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Regular Article |
Upregulate Gene Expression of Members of the 1-8 Family in the Bovine Uterus1
a Department of Animal Science, University of Wyoming, Laramie, Wyoming 82071
b Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
ABSTRACT
The 1-8 family (1-8U, 1-8D, Leu-13/9-27) of interferon (IFN)-inducible genes encodes proteins that are components of multimeric complexes involved with transduction of antiproliferative and homotypic adhesion signals. Human 1-8 family members are highly similar and are regulated by type 1 and type 2 IFNs. Because the bovine uterus is bathed in conceptus-derived IFN
during early pregnancy, it was hypothesized that members of the 1-8 family were upregulated in the bovine uterus during early pregnancy. Oligonucleotide primers were designed based on human and rat 1-8U and Leu-13 cDNAs and used in reverse transcription polymerase chain reactions to amplify bovine cDNAs from endometrial RNA. The bovine 1-8U cDNA was sequenced, found to be 84% identical to the human 1-8U, and used to screen a bovine endometrial cDNA library to isolate the full-length 1-8U and Leu-13 cDNAs. The inferred amino acid sequences of bovine 1-8U and Leu-13 were 72% and 73% identical to their respective human counterparts. Bovine 1-8U and Leu-13 retain an amino acid motif that is conserved in other 1-8 family members and in some ubiquitin-conjugating enzymes (E2s). This motif is critical for function of E2s in covalently linking ubiquitin to targeted proteins. Northern blotting revealed that bovine endometrial 1-8U and Leu-13 mRNAs were upregulated on Day 15 of pregnancy (P < 0.0001) and continued to accumulate through Day 18 of pregnancy (P < 0.05) when compared with endometrium from nonpregnant cows. The bovine 1-8U and Leu-13 mRNAs were also upregulated (P < 0.05) by IFN (25 nM) within 3 h, continued to accumulate through 12 h, and reached a plateau at 12–24 h in cultured bovine endometrial cells. In situ hybridization revealed that mRNAs encoding 1-8 family members were heavily localized to glandular epithelium but also were present to a lesser extent in the luminal epithelium and stroma. The temporal upregulation of 1-8U and Leu-13 mRNAs by pregnancy and IFN and tissue distribution of these mRNAs paralleled closely that of the ubiquitin homolog, IFN-stimulated gene product 17. These IFN-induced proteins probably work together to prepare the endometrium for adhesion of the developing conceptus.
cytokines, implantation, pregnancy, uterus
INTRODUCTION
The bovine conceptus secretes interferon (IFN)- during the peri-implantation period [1, 2]. It is generally accepted that IFN is the maternal recognition of pregnancy signal in ruminants. In cattle, IFN functions to limit the release of the luteolysin prostaglandin F2
(PGF), thereby rescuing the corpus luteum from regression (reviewed in [2, 3]). In this way, continued exposure of the endometrium to progesterone supports the processes of adhesion, implantation, placentation, and embryogenesis and prevents the ensuing estrous cycle. Interferon- also induces the expression of numerous uterine proteins. One of these uterine proteins is the ubiquitin homolog IFN-stimulated gene product 17 (ISG17). ISG17 becomes covalently linked to targeted intracellular proteins [4], is released from endometrial cells [5], and may function as a paracrine modulator [6].
Bovine ISG17, also known as ubiquitin cross-reactive protein [5, 7], is the ortholog of human ISG15 [8, 9]. The difference in nomenclature is real versus relative mass. The bovine ISG17 gene [7] encodes a protein of 17 kDa that migrates to an apparent molecular weight of 17 000 on polyacrylamide gels. Human and mouse genes encode a pre-ISG15 that is processed [10, 11] to yield a mature 17-kDa protein that migrates to an apparent molecular weight of 15 000 on polyacrylamide gels. ISG15 has an extracellular cytokine role in inducing proliferation of natural killer cells and non-major histocompatibility complex-restricted cytotoxicity [12]. Also, ISG15 [13] and ISG17 [6] induce release of IFN
by cultured peripheral blood mononuclear cells. These ISGs encode two ubiquitin-like (
30% identity) domains [7, 14] that terminate in C-terminal LRGG amino acid residues that are required for the covalent attachment of ubiquitin to cellular proteins. Proteins coupled to ubiquitin often are degraded through the 26S proteasome [15]. ISG15 also becomes covalently attached to targeted proteins through a pathway that includes the C-terminal LRGG amino acid residues but is distinct from that described for ubiquitin [16]. The fate of conjugates appears to be different for ISG15 than for ubiquitin. For example, proteins conjugated to ISG17 [4] continue to accumulate rather than disappear via degradation through the 26S proteasome. Another major difference between the pathways is that neither ubiquitin nor its conjugates are induced by IFN or by pregnancy [4].
Other IFN-induced proteins have been identified, but function in the endometrium during early pregnancy is unknown. These proteins may play an important role in establishing communication between the mother and embryo and in preparing the uterus for implantation and include granulocyte chemotactic protein 2 [17, 18], the GTPase Mx [19, 20], 2',5'-oligoadenylate synthetase [21], and the 1-8 family described here.
Three functional members of the 1-8 gene family have been isolated on a genomic DNA fragment of less than 18 kilobases (kb) in the human [22]: 1-8U, 1-8D, and Leu-13/9-27. The promoter of each family member contained multiple IFN-stimulated response elements (ISREs) [23]. 1-8U and 1-8D are exclusively induced by type 1 IFNs (, ß, 
), and Leu-13 gene expression is promoted by type 1 and type 2 IFNs () [24, 25]. Leu-13 was originally identified as a 16-kDa protein that was localized to the surface of normal T cells [26]. Treatment of T cells with anti-Leu-13 monoclonal antibody caused homotypic aggregation of these cells [26]. Leu-13 also has been immunolocalized to adult human endothelium of major organs and to epithelium of renal proximal tubules, cervix, and esophagus [26]. These observations indicate that the 1-8 proteins modulate cellular growth and adhesion.
The molecular events of implantation in ruminants include regulation of cellular growth and adhesion. The roles of IFN and induced endometrial proteins in this process are largely unknown because most uterine proteins induced or upregulated by IFN have yet to be examined in detail. The interaction between and the relationship of function of these IFN-induced proteins in the endometrium during pregnancy also have not been described. The current experiments were designed to test the hypothesis that members of the 1-8 family of genes are expressed in the bovine endometrium in response to pregnancy and recombinant (r) IFN. The objectives were to clone and sequence full-length bovine 1-8 cDNAs and to determine the temporal and spatial changes in mRNA expression during corresponding times of the estrous cycle and early pregnancy and in bovine endometrial (BEND) cells in response to rIFN. Long-term goals include clarifying the function of these IFN-induced proteins and their relationship to ISG17 and its conjugates in the endometrium during early pregnancy.
MATERIALS AND METHODS
Animal and Cell Culture Models
Estrous cycles in cows were synchronized using synthetic prostaglandin F2 (Lutalyse; Upjohn Co, Kalamazoo, MI), and cows were observed for signs of estrus. The day of estrus was defined as Day 0 of a 21-day estrous cycle. Cows assigned to the pregnant group were artificially inseminated about 12 h after standing estrus. Cows assigned to the nonpregnant group were not exposed to semen. For both pregnant and nonpregnant cows, the uterine horn and ovary ipsilateral to the corpus luteum were surgically removed as approved by the University of Wyoming Animal Care and Use Committee. Uterine horn cross sections were collected and prepared for in situ hybridization. For Northern blotting, endometrial tissue was dissected from uterine horns obtained from nonpregnant (Days 0, 12, 15, and 18) and pregnant (Days 12, 15, and 18) cows (three cows on each day). Tissue was snap frozen in liquid nitrogen and stored at -80°C.
BEND cells in primary culture [18] were used to study the induction of 1-8 mRNA by rIFN. The BEND cells were developed at the University of Wyoming and are currently available from the American Type Culture Collection (Manassas, VA).
Reverse Transcription Polymerase Chain Reaction Amplification of Bovine 1-8U and Leu-13 cDNA
BEND cells (9 x 106) were cultured in flasks (T75) in the presence (25 nM) or absence of rIFN for 24 h as described elsewhere [18]. Cells were harvested via scraping and centrifugation, and total cellular RNA was isolated using Tri Reagent (Sigma Chemical Co., St. Louis, MO). RNA (1 µg) was reverse transcribed and amplified by the polymerase chain reaction (PCR) using the GeneAmp RNA PCR kit (Perkin Elmer, Branchburg, NJ). Primers for 1-8U (5'-GATGTTCAGGCACTTGGCGGT, 5'-CTGCTGCCTGGGCTTCAT), and Leu-13 (5'-CAGGGCCCAGATGTTCAGGCA, 5'-GTCTGGTCCCTGTTCAA) were designed from human and rat consensus sequences (EMBL/GenBank accessions J04164, X57352, X61381, and AF164039). Amplified PCR products were subcloned into pBluescript (Stratagene, La Jolla, CA) and sequenced using the dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA). Cycle sequencing was performed using an Ericomp thermocycler (25 cycles of 95°C for 30 sec, 50°C for 15 sec, and 60°C for 4 min).
