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Teratogen-induced activation of caspase-9 and the mitochondrial apoptotic pathway in early postimplantation mouse
Sally A Little1, Philip E Mirkes
1Birth Defects Research Laboratory, University of Washington, Seattle, Washington 98195, USA.
Abstract:
Previously we showed that teratogen-induced cell death in mouse embryos is apoptotic in nature, i.e., involves the release of cytochrome c from mitochondria and the subsequent activation of caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and internucleosomal DNA fragmentation. Herein we show that hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine also activate caspase-9, the apical caspase in the mitochondrial apoptotic pathway. Activation of procaspase-9 is associated with the cleavage of this proenzyme and the generation of two forms of the large subunit, primarily a 39-kDa subunit (p39) but also a lesser amount of a 37-kDa subunit (p37). We also present data that support the idea that the teratogen-induced formation of the p37 subunit in vivo occurs by the cytochrome c-mediated processing of procaspase-9, whereas the p39 subunit is formed by an amplification loop involving caspase-3. We also previously showed that the release of cytochrome c, activation of caspase-3, cleavage of PARP, and DNA fragmentation are blocked in cells of the developing heart, which are resistant to teratogen-induced cell death. We now show that this block in the mitochondrial apoptotic pathway in heart cells extends to the activation of procaspase-9. Thus, our cumulative data indicate that hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine induce cell death in Day 9 mouse embryos by activating the mitochondrial apoptotic pathway. In addition, our data suggest that cells of the Day 9 mouse embryo that are resistant to teratogen-induced cell death possess multiple mechanisms for inhibiting the mitochondrial apoptotic pathway after a teratogenic exposure.
Insights
Teratogens trigger apoptosis in mouse embryos via the mitochondrial pathway, activating caspase-9. Resistant heart cells block this pathway at multiple points, preventing cell death.
Area of Science:
- Developmental Biology
- Cell Biology
- Toxicology
Background:
- Teratogen-induced embryonic cell death is apoptotic, involving mitochondrial cytochrome c release and caspase-3 activation.
- The mitochondrial apoptotic pathway is crucial for normal embryonic development and response to toxic insults.
Purpose of the Study:
- To investigate the role of caspase-9 activation in teratogen-induced apoptosis in mouse embryos.
- To identify mechanisms of resistance to teratogen-induced cell death in developing heart cells.
Main Methods:
- Exposure of Day 9 mouse embryos to teratogens (hyperthermia, 4-hydroperoxycyclophosphamide, staurosporine).
- Analysis of caspase-9 activation, procaspase-9 cleavage products (p39 and p37), and cytochrome c release.
- Comparison of apoptotic pathway activation in sensitive embryonic cells versus resistant heart cells.
Main Results:
- Hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine activate caspase-9 in mouse embryos.
- Teratogen-induced p37 caspase-9 subunit formation is cytochrome c-dependent, while p39 formation involves a caspase-3 amplification loop.
- Resistant heart cells exhibit a block in caspase-9 activation, upstream of caspase-3 activation.
Conclusions:
- Teratogens induce embryonic cell death by activating the mitochondrial apoptotic pathway, including caspase-9.
- Developing heart cells employ multiple resistance mechanisms to inhibit the mitochondrial apoptotic pathway following teratogenic exposure.
