GSH-Px mRNA Research Articles

Transgenic glutathione peroxidase mouse models for neuroprotection studies.

Seleno-glutathione peroxidase (GSHPx) is considered to be the major enzymatic activity in charge of removing excess cytosolic and mitochondrial H2O2 in most tissues including brain. Intracellular GSHPx activity is therefore hypothesized to be one important factor that contributes to minimize hydroxyl radical formation via Fenton-type reactions. An animal model was developed to challenge this hypothesis in vivo and evaluate the role of GSHPx in hydroperoxide metabolism and oxidative stress homeostasis. Three lines of transgenic mice, homozygous for the integration of 1 to 3 GSHPx transgene copies, have been generated. The transgene was placed under transcriptional control of a metallothionein promoter (hMT-IIA). This promoter was chosen because metallothionein expression, normally low in most tissues, can be induced by several inflammatory cytokines, protein kinase C activators, and stress agents including heavy metals. The data reported here provide information on the constitutive expression of GSHPx mRNA and enzyme in various brain regions of healthy untreated adult tg-MT-GPx mice. Northern and/or Western analysis indicated that transgenic GSHPx was expressed constitutively in all brain regions investigated in tg-MT-GPx-6 mice, including the cerebral cortex, brainstem, hippothalamus, cerebellum, substantia nigra, and striatum. Similar results were obtained with the two other transgenic lines, tg-MT-GPx-11 and -13. Depending on the brain region, the GSHPx immunoreactivity detected in tissue extracts with an immunoaffinity-purified polyclonal antibody was about 2- to 5-fold stronger in transgenic extracts than in their non-tg counterparts (western blots). In contrast, the corresponding increase in GSHPx activity measured in these extracts was smaller, for example, about 1.5-fold in transgenic mesencephalon. Immunocytochemical data indicated that GSHPx-like staining was distinctly more intense in transgenic midbrain brain sections than in corresponding non-tg sections. Interestingly, only a subset of the cells displayed higher density staining that most likely reflects increased amounts of GSHPx protein. This observation suggests that the stained cells, not yet identified, may have larger GSHPx activity increments than the cell-average increments measured in tissue extracts. Current work is in progress to determine whether transgenic GSHPx expression may be induced by inflammatory processes or perturbations of heavy metal metabolism.

Effects of Nerve Growth Factor on Glutathione Peroxidase and Catalase in PC 12 Cells

Nerve growth factor (NGF) is a member of the neurotrophin family and is required for the survival and maintenance of peripheral sympathetic and sensory ganglia. In the CNS, NGF regulates cholinergic expression by basal forebrain cholinergic neurons. NGF also stimulates cellular resistance to oxidative stress in the PC12 cell line and protects PC12 cells from the toxic effects of reactive oxygen species. The hypothesis that NGF protection involves changes in antioxidant enzyme expression was tested by measuring its effects on catalase and glutathione peroxidase (GSH Px) mRNA expression in PC12 cells. NGF increased catalase and GSH Px mRNA levels in PC12 cells in a time- and dose-dependent manner. There was also a corresponding increase in the enzyme activities of catalase and GSH Px. Thus, NGF can provide cytoprotection to PC12 cells by inducing the free radical scavenging enzymes catalase and GSH Px.

Tissue-specific expression of glutathione peroxidase gene in guinea pigs

Glutathione peroxidase (GSH-Px), a selenocysteine-containing enzyme, is generally considered to be important in protecting animals from oxidative injury. However, guinea pigs have very low GSH-Px activity in major tissues such as liver and kidney, while the activity in the erythrocytes is as high as that of mice or rats. The present study attempted to clarify which step in the gene expression of GSH-Px is responsible for the tissue specific regulation of GSH-Px activity in guinea pigs. Northern blot analysis showed clear signals of GSH-Px mRNA in the reticulocytes and erythroblast-enriched bone marrow cells of guinea pigs, while it was barely detectable in the liver, kidney and heart. Using the nuclear run-on assay, we confirmed that the difference in GSH-Px mRNA levels among tissues of guinea pigs results primarily from the difference in the transcription rate of the GSH-Px gene. Thus, the guinea pig may be a good model for studying the factors regulating the tissue-specific gene expression of this selenoenzyme as well as its essential role.

Regulation of glutathione peroxidase mRNA level by dietary selenium manipulation

Glutathione peroxidase (GSH-Px) contains selenium at its active site as a selenocysteine moiety. We have shown that feeding mice a selenium-deficient diet for a long period caused a large decrease in the GSH-Px mRNA level as well as in GSH-Px activity both in the liver and kidneys (Toyoda, H., Himeno, S. and Imura, N. (1989) Biochim. Biophys. Acta 1008, 301–308). In the present study, the transcription rate of the GSH-Px gene was determined by a nuclear run-on assay using liver nuclei of mice fed a selenium-deficient or selenium-adequate diet. The results clearly demonstrate that the transcription rate of the GSH-Px gene was not changed by dietary selenium manipulation, indicating that dietary selenium regulates the level of GSH-Px mRNA in the post-transcriptional step.

