Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis

Abstract

Calpains and caspases are two cysteine protease families that play important roles in regulating pathological cell death. Here, we report that m-calpain may be responsible for cleaving procaspase-12, a caspase localized in the ER, to generate active caspase-12. In addition, calpain may be responsible for cleaving the loop region in Bcl-xL and, therefore, turning an antiapoptotic molecule into a proapoptotic molecule. We propose that disturbance to intracellular calcium storage as a result of ischemic injury or amyloid beta peptide cytotoxicity may induce apoptosis through calpain- mediated caspase-12 activation and Bcl-xL inactivation. These data suggest a novel apoptotic pathway involving calcium-mediated calpain activation and cross-talks between calpain and caspase families.

Figure 1

The expression of caspase-12. (A) A Western blot of caspase-12. Anti–caspase-1 and antitubulin antibodies were used as controls. Caspase-12 is constitutively expressed in postnatal day 4 mouse tissues. (B) Caspase-12 localizes on the outer membrane of the ER. IP3R, a transmembrane protein, was readily digested by trypsin as that of caspase-12, whereas ER lumenal proteins, grp78, grp94, and calreticulin were protected against trypsin digestion. ut, untreated.

Figure 2

The requirement of calpain for caspase-12 activation in vivo. (A) Caspase-12 and Bcl-xL are cleaved in glial cells under oxygen and glucose deprivation (OGD, 9-h treatment), but not under hypoxia condition (oxygen deprivation, OD). ER stress is induced by OGD but not by OD, as indicated by the induction of BiP in OGD but not OD-treated cells. Antitubulin was used as a control (bottom). UT; untreated. (B) Caspase-12 is cleaved in glial cells treated by ER stress–inducing agents (thapsigargin, 2 μM; A23187, 2 μM), which induces ER stress as indicated by BiP induction. (C and D) The effects of pan-caspase inhibitor (zVAD), and calpain inhibitor (calpain inhibitors I, II, and E64d) in glial cells treated by OGD. Calpain inhibitors (calpain inhibitors I and II; CI-I and -II, respectively), but not z-VAD, inhibited caspase-12 and Bcl-xL cleavages (C and D). zVAD and calpain inhibitor partially protected glial cell death induced by OGD (D). Calpain activation correlates with caspase-12 cleavage activity. Endogenous calpain substrate, α-spectrin, was cleaved to 145- and 150-kD fragments after OGD treatment (D), which indicates calpain activation. (E) Fibrillar Aβ peptide induced caspase-12 cleavage, and calpain inhibitors prevented caspase-12 cleavage in primary cortical neurons. The cleavage product of caspase-12 was detected by Western blotting after 48 h of treatment with 40 μM fibrillar Aβ (25-35). Arrows indicate cleaved caspase-12 fragment. no suppl: no supplement.

Figure 2

The requirement of calpain for caspase-12 activation in vivo. (A) Caspase-12 and Bcl-xL are cleaved in glial cells under oxygen and glucose deprivation (OGD, 9-h treatment), but not under hypoxia condition (oxygen deprivation, OD). ER stress is induced by OGD but not by OD, as indicated by the induction of BiP in OGD but not OD-treated cells. Antitubulin was used as a control (bottom). UT; untreated. (B) Caspase-12 is cleaved in glial cells treated by ER stress–inducing agents (thapsigargin, 2 μM; A23187, 2 μM), which induces ER stress as indicated by BiP induction. (C and D) The effects of pan-caspase inhibitor (zVAD), and calpain inhibitor (calpain inhibitors I, II, and E64d) in glial cells treated by OGD. Calpain inhibitors (calpain inhibitors I and II; CI-I and -II, respectively), but not z-VAD, inhibited caspase-12 and Bcl-xL cleavages (C and D). zVAD and calpain inhibitor partially protected glial cell death induced by OGD (D). Calpain activation correlates with caspase-12 cleavage activity. Endogenous calpain substrate, α-spectrin, was cleaved to 145- and 150-kD fragments after OGD treatment (D), which indicates calpain activation. (E) Fibrillar Aβ peptide induced caspase-12 cleavage, and calpain inhibitors prevented caspase-12 cleavage in primary cortical neurons. The cleavage product of caspase-12 was detected by Western blotting after 48 h of treatment with 40 μM fibrillar Aβ (25-35). Arrows indicate cleaved caspase-12 fragment. no suppl: no supplement.

Figure 2

The requirement of calpain for caspase-12 activation in vivo. (A) Caspase-12 and Bcl-xL are cleaved in glial cells under oxygen and glucose deprivation (OGD, 9-h treatment), but not under hypoxia condition (oxygen deprivation, OD). ER stress is induced by OGD but not by OD, as indicated by the induction of BiP in OGD but not OD-treated cells. Antitubulin was used as a control (bottom). UT; untreated. (B) Caspase-12 is cleaved in glial cells treated by ER stress–inducing agents (thapsigargin, 2 μM; A23187, 2 μM), which induces ER stress as indicated by BiP induction. (C and D) The effects of pan-caspase inhibitor (zVAD), and calpain inhibitor (calpain inhibitors I, II, and E64d) in glial cells treated by OGD. Calpain inhibitors (calpain inhibitors I and II; CI-I and -II, respectively), but not z-VAD, inhibited caspase-12 and Bcl-xL cleavages (C and D). zVAD and calpain inhibitor partially protected glial cell death induced by OGD (D). Calpain activation correlates with caspase-12 cleavage activity. Endogenous calpain substrate, α-spectrin, was cleaved to 145- and 150-kD fragments after OGD treatment (D), which indicates calpain activation. (E) Fibrillar Aβ peptide induced caspase-12 cleavage, and calpain inhibitors prevented caspase-12 cleavage in primary cortical neurons. The cleavage product of caspase-12 was detected by Western blotting after 48 h of treatment with 40 μM fibrillar Aβ (25-35). Arrows indicate cleaved caspase-12 fragment. no suppl: no supplement.

