Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing protein that catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides using glutathione as a reducing agent (GSH). Our goals are to overexpress GPx-3, determine its activity, and study its regulation. The complex expression of selenoproteins requires specific translational cofactors (SBP2, SelD, and tRNASec), adequate organified selenium, and the selenocysteine insertion sequence (SECIS), which facilitate read-through of the UGA codon for selenocysteine (Sec) and incorporation in the growing polypeptide, rather than premature termination of translation. GPx-3 cDNA and the SECIS were cloned into a pcDNA 4/V5 His C vector. This vector contains a unique C terminal His tag important for purification of GPx-3. Stable Cos 7 cell lines of the wild type, SECIS containing mutant 3′UTR fragments, and a Sec → Cys mutant were also generated and grown in adequate sodium selenite to ensure optimal organification and selenocysteine synthesis. mRNA expression of endogenous and recombinant forms of GPx-3 were detected by RT-PCR in Cos 7 cells. Immunoprecipitation with anti His C term antibody followed by Western blot with anti V5 antibody showed detection of both the wild type and the selenocysteine UGA mutant protein. Mutating the UGA codon of GPx-3 allows for translational read-through without the requirement for translational cofactors. Support: NIH HL61795, HL58976, PO1 HL81587
Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing protein with antioxidant properties. GPx-3 plays an important role in plasma against oxidant stress by scavenging reactive oxygen species. A deficiency of this enzyme has been associated with platelet dependent thrombosis. We recently developed an animal model to assess platelet function in GPx-3 deficient mice. We hypothesized that GPx-3 deficiency induces platelet activation in vivo . GPx-3 (−/−) mice showed an attenuated bleeding time compared with wild-type mice (94.5 ± 28.8 s versus 153.4 ± 32.3, P<0.05). We also noted an increase in the plasma levels of soluble P-selectin, a marker of platelet activation and prothrombotic activity, in GPx-3 (−/−) mice compared with wild-type mice (137.8 ± 12.3 ng/ml plasma versus 101.5 ± 8.8, P<0.05). Cyclic GMP, a key intracellular second messenger molecule and marker for activation of soluble guanylyl cyclase by nitric oxide, was decreased in the plasma of GPx-3 (−/−) mice compared with wild-type mice (5.38 ± 1.75 pmol/ml plasma versus 23.67 ± 3.59, P<0.001), consistent with less bioactive NO in GPx-3 (−/−) mice. ADP was infused into the right ventricle of mice to induce platelet aggregation in the pulmonary vasculature; this assay resulted in higher pulmonary artery pressure in GPx-3 (−/−) compared with wild-type mice suggesting a more robust platelet activation response in the GPx-3 (−/−) mice. To confirm this interpretation, histological sections from the pulmonary vasculature of GPx-3 (−/−) compared with wild-type mice showed increased thrombi per 7.5 mm 2 section normalized to wild-type mice based on staining intensity for P-selectin (1.7 ± 0.4 versus 1.0 ± 0.1, P<0.001), as well as a higher percentage of occluded vessels (0.82 ± 0.16 % versus 0.54 ± 0.21, P<0.05). These findings demonstrate that GPx-3 deficiency causes platelet activation resulting in a prothrombotic state. These data illustrate the importance of this plasma antioxidant enzyme in regulating platelet activity and platelet-dependent thrombosis.
Interspersed between cardiac myocytes, cardiac fibroblasts serve mainly as a structural support during ventricular wall thickening from embryogenesis until adulthood. Cardiac fibroblasts, however, may also serve as a source of mitogens, extracellular matrix proteins, cytokines, and growth factors that could affect the phenotype of the cardiac myocyte. The crosstalk between cardiac fibroblasts and myocytes is important during cardiac development and remodeling in response to injury. The cell-to-cell communication involves paracrine signals (cytokines and growth factors), direct interactions (connexins and cadherins) as well as indirect interactions (integrin signaling through the extracellular matrix). In this review, known cardiac fibroblast-cardiac myocyte signaling pathways are briefly examined and their effect on the heart during disease progression is discussed. Furthermore, speculations are made regarding the possibility that vascular endothelial growth factor B can serve as an important signaling molecule between cardiac fibroblasts and cardiac myocytes and could promote cardiac function in compromised hearts.
Background— Heart failure is a growing cause of morbidity and mortality. Cardiac phosphatidylinositol 3-kinase signaling promotes cardiomyocyte survival and function, but it is paradoxically activated in heart failure, suggesting that chronic activation of this pathway may become maladaptive. Here, we investigated the downstream phosphatidylinositol 3-kinase effector, serum- and glucocorticoid-regulated kinase-1 (SGK1), in heart failure and its complications. Methods and Results— We found that cardiac SGK1 is activated in human and murine heart failure. We investigated the role of SGK1 in the heart by using cardiac-specific expression of constitutively active or dominant-negative SGK1. Cardiac-specific activation of SGK1 in mice increased mortality, cardiac dysfunction, and ventricular arrhythmias. The proarrhythmic effects of SGK1 were linked to biochemical and functional changes in the cardiac sodium channel and could be reversed by treatment with ranolazine, a blocker of the late sodium current. Conversely, cardiac-specific inhibition of SGK1 protected mice after hemodynamic stress from fibrosis, heart failure, and sodium channel alterations. Conclusions— SGK1 appears both necessary and sufficient for key features of adverse ventricular remodeling and may provide a novel therapeutic target in cardiac disease.
