Selenoproteins in the Bovine Mammary Gland. Regulation of mRNA and Protein Expression

University dissertation from Pure and Applied Biochemistry, Faculty of Engineering LTH. P.O. Box 124, 221 00 Lund

Abstract: Selenium is a micronutrient that is essential for many important life processes due to the action of the specific selenoproteins containing one or more of the 21st amino acid, selenocysteine. Twenty-five selenoprotein genes have been found in the human genome but the function of many of them is not yet known. Some of the characterised selenoproteins have, however, antioxidant properties. Selenium deficiency or low selenium status has been linked to various diseases in humans such as certain types of cancer, and a number of selenium deficiency symptoms are known in farm animals. When the concentration of selenium in the soil is low, as it is in Sweden, selenium supplements are often added to cow's feed to increase the levels of selenium in cow tissues which also leads to higher levels in milk. In the Swedish diet, cow's milk accounts for about one fifth of the selenium intake of adults. The general aims of the present study were to investigate which selenoproteins are found in bovine mammary cells and tissue, to find out how mRNA and protein expression are regulated, and to explore the possible role of selenoproteins in milk formation and mammary gland physiology. Biomarkers of selenium status and mechanisms of action of selenium supplements in cancer prevention were also reviewed. Messenger RNA (mRNA) was demonstrated for selenoprotein P, thioredoxin reductase 1 and for glutathione peroxidase (GPx) 1, 3 and 4 in bovine mammary tissue, and all but GPx 3 were also found to be expressed in the bovine mammary cell line MAC-T. It was also found that selenium status is an important regulator of selenoprotein activity and expression in bovine mammary tissue and cells, but that other factors are also involved. The effect of the vitamin A metabolite retinoic acid (RA) and the lactogenic hormones prolactin, insulin and hydrocortisone on mRNA expression of selenoproteins in MAC-T cells was also investigated. RA was found to increase the expression of GPx 1 and thioredoxin reductase 1. It was also found that insulin decreased the expression of selenoprotein P, and a combination of insulin and hydrocortisone increased the expression of GPx 1. The selenoproteome of MAC-T cells was studied by labelling with radioactive 75Se and examination by 2D electrophoresis, autoradiography and MALDI-TOF. MAC-T cells were shown to express a number of selenoproteins, the cytosolic GPx 1 being the most abundant. The effects of RA on the selenoprotein levels in the cell line were also studied. The concentration of 75Se in total cell protein was reduced in cells cultured with RA compared with control cells, and the opposite was seen in extracellular proteins. RA treatment of MAC-T cells also led to changes in the levels of several non-selenoproteins in the cells, for example, decreased levels of proteins associated with cancer, such as heat shock proteins 70:8 and 27:1, and keratin 8. Other proteins decreased in the MAC-T cells by RA may be associated with mammary gland development and milk formation. In summary, this study shows that a number of selenoproteins could be detected in bovine mammary tissue and cells. It was also demonstrated that the expression of these proteins can be affected by selenium status, retinoic acid and hormones.

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