| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 279, Issue 9, 8011-8017, February 27, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
¶
From the
Section on the Molecular Biology of Selenium, Basic Research Laboratory, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland, 20892, Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588, ||Laboratory of Molecular Genetics, School of Biological Sciences, Seoul National University, Seoul 151-742, South Korea, and **Pathology Histotechnology Laboratory, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, Frederick, Maryland 21702
Received for publication, September 22, 2003 , and in revised form, December 2, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Selenoproteins are most certainly responsible for many of the health benefits of selenium. Humans encode 25 selenoproteins in their genome and mice 24, but the functions of only about half of these proteins are now known (2). Determining the identity and functions of selenoproteins is essential to understanding the role of selenium in human health. Selenium makes its way into protein as the amino acid selenocysteine (Sec) (reviewed in Ref. 3). Sec has its own code word, UGA, and its own tRNA designation, Sec tRNA[Ser]Sec. Sec is biosynthesized on its tRNA after the tRNA is aminoacylated initially with serine by seryl-tRNA synthetase. The presence of a stem-loop structure, designated a Sec insertion sequence element, in selenoprotein mRNAs dictates a UGA codon within an open reading frame to function as Sec and not as stop (4). In addition, much of the Sec protein insertion machinery is unique to this amino acid, and in mammals, there is a specific Sec insertion sequence-binding protein, SBP2 (5), and a specific elongation factor, EFsec (6, 7), required for Sec insertion into protein.
Selenoproteins are the only known class of proteins for which expression is determined by the presence of a single tRNA. Thus, manipulating the expression of Sec tRNA[Ser]Sec can perturb the expression of selenoproteins, which in turn provides an important tool in elucidating the biological functions of the various members of this class of proteins and their potential roles in promoting better health. One approach to elucidating the cellular roles of selenoproteins is to knock out the corresponding gene. Indeed, several laboratories have targeted specific selenoproteins for removal from the mouse genome, including glutathione peroxidases 1, 2, and 4 (GPx1 (8), GPx2 (9), GPx4 (10)), thyroid hormone deiodinase 2 (DIO2) (11), and selenoprotein P (SelP)1 (12, 13). These studies have provided insights into the roles of these selenoproteins in development and cellular metabolism.
In a different approach, and to provide alternative models for examining the roles of selenium in health, we initially generated a transgenic mouse carrying a mutant Sec tRNA[Ser]Sec transgene wherein the expressed tRNA lacked a highly modified base, N6-isopentenyladenosine (i6A), at position 37 (14). The i6A-tRNA[Ser]Sec-deficient mice manifested a reduction in selenoproteins that occurred in a protein- and tissue-specific manner. More recently, we generated a conditional knockout of the Sec tRNA[Ser]Sec gene encoding flanking loxP sites whereby the gene is receptive to removal by the Cre recombinase, which may be under the control of promoters targeted for specific organs or tissues (15). Mice that are homozygous for this floxed allele, designated Trspfl/fl, were crossed to transgenic mice carrying the Cre recombinase under the control of two promoters targeting mammary epithelium. Neither Cre recombinant resulted in complete removal of Trsp. However, one of the promoters, MMTV-Cre, removed about 80% of the Trsp, which resulted in an altered selenoprotein expression in mammary epithelium (15) similar to that observed in mouse liver with the mutant transgene (14). However, no apparent phenotypic changes due to selenoprotein deficiency were detected. In the present study, Trsp was selectively removed from liver by mating floxed mice with transgenic mice carrying the Alb-Cre transgene (16, 17). Characterization of these mice provided important insights into selenoprotein synthesis and transport and identified an essential role of selenoproteins in liver function.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Identification of Trsp, Trspfl/fl, and Alb-Cre+/- and Selective Removal of Trsp in Liver—Mice carrying homozygous floxed Trsp (Trspfl/fl) were identified by PCR analysis of tail DNA as described previously (15), and the Alb-Cre transgene was identified by PCR analysis of tail DNA with primers 5'-ACCTGAAGATGTTCGCGATTATCT-3' and 5'-ACCGTCAGTACGTGAGATATCTT-3', which resulted in a 370 -bp fragment (16). Heterozygous floxed Trsp-heterozygous albumin Cre (Trspfl/+-Alb-Cre+/-) mice were generated by mating Trspfl/fl mice with Alb-Cre+/- mice, selecting for the appropriate offspring, and then mating the resulting mice heterozygous for both genes to obtain homozygous floxed Trsp-heterozygous albumin Cre (Trspfl/fll-Alb-Cre+/-), Trspfl/+-Alb-Cre+/-, and Trspfl/fll offspring for Sec tRNA[Ser]Sec, selenoprotein, blood, and pathological analyses.
