Programmed cell death in cortical chick embryo astrocytes is associated with activation of protein kinase PK60 and ceramide formation.
Embryonic astrocytes respond readily to serine/threonine kinase regulation in terms of cytoskeleton assembly, mitotic activity, and cell fate. We now present evidence that these responses include apoptosis. Staurosporine induced apoptosis in astrocyte cultures derived from chick embryo cerebral hemispheres, as assayed both by immunocytochemical detection of new 3-hydroxy DNA ends and production of 200-bp DNA fragment laddering. Staurosporine treatment also resulted in the prolonged (>24 h) activation of a 60-kDa serine/threonine protein kinase (PK60), increased ceramide formation (fourfold after 24 h), increased glutamine synthetase activity, and significant apoptosis (40%) after 24 h. PK60 was shown to be cytoskeleton associated and its activity, as measured by phosphorylation of myelin basic protein, was rapid, increased for up to 3 h, and was stable for at least 24 h. Other protein kinase C inhibitors, H8, sphingosine, calphostin C, or the protein kinase A inhibitor KT5720 did not induce either PK60 activation or apoptosis. The dose-dependent increase in [3H]palmitate labeling of ceramide and a specific decrease in labeling of its precursor sphingomyelin were not blocked by the biosynthetic inhibitor fumonisin beta1 but were increased (in a dose-dependent manner) by the coaddition of the ceramidase inhibitor oleoylethanolamine. Exogenous addition of C2-ceramide induced apoptosis but did not activate PK60. These results suggest that apoptosis in embryonic astrocytes involves pathways similar to those described in other cell types and that the activation of PK60 and formation of ceramide are early events in the pathway.
Mangoura
D
D
Department of Pediatrics, University of Chicago Medical School, Illinois 60637, USA.
Dawson
G
G
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001253:Astrocytes / Q000187:drug effects / Q000502:physiology*; D002518:Ceramides / Q000096:biosynthesis*; D002540:Cerebral Cortex / Q000166:cytology / Q000196:embryology / Q000502:physiology*; D002642:Chick Embryo; D003599:Cytoskeleton / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D063388:Endocannabinoids; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004983:Ethanolamines / Q000494:pharmacology; D009829:Oleic Acids; D010770:Phosphotransferases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors
D002518:Ceramides; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D004983:Ethanolamines; D009829:Oleic Acids; C033595:N-oleoylethanolamine; D010770:Phosphotransferases; C109922:protein kinase PK60; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine