treated HEI-OC1
PHYSIOLOGICAL SIGNIFICANCE OF SELECTIVE DEGRADATION OF P62 BY AUTOPHAGY IN AUDITORY CELLS !
PP063
1Ken
Hayashi, 2Katsuaki Dan, 3Fumiyuki Goto, 4Yasuyuki Nomura, 3Sho Kanzaki, 3Kaoru Ogawa 1. Department of Otolaryngology, Shinkawa Clinic, Kanagawa, Japan, 2. Collaborative Research Resources, Core Instrumentation Facility, Keio University, Tokyo, Japan, " 3. Department of Otolaryngology, Keio University, Tokyo, Japan, 4. Department of Otolaryngology, Nihon University, Tokyo, Japan
ABSTRACT
INTRODUCTION
RESULTS
Introduction and aim of this study: Oxidative stress and redox balance affect auditory cellular functions, including signaling pathways. This, in turn, may cause induction of autophagy or necrosis. The Nrf2/Keap1 signaling pathway is the main pathway responsible for cell defense against oxidative stress and maintaining the cellular redox balance at physiological levels. The relation between Nrf2/Keap1 signaling and regulation of autophagy and necrosis in auditory cells is not well understood. Thus, the purposes of this study were to elucidate how Keap1 and Nrf2 provide molecular foundation for a possible crosstalk between autophagy and necrosis pathways in auditory cells. Methods: We used auditory cell line (HEI-OC1) in this study. The viability of HEI-OC1 was determined by cell viability assays. The samples after treatment of HEI-OC1 were analyzed by a flow cytometer. Immunofluorescent confocal laser microscopy was used. Western blot was performed. HEI-OC1 was transfected with Nrf2, Keap1 and p62 siRNA. Results: After H2O2 treatment, not apoptotic cell but necrotic cell was detected by FACscan analysis. The pretreatment of HEI-OC1 with rapamycin protect against H2O2 –induced necrotic cell death. After treatment with rapamycin, the accumulation of LC3-I/II ratio and the expression of GFP-LC3 were observed in HEI-OC1. The knockdown of p62 by siRNA, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62mediated Nrf2 up regulation is Keap1 dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in auditory cells. Conclusion: We demonstrated a promotion of autophagy through the mTOR signaling pathway to enhance cell survival in auditory cells under oxidative stress. A crosstalk between Nrf2/Keap1 pathway in the autophagy related p62 are also presented. The selective autophagy substrate p62 activates the oxidative stress responsive factor Nrf2 through inactivation of keap1.
Autophagy is a highly conserved self-eating process and one of the major protein degradation systems induced by a decrease of intracellular adenosine triphosphate (ATP). Under different type of stress, such as nutrient depletion, oxidative stress and metabolic stress, autophagy is induced to produce metabolic substrates that meet bioenergetic needs of cells and to prevent cell death. On the other hand, autophagy has been suggested to be a cell death mechanism, leading to autophagic cell death or type II programmed cell death " Oxidative stress, perturbations in the cellular thiol level and redox balance, affects many cellular functions, including signaling pathways. This, in turn, may cause the induction of autophagy, necrosis or apoptosis (1). The Nrf2/Keap1 signaling pathway is the main pathway responsible for cell defense against oxidative stress and maintaining the cellular redox balance at physiological levels (2). The relation between Nrf2/Keap1 signaling and regulation of apoptosis, necrosis and autophagy is not well understood. " Current studies have reported that oxidative stress may play a crucial role in the pathogenesis of a variety of inner ear diseases (3)(4). However, the molecular mechanism for inner ear protective efficacy of antioxidants remains unclear. Recently, Marino et al reported an essential role for autophagy in inner ear development and equilibrioception using mice lacking the protease autophagy-related 4B (Atg4b-/- mice) (5). Namely, there is a possibility that autophagy has an inner ear protective efficacy." The loss of adenosine 5ʻ-triphosphate (ATP) has been reported to be an early step after initiation of H2O2-induced oxidative stress in non-neuronal and neural systems. Teepker et al. reported that ATP-decline under H2O2-induced oxidative stress might point to a relevant ATP consumption related to apoptosis. On the other hand, Zhang et al described that oxidative stress induces parallel autophagy and mitochondria dysfunction in human Glioma cells. It has also been reported that autophagic neuron cell death has been observed under condition of ATP deletion, such as ischemia and exposure to rotenone, a mitochondrial complex I inhibitor. Meanwhile, it was reported that ATPinduced autophagy was verified in human monocytes/macrophages。 This time, we discuss determine whether hydrogen peroxide (H2O2) can induce programmed cell death in auditory cells (HEI-OC1) and to identify the role of autophagy induced by oxidative stress and ATP in vitro, and how Keap1 protein and its direct interaction with autophagy molecular hub, p62 provide a molecular foundation for a possible cross talk between Nrf2/keap1, apoptosis, necrosis and autophagy.