Screening the cDNA Library
A 
ZAP II cDNA library was constructed from mRNA isolated from a bovine endometrial primary cell culture [27]. A total of 500 000 plaque-forming units (pfu) were screened by first mixing phage (5000 pfu per 150-mm plate) with XL1 Blue Escherichia coli cells (optical density = 0.2; Clontech, Palo Alto, CA) in 10 mM MgSO4. After a 30-min incubation (37°C), infected cells were added to top agar (8 ml, 0.7% agarose, 10 mM MgSO4) and immediately poured over Luria-Bertani agar plates. Plates were incubated at 37°C for 12 h. Plaques were lifted onto nylon membranes (0.2 µm; Micron Separations, Westborough, MA) and hybridized using standard procedures [28] with the partial length radiolabeled 1-8U cDNA (derived from reverse transcription [RT]-PCR). Plaques were purified with a secondary screening. Positive plaques were selected and then eluted overnight in SM buffer (0.1 M NaCl, 0.05 M Tris, pH 7.5, 8 mM MgSO4). Phage were amplified, and the SK- plasmid was excised using the R408 helper phage. Four plasmids containing inserts of the appropriate size were selected for amplification and sequencing. Sequences were aligned and compared with human 1-8 family members (X57351, J04164).
Gene Expression of 1-8 Family Members
Gene expression of 1-8 family members was determined using Northern blotting. Ten micrograms of total cellular RNA was denatured (5 min, 70°C), electrophoresed in a 1.5% agarose-formaldehyde gel, and passively transferred to nylon membranes (0.2 µm) by capillary blotting. Membranes were baked (2 h, 80°C) and prehybridized (50% formamide, 5x saline-sodium citrate [SSC], 50 mM NaPO4, 5x Denhardt solution, 0.1% SDS, 0.1 mg/ml salmon sperm DNA) for 3 h at 42°C. Blots were hybridized (15 h, 42°C) by adding the RT-PCR amplified 1-8U cDNA randomly primed with 50 µCi deoxycytidine 5' [-32P]triphosphate (3000 Ci/mmol; Pharmacia Amersham, Piscataway, NJ) to the prehybridization solution. Blots were washed as described previously [29] and exposed to x-ray film for 4 days. Blots were reprobed with radiolabeled cDNA for murine 18S rRNA (Ambion, Austin, TX) to ensure equal loading. Autoradiographic signals were quantified using UnScan-It Automated Digitizing System, Version 5.1 (Silk Scientific Corp., Orem, UT). To determine bovine (b) 1-8U and bLeu-13 gene expression, sequence-specific probes were generated from the 3' regions of each clone, which retained less than 20% nucleotide sequence similarity. Double digestion (SacI/XhoI) of the b1-8U clone resulted in a 116-base pair (bp) fragment corresponding to nucleotide positions 463–579. Double digestion (AccI/XhoI) of the bLeu-13 clone resulted in a 205-bp fragment corresponding to nucleotide positions 417–622. Each DNA probe was tested for the ability to hybridize with the b1-8U and bLeu-13 full-length cDNAs (EcoRI/XhoI) via Southern blotting using standard procedures [28].
In Situ Hybridization
Uterine horn cross sections were fixed in 4% paraformaldehyde (4% paraformaldehyde in PBS, pH 7.2) for 24 h (25°C). Sections were transferred to 70% ethanol, which was changed for three consecutive days. Tissues were dehydrated and then infiltrated and embedded in paraffin. Serial cross sections (6 µm) were prepared. Tissue sections were postfixed in 4% paraformaldehyde in PBS and digested (8 min, 37°C) with Proteinase K (20 µg/ml) dissolved in digestion buffer (50 mM Tris, 5 mM EDTA, pH 8). Sections were hybridized with radiolabeled sense and antisense cRNA probes transcribed in vitro with uridine 5' [-35S]thiotriphosphate (>1000 Ci/mmol; Pharmacia Amersham). Antisense and sense cRNA probes were constructed from the 1-8U plasmid. Radiolabeled cRNA probes (3 x 106 cpm/slide) were denatured in hybridization buffer (50% formamide, 0.3 M NaCl, 20 mM Tris, pH 8, 5 mM EDTA, 10 mM NaPO4, 1x Denhardt solution, 10% dextran sulfate, 0.5 mg/ml yeast RNA, 100 mM dithiothreitol) for 10 min at 70°C. Hybridization solution (75 µl) was added, and a coverslip was gently placed on top. Slides were incubated in a humidified chamber (50% formamide, 2x SSC) at 55°C for 15 h. Washing conditions were identical to those described by Johnson et al. [30]. Slides were dipped in Kodak NTB-2 liquid emulsion, air dried, and held at 4°C for 4 days. Slides were developed and counterstained with 0.025% Giemsa stain and then coverslipped.
