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Reproductive Biology Program (K.J.A., L.A.R., T.R.H.), Department of Animal Science, Department of Veterinary Science (E.L.B.), University of Wyoming, Laramie, Wyoming 82071; and Genes & Development Research Group (B.M.B., J.C.C.), Departments of Biochemistry & Molecular Biology and Obstetrics & Gynaecology (J.C.C.), University of Calgary, Calgary, Alberta, Canada T2N 4N1
Address all correspondence and requests for reprints to: Thomas R. Hansen, Reproductive Biology Program, University of Wyoming, Laramie, Wyoming 82071-3684. E-mail: thansen{at}uwyo.edu.
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Abstract |
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. Isg15 is one of few gene products identified in murine implantation sites to require presence of the conceptus and not simply differentiation of the stroma. In vitro data support the inference that the pregnancy-specific inducer of uterine Isg15 is a type 1 IFN or a cytokine that signals through a similar pathway. |
Introduction |
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(6, 9). The ISG15 mRNA is localized heavily to glandular epithelium and is present in a more diffuse staining pattern in luminal epithelial, stromal, and myometrial tissues (10). The ISG15 proteins are recognized by antisera against ubiquitin (5, 6) and share limited (
30%) amino acid sequence identity with a tandem ubiquitin repeat (3, 7, 8). Thus, these proteins have also been called ubiquitin cross-reactive protein or UCRP. Human (3, 5, 11) and bovine (9) ISG15 become conjugated to intracellular proteins through the conserved C-terminal Leu-Arg-Gly-Gly amino acids. The pathway for conjugation of ISG15 to target proteins is distinct from that described for ubiquitin (11). Proteins conjugated to ubiquitin are either modified or directed to the proteasome where the targeted protein is degraded and ubiquitin is recycled, but the fate of proteins covalently modified by ISG15 has not been determined.
Antiviral activity in mouse reproductive tissues consistent with IFN action was reported as early as 1980 (12, 13). This antiviral activity was confirmed in the mouse placenta (14, 15) but may not be due to a classical IFN-ß or IFN-
(16). However, more recent findings indicate that IFN--induced genes are expressed in mouse embryos (17) and endometrium (18). IFN- and its receptor have been described in mouse uterine macrophages by d 7 and in uterine natural killer cells by d 9 (19). The IFN- mRNA and protein also have been localized to luminal and glandular epithelia, natural killer cells, macrophages, placental trophoblast cells, and degenerating metrial gland cells in the pregnant mouse uterus (20). Mice deficient in IFN- and IFN- receptor fail to initiate normal pregnancy-induced modification of decidual arteries and display necrosis of the decidua (21). In addition, uterine natural killer cells are the main source of IFN- on the mesometrial side of the uterus, but cells other than natural killer cells or T cells also produce IFN- in the mouse uterus (21). Likewise, gene expression microarray screens have revealed the presence of IFN-ß and several IFN-induced genes in implantation sites (22).
Despite the fact that an IFN- is not produced by conceptuses in nonruminant species, ISG15 expression appears in human endometrium during pregnancy (23). The present study was undertaken to determine whether Isg15 is expressed in the mouse uterus after the onset of implantation. We tested the hypothesis that up-regulation of Isg15 in the murine uterus required presence of the conceptus and was not simply a response to decidualization of the stroma. Finally, we determined whether type 1 IFN (e.g. or ß) is a potential regulator of Isg15 in decidual cells.
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Materials and Methods |
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). To obtain pseudopregnant mice, females were placed with vasectomized males. Mice were killed by cervical dislocation on d 3.5, 4.5, 7.5, and 9.5 of pregnancy and pseudopregnancy, and the uteri were dissected. Uterine horns undergoing artificially induced decidualization were obtained from ovariectomized hormone-treated mice as described previously (24). Briefly, sesame oil was injected in the lumen of uteri sensitized for a deciduogenic stimulus and the uterine horns were dissected 3 d later. These experiments using animals were approved by the University of Wyoming Institutional Animal Care and Use Committee (assurance no. A-3216-01).
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; 25 nM; 1.1 x 108 IU/mg; from R. M. Roberts, University of Missouri, Columbia, MO) for 24 h. The RNA was reverse transcribed with an oligo deoxythymidine primer. Isg15-specific primers (5'-AAGCTCAGCCAGAACTGGTCT-3'; 5'-ATGGCCTGGGACCTAAAGGTGAA-3') were designed based on the published sequence (accession no. NM_015783) and used to amplify a partial Isg15 cDNA using PCR (35 cycles of 95 C, 1 min, 60 C, 1 min, and a single 72 C extension cycle, 7 min). The Isg15 cDNA was cloned into the Zeroblunt vector (Invitrogen, Carlsbad, CA). The 18S ribosomal cDNA was from Ambion, Inc. (Austin, TX) and the Hand2 probe was generated as previously described (25).
