Selenium is an essential micronutrient in the diet of humans and other mammals. Many health benefits have been attributed to selenium that include preventing various forms of cancer (e.g., colon cancer, prostate cancer, lung cancer and liver cancer), heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus (HIV)-positive patients, slowing the aging process, and having roles in mammalian development, including male reproduction and immune function. Numerous human clinical trails have been undertaken in recent years to assess the role of this element in cancer prevention, delaying the progression of AIDS, etc., at a cost of billions of dollars, but little is known about the mechanism of how selenium acts at the metabolic level in mammals to exert these many health benefits. We propose that the health benefits of selenium are due largely to its presence in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec). Our program therefore focuses on: 1) developing mouse models to assess the role of selenium and selenoproteins in cancer prevention and development, 2) characterizing and elucidating the function of various selenoproteins and their roles in cancer prevention and development, and 3) identifying the means by which Sec is biosynthesized and incorporated into protein. The project discussed herein examines our research on the development of various mouse models for determining the role of selenium in cancer prevention and development. During the past year, we focused our attention on further characterizing 5 of our mouse models that included: 1) knockout of the Sec tRNA gene (designated Trsp) and consequently the loss of selenoprotein expression in (a) T cells, (b) macrophage, (c) epidermal skin tissue, and (d) liver, and subsequent rescue or partial rescue of selenoprotein expression with a wild-type or mutant Trsp transgene; and 2) elucidation of the role of STAF, a transcription-activating factor for a number of RNA Pol II- and RNA Pol III-dependent genes, including the Sec tRNA gene, on the expression of Sec tRNA by deleting its binding site (the Activator Element or AE) within the upstream regulatory region of Trsp. In the past year, our investigation of the molecular mechanism that links selenium to T cell immunity found that selenoprotein deficiency in these cells leads to oxidant hyperproduction, thereby suppressing their proliferation in response to T cell receptor stimulation. Our findings provided novel insights into the immune function of selenium and physiological antioxidants. On the other hand, removal of selenoproteins in macrophages showed an accumulation of ROS levels in selenoprotein KO macrophages compared to wild-type controls. KO macrophages also exhibit impaired invasiveness based on the matrigel invasion assay. Microarray analysis has identified altered expression of several extracellular matrix and fibrosis-associated genes. These results have been verified by qPCR analysis. The altered gene expression does not appear to be a result of increased ROS levels, as treatment with NAC had little effect on altered gene expression. Removal of selenoproteins in skin epidermal cells resulted in a runt phenotype and premature death of the progeny. The KO mice had alopecia along with a flaky and fragile skin. Histological studies on the KO mice reveal epidermal hyperplasia along with changes in hair follicle appearance, wherein the hair cycle was disturbed with an early regression of hair follicles. These observations highlight a hitherto unknown role of selenoproteins in cutaneous development. We also developed a mouse model in which the hepatic selenoprotein population was targeted for removal by disrupting Trsp and then selenoprotein expression was restored or partially restored by introducing wild-type or mutant transgenes. Two mutant tRNAs lacking the 2-methylribose at position 34 (Um34) supported expression of housekeeping selenoproteins (e.g., thioredoxin reductase 1) in liver, but not stress-related proteins (e.g., glutathione peroxidase 1) providing further evidence from our laboratory that Um34 is responsible for stress-related selenoprotein synthesis, whereas the Sec tRNA isoform without Um34 is responsible largely for housekeeping selenoprotein synthesis. The role of STAF in regulating expression of Sec tRNA and selenoproteins was examined. We generated mice in which the STAF binding site, designated AE, that is located upstream of Sec tRNA gene was deleted. The level of Sec tRNA was unaffected or slightly elevated in heart and testis, but reduced in liver, kidney, lung and spleen, brain and muscle compared to the corresponding organs in control mice. Also, the ratio of the two isoforms of Sec tRNA that differ by methylation at position 34 (Um34) was altered significantly, and the Um34-containing form was substantially reduced in all tissues examined. Selenoprotein expression in these animals was most affected in tissues in which the Sec tRNA levels were most severely reduced. The mice manifested a neurological disorder phenotype and their lifespan was substantially reduced. Our results indicate that STAF influences selenoprotein expression by controlling Sec tRNA gene synthesis in an organ-specific manner and by down-regulating Sec tRNA expression and modification status.
Showing the most recent 10 out of 47 publications