Statistics
Assignment to treatments was made at random. Data were subjected to least-squares factorial analysis of variance using the general linear models procedures of the Statistical Analyses System [31] followed by protected (P < 0.05) t-tests for paired comparisons. Main effects were pregnancy status (nonpregnant vs. pregnant) and day (0, 12, 15, 18) and associated interaction (pregnancy x day) or IFN treatment (0 or 25 nM) and time (0, 3, 6, 12, 24, 48 h) and associated interaction (IFN x time). The results are expressed as the mean ± SEM.
RESULTS
RT-PCR Amplification of b1-8U and bLeu-13 cDNAs
Nucleotide sequencing revealed that the partial b1-8 PCR fragments generated using human (h) and rat consensus primers were highly similar (81% and 83%) to h1-8U and hLeu-13. These data represent the first evidence that BEND cells treated with IFN transcribe mRNAs for members of the 1-8 family.
cDNA Library Screening
Using the radiolabeled 1-8U PCR fragment (114 bp) as a probe, 500 000 plaques from a bovine endometrial cDNA library [27] were screened to isolate full-length b1-8U and bLeu-13 clones. Because this radiolabeled 1-8U cDNA probe shared high nucleotide sequence identity with h1-8U and hLeu-13, we anticipated that multiple members of the 1-8 family would be isolated following this primary screen. One hundred positive plaques were isolated, 20 of which were purified by a secondary screening. After excising 20 SK- plasmids from phage, four clones with inserts of approximately 600 bp were further characterized. DNA sequencing revealed that three clones (b1-8a–c) encoded b1-8U and one clone (b1-8d) encoded bLeu-13.
Nucleotide Sequence of b1-8U
The b1-8U cDNA (EMBL/GenBank accession AF272041) is 579 bp in length and contains an open reading frame of 146 codons. It differs from the h1-8U cDNA (EMBL/GenBank accession X57352), which contains additional nucleotides in the 3' and 5' untranslated regions but has only 133 codons in the coding region. The translation initiation site was assigned to an in-frame ATG codon 49 bases from the 5' end [32]. The first in-frame stop codon, TAG, is located at positions 487–489, and the putative polyadenylation site [33] begins at position 556. The number of codons within the coding region differs between species in that the b1-8U cDNA contains an additional 13 codons at the 3' end. When comparing b1-8U and bLeu-13, the untranslated regions retain only 38% and 20% identity at the 5' and 3' ends, respectively. A comparison of sequence identities is shown in Table 1.
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Nucleotide Sequence of bLeu-13
The bLeu-13 cDNA (EMBL/GenBank accession AF272042) is 622 bp in length and, like hLeu-13 (EMBL/GenBank accession J04164), contains an open reading frame of 125 amino acids. The 5' and 3' untranslated regions of bLeu-13 are truncated and highly divergent from hLeu-13. The putative translation initiation site is located at position 87. An in-frame TAG stop codon was identified at positions 461–463. The position of this codon was conserved in the hLeu-13 sequence. The putative polyadenylation hexanucleotide [33] of bLeu-13 aligned with hLeu-13 at positions 601–606. Nucleotide sequences of b1-8U and bLeu-13 were 96% identical.
Amino Acid Sequence Analysis of b1-8U and bLeu-13
Figure 1A shows a comparison of the inferred amino acid sequences of known bovine, human, rat, and mouse 1-8 family members. Bovine 1-8U and bLeu-13 are 146 and 125 amino acids in length, respectively. Table 1 shows the amino acid similarities between b1-8U, bLeu-13, and other 1-8 family members. Amino acids 94–108 of b1-8U and 73–87 bLeu-13 (Fig. 1B) retain critical residues found in the active sites of human E2 ubiquitin conjugation enzymes. Each conjugating enzyme retains a cysteine residue that is required for thioester bond formation with activated ubiquitin.
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Computer analysis indicated that both b1-8U and bLeu-13 lack a signal sequence [34]. The predicted molecular masses for b1-8U and bLeu-13 are 15.7 kDa and 14.0 kDa, respectively. Profiling analysis indicated that b1-8U contains two putative N-linked glycosylation sites at amino acid residues 2 and 127 and three putative myristylation sites at residues 11, 33, and 114 [35]. Bovine Leu-13 contains putative myristylation sites at residues 2 and 93 but lacks sites for N-linked glycosylation. Putative protein kinase C phosphorylation sites were also found at multiple positions on each protein. Both proteins were predicted [36] to contain two large alpha helical regions. These regions include amino acids 60–79 and 107–132 of b1-8U and 38–58 and 86–115 of bLeu-13. These regions were also predicted to be transmembrane regions (Fig. 2; http://www.biokemi.su.se/server/DAS/) and are highly conserved between bovine and human 1-8 family members. This prediction was confirmed by TopPred 2 [37].
