Teratogen-induced activation of caspase-6 and caspase-7 in early postimplantation mouse embryos.
BACKGROUND
Previous work has shown that teratogens such as hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway. Key to the activation of this pathway is the activation of a caspase cascade involving the cleavage-induced activation of an initiator procaspase, caspase-9, and the downstream effector procaspase, caspase-3. For example, procaspase-3, an inactive proenzyme of 32 kDa is cleaved by activated caspase-9 to generate a large subunit of approximately 17 kDa and a small subunit of approximately 10 kDa. In turn, caspase-3 is known to target a variety of cellular proteins for proteolytic cleavage as part of the process by which dying cells are eliminated. Previous work has also shown that neuroepithelial cells are sensitive to teratogen-induced activation of this pathway and subsequent cell death whereas cells of the heart are resistant. Although caspase-3 is a key effector caspase activated by teratogens, two other effector caspases, caspase-6 and caspase-7, are known; however, their role in teratogen-induced cell death is unknown.


METHODS
Because cleavage-induced generation of specific subunits is the most specific assay for activation of caspases, we have used antibodies that recognize the procaspase and one of its active subunits and a Western blot approach to assess the activation of caspase-6 and caspase-7 in day 9 mouse embryos (or heads, hearts and trunks isolated from whole embryos) exposed to HS, 4CP, and ST. To probe the relationship between teratogen-induced activation of caspase-9/caspase-3 and the activation of caspase-6/caspase-7, we used a mitochondrial-free embryo lysate with or without the addition of cytochrome c, recombinant active caspase-3, or recombinant active caspase-9.


RESULTS
Western blot analyses show that these three teratogens, HS, 4CP, and ST, induce the activation of procaspase-6 (appearance of the 13 kDa subunit, p13) and caspase-7 (appearance of the 19 kDa subunit, p19) in day 9 mouse embryos. In vitro studies showed that both caspase-6 and caspase-7 could be activated by the addition of cytochrome c to a lysate prepared from untreated embryos. In addition, caspase-6 could be activated by the addition of either recombinant caspase-3 or caspase-9 to a lysate prepared from untreated embryos. In contrast, caspase-7 could be activated by addition of recombinant caspase-3 but only minimally by recombinant caspase-9. Like caspase-9/caspase-3, caspase-6 and caspase-7 were not activated in hearts isolated from embryos exposed to these three teratogens.


CONCLUSIONS
HS, 4CP and ST induce the cleavage-dependent activation of caspase-6 and caspase-7 in day 9 mouse embryos. Results using DEVD-CHO, a caspase-3 inhibitor, suggest that teratogen-induced activation of caspase-6 is mediated by caspase-3. In addition, our data suggest that caspase-7 is activated primarily by caspase-3; however, we cannot rule out the possibility that this caspase is also activated by caspase-9. Finally, we also show that teratogen-induced activation of caspase-6 and caspase-7 are blocked in the heart, a tissue resistant to teratogen-induced cell death.
Little
S A
SA
Birth Defects Research Laboratory, Division of Genetics and Development, Department of Pediatrics, University of Washington, Seattle, WA 98195, USA.
Kim
W K
WK
Mirkes
P E
PE
D000818:Animals; D017209:Apoptosis; D001755:Blastocyst / Q000187:drug effects*; D015153:Blotting, Western; D053178:Caspase 6; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism*; D003520:Cyclophosphamide / Q000031:analogs & derivatives*; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005334:Fever; D051379:Mice; D008954:Models, Biological; D009842:Oligopeptides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D019311:Staurosporine / Q000494:pharmacology; D013723:Teratogens
D004791:Enzyme Inhibitors; D009842:Oligopeptides; D011994:Recombinant Proteins; D013723:Teratogens; C108637:aspartyl-glutamyl-valyl-aspartal; D003520:Cyclophosphamide; D045304:Cytochromes c; C505516:Casp6 protein, mouse; C505524:Casp7 protein, mouse; D053178:Caspase 6; D053179:Caspase 7; D020169:Caspases; D019311:Staurosporine; C011272:perfosfamide