The p66Shc protein controls redox signaling and oxidation-dependent DNA damage in human liver cells.
The p66Shc protein mediates oxidative stress-related injury in multiple tissues. Steatohepatitis is characterized by enhanced oxidative stress-mediated cell damage. The role of p66Shc in redox signaling was investigated in human liver cells and alcoholic steatohepatitis. HepG2 cells with overexpression of wild-type or mutant p66Shc, with Ser36 replacement by Ala, were obtained through infection with recombinant adenoviruses. Reactive oxygen species and oxidation-dependent DNA damage were assessed by measuring dihydroethidium oxidation and 8-hydroxy-2'-deoxyguanosine accumulation into DNA, respectively. mRNA and protein levels of signaling intermediates were evaluated in HepG2 cells and liver biopsies from control and alcoholic steatohepatitis subjects. Exposure to H2O2 increased reactive oxygen species and phosphorylation of p66Shc on Ser36 in HepG2 cells. Overexpression of p66Shc promoted reactive oxygen species synthesis and oxidation-dependent DNA damage, which were further enhanced by H2O2. p66Shc activation also resulted in increased Erk-1/2, Akt, and FoxO3a phosphorylation. Blocking of Erk-1/2 activation inhibited p66Shc phosphorylation on Ser36. Increased p66Shc expression was associated with reduced mRNA levels of antioxidant molecules, such as NF-E2-related factor 2 and its target genes. In contrast, overexpression of the phosphorylation defective p66Shc Ala36 mutant inhibited p66Shc signaling, enhanced antioxidant genes, and suppressed reactive oxygen species and oxidation-dependent DNA damage. Increased p66Shc protein levels and Akt phosphorylation were observed in liver biopsies from alcoholic steatohepatitis compared with control subjects. In human alcoholic steatohepatitis, increased hepatocyte p66Shc protein levels may enhance susceptibility to DNA damage by oxidative stress by promoting reactive oxygen species synthesis and repressing antioxidant pathways.
Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology, Andrology and Metabolic Diseases, University of Bari Aldo Moro, Bari, Italy;
Sebastio
S
Perrini
Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology, Andrology and Metabolic Diseases, University of Bari Aldo Moro, Bari, Italy;
Federica
F
Tortosa
Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology, Andrology and Metabolic Diseases, University of Bari Aldo Moro, Bari, Italy;
Annalisa
A
Natalicchio
Department of Medical Biochemistry, Biology and Physics, University of Bari Aldo Moro, Bari, Italy;
Consiglia
C
Pacelli
Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology, Andrology and Metabolic Diseases, University of Bari Aldo Moro, Bari, Italy;
Angelo
A
Cignarelli
Department of Biochemical Sciences and Human Oncology, Clinica Medica "A. Murri," University of Bari Aldo Moro, Bari, Italy;
Vincenzo O
VO
Palmieri
Cristina
C
Caccioppoli
Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology, Andrology and Metabolic Diseases, University of Bari Aldo Moro, Bari, Italy;
Francesca
F
De Stefano
Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology, Andrology and Metabolic Diseases, University of Bari Aldo Moro, Bari, Italy;
Stefania
S
Porro
Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology, Andrology and Metabolic Diseases, University of Bari Aldo Moro, Bari, Italy;
Anna
A
Leonardini
Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology, Andrology and Metabolic Diseases, University of Bari Aldo Moro, Bari, Italy;
Romina
R
Ficarella
Department of Emergency and Organ Transplantation, General Surgery and Liver Transplantation, University of Bari Aldo Moro, Bari, Italy; and.
Michele
M
De Fazio
Department of Medical Biochemistry, Biology and Physics, University of Bari Aldo Moro, Bari, Italy;
Tiziana
T
Cocco
Department of Emergency and Organ Transplantation, General Surgery and Liver Transplantation, University of Bari Aldo Moro, Bari, Italy; and Azienda Sanitaria Locale Bari, Ospedale M. Sarcone, Terlizzi (BA), Italy.
Francesco
F
Puglisi
Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology, Andrology and Metabolic Diseases, University of Bari Aldo Moro, Bari, Italy;
Luigi
L
Laviola
Department of Biochemical Sciences and Human Oncology, Clinica Medica "A. Murri," University of Bari Aldo Moro, Bari, Italy;
Giuseppe
G
Palasciano
Department of Emergency and Organ Transplantation, Section on Internal Medicine, Endocrinology, Andrology and Metabolic Diseases, University of Bari Aldo Moro, Bari, Italy; francesco.giorgino@uniba.it.
Francesco
http://orcid.org/0000-0002-2728-7721
F
Giorgino
D018929:Cell Culture Techniques; D004249:DNA Damage; D005235:Fatty Liver, Alcoholic / Q000378:metabolism*; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D051058:Oncogene Protein v-akt / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1
C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D017382:Reactive Oxygen Species; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D051058:Oncogene Protein v-akt
Akt; FoxO3a; Nrf2; alcoholic steatohepatitis; hepatocyte; oxidative stress; p66Shc