Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis

Abstract

Background: Apoptosis is a form of programmed cell death that is regulated by the Bcl-2 family and caspase family of proteins. The caspase cascade responsible for executing cell death following cytochrome c release is well described; however the distinct roles of caspases-9, -3 and -7 during this process are not completely defined.

Results: Here we demonstrate several unique functions for each of these caspases during cell death. Specific inhibition of caspase-9 allows for efficient release of cytochrome c, but blocks changes in mitochondrial morphology and ROS production. We show that caspase-9 can cleave Bid into tBid at amino acid 59 and that this cleavage of Bid is required for ROS production following serum withdrawal. We also demonstrate that caspase-3-deficient MEFs are less sensitive to intrinsic cell death stimulation, yet have higher ROS production. In contrast, caspase-7-deficient MEFs are not resistance to intrinsic cell death, but remain attached to the ECM.

Conclusions: Taken together, these data suggest that caspase-9 is required for mitochondrial morphological changes and ROS production by cleaving and activating Bid into tBid. After activation by caspase-9, caspase-3 inhibits ROS production and is required for efficient execution of apoptosis, while effector caspase-7 is required for apoptotic cell detachment.

Figure 1

Bid is cleaved in a caspase-9-dependent fashion during IL-3 withdrawal and is necessary for ROS production. (A-B) FL5.12 Neo (±100 μM BocD-fmk), Bcl-x_L, Casp9DN and CrmA cells were cultured in the presence or absence of IL-3 for 24 h. (A) Bid levels were determined by western blot and viability was determined. (B) Loss of membrane potential was determined by flow cytometry (filled histogram (+)IL-3, black line (-)IL-3, grey line (-)IL-3 +BocD). (C)In vitro translated Bid, Bid^D98A, Bid^D75A and Bid^D59A were subjected to cleavage by increasing levels of caspase-9 for 90 min. Bid cleavage was determined by western blot. (D) Bid and Bid^D59A were expressed in Bid^-/- MEFs by retroviral transduction and Bid levels were determined by western blot (Bid expression from same blot). (E) Bid^-/- pBabe, Bid and Bid^D59A MEFs were withdrawn from serum for 12 hr. ROS production was determined by flow cytometry. (light grey line (+)FBS, dark grey line (-)FBS, black line (-)FBS +BocD). Treatment with 50 μg/ml antimycin A for 30 min was used as a positive control (red line). Data are representative of at least 3 independent experiments.

Figure 2

Caspase-7 and-3 have distinct effects on ROS production during apoptosis. (A) Expression levels of effector caspases in Casp KO MEFs were determined by western blot. (B) WT, Casp7^-/-, Casp3^-/- and Casp3^-/-7^-/- MEFs were withdrawn from serum for 12 hr. ROS production was determined by flow cytometry. Treatment with 50 μg/ml antimycin A for 30 min was used as a positive control. Data are representative of at least 3 independent experiments.

Figure 3

Caspase-7 and-3 have distinct functions during apoptosis. (A-B) WT, Casp7^-/-, Casp3^-/- and Casp3^-/-7^-/- MEFs were withdrawn from serum for 4 days. (A) At the indicated time points, cell death was determined by Annexin V-FITC staining. (B) At the indicated time points, percent detachment was determined by separating non-adherent from adherent cells and counting with a hemocytometer. Data are presented as the mean ± SEM of at least 3 independent experiments. (C) Casp7^−/− MEFs were grown on glass coverslips for 24 hr and then were washed with PBS and the medium was replaced with full medium or serum free medium for 48 hr. After, cells were fixed, stained for actin and DNA and visualized by fluorescent microscopy.

Figure 4

Reconstitution of caspase-deficient MEFs with caspase-3 or caspase-7 rescues the WT phenotype. (A) Caspase-deficient MEFs were reconstituted with the appropriate caspase by retroviral transduction and caspase expression was determined by western blot. (B) WT, Casp3^-/- pBabe and Casp3^-/- C3 MEFs were withdrawn from serum for 4 days. At the indicated time points, cell death was determined by Annexin V-FITC staining. Data are presented as the mean ± SEM of at least 3 independent experiments. (C) WT, Casp7^-/- pBabe and Casp7^-/- C7 MEFs were withdrawn from serum for 4 days. At the indicated time points, percent detachment was determined by separating non-adherent from adherent cells and counting with a hemocytometer. Data are presented as the mean ± SEM of at least 3 independent experiments.

Figure 5

Model of caspase activation downstream of cytochrome c release during apoptosis. Cell death signals induce MOMP, which leads to cytochrome c release and the activation of caspase-9. Caspase-9 can cleave and activate Bid, caspase-7 and caspase-3. tBid can remodel the mitochondria and make conditions favorable for ROS production, which is enhanced by caspase-7 and inhibited by caspase-3.