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Regular Article |
a Center for Animal Biotechnology and Genomics, Department of Animal Science, Texas A&M University, College Station, Texas 77843
b Departments of Medicine, Molecular and Cellular Biology, and Immunology, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) is the pregnancy recognition signal produced by the conceptus trophectoderm and acts in a paracine manner on the ovine endometrium to increase expression of IFN-stimulated genes primarily in the stroma and deep glandular epithelium, including IFN regulatory factor-1 (IRF-1). The roles of Stat1, Stat2, and IRF-9 in IFN regulation of IRF-1 expression were determined using human stromal fibroblasts lacking specific IFN signaling components or complemented with specific Stat1 mutants. In parental (2fTGH) cells treated with IFN, Stat1
/ß was tyrosine phosphorylated by 15 min, and IRF-1 mRNA and protein increased from 0 to 6 h, was maximal at 6 h, and decreased to 24 h. In contrast, IFN did not affect IRF-1 expression in Stat1- and Stat2-deficient cells or in Stat1-deficient cells complemented with Stat1 Y701Q or Stat1 R602L mutants. In Stat1-deficient cells complemented with the Stat1 S727A mutant, Stat1, or Stat1ß and treated with IFN, IRF-1 increased from 0 to 6 h, was maximal at 6 h, and decreased thereafter. In IRF-9-deficient cells stimulated with IFN, IRF-1 increased from 0 to 6 h but did not exhibit the sharp decline from 6 to 12 h observed in other cells. Collectively, results indicate that IFN effect on IRF-1 expression is primarily regulated by tyrosine-phosphorylated Stat1 or Stat1ß dimers, whereas the decline of IRF-1 after 6 h of IFN treatment is regulated by IRF-9.
gene regulation, mechanisms of hormone action, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), a unique member of the type I IFN family [1], is produced exclusively by the trophectoderm of ruminant (i.e., sheep, cattle, and goat) conceptuses and is the signal for maternal recognition of pregnancy [2]. The ovine conceptus secretes IFN between Days 11 and 20–24 postmating [3, 4], and peak production is between Days 15 and 17 when a single conceptus produces more than 106 antiviral units/day [5]. In the ovine uterus, IFN acts in a paracrine manner on the endometrium to suppress transcriptional upregulation of estrogen receptor alpha and oxytocin receptor genes in the luminal epithelium (LE) and superficial glandular epithelium (GE) [6–8]. 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 [2].
Available evidence suggests that the actions of IFN on the endometrium are mediated by the ovine type I IFN receptor that is not appreciably different from those in other species [9]. Analyses of bovine and ovine endometrial cells and human stromal fibroblasts indicate that positive effects of IFN are mediated primarily by a signal transduction pathway involving activation of the signal transducers and activators of transcription (Stat) proteins [10–12]. IFN induces rapid tyrosine phosphorylation and nuclear translocation of Stats 1, 2, 3, 5a/b, and 6, but only Stat1 and Stat2 remain persistently phosphorylated and in the nucleus upon long-term stimulation of cells with IFN [11]. IFN activation of Stats 1 and 2 results in the formation of 2 transcription factors, gamma-activated factor (GAF) and IFN-stimulated gene (ISG) factor 3 (ISGF3) [11, 12]. GAF is a Stat1 homodimer [13, 14] that regulates transcription of genes containing a gamma-activated sequence (GAS) element such as IFN regulatory factor (IRF) 1 [15]. ISGF3 is a heterotrimer consisting of Stat1, Stat2, and IRF-9 [16] that regulates transcription of genes containing IFN-stimulated response elements (ISREs), such as Stat1, Stat2, IRF-9 and 2',5'-oligoadenylate synthetase (OAS) [17, 18]. In ovine endometrial cells, IFN elicits persisent formation of ISGF3 [11]. Although GAF is formed within 20 min of IFN stimulation, the abundance of GAF declines after 6 h of treatment. The shift in transcription factor formation from GAF to ISGF3 was hypothesized to result from concomitant increases in expression of IRF-9 and Stat2, which are also ISGs that are increased by IFN [11, 12].
IFN increases or induces expression of a number of ISGs in the ovine endometrium in vivo or ovine endometrial and human fibroblast cells in vitro, including Stats 1 and 2 [7, 10–12, 19], IRF-9 [11, 19], OAS [12, 20], ISG17 (also known as ubiquitin cross-reactive protein) [11, 21, 22], and IRF-1 [10, 11, 19, 20, 22]. The increases in expression of these ISGs in the ovine endometrium in vivo by IFN is primarily observed in the stromal fibroblasts and middle to deep GE. Expression of IRF-2, a potent repressor of ISG expression, in the LE and superficial GE appears to restrict IFN-induced increases in ISG expression to the endometrial stroma and middle to deep GE [19]. Using 2fTGH stromal fibroblast cells lacking Stat1, Stat2, and IRF-9, IFN induction of Stat1, Stat2, IRF-9, 40/46-kDa OAS, and 69/71-kDa OAS expression was found to require Stat1, Stat2, and IRF-9, which compose ISGF3 [12]. Given that effects of IFN on ISG expression in the ovine endometrial stroma in vivo are similar to those in 2fTGH human fibroblast cells in vitro [12, 19], the 2fTGH cell line and its derivatives represent a useful model to determine the role of specific signaling molecules in the IFN signal transduction pathway.
IRF-1 is an early gene that is induced by both type I and type II IFNs and regulates gene transcription by binding to an ISRE or the closely related IRF element [23]. In some cases, IRF-1 cooperates with ISGF3 to induce or maintain ISG expression [24–26] or with GAF to increase transcription of ISGs in response to the type II IFN
[27, 28]. In ovine endometrial cells treated with IFN, increases in IRF-1 expression preceded increases in expression of other ISGs, including Stat1, Stat2, IRF-9, and ISG17 [11]. The promoter of the human and rat IRF-1 genes contain a GAS element(s) that mediates induction by IFN [15]. However, the precise signaling components regulating IFN induction of IRF-1 are not known. The objective of this study was to utilize the 2fTGH cell line and its derivatives, lacking Stat1, Stat2, or IRF-9, and to utilize Stat1-deficient cells complemented with various mutations of Stat1 to understand the specific roles of Stat1, Stat2, and IRF-9 in the signal transduction pathway whereby IFN regulates IRF-1 gene expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The 2fTGH, U2A, U3A, U6A, U3A-701, U3A-727, U3A-SH2, U3A-p84, and U3A-p91 cells have been described previously [29–31], and their characteristics are summarized in Table 1. The 2fTGH cells were maintained in basal medium containing Dulbecco modified Eagle medium with F-12 salts (DMEM-F12; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum and penicillin/streptomycin/amphotericin B (Gibco-BRL, Rockville, MD). The U2A, U3A, and U6A cells were maintained in basal medium with hygromycin B (250 µg/ml; Sigma-Aldrich). The U3A-701, U3A-727, U3A-SH2, U3A-p84, and U3A-p91 cells were maintained in basal medium with hygromycin B and G418-sulfate (400 µg/ml, Geneticin; Gibco-BRL).
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Recombinant ovine IFN (roIFN) was prepared and assayed for biological activity as described previously [32]. The biological activity of roIFN was determined in antiviral units (AVU), and the specific activity of the roIFN preparation was high (109 AVU/mg protein). Antibodies used in these experiments were mouse anti-Stat1 (no. S21120) from Transduction Laboratories (Lexington, KY), rabbit anti-phospho-Stat1 (no. 9171S) from Cell Signaling Technologies (Beverly, MA), rabbit anti-IRF-1 (no. sc-497X) from Santa Cruz Biotechnology (Santa Cruz, CA), and peroxidase-conjugated goat anti-rabbit (no. 474-1506) and goat anti-mouse (no. 474-1806) IgG from Kirkegaard & Perry Laboratories (Gaithersburg, MD).
Western Blot Analysis
Monolayer cultures were grown to approximately 80% confluence on 100-mm tissue culture plates. For Stat1 and phosphotyrosine Stat1 (p-Stat1) Western blots, 2fTGH cells were left untreated as a control or were treated with roIFN (104 AVU/ml) for 0.25, 0.5, 0.75, 1, 3, 6, 12, or 24 h. For IRF-1 Western blots, cells were left untreated as a control or were treated with roIFN (104 AVU/ml) for 1, 3, 6, 12, 24, or 48 h. The dose of roIFN used in cell culture studies was derived from that used in previous studies [8, 10–12, 20]. These cell culture studies were replicated in 3 independent experiments.
