Gonzalo González de Buitrago

Implication of calpain in caspase activation during B cell clonal deletion.

In the absence of costimulating signals, B cell receptor (BCR) crosslinking on immature B cells triggers the apoptotic cell death program. In the WEHI‐231 B cell lymphoma model, anti‐IgM crosslinking triggers activation of caspase‐7 independently of caspase‐8, followed by apoptosis. Two main mechanisms for caspase‐7 activation have been proposed: (i) caspase‐8 recruitment to death receptors (Fas or tumour necrosis factor); and (ii) changes in mitochondrial membrane permeability and cytochrome c release, which activate caspase‐9. Here we report that caspase‐7 activation induced by BCR crosslinking is independent of caspase‐8 and cytochrome c translocation from mitochondria to the cytosol, as well as of mitochondrial depolarization. In addition, in a cell‐free system, the S‐100 fraction of anti‐IgM‐treated WEHI‐231 cells induces a caspase activation pattern different from that activated by cytochrome c and dATP. We demonstrate that calpain specifically triggers activation and processing of caspase‐7 both in vitro and in vivo, and that both processes are inhibited by calpain inhibitors. Furthermore, calpain activation is associated with decreased expression levels of calpastatin, which is upregulated by CD40 ligation. These data confirm a role for calpain during BCR crosslinking, which may be critical for cell deletion by apoptosis during B cell development and activation.

Secreted MMP9 promotes angiogenesis more efficiently than constitutive active MMP9 bound to the tumor cell surface

Association of matrix metalloprotease 9 (MMP9) to the cell membrane is considered important in tumor growth and angiogenesis. To dissect this regulatory mechanism, we generated raft and non-raft MMP9 chimeras to force membrane expression in the MCF-7 human breast carcinoma cell line. MMP9 targeting to non-raft cell surface domains rendered a constitutive active membrane MMP9 form, suggesting a contribution by the lipid environment in MMP activation. We generated human breast cancer xenograft models using MCF-7 cells overexpressing secreted and membrane-anchored MMP9. The non-raft MMP9 chimera was constitutively active at the cell membrane in xenografts, but this activation did not correlate with an increase in MMP9-induced angiogenesis. Capillary number and vessel perimeter were specifically increased only in tumors overexpressing wild-type MMP9 (the secreted form); this increase was inhibited when tumors were induced in doxycycline-treated mice. Xenografts from tumor cells overexpressing wild-type MMP9 showed increased vascular endothelial growth factor (VEGF)/VEGFR2 receptor association, which was also dependent on MMP9 activity. These observations indicate that membrane location can influence MMP9 activity in vitro and in vivo, and confirm the relevance of stromal-associated, but not tumor-bound MMP9 in mediating tumor-induced angiogenesis.

Inhibition of programmed cell death impairs in vitro vascular-like structure formation and reduces in vivo angiogenesis

Tissue remodeling during embryonic development and in the adult organism relies on a subtle balance between cell growth and apoptosis. As angiogenesis involves restructuring of preexisting endothelium, we examined the role of apoptosis in new vessel formation. We show that apoptosis occurs before capillary formation but not after vessels have assembled. Using the human umbilical vein endothelial cell (HUVEC) in vitro Matrigel angiogenesis model, we show that vascular‐like structure formation requires apoptotic cell death through activation of a caspasedependent mechanism and mitochondrial cytochrome c release. Vascular‐like structure formation was further blocked by caspase inhibitors such as z‐VAD or AcD‐EVD‐CHO, using HUVEC and human lung microvascular endothelial cells. Overexpression of anti‐apoptotic human Bcl‐2 or baculovirus p35 genes in HUVEC altered endothelial cell rearrangement during in vitro angiogenesis, causing impaired vessel‐like structure formation. Caspase inhibitors blocked VEGF‐ or bFGF‐induced HUVEC angiogenesis on 2‐ or 3‐D collagen gels, respectively, confirming that apoptosis was not the result of nonspecific cell death after seeding on the matrix. In an in vivo angiogenesis assay, caspase inhibitors blocked VEGF‐dependent vascular formation at the alignment step, as demonstrated histologically. This evidence indicates that endothelial cell apoptosis may be relevant for precise vascular tissue rearrangement in in vitro and in vivo angiogenesis.—Segura, I., Serrano, A., González de Buitrago, G., González, M. A., Abad, J. L., Clavería, C., Gómez, L., Bernad, A., Martínez‐A, C., Riese, H. H. Inhibition of programmed cell death impairs in vitro vascular‐like structure formation and reduces in vivo angiogenesis. FASEB J. 16, 833–841 (2002)

African Swine Fever Virus IAP Homologue Inhibits Caspase Activation and Promotes Cell Survival in Mammalian Cells

ABSTRACT African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis.

