OBJECTIVETo investigate whether the deletion of PTEN affects the expression of Cu/Zn SOD and the related biology.

METHODSProtein and mRNA expression levels of PTEN, P-Akt, Cu/Zn SOD in the control immortalized wild type mouse embryonic fibroblast cells (PTEN+/+) and PTEN-null cells (PTEN-/-) were evaluated by Western blot and Northern blot respectively. The level of superoxide anions were detected using fluorescent probes. The DNA damage was documented by single cell alkalescence gel assay. MTT was used to study the effect of H2O2 on the proliferation of cells.

RESULTSThe expression of Cu/Zn SOD was down regulated at both protein and mRNA levels, and the level of superoxide anions increased in the PTEN-null cells (PTEN-/-). The phosphorylation level of Akt kinase was up-regulated and the antiproliferative effect of H2O2 decreased in PTEN-/- cells. Furthermore, DNA damage was observed significantly severer in both the blank control and H2O2 treated groups than that in the PTEN+/+ cells.

CONCLUSIONSDeletion of PTEN affects the expression of Cu/Zn SOD. As a result, reactive oxygen species (ROS) keep at a high level, along with decrease of accumulated oxidative damage and the antiproliferative effect of ROS.

Animals ; Copper ; chemistry ; Down-Regulation ; Fibroblasts ; pathology ; Gene Expression Regulation ; genetics ; Hydrogen Peroxide ; metabolism ; Mice ; Mice, Knockout ; PTEN Phosphohydrolase ; deficiency ; genetics ; Phosphorylation ; Reactive Oxygen Species ; Sequence Deletion ; Superoxide Dismutase ; genetics ; metabolism ; Zinc ; chemistry

Using forward and reverse genetics and global gene expression analyses, we explored the crosstalk between the IκB kinase β (IKKβ) and the transforming growth factor β (TGFβ) signaling pathways. We show that in vitro ablation of Ikkβ in fibroblasts led to progressive ROS accumulation and TGFβ activation, and ultimately accelerated cell migration, fibroblast-myofibroblast transformation and senescence. Mechanistically, the basal IKKβ activity was required for anti-oxidant gene expression and redox homeostasis. Lacking this activity, IKKβ-null cells showed ROS accumulation and activation of stress-sensitive transcription factor AP-1/c-Jun. AP-1/c-Jun activation led to up-regulation of the Tgfβ2 promoter, which in turn further potentiated intracellular ROS through the induction of NADPH oxidase (NOX). These data suggest that by blocking the autocrine amplification of a ROS-TGFβ loop IKKβ plays a crucial role in the prevention of fibroblast-myofibroblast transformation and senescence.
Adenoviridae ; genetics ; Animals ; Autocrine Communication ; physiology ; Cell Line ; Cell Movement ; Cellular Senescence ; Genetic Vectors ; genetics ; metabolism ; I-kappa B Kinase ; deficiency ; genetics ; metabolism ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Mice ; Myofibroblasts ; cytology ; metabolism ; NADPH Oxidases ; metabolism ; Oxidative Stress ; Promoter Regions, Genetic ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Superoxide Dismutase ; genetics ; metabolism ; Transcription Factor AP-1 ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Up-Regulation

OBJECTIVEThis report aims to describe the oxidative damage profile in brain of presenilin1 and presenilin2 conditional double knockout mice (dKO) at both early and late age stages, and to discuss the correlation between oxidative stress and the Alzheimer-like phenotypes of dKO mice.

METHODSThe protein level of Abeta(42) in dKO cortex and free 8-OHdG level in urine were measured by ELISA. Thiobarbituric acid method and spectrophotometric DNPH assay were used to determine the lipid peroxidation and protein oxidation in cortex, respectively. SOD and GSH-PX activities were assessed by SOD Assay Kit-WST and GSH-PX assay kit, separately.

RESULTSSignificant decrease of Abeta(42) was verified in dKO cortex at 6 months as compared to control mice. Although lipid peroxidation (assessed by MDA) was increased only in dKO cortex at 3 months and protein oxidation (assessed by carbonyl groups) was basically unchanged in dKO cortex, ELISA analysis revealed that free 8-OHdG, which was an indicator of DNA lesion, was significantly decreased in urine of dKO mice from 3 months to 12 months. Activities of SOD and GSH-PX in dKO and control cortices showed no statistical difference except a significant increase of GSH-PX activity in dKO mice at 9 months.

CONCLUSIONOxidative damage, especially DNA lesion, was correlated with the neurodegenerative symptoms that appeared in dKO mice without the deposition of Abeta(42). Triggers of oxidative damage could be the inflammatory mediators released by activated microglia and astrocytes.

Age Factors ; Alzheimer Disease ; genetics ; metabolism ; physiopathology ; Amyloid beta-Peptides ; urine ; Animals ; Deoxyguanosine ; analogs & derivatives ; urine ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; methods ; Glutathione ; metabolism ; Hydrazines ; metabolism ; Lipid Peroxidation ; genetics ; Malondialdehyde ; metabolism ; Mice ; Mice, Inbred CBA ; Mice, Knockout ; physiology ; Oxidation-Reduction ; Oxidative Stress ; physiology ; Peptide Fragments ; urine ; Presenilin-1 ; deficiency ; Presenilin-2 ; deficiency ; Spectrophotometry, Atomic ; methods ; Superoxide Dismutase ; metabolism

Cytoplasmic Cu/Zn superoxide dismutase (SOD1) is an antioxidant enzyme that converts superoxide to hydrogen peroxide in cells. Its spatial distribution matches that of superoxide production, allowing it to protect cells from oxidative stress. SOD1 deficiencies result in embryonic lethality and a wide range of pathologies in mice, but little is known about normal SOD1 protein expression in developing embryos. In this study, the expression pattern of SOD1 was investigated in post-implantation mouse embryos and extraembryonic tissues, including placenta, using Western blotting and immunohistochemical analyses. SOD1 was detected in embryos and extraembryonic tissues from embryonic day (ED) 8.5 to 18.5. The signal in embryos was observed at the lowest level on ED 9.5-11.5, and the highest level on ED 17.5-18.5, while levels remained constant in the surrounding extraembryonic tissues during all developmental stages examined. Immunohistochemical analysis of SOD1 expression on ED 13.5-18.5 revealed its ubiquitous distribution throughout developing organs. In particular, high levels of SOD1 expression were observed in the ependymal epithelium of the choroid plexus, ganglia, sensory cells of the olfactory and vestibulocochlear epithelia, blood cells and vessels, hepatocytes and hematopoietic cells of the liver, lymph nodes, osteogenic tissues, and skin. Thus, SOD1 is highly expressed at late stages of embryonic development in a cell- and tissue-specific manner, and can function as an important antioxidant enzyme during organogenesis in mouse embryos.
Animals ; Cerebral Cortex/embryology/enzymology ; Copulation ; Cytoplasm/*enzymology ; Embryonic Development/*physiology ; Female ; Immunohistochemistry ; Lung/embryology/enzymology ; Male ; Mice ; Mice, Inbred ICR ; Organogenesis/*physiology ; Pregnancy ; Stomach/embryology/enzymology ; Superoxide Dismutase/deficiency/genetics/*metabolism