Whole cell extracts were prepared and analyzed by Western blotting as previously described [12], except for the p-Stat1 Western blots in which the nitrocellulose filters were blocked and primary antibody diluted in 5% BSA with TBST (Tris-buffered saline and 0.1% Tween-20). Prestained molecular weight standards (Bio-Rad Laboratories, Burlingame, CA) were included on each gel. Stat1 and p-Stat1 antibodies were diluted according to the manufacturer's recommendation. The IRF-1 antibody was diluted 1:1000 in 2% (wt/vol) nonfat milk-TBST. Western blots were quantified by scanning densitometry using a GS-690 Imaging Densitometer and MultiAnalyst software (Bio-Rad Laboratories, Hercules, CA) as described previously [12].
Semiquantitative Reverse Transcription Polymerase Chain Reaction
The 2fTGH cells were grown to approximately 80% confluence in 6-well tissue culture plates (Nunc, Naperville, IL) and then left untreated or were treated with roIFN (104 AVU/ml) for 0.25, 0.5, 0.75, 1, 3, 6, 12, or 24 h. Treatments at each time point were replicated in triplicate. Total cellular RNA was isolated using Trizol (Gibco BRL, Grand Island, NY) according to the manufacturer's protocol. Expression of IRF-1 mRNA was assessed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) as previously described [33]. The cDNA was synthesized from 5 µg total cellular RNA using random (Life Technologies, Gaithersburg, MD) and oligo(dT) primers with SuperScript II Reverse Transcriptase (Life Technologies). Newly synthesized cDNA was acid-ethanol precipitated, resuspended in 20 µl water, and stored at -20°C. The cDNAs were diluted (1:10) with water prior to PCR. IRF-1 primers were developed to amplify a 502-base pair (bp) region of the published human IRF-1 cDNA (GenBank accession no. NM002198) as described previously [19]. The sequences of the forward and reverse IRF-1 primers were 5'-TCC ACC TCT CAC CAA GAA-3' and 5'-TTC TGG CTC CTC CTT ACA GC-3', respectively. The ß-actin primers were developed to amplify a 420-bp region of the published ovine ß-actin cDNA (GenBank accession no. U39357). The sequences of the ß-actin primers were 5'-CAT CCT GAC CCT CAA GTA CCC-3' and 5'-GTG GTG GTG AAG CTG TAG CC-3'.
The PCRs were performed using AmpliTaq DNA polymerase (PE Applied Biosystems, Foster City, CA) and Optimized Buffer F (Invitrogen, Carlsbad, CA) for IRF-1 or Optimized Buffer D (Invitrogen) for ß-actin. The amount of cDNA template, annealing temperature, and number of PCR cycles were optimized as previously described [33] using cDNA derived from 2fTGH cells treated with IFN for 3 h to ensure that PCR conditions were within the linear range of amplification for each primer pair. IRF-1 and ß-actin PCRs contained 3 µl and 1 µl of cDNA, respectively. The IRF-1 PCR reaction consisted of 95°C for 30 sec, 57°C for 30 sec, and 72°C for 30 sec. The ß-actin PCR consisted of 95°C for 30 sec, 55°C for 1 min, and 72°C for 1 min. The optimized number of PCR cycles for IRF-1 and ß-actin was determined to be 30 and 26, respectively. Following PCR, 20 µl of each reaction was analyzed by agarose gel electrophoresis, and PCR products were visualized using ethidium bromide. The relative amount of DNA present was quantified by measuring the intensity of light emitted from bands of the corrrect size under ultraviolet light using an AlphaImager (Alpha Innotech Corp., San Leandro, CA). The ß-actin values were used as a covariate in statistical analyses to correct for differences in the amount of cDNA template between samples.