Isolation, Fractionation, and Identification of Specific tRNA Isoforms—Total tRNA was isolated from tissues (18) and fractionated by RPC-5 chromatography (19) or by polyacrylamide gel electrophoresis as described (14). Sec tRNA[Ser]Sec and serine tRNASer1 were identified by northern blotting and quantitated as given (14).
Labeling of Selenoproteins and GPx1 and TR1 Assays—Mice with genotypes Trspfl/fll-Alb-Cre+/-, Trspfl/+-Alb-Cre+/-, and Trspfl/fll were labeled with 75Se, tissues and organs excised, proteins extracted and electrophoresed, gels stained with Coomassie Blue, and proteins transferred to nylon membranes. The resulting transblots were exposed to a PhosphorImager as described (14, 15, 20).
GPx1 activity was assayed directly, and TR1 activity was assayed after enrichment on ADP-Sepharose as described (14). A major protein differentially expressed between knockout and control livers was identified by N-terminal Edman degradation at the University of Nebraska-Lincoln proteomics core facility.
Pathology Evaluation—Mice were sacrificed using CO2 inhalation. Necropsy examination was performed on a subset of mice that were sacrificed or that died spontaneously. The total numbers of mice examined were as follows: Trspf1/f1, scheduled sacrifice, 7 males and 0 females; Trspf1/f1-Alb-Cre+/-, scheduled sacrifice, 4 males and 2 females; and Trspf1/f1-AlbCre+/-, spontaneous death/clinically ill, 3 males and 6 females. A comprehensive set of organs and tissues was collected and fixed in 10% buffered neutral formalin. Tissues were paraffin-embedded, sectioned at 5 µm, and stained with hematoxylin and eosin. The TUNEL assay (Apoptag, Serologicals Corp.) was performed on sections of liver from all mice. Prussian blue stain for iron was performed on samples of necrotic fat.
Blood and Selenium Analyses—Blood samples were taken from mice prior to necropsy by cardiac puncture. The serum was obtained by centrifugation and used for determining blood chemistries (run by the Pathology/Histotechnology Laboratory) using standard techniques. The analytes tested were urea nitrogen, total protein, albumin, aspartate transaminase, alanine transaminase, alkaline phosphatase, 
-glutamyl transpeptidase, and total bilirubin.
To determine the levels of selenium in the form of low molecular weight selenocompounds or selenoproteins, 300 mg of liver were homogenized in 5 ml of a lysis buffer (14, 15, 20), and proteins were precipitated in trichloroacetic acid and collected as described previously (21). Selenium levels in the extracts, pellets, and trichloroacetic acid supernatants and in all other tissues and organs were determined by the Oscar E. Olsen Biochemistry Laboratories at South Dakota State University.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
Selenoprotein Expression—The expression of selenoproteins in liver, kidney, plasma, brain and testes of Trspfl/fl, Trspfl/+-Alb-Cre+/-, and Trspfl/fl-Alb-Cre+/- mice was assessed by 75Se labeling of the corresponding mice and by examining the resulting labeled proteins from these tissues following gel electrophoresis. Coomassie Blue-stained gels of total proteins from livers of these mice appeared similar (Fig. 2, lower panels) with the exception of an enriched band in the extract of Trspfl/fl-Alb-Cre+/- mice (designated by an arrow). We extracted this 25-kDa band from gels and sequenced the first 10 residues, PMILGYWNVR. This sequence is identical to that of mouse glutathione S-transferase, demonstrating that the expression of this enzyme is elevated in the liver of selenoprotein-deficient mice.
|
GPx1 and TR1, which are major selenoproteins expressed in the liver, often exhibit changes in expression that are opposite to each other (14, 15, 27). To confirm the decreased expression of these two selenoproteins in Trsp knockout liver, we assayed GPx1 and TR1 activities in mouse lines carrying the Trspfl/fl and Trspfl/fl-Alb-Cre+/- genotypes. Consistent with the 75Se labeling assays, the liver of Trspfl/fl-Alb-Cre+/- mice had less than 5% of the GPx1 activity of that found in the floxed control mouse, whereas the activities in kidney were virtually identical in these two mouse lines (data not shown). TR1 activity was similarly reduced in the liver of the Trspfl/fl-Alb-Cre+/- mice (data not shown), but TR1 levels were virtually unchanged (see Fig. 2 and its legend), providing further evidence that the protein is terminated at the penultimate UGA Sec codon.
Two major selenoproteins, SelP and GPx3, have been described in plasma (Ref. 22 and references therein). SelP has been reported to be synthesized in the liver and transported to plasma (23, 24) and to be imported into the brain and testes (25), whereas the kidney-proximal tubules are the major source of plasma GPx3 (26). Brain has been reported to express SelP mRNA, but not testes, suggesting that SelP is also synthesized in brain but not testes (25). The SelP level was reduced about 75% in plasma in Trspfl/fl-Alb-Cre+/- mice as shown in Fig. 2, "Plasma" panel. To verify that the major labeled band observed in plasma was SelP, a membrane containing the 75Se-labeled liver extracts from these three mouse lines was exposed to antibodies for SelP (Fig. 2, see small panel (insert) attached to the right of the Plasma panel). The Western blot shows that the first and second lanes (Fig. 2) with plasma of Trspfl/fl and Trspfl/+-Alb-Cre+/- mice yielded a positive signal with SelP antibodies, but that the lane with plasma from Trspfl/fl-Alb-Cre+/- mice did not respond to SelP antibodies. These results demonstrate that the major 75Se-labeled band in plasma is SelP (see also Ref. 22). It should also be noted that SelP antibodies do not detect SelP in tissues.2 In contrast, the Coomassie Blue-stained gels of total proteins from plasma of the three mouse lines appeared to be similar. The 75Se-selenoprotein labeling pattern observed in brain was similar in the different genetic backgrounds of each animal examined, whereas in testes a band that migrates at the expected position of SelP was reduced. The labeling pattern of GPx3 in plasma suggests that its level may not be affected, providing further evidence that its presence in this tissue is the result of its synthesis in kidney (26).
Selenoprotein mRNA Analysis—Because the selective loss of Trsp in liver affects the expression of selenoproteins at the translation step, we examined the amounts of several selenoprotein mRNAs in mouse lines with Trspfl/fl-Alb-Cre+/- and Trspfl/+-Alb-Cre+/- genotypes. Of the selenoprotein mRNAs examined, the level of GPx1, and possibly that of SelP, appeared to be reduced in liver of Trspfl/fl-Alb-Cre+/- mice, whereas those of GPx4, SPS2, D1, and TR1 appeared to be similar as compared with the corresponding levels in Trspfl/+-Alb-Cre+/- mice (Fig. 3). The instability of GPx1 mRNA in liver not expressing this protein due to selenium deficiency generated by diet (28) or by genetic analysis (14) has been noted in other studies.
|
65 days for males and females. A subset of mice were for pathological analysis. Findings were markedly different in Trspf1/f1-Alb-Cre+/- mice, which were found dead or were sacrificed when clinically ill compared with scheduled sacrifices of clinically normal mice. Spontaneous deaths and moribund mice had two major lesions with 100% incidence: hepatocellular degeneration/necrosis and necrosis of peritoneal/retroperitoneal fat. In contrast, neither of these phenotypes was present in clinically healthy Trspf1/f1-Alb-Cre+/- mice. Many of the hepatocytes were swollen with an indistinctly vacuolated cytoplasm as shown in Fig. 4A. A number of these swollen cells had karyorrhectic nuclei. Other hepatocytes had undergone massive coagulative necrosis, and there was no apparent lobular pattern to this damage. Some of the necrotic hepatocytes were mineralized (dystrophic calcification). Livers in two females had foci of birefringent crystals (data not shown). Foci of subacute inflammation were present in damaged livers but were less extensive than expected with the large amount of cell death. The majority of hepatocytes that had karyorrhectic or more normal nuclei were positive for DNA laddering (apoptosis) with the TUNEL technique (Fig. 4B). Their nuclei did not have typical apoptotic morphology of shrinkage and pyknosis. The DNA laddering was confirmed by direct examination of the DNA (Fig. 4C), but none of the abnormal liver cells manifested enriched p53 levels as determined by direct assay (data not shown). Liver and fat lesions were absent in sacrificed Trspf1/fl mice, which served as age-matched controls.
|
Selenium Levels and Blood Chemistries—Selenium levels were determined in the liver, kidney, plasma, brain, and testes of Trspfl/fl, Trspfl/+-Alb-Cre+/-, and Trspfl/fl-Alb-Cre+/- mice. Substantially reduced levels of this element were found in liver and plasma of Trspfl/fl-Alb-Cre+/- mice (Fig. 5). Slightly lower selenium levels were observed in testes and brain of Trspfl/fl-Alb-Cre+/- mice, compared with Trspfl/fl mice, but these amounts did not appear to be significantly lower in these tissues of liver Trsp knockout mice (see "Discussion").
|
|
-glutamyl transpeptidase, and total bilirubin) were performed on clinically normal mice (ages 49–76 days) of all three genotypes: Trspf1/f1 (total number of animals = 3), Trspf1/+-Alb-Cre+/- (3 animals), and Trspf1/f1-Alb-Cre+/- (6 animals). Interestingly, there were no significant differences in the eight parameters tested among the different genotypes (data not shown). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
65 days. One characteristic of mice that fail to make selenoproteins in hepatocytes is that their liver is selenium-deficient. Reduced selenium levels have also been observed in the liver of GPx1 gene knockout mice suggesting that this selenoprotein provides a major source of selenium in this organ (8). We find that the low molecular weight selenocompounds constitute about one-third of the selenium in liver, and this amount varied only slightly in selenoprotein-deficient mice. Therefore, most of the selenium in the liver is accounted for by selenium-containing proteins, as specific excision of the Sec tRNA[Ser]Sec gene resulted in disrupted selenoprotein synthesis and a concomitant decrease in liver selenium levels but with little or no change in the low molecular weight selenocompounds.
Selenium levels appeared to be similar in brain as well as testes in mice that were either liver Trsp- or Trsp+. Selenium levels in the plasma of these mice, however, were reduced, suggesting that adequate amounts of this element in plasma are largely determined by SelP content, which supports the earlier observation of Burk et al. (22).
Our results confirm earlier reports that SelP is primarily synthesized in liver (23, 24). However, the finding that SelP is still present in plasma in the liver of Trsp- mice suggests that other organs contribute to the occurrence of SelP in the plasma, at least under conditions of disrupted SelP synthesis in the liver. Our finding that SelP levels are not decreased in brain of liver Trsp- mice, but they are decreased in testes, provides strong evidence that the former organ synthesizes its own SelP but the latter does not (see also 23,24). Enriching the diet with selenium in SelP knockout mice restores the neurologically defective phenotype observed in these animals to almost normal (12, 13), providing further evidence that the role of SelP, at least in part, is to transport selenium to various organs (see Ref. 30 for review). Interestingly, the finding of widespread necrosis of peritoneal and retroperitoneal fat in the absence of pancreatic damage in this study suggests that this circulating selenoprotein may also have a role in adipocyte homeostasis.
An interesting observation regarding the loss of selenoprotein expression and/or selenium deficiency in the liver of Trsp- mice is that the glutathione S-transferase levels were elevated, suggesting that this genotype may have up-regulated a detoxification program. Because many selenoproteins have roles as antioxidants (3), it would then seem that an interplay may exist between selenoproteins and other proteins involved in stress-related phenomena whereby they can compensate for each other.
Clinically ill mice used in this study had severe hepatocellular swelling and necrosis. DNA laddering was observed in the ill mice, suggesting that numerous cells were undergoing apoptosis. A TUNEL assay confirmed the presence of apoptotic cells. However, a direct assay of p53 showed that the levels of this protein were not elevated in liver cells of ill mice. Although enhanced expression of p53 often accompanies apoptosis, this is not true in all cases (e.g. see Ref. 31 and references therein), and apparently liver cells lacking selenoprotein expression undergo apoptosis without enhanced p53 expression.
An interesting feature of the removal of selenoprotein biosynthesis in hepatocytes is that liver requires their expression for proper function. In addition to demonstrating an essential role of selenoproteins in liver function, the present study has also provided new insights into the synthesis of SelP, its transport to other tissues, and a possible new function of this selenoprotein in fat metabolism.
|
FOOTNOTES |
|---|
¶ Current address: The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104.
To whom correspondence should be addressed: NCI, National Institutes of Health, Bldg. 37, Rm. 6016, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-2797; Fax: 301-435-4957; E-mail: hatfield{at}dc37a.nci.nih.gov.
1 The abbreviations used are: SelP, selenoprotein P; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; GPx, glutathione peroxidase; RPC-5, reverse phase chromatographic column-5.
