Takashi Momoi

Deficiency in caspase-9 or caspase-3 induces compensatory caspase activation

Dysregulation of apoptosis contributes to the pathogenesis of many human diseases. As effectors of the apoptotic machinery, caspases are considered potential therapeutic targets. Using an established in vivo model of Fas-mediated apoptosis, we demonstrate here that elimination of certain caspases was compensated in vivo by the activation of other caspases. Hepatocyte apoptosis and mouse death induced by the Fas agonistic antibody Jo2 required proapoptotic Bcl-2 family member Bid and used a Bid-mediated mitochondrial pathway of caspase activation; deficiency in caspases essential for this pathway, caspase-9 or caspase-3, unexpectedly resulted in rapid activation of alternate caspases after injection of Jo2, and therefore failed to protect mice against Jo2 toxicity. Moreover, both ultraviolet and gamma irradiation, two established inducers of the mitochondrial caspase-activation pathway, also elicited compensatory activation of caspases in cultured caspase-3−/− hepatocytes, indicating that the compensatory caspase activation was mediated through the mitochondria. Our findings provide direct experimental evidence for compensatory pathways of caspase activation. This issue should therefore be considered in developing caspase inhibitors for therapeutic applications.

Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD

Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)–induced cell death. We have now identified human TNF receptor type 1 (TNFR1)–associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1–270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.

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.

Caspase-12 processing and fragment translocation into nuclei of tunicamycin-treated cells

Excess endoplasmic reticulum (ER) stress induces processing of caspase-12, which is located in the ER, and cell death. However, little is known about the relationship between caspase-12 processing and cell death. We prepared antisera against putative caspase-12 cleavage sites (anti-m12D318 and anti-m12D341) and showed that overexpression of caspase-12 induced autoprocessing at D318 but did not induce cell death. Mutation analysis confirmed that D318 was a unique autoprocessing site. In contrast, tunicamycin, one of the ER stress stimuli, induced caspase-12 processing at the N-terminal region and the C-terminal region (both at D318 and D341) and cell death. Anti-m12D318 and anti-m12D341 immunoreactivities were located in the ER of the tunicamycin-treated cells, and some immunoreactivities were located around and in the nuclei of the apoptotic cells. Thus, processing at the N-terminal region may be necessary for the translocation of processed caspase-12 into nuclei and cell death induced by ER stress. Some of the caspase-12 processed at the N-terminal and C-terminal regions may directly participate in the apoptotic events in nuclei.

Prevention of Hepatocellular Carcinoma Development Associated with Chronic Hepatitis by Anti-Fas Ligand Antibody Therapy

A persistent immune response to hepatitis viruses is a well-recognized risk factor for hepatocellular carcinoma. However, the molecular and cellular basis for the procarcinogenic potential of the immune response is not well defined. Here, using a unique animal model of chronic hepatitis that induces hepatocellular carcinogenesis, we demonstrate that neutralization of the activity of Fas ligand prevented hepatocyte apoptosis, proliferation, liver inflammation, and the eventual development of hepatocellular carcinoma. The results indicate that Fas ligand is involved not only in direct hepatocyte killing but also in the process of inflammation and hepatocellular carcinogenesis in chronic hepatitis. This is the first demonstration that amelioration of chronic inflammation by some treatment actually caused reduction of cancer development.

Caspase-3-deficiency induces hyperplasia of supporting cells and degeneration of sensory cells resulting in the hearing loss ☆

Caspase-3 is one of the cystein proteases that play essential roles in programmed cell death. As such, brain development is profoundly affected by caspase-3-deficiency, resulting in hyperplasia and abnormal cell organization (Kuida et al., Nature 1996;384:368-372). In the present study, we used caspase-3 (-/-) mice to show that caspase-3 deficiency results in severe hearing loss, hyperplasia of supporting cells and degeneration of sensory hair cells. The greater epithelial ridge, a remnant of the primordial organ of Corti, persists throughout all of the turns of cochlea in 2-week-old caspase-3 (-/-) mice, which indicates that the morphology of the cochlea is immature. The number of border cells, that develop from the greater epithelial ridge and are one of the supporting cells of the inner hair cell, increase significantly in both 2- and 5-week-old caspase-3 (-/-) mice. On the other hand, abnormal fused stereocilia can be seen in both 2- and 5-week-old caspase-3 (-/-) mice, and disarrangement and loss of sensory hair cells are observed in 5-week-old caspase-3 (-/-) mice. Taken together, both hyperplasia and degeneration occur simultaneously in the inner ear of the caspase-3 (-/-) mice, suggesting that caspase-3-dependent apoptosis is necessary for the development and formation of a properly functioning auditory system in mammals.

bFGF inhibits the activation of caspase-3 and apoptosis of P19 embryonal carcinoma cells during neuronal differentiation

P19 embryonal carcinoma (EC) cells undergo apoptosis during neuronal differentiation induced by all-trans retinoic acid (RA). Caspase-3-like proteases are activated and involved in the apoptosis of P19 EC cells during neuronal differentiation. Recently it has been shown that growth factor signals protect against apoptosis by phosphorylation of Bad. Phosphorylated Bad, an apoptotic member of the Bcl-2 family, cannot bind to Bcl-xL and results in Bcl-xL homodimer formation and subsequent antiapoptotic activity. In the present study, we demonstrate that this system is used generally to protect against apoptosis during neuronal differentiation. Bcl-xL inhibited the activation of caspase-3-like proteases. Basic fibroblast growth factor (bFGF) inhibited more than 90% of the caspase-3-like activity, inhibited processing of caspase-3 into its active form, and inhibited DNA fragmentation. bFGF activated phosphatidyl-inositol-3-kinase (PI3K) and stimulated the phosphorylation of Bad. Phosphorylation was inhibited by wortmannin, an inhibitor of PI3K and its downstream target Akt. Thus, Bad is a target of the FGF receptor-mediated signals involved in the protection against activation of caspase-3.

