Dual inhibition of protein phosphatase-1/2A and calpain rescues nerve growth factor-differentiated PC12 cells from oxygen-glucose deprivation-induced cell death.
In the present study, we examined how the cell survival signaling via cyclic AMP-responsive element binding protein (CREB) and Akt, and the cell death signaling via cystein proteases, calpain and caspase-3, are involved in oxygen-glucose deprivation (OGD) followed by reoxygenation (OGD/reoxygenation)-induced cell death in nerve growth factor (NGF)-differentiated PC12 cells. OGD/reoxygenation-induced cell death was evaluated by LDH release into the culture medium. The level of LDH release was low (9.0% +/- 4.1%) immediately after 4 hr of OGD (0 hr of reoxygenation), was significantly increased to 28.6% +/- 6.6% at 3 hr of reoxygenation, and remained at similar levels at 6 and 20 hr of reoxygenation, suggesting that reoxygenation at least for 3 hr resulted in the loss of cell membrane integrity. After 4 hr of OGD followed by 3 hr of reoxygenation, dephosphorylation of phosphorylated CREB (pCREB), but not phosphorylated Akt (pAkt), was induced. Under these conditions, calpain- but not caspase-3-mediated alpha-spectrin breakdown product was increased, indicating that OGD/reoxygenation also induced an increase in calpain activity. The restoration of pCREB by protein phosphatase (PP)-1/2A inhibitors or the inhibition of excessive activation of calpain by calpain inhibitor did not reduce OGD/reoxygenation-induced LDH release. Cotreatment with PP-1/2A and calpain inhibitors reduced OGD/reoxygenation-induced LDH release. The present study suggests that a balance in the phosphorylation and proteolytic signaling is involved in the survival of NGF-differentiated PC12 cells.
Nakajima
Takayuki
T
Laboratory of Cellular Cybernetics, Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Japan.
Wakasa
Takashi
T
Okuma
Yasunobu
Y
Inanami
Osamu
O
Nomura
Yasuyuki
Y
Kuwabara
Mikinori
M
Kawahara
Koichi
K
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D050882:CREB-Binding Protein / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000627:therapeutic use*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D005947:Glucose / Q000172:deficiency*; D000860:Hypoxia / Q000188:drug therapy* / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology; D009433:Neural Inhibition / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D051058:Oncogene Protein v-akt / Q000378:metabolism; D010100:Oxygen / Q000627:therapeutic use; D016716:PC12 Cells / Q000187:drug effects; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054645:Protein Phosphatase 1; D051381:Rats; D012694:Serine / Q000378:metabolism; D013049:Spectrin / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D013997:Time Factors; D016875:tau Proteins / Q000378:metabolism
D004791:Enzyme Inhibitors; D016875:tau Proteins; D013049:Spectrin; D013912:Threonine; D012694:Serine; D020932:Nerve Growth Factor; D007770:L-Lactate Dehydrogenase; D050882:CREB-Binding Protein; D051058:Oncogene Protein v-akt; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D002154:Calpain; D005947:Glucose; D010100:Oxygen