Involvement of TLCK-sensitive serine protease in colchicine-induced cell death of sympathetic neurons in culture.
Superior cervical ganglion (SCG) cells from neonatal rats underwent apoptosis upon treatment with colchicine, a microtubule-disrupting agent. Western blotting and activity measurements showed that caspase-3 was indeed activated, but its peptide inhibitor (Ac-DEVD-CHO) neither suppressed nuclear fragmentation nor rescued the neurons from cell death. z-VAD-fmk, the general inhibitor of caspases, prevented nuclear fragmentation and delayed the cell death. Moreover, N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), but not N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK), prevented nuclear fragmentation and provided neuronal protection as well. The combination of both z-VAD-fmk and TLCK provided a long-term neuronal protection (>4 days), whereas neither one alone could do so, suggesting that there are both caspase-dependent and -independent pathways. TLCK-sensitive serine protease is also likely to act upstream of caspase-3 in a caspase-dependent pathway. Electron microscopic observations demonstrated that z-VAD-fmk suppressed nuclear fragmentation and improved mitochondrial swelling, but failed to prevent vesicular formation, which resulted in a slowly-occurring necrosis. More importantly, TLCK effectively blocked this abundant vesicular formation along with suppressing chromatin condensation. Thus, the combination of both conferred a nearly normal morphology, which is consistent with the results of cell survival experiments. These findings clearly indicate that TLCK-sensitive serine protease plays multiple roles in caspase-dependent and -independent pathways of colchicine-induced cell death, and suggest a novel mechanism underlying a necrotic pathway involving ER swelling and vesicular formation.
Mitsui
C
C
Molecular Neurobiology Laboratory, Hokkaido University, Graduate School of Science, Sapporo, Japan.
Sakai
K
K
Ninomiya
T
T
Koike
T
T
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D003994:Bucladesine / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D003078:Colchicine / Q000494:pharmacology; D004791:Enzyme Inhibitors; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008868:Microtubule Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009336:Necrosis; D009420:Nervous System / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D009474:Neurons / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D018696:Neuroprotective Agents / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012697:Serine Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D017783:Superior Cervical Ganglion / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology
D004791:Enzyme Inhibitors; D008868:Microtubule Proteins; D018696:Neuroprotective Agents; D011500:Protein Synthesis Inhibitors; D015842:Serine Proteinase Inhibitors; D014107:Tosyllysine Chloromethyl Ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D003994:Bucladesine; D012697:Serine Endopeptidases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D011188:Potassium; D003078:Colchicine