Trans-Activation Functions of the Sp-Related Nuclear Factor, Basic Transcription Element-Binding Protein, and Progesterone Receptor in Endometrial Epithelial Cells*

Abstract

The present study examined the trans-activation potential of basic transcription element-binding protein (BTEB), a recently identified member of the Sp family of GC box-binding transcription factors, on the expression of the gene encoding the pregnancy-associated, epithelial-specific, and progesterone (P)-induced porcine uterine endometrial secretory protein, uteroferrin (UF). Endometrial expression of BTEB, P receptor (PR), and UF genes was analyzed by RT-PCR as a function of pregnancy stage and cell type and was correlated with the levels of endometrial BTEB that were quantified by Western blot and/or electrophoretic mobility shift assay. PR, BTEB, and UF messenger RNAs (mRNAs) were present in early (day 12) and mid(day 60) pregnancy pig endometrium, although expression levels varied for each mRNA (UF, day 12 << day 60; PR and BTEB, day 12 = day 60). Within the endometrium, glandular epithelial (GE) cells manifested higher amounts of UF mRNA than stromal fibroblastic cells, whereas both cell types had comparable amounts of BTEB and PR mRNAs. Expression of BTEB, however, was limited to endometrial GE cells. A BTEB expression vector (pcDNA-3BTEB) was used to examine the effect of increased BTEB protein on UF gene expression and promoter activity in primary cultures of pig endometrial GE cells. Cells transiently transfected with pcDNA-3BTEB had 2-fold higher UF mRNA levels than those transfected with the empty expression vector (pcDNA-3). Further, cells cotransfected with a UF promoter-luciferase (-1935UF-Luc) reporter gene and the BTEB expression vector had 2-fold higher Luc activity than those cotransfected with reporter gene and pcDNA-3. This effect of BTEB was not observed in transfected endometrial stromal fibroblastic cells, but was apparent in the human endometrial epithelial carcinoma cell lines ECC-1 and Hec-1-A, which exhibit low levels of BTEB protein and low or undetectable PR mRNA levels, respectively. The respective contributions of BTEB and PR to the modulation of UF promoter activity were examined by cotransfection of Hec-1-A and ECC-1 cells with expression plasmids for BTEB and PR and one of two UF promoter constructs (-831UF-Luc or -1935UF-Luc) in the absence or presence of P. The increase in UF promoter activity with BTEB was mimicked by PR in a P-dependent manner in both cell lines. The combined effect of PR/P and BTEB appeared additive in Hec-1-A cells and was synergistic in ECC-1 cells. These results highlight the cell context dependence of the trans-activation potential of BTEB and suggest its unique role, in concert with PR, in directing the temporal expression of endometrial epithelial genes of pregnancy.

Selective Interactions of Krüppel-like Factor 9/Basic Transcription Element-binding Protein with Progesterone Receptor Isoforms A and B Determine Transcriptional Activity of Progesterone-responsive Genes in Endometrial Epithelial Cells

“…We also showed that BTEB1 transactivates gene promoters containing GC-rich recognition motifs (25,26), but in the presence of ligand-activated PR-B, the functional complex formed between the PR-B dimer and BTEB1 favored the induction of PRE-containing promoters (24). Because the relative levels of PR-A, PR-B and BTEB1 are tightly regulated in normal uterine endometrium, and deviations in PR isoform ratios can affect tissue responsiveness to P, we examined whether BTEB1 alters the transactivity of PR-A for different promoters.…”

Expression and transcriptional regulation of caspase-14 in simple and complex epithelia

“…Quantitative determination of luciferase activity (measured as relative light units) in the cell lysates was normalized to the protein concentration of cell lysate. 36 …”

Inhibition of Vascular Endothelial Growth Factor Expression in HEC1A Endometrial Cancer Cells through Interactions of Estrogen Receptor α and Sp3 Proteins

“…Sp1 physically and functionally interacts with several nuclear receptor superfamily proteins including ER␣ (53)(54)(55)(56)(57)(58)(59)(60)(61)65), retinoic acid receptors, chicken ovalbumin upstream promoter transcription factor (COUP-TF), and steroidgenic factor-1, and the progesterone receptor interacts with Sp1 and basal transcription element binding protein (an Sp family member) to modulate gene expression through GC-rich promoter elements (93)(94)(95)(96)(97)(98)(99). This paper describes a novel ER␣/Sp3 pathway for hormone-mediated decreased gene expression through a G/GCrich element in the VEGF gene promoter, and this mechanism may be important for hormone-mediated down-regulation of other genes.…”