Shiro Kanamori

Regulation of a novel pathway for cell death by lysosomal aspartic and cysteine proteinases.

PC12 cells undergo apoptosis when cultured under conditions of serum deprivation. In this situation, the activity of caspase-3-like proteinases was elevated, and the survival rate could be maintained by treatment with acetyl-DEVD-cho, a specific inhibitor of caspase-3. In a culture of PC12 cells treated with acetyl-DEVD-cho, where caspase-3-like proteinases are not activated, CA074, a specific inhibitor of cathepsin B induced active death of the cells. Cathepsin B antisense oligonucleotides showed a similar effect to CA074 on the induction of active cell death. By double staining of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and activated caspase-3, the dying cells treated with CA074 were positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling staining but negative for activated caspase-3. Ultrastructurally, the cells were relatively large and had nuclei with chromatin condensation. The initiation of cell death by CA074 or the cathepsin B antisense were inhibited by the addition of pepstatin A, a lysosomal aspartic proteinase inhibitor, or by cathepsin D antisense. To examine whether this cell death pathway was present in cell types other than PC12 cells, we analysed dorsal root ganglion neurons obtained from rat embryos on the 15th gestational day, a time when they require nerve growth factor for survival and differentiation in culture. When cultured in the absence of nerve growth factor, the neurons survived in the presence of acetyl-DEVD-cho or acetyl-YVAD-cho. Under these conditions, CA074 reduced the survival rate of the neurons, which was subsequently restored by the further addition of pepstain A. These results suggest that a novel pathway for initiating cell death exists which is regulated by lysosomal cathepsins, and in which cathepsin D acts as a death factor. We speculate that this death-inducing activity is normally suppressed by cathepsin B.

Identification and Analysis of the K5 Gene of Kaposi's Sarcoma-Associated Herpesvirus

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8), belongs to the gammaherpesvirus subfamily and encodes ∼80 open reading frames (ORFs). Among them are a few candidates for immediate-early genes (e.g., K5). We developed a monoclonal antibody (MAb), 328C7, against the K5 antigen. This MAb reacted with the K5 gene product by immunoscreening of a cDNA library from BCBL-1 cells, and this result was confirmed by transfection of the K5 ORF into Cos-7 cells. After induction of lytic infection by treatment with 12-O-tetradecanoylphorbol-13-acetate, MAb 328C7 reacted with an antigen in the cytoplasm of BCBL-1 and BC-3 cells as early as after 4 h of induction. Immunoelectron microscopy showed that the K5 antigen was situated mainly in the endoplasmic reticulum but was not present on the virion or in the nucleus. Northern blotting with a K5-specific probe revealed a single transcript of 1.2 kb, while Western blotting showed the antigen to be a 36-kDa polypeptide. The 5′ and 3′ ends were then determined by rapid amplification of cDNA, followed by sequencing of RACE products, and a splice was revealed upstream of the K5 ORF. K5 expression was unaffected by the respective DNA and protein synthesis inhibitors phosphonoformic acid and cycloheximide plus actinomycin D, confirming its immediate-early nature. Transient-transfection assays showed that the K5 promoter was transactivated by ORF 50 (KSHV Rta), a homolog of Epstein-Barr virus Rta, but the K5 gene product exhibited no transregulation of its own promoter or those of DNA polymerase and the human immunodeficiency virus type 1 long terminal repeat. This is the first such analysis of an immediate-early gene product; determination of its specific biological function requires further investigation.

Selective localization of Bcl-2 to the inner mitochondrial and smooth endoplasmic reticulum membranes in mammalian cells.

Bcl-2, an anti-apoptotic protein, is believed to be localized in the outer mitochondrial membrane, endoplasmic reticulum, and nuclear envelope. However, Bcl-2 has also been suggested as playing a role in the maintenance of mitochondrial membrane potential, indicating its possible association with the inner mitochondrial membrane. We therefore further examined the exact localization of Bcl-2 in mitochondria purified from wild-type and bcl-2-transfected PC12 cells and pre- and postnatal rat brains. Double immunostaining demonstrated that Bcl-2 was co-localized with subunit β of F1F0ATPase in the inner mitochondrial membrane. Biochemical analysis of isolated mitochondria using digitonin and trypsin suggests an association of Bcl-2 with the inner mitochondrial membrane. More interestingly, the majority of Bcl-2 disappeared from the inner membrane of mitochondria when cultured under serum deprivation. These results suggest that Bcl-2 acts as an anti-apoptotic regulator by localizing mainly to the inner mitochondrial and smooth ER membranes. Cell Death and Differentiation (2000) 7, 666–674

Different Distribution Patterns of the Two Mannose 6-phosphate Receptors in Rat Liver

