Caspase-3 mediated cleavage of HsRad51 at an unconventional site.

The human recombinase HsRad51 is cleaved during apoptosis. We have earlier observed cleavage of the 41-kDa full-length protein into a 33-kDa product in apoptotic Jurkat cells and in in vitro translated HsRad51 after treatment with activated S-100 extract. In this study, site-directed mutagenesis was used for mapping of the cleavage site to AQVD274 downward arrow G, which does not correspond to a conventional caspase cleavage site. The absence of HsRad51 cleavage in staurosporine-treated apoptotic MCF-7 cells, which lack caspase-3, indicates that caspase-3 is essential for HsRad51 cleavage in vivo. Cleavage into the 33-kDa fragment was generated by recombinant caspase-3 and -7 in in vitro translated wild type HsRad51, but not in the HsRad51 AQVE274 downward arrow G mutant. Similarly, HsRad51 of Jurkat cell extracts was cleaved into the 33-kDa product by recombinant caspase-3, whereas caspase-7 failed to cleave endogenous HsRad51. The cleavage of in vitro translated wild type and AQVE274 downward arrow G mutant HsRad51 as well as of endogenous HsRad51 also gave rise to a smaller fragment, which corresponds in size to a recently reported DVLD187 downward arrow N HsRad51 cleavage product. In Jurkat cell extracts, the AQVD274 downward arrow G and DVLD187 downward arrow N cleavage products of HsRad51 appeared at equal concentrations of caspase-3. Moreover both fragments were generated by induction of apoptosis in MDA-MB 157 cells with staurosporine and in Jurkat cells with camptothecin. Thus, two sites in the HsRad51 sequence are targets for caspase cleavage both in vitro and in vivo.     

Protein kinase Cepsilon activates protein kinase B/Akt via DNA-PK to protect against tumor necrosis factor-alpha-induced cell death

We have previously shown that protein kinase Cepsilon (PKCepsilon) protects breast cancer cells from tumor necrosis factor-alpha (TNF)-induced cell death. In the present study, we have investigated if the antiapoptotic function of PKCepsilon is mediated via Akt and the mechanism by which PKCepsilon regulates Akt activity. TNF caused a transient increase in Akt phosphorylation at Ser473 in MCF-7 cells. Overexpression of PKCepsilon in MCF-7 cells increased TNF-induced Akt phosphorylation at Ser473 resulting in its activation. Knockdown of PKCepsilon by small interfering RNA (siRNA) decreased TNF-induced Akt phosphorylation/activation and increased cell death. Introduction of constitutively active Akt protected breast cancer MCF-7 cells from TNF-mediated cell death and partially restored cell survival in PKCepsilon-depleted cells. Depletion of Akt in MCF-7 cells abolished the antiapoptotic effect of PKCepsilon on TNF-mediated cell death. Akt was constitutively associated with PKCepsilon and DNA-dependent protein kinase (DNA-PK), and this association was increased by TNF treatment. Overexpression of PKCepsilon enhanced the interaction between Akt and DNA-PK. Knockdown of DNA-PK by siRNA inhibited TNF-induced Akt phosphorylation and the antiapoptotic effect of Akt and PKCepsilon. These results suggest that PKCepsilon activates Akt via DNA-PK to mediate its antiapoptotic function. Furthermore, we report for the first time that DNA-PK can regulate receptor-initiated apoptosis via Akt.     

Caspase-3-mediated processing of poly(ADP-ribose) glycohydrolase during apoptosis

Poly(ADP-ribose) glycohydrolase (PARG) is responsible for the catabolism of poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerase (PARP-1) and other PARP-1-like enzymes. In this work, we report that PARG is cleaved during etoposide-, staurosporine-, and Fas-induced apoptosis in human cells. This cleavage is concomitant with PARP-1 processing and generates two C-terminal fragments of 85 and 74 kDa. In vitro cleavage assays using apoptotic cell extracts showed that a protease of the caspase family is responsible for PARG processing. A complete inhibition of this cleavage was achieved at nanomolar concentrations of the caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting the involvement of caspase-3-like proteases. Consistently, recombinant caspase-3 efficiently cleaved PARG in vitro, suggesting the involvement of this protease in PARG processing in vivo. Furthermore, caspase-3-deficient MCF-7 cells did not show any PARG cleavage in response to staurosporine treatment. The cleavage sites identified by site-directed mutagenesis are DEID(256) downward arrow V and the unconventional site MDVD(307) downward arrow N. Kinetic studies have shown similar maximal velocity (V(max)) and affinity (K(m)) for both full-length PARG and its apoptotic fragments, suggesting that caspase-3 may affect PARG function without altering its enzymatic activity. The early cleavage of both PARP-1 and PARG by caspases during apoptosis suggests an important function for poly(ADP-ribose) metabolism regulation during this cell death process.     

