Upstream open reading frames regulate the translation of the multiple mRNA variants of the estrogen receptor alpha

It is by now well established that the estrogen receptor alpha (ER alpha) is transcribed from multiple promoters. One direct consequence of multiple promoters is the generation of mRNA variants with different 5'-untranslated regions (5'-UTRs). However, the potential roles of these individual mRNA variants are not known. All 5'-UTRs of ER alpha contain between one and six upstream open reading frames. In this study the effect of the 5'-UTRs of major human and mouse ER alpha mRNA variants on translation was evaluated. Some of the 5'-UTRs were found to strongly inhibit translation of the downstream open reading frame. Mutation of the upstream AUG codons partially or completely restored translation efficiency. A toeprinting analysis and assessment of the contribution of each AUG codon to the inhibitory effect on translation showed that leaky scanning and reinitiation occurs with these mRNAs. In conclusion, the upstream open reading frames in the 5'-UTRs of ER alpha mRNAs have the potential to regulate estrogen receptor alpha expression.     

Tissue-specific expression of the human alpha 1-antitrypsin gene is controlled by multiple cis-regulatory elements.

Human alpha 1-antitrypsin (AAT) is expressed in the liver, and a 318 bp fragment immediately flanking the CAP site of the gene was found to be sufficient to drive the expression of a reporter gene (CAT) specifically in hepatoma cells. The enhancing activity however, was orientation-dependent. The DNA fragment was separated into a distal region and a proximal region. A "core enhancer" sequence GTGGTTTC is present within the distal region and is capable of activity enhancement in both orientations when complemented by the proximal region in the sense orientation. The results strongly suggest that there are multiple cis-acting elements in the human AAT gene that confer cell specificity for its expression. Nuclear proteins prepared from the hepatoma cells bound specifically to the proximal region in a band-shifting assay that was resistant to competition by the globin promoter DNA. Foot-printing analysis showed a protected domain within the proximal region that contains a nearly perfect palindromic sequence TGGTTAATATTCACCA, which may be important in the regulation of AAT expression in the liver.     

Sex differences in progesterone receptor immunoreactivity in neonatal mouse brain depend on estrogen receptor alpha expression

Around the time of birth, male rats express higher levels of progesterone receptors in the medial preoptic nucleus (MPN) than female rats, suggesting that the MPN may be differentially sensitive to maternal hormones in developing males and females. Preliminary evidence suggests that this sex difference depends on the activation of estrogen receptors around birth. To test whether estrogen receptor alpha (ER alpha) is involved, we compared progesterone receptor immunoreactivity (PRir) in the brains of male and female neonatal mice that lacked a functional ER alpha gene or were wild type for the disrupted gene. We demonstrate that males express much higher levels of PRir in the MPN and the ventromedial nucleus of the neonatal mouse brain than females, and that PRir expression is dependent on the expression of ER alpha in these regions. In contrast, PRir levels in neocortex are not altered by ER alpha gene disruption. The results of this study suggest that the induction of PR via ER alpha may render specific regions of the developing male brain more sensitive to progesterone than the developing female brain, and may thereby underlie sexual differentiation of these regions.     

Tissue-specific regulation of rat estrogen receptor mRNAs

The estrogen receptor (ER) is present in a wide variety of mammalian tissues and is required for physiological estrogen responses, including estrogen-induced tissue-specific changes in gene expression. We studied the estrogen regulation of the mRNAs encoding the ER in rat uterus, liver, and pituitary. Ovariectomized (21-28 day post surgery) female CD-1 rats were injected daily with 17 beta-estradiol (E2, 10 micrograms/100 g BW) for 0, 1, or 4 h, 1, 3, or 7 days and compared with intact controls. Steady-state levels of ER mRNA were quantified using a human ER cDNA probe. Only one hybridizing species of approximately 6.2 kilobase (kb) was detected in uterine and liver RNA, similar to that observed in MCF7 human breast cancer cells. However, the ER mRNA regulation by E2 differed in direction depending on the tissue examined. In uterus, ER mRNA increased 3- to 6-fold after ovariectomy, and returned to intact levels within 24 h of E2 replacement. In contrast, liver ER mRNA declined 1.5- to 3-fold after ovariectomy and returned to intact levels after 1-3 days of E2. In pituitary tissue two hybridizing forms of ER mRNA were observed, with one species migrating at 6.2 kb, equivalent to the form in other tissues, and a second smaller species at approximately 5.5 kb. The lower molecular weight species varied somewhat in abundance from animal to animal, averaging about 20% of the intensity of the 6.2 kb band. The ER mRNA forms were regulated positively with E2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue-specific expression of the rat alpha 2u globulin gene family.

