Takeshi Mukasa

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.

DETECTION OF ACTIVATED CASPASE-3 (CPP32) IN THE VERTEBRATE NERVOUS SYSTEM DURING DEVELOPMENT BY A CLEAVAGE SITE-DIRECTED ANTISERUM

We previously demonstrated that Caspase-3 is highly expressed in dorsal root ganglia and trigeminal ganglia of mouse embryos [T. Mukasa, K. Urase, Y.M. Momoi, I. Kimura, T. Momoi, Specific expression of CPP32 in sensory neurons of mouse embryos and activation of CPP32 in the apoptosis induced by a withdrawal of NGF, Biochem. Biophys. Res. Commun., 231 (1997) 770-774.]. Since, however, Caspases are processed into active form during apoptosis, it is difficult to examine the involvement of activated Caspases in naturally occurring cell death during development by immunohistochemical staining or in situ hybridization method. We prepared a cleavage site-directed antiserum against Caspase-3 (anti-p20/17). This antiserum reacted with fragment (p20/17) of Caspase-3, but not proCaspase-3 (p32), proCaspase-7 (p34) and its cleaved fragment (p24). We examined the relationship between the activation of Caspase-3 and the appearance of the naturally occurring apoptotic cells in the nervous system during development. In the trigeminal ganglia and dorsal root ganglia, the expression of Caspase-3 mRNA was maximal before the appearance of p20/17-positive cells and apoptotic cells. In the mouse brain, many p20/17-positive cells and apoptotic cells were observed in the neuroepithelium in the early developmental stages, but very few p20/17-positive cells were detected in postmitotic neurons in the cerebral cortex although Caspase-3 mRNA was expressed highly. Caspase-3 is activated mainly during apoptosis of neuroepithelial cells in the early developmental stages but not of mature neurons at postnatal stages.

The analysis of naturally occurring neuronal cell death by cleavage site-directed antiserum against Caspases

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.