| Budget Amount *help |
¥11,500,000 (Direct Cost: ¥11,500,000)
Fiscal Year 2001: ¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 2000: ¥6,100,000 (Direct Cost: ¥6,100,000)
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| Research Abstract |
It is well known that PI-3k pathway is deeply involved in the regulation of apoptosis. Overexpression of PI-3k or Akt kinase showed the protection of the cells against staurosporine-induced apoptosis without increase of ceramide formation, and anti-inflammatory cytokines, IL-10 and IL-13 was suggested to inhibit TNF-a induced ceramide formation through activation of PI-3k in rat primary astrocytes. As a mechanism by which PI-3k signalling inhibits ceramide generation the inhibition of Smase was suggested because IGF-1/PI-3k inhibited bacterial SMase-induced ceramide generation. Herein, it has not been elucidated whether stress-induced ceramide generation is inhibited by IGF-1/PI-3k anti-apoptotic signalling. Therefore, we recently examined whether ceramide generation is inhibited by IGF-1 in heat-shock (HS) -induced apoptosis. It was shown that ceramide generation induced by HS at 43 degree for 30 min was not affected by 100 ng/ml IGF-1 in HL-60 cells when IGF-1 restored HS-induced apo
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ptosis by approximately 50 %. However, HS-induced caspase-3 activation and oxidative damage judged by lipid peroxidation was inhibited by IGF-1 and this inhibition by IGF-1 of caspase-3 and oxidative damage was recovered in the presence of a PI-3k inhibitor wortmannin. These results suggested that PI-3k-dependent anti-apoptotic signalling acts not only upstream of ceramide but also downstream of ceramide including activation of caspase and oxidative damage.
Since ceramide content in mitochondria is increased by stress-induced apoptosis, IGF-1 may inhibit caspase-3 activation by abrogating ceramide-induced increase of mitochondrial membrane potential to release oxidative damage. HS-increased ceramide signalling was inhibited by IGF-1/PI-3k at the step for caspase-3 activation probably through inhibition of caspase-9 and activation of Bcl-2 via Akt kinase action and generation of oxidative damage was subsequently blocked. How does ceramide induced caspase-3 increase oxidative damage? We investigated the relation of easpase and reactive oxygen intermediates (ROI) scavenger system. Although ceramide was reported to decrease glutathione, we found that C2-ceramide inhibited catalase activity in a dose- and time-dependent manner and that this inhibition of catalase by ceramide was restored by IGF-1 and overexpression of PI-3k in HL-60 cells (manuscript prepared). Since ceramide-induced apoptosis was suppressed by various kinds of ROI scavengers, glutathione, catalase and SOD, execution of ceramide-mediated apoptosis through oxidative damage may be regulated by caspase-3-dependent ROI scavenger system. In sum mary, these results suggest that IGF-1/PI-3 kinase inhibited C2-ceramide-induced apoptosis due to relieving oxidative damage, which resulted from the inhibition of catalase by activated caspase-3. In the future, analysis about the topologkal mechanism by which ceramide connects to PI-3k signalling would be required to understand the precise, physiological relation of ceramide-mediated apoptosis and PI-3k/Akt kinase-dependent cell survival. Less
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