The stress-activated protein kinases c-Jun-activated kinase (JNK) and p38 are implicated in neuronal apoptosis. Early studies in cell lines suggested a requirement for both in the apoptosis induced by withdrawal of nerve growth factor. However, studies in neuronal cells typically implicate JNK but not p38 in apoptosis. In some cases, p38 is implicated, but the role of JNK is undefined. It remains unclear whether p38 and JNK have differing roles dependent on cell type, apoptotic stimulus, or mechanism of cell death or whether they are redundant and each sufficient to induce identical forms of cell death. We investigate the relative roles of these protein kinases in different death mechanisms in a single system, cultured cerebellar granule neurons. Apoptosis induced by withdrawal of trophic support and glutamate are mechanistically different in terms of caspase activation, DNA fragmentation profile, chromatin morphology, and dependence on de novo gene expression. Caspase-independent apoptosis induced by glutamate is accompanied by strong activation of p38, and dominant negatives and inhibitors of the p38 pathway prevent this apoptosis. In contrast, withdrawal of trophic support induces caspase-dependent death accompanied by JNK-dependent phosphorylation of c-Jun, and inhibition of JNK is sufficient to prevent the death induced by withdrawal of trophic support. Inhibition of p38 does not block withdrawal of trophic support-induced death, nor does inhibition of JNK block glutamate-induced death. We propose that mechanistically different forms of apoptosis have differing requirements for p38 and JNK activities in neurons and demonstrate that only inhibition of the appropriate kinase will prevent neurons from undergoing apoptosis.Stress-activated protein kinases (SAPKs) 1 are believed to play an obligatory role in neuronal cell death; however, the relative roles of the JNK and p38 kinase groups have been unclear. Initial studies in the PC12 cell line suggested a role for both JNK and p38 in caspase-dependent cell death induced by withdrawal of nerve growth factor (1). Subsequent studies in primary cultured neurons failed to demonstrate a requirement for p38 in apoptosis induced by deprivation of trophic support, whereas a role for JNK in developmental, trophic withdrawalinduced, excitotoxic, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced death has been substantiated in a variety of neuronal systems (2-7). There are proposals that p38 contributes to axotomy-induced apoptosis of retinal ganglion cells, excitotoxicity-induced apoptosis of cerebellar granule neurons, and ceramide-induced death of cortical neurons (8 -10). However, these reports rely on SB203580, the specificity of which has recently been called into question, since it inhibits other kinases more potently than it does p38 (11), and it discriminates poorly between p38 and certain JNK isoforms (12, 13). Furthermore, the nature of the cell death under investigation, whether apoptotic, necrotic, or intermediate, is often unclear. Whether JNK and p38 ...

Disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal DNA fragments represents two major periodicities of DNA fragmentation during apoptosis. These are thought to originate from the excision of DNA loop domains and from the cleavage of nuclear DNA at the internucleosomal positions, respectively. In this report, we demonstrate that different apoptotic insults induced apoptosis in NB-2a neuroblastoma cells that was invariably accompanied by the formation of HMW DNA fragments of about 50 -100 kb but proceeded either with or without oligonucleosomal DNA cleavage, depending on the type of apoptotic inducer. We demonstrate that differences in the pattern of DNA fragmentation were reproducible in a cell-free apoptotic system and develop conditions that allow in vitro separation of the HMW and oligonucleosomal DNA fragmentation activities. In contrast to apoptosis associated with oligonucleosomal DNA fragmentation, the HMW DNA cleavage in apoptotic cells was accompanied by down-regulation of caspase-activated DNase (CAD) and was not affected by z-VAD-fmk, suggesting that the caspase/CAD pathway is not involved in the excision of DNA loop domains. We further demonstrate that nonapoptotic NB-2a cells contain a constitutively present nuclease activity located in the nuclear matrix fraction that possessed the properties of topoisomerase (topo) II and was capable of reproducing the pattern of HMW DNA cleavage that occurred in apoptotic cells. We demonstrate that the early stages of apoptosis induced by different stimuli were accompanied by activation of topo II-mediated HMW DNA cleavage that was reversible after removal of apoptotic inducers, and we present evidence of the involvement of topo II in the formation of HMW DNA fragments at the advanced stages of apoptosis. The results suggest that topo II is involved in caspase-independent excision of DNA loop domains during apoptosis, and this represents an alternative pathway of apoptotic DNA disintegration from CAD-driven caspase-dependent oligonucleosomal DNA cleavage.At the higher level of chromatin compaction, nuclear DNA is arranged into loop domains by periodical attachment of the chromatin fiber to the nuclear matrix (1, 2). The domain level of chromatin organization is supported by the interaction of specific DNA sequences, matrix/scaffold attachment regions, with nuclear matrix proteins (3). The chromatin loops represent the basic structural components of higher-order chromatin folding, which is maintained during the cell cycle and in differentiated cells (3-5).Disintegration of nuclear DNA into nucleosome-sized fragments represents a classical manifestation of apoptosis (6). In addition, another type of DNA cleavage during apoptosis has been reported to yield a set of the high molecular weight (HMW) 1 DNA fragments of about 50 -100 kb (7). The formation of HMW DNA fragments is widely thought to result from the excision of DNA loop domains at the positions of their attachment to the nuclear matrix (8, 9) and is considered to be an initial...

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Transforming growth factors b (TGF-bs) are multifunctional cytokines that modulate cell growth, differentiation and apoptosis. Numerous effects initiated by TGF-bs in vitro have been described, but the role of TGF-b targeting and activation under physiological conditions has gained very little attention and understanding. We report here that apoptosis of human umbilical vein endothelial cells (HUVECs) is accompanied by release of truncated large latent TGF-b complexes from the pericellular matrix followed by activation of TGF-b. The activation of TGF-b during apoptosis was accompanied by enhanced secretion of b1-LAP protein, and apoptotic HUVECs acquired the capacity to induce the release of latent TGF-bbinding proteins (LTBPs) from extracellular matrices. Activated TGF-b, in turn, attenuated apoptotic death of HUVECs. Current results indicate that the activation of TGF-b accompanies the apoptosis of HUVECs, and may play a protective feedback role against apoptotic cell death. The results suggest a role for TGF-b as a putative extracellular modulator of apoptosis.

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