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Fas induces cytoplasmic apoptotic responses and activation of the MKK7-JNK/SAPK and MKK6-p38 pathways independent of CPP32-like proteases.

Toyoshima F, Moriguchi T, Nishida E - J. Cell Biol. (1997)

Bottom Line: Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells.These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself.Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-01, Japan.

ABSTRACT
IL-1beta converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1-like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.

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Inhibition of CPP32-like proteases did not block Fas-induced morphological changes in  KB cells. (A) KB cells were preincubated for 1 h with or without ICE-family protease inhibitors;  Z-DEVD-FK (50 μM) or Z-VAD-FK (50 μM) in the presence of 50 μg/ml cycloheximide. The  cells were then treated with anti-Fas mAb (CH-11, 150 ng/ml) for 4 and 8 h. Phase contrast images  are shown. (B) KB cells were treated as in A. After incubation for 8 h, the cells were fixed and  stained with DAPI and phalloidin. (C) KB cells were preincubated for 1 h with or without  Z-DEVD-FK (50 μM) in the presence of 50 μg/ml cycloheximide. The cells were then treated  with anti-Fas mAb (CH-11, 150 ng/ml) for 8 h, and collected and lysed. A portion of the cell lysate  was analyzed by immunoblotting with anti-actin mAb (A-4700; Sigma) that recognizes the  COOH-terminal region of actin. An arrow and an arrowhead indicate an intact actin molecule  and a 15-kD fragment of actin, respectively.
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Figure 7: Inhibition of CPP32-like proteases did not block Fas-induced morphological changes in KB cells. (A) KB cells were preincubated for 1 h with or without ICE-family protease inhibitors; Z-DEVD-FK (50 μM) or Z-VAD-FK (50 μM) in the presence of 50 μg/ml cycloheximide. The cells were then treated with anti-Fas mAb (CH-11, 150 ng/ml) for 4 and 8 h. Phase contrast images are shown. (B) KB cells were treated as in A. After incubation for 8 h, the cells were fixed and stained with DAPI and phalloidin. (C) KB cells were preincubated for 1 h with or without Z-DEVD-FK (50 μM) in the presence of 50 μg/ml cycloheximide. The cells were then treated with anti-Fas mAb (CH-11, 150 ng/ml) for 8 h, and collected and lysed. A portion of the cell lysate was analyzed by immunoblotting with anti-actin mAb (A-4700; Sigma) that recognizes the COOH-terminal region of actin. An arrow and an arrowhead indicate an intact actin molecule and a 15-kD fragment of actin, respectively.

Mentions: We next examined other apoptotic responses in KB cells. KB cells were preincubated with or without each of peptide inhibitors, Z-VAD-FK or Z-DEVD-FK, for 1 h in the presence of 50 μg/ml cycloheximide. The cells were then treated with anti-Fas mAb (CH-11, 150 ng/ml). In the absence of peptide inhibitors, dramatic morphological changes, rounding and shrinkage of the cell and membrane blebbing, were induced 3–4 h after Fas stimulation, and almost all the cells became detached from the dish and fragmented into apoptotic bodies within 8 h (Fig. 7 A, anti-Fas). In the presence of Z-VAD-FK, all of the Fas-induced cellular responses were completely inhibited (Fig. 7, A and B, Z-VAD-FK + anti-Fas). In the presence of Z-DEVD-FK, cellular shrinkage and membrane blebbing were induced by Fas stimulation, like in the absence of the inhibitor, but the cells were not detached from the dish and eventually died without any fragmentation of the cell (Fig. 7 A, Z-DEVD-FK + anti-Fas). When KB cells were fixed 8 h after Fas stimulation, and stained with DAPI and phalloidin, the Fas-induced chromatin condensation and disruption of actin network were clearly observed (Fig. 7 B, anti-Fas). Z-VAD-FK completely blocked either response; the cells exhibited normal nuclear morphology and normal actin network (Fig. 7 B, Z-VAD-FK + anti-Fas). Importantly, Z-DEVD-FK inhibited strongly the Fas-induced chromatin condensation but did not block the disruption of actin network; the cells thus showed only partial chromatin condensation and no actin network (Fig. 7 B, Z-DEVD-FK + anti-Fas). Essentially the same results were obtained with Ac-DEVD-CHO (data not shown).