In situ hybridization
After fixation in 4% paraformaldehyde in PBS, tissues were dehydrated and then embedded in paraffin for sectioning. The in situ hybridization and detection methods have been described elsewhere (24) using digoxygenin reagents from Roche (Indianapolis, IN). Digoxygenin-labeled Isg15 and Hand2 antisense and sense riboprobes were prepared using T3 or T7 polymerase. Positive hybridization signal was purple, and sections were counterstained with nuclear fast red.
Antirecombinant mouse Isg15 antibody
The coding region of the murine Isg15 cDNA (NM_015783) was amplified using 5' (5'-CTAGGATCCATGGCCTGGGACCTAAAG-3') and 3' (5'-TGTGAATTCCTACCCACCCCTCAGGCG-3') oligonucleotide primers from RNA isolated from d 7.5 pregnant mouse uterus. The Isg15 cDNA was amplified using RT-PCR (35 cycles of 95 C, 1 min, 60 C, 1 min, and a single 72 C extension cycle, 7 min). The amplicon was sequenced to confirm 100% identity with murine Isg15 cDNA and subcloned into pGEX-4T-1 expression vector (Amersham Pharmacia Biotech, Piscataway, NJ) for subsequent transformation into Bl21-RIL Escherichia coli. Expressed glutathione-S-transferase (GST)-Isg15 fusion protein was extracted from Escherichia coli and isolated by binding to glutathione (GSH)-Sepharose (Amersham Pharmacia Biotech). Recombinant mouse Isg15 was cleaved from the GST-Isg15 fusion protein using thrombin and used to immunize rabbits using standard procedures (9). Rabbit serum was tested for presence of anti-Isg15 antibodies using ELISA (10) and Western blot (6, 9).
Cell culture
Mouse endometrial decidual cells, isolated from 72 h deciduomas as described previously (26), were cultured in DMEM containing 10% charcoal-stripped and heat-inactivated fetal calf serum for 24 h before treatment with IFNs. Mouse 3T3 (embryo fibroblast) and L929 (fibroblast) cells were cultured in MEM-Eagle’s Modification with 10% FBS (Sigma, St. Louis, MO). Cells were treated without (control) or with 1000 U/ml (Calbiochem) murine IFN- (4.3 x 106 U/mg), IFN-ß (1.2 x 107 U/mg), or IFN- (1.02 x 107 U/mg) for 24 h. IFNs were added to cultures based on equal antiviral activity to adjust for differences in bioactivity. In a separate experiment, IFNs also were added to cultures on a 15 nM basis to allow for interaction with IFN receptors on an equimolar basis regardless of bioactivity in an antiviral assay.
Western blots
Cells were collected into Laemmli buffer and homogenized as described previously (6, 8). Cellular proteins (20 µl lysate per lane) were separated using 1D-PAGE, and then transferred to 0.2-µm nitrocellulose membranes in single strength Towbin buffer (6, 8). Isg15 and its conjugates were detected using a polyclonal antibody (C2; 1:30,000) against recombinant murine Isg15 (described herein). Alkaline phosphatase conjugated second antibody against rabbit was used at a 1:10,000 dilution (Promega Corp., Madison, WI). Immunoreacting bands were visualized using nitro blue tetrazolium and 5-bromo-4-chloro-2-indolyl phosphate substrate solution (Promega Corp.). Western blots were scanned and quantitated using UNSCANIT software (Silk Scientific, Orem, UT). When using the antimouse Isg15 antibody, the immunoreacting band at 15-kDa represented free (unconjugated) Isg15. Immunoreacting bands more than 30 kDa represented those that became covalently attached to Isg15 (conjugated) in response to IFN treatment.
Statistical analysis
Effects of day (3.5, 4.5, 7.5, 9.5) and pregnancy status (pseudopregnant, pregnant) on Isg15 mRNA were examined using factorial ANOVA (SAS Institute, Inc., Cary, NC) to test the interaction, followed by t test (protected; P < 0.05) on preplanned comparisons to examine effect of pregnancy on each day examined. Effects of IFN (, ß, ) on free and conjugated Isg15 were examined using ANOVA, and protected preplanned t test when comparing each IFN treatment with controls. In cases where residual error was not normally distributed, data were log transformed and then analyzed as described above. This transformation of the data and subsequent analysis did not change interpretation of results.