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Gene Expression of 1-8 Family Members
The 114-bp 1-8 cDNA probe generated by RT-PCR hybridized to both b1-8U and bLeu-13 full-length cDNAs (Fig. 3A). This result was expected because the sequences are 98% identical in the region amplified as the RT-PCR product. Unique nucleotide sequences within b1-8U and bLeu-13 cDNAs were identified and confirmed using Southern blot analysis. These truncated "specific" cDNA probes were used to study the expression of individual 1-8 family members. Probes generated from b1-8U and bLeu-13 in nontranslated 3' regions hybridized only to corresponding full-length cDNAs (Fig. 3, B and C).
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Northern blot analysis was performed (Fig. 4A) and quantitated (Fig. 4B) to identify differences in amount of the mRNA transcripts for b1-8 family members during the estrous cycle and early pregnancy (time x status interaction, P < 0.0001). Blots were hybridized with a radiolabeled cDNA probe common to b1-8 family members. Bovine 1-8 transcripts were detected only on Days 15 and 18 of pregnancy and were absent on Day 12 of pregnancy and during the estrous cycle. Quantitation of these data revealed that expression of b1-8 mRNAs is elevated (P < 0.0001) in pregnant when compared with nonpregnant cows (Fig. 4B). Detection of 18S rRNA was similar across all samples evaluated using Northern blots. Because no differences in rRNA were detected for any Northern blot, normalization was not deemed necessary.
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To determine whether IFN was the pregnancy-specific factor that induced the expression of all b1-8 mRNAs, BEND cells were treated with 0 or 25 nM rIFN (Fig. 4, C and D; time x treatment interaction, P < 0.0001). Expression of b1-8 mRNAs was detected at very low levels in untreated BEND cells (Fig. 4D). Recombinant IFN upregulated (P < 0.001) the expression of b1-8 mRNAs starting at 3 h. Levels of b1-8 mRNA peaked at 12 h but declined by 35% at 48 h.
Figure 5 shows quantitation of b1-8U (Fig. 5, A and B) and bLeu-13 (Fig. 5, C and D) mRNA expression during the estrous cycle and early pregnancy (time x status interaction, P < 0.05) and in BEND cells treated with rIFN (time x treatment interaction, P < 0.05). Messenger RNA-specific probes were used during hybridization (see Fig. 3). Both b1-8U and bLeu-13 were upregulated (P < 0.001) in the endometrium of pregnant cows on Days 15 and 18 when compared with corresponding days of the estrous cycle. Likewise, b1-8U and bLeu-13 were upregulated (P < 0.001) in BEND cells in response to rIFN. Unlike b1-8U, bLeu-13 expression significantly declined (P < 0.05) between 24 and 48 h in BEND cells (Fig. 5D). Based on pixel density, b1-8U was expressed at higher (P < 0.05) levels than bLeu-13.
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A 6-kb transcript was detected in BEND cells that hybridized with the b1-8U-specific probe (Fig. 6A). The b1-8U-like transcript did not appear until 6 h and remained elevated through the remainder of the time course (Fig. 6B). This transcript was not detected in RNA preparations obtained from the endometrium or in untreated BEND cells. No upper transcript was detected when blots were probed with the bLeu-13-specific probe.
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In Situ Hybridization
In situ hybridization was used to study the cellular localization of mRNA for b1-8 family members within the endometrium of Day 17 nonpregnant and pregnant cows (Fig. 7). The full-length b1-8U clone was used to generate sense and antisense cRNA probes. Because of the significant sequence homology between b1-8 family members, it is likely that the antisense probe hybridized to all 1-8 family members. Bovine 1-8 family members were largely localized to the glandular epithelium within the endometrium of pregnant cows and to a lesser degree to the luminal epithelium, stroma, and myometrium. As was shown with Northern blotting, endometrium from nonpregnant cows expressed little or no b1-8 mRNAs. Actin was used as a positive control and was localized to all cells indiscriminately in both nonpregnant and pregnant uterine sections.
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DISCUSSION
The major secretory product generated by the preimplantation bovine conceptus is IFN [1–3]. Interferon- is secreted from Day 15 to Day 26 postconception, with maximal secretion occurring around Day 18 [3]. The pulsatile release of uterine-derived PGF is attenuated in response to IFN [3]. Because IFN attenuates PGF, the corpus luteum continues to generate progesterone, which is critical for the maintenance of pregnancy. Several endometrial proteins are induced by IFN. Here, we discuss the cloning, sequencing, and temporal and spatial mRNA expression of two members of the 1-8 family of IFN-inducible genes in the bovine endometrium.