Statistical Analyses
Integrated optical density (Westerns) and light intensity (RT-PCR) measurements were subjected to least squares (LS) ANOVA using the general linear models procedures of the Statistical Analysis System (version 8.1 for Windows; SAS Institute, Cary, NC) as described previously [12]. The model used in the LS-ANOVA included time (hours after IFN treatment) and replicate as sources of variation. For Western blots, the initial measurement of band optical density at Time 0 was used as a covariate. For RT-PCR, the intensity of light emitted from ß-actin PCR products was used as a covariate. The least square means (LSMs) and SEMs illustrated in the figures were derived from this analysis. If a significant effect of IFN treatment was detected (P < 0.05), the data for each individual protein were analyzed by LS regression analysis. In these analyses, time was considered a continuous and independent source of variation, and replicate was a dependent source. The initial measurement of band optical density at Time 0 or light intensity of ß-actin PCR products was used as a covariate in regression analyses. To compare the effects of IFN treatment on expression of the 51-kDa and 47-kDa IRF-1, data from 0 and 6 h were analyzed by LS ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in 2fTGH Cells
In the parental 2fTGH fibroblasts, tyrosine-phosphorylated Stat1 and Stat1ß were not detected at 0 h in untreated cells. Treatment of 2fTGH cells with IFN induced tyrosine phosphorylation of Stat1/ß within 15 min. Stat1/ß remained tyrosine phosphorylated from 15 min to 24 h in response to IFN treatment. Although the overall amount of tyrosine phosphorylated Stat1/ß declined between 6 and 12 h, the total amount of phosphorylated and unphosphorylated Stat1/ß protein increased between 6 h and 24 h of IFN treatment (Fig. 1).
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IFN Induces a Transient Increase in IRF-1 mRNA and Protein in Parental 2fTGH Fibroblasts
The effect of PCR cycle number of amplification of IRF-1 and ß-actin partial cDNAs is illustrated in Figure 2A. Based on these experiments, the optimum number of PCR cycles for IRF-1 and ß-actin amplification was determined to be 30 and 26, respectively. Stimulation of 2fTGH cells with IFN increased steady-state levels of IRF-1 mRNA from 0 to 6 h (cubic; P < 0.001) (Fig. 2, B and C). IRF-1 mRNA levels reached a maximum at 6 h and declined thereafter; however, IRF-1 mRNA levels at 24 h post-IFN treatment were still greater than those at 0 h.
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Western blot analyses revealed an immunoreactive protein of approximately 51 kDa in 2fTGH whole cell extracts using the rabbit anti-IRF-1 antibody (Fig. 3). The exposure of the chemiluminescent Western blot in Figure 3 was timed to ensure linearity of signal. A longer exposure of the Western blots revealed low levels of another immunoreactive protein of approximately 47 kDa (data not shown) that can be observed in other figures. The 51-kDa form of IRF-1 is the hyperphosphorylated 47-kDa IRF-1 protein. No background was observed in negative controls, in which the primary antibody was omitted or substituted with normal rabbit IgG (data not shown).
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Similar to results of IRF-1 mRNA analyses, treatment of 2fTGH cells with IFN elicited an increase (cubic effect of time, P < 0.001) in IRF-1 protein levels (Fig. 3). IRF-1 protein levels increased 11.9-fold between 0 and 6 h and then declined; however, IRF-1 protein levels at 24 h post-IFN treatment were still 3.5-fold higher than those at 0 h.
IRF-9 Plays a Role in Responsiveness of the IRF-1 Gene to IFN
In U2A (IRF-9 deficient) cells (Fig. 4), 2 distinct immunoreactive IRF-1 proteins with calculated molecular masses of 47 and 51 kDa were detected in Western blots. The 47-kDa form of IRF-1 could also be detected in the lane containing extracts from the 2fTGH parental cells because of the longer exposure of the Western blots to film. IFN treatment of U2A cells increased (cubic, P < 0.001) expression of 51-kDa IRF-1 but did not increase (P > 0.10) expression of 47-kDa IRF-1 (Fig. 4). The relative levels of 51-kDa IRF-1 increased from 0 to 6 h, remained maximal from 6 to 12 h, and then decreased slightly from 12 to 24 h post-IFN treatment. Between 0 and 6 h post-IFN stimulation, a 10.9-fold increase in expression of the 51-kDa IRF-1 (P < 0.01) was detected.
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Stat1 and Stat2 Are Required for IFN Induction of IRF-1 Expression
In U3A (Stat1 deficient) cells (Fig. 5, A and B) and U6A (Stat2 deficient) cells (Fig. 5, C and D), expression of the 51- and 47-kDa forms of IRF-1 was not affected (P = 0.132 and 0.844, respectively) by IFN treatment.
