Expression and regulatory function of the transcription factor Sp1 in the uterine endometrium at early pregnancy: implications for epithelial phenotype

The uterus during early pregnancy synthesizes a complex array of signaling molecules with specific spatial and temporal modes of expression and which are critical for embryo implantation and subsequent development. The mechan ism(s) underlying the differential pattern of synthesis of these pregnancy- associated proteins is not understood very well. The present study evaluate d the expression and trans-activation potential of the transcription factor Sp1 in the early pregnancy porcine endometrium to determine its temporal a nd functional association with the endometrial epithelial-specific genes en coding the transplacental iron-transport protein uteroferrin (UF) and an Sp -family member, basic transcription element-binding (BTEB) protein. Two ide ntical Sp1 clones (717 bp) were isolated from a porcine endometrial cDNA li brary by polymerase chain reaction (PCR). The nucleotide sequence of these clones encodes a partial protein sequence of 238 amino acids encompassing t he Zn-finger region and had significant identities with the corresponding r egions in the rat and human proteins. By using a specific antibody raised a gainst human Sp1, porcine endometrial Sp1 was found to exhibit a molecular weight of 110 kDa, was localized predominantly in the nuclei of glandular a nd luminal epithelial cells, and appeared to exist as a phosphorylated prot ein. Northern blot analysis demonstrated three distinct size transcripts of approximately 3.5, 5, and 8 kb for endometrial Sp1. The expression of Sp1 mRNA and protein, determined by RT-PCR and by its ability to bind Sp1 conse nsus motif in gel mobility shift assays, respectively, overlapped with, but did not parallel that of UF mRNA during early pregnancy. The effect of inc reased Sp1 expression on UF gene promoter activity was examined using a hum an Sp1 expression vector that was transiently transfected into primary cult ures of pig endometrial glandular epithelial cells. Sp1 increased (P < 0.05 ) the promoter activities of various UF promoter-luciferase reporter constr ucts by 2 to 4-fold, over those transfected with empty expression vector. C o-transfection of a BTEB expression vector with the Sp1 expression vector m odified the effect of Sp1 on UF promoter activity in the shortest construct . These results suggest that Sp1 mediates the regulation of endometrial epi thelial gene expression during pregnancy, and that this function is likely altered in vivo by co-expression of other family members, including BTEB. ( C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.