Much of what is known about the function of this family of proteins stems from limited work on hLeu-13 after it was first identified as a 16-kDa T-cell surface antigen [25]. Leu-13 is expressed in many tissues, including the vasculature, epithelia of the renal proximal tubules, nonkeratinized basal epithelia of the cervix, esophagus, and medullary thymocytes, and placental trophoblasts [26]. Leu-13 is physically associated with other cell surface membrane proteins in B cells [38, 39]. This complex, referred to as the molecular facilitator, consists of CD19, CD21, CD81/TAPA-1, and Leu-13. Signal transduction generated through the cytoplasmic domain of CD19 is essential for B-cell development and function [39]. Although CD19 is the dominant signaling component of the complex [40], activating antibodies that bind other components of the molecular facilitator induce similar biologic responses, including induction of homotypic adhesion and increases in intracellular calcium in leukocytes [41]. Moreover, hLeu-13 may play a role in the antiproliferative effects of IFNs because membrane fractions enriched with Leu-13 inhibit cell growth [41, 42]. Functions for other members of the 1-8 family have not been described.
Retention of structural similarity between family members would suggest that the proteins have related functions. However, some amino acid differences occur at the carboxyl and amino termini of b1-8 family members, which may contribute to subtle differences in function or cellular localization. Prediction data described herein for the identification of myristylation sites and transmembrane regions corroborate data collected by others [22, 25, 42], suggesting that members of the 1-8 family are associated with membranes. There are sufficient amino acids in the predicted carboxyl terminal transmembrane domains of b1-8U (N107-I132) and bLeu-13 (N86-V115) to traverse a phospholipid bilayer two times. Based on prediction analysis, transmembrane domains typically consist of 20 or more amino acids. In addition, the amino acids that are found at either end of the putative transmembrane domain are such that they help to stabilize the protein within the phospholipid bilayer. Additionally, internal transmembrane amino acids are typically hydrophobic in nature. The prediction program used herein recognized these features and "predicted" that the 1-8 family members retain transmembrane domains in the carboxyl terminal region. Because the 1-8 family members have two such domains, this would place both the C- and N-termini on the same side of the membrane. Identification of multiple phosphorylation sites suggests that 1-8 family members are allosterically regulated, although the functional importance of these sites has not been determined.
A general cDNA probe that recognized multiple members of the 1-8 family was used for Northern blotting to determine temporal mRNA expression of the 1-8 family in the endometrium and in BEND cells treated with rIFN. Similar to bISG17 mRNA [43], expression of the 1-8 mRNA family was induced in the endometrium of Day 15 and Day 18 pregnant cows. Expression within the endometrium was highest on Day 18, a time when conceptus-derived IFN secretion naturally peaks. Messenger RNAs for the 1-8 family were also upregulated in BEND cells treated with 25 nM rIFN. The concentration of rIFN used for treatment is consistent with that used by our laboratory [18, 43] and others [44] and represents physiologic levels of cytokines. Untreated BEND cells and endometrium from nonpregnant cows showed limited expression of b1-8U and bLeu-13 mRNAs. ISG17 mRNA is also present at very low levels in nonpregnant cows and in untreated BEND cells. The 1-8 family of proteins and ISG17 may be involved in normal cellular processes in endometrium from nonpregnant cows. Evidence is provided here that IFN is the product of pregnancy that regulates mRNA expression of 1-8 family members.
Specific probes constructed from the 3' untranslated regions of b1-8U and bLeu-13 cDNAs were used to study the independent expression of b1-8U and bLeu-13 in the endometrium and BEND cells treated with rIFN. There was little difference in the timing of expression of the two 1-8 family members. This result was somewhat unexpected because h1-8U and hLeu-13 promoters are quite different. The h1-8U promoter possesses three ISREs, whereas the hLeu-13 promoter has only one ISRE [22]. Each promoter differs in the number and type of other response elements. Although h1-8U is exclusively regulated by type 1 IFNs, hLeu-13 is the only gene known to be regulated by both types (1 and 2) of IFNs [22].
A 6-kb transcript also was detected with the b1-8U-specific cDNA probe in IFN-treated BEND cells. This transcript was not detected in endometrial preparations. The larger transcript may represent an unprocessed or alternatively spliced form of b1-8U or it may be a homologous transcript. The larger transcript is temporally regulated in BEND cells by IFN in a manner similar to that of b1-8U mRNA. The larger transcript is found in BEND cells following culture with IFN but not in the endometrium from pregnant cows possibly because BEND cells represent a highly enriched epithelial cell population [45] and may be a highly enriched source for the larger mRNA when compared with the more complex endometrial tissue. A b1-8U antisense riboprobe was generated to determine the source of 1-8 mRNA in endometrial tissue. Because of the considerable homology between family members, this probe likely hybridized with many members of the 1-8 family. The 1-8 family of mRNAs is distributed primarily in the glandular epithelium. Hybridization signal was also observed in the superficial stroma and to a much lesser degree in the luminal epithelium. This pattern of expression for the 1-8 family is identical to that described for ISG17 in both sheep [30] and cows [29]. Expression of these IFN-induced proteins occurs in stromal tissue even though IFN does not appear to breech the basement membrane of the overlying epithelium and is not found in surrounding venous blood [46, 47]; yet, both the epithelial and stromal components of the endometrium are responsive to treatment with rIFN. Methods used to detect IFN outside the lumen of the uterus may not be sensitive enough to measure small amounts of IFN that may diffuse across the basement membrane.
