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Multiple polyphosphoinositide pathways regulate apoptotic signalling in a dorsal root ganglion derived cell line
R Goswami1, S A Dawson, G Dawson
1Department of Pediatrics, University of Chicago, Illinois 60637, USA.
Journal of Neuroscience Research
|February 5, 2000
Summary
Polyphosphoinositides regulate cell survival and apoptosis. Inhibiting phosphatidylinositol-3 kinase (PI-3K) triggers apoptosis, which is worsened by lowered cyclic AMP (cAMP) levels and PtdIns(4,5)P2 hydrolysis.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Polyphosphoinositides are crucial for cell signaling, protein anchoring, organelle transport, cytoskeleton organization, and cell survival.
- Phosphatidylinositol-3 kinase (PI-3K) synthesizes polyphosphoinositides like Ptd(3,4)InsP2 and PtdIns(3,4,5)P3, which are vital for cell survival via Akt kinase activation.
- Dysregulation of polyphosphoinositide pathways is implicated in various cellular processes, including apoptosis.
Purpose of the Study:
- To investigate the role of polyphosphoinositides in apoptosis regulation.
- To elucidate the mechanisms by which cyclic AMP (cAMP) levels and PtdIns(4,5)P2 hydrolysis influence apoptosis induced by PI-3K inhibition.
- To explore the signaling pathways involved in opioid-mediated PtdIns(4,5)P2 hydrolysis.
Main Methods:
- Utilized PI-3K inhibitors (wortmannin, LY294002) to induce apoptosis in F-11 dorsal root ganglion cells.
- Manipulated intracellular cAMP levels using analogs and opioid agonists.
- Stably transfected F-11 cells with a constitutively active PI-3K catalytic subunit.
- Stimulated PtdIns(4,5)P2 hydrolysis using bradykinin (BK) and opioid agonists.
- Assessed caspase-3 (CPP32) activation and apoptosis via biochemical assays.
Main Results:
- PI-3K inhibitors wortmannin and LY294002 induced time-dependent caspase-3 activation and apoptosis in F-11 cells.
- Lowering cAMP levels enhanced PI-3K inhibitor-induced apoptosis, while a cAMP analog (Bt2cAMP) was protective.
- Stably expressing active PI-3K conferred resistance to wortmannin/LY294002-induced apoptosis.
- Bradykinin and opioid agonists stimulated PtdIns(4,5)P2 hydrolysis, enhancing wortmannin-induced apoptosis.
- Opioid-enhanced PtdIns(4,5)P2 hydrolysis was pertussis toxin-sensitive, suggesting involvement of Gi/o proteins.
Conclusions:
- Receptor-mediated lowering of cAMP and PtdIns(4,5)P2 hydrolysis exacerbate apoptosis induced by PI-3K inhibitors.
- These pathways do not directly affect caspase-3 activity but modulate its activation.
- Multiple polyphosphoinositide pathways are involved in the intricate regulation of apoptosis.