Figure 2

The requirement of calpain for caspase-12 activation in vivo. (A) Caspase-12 and Bcl-xL are cleaved in glial cells under oxygen and glucose deprivation (OGD, 9-h treatment), but not under hypoxia condition (oxygen deprivation, OD). ER stress is induced by OGD but not by OD, as indicated by the induction of BiP in OGD but not OD-treated cells. Antitubulin was used as a control (bottom). UT; untreated. (B) Caspase-12 is cleaved in glial cells treated by ER stress–inducing agents (thapsigargin, 2 μM; A23187, 2 μM), which induces ER stress as indicated by BiP induction. (C and D) The effects of pan-caspase inhibitor (zVAD), and calpain inhibitor (calpain inhibitors I, II, and E64d) in glial cells treated by OGD. Calpain inhibitors (calpain inhibitors I and II; CI-I and -II, respectively), but not z-VAD, inhibited caspase-12 and Bcl-xL cleavages (C and D). zVAD and calpain inhibitor partially protected glial cell death induced by OGD (D). Calpain activation correlates with caspase-12 cleavage activity. Endogenous calpain substrate, α-spectrin, was cleaved to 145- and 150-kD fragments after OGD treatment (D), which indicates calpain activation. (E) Fibrillar Aβ peptide induced caspase-12 cleavage, and calpain inhibitors prevented caspase-12 cleavage in primary cortical neurons. The cleavage product of caspase-12 was detected by Western blotting after 48 h of treatment with 40 μM fibrillar Aβ (25-35). Arrows indicate cleaved caspase-12 fragment. no suppl: no supplement.

Figure 3

Cleavage of caspase-12 by calpain in vitro. (A) Cleavage of caspase-12 by cerebral cortex–soluble (S-100) proteins in a calcium-dependent manner. Caspase-12 is effectively cleaved in the presence of millimolar, but not micromolar, calcium. Activation of caspase-12 is inhibited by calcium chelators (EGTA and EDTA). (B) Caspase-12 and Bcl-xL are cleaved by purified m-calpain in vitro. (a) Caspase-12 is cleaved by m-calpain to generate three major fragments each ∼35 kD, which may autoactivate themselves to generate fragments of 20 and 10 kD (arrows), which may be the large and small subunits of caspase-12, respectively. The 20-kD fragment is detected by anti–caspase-12 p20 antibody after a longer incubation with calpain (b). (c) Bcl-xL is cleaved by m-calpain in vitro. The arrow indicates the cleaved product that was sequenced. (C) The cleavage products of caspase-12 in vivo are similar in sizes to those generated by in vitro cleavage assays (S100 and purified m-calpain). The arrows point to the procaspase-12 (arrows 1 and 2) and the cleaved products of caspase-12 (arrows 3–5). Cleaved products (arrows 3 and 5) were sequenced. Cleavage sites of 3 and 5 are T132/A133 and K158/T159, respectively. The large subunit of caspase-12 (p20) is produced in a time-dependent manner.

Figure 3

Cleavage of caspase-12 by calpain in vitro. (A) Cleavage of caspase-12 by cerebral cortex–soluble (S-100) proteins in a calcium-dependent manner. Caspase-12 is effectively cleaved in the presence of millimolar, but not micromolar, calcium. Activation of caspase-12 is inhibited by calcium chelators (EGTA and EDTA). (B) Caspase-12 and Bcl-xL are cleaved by purified m-calpain in vitro. (a) Caspase-12 is cleaved by m-calpain to generate three major fragments each ∼35 kD, which may autoactivate themselves to generate fragments of 20 and 10 kD (arrows), which may be the large and small subunits of caspase-12, respectively. The 20-kD fragment is detected by anti–caspase-12 p20 antibody after a longer incubation with calpain (b). (c) Bcl-xL is cleaved by m-calpain in vitro. The arrow indicates the cleaved product that was sequenced. (C) The cleavage products of caspase-12 in vivo are similar in sizes to those generated by in vitro cleavage assays (S100 and purified m-calpain). The arrows point to the procaspase-12 (arrows 1 and 2) and the cleaved products of caspase-12 (arrows 3–5). Cleaved products (arrows 3 and 5) were sequenced. Cleavage sites of 3 and 5 are T132/A133 and K158/T159, respectively. The large subunit of caspase-12 (p20) is produced in a time-dependent manner.

Figure 4

The cleavage sites in caspase-12 and Bcl-xL by m-calpain. (A) Caspase-12 is cleaved in at least two sites at T132/A133 and K158/T159. Bcl-xL is cleaved at one site, A60/D61. The cleavage site in Bcl-xL is close to that of caspase-1 or -3, (D61/S62 by caspase-1 and -3, D76/A77 by caspase-3; Clem et al. 1998; Fujita et al. 1998). The cleavage site of Bcl-xL is not conserved in Bcl-2. Underlines show sequence results. (B) Truncated (T159-N419) caspase-12 (t-caspase-12) expressed in bacteria is active in cleaving the wild-type caspase-12 but not D318E mutant. Caspase-12 activity is inhibited by zVAD. Arrow indicates the cleaved product of caspase-12. (C) t-Caspase-12 induced cell death in Rat1 cells. The percentage of cell death was determined by morphology 24 h after transfection. Data are averages (± SEM).