The estrogen receptor α Σ3 mRNA splicing variant is differentially regulated by estrogen and progesterone in the rat uterus

Abstract

The gene for estrogen receptor (ER ) has been shown to be under complex hormonal control and its activity can be regulated by mRNA alternative splicing. Here we examined the regulation of ER transcription and translation in the rat uterus by ovarian steroid hormones. We examined whether expression of ER mRNA splice isoforms is hormonally regulated in ovariectomized (OVX) and cycling rats. Adult OVX female rats were treated daily with 17-estradiol (E 2 ) (0·05 µg/rat or 5 µg/rat), progesterone (P 4 ) (1 mg/rat) or a combination of both hormones for 4 days. Animals were killed 24 h after the last injection and uterine horns were removed. In order to determine whether ER mRNA isoforms are differentially expressed under various physiological conditions, animals were evaluated at proestrus, estrus and diestrus. The ER protein and mRNA were detected by immunohistochemistry and comparative RT-PCR analysis respectively. The presence of ER mRNA isoforms was evaluated using a nested RT-PCR assay. In OVX control rats, ER mRNA and protein levels were high, demonstrating a constitutive expression of the ER gene in the uterus. When animals received P 4 or the high dose of E 2 , a significant decrease in both ER mRNA and protein was observed in the uterus. However, when rats were treated with the low dose of E 2 , only the ER protein was down-regulated; no changes were observed in ER mRNA expression. In addition to the full-length ER mRNA, OVX control rat uteri expressed three shorter transcripts: 3, 4 and 3,4 (lacking exon 3, exon 4, or both 3 and 4 respectively). Surprisingly, when OVX animals were treated with P 4 , the low dose of E 2 or a combination of both steroids, expression of the 3 isoform was completely abolished. During the estrous cycle, all ER mRNA splicing variants were detected at proestrus and estrus. However, in diestrus, significant low levels of the 3 isoform were observed. In summary, our results suggest a dose-dependent relationship between E 2 concentrations and the level of control in the ER transcription-translation cascade. Moreover, the alternative splicing of the ER primary transcript is influenced by the hormonal milieu, suggesting that these events could affect the estrogen responsiveness of the rat uterus during the estrous cycle.

Structure, tissue distribution and estrogen regulation of splice variants of the sea bream estrogen receptor α gene

“…2C). This structure is similar to that of the ΔE2,3 or ΔE3 variants detected in mammalian tissues (Pasqualini et al, 1999;Poola et al, 2000;Perlman et al, 2005;Varayoud et al, 2005), which lack both exons 2 and 3 or exon 3 only, and the in vitro translated human ERαΔE3 protein was unable to bind DNA or activate ERE-driven transcription but could bind E 2 , translocate to the nucleus and negatively affect wt-ERα transactivation activity at an ERE promoter (Bollig and Miksicek, 2000). In addition and like wt-ERα, the human ΔE3 protein could support transcriptional activation of promoters regulated by AP-1 and Sp-1 elements (Bollig and Miksicek, 2000;Koduri et al, 2006).…”

Neonatal exposure to bisphenol A modifies the abundance of estrogen receptor α transcripts with alternative 5′-untranslated regions in the female rat preoptic area

“…Since sexual differentiation of the hypothalamus is estrogen-dependent and mediated at least partly through ERa (Simerly et al 1997), BPA deregulation of this receptor could affect the natural estrogen-directed dimorphisms of POA circuits. In a previous work performed in our laboratory, it has been demonstrated that low and high doses of estrogen can differentially regulate the splicing of the rat ERa mRNA coding region (Varayoud et al 2005). Selective promoter usage and alternative mRNA splicing events are estrogen-dependent mechanisms of ERa gene variation, arising as sources of complexity in the endocrine disruptor's toxicology.…”

“…A standard immunohistochemical technique (avidin-biotinperoxidase) was used to visualize ERa immunostaining intensity and distribution following a previously described protocol (Munoz de Toro et al 1998, Varayoud et al 2005. Overnight incubation at 4 8C was performed by applying a diluted mouse monoclonal antibody raised to full-length recombinant human ERa (working dilution 1:200; clone 6F-11, Novocastra, Newcastle upon Tyne, UK), followed by incubation with a biotinylated secondary antibody (working dilution 1:80; Sigma-Aldrich).…”

“…Since ERa mRNA transcription and maturation could be differentially regulated by estrogens (Varayoud et al 2005), it is very interesting to know whether high and low doses of xenoestrogens can modify the differential usage of ERa gene promoters in the rat hypothalamus. Using rats exposed early in life, we examined the effects of DES and BPA over the control of ERa transcription and translation in the hypothalamic POA of prepubertal female rats.…”

In vivo xenoestrogenic actions of cadmium and arsenic in anterior pituitary and uterus

“…In uterus, many ERα-splicing variants have been reported (Varayoud et al 2005), including the aforementioned truncated variants. Noticeably, only iAs was able to augment full-length ERα expression in both tissues, whereas Cd did not modify it.…”