Figure 1. The cell viability of HEI-OC1 after treatment of H2O2
CONTACT Name: Ken Hayashi Organization name: Shinkawa Clinic Email: [email protected] Phone: +81-463-76-3341 Website: http://www.shinkawaclinic.com
Cell culture HEI-OC1 cells were provided by Dr. Kalinec (House Ear Institute). Cell viability Cell viability was calculated with Countess® (Invitrogen, Life Technologies, USA) after stain with trypan blue. " Protein extraction and Western blot analysis One hundred micrograms of protein was solubilized in Laemmi's sample buffer by boiling and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by i-Blot. " Reagents Hydrogen peroxide and rapamycin were purchased from Sigma. Antibody against LC3 (Dr. Komatsu), Antibody against Atg7 (Dr. Ueno), Antibody against Nrf2 (Santa Cruz BioTec), Antibody against Keap1 (ProteinTech Group), Antibody against p62 (PROGEN), Antibody against α-tublin (Bio Legend) were used. The GFP-LC3 plasmid was kindly provided by Dr. Komatsu. Atg7 siRNA was from Tomy Digital Biology." Detection of cell death Cell death (apoptosis or necrosis) was quantified by detecting surface exposure of phosphatidylserine in cells using Annexin V-FITC kit. " ATP measurement Intracellular ATP was measured by the luciferin/luciferase using a ATP measurement kit (Bioassay Systems; Hayward, CA, USA). " Transient transfection and confocal microscopy analysis Cells were transfected with 1 µg of the GFP-LC3 expression plasmid using the Amaxa electroporation system, according to the supplierʼs protocol. After 24h, cells were treated as designated. The transfected cells were observed using a confocal microscope (Olympus Fluoview 2000)." Electron Microscope! Cells were fixed for 30 min with ice-cold 3% glutaraldehyde in 0,1M cacodylate buffer, embedded in Epton, and processed for transmission electron microscopy by standard procedure. Representative areas were chosen for ultra-thin sectioning and examined on transmission electron microscope at x 6000 or x12000 magnification." Transient siRNA transfection Cells were transfected with 100nM Atg7, 100nM p62 and a non-targeting siRNA control using G-fectin according to the supplierʼs protocol. After 72h of transfection, cells were treated as designated.
Figure 7. The expression of GFP-LC3 and nuclear status in H2O2-treated HEI-OC1
HEI-OC1 treated with different concentrations of H 2 O 2 for 0-5h exhibited dose-and timedependent cell death.
80! 60! 0.5uM H2O2!
40!
1uM H2O2!
20! 0! 0h!
1h!
3h!
Figure 8. The expression of p62, Nrf2 and Keap1 in H2O2treated HEI-OC1
5h!
RESULTS Figure 13. The expression of p62, Nrf2 and Keap in Atg7 KD-HEIOC1
Both flattened nuclear and swelling of the cell body which indicate necrosis were observed by Hoechst 33342 staining after treatment with 1uM H2O2. On the other hand, dots of GFPLC3 which indicate autophagy were increased in the cytoplasm. This result shows that H2O2 treatment induce necrosis and autophagy inside HEI-OC1.
100!
Figure 2. Effects of H2O2 on the apoptosis or necrosis rates of HEI-OC1
The expression of p62 and Nrf2 increased after 5h treatment of H2O2. The expression of Keap1 increased after 24h treatment of H2O2.
LC3-I to LC3-II conversion induced by H2O2 was markedly blocked by the knockdown of Atg7. Knockdown of Atg7 down regulate the expression of p62, but didn’t affect the expression of Nrf2 and Keap1.
Figure 14. The cell viability of Atg7 knockdown HEI-OC1 after treatment of H2O2 60!
The suppression of autophagy by Atg7 knockdown promote the cell death induced by H2O2.
40! 1uM H2O2!
20!
H2O2 treatment induced necrosis cells to 14.4% after 15 minutes, and 91.3% after 60 minutes.