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Stat1 or Stat1ß Restores IFN-Induced IRF-1 Expression in Stat1-Deficient Cells
The ability of IFN to regulate IRF-1 expression was restored in U3A Stat1-deficient cells complemented with either Stat1 (p91) or Stat1ß (p84) (Fig. 6). Stat1ß contains a truncated C-terminal domain, thereby lacking some of the transactivation domain and a serine phosphorylation site [31]. Similar to 2fTGH cells, IRF-1 protein levels exhibited an increase (cubic, P < 0.001) in both cell lines upon treatment with IFN. Overall, levels of the 51-kDa IRF-1 increased to maximum at 6 h and then declined by 24 h; however, the overall levels of IRF-1 were greater at 24 h post-IFN treatment as compared with those at 0 h. IFN only increased expression of the 51-kDa IRF-1 form. At 6 h post-IFN treatment, the 51-kDa IRF-1 was 5.7-fold greater (P < 0.001) in U3A-p91 cells and 21.7-fold greater (P < 0.001) in U3A-p84 cells compared with values at 0 h.
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Tyrosine Phosphorylation of Stat1 and the SH2 Domain of Stat1 Are Critical for IFN-Induced IRF-1 Expression
In U3A Stat1-deficient cells complemented with the Stat1Y701Q mutant (U3A-701 cells) (Fig. 7, A and B) or the Stat1 R602L mutant (U3A-SH2 cells) (Fig. 7, C and D), IFN did not increase expression of either IRF-1 form (P = 0.062 and 0.087, respectively). The Stat1Y701Q mutant cannot be tyrosine phosphorylated, whereas the Stat1 R602L mutant inactivates a residue critical for SH2 function [31]. Both these mutants render Stat1 incapable of homodimerizing or heterodimerizing with Stat2. Thus, neither GAF nor ISGF3 can be formed in these cells.
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Serine Phosphorylation of Stat1 Is Not Critical for IFN-Induced IRF-1 Expression
Serine phosphorylation of Stat1 has been observed to play a role in regulation of specific genes by other type I IFNs. In Stat1-deficient cells complemented with the Stat1 S727A mutant (U3A-727 cells) (Fig. 8), treatment with IFN increased (cubic, P < 0.001) expression of the 51-kDa IRF-1 to maximal levels at 6 h. However, IRF-1 levels rapidly declined after 6 h. Between 0 and 6 h post-IFN treatment, expression of the 51-kDa IRF-1 increased 5.1-fold (P < 0.001), whereas expression of the 47.4-kDa IRF-1 was unaffected (P = 0.655) by treatment. Relative levels of IRF-1 protein were not different at 0 h and 24 h post-IFN treatment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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induced rapid tyrosine phosphorylation of Stat1/ß, and the overall amount of tyrosine phosphorylated Stat1/ß was highly correlated with IRF-1 mRNA and protein expression. The mechanism(s) regulating the apparent decrease in overall levels of Stat1 tyrosine phosphorylation after 6 h of IFN treatment is not known. One possible explanation is the increased expression of genes that negatively regulate the type I IFN signaling pathways, including specific protein tyrosine phosphatases [34, 35], suppressor of cytokine signaling [36], Jak-binding protein [37], and/or protein inhibitor of activated Stat [38]. An alternative explanation for the apparent decrease in p-Stat1/ß after 6 h of chronic IFN stimulation is the large increase in total phosphorylated and unphosphorylated Stat1 and Stat2 protein observed in 2fTGH cells after 6 h of IFN treatment. In all cell lines except 2fTGH, IRF-1 was found in 2 forms with different molecular masses. The 51-kDa form is likely a hyperphosphorylated form of IRF-1, because casein kinase II can phosphorylate IRF-1 on 2 clusters of serine residues, which enhances transactivational capacity of IRF-1 [39]. The 47-kDa IRF-1 exists in 2fTGH cells, as seen in the positive controls for the other cell lines (e.g., Fig. 4A). However, the 47-kDa IRF-1 was not visible in the Western blot analysis of the 2fTGH cells (Fig. 3) because of the short exposure time necessary to achieve a linear signal for the 51-kDa IRF-1.
