We reveal a novel pro-survival role for mammalian p38α in response

We reveal a novel pro-survival role for mammalian p38α in response to H2O2 which involves an up-regulation of antioxidant defenses. response allows WT cells to maintain an efficient activation of the mTOR/p70S6K pathway. Accordingly the loss of p38α leads to ROS accumulation in response to H2O2 which causes cell death and inactivation of mTOR/p70S6K signaling. This can be rescued by either p38α re-expression or treatment with the antioxidants (16) showed that Akt activation sensitized cells to oxidative stress through down-regulation of ROS scavengers leading to the accumulation of intracellular ROS and cell death. Therefore we hypothesized that p38α through inhibition of Akt might allow a proper expression CYT997 (Lexibulin) of antioxidant genes and cell survival. Thus we analyzed the precise function of p38α in the regulation of the cell fate using nontransformed WT and p38α?/? MEFs exposed to oxidative stress. Furthermore we explored the mechanisms involved in the regulation of antioxidant responses in the context of Akt/mTOR signaling as well as other pathways linked to ROS level regulation. EXPERIMENTAL PROCEDURES Cell Lines Culture Conditions and Inhibitors WT and p38α-deficient MEFs immortalized either by passages or by LTAg (Large T Antigen) expression were grown in DMEM supplemented with 10% FBS (Invitrogen) at 37 °C CYT997 (Lexibulin) in a humidified atmosphere with 5% CO2. For signaling experiments confluent cells were stimulated with 0.1-1 mm H2O2 for 20 min. For cell death analysis growing cells were treated with 0.1-1 mm H2O2 for 6-24 h. The mTORC1 inhibitor rapamycin was used at a concentration of 1-10 μm. Treatment with Antioxidants Actinomycin D and MG-132 The cells were treated with the following antioxidants to decrease intracellular levels of ROS and/or to metabolize H2O2: 50 units/ml of catalase (Sigma; C-1345) and 2.5 mm luciferase (Clontech) (100-500 ng). Then cells were treated with H2O2 0.5 mm for 4 and 8 h and lysed using the passive lysis buffer from Promega. Luciferase activity was detected with a luminometer (Molecular Devices Spectramax M5E) using a dual luciferase reporter kit from Promega following the manufacturer’s instructions. Luciferase activity was normalized to luciferase activity. Western Blot Analysis Western blot analysis was carried out as previously described using total cell extracts (15). Proteins were separated by electrophoresis using Anderson gels (or SDS-PAGE gels) and CYT997 (Lexibulin) transferred to nitrocellulose membranes that were probed with the following antibodies: Akt (Cell Signaling; 9272) catalase (Sigma; C-0979) p70S6K (Cell Signaling; 9202) p38α (Santa Cruz; sc-535) phospho-acetyl-CoA-carboxylase (Cell Signaling; 3661) phospho-Akt (Cell Signaling; 9271) phospho-AMPK (Cell Signaling; 2531) phospho-MKK3/6 (Cell Signaling; 9231) SOD-2 (Upstate Biotech; 06-984) TSC-1 (Cell Signaling; 4906) phospho-TSC-2 (Cell Signaling; 3615) phospho-p38 (Cell Signaling; 9211) phospho-p70S6K (Cell Signaling; 4376) anti-HA Clone 16 B12 (Covance; MMS-101P) phospho-ATF-2 (Cell Signaling; 9221) ATF-2 (Cell Signaling; 9226) and α-tubulin (Sigma; T-5168). Catalase and SOD Activity Assays Catalase activity was measured by quantification of peroxide decomposition in a 50 mm phosphate buffer at pH 7 containing 3 mm H2O2. This was monitored spectrophotometrically at 240 nm. SOD activity from cell extracts was quantified using a kit (BioVision reference number K335-100) where WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2 4 tetrazolium monosodium salt) is the CR6 substrate. WST-1 produces a water-soluble formazan dye upon its reduction with superoxide anion which can be monitored spectrophotometrically at 450 nm. The rate of the reduction is linearly related to the xanthine oxidase activity and inhibited by SOD so the IC50 of SOD is determined as a measure of SOD activity. CYT997 (Lexibulin) RT-PCR and RT-Quantitative PCR Analysis After the isolation of total RNA with RNeasy Mini kit (Qiagen; 74104) 1 μg of RNA was reverse transcribed with SuperScrip III RT kit (Qiagen; 18080) to generate cDNA. Then PCR analysis was performed using specific primers: for SOD-1: forward 5 and reverse 5 (n141-n160 and n554-n573 respectively; accession number 000082.5); and for SOD-2: forward 5 and reverse 5 (n646-n665 and n751-n770 respectively; accession number 000083.5). The amplified bands were normalized using internal control: GAPDH forward 5 and reverse 5 TGG-3′ in the same PCR. The conditions for the PCR were: 94 °C for 45 s 55 °C for 45 s and 72 °C for 1 min for.