Downregulation of Bid is associated with PKCepsilon-mediated TRAIL resistance

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent as it selectively kills tumor cells but spares normal cells. Resistance to TRAIL by tumor cells limits its therapeutic use. We have previously shown that protein kinase C-epsilon (PKCepsilon) acts as an antiapoptotic protein in MCF-7 breast cancer cells. In the present study, we have investigated the mechanism(s) by which PKCepsilon contributes to TRAIL resistance. Overexpression of PKCepsilon inhibited caspase-8 and -9 activation, release of mitochondrial cytochrome c and cell death induced by TRAIL, but did not interfere with the recruitment of caspase-8 to the death-inducing signaling complex. Knockdown/inhibition of PKCepsilon resulted in enhanced sensitivity to TRAIL. The level of Bcl-2 was increased and Bid was decreased by PKCepsilon at both the protein and mRNA level but PKCepsilon had no effect on Bax. Knockdown of Bcl-2 by siRNA reversed TRAIL resistance in PKCepsilon-overexpressing cells, whereas depletion of Bid contributed to TRAIL resistance in MCF-7 cells. A decrease in Bid content was also associated with inhibition of TRAIL-induced caspase-8 activation. Furthermore, PKCepsilon depletion or overexpression of DN-PKCepsilon was associated with a decrease in Bcl-2 protein level. Thus, our results suggest that PKCepsilon acts upstream of mitochondria and mediates TRAIL resistance via both Bcl-2 and Bid in MCF-7 cells.     

Prostate apoptosis response 4 (Par-4), a novel substrate of caspase-3 during apoptosis activation

Prostate apoptosis response 4 (Par-4) is a ubiquitously expressed proapoptotic tumor suppressor protein. Here, we show for the first time, that Par-4 is a novel substrate of caspase-3 during apoptosis. We found that Par-4 is cleaved during cisplatin-induced apoptosis in human normal and cancer cell lines. Par-4 cleavage generates a C-terminal fragment of ~25 kDa, and the cleavage of Par-4 is completely inhibited by a caspase-3 inhibitor, suggesting that caspase-3 is directly involved in the cleavage of Par-4. Caspase-3-deficient MCF-7 cells do not show Par-4 cleavage in response to cisplatin treatment, and restoration of caspase-3 in MCF-7 cells produces a decrease in Par-4 levels, with the appearance of a cleaved fragment. Additionally, knockdown of Par-4 reduces caspase-3 activation and apoptosis induction. Site-directed mutagenesis reveals that Par-4 cleavage by caspase-3 occurs at an unconventional site, EEPD(131)↓G. Interestingly, overexpression of wild-type Par-4 but not the Par-4 D131A mutant sensitizes cells to cisplatin-induced apoptosis. Upon caspase-3 cleavage, the cleaved fragment of Par-4 accumulates in the nucleus and displays increased apoptotic activity. Overexpression of the cleaved fragment of Par-4 inhibits IκBα phosphorylation and blocks NF-κB nuclear translocation. We have identified a novel specific caspase-3 cleavage site in Par-4, and the cleaved fragment of Par-4 retains proapoptotic activity.     

Selective degradation of the PKC-epsilon isoform during cell death in AKR-2B fibroblasts

The protein kinase C (PKC) family of serine/threonine protein kinases is involved in intracellular signals that regulate growth, differentiation, and apoptosis. AKR-2B cells express the PKC isoforms alpha, gamma, epsilon, lambda, mu, und zeta (J. Hoppe, R. Sch?fer, V. Hoppe, and A. Sachinidis, Cell Death Differ. 6, 546-556). Here we show that during serum starvation only PKC-epsilon was cleaved. An N-terminal fragment of 42 kDa remained associated with subcellular components, presumably the Golgi apparatus. The C-terminal part (catalytic domain) was further degraded and was no longer detectable in vivo. As published before, the activation of the DEVDase in AKR-2B cells is prevented by numerous agents like PDGF, TPA, and DEVD.cmk (R. Sch?fer, D. Karbach, and J. Hoppe, Exp. Cell Res. 240, 28--39). All these agents completely prevented PKC-epsilon cleavage, indicating a tight correlation between DEVDase activity and PKC-epsilon cleavage. By using recombinant caspase-3 or highly purified DEVDase from cytosolic extracts we localized by Edman degradation the cleavage site in recombinant PKC-epsilon to asp383 in the hinge region between regulatory and catalytic domains. The corresponding tetrapeptide sequences SSPD and SATD for human and mouse PKC-epsilon, respectively, are unusual for caspase-3. Expression of the catalytic domain or of the cleavage-resistant mutant D383A had no effect on cell death in AKR-2B cells.     

Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2.

Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.     

Caspase-3-mediated cleavage of Akt: involvement of non-consensus sites and influence of phosphorylation

Here, we show for the first time that Akt1 is cleaved in vitro at the caspase-3 consensus site DQDD(456) downward arrow SM. Our data suggest QEEE(116) downward arrow E(117) downward arrow MD, EEMD(119) downward arrow, TPPD(453) downward arrow QD and DAKE(398) downward arrow IM as novel non-consensus caspase-3 cleavage sites. More importantly, we demonstrate that phosphorylation of Akt1 modulates its cleavage in a site-specific manner: Resistance to cleavage at site DAKE(398) (within the kinase domain) in response to phosphorylation suggests a possible mechanism by which the anti-apoptotic role of Akt1 is regulated. Our result is important in biological models which rely on Akt1 for cell survival.     

Caspase-cleaved TRAF1 negatively regulates the antiapoptotic signals of TRAF2 during TNF-induced cell death

Tumor necrosis factor (TNF) signaling leads to pleiotropic responses in a wide range of cell types, in part by activating antiapoptotic and proapoptotic pathways. Previous studies have suggested that TNF receptor-associated factor (TRAF) 2 can mediate crucial antiapoptotic signals during TNF stimulation. However, it is unclear how the antiapoptotic signals via TRAF2 in TNF-R1 signaling is regulated. Here we show that TRAF1 is cleaved by caspase-8 into two fragments during apoptosis induced by TNF. Overexpression of the C-terminal cleavage product, TRAF1-c, increased TNF-induced cell death of hybridoma T cells. Importantly, we demonstrate that the cleavage product of TRAF1 coimmunoprecipitates with TRAF2 that is released from the TNF-R1 complex in response to prolonged TNF treatment. These results indicate that caspase-dependent cleavage of TRAF1 generates TRAF1-c fragments that are able to bind TRAF2, and then sequester TRAF2 from the TNF-R1 complex, rendering cells, at least in part, sensitive to TNF.     

Structural determinants of procryptdin recognition and cleavage by matrix metalloproteinase-7

The bactericidal activity of mouse Paneth cell alphadefensins, or cryptdins, is dependent on processing of cryptdin precursors (pro-Crps) by matrix metalloproteinase-7 (MMP-7) (Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., Lopez-Boado, Y. S., Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and Parks, W. C. (1999) Science 286, 113-117). To investigate the mechanisms of pro-Crp processing by this enzyme, recombinant pro-Crp4, a His-tagged chimeric pro-Crp (pro-CC), and site-directed mutant precursors of each were digested with MMP-7, and the cleavage products were analyzed by NH(2)-terminal peptide sequencing. Proteolysis of pro-Crp4 with MMP-7 activated in vitro bactericidal activity to the level of the mature Crp4 peptide by cleaving pro-Crp4 at Ser(43) downward arrow Ile(44) and Ala(53) downward arrow Leu(54) in the proregion and near the Crp4 peptide NH(2) terminus between Ser(58) downward arrow Leu(59). Because the Crp4 NH(2) terminus occurs at Gly(61), not Leu(59), MMP-7 is necessary but insufficient to complete the processing of Crp4. Crp activating proteolysis at S58 downward arrow L59 was unaffected by I44S/I44D or L54S/L54D loss-of-function mutations in pro-Crp4, and a (L59S)-pro-CC mutant was cleaved normally at Ser(43) downward arrow Val(44) and Ser(53) downward arrow Leu(54) sites but not at the peptide NH(2) terminus. C57BL/6 mice contain an abundant (L59S)-Crp4 mutant peptide with Leu(54) at its NH(2) terminus resulting from Ala(53) downward arrow Leu(54) cleavage and loss-of-function at the Ser(58) downward arrow Ser(59) cleavage site. Thus, alpha-defensins resulting from mutations at MMP-7 cleavage sites exist in mouse populations. A pro-CC substrate containing both L54S and L59S mutations resisted cleavage at Ser(43) downward arrow Val(44) completely, showing that cleavage at one or both downstream sites must precede proteolysis at Ser(43) downward arrow Val(44). These findings show that MMP-7 activation of pro-Crps can occur without proteolysis of the proregion, and prosegment fragmentation depends, at least in part, on the release of the Crp peptide from the precursor.     

Caspase-3 is necessary and sufficient for cleavage of protein synthesis eukaryotic initiation factor 4G during apoptosis.

Induction of apoptosis BJAB cells is accompanied by the rapid cleavage of protein synthesis eukaryotic initiation factor 4G and the appearance of a fragment of approximately 76 kDa. Inhibition of apoptotic proteases (caspases) has previously been shown to prevent the cleavage of eukaryotic initiation factor 4G. In MCF-7 breast carcinoma cells, which are deficient in caspase-3, eukaryotic initiation factor 4G is not cleaved but in vivo expression of caspase-3 restores eukaryotic initiation factor 4G cleavage following induction of apoptosis. Recombinant caspase-3 can also cleave eukaryotic initiation factor 4G to yield the 76 kDa fragment both in cell extracts and when the eukaryotic initiation factor 4G is presented in a purified eukaryotic initiation factor 4F complex. These results indicate that caspase-3 activity is necessary and sufficient for eukaryotic initiation factor 4G degradation.     

Altered Processing of Amyloid Precursor Protein in Cells Undergoing Apoptosis

Altered proteolysis of amyloid precursor protein is an important determinant of pathology development in Alzheimer''s disease. Here, we describe the detection of two novel fragments of amyloid precursor protein in H4 neuroglioma cells undergoing apoptosis. Immunoreactivity of these 25–35 kDa fragments to two different amyloid precursor protein antibodies suggests that they contain the amyloid-β region and an epitope near the C-terminus of amyloid precursor protein. Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases. Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results. Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism. Molecular weight prediction and immunoreactivity of the fragments generated suggested that such fragments could not be generated by cleavage at any previously identified caspase, secretase, or calpain site on amyloid precursor protein. Bioinformatic analysis of the amino acid sequence of amyloid precursor protein revealed that fragments fitting the observed size and immunoreactivity could be generated by either cleavage at a novel, hitherto unidentified, caspase site or at a previously identified matrix metalloproteinase site in the extracellular domain. Proteolytic cleavage at any of these sites leads to a decrease in the generation of α-secretase cleaved secreted APP, which has both anti-apoptotic and neuroprotective properties, and thus may contribute to neurodegeneration in Alzheimer''s disease.     

Caspase-3 is required in the apoptotic disintegration of the nuclear matrix

Apoptotic breakdown of cellular structures is largely mediated by caspases. One target of degradation is a proteinaceous framework of the nucleus termed the nuclear matrix. We compared the apoptotic changes of the nuclear matrix in staurosporine-treated caspase-3-deficient MCF-7 cells transfected with intact CASP-3 gene (MCF-7c3) or an empty vector (MCF-7v) as a control. Nuclear Mitotic Apparatus protein (NuMA), lamin A/C and lamin B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. In both cell lines, staurosporine induced rapid cytoplasmic shrinkage and partial chromatin condensation. MCF-7c3 cells formed apoptotic bodies, whereas MCF-7v cells did not. NuMA and lamins were actively cleaved in MCF-7c3 cells following caspase-3 activation, but only minimal or no cleavage was detected in MCF-7v cells. Interestingly, lamin B but not lamin A/C was relocated into cytoplasmic granules in apoptotic MCF-7v cells. Pancaspase inhibitor, z-VAD-fmk, prevented the apoptotic changes, while caspase-3 inhibitor, z-DEVD-fmk, induced lamin B granules in both cell lines. These results show that caspase-3 is involved in the cleavage of NuMA and lamins either directly or by activating other proteases. This may be essential for disintegration of the nuclear structure during apoptosis.     

