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Uterine expression of the mRNA encoding antileukoproteinase (ALP) is highest in pig uterus during mid- to late pregnancy, suggesting a stage of pregnancy-dependent role for this elastase/cathepsin G protease inhibitor in feto-maternal interactions. To examine a potential relationship between uterine synthesis of ALP and the type of placentation in mammalian species, the expression of ALP mRNA and/or protein in pregnant mares, cows, rats, and mice was evaluated. Genomic DNA and mRNA hybridization analyses were performed using a porcine ALP cDNA as probe. The concentration of ALP protein in reproductive tissues was determined by RIA using a polyclonal antibody raised against a synthetic peptide (ALP 16P) corresponding to amino acid residues 21-36 of the porcine ALP protein. A single ALP mRNA transcript of approximately 0.8 kb in length was detected in equine and bovine uterine tissues. The relative abundance of ALP mRNA in equine endometrium increased between days 125-170 (mid-pregnancy), and then decreased by day 215 of pregnancy. Similarly, the steady state levels of ALP mRNA in bovine endometrium and myometrium were higher during mid- to late than during early pregnancy. The levels of ALP mRNA in bovine fetal cotyledon were low and did not change significantly with stage of pregnancy. No hybridization was detected to pregnant rat endometrial tissues, although high stringency Southern blot analysis of porcine, bovine, and rat genomic DNAs using porcine ALP cDNA as probe predicted a high degree of nucleotide sequence homology in their respective ALP genes.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA-protein interactions within two putative regulatory regions distal from the transcription initiation site of the porcine uteroferrin (UF) gene were characterized. These regions, termed XB (-1,600 to -1,129 bp) and AB (-1,128 to -893 bp) exhibited transcriptional enhancer activities within the context of the heterologous SV40 promoter, that were specific to endometrial cells. DNase I and gel-shift assays demonstrated that both fragments contain a heptamer motif TGCTAGA that binds a nuclear protein present in crude and DEAE-fractionated nuclear extracts from porcine endometrium of pregnancy. This heptad sequence, designated as endometrial-associated sequence (EAS), is different from previously described nuclear protein-binding consensus sequences. Mutations in the heptamer motif abolished binding to the nuclear factor, as detected by gel-shift assays. The endometrial nuclear protein that interacts with the heptamer was characterized by Southwestern and UV cross-linking analysis. The protein has an approximate M(r) of 80 kD, is basic (pI 7.7-8.6) and is present in pig endometrium throughout pregnancy. The functional relevance of this DNA-binding protein in the control of UF gene transcription in the endometrium is discussed.

The apparent preferential expression of the elastase/cathepsin G protease inhibitor antileukoproteinase (ALP) in endometrium of species with epitheliochorial placenta suggests mechanisms of transcriptional regulation unique to these mammalian species. To begin to define the cis-acting regulatory elements involved in the endometrial transcription of the ALP gene, the porcine ALP gene was isolated and characterized. The porcine gene spans at least 13 kb and consists of 5 exons and 4 introns. This genomic structure, except for an additional exon, is similar to that of the human gene where the first three exons encode the signal peptide, trypsin/cathepsin G binding region, and elastase binding region, respectively. The positions of the 16 cysteine residues in exons 2 and 3 of the human gene are conserved in the porcine gene. The porcine gene contains a TATA box at -29 nucleotide (nt), and sequences with limited homology to those which might bind the transcription factors AP-1, AP-2, Sp-1 and Oct-1. The functional promoter activity of the ALP-5' flanking DNA was examined using chimeric ALP-chloramphenicol acetyl transferase (CAT) DNA constructs, after transient transfection in human (ECC-1, Ishikawa) and rabbit (HRE-H9) endometrial and human trophoblastic (JEG-3) cell lines. A 887 nt fragment of the ALP-5'-flanking region (-887ALP-pCAT-E) was active in these cell lines, with the highest promoter activity observed in the ECC-1. Progressive 5' deletion of the 887 nt fragment up to -243 nt had no effect on CAT gene expression in all cell lines, relative to the longest construct.(ABSTRACT TRUNCATED AT 250 WORDS)