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Results |
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). Only very low levels of Isg15 mRNA were detected on d 3.5 and 4.5 (the start of implantation). However, levels of Isg15 mRNA significantly (P < 0.05) increased 4.5- and 11-fold on d 7.5 and 9.5 of pregnancy, respectively. A similar increase in Isg15 mRNA expression was not detected in the uteri of pseudopregnant mice, indicating that the increase in Isg15 transcript levels on d 7.5 and 9.5 actually required the presence of an implanted conceptus and was not simply due to maternal hormonal influences.
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). The low levels of Isg15 mRNA in deciduomas were not different from levels in the nonpregnant uterus and were approximately 10-fold lower (P < 0.05) than in decidua from the implantation segments of a pregnant uterus (Fig. 3). Neither Hand2 nor 18S rRNA differed in decidua when compared with deciduoma.
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). Isg15 mRNA was minimally detectable using in situ hybridization in deciduomas (Fig. 4). This is in contrast to heavy staining of Isg15 mRNA in antimesometrial regions of uterine cross-sections in response to the implanting embryo on d 7.5 of pregnancy. Further studies revealed that Isg15 mRNA was only detected in implantation sites of the uterus and not in interimplantation sites (Fig. 4). Thus, the increase in Isg15 mRNA was localized to areas of the endometrium near the conceptus. It is concluded that an artificial deciduogenic stimulus fails to cause an increase in Isg15 expression and that the conceptus has a direct role in augmenting Isg15 gene expression.
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). Recombinant murine Isg15 was purified using GST-GSH affinity, cleaved from GST using thrombin and used to generate polyclonal antibodies that detected recombinant Isg15 but not ubiquitin on Western blots (Fig. 5B). Note also that this antibody showed weak cross-reactivity with recombinant human ISG15.
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). IFNs were added based on antiviral units (1000 U/ml) to equalize bioactivity or based on mass (15 nM) to equalize interaction with IFN receptors. Regardless of method of IFN treatment, the results were similar (Figs. 6 and 7). Mouse decidual cells (as well as 3T3 and L929 cells which were used as positive controls) produced free (unconjugated) and conjugated Isg15 in response to murine IFN- and IFN-ß. However, the level of free Isg15 was not different in IFN--treated compared with control cells. This lack of response of Isg15 to IFN- in L929 cells was associated with a lack of an increase in conjugated Isg15. However, 3T3 and uterine stromal cells treated with IFN- had higher levels of conjugated Isg15 when compared with controls. Perhaps Isg15 was induced by IFN- in these cells but became rapidly conjugated to targeted proteins so that little free Isg15 remained. Regardless, all cells synthesized more Isg15 and the Isg15 became conjugated to more proteins in response to the murine type 1 when compared with type 2 IFN.
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Discussion |
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The uterine response to the invading conceptus could be viewed as inflammatory. The inflammatory and angiogenic responses induced by the implanting conceptus include up-regulation of cytokines and other proinflammatory effectors. Isg15 is induced by IFNs and, possibly other cytokines as part of antiproliferative, antiviral, and inflammatory responses. Thus, it was hypothesized that Isg15 might be induced in the murine uterus in response to the invasive and inflammatory nature of the implanting murine conceptus.
The absence of Isg15 expression in the deciduoma could have been due to the failure of formation of normal antimesometrial decidual cells. To address this, we examined the expression of the Hand2 gene. Hand2 encodes a basic helix-loop-helix transcription factor that is expressed in the decidua and heart (25, 28), and is critical for embryonic heart development (28). In contrast to Isg15, steady-state levels of Hand2 mRNA were similar between decidua and deciduoma. In the normal decidua, Hand2 is expressed in the antimesometrial zone in a pattern overlapping that of Isg15. In the deciduoma, Hand2 mRNA was detected in a similar antimesometrial pattern. It is concluded that antimesometrial cells form correctly in deciduomas because Hand2 mRNA was similar between decidua and deciduoma.
Attenuated Isg15 expression in the deciduoma could be attributed to a lack of a direct effect of the conceptus in up-regulating Isg15 expression. This is in contrast to most genes that are induced in both the decidua and deciduoma. In fact, to our knowledge, there is evidence of only one other gene (fibroblast growth factor 2) that requires the presence of a conceptus for expression in the rodent uterus (29).
