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 mechanism(s) underlying the differential pattern of synthesis of these pregnancy-associated proteins is not understood very well. The present study evaluated the expression and trans-activation potential of the transcription factor Sp1 in the early pregnancy porcine endometrium to determine its temporal and functional association with the endometrial epithelial-specific genes encoding the transplacental iron-transport protein uteroferrin (UF) and an Sp-family member, basic transcription element-binding (BTEB) protein. Two identical Sp1 clones (717 bp) were isolated from a porcine endometrial cDNA library by polymerase chain reaction (PCR). The nucleotide sequence of these clones encodes a partial protein sequence of 238 amino acids encompassing the Zn-finger region and had significant identities with the corresponding regions in the rat and human proteins. By using a specific antibody raised against human Sp1, porcine endometrial Sp1 was found to exhibit a molecular weight of 110 kDa, was localized predominantly in the nuclei of glandular and luminal epithelial cells, and appeared to exist as a phosphorylated protein. 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 consensus motif in gel mobility shift assays, respectively, overlapped with, but did not parallel that of UF mRNA during early pregnancy. The effect of increased Sp1 expression on UF gene promoter activity was examined using a human Sp1 expression vector that was transiently transfected into primary cultures of pig endometrial glandular epithelial cells. Sp1 increased (P < 0.05) the promoter activities of various UF promoter-Luciferase reporter constructs by 2 to 4-fold, over those transfected with empty expression vector. Co-transfection of a BTEB expression vector with the Sp1 expression vector modified the effect of Sp1 on UF promoter activity in the shortest construct. These results suggest that Sp1 mediates the regulation of endometrial epithelial gene expression during pregnancy, and that this function is likely altered in vivo by co-expression of other family members, including BTEB.
Simmen
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RC
Department of Animal Science, Interdisciplinary Concentration in Animal Molecular and Cell Biology, University of Florida, Gainesville 3261-0910, USA. simmen@animal.ufl.edu
Zhang
X L
XL
Zhang
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D
Wang
Y
Y
Michel
F J
FJ
Simmen
F A
FA
D000135:Acid Phosphatase; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D004717:Endometrium / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes; D051741:Kruppel-Like Transcription Factors; D008667:Metalloproteins / Q000235:genetics; D008969:Molecular Sequence Data; D010641:Phenotype; D011247:Pregnancy; D011270:Pregnancy, Animal / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D016329:Sp1 Transcription Factor / Q000235:genetics* / Q000378:metabolism*; D013045:Species Specificity; D013552:Swine; D000071681:Tartrate-Resistant Acid Phosphatase; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation
D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D007527:Isoenzymes; C489676:KLF9 protein, human; D051741:Kruppel-Like Transcription Factors; D008667:Metalloproteins; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C077017:Klf9 protein, rat; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase