Role of the life span determinant P66shcA in ethanol-induced liver damage

Abstract

Mice lacking the 66 kDa isoform of the adapter molecule shcA (p66 shcA ) display increased resistance to oxidative stress and delayed aging. In cultured cell lines, p66 promotes formation of Reactive Oxygen Species (ROS) in mitochondria, and apoptotic cell death in response to a variety of pro-oxidant noxious stimuli. As mitochondrial ROS and oxidative cell damage are clearly involved in alcohol-induced pathology, we hypothesized that p66 may also have a role in ethanol. In vivo, changes observed in p66 þ / þ mice after 6-week exposure to ethanol in the drinking water, including elevated serum alanine aminotransferase (ALT), liver swelling and evident liver steatosis, were significantly attenuated in p66À/À mutant mice. Biochemical analysis of liver tissues revealed induction of the p66 protein by ethanol, whereas p66-deficient livers responded to alcohol with a significant upregulation of the mitochondrial antioxidant enzyme MnSOD, nearly absent in control mice. Evidence of an inverse correlation between expression level of p66 and protection from alcohol-induced oxidative stress was also confirmed in vitro in primary hepatocytes and in HepG2-E47 cells, an ethanol-responsive hepatoma cell line. In fact, MnSOD upregulation by exposure to ethanol in vitro was much more pronounced in p66KO versus wild-type isolated liver cells, and blunted in HepG2 cells overexpressing p66shc. p66 overexpression also prevented the activation of a luciferase reporter gene controlled by the SOD2 promoter, indicating that p66 repression of MnSOD operates at a transcriptional level. Finally, p66 generated ROS in HepG2 cells and potentiated oxidative stress and mitochondrial depolarization by ethanol. Taken together, the above observations clearly indicate a role for p66 in alcohol-induced cell damage, likely via a cell-autonomous mechanism involving reduced expression of antioxidant defenses and mitochondrial dysfunction.

Role of Mitochondria in Alcoholic Liver Disease

“…p66Shc is a signaling adapter protein known to directly stimulate hydrogen peroxide generation by transferring electrons to cytochrome c [50]. In this regard, increased hepatic p66Shc expression has been reported in mice treated with alcohol in the drinking water and the deletion of p66Shc ameliorated alcohol-induced hepatic steatosis, oxidative stress and cell injury [51]. Intriguingly, these effects in the p66Shc null mice were accompanied by increased expression of MnSOD, raising the question of whether the protection in the null mice was due to the lack of direct generation of hydrogen peroxide by p66Shc or indirectly via increased antioxidant defenses.…”

“…However, the role of alcohol on MnSOD regulation is controversial, with findings indicating increased expression [52], no change [51, 53] or decreased expression [54]. Moreover, homozygous mutations in the MnSOD gene associated with increased mitochondrial localization has been shown to be a risk for severe ALD in humans [55].…”

Deletion of p66Shc in mice increases the frequency of size‐change mutations in the lacZ transgene

“…Notably, a critical role of p66Shc in the propagation of apoptotic signals has been documented both in cell culture upon a variety of stimuli, including irradiation, oxidants, anticancer drugs, hyperglycemia, calcium overload, amyloid or HIV proteins (see for review Migliaccio et al , 2006) and has also been observed in vivo . In fact, p66KO mice show markedly reduced signs of tissue damage and apoptosis after ischemia (Zaccagnini et al , 2004; Carpi et al , 2009), hypercholesterolemic diet (Napoli et al , 2003), diabetes (Rota et al , 2006; Pugliese et al , 2006; Fadini et al , 2010), encephalitis (Su et al , 2012), hepatectomy (Haga et al , 2010) and challenges with angiothensin II (Graiani et al , 2005; Sun et al , 2010), paraquat (Migliaccio et al , 1999), ethanol (Koch et al , 2008) or chloride carbide (Giorgio et al , 2005). Consistently p66KO mice are resistant to degenerative diseases and show signs of retarded aging (Cosentino et al , 2004; Pugliese et al , 2006; Pesaresi et al , 2011).…”

Investigation of the spatial distribution of glutathione redox-balance in live cells by using Fluorescence Ratio Imaging Microscopy

“…While traditional biochemical analysis of tissue and cell extracts and traditional imaging techniques suffer from several technical limitations, when seeking to determine the redox environment, Fluorescence Ratio Imaging Microscopy allows one to determine intracellular redox states by making it possible to cancel out most or all of the possible variability caused by instrument efficiency and effective dye content (Bright et al, 1987;Bizzarri et al, 2006;Gross and Loew, 1994;Luby-Phelps et al, 1993;Dooley et al, 2004). Recently, it has been shown that rxYFP can be employed ratiometrically to measure the fraction R of reduced rxYFP at equilibrium Koch et al, 2008): monitor any oxidative/reductive state of the protein, independent of any possible experimental artifacts (dye concentration, photobleaching, intensity fluctuations, etc.). By mapping R over the whole microscope scanning field, images representative of the GSH:GSSG redox status, at a resolution of 200 nm (the minimum confocal voxel size) can be created .…”