title: Inhibition of wild-type p66ShcA in mesangial cells prevents glycooxidant-dependent FOXO3a regulation and promotes the survival phenotype.
abstract: Hyperglycemia triggers an exponential increase in reactive oxygen species (ROS) at the cellular level. Here, we demonstrate induction of the oxidant-resistant phenotype in mesangial cells by silencing the wild-type (WT) p66ShcA gene. Two approaches were employed to inhibit WTp66ShcA in SV40 murine mesangial cells and normal human mesangial cells: transient transfection with isoform-specific p66ShcA short-intervening RNA and stable transfection with mutant 36 p66ShcA expression vector. At high ambient glucose (HG), p66ShcA-deficient cells exhibit resistance to HG-induced ROS generation and attenuation in the amplitude of the kinetic curves for intracellular ROS metabolism, indicative of the pivotal role of WTp66ShcA in the generation of HG oxidant stress. We next examined phosphorylation and subcellular distribution of FKHRL1 (FOXO3a), a potent stress response regulator and downstream target of WTp66ShcA redox function. At HG, cell extracts of p66ShcA-deficient cells analyzed by immunoblotting show attenuation of FOXO3a phosphorylation at Thr-32, and indirect immunofluorescence of p66ShcA-deficient cells, cotransfected with HA-FOXO3a, show predominant HA-FOXO3a nuclear localization. Conversely, parental cells at HG show upregulation of phos-Thr-32 and nuclear export of HA-FOXO3a. To determine whether inhibition of cross talk between WTp66ShcA and FOXO3a confers protection against oxidant-induced DNA damage, DNA strand breaks (DSB) and apoptosis were examined. At HG, p66ShcA-deficient cells exhibit increased resistance to DSB and apoptosis, while parental cells show a striking increase in both parameters. We conclude that knockdown of WTp66ShcA redox function prevents HG-dependent FOXO3a regulation and promotes the survival phenotype.
authors.affiliation: Center for Neurovirology and Cancer Biology, Temple University, Philadelphia, Pennsylvania, USA.
authors.forename: Janaki
authors.initials: J
authors.lastname: Chintapalli
authors.forename: Shuo
authors.initials: S
authors.lastname: Yang
authors.forename: David
authors.initials: D
authors.lastname: Opawumi
authors.forename: Sunita Ray
authors.initials: SR
authors.lastname: Goyal
authors.forename: Nazia
authors.initials: N
authors.lastname: Shamsuddin
authors.forename: Ashwani
authors.initials: A
authors.lastname: Malhotra
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Reiss
authors.forename: Leonard G
authors.initials: LG
authors.lastname: Meggs
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors* / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000037:antagonists & inhibitors / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D050527:Mesangial Cells / Q000187:drug effects* / Q000502:physiology*; D051379:Mice; D010641:Phenotype; D034741:RNA, Small Interfering / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D005947:Glucose