Abstract
The immature rat uterus has been extensively used as an in vivo model system to study the molecular mechanisms of steroid hormone actions. In this study, we demonstrated the regulated expression of syndecan-3 in the rat uterus by the steroid hormone 17 beta-estradiol. Administration of a single physiological dose of 17 beta-estradiol (40 microg/kg) to ovariectomized immature animals induced a rapid and transient increase in uterine syndecan-3 mRNA. Transcript levels reached a peak elevation of 3-fold above saline control tissues 4 h after hormone administration. Inhibition of message up-regulation by actinomycin D but not cycloheximide indicated a hormone response dependent on RNA transcription but not new protein synthesis. The estrogenic ligands estriol and tamoxifen were also effective at raising syndecan-3 mRNA levels; however, nonestrogenic ligands, including progesterone, 5 alpha-dihydrotestosterone, and dexamethasone, failed to stimulate a change in mRNA levels. Hormone-induced changes in mRNA led to transient changes in syndecan-3 protein content and significant alteration in the temporal and spatial expression in endometrial epithelial cells. Collectively, these data show that the steroid hormone 17 beta-estradiol, regulates transcription of the syndecan-3 gene in the uterus via an estrogen receptor-dependent mechanism. This estrogen-regulated expression of syndecan-3 may play an important role in changes in tissue ultrastructure crucial for proper uterine growth.
Highlights
It has been known for some time that ovarian hormones induce changes in expression of surface proteins on epithelial cells lining the uterine lumen believed to be crucial for blastocyst reception [1]
The immature rat uterus has been widely used as a model system to study the biochemical and molecular mechanisms of steroid hormone action
The early responses induced by E2 are characterized by altered expression of genes encoding for transcription factors [30], oncogenes [31,32,33], and growth factors and their receptors [34, 35]
Summary
Northern blots were prehybridized at 65 °C in a solution containing 5ϫ saline/sodium phosphate/EDTA, 50% formamide, 0.5% SDS, 100 g/ml denatured salmon sperm DNA, and 5ϫ Denhardt’s reagent. Prehybridized blots were incubated at 65 °C in a hybridization solution (5ϫ saline/sodium phosphate/EDTA, 0.5% SDS, 100 g/ml denatured salmon sperm DNA, 5ϫ Denhardt’s reagent, 10% dextran sulfate) and the 32P-labeled probe (ϳ1.2–2.1 ϫ 109 dpm/g DNA). Samples were applied to a 6% SDS-polyacrylamide gel and resolved by electrophoresis (Mini Protean II apparatus; Bio-Rad) at 100 V in a buffer containing 2.5 mM Tris base (pH 8.3), 192 mM glycine, and 0.1% SDS. Membranes were again washed and processed for detection of syndecan-3 protein by incubation in an appropriate substrate solution containing a luminol compound (SuperSignal Substrate; Pierce) for chemiluminescent signal development.
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