Modulation of glutathione peroxidase expression by selenium: effect on Human MCF-7 breast cancer cell transfectants expressing a cellular glutathione peroxidase cDNA and doxorubicin-resistant MCF-7 cells

We have studied the effect of selenium on the expression of a cellular glutathione peroxidase, GSHPx-1, in transfected MCF-7 cells and in doxorubicin-resistant (Adrr) MCF-7 cells. A GSHPx-1 cDNA with a Rous Sarcoma virus promoter was transfected into a human mammary carcinoma cell line, MCF-7, which has very low endogenous cytosolic glutathione (GSH) peroxidase activity and no detectable message. The transfectant with the highest GSH peroxidase activity among the isolates, MCF-7H6, was characterized. Adrr MCF-7 cells, a subline of MCF-7 cells, also has elevated GSH peroxidase activity. GSH peroxidase expressed by MCF-7H6 and Adrr MCF-7 cells is similar to the endogenous GSHPx-1 based on molecular weight, immunoreactivity, and metabolic labeling with 75Se. MCF-7H6 and Adrr MCF-7 cells grown in Se-deficient media had 2.6 +/- 2.4 (mean +/- S.D.) and 4.2 +/- 3.6 units/mg protein of GSH peroxidase specific activity, respectively. Se supplementation increased GSH peroxidase activity in a concentration- and time-dependent fashion. Enzymatic activity reached a level of 164 +/- 62 in MCF-7H6 cells and 114 +/- 27 in Adrr MCF-7 cells within 5 days of growth in media supplemented with 30 nM Se. Northern analysis revealed that Se-deficient MCF-7H6 cells expressed 2.1 +/- 0.4-fold less GSHPx-1 mRNA than their Se-sufficient counterparts. Similarly, Se-deficient Adrr MCF-7 cells expressed 3.3 +/- 1.8-fold less GSHPx-1 mRNA than their Se-supplemented counterparts after the quantity of mRNA was normalized with beta-actin. These studies suggest that modulation of GSH peroxidase activity by Se in both MCF-7H6 transfectants expressing pRSV-GSHPx-1 and Adrr MCF-7 cells expressing endogenous GSHPx-1 occurs largely at the translational level, and to a lesser degree at the level of mRNA, possibly by stabilizing GSHPx-1 mRNA since the transfected cDNA in MCF-7H6 cells has only 5 nucleotides 5' to the AUG initiation codon.

The regulation of glutathione peroxidase gene expression relevant to species difference and the effects of dietary selenium manipulation

Glutathione peroxidase (GSH-Px) contains selenium (Se) as selenocysteine in the active site of the enzyme. GSH-Px activities in the cytosol of all guinea-pig tissues examined were extremely low compared with those in mice and rats, while Se concentrations in tissues were almost the same among three animal species. In addition, no GSH-Px mRNA was detectable in any tissues of guinea-pigs, although the guinea-pig had the same copy number (probably a single copy) of the GSH-Px gene in its genomic DNA as that of the mouse and rat, suggesting that the species difference of GSH-Px activity observed in rodents might be due to incapability of gene transcription. On the other hand, feeding of mice with Se-deficient diet for 6 weeks resulted in a remarkable decrease in GSH-Px mRNA as well as GSH-Px activity both in the liver and kidneys. The detailed time-course experiment revealed that the drop in GSH-Px activity preceded the decrease in the mRNA level in Se-depleted mice and the mRNA level recovered rapidly in contrast to the slow rate of increase in the enzyme activity in Se-repleted mice. These results suggested that the alteration in GSH-Px activity in mice subjected to dietary Se manipulation is attributable not only to transcriptional but also to post-transcriptional regulation.

The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the 'termination' codon, TGA.

Glutathione peroxidase (GSHPx) is an important selenium-containing enzyme which protects cells from peroxide damage and also has a role in leukotriene formation. We report the identification of a genomic recombinant as encoding the entire mouse GSHPx gene. Surprisingly, the selenocysteine in the active site of the enzyme is encoded by TGA: this has been confirmed by primer extension/dideoxy sequencing experiments using reticulocyte mRNA. The same site of transcription initiation is used in three tissues in which the GSHPx mRNA is expressed at high levels (erythroblast, liver and kidney). Like some other regulated 'house-keeping' genes, the GSHPx gene has Sp1 binding site consensus sequences but no 'ATA' and 'CAAT' consensus sequences upstream of the transcription initiation site. Moreover, there is a cluster of two Sp1 binding site consensus sequences and two SV40 core enhancer sequences in the 3' region of the gene, close to the previously mapped position of a DNase I-hypersensitive site found only in tissues expressing the GSHPx mRNA at high levels.