Activation and Caspase-mediated Inhibition of PARP: A Molecular Switch between Fibroblast Necrosis and Apoptosis in Death Receptor Signaling

Abstract

Death ligands not only induce apoptosis but can also trigger necrosis with distinct biochemical and morphological features. We recently showed that in L929 cells CD95 ligation induces apoptosis, whereas TNF elicits necrosis. Treatment with anti-CD95 resulted in typical apoptosis characterized by caspase activation and DNA fragmentation. These events were barely induced by TNF, although TNF triggered cell death to a similar extent as CD95. Surprisingly, whereas the caspase inhibitor zVAD prevented CD95-mediated apoptosis, it potentiated TNF-induced necrosis. Cotreatment with TNF and zVAD was characterized by ATP depletion and accelerated necrosis. To investigate the mechanisms underlying TNF-induced cell death and its potentiation by zVAD, we examined the role of poly(ADP-ribose)polymerase-1 (PARP-1). TNF but not CD95 mediated PARP activation, whereas a PARP inhibitor suppressed TNF-induced necrosis and the sensitizing effect of zVAD. In addition, fibroblasts expressing a noncleavable PARP-1 mutant were more sensitive to TNF than wild-type cells. Our results indicate that TNF induces PARP activation leading to ATP depletion and subsequent necrosis. In contrast, in CD95-mediated apoptosis caspases cause PARP-1 cleavage and thereby maintain ATP levels. Because ATP is required for apoptosis, we suggest that PARP-1 cleavage functions as a molecular switch between apoptotic and necrotic modes of death receptor-induced cell death. INTRODUCTIONTwo forms of cell death, namely apoptosis and necrosis, are distinguished by morphological and biochemical features. Although apoptosis accounts for most of physiological cell death, necrosis is usually induced in pathological situations by accidental and acute damage to cells (Kerr et al., 1972;Wyllie et al., 1980). Necrosis is characterized by cell swelling and disruption of the cell membrane, leading to the release of the cellular content, which may result in an inflammatory response (Fiers et al., 1999). In contrast, apoptosis is a tightly regulated process controlled by a hierarchical set of molecules that were originally identified in Caenorhabditis elegans and later in mammalian cells (Cohen, 1997;Cryns and Yuan;Los et al., 1999). The apoptotic cascade has been intensively studied for death receptors such as TNF receptor-1 (TNF-R1) and CD95 (APO-1/Fas) . After death ligands bind to their cognate receptors, several prominent biochemical events occur including the proteolytic activation of caspases as the critical executioners of apoptosis and the internucleosomal fragmentation of DNA that is mediated by cleavage of DNA fragmentation factor (DFF45/ICAD) (Los et al., 1995;Muzio et al., 1996;Liu et al., 1997;Enari et al., 1998).Another characteristic event of apoptosis is the proteolytic cleavage of poly(ADP-ribose)polymerase-1 (PARP-1), a nuclear enzyme involved in DNA repair, DNA stability, and transcriptional regulation. Caspases, in particular caspase-3 and -7, cleave the 116-kDa form of PARP-1 at the DEVD site to generate a 85-and a 24-kDa fragment (Kaufmann et al., 1993;L...

Involvement of lysosomal cathepsins in the cleavage of DNA topoisomerase I during necrotic cell death

“…Treatment of L929 murine fibroblasts with TNF␣ induces a relatively slow death in which both apoptotic and necrotic cells can be observed (22)(23)(24)(25). Blockade of caspase activation in these cells with Z-VAD-FMK dramatically potentiates TNF␣-mediated necrosis (22), whereas inhibition of transcription or translation with actinomycin D or cycloheximide, respectively, in the presence of TNF␣ shifts cell death toward apoptosis (22)(23)(24)(25). Incubation of L929 cells with TNF␣ (10 ng/ml) led to a gradual loss of cytoplasmic membrane integrity, characteristic of necrosis, as measured by the ability of cells to take up trypan blue ( Figure 1A).…”

Autophagy, Apoptosis, Mitoptosis and Necrosis: Interdependence Between Those Pathways and Effects on Cancer

“…This is particularly frequent when a cell for one or other reason (i.e. low ATP-level) is unable to die by apoptosis (Los et al 2002;Skulachev 2006). Necrosis is typically not associated with activation of caspases (it rather may be a result of their inhibition) (Los et al 2002); it is characterized by swelling of the endoplasmic reticulum, mitochondria, and cytoplasm, with subsequent rupture of the plasma membrane and lysis of the cells (Schweichel and Merker 1973).…”

“…Beyond this, necrosis is often associated with the disintegration of the dynamic plasma membrane of the dying cell (Leist and Jaattela 2001). Inflammatory reactions are frequently triggered in response to necrosis (Holmes 1856;Los et al 2002). The induction of necrosis usually takes place in pathological situations of damage to cells, either in an accidental or an acute manner (Kerr et al 1972;Wyllie et al 1980).…”

“…Several forms of cell death may typically be induced within the same tissue although apoptosis is the fastest, while other forms, like necrosis or autophagy, only become visible when apoptosis is inhibited (Los et al 2002;Martinet et al 2006). Apoptosis is also frequently called programmed cell death (PCD), although some researchers reserve the term PCD only to developmental process-related death.…”

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“…Despite a largely unclear mechanism, PARP (poly(ADP‐ribose) polymerase 1)‐mediated signaling is believed to facilitate the interconnection between autophagy and necrosis (Chaabane, User, El‐Gazzah, Jaksik, & Sajjadi, 2013; Los, Mozoluk, Ferrari, Stepczynska, & Stroh, 2002). The activation of PARP either promotes transcription of autophagy components or induces ATP depletion and subsequent necrotic cell death.…”