Activation of atypical protein kinase C zeta by caspase processing and degradation by the ubiquitin-proteasome system

Atypical protein kinase C zeta (PKCzeta) is known to transduce signals that influence cell proliferation and survival. Here we show that recombinant human caspases can process PKCzeta at three sites in the hinge region between the regulatory and catalytic domains. Caspase-3, -6, -7, and -8 chiefly cleaved human PKCzeta at EETD downward arrowG, and caspase-3 and -7 also cleaved PKCzeta at DGMD downward arrowG and DSED downward arrowL, respectively. Processing of PKCzeta expressed in transfected cells occurred chiefly at EETD downward arrowG and DGMD downward arrowG and produced carboxyl-terminal polypeptides that contained the catalytic domain. Epitope-tagged PKCzeta that lacked the regulatory domain was catalytically active following expression in HeLa cells. Induction of apoptosis in HeLa cells by tumor necrosis factor alpha plus cycloheximide evoked the conversion of full-length epitope-tagged PKCzeta to two catalytic domain polypeptides and increased PKCzeta activity. A caspase inhibitor, zVAD-fmk, prevented epitope-tagged PKCzeta processing and activation following the induction of apoptosis. Induction of apoptosis in rat parotid C5 cells produced catalytic domain polypeptides of endogenous PKCzeta and increased PKCzeta activity. Caspase inhibitors prevented the increase in PKCzeta activity and production of the catalytic domain polypeptides. Treatment with lactacystin, a selective inhibitor of the proteasome, caused polyubiquitin-PKCzeta conjugates to accumulate in cells transfected with the catalytic domain or full-length PKCzeta, or with a PKCzeta mutant that was resistant to caspase processing. We conclude that caspases process PKCzeta to carboxyl-terminal fragments that are catalytically active and that are degraded by the ubiquitin-proteasome pathway.     

Notch1 antiapoptotic activity is abrogated by caspase cleavage in dying T lymphocytes

Excessive signaling via the Notch1 receptor inhibits apoptosis in T lymphocytes. Since several antiapoptotic proteins are cleaved by caspases during cell death, we investigated whether Notch1 was a caspase substrate. Results demonstrate that the intracellular domain of Notch1 (NICD) is cleaved into six fragments during apoptosis in Jurkat cells or peripheral T lymphocytes. Notch1 cleavage is prevented by the caspase inhibitors DEVD-fmk and VEID-fmk or by Bcl-2 expression. Caspase-3 and caspase-6 cleave the NICD into six fragments using sites located within the NF-kappaB binding domain, the ankyrin repeats and the transactivation domain. Notch1 cleavage correlates with the loss of HES-1 expression in apoptotic T cells. Notch1 fragments cannot inhibit activation-induced cell death in a T-cell hybridoma, confirming the abrogation of Notch1 antiapoptotic activity by caspases. The ability of the NICD but not the fragments to antagonize Nur77 activity supports a role for this factor in Notch1 antiapoptotic function.     

Hyperthermia engages the intrinsic apoptotic pathway by enhancing upstream caspase activation to overcome apoptotic resistance in MCF-7 breast adenocarcinoma cells

Febrile hyperthermia enhanced TNF-stimulated apoptosis of MCF-7 cells and overcame resistance in a TNF-resistant, MCF-7 variant (3E9), increasing their TNF-sensitivity by 10- and 100-fold, respectively. In either cell line, the hyperthermic potentiation was attributable to increased apoptosis that was totally quenched by caspase inhibition. In MCF-7 cells, hyperthermic potentiation of apoptosis was associated with sustained activation of upstream caspases in response to TNF and more prominent engagement of the intrinsic apoptotic pathway. Apoptotic enhancement by hyperthermia was primarily mediated by caspase-8 activation, as the specific inhibitor, Z-IETD, blocked cell death, whereas direct engagement of the intrinsic apoptotic pathway (with doxorubicin) was not affected. In 3E9 cells, hyperthermia alone induced activation of caspase-8, and was further enhanced by TNF. In 3E9 cells, hyperthermia caused TNF-dependent loss of mitochondrial membrane potential and activation of capspase-9 that was initiated and dependent on upstream caspases. MCF-7 and 3E9 cells were equally sensitive to exogenous C(6)-ceramide, but mass spectroscopic analysis of ceramide species indicated that total ceramide content was not enhanced by TNF and/or hyperthermia treatment, and that the combination of TNF and hyperthermia caused only modest elevation of one species (dihydro-palmitoyl ceramide). We conclude that febrile hyperthermia potentiates apoptosis of MCF-7 cells and overcomes TNF-resistance by sustained activation of caspase-8 and engagement of the intrinsic pathway that is independent of ceramide flux. This report provides the first evidence for regulation of caspase-dependent apoptosis by febrile hyperthermia.     

Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes

We previously demonstrated that tumour necrosis factor (TNF)-induced ceramide production by endosomal acid sphingomyelinase (A-SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase-9 and caspase-3 activation. The mechanism of TNF-mediated A-SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF-induced A-SMase activation depends on functional caspase-8 and caspase-7 expression. The active forms of all three enzymes, caspase-8, caspase-7 and A-SMase, but not caspase-3, colocalize in internalized TNF receptosomes. While caspase-8 and caspase-3 are unable to induce activation of purified pro-A-SMase, we found that caspase-7 mediates A-SMase activation by direct interaction resulting in proteolytic cleavage of the 72-kDa pro-A-SMase zymogen at the non-canonical cleavage site after aspartate 253, generating an active 57 kDa A-SMase molecule. Caspase-7 down modulation revealed the functional link between caspase-7 and A-SMase, confirming proteolytic cleavage as one further mode of A-SMase activation. Our data suggest a signalling cascade within TNF receptosomes involving sequential activation of caspase-8 and caspase-7 for induction of A-SMase activation by proteolytic cleavage of pro-A-SMase.     

LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway

We previously showed (Gastroenterology 123: 206-216, 2002) that lysophosphatidic acid (LPA) protects and rescues rat intestinal epithelial cells (IEC-6) from apoptosis. Here, we provide evidence for the LPA-elicited inhibition of the mitochondrial apoptotic pathway leading to attenuation of caspase-3 activation. Pretreatment of IEC-6 cells with LPA inhibited campothecin-induced caspase-9 and caspase-3 activation and DNA fragmentation. A caspase-9 inhibitor peptide mimicked the LPA-elicited antiapoptotic activity. LPA elicited ERK1/ERK2 and PKB/Akt phosphorylation. The LPA-elicited antiapoptotic activity and inhibition of caspase-9 activity were abrogated by pertussis toxin, PD 98059, wortmannin, and LY 294002. LPA reduced cytochrome c release from mitochondria and prevented activation of caspase-9. LPA prevented translocation of Bax from cytosol to mitochondria and increased the expression of the antiapoptotic Bcl-2 mRNA and protein. LPA had no effect on Bcl-xl, Bad, and Bak mRNA or protein expression. These data indicate that LPA protects IEC-6 cells from camptothecin-induced apoptosis through G(i)-coupled inhibition of caspase-3 activation mediated by the attenuation of caspase-9 activation due to diminished cytochrome c release, involving upregulation of Bcl-2 protein expression and prevention of Bax translocation.     

Reduced cellular Ca availability enhances TDP-43 cleavage by apoptotic caspases

Accumulation of transactive response DNA binding protein (TDP-43) fragments in motor neurons is a post mortem hallmark of different neurodegenerative diseases. TDP-43 fragments are the products of the apoptotic caspases-3 and -7. Either excessive or insufficient cellular Ca²⁺ availability is associated with activation of apoptotic caspases. However, as far as we know, it is not described whether activation of caspases, due to restricted intracellular Ca²⁺, affects TDP-43 cleavage. Here we show that in various cell lineages with restricted Ca²⁺ availability, TDP-43 is initially cleaved by caspases-3 and -7 and then, also by caspases-6 and -8 once activated by caspase-3. Furthermore, we disclose the existence of a TDP-43 caspase-mediated fragment of 15 kDa, in addition to the well-known fragments of 35 and 25 kDa. Interestingly, with respect to the other two fragments this novel fragment is the major product of caspase activity on murine TDP-43 whereas in human cell lines the opposite occurs. This outcome should be considered when murine models are used to investigate TDP-43 proteinopathies.     

Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c.

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.