CHROMOSOMAL ORGANIZATION OF THE GENE ENCODING PORCINE ANTILEUKOPROTEINASE AND FUNCTIONAL-ANALYSIS OF THE PROMOTER REGION IN ENDOMETRIAL AND PLACENTAL CELLS

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' Banking DNA was examined using chimeric ALP-chloramphenicol acetyl transferase (CAT) DNA constructs. after transient transfection in human (ECC-I, 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 cel 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. Results suggest that the approximately 240 bp fragment most proximal to the transcription initiation site confers basal and limited endometrial tissue-specific promoter activity to the ALP gene 5'-flanking region. These studies also establish the ECC-1 cell line as an in vitro model system to elucidate the control of ALP gene transcription in the endometrium.