Members of the b1-8 family retain amino acids critical for the function of ubiquitin E2-conjugating enzymes [48]. Of particular importance is the cysteine residue within the active site. This residue forms a thioester bond with ubiquitin. Ubiquitin-conjugating enzymes are critical for substrate recognition. Multiple E2 isoforms exist, and each one is responsible for the transfer of ubiquitin to specific subsets of proteins [48]. Ubiquitination may target proteins for degradation by the proteasome or may alter function of the protein. Although ubiquitin levels do not change in the bovine endometrium in response to IFN, the ubiquitin homolog ISG17 is upregulated [30, 43]. Bovine ISG17 is a 17-kDa protein consisting of two ubiquitin-like domains. Like ubiquitin, ISG17 is found in free form but also becomes covalently linked to targeted proteins within the endometrium of pregnant cows [4]. Unpublished results (Hansen Laboratory) obtained from the transfection of BEND cells provide preliminary evidence that the conjugation of ISG17 to targeted proteins is dependent upon IFN. It is likely that the enzymes required for this process are regulated, either transcriptionally or posttranslationally, by IFN. Because b1-8U and bLeu-13 retain a conserved E2 motif and they are regulated by IFN in parallel with ISG17, we hypothesize that members of the 1-8 family function as novel E2-like enzymes that facilitate formation of an isopeptide bond between bISG17 and targeted cytosolic proteins. Alternatively, members of the 1-8 family may be involved with the release of ISG17 from the endometrium. ISG17 is present in uterine flushings from Day 18 pregnant cows [5]. However, ISG17 does not possess a signal sequence typically associated with secreted proteins. If members of the 1-8 family are localized to the plasma membrane as predicted, they also might function to shuttle ISG17 across the membrane. Future experiments will test these hypotheses.
The present work demonstrates that the 1-8 family of genes is upregulated in the bovine uterus in response to pregnancy and IFN. Two family members, b1-8U and bLeu-13, were cloned and sequenced, and mRNAs were localized primarily in the glandular epithelium but also to a lesser degree in the luminal epithelium and stroma. Endometrial expression of b1-8 family members parallels expression of the ubiquitin homolog ISG17. After generating recombinant b1-8U and bLeu-13 and antibodies against these proteins, future experiments will be designed to study the colocalization of b1-8 family members and ISG17 and to test the hypothesis that b1-8 family members are E2-like conjugating enzymes. The hypothesis that the 1-8 proteins are involved with adhesion of the conceptus to the endometrium in preparation for final development of the placental exchange organ also will be tested. Because 1-8 proteins function in adhesion, wound healing, and inflammatory responses, it is likely that they are induced by IFN and mediate similar uterine responses during adhesion of the conceptus and development of the placentome.
ACKNOWLEDGMENTS
The authors thank Dr. R. Michael Roberts (University of Missouri) for the generous gift of rbIFN.
FOOTNOTES
1 Supported in part by NIH grant R01-32475-6 and USDA 97-02406 awarded to T.R.H. Research contributed in part to the 2000 SSR Trainee Research Award and to requirements for the Ph.D. degree at the University of Wyoming (J.K.P.). 
2 Correspondence. FAX: 302 766 2355;thansen{at}uwyo.edu
3 Current Address: Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114.
Accepted: June 28, 2001.
Received: May 8, 2001.
