Eiichi Tani

Proteasome inhibitors induce mitochondria-independent apoptosis in human glioma cells

The proteasome inhibitors lactacystin and AcLLNal induced p53‐independent apoptosis in two human glioma cell lines, and the apoptosis was accompanied by up‐regulation of immunoreactive wild‐type p53, p21Waf1, Mdm2, and p27Kip1. Pretreatment with cycloheximide decreased the induction of cell death independently of p53 protein status, suggesting that the up‐regulation of short‐lived proteins is associated with proteasome inhibitor‐induced apoptosis. Caspase‐3‐like proteases were activated in the proteasome inhibitor‐mediated apoptosis, and the induction of cell death was inhibited more effectively in the presence of z‐VAD.fmk than in the presence of Ac‐DEVD.fmk, suggesting that caspases other than caspase‐3 are involved. Nonetheless, there were no significant alterations in levels of immunoreactive Bcl‐2, Bcl‐xL, Bax, Bad, and Bak, nor any evidence of cytochrome c release into cytosol and dissipation of ΔΨ m. Thus, the proteasome inhibitor‐induced apoptosis is mediated by a mitochondria‐independent mechanism, and the once activated caspase‐3 does not cause the cytochrome c release and the ΔΨ m disruption.

Activation of Stress-activated Protein Kinase/c-Jun NH2-terminal Kinase and p38 Kinase in Calphostin C-induced Apoptosis Requires Caspase-3-like Proteases but Is Dispensable for Cell Death

Apoptosis was induced in human glioma cell lines by exposure to 100 nm calphostin C, a specific inhibitor of protein kinase C. Calphostin C-induced apoptosis was associated with synchronous down-regulation of Bcl-2 and Bcl-xL as well as activation of caspase-3 but not caspase-1. The exposure to calphostin C led to activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) and p38 kinase and concurrent inhibition of extracellular signal-regulated kinase (ERK). Upstream of ERK, Shc was shown to be activated, but its downstream Raf1 and ERK were inhibited. The pretreatment with acetyl-Tyr-Val-Ala-Asp-aldehyde, a relatively selective inhibitor of caspase-3, or benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk), a broad spectrum caspase inhibitor, similarly inhibited calphostin C-induced activation of SAPK/JNK and p38 kinase as well as apoptotic nuclear damages (chromatin condensation and DNA fragmentation) and cell shrinkage, suggesting that caspase-3 functions upstream of SAPK/JNK and p38 kinase, but did not block calphostin C-induced surface blebbing and cell death. On the other hand, the inhibition of SAPK/JNK by transfection of dominant negative SAPK/JNK and that of p38 kinase by SB203580 induced similar effects on the calphostin C-induced apoptotic phenotypes and cell death as did z-VAD.fmk and acetyl-Tyr-Val-Ala-Asp-aldehyde, but the calphostin C-induced PARP cleavage was not changed, suggesting that SAPK/JNK and p38 kinase are involved in the DNA fragmentation pathway downstream of caspase-3. The present findings suggest, therefore, that the activation of SAPK/JNK and p38 kinase is dispensable for calphostin C-mediated and z-VAD.fmk-resistant cell death.

Three Distinct Phases of Fodrin Proteolysis Induced in Postischemic Hippocampus: Involvement of Calpain and Unidentified Protease

BACKGROUND AND PURPOSE Fodrin, a neuronal cytoskeleton protein, is proteolyzed by calpain after ischemic insult. We examined proteolysis of fodrin induced by global forebrain ischemia in gerbil hippocampus in spatial terms by using the antibody specific to the calpain-proteolyzed form of fodrin. METHODS In gerbils, a 10-minute forebrain ischemia was produced by occlusion of both carotid arteries. After recirculation, the hippocampus was processed for immunohistochemical and immunoblot study with the antibody against the calpain-proteolyzed form of fodrin. Additionally, short-term ischemia was studied to find the threshold of fodrin proteolysis. RESULTS Three phases of fodrin proteolysis distinct in chronology and distribution arose: (1) an early predegeneration phase in the molecular layer and stratum oriens of the CA1 and CA3 sectors within the first 15 minutes, which lasted up to 4 hours; (2) a late predegeneration phase in the whole CA1 sector, except for the pyramidal cells, between 12 hours and 2 days; and (3) a postdegeneration phase in the cytoplasm of the CA1 neurons, which arose in 3 to 7 days. A 4-minute (not a 3-minute) forebrain ischemia induced the late predegeneration phase of fodrin proteolysis and delayed neuronal death in CA1. Immunoblotting showed that the primary product of calpain action was further proteolyzed by an unidentified protease. CONCLUSIONS Calpain induced proteolysis of fodrin in ischemic hippocampus, and the late predegeneration phase of the proteolysis was closely associated with the delayed neuronal death in the CA1 sector. Calpain and another protease may play a role in the development of neuronal death after transient forebrain ischemia.