0! Atg7 normal! Atg7 KD!
Figure 9. The induction of LC3-II induced by Rapamycin treatment
Figure 15. Cell structure and the expression of GFP-LC-3 in p62 KD HEI-OC1
Rapamycin treatment increased the levels of LC3-II.
Figure 3. The intracellular ATP level after treatment of H2O2 in HEI-OC1
The aggregates were confirmed in cytoplasm of p62 KD HEIOC1, but nuclear structure was remained. Dot of GFP-LC3 wasnʼt confirmed in p62 KD HEI-OC1.
ATP level! 120!
Intracellular ATP level was decreased dramatically after treatment of H2O2.
100! 80! 60!
MATERIALS AND METHODS
RESULTS
ATP level"
Figure 10. The punctuated distribution of GFP-LC3 in Rapamycin treated-HEI-OC1
40! 20! 0! 0!
10!
20!
30!
Dots of green fluorescence were increased in HEIOC1 cells after 1h of 10 nM Rapamycin treatment in HEI-OC1.
Figure 4. Accumulation of LC3-II induced by H2O2 treatment and nutrient free
Figure 16. The cell viability of p62 knockdown HEI-OC1 after treatment of H2O2 60!
The knockdown of p62 promote the cell death induced by H2O2.
40! 1uM H2O2!
20! 0! p62 normal!
H2O2 treatment resulted in time-dependent accumulation of LC3-II as well as nutrient free.
Figure 5. The expression of GFP-LC3 in H2O2 treatment and nutrient free in HEI-OC1
DISCUSSION and CONCLUSIONS Figure 11. The morphological change under Transmission Electron Microscope Culture for 1h in 10μM Rapamycin resulted in the appearance of abundant autophagic vacuoles (arrow).
Dots of green fluorescence of GFP-LC3 were increased in HEI-OC1 cells treated in nutrient free medium for 24 hours or with 1uM H2O2 for 1 hour.
Figure 12. The cell viability of H2O2-treated HEI-OC1 after treatment of Rapamycin Figure 6. The morphological change under Transmission Electron Microscope Control cells exhibited normal nuclei with uniform and finely dispersed chromatin, surrounded by cytoplasm with normal appearing mitochondria. In contrast, culture for 24h in nutrient free medium or 3h in 1μM H2O2 resulted in the appearance of abundant autophagic vacuoles (arrows).
p62 KD!
100! 80! 60!
H2O2!
40!
H2O2+Rapa mycin!
20! 0! 0h!
1h!
3h!
5h!
Pretreatment of Rapamycin an hour before the treatment of H2O2 could obviously recover the cell viabilities of the H2O2-treated HEI-OC1. The cell viabilities after 5 hour of treatment of 1uM H2O2 were 32.0 % in Rapamycin pretreated-HEI-OC1. On the other hand, the cell viabilities of H2O2-treated HEI-OC1 decreased in a similar manner as when Rapamycin was added concurrently with H2O2.
In conclusion, our study demonstrated that autophagy serves as a cell survival mechanism to protect against H2O2-indced cell death in auditory cells. We demonstrated a promotion of autophagy through the mTOR signaling pathway to enhance cell survival in auditory cells under oxidative stress. Our findings are believed to bear important implications for understanding the complex relationship among oxidative stress, autophagy and cell death. A crosstalk between Nrf2/Keap1 pathway in the autophagy related p62 are also presented. The selective autophagy substrate p62 activates the oxidative stress responsive factor Nrf2 through inactivation of keap1.