Fas induces cytoplasmic apoptotic responses and activation of the MKK7-JNK/SAPK and MKK6-p38 pathways independent of CPP32-like proteases.

Toyoshima F, Moriguchi T, Nishida E - J. Cell Biol. (1997)

Inhibition of CPP32-like proteases did not block Fas-induced morphological changes in  KB cells. (A) KB cells were preincubated for 1 h with or without ICE-family protease inhibitors;  Z-DEVD-FK (50 μM) or Z-VAD-FK (50 μM) in the presence of 50 μg/ml cycloheximide. The  cells were then treated with anti-Fas mAb (CH-11, 150 ng/ml) for 4 and 8 h. Phase contrast images  are shown. (B) KB cells were treated as in A. After incubation for 8 h, the cells were fixed and  stained with DAPI and phalloidin. (C) KB cells were preincubated for 1 h with or without  Z-DEVD-FK (50 μM) in the presence of 50 μg/ml cycloheximide. The cells were then treated  with anti-Fas mAb (CH-11, 150 ng/ml) for 8 h, and collected and lysed. A portion of the cell lysate  was analyzed by immunoblotting with anti-actin mAb (A-4700; Sigma) that recognizes the  COOH-terminal region of actin. An arrow and an arrowhead indicate an intact actin molecule  and a 15-kD fragment of actin, respectively.
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Figure 7: Inhibition of CPP32-like proteases did not block Fas-induced morphological changes in KB cells. (A) KB cells were preincubated for 1 h with or without ICE-family protease inhibitors; Z-DEVD-FK (50 μM) or Z-VAD-FK (50 μM) in the presence of 50 μg/ml cycloheximide. The cells were then treated with anti-Fas mAb (CH-11, 150 ng/ml) for 4 and 8 h. Phase contrast images are shown. (B) KB cells were treated as in A. After incubation for 8 h, the cells were fixed and stained with DAPI and phalloidin. (C) KB cells were preincubated for 1 h with or without Z-DEVD-FK (50 μM) in the presence of 50 μg/ml cycloheximide. The cells were then treated with anti-Fas mAb (CH-11, 150 ng/ml) for 8 h, and collected and lysed. A portion of the cell lysate was analyzed by immunoblotting with anti-actin mAb (A-4700; Sigma) that recognizes the COOH-terminal region of actin. An arrow and an arrowhead indicate an intact actin molecule and a 15-kD fragment of actin, respectively.
Mentions: We next examined other apoptotic responses in KB cells. KB cells were preincubated with or without each of peptide inhibitors, Z-VAD-FK or Z-DEVD-FK, for 1 h in the presence of 50 μg/ml cycloheximide. The cells were then treated with anti-Fas mAb (CH-11, 150 ng/ml). In the absence of peptide inhibitors, dramatic morphological changes, rounding and shrinkage of the cell and membrane blebbing, were induced 3–4 h after Fas stimulation, and almost all the cells became detached from the dish and fragmented into apoptotic bodies within 8 h (Fig. 7 A, anti-Fas). In the presence of Z-VAD-FK, all of the Fas-induced cellular responses were completely inhibited (Fig. 7, A and B, Z-VAD-FK + anti-Fas). In the presence of Z-DEVD-FK, cellular shrinkage and membrane blebbing were induced by Fas stimulation, like in the absence of the inhibitor, but the cells were not detached from the dish and eventually died without any fragmentation of the cell (Fig. 7 A, Z-DEVD-FK + anti-Fas). When KB cells were fixed 8 h after Fas stimulation, and stained with DAPI and phalloidin, the Fas-induced chromatin condensation and disruption of actin network were clearly observed (Fig. 7 B, anti-Fas). Z-VAD-FK completely blocked either response; the cells exhibited normal nuclear morphology and normal actin network (Fig. 7 B, Z-VAD-FK + anti-Fas). Importantly, Z-DEVD-FK inhibited strongly the Fas-induced chromatin condensation but did not block the disruption of actin network; the cells thus showed only partial chromatin condensation and no actin network (Fig. 7 B, Z-DEVD-FK + anti-Fas). Essentially the same results were obtained with Ac-DEVD-CHO (data not shown).

Bottom Line: Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells.These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself.Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-01, Japan.

ABSTRACT
IL-1beta converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1-like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.

Show MeSH
Related in: MedlinePlus