GPx‐3 is a selenocysteine‐containing protein that catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides using glutathione as a reducing agent (GSH). Our goals are to overexpress GPx‐3, determine its activity, and study its regulation. The complex expression of selenoproteins requires specific translational cofactors (SBP2, SelD, and tRNA Sec ), adequate selenium, and the selenocysteine insertion sequence (SECIS), which facilitate read‐through of the UGA codon for selenocysteine (Sec) and incorporation in the growing polypeptide. Stable Cos 7 cell lines of GPx‐3 cDNA/SECIS, His/V5 tagged wild type, SECIS containing mutated 3'UTR fragments, and a Sec → Cys mutant were generated and grown in medium with adequate sodium selenite. Purification was accomplished by immobilized metal ion affinity chromatography with nickel Sepharose that binds GPx‐3 His tagged protein, and followed by Western blot using an anti‐V5 antibody, and by His‐tag ELISA. The mRNA stability of both the wild type and Cys mutant were examined by Real‐Time PCR. Translation of the wild type GPx‐3 was much less efficient than that of the Cys mutant. Mass spectrometry confirmed GPx‐3 identity. The 3' UTR of GPx‐3 is important in regulating its mRNA stability and protein expression. Support: HL61795, HV28718, and HL81857
Plasma glutathione peroxidase (GPx-3)-deficiency increases extracellular oxidant stress, decreases bioavailable nitric oxide, and promotes platelet activation. The aim of this study is to identify polymorphisms in the GPx-3 gene, examine their relationship to arterial ischemic stroke (AIS) in a large series of children and young adults, and determine their functional molecular consequences.We studied the GPx-3 gene promoter from 123 young adults with idiopathic AIS and 123 age- and gender-matched controls by single-stranded conformational polymorphism and sequencing analysis. A second, independent population with childhood stroke was used for a replication study. We identified 8 novel, strongly linked polymorphisms in the GPx-3 gene promoter that formed 2 main haplotypes (H1 and H2). The transcriptional activity of the 2 most prevalent haplotypes was studied with luciferase reporter gene constructs.The H2 haplotype was over-represented in both patient populations and associated with an independent increase in the risk of AIS in young adults (odds ratio=2.07, 95% CI=1.03 to 4.47; P=0.034) and children (odds ratio=2.13, 95% CI=1.23 to 4.90; P=0.027). In adults simultaneously exposed to vascular risk factors, the risk of AIS approximately doubled (odds ratio=5.18, 95% CI=1.82 to 15.03; P<0.001). Transcriptional activity of the H2 haplotype was lower than that of the H1 haplotype, especially after upregulation by hypoxia (normalized relative luminescence: 3.54+/-0.32 versus 2.47+/-0.26; P=0.0083).These findings indicate that a novel GPx-3 promoter haplotype is an independent risk factor for AIS in children and young adults. This haplotype reduces the gene's transcriptional activity, thereby compromising gene expression and plasma antioxidant and antithrombotic activities.
GPx‐3 scavenges reactive oxygen species in plasma by catalyzing the reduction of hydrogen peroxide and lipid hydroperoxides using glutathione (GSH) as a reducing co‐substrate. We compared the specific activity of wild type (WT) and Sec73Cys mutant recombinant GPx‐3 (rGPx‐3). rGPx‐3 WT, Sec73Cys, and empty vector cDNAs were transfected in Cos7 cells in the presence of sodium selenite. rGPx‐3 protein was isolated by His‐Gravi Trap purification, determined by Western blot under reducing and non‐reducing conditions, and compared to native plasma GPx‐3. GPx‐3 activity was assessed by a chromogenic assay, a redox‐sensitive fluorescence based endpoint assay, and an in‐gel assay. Western blot demonstrated a 28 kDa band under reducing conditions for WT and Sec73Cys rGPx‐3 and an additional ∼112 kDa band under non‐reducing conditions. An ∼2.5‐fold increase in tetramer‐to‐monomer ratio for Sec73Cys rGPx‐3 was observed compared to WT rGPx‐3. Native plasma GPx‐3 resulted in a tetramer‐to‐monomer ratio of 2.5. The enzyme activity of WT rGPx‐3 was ~3‐fold higher than Sec73Cys with GSH (ANOVA, p<0.01) using the chromogenic assay, and ~2‐fold higher with the redox‐sensitive fluorescence based endpoint assay (ANOVA, p<0.005). The in‐gel assay also showed activity. WT rGPx‐3 is more catalytically active than Sec73Cys. Quaternary structure does not seem to influence intrinsic activity. Support: HL61795, HL28178, HL81587
