Novel estrogen receptor beta transcript variants identified in human breast cancer cells affect cell growth and apoptosis of COS-1 cells
Introduction
Two estrogen receptor types, named ERα and ERβ, have been found to be the major mediators of a variety of biological functions of estrogens (Pettersson and Gustafsson, 2001, Gustafsson, 1999, Osborne and Schiff, 2005). Although particularly the molecular mechanisms of ERβ function are still poorly elucidated, it is becoming increasingly clear that both receptor types are responsible for different biological functions, as indicated by their specific expression patterns and different effects of their gene knockout (Couse et al., 2000, Merchenthaler and Shugrue, 1999). Recent studies have shown that both ERα or ERβ homodimers and ERα/β heterodimers regulate unique sets of E2-responsive genes (Monroe et al., 2005). Besides their different physiological functions, recent studies suggested that ERβ, in contrast to ERα, might act as a tumor suppressor (Bardin et al., 2004, Lazennec et al., 2001), whereas other studies came to contrary conclusions (Burns et al., 2003). Given that ERβ is able to counteract ERα-signaling in some settings, loss of ERβ is thought to enhance ERα-mediated proliferation of hormone-dependent cancer cells (Lindberg et al., 2003). Furthermore, recent studies suggested that ERβ signaling might affect cellular apoptosis (Cheng et al., 2004). Another feature of both ERs is the variety of their mRNA isoforms resulting from differential splicing (Poola et al., 2002a, Poola et al., 2002b, Herynk and Fuqua, 2004, Price et al., 2000, Speirs et al., 2000). The so far identified ERβ splice variants are characterized by alternative 3′-exons (ERβ2, ERβ3, ERβ4, ERβ5) or by deletion of single or multiple exons (e.g. ERβΔ2, ERβΔ5/6). Some of these mRNA isoforms were demonstrated to code for ERβ proteins which are characterized by impaired estrogen or DNA binding or altered cofactor interaction (Zhao et al., 2005, Sierens et al., 2004, Price et al., 2001). The emerging picture of multiple ERβ mRNA isoforms, and thus also the multitude of differentially built proteins, strongly suggests their synthesis to be considered as another level of complexity of estrogen signaling.
In this study, we succeeded in molecular cloning of two novel ERβ transcript variants from ERα-negative MDA-MB-231 breast cancer cells. To analyze their function in comparison to ERβ1, we engineered COS-1 cells stably expressing these ERβ isoforms and examined their effects on cellular proliferation, apoptosis, motility and gene regulation.
Section snippets
Materials
Phenol red-free DMEM culture medium was obtained from Invitrogen (Karlsruhe, Germany), FCS was purchased from PAA (Pasching, Austria). 17-β estradiol, 4-OH tamoxifen, staurosporine and serum replacement 2 (SR2) were obtained from Sigma (Deisenhofen, Germany), pure antiestrogen ICI 182,780, ERα agonist PPT and ERβ agonist diarylpropionitrile (DPN) were purchased from Tocris (Ellisville, USA). COS-1 African green monkey kidney cells and MDA-MB-231 breast cancer cells were obtained from American
Molecular cloning of novel ERβ splice variants
An attempt to clone the open reading frame of human ERβ1 by means of RT-PCR from ERα-negative, but ERβ-positive MDA-MB-231 breast cancer cells using PCR primers ERβ-F1 binding in the 5′ UTR at exon 0 and ERβ-F2 binding in the 3′ UTR at exon 8 of ERβ1 resulted not only in amplification of the expected 1.7 kb ERβ1 cDNA fragment, but also in generation of PCR products of unexpected size (Fig. 1a). Two of these amplicons, which exhibited an approximate size of 1.3 and 1.2 kb in 0.8% agarose gel
Discussion
We report identification of two novel estrogen receptor β splice variants in MDA-MB-231 breast cancer cells and the results of an attempt to characterize their function in regulation of cell growth, apoptosis, motiliy and gene expression. MDA-MB-231 breast cancer cells are ERα-negative but express ERβ1, ERβ2 and various exon-skipped isoforms like ERβΔ2, ERβΔ5 and ERβΔ25 (Poola et al., 2002a, Poola et al., 2002b, Herynk and Fuqua, 2004). Given that estradiol is not able to trigger any typical
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