Cytotoxic Fragment of Amyloid Precursor Protein Accumulates in Hippocampus After Global Forebrain Ischemia

We developed an antibody specific to β-amyloid precursor protein (βAPP) fragments possessing the exact amino terminus of the β-amyloid peptide and examined its induction in postischemic hippocampus. In control hippocampus, this APP fragment was lightly observed in pyramidal neurons of CA sectors and dentate granule cells. Transient forebrain ischemia enhanced accumulation of the APP fragment in CA1 pyramidal neurons. Seven days after the ischemia, while the APP fragment was still observed in dentate granule cells and CA3 neurons, it disappeared in dead CA1 neurons. While astrocytes did not show in any immunoreactivity throughout the experiment, those in the CA1 sector showed moderate immunoreactivity 7 days after the ischemia. The APP fragment has a cytotoxic effect on cultured neurons. These results suggest that the accumulation of the cytotoxic APP fragment in CA1 neurons may play a role in the development of delayed neuronal death after the ischemic insult.

Immunohistochemical study of fibronectin in human glioma and meningioma

SummaryThe presence of fibronectin (FN) and glial fibrillary acidic protein (GFAP) in two astrocytomas, 17 glioblastomas, and five meningiomas was studied by indirect immunoproxidase staining of formalin-fixed and paraffin-embedded surgical specimens. Angiogenesis in tumor was scored by the microscopic angiogenesis grading system (MAGS), and plasma FN levels were measured by single radial immunodiffusion. In astrocytomas and glioblastomas, GFAP-positive tumor cells had no FN expression and FN was confined to proliferating vessel walls and the leptomeninges, showing a mutually exclusive FN and GFAP expression. GFAP-positive tumor cells were occasionally surrounded by a network of FN-positive matrix produced by cells derived from the leptomeninges or blood vessels. In meningiomas, FN expression was found in vessel walls and meningioma cells including whorl formations and psammoma bodies. In general, deep immunoperoxidase staining for FN was shown in the endothelial cells and the psammoma bodies. Plasma FN levels were correlated significantly not to the degree of leptomeningeal proliferation but to the MAGS scores in gliomas.

Calpain inhibitor entrapped in liposome rescues ischemic neuronal damage

Transient forebrain ischemia induces activation of calpain and proteolysis of a neuronal cytoskeleton, fodrin, in gerbil hippocampus. This phenomenon precedes delayed neuronal death in hippocampal CA1 neurons. We examined effects of a calpain inhibitor on delayed neuronal death after transient forebrain ischemia. In gerbils, a selective calpain inhibitor entrapped in liposome was given transvenously and 30 min later, 5-min forebrain ischemia was produced by occlusion of both common carotid arteries. On day 7, CA1 neuronal damage was examined in the hippocampal slices stained with cresyl violet. Calpain-induced proteolysis of fodrin was also examined by immunohistochemistry and immunoblot. Additionally, to assure entrapment of the inhibitor by CA1 neurons, the inhibitor-liposome complex was labeled with FITC and given to gerbils. Fluorescence in the hippocampal slices was examined by confocal laser scanning microscope. Selective CA1 neuronal damage induced by forebrain ischemia was prevented by administration of the inhibitor in a dose-dependent manner. Calpain-induced proteolysis of fodrin was also extinguished by the calpain inhibitor in a dose-dependent manner. Bright fluorescence of the FITC-labeled inhibitor was observed in the CA1 neurons. The data show an important role of calpain in the development of the ischemic delayed neuronal death. Calpain seems to produce neuronal damage by degrading neuronal cytoskeleton. Our data also show a palliative effect of the calpain inhibitor on the neurotoxic damage, which offers a new and potent treatment of transient forebrain cerebral ischemia.

Freeze-fracture of capillary endothelium in rat brain.

SummaryThe rat brain capillary was studied with freeze-fracture technique. The attached plasmalemmal vesicles were quite few in number on the luminal front and sometimes numerous on the contraluminal side. The fracture appearance of some tight junctions showed interconnecting ridges on face A and complementary furrows devoid of particles on face B, comparable to the common tight junction in the normal epithelia. Other tight junctions revealed a preferential disposition of quasicontinuous rows of particles on shallow furrows of face B, resembling the tight junctional strands of capillary endothelium in non-cerebral tissues. Either behavior is probably due to the difference in the fracture plane around the single fibril. In addition, the tight junctional strand could surround the perimeter of the endothelial cell completely although the exposed strand of tight junction was limited in length.