REFERENCES 1. Taguchi K, Motoshita H, Yamamoto M. Molecular mechanisms of the Keap1-Nrf2 pathway in stress response and cancer evolution. Genes to Cells, 2011; 16: 123-140." 2. Fan W, Tang Z, Chen D, et al. Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy. Autophagy, 2010; 6 (5): 614-21." 3. Morizane I, Hakuba N, Hyodo J, et al. Ischemic damage increases nitric oxide production via inducible nitric oxide synthase in the cochlea. Neurosci Lett. 2005;391:62-67." 4. Kujoth GC, Hiona A, Pugh TD, et al. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammarian aging. Science. 2005;309:481-484." 5. Mariño G, Fernández AF, Cabrera S, Lundberg YW, Cabanillas R, Rodríguez F, Salvador-Montoliu N, Vega JA, Germanà A, Fueyo A, Freije JM, López-Otín C. Autophagy is essential for mouse sense of balance. J Clin Invest. 2010;120(7):2331-44. "
PP063
1Ken
Hayashi, 2Katsuaki Dan, 3Fumiyuki Goto, 4Yasuyuki Nomura, 3Sho Kanzaki, 3Kaoru Ogawa 1. Department of Otolaryngology, Shinkawa Clinic, Kanagawa, Japan, 2. Collaborative Research Resources, Core Instrumentation Facility, Keio University, Tokyo, Japan, " 3. Department of Otolaryngology, Keio University, Tokyo, Japan, 4. Department of Otolaryngology, Nihon University, Tokyo, Japan
ABSTRACT
INTRODUCTION
RESULTS
Introduction and aim of this study: Oxidative stress and redox balance affect auditory cellular functions, including signaling pathways. This, in turn, may cause induction of autophagy or necrosis. The Nrf2/Keap1 signaling pathway is the main pathway responsible for cell defense against oxidative stress and maintaining the cellular redox balance at physiological levels. The relation between Nrf2/Keap1 signaling and regulation of autophagy and necrosis in auditory cells is not well understood. Thus, the purposes of this study were to elucidate how Keap1 and Nrf2 provide molecular foundation for a possible crosstalk between autophagy and necrosis pathways in auditory cells. Methods: We used auditory cell line (HEI-OC1) in this study. The viability of HEI-OC1 was determined by cell viability assays. The samples after treatment of HEI-OC1 were analyzed by a flow cytometer. Immunofluorescent confocal laser microscopy was used. Western blot was performed. HEI-OC1 was transfected with Nrf2, Keap1 and p62 siRNA. Results: After H2O2 treatment, not apoptotic cell but necrotic cell was detected by FACscan analysis. The pretreatment of HEI-OC1 with rapamycin protect against H2O2 –induced necrotic cell death. After treatment with rapamycin, the accumulation of LC3-I/II ratio and the expression of GFP-LC3 were observed in HEI-OC1. The knockdown of p62 by siRNA, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62mediated Nrf2 up regulation is Keap1 dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in auditory cells. Conclusion: We demonstrated a promotion of autophagy through the mTOR signaling pathway to enhance cell survival in auditory cells under oxidative stress. A crosstalk between Nrf2/Keap1 pathway in the autophagy related p62 are also presented. The selective autophagy substrate p62 activates the oxidative stress responsive factor Nrf2 through inactivation of keap1.
Autophagy is a highly conserved self-eating process and one of the major protein degradation systems induced by a decrease of intracellular adenosine triphosphate (ATP). Under different type of stress, such as nutrient depletion, oxidative stress and metabolic stress, autophagy is induced to produce metabolic substrates that meet bioenergetic needs of cells and to prevent cell death. On the other hand, autophagy has been suggested to be a cell death mechanism, leading to autophagic cell death or type II programmed cell death " Oxidative stress, perturbations in the cellular thiol level and redox balance, affects many cellular functions, including signaling pathways. This, in turn, may cause the induction of autophagy, necrosis or apoptosis (1). The Nrf2/Keap1 signaling pathway is the main pathway responsible for cell defense against oxidative stress and maintaining the cellular redox balance at physiological levels (2). The relation between Nrf2/Keap1 signaling and regulation of apoptosis, necrosis and autophagy is not well understood. " Current studies have reported that oxidative stress may play a crucial role in the pathogenesis of a variety of inner ear diseases (3)(4). However, the molecular mechanism for inner ear protective efficacy of antioxidants remains unclear. Recently, Marino et al reported an essential role for autophagy in inner ear development and equilibrioception using mice lacking the protease autophagy-related 4B (Atg4b-/- mice) (5). Namely, there is a possibility that autophagy has an inner ear protective efficacy." The loss of adenosine 5ʻ-triphosphate (ATP) has been reported to be an early step after initiation of H2O2-induced oxidative stress in non-neuronal and neural systems. Teepker et al. reported that ATP-decline under H2O2-induced oxidative stress might point to a relevant ATP consumption related to apoptosis. On the other hand, Zhang et al described that oxidative stress induces parallel autophagy and mitochondria dysfunction in human Glioma cells. It has also been reported that autophagic neuron cell death has been observed under condition of ATP deletion, such as ischemia and exposure to rotenone, a mitochondrial complex I inhibitor. Meanwhile, it was reported that ATPinduced autophagy was verified in human monocytes/macrophages。 This time, we discuss determine whether hydrogen peroxide (H2O2) can induce programmed cell death in auditory cells (HEI-OC1) and to identify the role of autophagy induced by oxidative stress and ATP in vitro, and how Keap1 protein and its direct interaction with autophagy molecular hub, p62 provide a molecular foundation for a possible cross talk between Nrf2/keap1, apoptosis, necrosis and autophagy.
Figure 1. The cell viability of HEI-OC1 after treatment of H2O2
CONTACT Name: Ken Hayashi Organization name: Shinkawa Clinic Email: [email protected] Phone: +81-463-76-3341 Website: http://www.shinkawaclinic.com
Cell culture HEI-OC1 cells were provided by Dr. Kalinec (House Ear Institute). Cell viability Cell viability was calculated with Countess® (Invitrogen, Life Technologies, USA) after stain with trypan blue. " Protein extraction and Western blot analysis One hundred micrograms of protein was solubilized in Laemmi's sample buffer by boiling and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by i-Blot. " Reagents Hydrogen peroxide and rapamycin were purchased from Sigma. Antibody against LC3 (Dr. Komatsu), Antibody against Atg7 (Dr. Ueno), Antibody against Nrf2 (Santa Cruz BioTec), Antibody against Keap1 (ProteinTech Group), Antibody against p62 (PROGEN), Antibody against α-tublin (Bio Legend) were used. The GFP-LC3 plasmid was kindly provided by Dr. Komatsu. Atg7 siRNA was from Tomy Digital Biology." Detection of cell death Cell death (apoptosis or necrosis) was quantified by detecting surface exposure of phosphatidylserine in cells using Annexin V-FITC kit. " ATP measurement Intracellular ATP was measured by the luciferin/luciferase using a ATP measurement kit (Bioassay Systems; Hayward, CA, USA). " Transient transfection and confocal microscopy analysis Cells were transfected with 1 µg of the GFP-LC3 expression plasmid using the Amaxa electroporation system, according to the supplierʼs protocol. After 24h, cells were treated as designated. The transfected cells were observed using a confocal microscope (Olympus Fluoview 2000)." Electron Microscope! Cells were fixed for 30 min with ice-cold 3% glutaraldehyde in 0,1M cacodylate buffer, embedded in Epton, and processed for transmission electron microscopy by standard procedure. Representative areas were chosen for ultra-thin sectioning and examined on transmission electron microscope at x 6000 or x12000 magnification." Transient siRNA transfection Cells were transfected with 100nM Atg7, 100nM p62 and a non-targeting siRNA control using G-fectin according to the supplierʼs protocol. After 72h of transfection, cells were treated as designated.
Figure 7. The expression of GFP-LC3 and nuclear status in H2O2-treated HEI-OC1
HEI-OC1 treated with different concentrations of H 2 O 2 for 0-5h exhibited dose-and timedependent cell death.
80! 60! 0.5uM H2O2!
40!
1uM H2O2!
20! 0! 0h!
1h!
3h!
Figure 8. The expression of p62, Nrf2 and Keap1 in H2O2treated HEI-OC1
5h!
RESULTS Figure 13. The expression of p62, Nrf2 and Keap in Atg7 KD-HEIOC1
Both flattened nuclear and swelling of the cell body which indicate necrosis were observed by Hoechst 33342 staining after treatment with 1uM H2O2. On the other hand, dots of GFPLC3 which indicate autophagy were increased in the cytoplasm. This result shows that H2O2 treatment induce necrosis and autophagy inside HEI-OC1.
100!
Figure 2. Effects of H2O2 on the apoptosis or necrosis rates of HEI-OC1
The expression of p62 and Nrf2 increased after 5h treatment of H2O2. The expression of Keap1 increased after 24h treatment of H2O2.
LC3-I to LC3-II conversion induced by H2O2 was markedly blocked by the knockdown of Atg7. Knockdown of Atg7 down regulate the expression of p62, but didn’t affect the expression of Nrf2 and Keap1.
Figure 14. The cell viability of Atg7 knockdown HEI-OC1 after treatment of H2O2 60!
The suppression of autophagy by Atg7 knockdown promote the cell death induced by H2O2.
40! 1uM H2O2!
20!
H2O2 treatment induced necrosis cells to 14.4% after 15 minutes, and 91.3% after 60 minutes.
0! Atg7 normal! Atg7 KD!
