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Specific activation of the cysteine protease CPP32 during the negative selection of T cells in the thymus.

Alam A, Braun MY, Hartgers F, Lesage S, Cohen L, Hugo P, Denis F, Sékaly RP - J. Exp. Med. (1997)

Bottom Line: This was observed both in anti-CD3- and dexamethasone-induced apoptosis.Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD.While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Immunologie, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7, Canada.

ABSTRACT
Cysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide-MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.

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In isolated thymocytes, CPP32 is activated  during apoptosis induced by  crosslinking of CD3 or exposure  to dexamethasone. Time course  of CPP32 activation (A) and  cleavage of PARP (B) in thymocytes cultured at 4°C, or at  37°C alone, with immobilized  anti-CD3 mAb or with dexamethasone for the indicated time.  Cell lysates were electrophoresed,  and substrate cleavage was visualized by immunoblotting as described in Materials and Methods. The results presented in this  figure are representative of two  independent experiments.
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Figure 2: In isolated thymocytes, CPP32 is activated during apoptosis induced by crosslinking of CD3 or exposure to dexamethasone. Time course of CPP32 activation (A) and cleavage of PARP (B) in thymocytes cultured at 4°C, or at 37°C alone, with immobilized anti-CD3 mAb or with dexamethasone for the indicated time. Cell lysates were electrophoresed, and substrate cleavage was visualized by immunoblotting as described in Materials and Methods. The results presented in this figure are representative of two independent experiments.

Mentions: To determine whether the activation of CPP32 is a common feature of different apoptotic pathways in thymocytes, Western blots with anti-CPP32 rabbit serum were performed in two independent experiments on total cell lysates obtained from thymocyte cultures subjected to different apoptotic stimuli. As mentioned earlier, activation of CPP32 results from the proteolytic cleavage of the proenzyme form p32 into two subunits p17 and p12 (5). Results illustrated in Fig. 2 A show that anti-CD3 or dexamethasone treatments were able to activate CPP32. Indeed, the presence of the p17 subunit was noticed as early as after 12 h of culture with immobilized anti-CD3 and its abundance increased progressively thereafter. Dexamethasone induced a rapid and massive emergence of the p17 band as early as 6 h after the initiation of culture. A direct correlation between the appearance of the active form of CPP32 and the onset of apoptosis was observed for both anti-CD3 and dexamethasone treatments. In addition, the levels of activated CPP32 were directly proportional to the number of cells undergoing apoptosis (Fig. 1 and 2 A). Altogether these results show the activation of CPP32 as an integral component of these apoptotic pathways. In contrast, spontaneous cell death that occurred at 37°C did not induce detectable levels of CPP32 processing as assessed by the complete absence of the p17 subunit even upon 24 h of culture and overexposure of the autoradiogram (Fig. 2 A and data not shown). The lack of CPP32 processing during spontaneous apoptosis was not due to lower cell death than in cultures with anti-CD3. Indeed, even at equal levels of cell death for spontaneous apoptosis and for anti-CD3 treatment (after 24 and 18 h, respectively) (Fig. 1 C), CPP32 activation still distinguished anti-CD3–mediated apoptosis (Fig. 2 A).


Specific activation of the cysteine protease CPP32 during the negative selection of T cells in the thymus.

Alam A, Braun MY, Hartgers F, Lesage S, Cohen L, Hugo P, Denis F, Sékaly RP - J. Exp. Med. (1997)

In isolated thymocytes, CPP32 is activated  during apoptosis induced by  crosslinking of CD3 or exposure  to dexamethasone. Time course  of CPP32 activation (A) and  cleavage of PARP (B) in thymocytes cultured at 4°C, or at  37°C alone, with immobilized  anti-CD3 mAb or with dexamethasone for the indicated time.  Cell lysates were electrophoresed,  and substrate cleavage was visualized by immunoblotting as described in Materials and Methods. The results presented in this  figure are representative of two  independent experiments.
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Related In: Results  -  Collection

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Figure 2: In isolated thymocytes, CPP32 is activated during apoptosis induced by crosslinking of CD3 or exposure to dexamethasone. Time course of CPP32 activation (A) and cleavage of PARP (B) in thymocytes cultured at 4°C, or at 37°C alone, with immobilized anti-CD3 mAb or with dexamethasone for the indicated time. Cell lysates were electrophoresed, and substrate cleavage was visualized by immunoblotting as described in Materials and Methods. The results presented in this figure are representative of two independent experiments.
Mentions: To determine whether the activation of CPP32 is a common feature of different apoptotic pathways in thymocytes, Western blots with anti-CPP32 rabbit serum were performed in two independent experiments on total cell lysates obtained from thymocyte cultures subjected to different apoptotic stimuli. As mentioned earlier, activation of CPP32 results from the proteolytic cleavage of the proenzyme form p32 into two subunits p17 and p12 (5). Results illustrated in Fig. 2 A show that anti-CD3 or dexamethasone treatments were able to activate CPP32. Indeed, the presence of the p17 subunit was noticed as early as after 12 h of culture with immobilized anti-CD3 and its abundance increased progressively thereafter. Dexamethasone induced a rapid and massive emergence of the p17 band as early as 6 h after the initiation of culture. A direct correlation between the appearance of the active form of CPP32 and the onset of apoptosis was observed for both anti-CD3 and dexamethasone treatments. In addition, the levels of activated CPP32 were directly proportional to the number of cells undergoing apoptosis (Fig. 1 and 2 A). Altogether these results show the activation of CPP32 as an integral component of these apoptotic pathways. In contrast, spontaneous cell death that occurred at 37°C did not induce detectable levels of CPP32 processing as assessed by the complete absence of the p17 subunit even upon 24 h of culture and overexposure of the autoradiogram (Fig. 2 A and data not shown). The lack of CPP32 processing during spontaneous apoptosis was not due to lower cell death than in cultures with anti-CD3. Indeed, even at equal levels of cell death for spontaneous apoptosis and for anti-CD3 treatment (after 24 and 18 h, respectively) (Fig. 1 C), CPP32 activation still distinguished anti-CD3–mediated apoptosis (Fig. 2 A).

Bottom Line: This was observed both in anti-CD3- and dexamethasone-induced apoptosis.Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD.While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Immunologie, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7, Canada.

ABSTRACT
Cysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide-MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.

Show MeSH
Related in: MedlinePlus