Complete nucleotide sequence of the penicillin acylase gene from Kluyvera citrophila

The penicillin acylase (PAC) from Kluyvera citrophila ATCC21285 has been purified to homogeneity and found to be composed of two non-identical subunits of 23 and 62 kDa, in contrast with the previous findings [Shimizu et al., Agr. Biol. Chem. 39 (1975) 1655-1661]. The nucleotide (nt) sequence of the K. citrophila pac gene contained in the 3-kb PvuI-HindIII fragment of pKAP1 [García and Buesa, J. Biotechnol. 3 (1986) 187-195] has been determined, showing that it encodes a protein of 844 amino acid (aa) residues. The aa analysis of the N-terminal and C-terminal sequences of the purified subunits showed that they were derived from a common precursor protein of 93.5 kDa, from which a signal peptide of 26 aa, responsible for the periplasmic translocation of the protein, and an internal connecting polypeptide of 54 aa, have been removed in the maturation of the PAC. The comparison of the nt sequences of the pac genes from K. citrophila and Escherichia coli ATCC11105 [Schumacher et al., Nucl. Acids Res. 14 (1986) 5713-5727] revealed 80% homology, suggesting a common ancestral pac gene origin. The results reported here should allow investigation of the unusual mechanism of maturation of this prokaryotic protein, as well as manipulation, using DNA recombinant techniques, of the catalytic properties of this industrially important enzyme.

Caspase activation by BCR cross-linking in immature B cells: differential effects on growth arrest and apoptosis

The B cell lymphoma WEHI‐231 has been used as a model to study immature B cell tolerance, based on its capacity to undergo growth arrest and programmed cell death on B cell receptor (BCR) cross‐linking. Using this model to identify the molecular mechanisms underlying these processes, we found that BCR cross‐linking results in the selective activation of caspase 7/Mch3, but not of the other two members of the CPP32 family, caspase 2/Nedd2 and caspase 3/CPP32. This was evidenced by the induction of proteolytic activity against the substrate for the CPP32 subfamily of caspases (z‐DVED‐AMC) in vitro, as well as PARP proteolysis in vivo and by the processing of the 35 kDa Mch3 into a 32 kDa species, which was later further proteolyzed. The general caspase inhibitor z‐VAD‐fmk, but not the CPP32 family inhibitor Ac‐DEVD‐CHO, blocked anti‐μ‐induced apoptosis, indicating that a caspase not belonging to the CPP32‐like family is also implicated in anti‐μ‐triggered apoptosis. In contrast, z‐VAD‐fmk was not able to counteract growth arrest induced by anti‐μ, treatment, suggesting that caspase activation is not necessary for induction of growth arrest. Neither of the inhibitors prevented Mch3 processing; however, z‐VAD‐fmk prevented proteolysis of the p32 subunit, suggesting that further processing of this subunit is associated with apoptosis. Bcl‐2 overexpression prevented anti‐μ. induction of CPP32‐like activity and apoptosis, and blocked further processing of the Mch3 p32 subunit. In contrast, CD40 stimulation completely blocked the appearance of the p32 subunit in addition to blocking CPP32‐like activity and apoptosis induced by BCR cross‐linking. Moreover, only CD40 stimulation was able to prevent anti‐μ‐induced growth arrest, which was correlated with inhibition of retino‐blastoma and of cyclin A down‐regulation. In splenic B cells, Mch3 is also specifically proteolyzed ex vivo after induction of apoptosis by BCR cross‐linking, demonstrating the specific involvement of caspase 7/Mch3 in apoptosis induced in B cell tolerance.—Brás, A., Ruiz‐Vela, A., González de Buitrago, G., Martínez‐A., C. Caspase activation by BCR cross‐linking in immature B cells: differential effects on growth arrest and apoptosis. FASEB J. 13, 931–944 (1999)

Nuclear Apaf‐1 and cytochrome c redistribution following stress‐induced apoptosis

Apoptotic protease activating factor‐1 (Apaf‐1) and cytochrome c are cofactors critical for inducing caspase‐9 activation following stress‐induced apoptosis. One consequence of caspase‐9 activation is nuclear–cytoplasmic barrier disassembly, which is required for nuclear caspase‐3 translocation. In the nucleus, caspase‐3 triggers proteolysis of the caspase‐activated DNA nuclease (CAD) inhibitor, causing CAD induction and subsequent DNA degradation. Here we demonstrate that apoptotic cells show perinuclear cytochrome c aggregation, which may be critical for nuclear redistribution of cytochrome c and Apaf‐1. We thus indicate that the nuclear redistribution of these cofactors concurs with the previously reported caspase‐9‐induced nuclear disassembly, and may represent an early apoptotic hallmark.

Death Inducer-Obliterator 1 Triggers Apoptosis after Nuclear Translocation and Caspase Upregulation

ABSTRACT Death inducer-obliterator 1 (DIO-1) is a gene that is upregulated early in apoptosis. Here we report that in healthy cells, the DIO-1 gene product was located in the cytoplasm, where it formed oligomers. After interleukin-3 starvation or c-Myc-induced apoptosis in serum-free conditions, DIO-1 translocated to the nucleus, where it upregulated caspase levels and activity. A nuclear localization signal deletion mutant (DIO-1ΔNLS) was unable to translocate to the nuclear compartment in the absence of interleukin-3 and failed to upregulate procaspase levels or trigger cell death. In addition, cells stably expressing DIO-1ΔNLS were protected from apoptosis induced by interleukin-3 withdrawal. These results indicate that DIO-1 has a relevant role in regulating the early stages of cell death.