The rat alpha 2u globulin gene family encodes approximately 20 low-molecular-weight (20,000) proteins with pIs ranging from 4.5 to 7.9. alpha 2u globulin protein isoforms were detected in the liver and in the submaxillary, lachrymal, preputial, and mammary glands of Sprague-Dawley rats. The hormonal and developmental regulation of alpha 2u globulin synthesis in each of these tissues was unique, and it appears that different alpha 2u gene sets were transcribed in the various tissues.     

Polymorphisms of estrogen receptor alpha gene in endometrial cancer

It is hypothesized that polymorphisms of estrogen receptor-alpha (ERalpha) gene are involved in endometrial cancer. To test this hypothesis, the genotype distributions of six different loci (codon 10 T-->C, codon 87 G-->C, codon 243 C-->T, codon 325 C-->G, codon 594 G-->A, and intron 1 C-->G) of the ERalpha gene were investigated and their association with endometrial cancer was determined. The DNA from 113 cases of human endometrial cancer was analyzed by sequence-specific polymerase chain reaction. The relative risk of variant genotype was calculated by comparison with 200 healthy controls. The frequency of variant genotype on codon 10 was significantly lower in endometrial cancer patients as compared to controls. Nine of 113 endometrial cancer patients (8.0%) showed genotype 10C/C compared to 27 of 200 healthy controls (13.5%). The relative risk of genotype 10C/C was calculated as 0.44, compared to wild-type. Forty-five of 113 endometrial cancer patients (39.8%) showed genotype T/C on codon 10 compared to 111 of 200 healthy controls (55.5%). The relative risk of genotype 10T/C was calculated as 0.67, compared to wild-type. The polymorphism on codon 87 was not detected both in endometrial cancer patients and in healthy control. Other loci, intron 1, and codons 243, 325, and 594, did not show a correlation with endometrial cancer. The frequency of alleles on codon 10 was also significantly lower in endometrial cancer patients as compared to controls. Sixty-three of 226 alleles (27.9%) of endometrial cancer patients showed allele C compared to 165 of 400 (41.2%) of healthy controls. The relative risk of allele 10C was calculated as 0.67, compared to wild-type. Other loci, intron 1, and codons 243, 325, and 594, did not show a difference between cancer patients and controls. All genotype and allelic distributions were in accordance with the Hardy-Weinberg equilibrium. The present study demonstrates for the first time a protective effect of 10C allele against endometrial cancer. Thus, inherited alterations in ERalpha may be associated with changes in estrogen metabolism and thereby may possibly explain inter-individual differences in disease incidences of endometrial cancer.     

Hamster estrogen receptor cDNA: cloning and mRNA expression

Estrogen-induced hamster kidney tumor model serves as a useful model to study the biochemical and molecular mechanisms of hormonal carcinogenesis. In this model, we have demonstrated an increased expression of estrogen receptor mRNA and protein in estrogen-treated kidneys and in estrogen-induced tumors. The sequence information for hamster estrogen receptor gene is not known and has been investigated in this study. A hamster uterus cDNA library was constructed and the 5'-region of the hamster estrogen receptor cDNA cloned from this library using polymerase chain reaction (PCR) methodology. Additionally, hamster kidney polyadenylated RNA was reverse transcribed and PCR amplified using primers that were designed based on maximum homology between mouse, rat and human estrogen receptor cDNAs. These PCR amplified fragments were cloned into plasmid vectors and clones with the expected size of the insert subjected to Southern blot analysis using human estrogen receptor cDNA as a probe. The positive clones on Southern blot analysis and the PCR amplified products from these clones were subjected to DNA sequence analysis. Using this strategy, a full length, 1978 bp hamster estrogen receptor cDNA has been cloned which shows 87% homology with human, 90% with rat and 91% with mouse estrogen receptor cDNA. The deduced amino acid shares 88% homology with human, and 93% with rat and mouse estrogen receptors. Hamster estrogen receptor domain C (DNA binding domain) shows a 100% homology with a similar domain from mouse, rat, human, pig, sheep, horse and chicken estrogen receptor (Genebank reference ID: AF 181077).