Figure 9. The induction of LC3-II induced by Rapamycin treatment
Figure 15. Cell structure and the expression of GFP-LC-3 in p62 KD HEI-OC1
Rapamycin treatment increased the levels of LC3-II.
Figure 3. The intracellular ATP level after treatment of H2O2 in HEI-OC1
The aggregates were confirmed in cytoplasm of p62 KD HEIOC1, but nuclear structure was remained. Dot of GFP-LC3 wasnʼt confirmed in p62 KD HEI-OC1.
ATP level! 120!
Intracellular ATP level was decreased dramatically after treatment of H2O2.
100! 80! 60!
MATERIALS AND METHODS
RESULTS
ATP level"
Figure 10. The punctuated distribution of GFP-LC3 in Rapamycin treated-HEI-OC1
40! 20! 0! 0!
10!
20!
30!
Dots of green fluorescence were increased in HEIOC1 cells after 1h of 10 nM Rapamycin treatment in HEI-OC1.
Figure 4. Accumulation of LC3-II induced by H2O2 treatment and nutrient free
Figure 16. The cell viability of p62 knockdown HEI-OC1 after treatment of H2O2 60!
The knockdown of p62 promote the cell death induced by H2O2.
40! 1uM H2O2!
20! 0! p62 normal!
H2O2 treatment resulted in time-dependent accumulation of LC3-II as well as nutrient free.
Figure 5. The expression of GFP-LC3 in H2O2 treatment and nutrient free in HEI-OC1
DISCUSSION and CONCLUSIONS Figure 11. The morphological change under Transmission Electron Microscope Culture for 1h in 10μM Rapamycin resulted in the appearance of abundant autophagic vacuoles (arrow).
Dots of green fluorescence of GFP-LC3 were increased in HEI-OC1 cells treated in nutrient free medium for 24 hours or with 1uM H2O2 for 1 hour.
Figure 12. The cell viability of H2O2-treated HEI-OC1 after treatment of Rapamycin Figure 6. The morphological change under Transmission Electron Microscope Control cells exhibited normal nuclei with uniform and finely dispersed chromatin, surrounded by cytoplasm with normal appearing mitochondria. In contrast, culture for 24h in nutrient free medium or 3h in 1μM H2O2 resulted in the appearance of abundant autophagic vacuoles (arrows).
p62 KD!
100! 80! 60!
H2O2!
40!
H2O2+Rapa mycin!
20! 0! 0h!
1h!
3h!
5h!
Pretreatment of Rapamycin an hour before the treatment of H2O2 could obviously recover the cell viabilities of the H2O2-treated HEI-OC1. The cell viabilities after 5 hour of treatment of 1uM H2O2 were 32.0 % in Rapamycin pretreated-HEI-OC1. On the other hand, the cell viabilities of H2O2-treated HEI-OC1 decreased in a similar manner as when Rapamycin was added concurrently with H2O2.
In conclusion, our study demonstrated that autophagy serves as a cell survival mechanism to protect against H2O2-indced cell death in auditory cells. We demonstrated a promotion of autophagy through the mTOR signaling pathway to enhance cell survival in auditory cells under oxidative stress. Our findings are believed to bear important implications for understanding the complex relationship among oxidative stress, autophagy and cell death. A crosstalk between Nrf2/Keap1 pathway in the autophagy related p62 are also presented. The selective autophagy substrate p62 activates the oxidative stress responsive factor Nrf2 through inactivation of keap1.
REFERENCES 1. Taguchi K, Motoshita H, Yamamoto M. Molecular mechanisms of the Keap1-Nrf2 pathway in stress response and cancer evolution. Genes to Cells, 2011; 16: 123-140." 2. Fan W, Tang Z, Chen D, et al. Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy. Autophagy, 2010; 6 (5): 614-21." 3. Morizane I, Hakuba N, Hyodo J, et al. Ischemic damage increases nitric oxide production via inducible nitric oxide synthase in the cochlea. Neurosci Lett. 2005;391:62-67." 4. Kujoth GC, Hiona A, Pugh TD, et al. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammarian aging. Science. 2005;309:481-484." 5. Mariño G, Fernández AF, Cabrera S, Lundberg YW, Cabanillas R, Rodríguez F, Salvador-Montoliu N, Vega JA, Germanà A, Fueyo A, Freije JM, López-Otín C. Autophagy is essential for mouse sense of balance. J Clin Invest. 2010;120(7):2331-44. "