Two mannose 6-phosphate receptors, cation-dependent and -independent receptors (CDMPR and CIMPR), play an important role in the intracellular transport of lysosomal enzymes. To investigate functional differences between the two in vivo, their distribution was examined in the rat liver using immunohistochemical techniques. Positive signals corresponding to CIMPR were detected intensely in hepatocytes and weakly in sinusoidal Kupffer cells and interstitial cells in Glissons capsule. In the liver acinus, hepatocytes in the perivenous region showed a more intense immunoreactivity than those in the periportal region. On the other hand, positive staining of CDMPR was detected at a high level in Kupffer cells, epithelial cells of interlobular bile ducts, and fibroblast-like cells, but the corresponding signal was rather weak in hepatocytes. In situ hybridization analysis also revealed a high level of expression of CIMPR mRNAs in hepatocytes and of CDMPR mRNA in Kupffer cells. By double immunostaining, OX6-positive antigen-presenting cells in Glissons capsule were co-labeled with the CDMPR signal but were only faintly stained with anti-CIMPR. These different distribution patterns of the two MPRs suggest distinct functional properties of each receptor in liver tissue.

Predominant expression of the short form of GGA3 in human cell lines and tissues

Three GGAs (GGA1-3) were found in humans, among which GGA3 has short and long forms of spliced variants (GGA3-S and GGA3-L). The present study analyzed expression patterns of both GGA3 variants in human tissues and cell lines. Western blot analysis revealed that the brain contained both GGA3-S and -L, while other tissues and cell lines examined predominantly expressed GGA3-S. By double immunofluorescence microscopy, GGA1 and GGA3 were localized with slightly different patterns in both the trans-Golgi network (TGN) and peripheral region. When the dominant-negative mutant, VHS-GAT domain, of GGA1 or GGA3-L was overexpressed, TGN-associated GGA1 was redistributed into the cytoplasm. However, the GGA3 distribution was not affected by the expression of either VHS-GAT domain. These results indicate that GGA3-S which would not be directly involved in the cargo protein recognition is predominantly expressed in human tissues except the brain and in cell lines.

Uncoupling protein 2 negatively regulates neurite extensions in PC12h cells

Uncoupling protein 2 (UCP2) distributes in many organs including the brain. Though recent reports suggest that UCP2 is involved in the neuroprotection and the regulation of neurosecretion, the roles of UCP2 in the central nervous systems remain largely unclear. In order to clarify the significance of UCP2 in the brain especially at developmental stage, subcellular localizations of rat UCP2 (rUCP2) in the developing cerebellar Purkinje cells were immunochemically examined. The rUCP2-like immunoreactivities observed axon or its terminal during axonal maturation. This result implies that rUCP2 contributes to the neurite development. In the PC12h cells overexpressing rUCP2 or active mutant of rUCP2, the neurite outgrowth was significantly inhibited along with a reduction of cellular ATP level. These findings suggest a possibility that UCP2 is involved in negative regulation of neurite extensions through repression of the energy supply.

Regulation of a novel pathway for apoptosis by lysosomal aspartic and cysteine proteinases

Some granule neurons naturally undergo apoptosis in the external granular layer of the postnatally developing rat cerebellum. We previously established an organotypic slice culture system as an experimental model for studying mechanisms of this apoptosis, in which deprivation of insulin or IGF-I analog induces apoptosis of external granular layer neurons. In the present study, we examined involvement of caspase-3 in this apoptosis using the slice culture system and an antibody specific for the active form of caspase-3. AC-DEVD-CHO, a peptide inhibitor of caspase-3-like proteases, partially prevented this apoptosis. Double staining by in situ nick end labeling and immunohistochemistry against the active caspase-3 revealed that the active caspase-3 is present in some of the apoptotic granule neurons. A similar staining pattern was also observed in the postnatal cerebellum in vivo. Thus, while caspase-3 was shown to be involved in apoptosis of some external granular layer neurons, there might exist a mechanism of apoptosis independent of caspase-3. Alternatively, activation of caspase-3 might peak before granule neurons become positive to the nick end labeling.

Anti-apoptotic function of BCL-2 requires localization in the inner mitochondrial membrane

To examine whether overexpression of Bcl-2 rescues motoneurons from programmed cell death in the developing chick spinal cord, we have generated adenovirus vectors capable of overexpressing Bcl-2. Following injection of Bcl-2 vectors into right leg buds of the chick embryos on embryonic day (E) 4.5-5 (mst 29, many motoneurons in the right lateral motor column (LMC) in lumbosacral segments strongly expressed Bcl-2 by E7. On E7 colchicine was injected into the same leg buds to induce motoneuron cell death due to depletion of target-derived neurotrophic factors. Virtually all motoneurons in the LMC died by El0 in the control embryos that received injection of colchicine without preceding injection of Bcl-2 vectors. By contrast, remaining motoneurons were observed in the LMC of the embryos that received injection of Bcl-2 vectors followed by injection of colchicine. Most of the remaining motoneurons expressed Bcl-2. These results suggest that Bcl-2 adenovirus vectors can be retrogradely transported from the periphery to cell bodies, and that overexpression of Bcl-2 can rescue motoneurons from cell death in viva.