Calpain activation is required for glutamate-induced p27 down-regulation in cultured cortical neurons.
Recent evidence suggests that cell cycle-related molecules play pivotal roles in multiple forms of cell death in post-mitotic neurons. Nevertheless, it remains unclear what molecular mechanisms are involved in the regulation of expression levels and activities of these molecules. We showed previously that treatment with extracellular glutamate decreases cyclin-dependent kinase inhibitor p27 before neuronal cell death. In this study, we demonstrate that reductions of both p27 and neuronal viability were dependent on activity of calpain, a Ca(2+)-dependent protease, but not on activity of caspase 3. Interestingly, the glutamate-induced reduction of p27 was not dependent on the ubiquitin-proteasome system. In fact, p27 was present only in the neuronal nucleus, whereas calpain 1, a ubiquitous calpain, was observed both in the neuronal nucleus and cytoplasm in control cultures. Glutamate treatment did not change the localization patterns of p27 and calpain 1. It reduced p27 expression level in the nucleus in a calpain-dependent manner. In vitro experiments using neuronal cell lysate and p27 recombinant protein revealed that p27 was degraded as a substrate of activated calpain 1. These results suggest that calpain(s), activated by glutamate treatment, degrade(s) p27 in the nucleus of neurons, which might promote aberrant cell cycle progression.
Akashiba
Hiroki
H
Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.
Matsuki
Norio
N
Nishiyama
Nobuyoshi
N
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D002154:Calpain / Q000378:metabolism* / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D003593:Cytoplasm / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D018698:Glutamic Acid / Q000494:pharmacology*; D007150:Immunohistochemistry; D009474:Neurons / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D025801:Ubiquitin / Q000378:metabolism
D025801:Ubiquitin; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018698:Glutamic Acid; D002154:Calpain; D053148:Caspase 3; D046988:Proteasome Endopeptidase Complex