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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. Kaneko-Tarui, L. Zhang, K. J Austin, L. E Henkes, J. Johnson, T. R Hansen, and J. K Pru Maternal and Embryonic Control of Uterine Sphingolipid-Metabolizing Enzymes During Murine Embryo Implantation Biol Reprod, October 1, 2007; 77(4): 658 - 665. [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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 S. Bauersachs, S. E Ulbrich, K. Gross, S. E M Schmidt, H. H D Meyer, H. Wenigerkind, M. Vermehren, F. Sinowatz, H. Blum, and E. Wolf Embryo-induced transcriptome changes in bovine endometrium reveal species-specific and common molecular markers of uterine receptivity. Reproduction, August 1, 2006; 132(2): 319 - 331. [Abstract] [Full Text] [PDF]
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D. D. Michael, I. M. Alvarez, O. M. Ocon, A. M. Powell, N. C. Talbot, S. E. Johnson, and A. D. Ealy Fibroblast Growth Factor-2 Is Expressed by the Bovine Uterus and Stimulates Interferon-{tau} Production in Bovine Trophectoderm Endocrinology, July 1, 2006; 147(7): 3571 - 3579. [Abstract] [Full Text] [PDF]
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M. E. Skaznik-Wikiel, T. Kaneko-Tarui, A. Kashiwagi, and J. K. Pru Sphingosine-1-Phosphate Receptor Expression and Signaling Correlate with Uterine Prostaglandin-Endoperoxide Synthase 2 Expression and Angiogenesis During Early Pregnancy Biol Reprod, March 1, 2006; 74(3): 569 - 576. [Abstract] [Full Text] [PDF]
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C. Klein, S. Bauersachs, S. E. Ulbrich, R. Einspanier, H. H.D. Meyer, S. E.M. Schmidt, H.-D. Reichenbach, M. Vermehren, F. Sinowatz, H. Blum, et al. Monozygotic Twin Model Reveals Novel Embryo-Induced Transcriptome Changes of Bovine Endometrium in the Preattachment Period Biol Reprod, February 1, 2006; 74(2): 253 - 264. [Abstract] [Full Text] [PDF]
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C. A. Gray, C. A. Abbey, P. D. Beremand, Y. Choi, J. L. Farmer, D. L. Adelson, T. L. Thomas, F. W. Bazer, and T. E. Spencer Identification of Endometrial Genes Regulated by Early Pregnancy, Progesterone, and Interferon Tau in the Ovine Uterus Biol Reprod, February 1, 2006; 74(2): 383 - 394. [Abstract] [Full Text] [PDF]
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M. M. Joyce, F. J. White, R. C. Burghardt, J. J. Muniz, T. E. Spencer, F. W. Bazer, and G. A. Johnson Interferon Stimulated Gene 15 Conjugates to Endometrial Cytosolic Proteins and Is Expressed at the Uterine-Placental Interface throughout Pregnancy in Sheep Endocrinology, February 1, 2005; 146(2): 675 - 684. [Abstract] [Full Text] [PDF]
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L. A. Rempel, B. R. Francis, K. J. Austin, and T. R. Hansen Isolation and Sequence of an Interferon-{tau}-Inducible, Pregnancy- and Bovine Interferon-Stimulated Gene Product 15 (ISG15)-Specific, Bovine Ubiquitin-Activating E1-Like (UBE1L) Enzyme Biol Reprod, February 1, 2005; 72(2): 365 - 372. [Abstract] [Full Text] [PDF]
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 K. I. Kim, N. V. Giannakopoulos, H. W. Virgin, and D.-E. Zhang Interferon-Inducible Ubiquitin E2, Ubc8, Is a Conjugating Enzyme for Protein ISGylation Mol. Cell. Biol., November 1, 2004; 24(21): 9592 - 9600. [Abstract] [Full Text] [PDF]
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M. H. Melner, N. A. Ducharme, A. R. Brash, V. P. Winfrey, and G. E. Olson Differential Expression of Genes in the Endometrium at Implantation: Upregulation of a Novel Member of the E2 Class of Ubiquitin-Conjugating Enzymes Biol Reprod, February 1, 2004; 70(2): 406 - 414. [Abstract] [Full Text] [PDF]
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J. Hemelaar, A. Borodovsky, B. M. Kessler, D. Reverter, J. Cook, N. Kolli, T. Gan-Erdene, K. D. Wilkinson, G. Gill, C. D. Lima, et al. Specific and Covalent Targeting of Conjugating and Deconjugating Enzymes of Ubiquitin-Like Proteins Mol. Cell. Biol., January 1, 2004; 24(1): 84 - 95. [Abstract] [Full Text] [PDF]
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V. Emond, L. A. MacLaren, S. Kimmins, J. A. Arosh, M. A. Fortier, and R. D. Lambert Expression of Cyclooxygenase-2 and Granulocyte-Macrophage Colony-Stimulating Factor in the Endometrial Epithelium of the Cow Is Up-Regulated During Early Pregnancy and in Response to Intrauterine Infusions of Interferon-{tau} Biol Reprod, January 1, 2004; 70(1): 54 - 64. [Abstract] [Full Text] [PDF]
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 B. Weinstock-Guttman, D. Badgett, K. Patrick, L. Hartrich, R. Santos, D. Hall, M. Baier, J. Feichter, and M. Ramanathan Genomic Effects of IFN-{beta} in Multiple Sclerosis Patients J. Immunol., September 1, 2003; 171(5): 2694 - 2702. [Abstract] [Full Text] [PDF]
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