Selective Removal of the Selenocysteine tRNA^[Ser]Sec Gene (Trsp) in Mouse Mammary Epithelium

Abstract

Mice homozygous for an allele encoding the selenocysteine (Sec) tRNA [Ser]Sec gene (Trsp) flanked by loxP sites were generated. Cre recombinase-dependent removal of Trsp in these mice was lethal to embryos. To investigate the role of Trsp in mouse mammary epithelium, we deleted this gene by using transgenic mice carrying the Cre recombinase gene under control of the mouse mammary tumor virus (MMTV) long terminal repeat or the whey acidic protein promoter. While both promoters target Cre gene expression to mammary epithelium, MMTV-Cre is also expressed in spleen and skin. Sec tRNA[Ser]Sec amounts were reduced by more than 70% in mammary tissue with either transgene, while in skin and spleen, levels were reduced only with MMTV-Cre. The selenoprotein population was selectively affected with MMTV-Cre in breast and skin but not in the control tissue, kidney. Moreover, within affected tissues, expression of specific selenoproteins was regulated differently and often in a contrasting manner, with levels of Sep15 and the glutathione peroxidases GPx1 and GPx4 being substantially reduced. Expression of the tumor suppressor genes BRCA1 and p53 was also altered in a contrasting manner in MMTV-Cre mice, suggesting greater susceptibility to cancer and/or increased cell apoptosis. Thus, the conditional Trsp knockout mouse allows tissue-specific manipulation of Sec tRNA and selenoprotein expression, suggesting that this approach will provide a useful tool for studying the role of selenoproteins in health.Selenium is an essential micronutrient in the diet of mammals and numerous other life forms (see reference 26 for a review). Many health benefits have been attributed to this element, including a role in the prevention of cancer (10) and heart disease and other cardiovascular and muscle disorders (11), in delaying the aging process (33) and the onset of AIDS in human immunodeficiency virus-positive patients (1), in male reproduction (17), in mammalian development (5), in immune function (33), and as an antiviral agent (2). Selenium is incorporated into protein in the form of selenocysteine (Sec), and Sec has its own tRNA (designated Sec tRNA[Ser]Sec ) and its own code word, UGA (26). Sec is indeed the 21st naturally occurring amino acid in the genetic code. Most certainly, the health benefits of selenium are due in large part to its presence in protein (26).Sec tRNA [Ser]Sec is the only known tRNA that governs the expression of an entire class of proteins, the selenoproteins (26). This provides a unique opportunity to study the expression of selenoproteins by manipulating the levels and characteristics of Sec tRNA [Ser]Sec . For example, the levels of numerous selenoproteins were reduced in a protein-and tissue-specific manner in transgenic mice carrying mutant Sec tRNA [Ser]Sec transgenes lacking the highly modified base isopentenyladenosine in its anticodon (37). Glutathione peroxidase 1 (GPx1) and thioredoxin reductases 1 (TR1) and 3 (TR3) were the most and least affected selenoproteins, respectively, and selenoprotein expre...

Selenoprotein expression is essential in endothelial cell development and cardiac muscle function

“…Removal of trsp from the mouse genome is embryonic lethal [16,17] resulting in death within the first few days post-coitum (dpc) [16]. These data further demonstrate that selenoproteins have a major role in mammalian development.…”

“…However, the mechanism of how and when selenoprotein expression is required in the development of various tissues and organs is largely unknown. Generation of a conditional knockout of trsp using loxP-Cre technology [17] has provided an important tool for carrying out a more detailed analysis of the role of selenoproteins in specific tissue and organ development. For example, targeted removal of trsp from mammary epithelium has shown that selenoprotein expression is altered when Cre is under the control of the mouse mammary tumor virus long terminal repeat promoter, but that there were no observed phenotypic changes [17].…”

“…Generation of a conditional knockout of trsp using loxP-Cre technology [17] has provided an important tool for carrying out a more detailed analysis of the role of selenoproteins in specific tissue and organ development. For example, targeted removal of trsp from mammary epithelium has shown that selenoprotein expression is altered when Cre is under the control of the mouse mammary tumor virus long terminal repeat promoter, but that there were no observed phenotypic changes [17]. Targeted removal of trsp from adult liver, however, demonstrated an essential requirement for selenoprotein expression in liver function following complete removal of the synthesis of this seleniumcontaining protein class [18].…”

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“…Selenium supplementation in cell culture as well as in animal models is known to modulate the relative ratios between the two isoforms by inducing the production of mcm 5 Um (70,129,162). The synthesis, function, and regulation of Sec tRNA [Ser]Sec have been reviewed extensively (40,55,128,131 of trsp leads to a selective reduction in selenoproteins such as GPx1, GPx4, and Sep 15, whereas TrxR1 levels are barely affected (190). In addition, expression of the breast cancersusceptibility gene product BRCA1 is decreased, whereas tumor-suppressor protein p53 is increased in these mice, suggesting a possible interplay between the DNA damage pathway and selenoproteins in breast cancer etiology.…”

Mouse models for assessing the role of selenoproteins in health and development

“…Rescue of the Atrsp mice with the mutant transgene, ~37trsp' afforded us with an opportunity of obtaining a mouse line with a mutant transgene wherein there is no background of host Sec and selenoprotein expression is therefore totally dependent on the mutant tRNA. G37trsp yielded a tRNA that lacked two base modifications, i6A37 and Urn34 (see above, Chapter 3 and [6,10]) and mice rescued with G37trspt lacked several selenoproteins including glutathione peroxidases 1 and 3, SelR and SelT [6,7]. Interestingly, we were not successful in rescuing selenoprotein synthesis in Atrsp mice with A34trspt (see reference to Carlson et a1 above).…”