Downregulation of microsomal glutathione-S-transferase 1 modulates protective mechanisms in differentiated PC12 cells
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ORIGINAL PAPER
Downregulation of microsomal glutathione-S-transferase 1 modulates protective mechanisms in differentiated PC12 cells Monika Sobczak & Tomasz Boczek & Antoni Kowalski & Magdalena Wiktorska & Jolanta Niewiarowska & Ludmila Zylinska
Received: 5 August 2013 / Accepted: 1 January 2014 # University of Navarra 2014
Abstract Microsomal glutathione-S-transferase 1 (Mgst1) plays a specific role in protection of cells against oxidative stress. In this study, we assayed the effect of Mgst1 downregulation on cells behavior using differentiated PC12 line, a widely accepted neuronal model system. We have developed stable transfected cells with downregulated Mgst1 (PC12_M), which were differentiated with 1 mM dibutyryl-cAMP (db-cAMP). Mgst1 reduction induced necrosis, decreased ATP amount, and increased thiobarbituric acid reacting substances (TBARS) content. However, in PC12_M cell population, we detected more intensive neuritogenesis than that in mock-transfected cells. Interestingly, total glutathione as well as GSH level were significantly higher than those in control PC12 line. Real-time PCR and Western blot analyses showed elevated expression of enzymes involved in glutathione metabolism—a rate-limiting γ-glutamylcysteine ligase and glutathione reductase. The present study shows for the first time that under stress conditions induced by Mgst1 downregulation, a rescue pathway can be activated and thereby
M. Sobczak : T. Boczek : A. Kowalski : L. Zylinska (*) Department of Molecular Neurochemistry, Medical University, 6/8 Mazowiecka Str., 92-215 Lodz, Poland e-mail: [email protected] M. Wiktorska : J. Niewiarowska Department of Molecular and Medical Biophysics, Medical University, 6/8 Mazowiecka Str., 92-215 Lodz, Poland
enables differentiated PC12 cells to survive. Since Mgst1expression was reported to decline with age, our results could represent a putative adaptive process during aging. It could also be an early mechanism protecting neuronal cells against some neurodegenerative insults. Keywords Glutathione . Microsomal glutathione-Stransferase 1 . Nrf2 . PC12 cells
Introduction The glutathione-S-transferases (GSTs) form a group of multigene isoenzymes involved in detoxification of xenobiotic and endobiotic compounds [26]. They are known as phase II detoxification enzymes protecting cellular compartments from reactive electrophiles [29]. GSTs have also been shown to modulate signaling pathways controlling apoptosis, cell proliferation, and differentiation and are related to several neuropathologies [6, 13]. Mgst1 exhibits both glutathione transferase and peroxidase activities and is activated by reactive oxygen and nitrogen species that distinguish this enzyme from other glutathione transferases [23]. Due to these peculiar properties, Mgst1 can protect cells against intermediates of lipids peroxidation [30]. Many studies have demonstrated that one of the crucial transcription factors activating antioxidant response is NF-E2-related factor 2 (Nrf2). Under stress conditions, more than 200 gen
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