immediately before implantation in a number of diverse species, including rats, humans and sheep.
In U3A cells and in the ovine endometrial LE, IFN increased expression of the Wnt7a gene. This is the first known report that any type of IFN regulates Wnt7a gene transcription either in vitro or in vivo. IFN did not increase Wnt7a gene expression in 2fTGH cells. This suggests that IFN induction of Wnt7a gene transcription in U3A and ovine endometrial LE is independent of STAT1-containing transcription factors. Wnt7a expression in endometrial LE increased between d 14 and 16 of pregnancy consistent with increasing IFN production by the conceptus. Indeed, intrauterine administration of roIFN increased Wnt7a expression in endometrial LE. Therefore, Wnt7a is a novel IFN-stimulated gene. Wnt genes are a large family of highly conserved, developmentally-related genes (39). The vertebrate Wnt genes are homologous to wingless, a Drosophila segment polarity gene that encodes a secreted molecule implicated in patterning and establishment of cell boundaries during embryogenesis. The vertebrate Wnt family is composed of 16 members and associated with cellular responses such as proliferation (40, 41), branching morphogenesis (42), cell fate determination or specification (43). Wnt7a gene not only guides the development of the anterior-posterior axis in the female reproductive tract, but also plays a critical role in uterine smooth muscle patterning and maintenance of adult uterine function (44, 45, 46). Expression of Wnt7a in uteri of adult mice (44) and humans (47) has been reported. In the adult mice, Wnt7a mRNA is expressed only within the LE of the uterus (44) and null mutation of Wnt7a results in several notable abnormalities in the Müllerian duct differentiation (45, 46, 48). Recently, some members of the Wnt signaling pathway (frizzled related protein (Fr-pHE; an inhibitor of Wnt action) and secreted frizzle-related protein) as well as IFI56 were found to be decreased in human endometrium during the window of implantation (49). However, Dickkopf-1 (an inhibitor of Wnt signaling) was markedly increased (49). Very little information is available on the transcriptional regulation of Wnt7a gene expression (50). However, a computerized search of the human promoter failed to find any putative IFN-responsive elements (Spencer, T. E., unpublished results). The present study supports the idea that IFN regulates Wnt7a gene expression through a novel STAT1-independent signaling pathway. Nonetheless, available results from a number of species support the role of Wnt family in conceptus-endometrial and endometrial epithelial-stromal interactions during the periimplantation period of pregnancy. The function of Wnt7a in the adult uterus is unknown.
Collectively, available results from the present study implicate a novel signal transduction pathway in IFN regulation of ISG12 and Wnt7a gene expression in the endometrial epithelium. Discovery of this signaling pathway that is independent of the classical JAK-STAT pathway will enhance our understanding of type I IFN functions in antiviral responses as well as pregnancy recognition and establishment in domestic animals as well as humans and rodents. Although the functions of ISGs in the uterus are not known, the precise implantation stage-specific expression of ISGs, such as ISG12, GBP-2 and their family members, in the endometrium across diverse species, including the rat, human, sheep and cow, supports a critical role of IFN signaling and ISGs as universal mediators of conceptus implantation (38, 51).
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Acknowledgments |
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Footnotes |
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S.K. and Y.C. contributed equally to the manuscript.
Present address for Y.C.: Department Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030.
Abbreviations: AVU, Antivirual units; CT, threshold cycle; ER, estrogen receptor ; GAF, activation factor; GBP-2, guanylate binding protein isoform I; GE, glandular epithelium; IFI56, IFN-inducible 56-kDa protein; IFN, interferon; ISG, IFN-stimulated genes; IRF-1, IFN regulatory factor-1; ISGF3, IFN-stimulated gene factor 3; LE, luminal epithelium; ß2MG, ß-2-microglobulin; MHC, major histocompatibility complex; OAS, 2',5'-oligoadenylate synthetase; OTR, oxytocin receptor; roIFN, recombinant ovine IFN; sGE, superficial ductal GE; STAT, signal transducer and activator of transcription.
Received May 29, 2003.
Accepted for publication August 12, 2003.
