Vascular ECE-1 mRNA expression decreases in response to estrogens

Abstract

DNA microarrays were used to identify new targets of estrogen in the vasculature. Ovariectomized rats were treated with estradiol, genistein or daidzein, for four days. [³³P]dCTP-labelled probes synthesized from mesenteric artery RNA were hybridized with DNA microarrays. Analysis of the microarray data identified endothelin converting enzyme-1 (ECE-1) as a gene whose expression was inhibited by treatment with estrogen, genistein, or daidzein. Semi-quantitative RT-PCR was used to confirm the data from the DNA microarrays. Reversal of the estrogen and phytoestrogen effect on ECE-1 expression by ICI 182,780 suggested that the inhibition was an estrogen receptor response. An inhibition of ECE-1 mRNA expression by estrogen or the phytoestrogens has not been previously reported. These data highlight the power of DNA microarray technology to identify new gene expression targets of estrogen in the vasculature. Moreover, the data suggest that genistein and daidzein inhibit ECE-1 expression by an estrogen receptor–mediated mechanism.

Introduction

Estrogen has been reported to provide protective effects to the vasculature by multiple mechanisms. For example, estrogen can decrease intimal hyperplasia of the vessel wall directly by decreasing mitosis Selzman et al., 1998a, Selzman et al., 1998b, Selzman et al., 1997. Estrogen can cause vasodilation directly by effects on ion channels in the vessel wall White et al., 1995, White et al., 2002, or indirectly by stimulating the expression of enzymes involved in vasodilatory pathways such as nitric oxide synthase (Weiner et al., 1994). Estrogen also reduces atherogenesis by effects on the lipid profile (Nathan and Chaudhuri, 1997) as well as through stimulation of endothelial relaxing factors such as nitric oxide Hayashi et al., 1992, Hayashi et al., 1995 and endothelium-derived hyperpolarizing factor (Liu et al., 2001). However, little is known about specific genes regulated by estrogen in the blood vessel wall.

Genistein and daidzein are isoflavones found in soybeans (Barnes, 1998). They bind to intracellular estrogen receptors and activate those receptors, thereby producing estrogenic effects Kuiper et al., 1998, Miksicek, 1994, Wang et al., 1996. Genistein also possesses potent tyrosine kinase inhibitory activity Akiyama et al., 1987, Kim et al., 1998, whereas daidzein lacks tyrosine kinase inhibitory effects (Akiyama et al., 1987). Many health benefits have been claimed for these soy-derived compounds; some claims are based on the estrogenic effects (vasculoprotection, Washburn et al., 1999) and others are based on the tyrosine kinase inhibitory effects of genistein (antioncogenic, Barnes, 1997).

Accordingly, the focus of this study was to identify new gene targets of estrogenic substances in the resistance arteries of the mesentery. Initial expression analysis was performed with DNA microarrays. These studies identified endothelin converting enzyme-1 (ECE-1) as an estrogen-regulated gene in the mesenteric arteries. ECE-1 is of interest because it catalyzes the conversion of pro-endothelin to the potent vasoconstrictor, endothelin-1 Kido et al., 1998, Russell et al., 1998. Follow-up confirmation of differences in gene expression were performed with semi-quantitative RT-PCR.