Epididymis response partly compensates for spermatozoa oxidative defects in snGPx4 and GPx5 double mutant mice

We report here that spermatozoa of mice lacking both the sperm nucleus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H(2)O(2)-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice.     

Molecular characterization of glutathione peroxidase gene from the liver of silver carp, bighead carp and grass carp

The cDNAs encoding glutathione peroxidase (GPx) were cloned and sequenced from the liver of three Chinese carps with different tolerance to hepatotoxic microcystins, phytoplanktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis), and herbivorous grass carp (Ctenopharyngodon idellus). Using genome walker method, a 750 bp 5'-flanking region of the silver carp GPx gene was obtained, and several potential regulatory elements were identified in the promoter region of the GPx gene. The silver carp GPx gene was widely expressed in all tissues examined. Despite phylogenetic analysis, assigning this newly described carp GPx to the group of mammalian GPx2, the carp GPx seems more similar to GPx1 from a physiological point of view. The constitutive expression pattern of the three carp liver GPx gene, shows a positive relationship with their tolerance to microcystins.     

Activation of the glutathione peroxidase 2 (GPx2) promoter by beta-catenin

GPx2, formerly named gastrointestinal glutathione peroxidase, is highly expressed in the proliferative area of the intestinal crypt-to-villus axis and in Paneth cells. Additionally, GPx2 is transiently up-regulated during development of gastrointestinal adenocarcinomas. Because both normal proliferation and differentiation of intestinal epithelial cells as well as carcinogenesis are regulated by the Wnt pathway, it was tested whether GPx2 may be a target of the beta-catenin/TCF complex which transfers Wnt signals. The GPx2 promoter contains five putative beta-catenin/TCF binding sites. Accordingly, the promoter was active in two cell lines with a constitutively active Wnt pathway, HepG2 and SW480, but not in BHK-21 cells in which the pathway is silent. Overexpression of beta-catenin/TCF activated the GPx2 promoter in all three cell lines. Overexpression of wild-type adenomatous polyposis coli (APC) in SW480 cells which harbor a mutated APC gene decreased basal GPx2 promoter activity. Truncation of the promoter identified one beta-catenin/TCF binding site that was sufficient for activation. Mutation of this site reduced the response to beta-catenin/TCF by more than 50%. These findings suggest a function of GPx2 in the maintenance of normal renewal of the intestinal epithelium. Whether up-regulation of GPx2 during carcinogenesis supports tumor growth or can rather be considered as a counteracting effect remains to be investigated.     

NF-kappaB signaling regulates functional expression of the MHC class I-related neonatal Fc receptor for IgG via intronic binding sequences

The neonatal Fc receptor for IgG (FcRn) functions to transport maternal IgG to a fetus or newborn and to protect IgG from degradation. Although FcRn is expressed in a variety of tissues and cell types, the extent to which FcRn expression is regulated by immunological and inflammatory events remains unknown. Stimulation of intestinal epithelial cell lines, macrophage-like THP-1, and freshly isolated human monocytes with the cytokine TNF-alpha rapidly up-regulated FcRn gene expression. In addition, the TLR ligands LPS and CpG oligodeoxynucleotide enhanced the level of FcRn expression in THP-1 and monocytes. Treatment of TNF-stimulated THP-1 cells with the NF-kappaB-specific inhibitor or overexpression of a dominant negative mutant inhibitory NF-kappaB (IkappaBalpha; S32A/S36A) resulted in down-regulation of FcRn expression. By using chromatin immunoprecipitation we identified three NF-kappaB binding sequences within introns 2 and 4 of the human FcRn gene. An EMSA confirmed the p50/p50 and/or p65/p50 complex (s) bound to intron 2- or 4-derived oligonucleotides containing putative NF-kappaB binding sequences, respectively. The intronic NF-kappaB sequences in combination with the promoter or alone regulated the expression of a luciferase reporter gene in response to TNF-alpha stimulation or overexpression of NF-kappaB p65 and p50. DNA looping interactions potentially occurred after the stimulation between intronic NF-kappaB sequences and the FcRn promoter as shown by a chromosome conformation capture assay. Finally, TNF-alpha stimulations enhanced IgG transport across an intestinal Caco-2 epithelial monolayer. Together, these data provide the first evidence that NF-kappaB signaling via intronic sequences regulates FcRn expression and function.     

Tumour suppressor PTEN enhanced enzyme activity of GPx,SOD and catalase by suppression of PI3K/AKT pathway in non-small cell lung cancer cell lines

Phosphates and tensin homologue deleted on chromosome 10 (PTEN) is a tumour suppressor gene which dephosphorilates phosphoinositol 3,4,5 triphosphates. Therefore PTEN can regulate PI3K/AKT pathway in cells. Because of promoter methylation or gene deletion, PTEN expression is commonly decreased or lost in non-small cell lung cancer (NSCLC) cell lines. Therefore, we hypothesized that PTEN could regulate the activity of superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx) and catalase. We first recreated PTENwt, G129R and G129E expressions in lung cell lines, in which endogenous PTEN expression was not detected. Then, we showed that PTEN could suppress AKT activity by its lipid phosphatase domain. We then examined the effect of recreated PTEN expressions in NSCLC cells. While PTENwt expression caused enhanced activity of SOD, GPx and catalase in transfected cells lines, neither G129R nor G129E expression effected enzyme activities. These results suggest that PTEN can up-regulate SOD, GPx and catalase activity by inhibition of PI3K/AKT pathway in NSCLC cell lines.     

A putative glutathione peroxidase of Drosophila encodes a thioredoxin peroxidase that provides resistance against oxidative stress but fails to complement a lack of catalase activity

Cellular defense systems against reactive oxygen species (ROS) include thioredoxin reductase (TrxR) and glutathione reductase (GR). They generate sulfhydryl-reducing systems which are coupled to antioxidant enzymes, the thioredoxin and glutathione peroxidases (TPx and GPx). The fruit fly Drosophila lacks a functional GR, suggesting that the thioredoxin system is the major source for recycling glutathione. Whole genome in silico analysis identified two non-selenium containing putative GPx genes. We examined the biochemical characteristics of one of these gene products and found that it lacks GPx activity and functions as a TPx. Transgene-dependent overexpression of the newly identified Glutathione peroxidase homolog with thioredoxin peroxidase activity (Gtpx-1) gene increases resistance to experimentally induced oxidative stress, but does not compensate for the loss of catalase, an enzyme which, like GTPx-1, functions to eliminate hydrogen peroxide. The results suggest that GTPx-1 is part of the Drosophila Trx antioxidant defense system but acts in a genetically distinct pathway or in a different cellular compartment than catalase.