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...

The Wnt/b-catenin signaling pathway is critical for normal mammalian development, the specification of epidermal cells and neoplastic transformation of intestinal epithelium. However, precise molecular information regarding cell-specific responses to b-catenin signaling has been limited. This question was addressed using a mouse model in which exon 3 of the b-catenin gene was deleted in several cell types with loxP-mediated recombination utilizing a Cre transgene under control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). The stabilization of b-catenin in prostate epithelium resulted in hyperplasias and extensive transdifferentiation into epidermal-like structures, which expressed keratins 1 and 6, filaggrin, loricrin and involucrin. The cell-specific loss of NKCC1 protein and reduced nuclear Stat5a is further suggestive of a loss of prostate epithelial characteristics. In addition to the prostate, hyperplasias and squamous metaplasias were detected in epithelia of the epididymis, vas deferens, coagulating gland, preputial gland and salivary gland. However, and in contrast to a recent study, no lesions reminiscent of highgrade prostate intraepithelial neoplasia were detected. Since b-catenin was activated in several cell types and impinged upon the viability of these mice, it was not possible to evaluate the cumulative effect over more than 3 months. To assess long-term consequences of b-catenin activation, mutant and control prostate tissues were transplanted into the mammary fat pads of wild-type males. Notably, squamous metaplasias, intra-acinous hyperplasia and possible neoplastic transformation were observed after a total of 18 weeks of b-catenin stimulation. This suggests that the transdifferentiation into squamous metaplasias is an early response of endodermderived cells to b-catenin, and that the development of intra-acinous hyperplasias or neoplastic foci is a later event.

The Wnt/b-catenin signaling pathway controls cell fate and neoplastic transformation. Expression of an endogenous stabilized b-catenin (DE3 b-catenin) in mammary epithelium leads to the transdifferentiation into epidermis-and pilar-like structures. Signaling molecules in the canonical Wnt pathway upstream from b-catenin induce glandular tumors but it is not clear whether they also cause squamous transdifferentiation. To address this question we have now investigated mammary epithelium from transgenic mice that express activating molecules of the Wnt pathway: Wnt10b, Int2/Fgf3, CK2a, DE3 b-catenin, Cyclin D1, and dominant negative (dn) GSK3b. Cytokeratin 5 (CK5), which is expressed in both mammary myoepithelium and epidermis, and the epidermis-specific CK1 and CK6 were used as differentiation markers. Extensive squamous metaplasias and widespread expression of CK1 and CK6 were observed in DE3 b-catenin transgenic mammary tissue. Wnt10b and Int2 transgenes also induced squamous metaplasias, but expression of CK1 and CK6 was sporadic. While CK5 expression in Wnt10b transgenic tissue was still confined to the lining cell layer, its expression in Int2 transgenic tissue was completely disorganized. In contrast, cytokeratin expression in CK2a, dnGSK3b and Cyclin D1 transgenic mammary tissues was similar to that in DE3 bcatenin tissue. In support of transdifferentiation, expression of hard keratins specific for hair and nails was observed in pilar tumors. These results demonstrate that the activation of Wnt signaling components in mammary epithelium induces not only glandular tumors but also squamous differentiation, possibly by activating LEF-1, which is expressed in normal mammary epithelium.

Mammary alveolar development during pregnancy is triggered by hormone signals. The prolactin receptor/Jak2/signal transducer and activator of transcription (Stat) 5 signal transduction pathway is the principal mediator of these cues and alveolar development is abrogated in its absence. The loss of the basic helix-loop-helix protein inhibitor of differentiation (Id)2 results in a similar defect. To investigate the role of Id2 in mammary epithelium, we performed structural and molecular analyses. Id2-null mammary epithelial cells were unable to form alveoli; the epithelial architecture was disorganized and dissimilar from early stages of alveologenesis in wild-type glands. The epithelial cells retained the ductal marker Na-K-Cl cotransporter (NKCC)1. Nuclear localization of Stat5a and down-regulation of NKCC1 was observed in some areas, indicating a limited response to pregnancy signals. The differentiation status of Id2-null tissue at term was further characterized with cDNA microarrays enriched in mammary specific sequences (mammochip). Some of the early differentiation markers for mammary epithelium were expressed in the Id2-null tissue, whereas genes that are expressed at later stages of pregnancy were not induced. From these results, we conclude that, in the absence of Id2, mammary epithelial development is arrested at an early stage of pregnancy.