Activation of Protein Kinases in Canine Basilar Artery in Vasospasm

Subarachnoid hemorrhage (SAH) often leads to a long-term narrowing of cerebral artery called vasospasm. To understand the molecular mechanisms in vasospasm, signal transduction of tyrosine kinase pathway and phosphorylation of myosin light chain (MLC) and calponin (CaP) in the basilar artery were studied. Vasospasm was produced in the canine basilar artery by a two-hemorrhage method, and vasocontraction was induced by a local application of KCl or serotonin to the basilar artery after a transclival exposure. Intracellular substrates of tyrosine kinase pathway, including Shc, Raf1, and extracellular-regulated kinases in the basilar artery, were activated after SAH, and the activation of Shc suggests stimulation of signal transductions from tyrosine kinase receptors, G-coupled receptors, or both. The activation of tyrosine kinase pathway in vasospasm also was supported by dose-dependent dilation of the spastic basilar artery on days 0 and 7 by topical application of genistein, a tyrosine kinase inhibitor, and associated marked inhibition of tyrosine phosphorylation of intracellular substrates, including Shc. In addition, the generation of protein kinase M, catalytic fragment of protein kinase Cα (PKCα), in vasospasm on days 0 and 7 was inhibited in response to genistein, indicating an inactivation of μ-calpain. It is suggested, therefore, that the reversal of vasospasm by genistein is closely associated with the restoration of intracellular Ca2+ levels. However, the increased activities of Raf1 and extracellular-regulated kinases in vasospasm were declined on day 7 compared with those on day 0 or 2, suggesting that the activation of tyrosine kinase pathway is more closely associated with the early stage of vasospasm than with the late stage of vasospasm. The analysis by pyrophosphate polyacrylamide gel electrophoresis (PPi-PAGE) demonstrated three MLC bands in vasospasm on days 2 and 7, as well as in KCl- and serotonin-induced vasocontraction. Since PPi-PAGE resolves smooth muscle MLC into three bands in the MLC kinase (MLCK)-mediated phosphorylation and into a single band in the PKC-mediated phosphorylation based on the phosphorylation state, the current results suggest that MLC in vasospasm is phosphorylated by MLCK but not by PKC. In basilar artery, CaP was significantly down-regulated, and in addition, significantly phosphorylated on serine and threonine residues only in vasospasm on days 2 and 7. Although the significance of CaP phosphorylations in vivo still is controversial, CaP down-regulation and phosphorylation may attenuate the inhibition of Mg2+-ATPase activity by CaP and induce a potential enhancement of smooth muscle contractility in delayed vasospasm. Since CaP is phosphorylated vivo by PKC, activated PKC in vasospasm may phosphorylate CaP. Thus, SAH stimulates tyrosine kinase pathway to increase intracellular Ca2+ and activate PKC, and the former activates MLCK to phosphorylate MLC, whereas the latter phosphorylates CaP but not MLC.

Proteasome inhibitors induce Fas-mediated apoptosis by c-Myc accumulation and subsequent induction of FasL message in human glioma cells

Proteasome inhibitors were shown previously to induce mitochondria‐independent and caspase‐3‐dependent apoptosis in human glioma cell lines by unknown mechanisms. Here, we showed that treatment with proteasome inhibitors, lactacystin or acetyl‐leucinyl‐leucinyl‐norleucinal, led to elevation of the steady‐state c‐Myc protein but not c‐myc mRNA, suggesting the accumulation of c‐Myc protein by proteasome inhibitors. In addition, the marked association of c‐Myc protein with ubiquitin by treatment with proteasome inhibitors indicated the involvement of proteasome in c‐Myc proteolysis and the stabilization of c‐Myc protein by proteasome inhibitors in vivo. The expression of Fas (also termed CD95 or APO‐1) mRNA, if analyzed by reverse transcriptase polymerase chain reaction assay, was found to occur constitutively, and increased slightly by the treatment with proteasome inhibitors. In contrast, the expression of Fas ligand (FasL) mRNA was markedly induced temporarily before the activation of caspase‐3 by the treatment. Agonistic anti‐Fas antibody (CH11) induced apoptotic cell death, suggesting the presence of a functional Fas receptor. In addition, proteasome inhibitor‐induced apoptosis was prevented by the addition of antagonistic anti‐FasL antibody (4A5) or z‐IETD.fmk, a potent inhibitor of caspase‐8, indicating the involvement of the Fas receptor–ligand apoptotic signaling system in proteasome inhibitor‐mediated apoptosis. Thus, it is suggested that proteasome inhibitors cause the accumulation of c‐Myc protein which induces transiently FasL message to stimulate the Fas receptor–ligand apoptotic signaling pathway.

Up- and down-regulation of calpain inhibitor polypeptide, calpastatin, in postischemic hippocampus

Based on our previous observation that transient forebrain ischemia induces calpain-catalyzed proteolysis in gerbil hippocampus in a region-specific manner, we examined the effect of ischemia on the quantity and localization of the endogenous calpain-specific inhibitor protein, calpastatin, in the tissue. Brief (5 min) forebrain ischemia followed by reperfusion induced an overall increase of calpastatin immunoreactivity in hippocampus, particularly in pyramidal cells, in 4 h as analyzed by Western blotting and immunohistochemistry. The amount of calpastatin, however, decreased to the preischemic level and lower in 24 h to 7 days due to proteolysis except in CA2 showing continuously elevated calpastatin immunoreactivity. Because calpastatin is not only a potent inhibitor but also a preferred substrate for calpain and because CA2 neurons are less vulnerable to ischemic stress than the adjacent CA1 neurons, these observations imply involvement of calpastatin in calpain regulation as a bait substrate and, possibly, in neuroprotection under ischemic conditions. Calpastatin may participate in the stress responses together with the previously known ischemia-induced stress proteins such as heat shock proteins.