p66shc-mediated toxicity of high-dose α-tocopherol in renal proximal tubule cells.
α-Tocopherol (TOC) is a widely used supplement known for its role as an antioxidant. Previously, we have shown that TOC elicits adaptive responses by upregulating the ERK/CREB/HO-1 pathway, which depends on its concentration in cultured renal proximal tubule cells (RPTCs). This suggests that high-dose TOC (hTOC) may elicit adverse effects via inflicting oxidative stress. Since the pro-oxidant p66shc is a major mediator of oxidant injury in various models of renal toxicants, we tested the hypothesis that hTOC elicits renal toxicity through activation of p66shc and consequent oxidative stress. RPTCs (NRK52E) were treated with high-dose TOC (hTOC; 400 nM) in cells where expression or mitochondrial cytochrome c-binding of p66shc was manipulated by genetic means. Intracellular production of reactive oxygen species (ROS), mitochondrial depolarization, and cell viability was also determined. Additionally, activation of the pro-survival ERK/CREB/HO-1 signaling and the p66shc promoter was determined via reporter luciferase assays. hTOC decreased cell viability via increasing ROS-dependent mitochondrial depolarization and suppressing the pro-survival ERK/CREB/HO-1 pathway via transcriptional activation of p66shc. Conversely, either knockdown of p66shc, mutation of its mitochondrial cytochrome c-binding site, or overexpression of ERK or HO-1 ameliorated adverse effects of hTOC and restored the pro-survival signaling. The pro-oxidant p66shc plays dual role in toxicity of high-dose TOC: it provokes oxidative stress and suppresses adaptive responses.
Department of Pediatrics, Division of Pediatric Nephrology, University of Mississippi Medical Center, 2500 N State St, Jackson, MS, 39216, USA. dreed@umc.edu.
Dustin K
DK
Reed
Department of Pediatrics, Division of Pediatric Nephrology, University of Mississippi Medical Center, 2500 N State St, Jackson, MS, 39216, USA.
Anthony
A
Carter
Department of Pediatrics, Division of Pediatric Nephrology, University of Mississippi Medical Center, 2500 N State St, Jackson, MS, 39216, USA.
Mehul
M
Dixit
Department of Pediatrics, Division of Pediatric Nephrology, University of Mississippi Medical Center, 2500 N State St, Jackson, MS, 39216, USA.
Istvan
I
Arany
D019943:Amino Acid Substitution; D000818:Animals; D000975:Antioxidants / Q000009:adverse effects*; D001665:Binding Sites; D002460:Cell Line; D002470:Cell Survival; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D019587:Dietary Supplements / Q000009:adverse effects; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D053078:Membrane Potential, Mitochondrial; D009154:Mutation; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D051381:Rats; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024502:alpha-Tocopherol / Q000009:adverse effects*
D000975:Antioxidants; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D045304:Cytochromes c; D024502:alpha-Tocopherol
Dietary supplements; HO-1; Kidney; Toxicity; Vitamin E; p66shc