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inhibits estrogen receptor up-regulation and estrogen-induced luteolysis in ewes. Endocrinology 136:4932–4944[Abstract]
promoter and functional regulation by ovine interferon . Endocrinology 142:2879–2887 inducible ubiquitin cross-reactive protein in the ovine uterus. Biol Reprod 61:312–318 and progesterone regulate ubiquitin cross-reactive protein expression in the ovine uterus. Biol Reprod 62:622–627 activates multiple STAT proteins and has complex effects on interferon-responsive gene transcription in ovine endometrial epithelial cells. Endocrinology 142:98–107
on 2', 5'-oligoadenylate synthetase expression in the ovine uterus. Biol Reprod 64:1392–1399 regulate major histocompatibility complex class I and beta2-microglobulin expression in the ovine uterus. Biol Reprod 68:1703–1710 (IFN) regulation of IFN-stimulated gene expression in cell lines lacking specific IFN-signaling components. Endocrinology 142:1786–1794 regulation of IRF-1. Biol Reprod 66:393–400 interferon to obtain mutations in the signaling pathway. Mol Cell Biol 9:4605–4612 by Picha pastoris. J Interferon Cytokine Res 16:119–126[Medline]
on the endometrium of cyclic ewes. Biol Reprod 61:464–470 interferon. Mol Cell Biol 15:975–982[Abstract]
-interferon-dependent signal transduction pathways may contribute to activation of transcription of the guanylate-binding protein gene. Mol Cell Biol 11:5147–5153 in the uterus during early pregnancy. J Reprod Fertil Suppl 54:329–339[Medline]This article has been cited by other articles:
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  G. Song, J. Kim, F. W. Bazer, and T. E. Spencer Progesterone and Interferon Tau Regulate Hypoxia-Inducible Factors in the Endometrium of the Ovine Uterus Endocrinology, April 1, 2008; 149(4): 1926 - 1934. [Abstract] [Full Text] [PDF]
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J. F. Oliveira, L. E. Henkes, R. L. Ashley, S. H. Purcell, N. P. Smirnova, D. N. R. Veeramachaneni, R. V. Anthony, and T. R. Hansen Expression of Interferon (IFN)-Stimulated Genes in Extrauterine Tissues during Early Pregnancy in Sheep Is the Consequence of Endocrine IFN-{tau} Release from the Uterine Vein Endocrinology, March 1, 2008; 149(3): 1252 - 1259. [Abstract] [Full Text] [PDF]
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 T. E Spencer, O. Sandra, and E. Wolf Genes involved in conceptus-endometrial interactions in ruminants: insights from reductionism and thoughts on holistic approaches Reproduction, February 1, 2008; 135(2): 165 - 179. [Abstract] [Full Text] [PDF]
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A. Kashiwagi, C. M. DiGirolamo, Y. Kanda, Y. Niikura, C. T. Esmon, T. R. Hansen, T. Shioda, and J. K. Pru The Postimplantation Embryo Differentially Regulates Endometrial Gene Expression and Decidualization Endocrinology, September 1, 2007; 148(9): 4173 - 4184. [Abstract] [Full Text] [PDF]
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Y. Chen, E. Antoniou, Z. Liu, L. B Hearne, and R M. Roberts A microarray analysis for genes regulated by interferon-{tau} in ovine luminal epithelial cells Reproduction, July 1, 2007; 134(1): 123 - 135. [Abstract] [Full Text] [PDF]
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K. Hayashi, R. C. Burghardt, F. W. Bazer, and T. E. Spencer WNTs in the Ovine Uterus: Potential Regulation of Periimplantation Ovine Conceptus Development Endocrinology, July 1, 2007; 148(7): 3496 - 3506. [Abstract] [Full Text] [PDF]
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G. Song, F. W Bazer, and T. E Spencer Pregnancy and interferon tau regulate RSAD2 and IFIH1 expression in the ovine uterus Reproduction, January 1, 2007; 133(1): 285 - 295. [Abstract] [Full Text] [PDF]
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G. Song, T. E. Spencer, and F. W. Bazer Progesterone and Interferon-{tau} Regulate Cystatin C in the Endometrium Endocrinology, July 1, 2006; 147(7): 3478 - 3483. [Abstract] [Full Text] [PDF]
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Y. Chen, J. A. Green, E. Antoniou, A. D. Ealy, N. Mathialagan, A. M. Walker, M. P. Avalle, C. S. Rosenfeld, L. B. Hearne, and R. M. Roberts Effect of Interferon-{tau} Administration on Endometrium of Nonpregnant Ewes: A Comparison with Pregnant Ewes Endocrinology, May 1, 2006; 147(5): 2127 - 2137. [Abstract] [Full Text] [PDF]
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J. G. W. Fleming, T. E. Spencer, S. H. Safe, and F. W. Bazer Estrogen Regulates Transcription of the Ovine Oxytocin Receptor Gene through GC-Rich SP1 Promoter Elements Endocrinology, February 1, 2006; 147(2): 899 - 911. [Abstract] [Full Text] [PDF]
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G. Song, T. E. Spencer, and F. W. Bazer Cathepsins in the Ovine Uterus: Regulation by Pregnancy, Progesterone, and Interferon Tau Endocrinology, November 1, 2005; 146(11): 4825 - 4833. [Abstract] [Full Text] [PDF]
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T. E Spencer, G. A Johnson, F. W Bazer, and R. C Burghardt Implantation mechanisms: insights from the sheep Reproduction, December 1, 2004; 128(6): 657 - 668. [Abstract] [Full Text] [PDF]
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S.-Z. Wang and R. M. Roberts Interaction of Stress-Activated Protein Kinase-Interacting Protein-1 with the Interferon Receptor Subunit IFNAR2 in Uterine Endometrium Endocrinology, December 1, 2004; 145(12): 5820 - 5831. [Abstract] [Full Text] [PDF]
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K. Hayashi, K. D. Carpenter, and T. E. Spencer Neonatal Estrogen Exposure Disrupts Uterine Development in the Postnatal Sheep Endocrinology, July 1, 2004; 145(7): 3247 - 3257. [Abstract] [Full Text] [PDF]
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T. E. Spencer, G. A. Johnson, R. C. Burghardt, and F. W. Bazer Progesterone and Placental Hormone Actions on the Uterus: Insights from Domestic Animals Biol Reprod, July 1, 2004; 71(1): 2 - 10. [Abstract] [Full Text] [PDF]
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 C. A. Gray, D. L. Adelson, F. W. Bazer, R. C. Burghardt, E. N. T. Meeusen, and T. E. Spencer Discovery and characterization of an epithelial-specific galectin in the endometrium that forms crystals in the trophectoderm PNAS, May 25, 2004; 101(21): 7982 - 7987. [Abstract] [Full Text] [PDF]
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