title: Essential role of PH domain and leucine-rich repeat protein phosphatase 2 in Nrf2 suppression via modulation of Akt/GSK3β/Fyn kinase axis during oxidative hepatocellular toxicity.
abstract: Instances of sustained oxidative activity have been shown to involve dysregulation of Nrf2-mediated transcriptional induction; however, mechanisms warranting Nrf2-repression remain unclear. In this study, using primary rat hepatocytes, we have attempted to identify factors that may negatively influence Nrf2 survival pathway. Though studies indicate a conspicuous association between Akt and Nrf2, a confirmatory link between the two is unaddressed. On inhibiting PI3K/Akt pathway, we observed compromised activities of antioxidant and detoxification enzymes culminating in oxidative cytotoxicity. This was accompanied by reduced nuclear retention of Nrf2 and its ARE binding affinity, increased Nrf2 ubiquitination and concurrent decline in its downstream targets. Moreover, Akt inhibition enhanced nuclear translocation as well as phosphorylation of Fyn kinase, an enzyme linked to Nrf2 degradation, by relieving GSK3β from phosphorylation-mediated repression. The involvement of Akt and Fyn kinase in influencing Nrf2 signaling was further confirmed in oxidatively stressed hepatocytes by using tert-butyl hydroperoxide (tBHP). tBHP-induced decrease in Nrf2 levels was associated with enhanced Fyn kinase phosphorylation, Fyn kinase nuclear translocation and decreased levels of phosphorylated GSK3β(Ser9) in a time-dependent manner. Interestingly, tBHP induced site-specific deactivation of Akt as only Akt(Ser473) phosphorylation was observed to be affected. Further, protein expression as well as nuclear localization of PHLPP2, a phosphatase specific for Akt(Ser473), was found to be significantly enhanced in tBHP-stressed hepatocytes. Silencing of PHLPP2 not only resulted in considerable restoration of Nrf2 signaling, enhanced Nrf2-ARE binding and reduced Nrf2 ubiquitination but also significantly suppressed tBHP-induced ROS generation and alterations in mitochondrial permeability. We infer that cellular PHLPP2 levels may aggravate oxidative toxicity by suppressing Nrf2/ARE transcriptional regulation via Akt(Se473)/GSK3β/Fyn kinase axis. The study indicates that PHLPP2 could serve as a new target for developing strategies to manage pathological conditions exacerbated due to oxidative stress.
authors.affiliation: 1] Academy of Scientific and Innovative Research, New Delhi, India [2] Herbal Research Section, Food Drug and Chemical Toxicology Division, Council of Scientific and Industrial Research-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, India.
authors.forename: F
authors.initials: F
authors.lastname: Rizvi
authors.affiliation: Herbal Research Section, Food Drug and Chemical Toxicology Division, Council of Scientific and Industrial Research-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, India.
authors.forename: S
authors.initials: S
authors.lastname: Shukla
authors.affiliation: 1] Academy of Scientific and Innovative Research, New Delhi, India [2] Herbal Research Section, Food Drug and Chemical Toxicology Division, Council of Scientific and Industrial Research-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, India.
authors.forename: P
authors.initials: P
authors.lastname: Kakkar
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D005609:Free Radicals / Q000378:metabolism; D055785:Gene Knockdown Techniques; D020868:Gene Silencing / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D022781:Hepatocytes / Q000378:metabolism* / Q000473:pathology; D042541:Intracellular Space / Q000378:metabolism; D008954:Models, Biological; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects; D020122:tert-Butylhydroperoxide / Q000633:toxicity
chemical_list: D000975:Antioxidants; D005609:Free Radicals; D051267:NF-E2-Related Factor 2; D020122:tert-Butylhydroperoxide; D051076:Proto-Oncogene Proteins c-fyn; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; C541124:PHLPP2 protein, rat; D010749:Phosphoprotein Phosphatases