Activation of Nrf2 Translation by a Keap1 Independent Mechanism

235 Cigarette Smoke Induced Emphysema and Pulmonary Hypertension Are Triggered by Tobacco Smoke Oxidants: Attenuation by Vitamin C Indranil Gupta1, Souradipta Ganguly1, Christine R. Rozanas2, Dennis J. Stuehr3, and Koustubh Panda1 1 University of Calcutta, India, 2GE Heathcare Life Sciences, USA, 3 Cleveland Clinic, USA Cigarette smoking causes emphysema, a fatal disease involving extensive structural and functional damage of the lung followed by pulmonary hypertension that restricts pulmonary circulation and causes right ventricular dysfunction of the heart. Using a guineapig model and human lung cells we show that oxidant(s) present in tobacco smoke not only cause direct oxidative damage of lung proteins, contributing to the major share of emphysematous lung injury, but are also responsible for the extensive pulmonary vascular remodelling that characterizes pulmonary hypertension. Tobacco-smoke oxidants activate the lung pro-inflammatory factor, Rtp801 which in turn stimulates nuclear-factor κB and consequent inducible nitric-oxide synthase (iNOS) mediated overproduction of nitric-oxide (NO) that contributes to lung protein nitration. However, lung-specific inhibition of iNOS with a iNOS-specific inhibitor, N6(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) solely restricts the observed lung protein nitration but fails to prevent or reverse the major tobacco-smoke induced oxidative lung injury, contrary to recent reports (Cell;147(2):293-305, 2011). In fact, inhibition of pulmonary iNOS also significantly accelerates lung peri-vascular collagen deposition by up-regulating the expression of arginase I, a key enzyme associated with vascular remodelling and pulmonary hypertension. In comparison, the dietary antioxidant, ascorbate or vitamin C, can substantially prevent emphysematous lung damage by inhibiting both tobacco-smoke induced lung protein oxidation as well as activation of pulmonary Rtp801 and nitration of lung proteins, that otherwise lead to increased proteolysis of such oxidized or nitrated proteins by endogenous lung proteases, causing emphysema. Vitamin C also restricts the up-regulation of matrix-metalloproteinase-9, the major lung protease involved in the proteolysis of such modified lung proteins during tobacco-smoke induced emphysema as well as arginase I, to prevent the subsequent peri-vascular collagen deposition and medial thickening of the pulmonary vessels causing pulmonary hypertension. Thus, our findings implicate tobacco-smoke oxidant(s) as the primary etiopathogenic factor behind both the emphysematous and vascular pathogenesis of the lung associated with smoking. More importantly, it demonstrates the potential of vitamin C to accomplish holistic prevention of both these major forms of tobacco-smoke induced lung damage Reference: Proc. Natl. Acad. Sci, USA,113(29):E4208-17, 2016

doi: 10.1016/j.freeradbiomed.2016.10.276 236 Hydrogen Sulfide Increases GSH Biosynthesis, Glucose Uptake and Utilization in Mouse Myotubes Rajesh Parsanathan1 and Sushil K Jain1 1 Louisiana State University Health Sciences Center, Shreveport, USA

and L-Cysteine (LC). LC undergoes enzymatic breakdown to produce hydrogen sulphide (H2S), a gasotransmitter that regulates glucose and lipid homeostasis. Using C2C12 mouse myoblast (differentiated into myotubes) as a model, this study investigates the hypothesis that beneficial effects of LC supplementation are mediated by the upregulation of H2S status in diabetic patients. Results show that exogenous administration of Sodium hydrosulfide (NaHS 10µM; 6 hours), a H2S donor, significantly (p<0.05) upregulate the mRNA levels of Cystathionine-γ-lyase (CSE), Solute Carrier Family 7 Member 11 (SLC7A11), GlutamateCysteine Ligase Catalytic Subunit (GCLC) and GlutamateCysteine Ligase Modifier Subunit (GCLM) mRNA levels as well as reduces ROS and increases glucose uptake and utilization in myoblast. Deficiency of endogenous H2S production induced by using CSE siRNA caused an increase in ROS, and down regulation of potent antioxidant GSH biosynthesis enzymes (GCLC, Glutathione Synthetase) but not GGLM. Further, CSE knockdown downregulate Glucose Transporter Type 4 (GLUT4) mRNA level and reduces glucose uptake in C2C12 myotubes. Taken together, these data suggest that the upregulation of physiological levels of H2S can have beneficial effects on glucose homeostasis and redox balance via GSH pathway. Further mechanistic studies are under investigation on how H2S regulate GSH biosynthesis and LCysteine transporter (SLC7A11) in the maintenance of glucose homeostasis. Supported by NIH/NCCIH RO1 AT007442

doi: 10.1016/j.freeradbiomed.2016.10.277 237 Activation of Nrf2 Translation by a Keap1 Independent Mechanism Oscar Perez-Leal1, Carlos A Barrero1, and Salim Merali1 Temple University, Philadelphia, USA

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Nuclear factor (erythroid-derived 2)-like 2 (nrf2) is the master regulator of the antioxidant response, and its function is tightly regulated at the transcription, translation, and post-translational level. It is well known that Keap1 regulates Nrf2 signaling but the Nrf2 regulation at the translation level and its chemical modulation is less documented. Here, we show a complementary way to promote an increase in the Nrf2 levels by overcoming its basal translational repression thus opening the possibility for the discovery of novel alternatives for promoting cellular protection against oxidative stress. We developed a novel reporter system that allowed for the identification of natural compounds present in the human diet as inducers of Nrf2 translation by a mechanism independent of Keap1-mediated degradation. Apigenin, resveratrol and piceatannol were identified as inducers of Nrf2 translation. More importantly, the newly synthesized Nrf2 overcomes keap1regulation, translocates to the nuclei and activates the antioxidant response. We conclude that Nrf2 translational control is physiologically relevant and can be regulated by apigenin. Also, targeting this mechanism could provide new insights into prevention and treatment of multiple diseases where oxidative stress plays a major role.

doi: 10.1016/j.freeradbiomed.2016.10.278

Diabetes is a leading cause of chronic diseases, such as heart disease. Diabetic patients have lower blood concentrations of H2S

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SfRBM / SFRRI 2016