Caspase activity is involved in, but is dispensable for, early motoneuron death in the chick embryo cervical spinal cord

We examined the role of caspases in the early programmed cell death (PCD) of motoneurons (MNs) in the chick embryo cervical cord between embryonic day (E) 4 and E5. An increase in caspase-3-like activity in MNs was observed at E4.5. Treatment with an inhibitor of caspase-3-like activity, Ac-DEVD-CHO, for 12 h blocked this increase and revealed that caspase-3-like activity is mainly responsible for DNA fragmentation and the nuclear changes during PCD but not for degenerative changes in the cytoplasm. When a more broad-spectrum caspase inhibitor was used (bocaspartyl (OMe)-fluoromethyl ketone, BAF), the appearance of degenerative changes in the cytoplasm was delayed by at least 12 h. However, following treatment with either Ac-DEVD-CHO or BAF for 24 h, the number of surviving healthy MNs did not differ from controls, indicating a normal occurrence of PCD despite the inhibition of caspases. These results suggest that caspase cascades that occur upstream of and are independent of the activation of caspase-3-like activity are responsible for the degenerative changes in the cytoplasm of dying cervical MNs. These data also suggest that, although one function of caspases may be to facilitate the kinetics of PCD, caspases are nonetheless dispensable for at least some forms of normal neuronal PCD in vivo.

Differential Effects of Bcl-2 Overexpression on Hippocampal CA1 Neurons and Dentate Granule Cells Following Hypoxic Ischemia in Adult Mice

In contrast to its known anti‐apoptotic activity in sympathetic neurons, immortal neuronal cell lines, and primary cultured immature neurons of the central nervous system (CNS), the role of Bcl‐2 in CNS neurons in the adult brain is poorly understood. In the present study, we examined effects of overexpression of Bcl‐2 on selective neuronal death of the hippocampal CA1 neurons and the dentate granule cells induced by hypoxic ischemia in adult transgenic mice overexpressing human Bcl‐2 under the control of neuron‐specific enolase (NSE‐hbcl‐2). At the light microscopic level, numbers of TUNEL‐positive cells with pyknotic nuclei were observed in the CA1 subfield of NSE‐hbcl‐2 transgenic mice, as well as that of wild‐type mice, after hypoxic ischemic insult, although the onset of neuronal death was apparently delayed in NSE‐hbcl‐2 transgenic mice. The electron microscopic studies showed that morphological changes of the degenerating CA1 neurons from both groups were clearly distinct from ordinary apoptosis. In contrast, a significant amount of degenerating dentate granule cells from wild‐type but not from transgenic mice had typical apoptotic nuclei by the treatment. The activation of caspase‐3 was detected in the dentate granule cells but not that of the CA1 neurons. These results indicate that the overexpression of Bcl‐2 effectively suppressed dentate granule cell apoptosis but only delayed cell death of the CA1 neurons induced by hypoxic ischemia, suggesting the occurrence of a non‐apoptotic, caspase‐3–independent mechanism for neuronal death in the CA1 subfield. J. Neurosci. Res. 57:1–12, 1999.

Mitosis and apoptosis in postnatal auditory system of the C3H/He strain

The mouse auditory neurons, hair cells and their supporting cells in the cochlea are considered to be generated mainly in the embryonic days and to be sustained throughout the whole life. In the present study, however, we observed that auditory ganglion cells in the spiral ganglia undergo apoptosis and mitosis in the suckling mouse (1- to 2-week-old C3H/HeJ mice) with a normal auditory system. In spiral ganglia at postnatal days 7 (P7) and 10 (P10), TUNEL (TdT-mediated dUTP nick-end labeling)-positive and morphologically apoptotic ganglion cells were found. Furthermore, by bromodeoxyuridine labeling, mitosis of auditory ganglion cells was found at P10 to P14. In a functional study of auditory brainstem response, we demonstrated that the C3H/HeJ mouse acquires the ability to hear airborne sound at P12 and this is the same time as the opening of their external acoustic meatus (EAM). These results indicate that C3H/HeJ auditory ganglion cells have the ability to proliferate even after opening of the EAM and the initial input of airborne sound. We found that postnatal apoptosis and mitosis after P7 also occurred in the greater epithelial ridge (GER) which is an important organ for maturation of the organ of Corti and is located around the inner hair cells. This indicates that GER cells are not only degenerated but also regenerated until their disappearance around P12. This is the first report on mammals to demonstrate that neuronal mitosis of spiral ganglion cells and that of GER cells occur not only in embryonic and neonatal development but also in postnatal development of the normal auditory system.