Induction of Isg15 gene expression in the endometrium is a conceptus-induced response that is conserved between species exhibiting major differences in mode of implantation. A similar increase in the expression of bovine and human ISG15 in the endometrium in response to the early conceptus has been previously described (6, 9, 23). Because implantation in the cow is superficial and quite different from that of humans and rodents (30), one might predict that a specific target like ISG15 would not show conserved expression across species. At first glance, this would certainly be reasonable in the case of ISG15 because it is IFN--inducible and IFN- is not thought to be expressed outside of the ruminant species. IFN- is produced by the conceptus in ruminants and appears to be critical for the establishment of early pregnancy (31). Although traces of antiviral activity (IFN-like activity), have been detected in murine embryos at the blastocyst stage, the mouse blastocyst does not produce a type 1 IFN that is equivalent to IFN- described in ruminants (16).
It is likely that a type 1 IFN (IFN- or -ß) mediates the induction of Isg15 in the mouse uterus. IFN- has only a minor effect on human ISG15 expression when compared with the more potent effects of IFN- and IFN-ß (32). However, IFN- did induce enough Isg15 that it became conjugated to proteins at higher levels than controls in cultured murine 3T3 and decidual cells. Thus, a minor, perhaps supplementary role of IFN- might be considered during implantation in the murine uterus. It is unclear at the moment whether the conceptus directly produces a type 1 IFN that acts on the decidual cells, or whether the conceptus produces another factor, which in turn stimulates local IFN release as an intermediate response.
The precise cellular function(s) of Isg15 are unknown. Human ISG15 and its conjugates are localized in a punctate cytoskeletal pattern similar to that observed for intermediate filament-associated proteins (i.e. cytokeratin and vimentin) in the cytoplasm in many human tissues (33, 34). Loeb and Haas (34) hypothesized that one function of human ISG15 might be to direct the association of otherwise soluble target proteins to these fibers. Vimentin filaments become phosphorylated in response to viral infection (35, 36). Thus, it also was suggested that ISG15 and its conjugates become associated with these filaments and potentially block binding or inhibit the action of viral proteins as part of an antiviral response. Human ISG15 was not associated with microtubules or actin fibers. Also, using immunocytochemical approaches, it has recently been reported that human ISG15 is absent in nonpregnant tissue but accumulates along with its conjugates in decidual cells during pregnancy (23). Because human ISG15 and its conjugates are present in many tissues and cell lines and found to be associated with the cytoskeletal network, some functions may be universal. The roles of ISG15 and its conjugates are unknown. Also, it is not known if ISG15 undergoes polymerization in a manner similar to ubiquitin.
The E1-activating enzyme for conjugation of ISG15 was recently described (37, 38). Some cell lines fail to form conjugates with ISG15 (39, 40). This might be caused by the deletion or mutation of the ISG15 E1 as a result of immortalization of the cells. For example, several lung cancer cell lines do not contain the ISG15-E1 and this might be related to carcinogenic properties of these cells (40). Also, some viruses specifically inhibit the conjugation of ISG15 and this might be related to host-cell suicide and inflammatory responses (37). In the present experiments, free, but not conjugated murine Isg15 was detected in cultured L929, 3T3, and decidual cells when using antibodies against human or bovine ISG15 (not shown). However, use of a new antibody against recombinant murine Isg15 revealed that both free and conjugated murine Isg15 could be detected in these cells. Thus, the Isg15 conjugating pathway appears to be intact and responsive to type 1 IFN in decidual cells. Many endometrial proteins become conjugated to ISG15 in response to pregnancy and IFN- in cattle (9), yet none have been identified to date. The identification of these proteins will be critical to understand the function of ISG15 during implantation.
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Footnotes |
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Abbreviations: GSH, Glutathione; GST, glutathione-S-transferase; Hand, basic helix-loop-helix transcription factor; IFN, interferon; ISG, IFN-stimulated gene; ISG15, ISG that encodes the ubiquitin-related protein; URCP, ubiquitin cross-reactive protein.
Received November 12, 2002.
Accepted for publication March 11, 2003.
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induce conjugation of bovine ubiquitin cross-reactive protein to cytosolic uterine proteins. Biol Reprod 58:898–904 inducible ubiquitin cross-reactive protein in the ovine uterus. Biol Reprod 61:312–318 and ß. Mol Reprod Dev 26:122–128[CrossRef][Medline]
-regulated gene in human and rat endometrium. Endocrinology 142:2390–2400 receptor gene in mouse uterine embryonic hematopoietic cells. J Leukoc Biol 55:617–625[Abstract]
gene expression in cycling and pregnant mouse uterus: temporal aspects and cellular localization. J Leukoc Biol 64:393–400[Abstract]
contributes to the normalcy of murine pregnancy. Biol Reprod 61:493–502This article has been cited by other articles:
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