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ACTIVE TRANSCRIPTION GLUTATHIONE PEROXIDASE

OF THE SELENIUM-DEPENDENT GENE IN SELENTUM-DEFICIENT

1431-1436

RATS

Mei Chang and C. Channa Reddy* Environmental Resources Research Institute and Department of Veterinary Science The Pennsylvania State University University Park, Pennsylvania 16802 Received

November

19,

1991

SUMMARY: Selenium-dependent glutathione peroxidase (Se-GSH-Px, Ec.1.11.1.9) is the best characterized selenoenzyme in higher animals. However, neither the mechanism whereby selenium (Se) becomes incorporated into the enzyme nor the level at which the expression of Se-GSH-Px gene is regulated by Se is fully understood. In the current investigation, we have determined the relative rates of the transcription of the Se-GSH-Px gene in purified liver nuclei isolated from rats fed on Se-supplemented or Se-deficient diets. No significant difference in the transcription rates appeared in these two groups. These results are consistent with the previous observations that active message for SeGSH-Px - that is, translatable mRNA for Se-GSH-Px - is present in Se-deficient tissues (Li et aZ.,J. Biol. Chem., 265, 108-113, 1990). The data also suggest that the alteration of SeGSH-Px activity and the corresponding protein and mRNA levels in rats subjected to dietary Se manipulation can be attributed only to post-transcriptional regulation. Q 1991 Academic Press, Inc. Selenium-GSH-Px catalyzes the two electron reduction of both Hz02 and organic hydroperoxides and thus plays an important role in eliminating potentially harmful peroxides formed by various enzymatic and nonenzymatic reactions in cells. The molecular enzymology of this remarkable protein has been recently reviewed (1). It is a tetramer of identical subunits with each monomer containing a gram atom of Se in the form of selenocysteine (Se-q). Ever since it was first discovered that Se is an essential component of Se-GSH-Px, there has been an intensive search for the identification of the chemical form and the mechanism of specific incorporation of the Se moiety into the enzyme protein (2). Recently, it has been reported in several systems, including the mammalian Se-GSH-Px, that Se-cys is encoded by the UGA termination (opal) codon (3-6). The evidence available so far suggests a cotranslational mechanism for the Se-q incorporation. An unusual seryl tRNA with UCA at the anticodon loop is acylated with serine and then converted to Se-cys, possibly through a phosphoserine intermediate, which is then incorporated into a nascent polypeptide chain (2,7,8). It is now clear that *To whom correspondence should be addressed.

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0006-291X/91 $1.50 Copyright 6 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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expression of Se-GSH-Px activity in tissues is regulated by Se. Our group and others have reported that progressive Se-deficiency resulted in a dramatic decrease in the level of immunoreactive Se-GSH-Px protein in parallel with a decrease in Se-GSH-Px activity (5,9). Both irt vifro translation experiments and RNA blot analysis, however, revealed that the mRNA corresponding to Se-GSH-Px is expressed in Se-deficient tissues (5,lO). These observations contradict the earlier reports which indicate that the Se-GSH-Px mRNA levels are reduced in livers of Se-deficient rats (11,12). To date, this discrepancy has not been clearly resolved. Furthermore, it is not fully understood how Se regulates the expression of Se-GSH-Px in mammalian tissues. In this article, we report the results of investigations into the transcription of the Se-GSH-Px gene in nuclei obtained from Sedeficient rat livers, which reveal that the transcriptional activity of the Se-GSH-Px gene is not affected by Se deficiency. MATERIALS

AND METHODS

Materials : Biotrans membranes (pore size; 0.2 pm) and [a-32P]UTP [specific activity, 13,000 Ci/mmol] were obtained from ICN Pharmaceuticals Inc., unlabeled NTPs were from Pharmacia LKB Biotechnology Inc., and bakers yeast RNA was from Sigma. Animals and Diets : Post-weaning male rats (5 rats in each group) of the Long-Evans hooded strain were fed either a torula yeast-based basal diet deficient in Se (Se-deficient group) or the same diet supplemented with 0.5 m Se as sodium selenite per kg diet (Sesupplemented group) as previously described (135 . After the animals had been fed the experimental diets for at least 8 weeks, rat livers from each group were used for the reparation of cytosolic fractions, and for the isolation of rat liver nuclei. Selenium-GSHK activity was measured as prevtously described (13). Nuclear Run-On Transcription Assav: Nuclei were isolated from rat liver as described by Blobel and Potter (14). The isolated nuclei were suspended in storage buffer containing 40% glycerol, 50 mM TrisHCl, pH 8.3, 5 mM MgCl , and 0.1 mM EDTA. Each aliquot (125 ~1) contained nuclei from about 1 g of liver. + he nuclei preparation was either used immediately or stored at -70°C for future transcription assays. In vitro transcription assays using isolated nuclei were erformed as previously described (15) which is a modification of the procedure of 6 roudin et al. (16) and M&night and PaImiter (17). The RNA was purified usin the acid guanidine thiocyanate-phenol-chloroform procedure (18) except that yeast Ra A (50 pg) was ad3Jed to each reaction along with 600 ~1 of uanidine thiocyanate solution. The purified [a- P RNA was resuspended in 100 ~1 o B the hybridization buffer (50% formamide, 0.5 d NaHzPOd, pH 7.2,7% SDS, 500 pg/ml yeast RNA) and transferred to 4-ml flint glass vials for hybridization. To examine the effects of a-amanitin on nuclear run-on assays, the nuclei were incubated for 30 min on ice with a-amanitin (1 kg/ml) before adding the transcription reaction components.

Hvbridization to Immobilized DNA: The cDNA clone pGPX1211, which is complementary to rat liver Se-GSH-Px mRNA (5), was used in this study to measure the transcriptional rates of the Se-GSH-Px ene. The plasmid DNA was linealized by EcoRI. Slot blots were prepared according to Ea fatos et a/. (19) with the modification suggested by Rohrbaugh et al. (20). The amount of the DNA in the blots was titrated to ensure that the mRNA-binding ca acity of the blots had not been exceeded under our experimental conditions. One pg o PDNA was subsequent1 used in the rest of the experiments. The blots were prehybridized in hybridization bu t?fer for at least 0.5 h bffore hybridization. The glass tubes containing 100 ,ul hybridization mixture (0.5 - 1.3 x 10 cpm) and the blots were incubated at 45°C for 72 h. One blot containing plasmid PUC 19, was used as a negative control. pGTR 261, a cDNA clone corn lementary to a rat liver glutathione Stransferase (GST) Ya subunit mRNA (21), and P8 R5, a human ty-,d-actin cDNA plasmid, 1432

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were employed as internal controls. After hybridization, the blots were washed (18) and digested with DNase-free RNaseA (10 &ml) and T1 RNase (1 CLglml) as previously described (15). The slot blots were then exposed to X-ra film with an intensifying screen at -70°C for 3-7 days. The autoradiograms were scanne (Ywith an LKE3 densitometer. To quantitatively compare the transcription rates of the Se-GSH-Px gene in Sesupplemented and Se-deficient rat liver a standard curve of the integrated area of densitometer scan vs the amount of radiation on the blot was constructed to ensure scanning capacity had not been exceeded under our experimental conditions. The data were subjected to an analysis of variance for two independent variables to determine if there was a dietary treatment effect. Student’s two-tailed t-test (22) was used to verify the statistical differences between the groups.

RESULTS

AND DISCUSSION

The expression of Se-GSH-Px protein in a given tissue appears to be under the influence of the Se concentration in that tissue. A dramatic decrease in the level of immunoreactive Se-GSH-Px protein as well as Se-GSH-Px activity has been observed in rats subjected to Se deficiency (5,9,10). Since the results of Northern (RNA) blot analysis on the corresponding mRNA levels are controversial, we have employed the nuclear runon transcription assay to measure the transcriptional activity of the Se-GSH-Px gene directly in the liver of both Se-sufficient and Se-deficient rats. The Se deficiency in the experimental group was confirmed by measuring the Se-GSH-Px activity of the liver and the blood from rats fed on the Se-deficient diets. As expected, the Se-GSH-Px was markedly reduced (to - 2% of the controls) in the Se-deficient group (data not shown). The incorporation of [~z-~~P]UTP into trichloroacetic acid-precipitable material was linear for at least 20 min. About 1.4% of the [a- 32P]UTP was incorporated at 20 min. As Figure 1 shows, when an equivalent amount of rat liver nuclei from both Sesupplemented and Se-deficient rats was used for in vitro transcription, the hybridization signals on the autoradiogram of these two samples had a similar intensity. The transcriptional activity of the Se-GSH-Px gene, as measured by quantitative densitometry and normalized by comparing it with the ly-p-actin signals, was virtually the same for both Se-supplemented and Se-deficient samples as shown in Table I. We did not observe any hybridization signals on the slot blots containing PUC 19 Vector DNA (see Fig. 1). The slot blots containing the cDNA clone for the GST Ya-size subunit served as internal controls. Our group had observed the transcriptional activation of the GST Ya and/or Yc genes by Se deficiency earlier (15). In the present study, the transcriptional activity of Ya Yc genes was consistently elevated in every Se-deficient sample (data not shown). As reported before (15), on the average, transcriptional activity of the Ya Yc genes was increased by about two times in the Se-deficient samples over the controls, indicating that the animals respond well to transcriptional stimulation caused by Se-deficiency. We assayed a-amanitin sensitivity to verity whether or not the transcriptional activity in Sedeficiency is RNA polymerase II-dependent. The transcriptional signals for the Se-GSHPx gene were greatly reduced at low levels of a-amanitin with no detectable bands on the blots (data not shown). 1433

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PVC19

SC-GSll-PX Fig.

Nuclearrun-on transcriotionof Se-GSH-Pxeene.s-32P-labelednuclear

RNA wasisolatedfrom the run-on transcriptionreactionand hybridizedto slot blotsat the plasmidindicated. After RNasetreatment,the slot blotswere quantitatedby autoradiographyand densitometry;+Se, nucleiisolatedfrom a Se-supplemented rat liver, -Se,nucleiisolatedfrom a Se-deficientrat liver.

The above results imply that Se primarily

regulates the expression of Se-GSH-Rx in higher animals at the post-transcriptional level. In general, this phenomenon appears to be conserved during the evolution for the genes encoding all Se-containing enzymes. For example, Se depletion causes a marked decrease in both the protein and enzyme activity levels of bacterial formate dehydrogenase, a well characterized prokaryote selenoprotein with Se-cys in its polypeptide chain. However, as in the case of mammalian SeGSH-Px, the levels of the corresponding mRNA for the formate dehydrogenase gene remained unaffected by Se deficiency (24), implying little likelihood of change in the

Table I

Effect of inadeauateSe nutrition on the transcrintionalactivitv of Se-GSH-Pzeeriein jsolatedrat liver nuclei. The sampleswere processedin sets. One Se-deficientand one Se-supplemented sample(one setat a time) were processedasdescribedunder methods. The hybridization signalson the slot blots were quantitated by autoradiographyand densitometry. The transcriptionalactivity of the selenium-supplemented sampleof each setwasarbitrarily givena value of 1.

Relative transcriptionalactivitya -Se

+ Se

1 -c 0.18

0.9025f 0.15b

a Valuesare meansf S.D. for 5 animals. b Not significantlydifferent from + Seanimals(~~0.05).

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transcriptional activity of this gene. Recently, nuclear run-on assays of the Se-GSH-Px gene have been performed with HL-60 cells as well as with mouse liver (25,26). No difference in the transcriptional activity was observed between the Se-deficient and Sesupplemented samples in these two systems. Interestingly enough, an abundance of Se in the tissue fails to increase the transcriptional activity of the Se-GSH-Px gene in guinea pig liver (27). Many steps are involved in the post-transcriptional processing, and any one of them might be subjected to regulation by tissue Se levels. The primary target, however, appears to be the cotranslational incorporation of Se-cys into the peptide chain of Secontaining enzymes. This step requires the generation of Se-cys-containing tRNA. The preformed Se-cys is not directly aminoacylated with a tRNA to give Se-cys tRNA, but it is generated in situ on a specific UGA suppressor tRNA. In bacteria, it.has been reported that the generation of Se-cys tRNA is a complex process involving many steps and requiring several gene products (2,7,28,29). The specific UGA suppressor tRNA, also known as phosphosetyl tRNA, has anticodons that recognize the UGA codon. It is initially esterified with L-serine by seryl tRNA ligase and then phosphorylated to Ophosphoseryl tRNA by a specific kinase (30). The 0-phosphoseryl tRNA is then transformed into Se-cys tRNA--presumably catalyzed by a 37 kD protein, a gene product of SeZ D (31)--but the chemical form of the Se intermediate involved in this process has not been identified. Nevertheless, the functional role of this new tRNA is to transport Secys to the UGA site on the mRNA for insertion into the nascent polypeptide chain. Therefore, it is likely that the tissue Se status influences the expression of Se-GSH-Px at the level of Se-cys-tRNA generation. Just how a cell responds to the absence of Se-cys tRNA during translation is still unclear. However, it does not stop transcribing the message for Se-GSH-Px, it may even accumulate higher levels of message--presumably due to reduced catabolism. Furthermore no translated or altered polypeptide product has yet been identified.

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Active transcription of the selenium-dependent glutathione peroxidase gene in selenium-deficient rats.

Selenium-dependent glutathione peroxidase (Se-GSH-Px, Ec.1.11.1.9) is the best characterized selenoenzyme in higher animals. However, neither the mech...
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