IFN induction of human IRF-1 gene transcription involves tyrosine phosphorylation of Stat1/ß homodimers that form GAF and transactivate a GAS element in the IRF-1 promoter [15]. IFN also induces tyrosine phosphorylation of Stat1/ß and formation of GAF, which binds to a GAS element and increases transcription of the rat IRF-1 promoter [11]. Treatment of different cell types with either IFN or prolactin elicits a biphasic induction in IRF-1 gene expression [40, 41]. However, IFN does not induce the same biphasic pattern of IRF-1 gene expression. This difference between IFN and the type II IFN and prolactin may be due to IRF-9. IRF-9 was originally termed ISGF3 (or p48), and the IRF-9 gene is IFN responsive and contains a novel -activated transcriptional element and nuclear factor kappa B (NF
B) element [42]. IRF-9 is induced by IFN in 2fTGH cells between 3 and 6 h after treatment [12] and is a major component of ISGF3. In the present study, IFN increased expression of IRF-1 from 0 to 6 h after treatment in IRF-9-deficient (U2A) cells. Unlike the parental (2fTGH) and other cell lines, IRF-1 protein levels did not exhibit the same pattern of decline between 6 and 24 h post-IFN treatment in U2A cells. Available results support the hypothesis that the lack of decline in IRF-1 expression is due to the deficiency of IRF-9. In the 2fTGH parental cells, IRF-9 can heterodimerize with Stat1-Stat2 dimers, but no competition exists between IRF-9 and p-Stat1 for dimerization with Stat2 in IRF-9-deficient U2A cells. Thus, Stat1 homodimers and Stat1-Stat2 heterodimers are persistently formed and are most likely responsible for continual activation of IRF-1 expression in U2A cells. Therefore, the observed decline in IRF-1 expression in 2fTGH and other cells after 6 h of IFN treatment is most likely due to a shift in formation of GAF to ISGF3 because of the increase in IRF-9 and Stat2 expression [12]. Although IRF-1 is induced in U2A cells by IFN and can bind to ISREs, IRF-1 is a weak transactivator of ISGs such as OAS [19, 26]. However, ample evidence exists that IRF-1 cooperates with ISGF3 and GAS to induce and/or maintain transcription of selected genes [26, 43], which may be the overall function of IRF-1 induced in the endometrial stroma and middle to deep GE by IFN during pregnancy in ruminants [19].
Stat1 and Stat2 are critical for type I IFN signaling, as indicated by the inability of IFN to regulate IRF-1 gene expression in Stat1- and Stat2-deficient cells in the present study. IFN did not increase expression of Stat1/ß, IRF-9, or OAS in Stat2-deficient U6A cells [12], because Stat2 is required for Stat1 tyrosine phosphorylation and GAF formation in response to IFN [30, 44]. The central role of Stat1 in type I and type II IFN signaling is due to GAF. GAF formation requires tyrosine phosphorylation of Stat1 and Stat2. The SH2 domain of Stat1 mediates interactions with other tyrosine-phosphorylated proteins such as Stat1 and Stat2 [45]. Mowen and David [46] reported that Stat1 containing a SH2 mutation was tyrosine phosphorylated but failed to enter the nucleus in response to IFN. Thus, neither functional GAF nor Stat1-Stat2 heterodimers can form in U3A-SH2 cells, offering an explanation for the inability of IFN to regulate IRF-1 expression in these cells. In the present study, IFN did tend to slightly increase IRF-1 expression in U3A-701 and U3A-SH2 cells. This observation suggests that IFN activates other signaling pathways, such as NFB, which is activated by IFN [47]. Given that the IRF-1 gene contains a composite GAS/B promoter element [48], it is tempting to speculate that IFN activation of NFB is responsible for the observed tendency of IFN to increase IRF-1 expression in cells that lack functional GAF formation.
Results from the present study indicate that tyrosine phosphorylation but not serine phosphorylation of Stat1 is required for IFN-induced IRF-1 expression. Tyrosine phosphorylation of Stat1 is required for strong dimerization, DNA binding, and transcriptional activation in response to type I IFN and type II IFN [49, 50]. The fact that IFN did not increase IRF-1 expression in U3A-701 cells illustrates the central role of Stat1 tyrosine phosphorylation in the type I IFN signaling pathway. Serine phosphorylation of Stat1 is required for maximal transactivation of GAS-containing genes in response to IFN [51]. In the present study, IFN induction of IRF-1 gene transcription was unaffected by the complementation of U3A cells with a Stat1 serine phosphoserine mutant (U3A-727 cells). This finding is consistent with previous results showing that IFN induces expression of ISGs, including Stat1, Stat2, IRF-9, and OAS, equally well in U3A727 cells and parental 2fTGH cells [12]. The fact that serine phosphorylation is not required for IFN-induced gene expression is consistent with the fact that Stat1ß, which lacks the C-terminal serine phosphorylation site, functions equally well as Stat1 in IFN induction of IRF-1 and other ISGs [12].