2 K. E. Hill and R. F. Burk, personal communication.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  L. Grumolato, H. Ghzili, M. Montero-Hadjadje, S. Gasman, J. Lesage, Y. Tanguy, L. Galas, D. Ait-Ali, J. Leprince, N. C. Guerineau, et al. Selenoprotein T is a PACAP-regulated gene involved in intracellular Ca2+ mobilization and neuroendocrine secretion FASEB J, June 1, 2008; 22(6): 1756 - 1768. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Suzuki, V. P. Kelly, H. Motohashi, O. Nakajima, S. Takahashi, S. Nishimura, and M. Yamamoto Deletion of the Selenocysteine tRNA Gene in Macrophages and Liver Results in Compensatory Gene Induction of Cytoprotective Enzymes by Nrf2 J. Biol. Chem., January 25, 2008; 283(4): 2021 - 2030. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. A. Carlson, M. E. Moustafa, A. Sengupta, U. Schweizer, R. Shrimali, M. Rao, N. Zhong, S. Wang, L. Feigenbaum, B. J. Lee, et al. Selective Restoration of the Selenoprotein Population in a Mouse Hepatocyte Selenoproteinless Background with Different Mutant Selenocysteine tRNAs Lacking Um34 J. Biol. Chem., November 9, 2007; 282(45): 32591 - 32602. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. A. Sheridan, N. Zhong, B. A. Carlson, C. M. Perella, D. L. Hatfield, and M. A. Beck Decreased Selenoprotein Expression Alters the Immune Response during Influenza Virus Infection in Mice J. Nutr., June 1, 2007; 137(6): 1466 - 1471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sengupta, B. A Carlson, V. J Hoffmann, V. N Gladyshev, and D. L Hatfield Conditional knockout of selenocysteine tRNA gene (trsp) in liver modulates lipoprotein metabolism FASEB J, April 1, 2007; 21(5): A114 - A114.
|
|
|
|
|
|
A. Nakayama, K. E. Hill, L. M. Austin, A. K. Motley, and R. F. Burk All Regions of Mouse Brain Are Dependent on Selenoprotein P for Maintenance of Selenium J. Nutr., March 1, 2007; 137(3): 690 - 693. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Riese, M. Michaelis, B. Mentrup, F. Gotz, J. Kohrle, U. Schweizer, and L. Schomburg Selenium-Dependent Pre- and Posttranscriptional Mechanisms Are Responsible for Sexual Dimorphic Expression of Selenoproteins in Murine Tissues Endocrinology, December 1, 2006; 147(12): 5883 - 5892. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-H. Yoo, X.-M. Xu, B. A. Carlson, V. N. Gladyshev, and D. L. Hatfield Thioredoxin Reductase 1 Deficiency Reverses Tumor Phenotype and Tumorigenicity of Lung Carcinoma Cells J. Biol. Chem., May 12, 2006; 281(19): 13005 - 13008. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Irons, B. A. Carlson, D. L. Hatfield, and C. D. Davis Both Selenoproteins and Low Molecular Weight Selenocompounds Reduce Colon Cancer Risk in Mice with Genetically Impaired Selenoprotein Expression J. Nutr., May 1, 2006; 136(5): 1311 - 1317. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sengupta, J. A Weaver, S. V Novoselov, D. E Fomenko, B. A Carlson, V. N Gladyshev, and D. L Hatfield A comparative analysis of selenoproteins and global gene expression in liver selenocysteine tRNA knockout mice and its rescued variants FASEB J, March 1, 2006; 20(4): A427 - A428. [Abstract] [Full Text]
|
|
|
|
|
|
L. Schomburg, C. Riese, M. Michaelis, E. Griebert, M. O. Klein, R. Sapin, U. Schweizer, and J. Kohrle Synthesis and Metabolism of Thyroid Hormones Is Preferentially Maintained in Selenium-Deficient Transgenic Mice Endocrinology, March 1, 2006; 147(3): 1306 - 1313. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. P. Kelly, T. Suzuki, O. Nakajima, T. Arai, Y. Tamai, S. Takahashi, S. Nishimura, and M. Yamamoto The Distal Sequence Element of the Selenocysteine tRNA Gene Is a Tissue-Dependent Enhancer Essential for Mouse Embryogenesis Mol. Cell. Biol., May 1, 2005; 25(9): 3658 - 3669. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Xia, K. E Hill, D. W Byrne, J. Xu, and R. F Burk Effectiveness of selenium supplements in a low-selenium area of China Am. J. Clinical Nutrition, April 1, 2005; 81(4): 829 - 834. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Morbitzer and T. Herget Expression of Gastrointestinal Glutathione Peroxidase Is Inversely Correlated to the Presence of Hepatitis C Virus Subgenomic RNA in Human Liver Cells J. Biol. Chem., March 11, 2005; 280(10): 8831 - 8841. [Abstract] [Full Text] [PDF]
|
|
|
|
|