The effects of IFN on IRF-1 expression were restored in Stat1-deficient cells complemented with either Stat1 or Stat1ß. Stat1 and Stat1ß are alternative splice variants of the Stat1 gene. Stat1ß differs from Stat1 by the lack of 38 C-terminal amino acids and does not activate transcription of certain IFN-responsive genes [50]. However, Stat1ß can associate with Stat2 and IRF-9 to form functional ISGF3 in response to IFN [52]. Complementation of U3A cells with Stat1 restored IFN and IFN signaling [29]; however, complementation of U3A cells with Stat1ß restored responsiveness only to IFN and not to IFN. Previous results indicate that IFN does increase or induce expression of Stat2, IRF-9, and OAS in both U3A-p84 and U3A-p91 cells [12]. Collectively, available results suggest that Stat1 and Stat1ß function equally well in formation of functional GAF and ISGF3 in response to IFN. Therefore, both type I and type II IFNs induce tyrosine phosphorylation, nuclear translocation, and DNA binding of Stat1ß [50, 52], but only type I IFNs can utilize Stat1ß to activate transcription. Given that Stat2 can heterodimerize with Stat1 and activate IRF-1 expression [53], Stat2 is the likely heterodimeric partner of Stat1ß that forms GAF and mediates IFN induction of IRF-1 expression in U6A cells. The inability of IFN to utilize Stat1ß adds another level to the divergence between the type I and type II IFN signaling pathways and the specificity of the type I IFN signaling pathway.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH grant 2R01-HD32534 (to F.W.B. and T.E.S.) and in part by NIH grants F32-HD08501 (to G.A.J.) and P30 ES09106. 
2 Correspondence: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 442C Kleberg Center, 2471 TAMU, Texas A&M University, College Station, TX 77843-2471. FAX: 979 862 2662; tspencer{at}tamu.edu
3 Current address: Department of Animal and Veterinary Science, Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-2330.
Accepted: September 18, 2001.
Received: August 14, 2001.
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) regulation of IFN-stimulated gene expression in cell lines lacking specific IFN-signaling components. Endocrinology 2001; 142::1786-1794This article has been cited by other articles:
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H. Khatib, R. L. Monson, V. Schutzkus, D. M. Kohl, G. J. M. Rosa, and J. J. Rutledge Mutations in the STAT5A Gene Are Associated with Embryonic Survival and Milk Composition in Cattle J Dairy Sci, February 1, 2008; 91(2): 784 - 793. [Abstract] [Full Text] [PDF]
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M. M. Joyce, J. R. Burghardt, R. C. Burghardt, R. N. Hooper, L. A. Jaeger, T. E. Spencer, F. W. Bazer, and G. A. Johnson Pig Conceptuses Increase Uterine Interferon-Regulatory Factor 1 (IRF1), but Restrict Expression to Stroma Through Estrogen-Induced IRF2 in Luminal Epithelium Biol Reprod, August 1, 2007; 77(2): 292 - 302. [Abstract] [Full Text] [PDF]
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K. J. Austin, A. L. Carr, J. K. Pru, C. E. Hearne, E. L. George, E. L. Belden, and T. R. Hansen Localization of ISG15 and Conjugated Proteins in Bovine Endometrium Using Immunohistochemistry and Electron Microscopy Endocrinology, February 1, 2004; 145(2): 967 - 975. [Abstract] [Full Text] [PDF]
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S. Kim, Y. Choi, F. W. Bazer, and T. E. Spencer Identification of Genes in the Ovine Endometrium Regulated by Interferon {tau} Independent of Signal Transducer and Activator of Transcription 1 Endocrinology, December 1, 2003; 144(12): 5203 - 5214. [Abstract] [Full Text] [PDF]
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Y. Choi, G. A. Johnson, T. E. Spencer, and F. W. Bazer Pregnancy and Interferon Tau Regulate Major Histocompatibility Complex Class I and {beta}2-Microglobulin Expression in the Ovine Uterus Biol Reprod, May 1, 2003; 68(5): 1703 - 1710. [Abstract] [Full Text] [PDF]
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