Nuclear export of LEI/L-DNase II by Crm1 is essential for cell survival

LEI/L-DNase II is the key protein of a caspase-independent pathway activated by serine proteases. LEI (Leukocyte elastase inhibitor), L-DNase II precursor, is a member of the clade B serpins (also called serpin b1). In its native conformation it inhibits several intracellular proteases and has an anti-apoptotic activity. Following a metabolic stress and the increase of protease activity in the cell, LEI is cleaved and transformed into L-DNase II (LEI-derived DNase II). This transformation is due to a conformational modification that exposes a nuclear localization signal and an endonuclease active site. In this paper we show that LEI can bind the exportin Crm1, and we identify on LEI a nuclear export signal involved in the control of LEI/L-DNase II nuclearization in healthy cells. Point mutation of this site increases the accumulation of the molecule in the nucleus and triggers cell death.     

L-DNase II, a Molecule That Links Proteases and Endonucleases in Apoptosis, Derives from the Ubiquitous Serpin Leukocyte Elastase Inhibitor

The most widely recognized biochemical change associated with the majority of apoptotic systems is the degradation of genomic DNA. Among the enzymes that may participate in this cleavage, the acidic cation-independent DNase II is a likely candidate since it is activated in many apoptotic cells. To better understand its role, we purified and sequenced a DNase II extracted from porcine spleen. Protein sequencing of random peptides demonstrated that this enzyme is derived from a ubiquitous serpin, the leukocyte elastase inhibitor (LEI), by an acidic-dependent posttranslational modification or by digestion with elastase. We call this novel enzyme L-DNase II. In vitro experiments with purified recombinant LEI show that the native form has no effect on purified nuclei whereas its posttranslationally activated form induces pycnosis and DNA degradation. Antibodies directed against L-DNase II showed, in different cell lines, an increased expression and a nuclear translocation of this enzyme during apoptosis. Since the appearance of the endonuclease activity results in a loss of the anti-protease properties of LEI, the transformation from LEI to L-DNase II may act as a switch of protease and nuclease pathways, each of which is activated during apoptosis.     

Regulation of poly(ADP-ribose) polymerase-1 functions by leukocyte elastase inhibitor/LEI-derived DNase II during caspase-independent apoptosis

Poly(ADP-ribose) polymerase-1 (PARP-1) is an important regulator of apoptosis. Its over-activation at the onset of apoptosis can inhibit the action of apoptotic endonucleases like caspase-activated DNase and DNAS1L3. Therefore, controlled PARP-1 proteolysis during caspase-dependent apoptosis is considered essential to promote DNA degradation. Yet, little is known about the interplay of PARP-1 and endonucleases that operate during caspase-independent cell death. Here we show that in the long-term cultured HeLa cells which undergo caspase-independent death, PARP-1 co-immunoprecipitates with leukocyte elastase inhibitor-derived DNase II (L-DNase II), an acid DNase implicated in this death pathway and activated by serine proteases. Our results indicate that, despite having putative poly(ADP-ribose)-acceptor sites, LEI/L-DNase II is neither significantly poly(ADP-ribosyl)ated nor inhibited by PARP-1 during caspase-independent apoptosis. Unexpectedly, caspase-independent apoptosis induced by hexa-methylene amiloride, LEI/L-DNase II can activate PARP-1 and promote its auto-poly(ADP-ribosyl)ation, thus inhibiting PARP-1 activity. Moreover, overexpression of LEI blocks the pro-survival effect of PARP-1 in this model of cell death. Our results provide the original evidence for a new mechanism of PARP-1 activity regulation in the caspase-independent death pathway involving LEI/L-DNase II.     

Acid DNases and their interest among apoptotic endonucleases

Apoptosis is characterized by cell shrinkage, nuclear condensation and internucleosomal DNA cleavage. Besides the central role of caspases and other proteases, cell death triggers DNA degradation so that DNases have an active role in apoptotic cell death. The best-characterized apoptotic DNase is CAD, a neutral Mg-dependent endonuclease. Its activity is regulated by its inhibitor, ICAD, which is cleaved by caspases. Other neutral DNases have been shown to cleave nuclear DNA in apoptotic conditions: endonuclease G, GADD. In cells, the cytosolic pH is maintained to 7.2, mostly due to the activity of the Na(+)/H(+) exchanger. In many apoptotic conditions, a decrease of the intracellular pH has been shown. This decrease may activate different acid DNases, mostly when pH decreases below 6.5. Three acidic DNases II are so far known: DNase II alpha, DNase II beta and L-DNase II, a DNase II, derived from the serpin LEI (Leukocyte Elastase Inhibitor). Their activation during cell death is discussed in this review.     

Apoptosis induced by Na+/H+ antiport inhibition activates the LEI/L-DNase II pathway

L-DNase II is derived from its precursor leucocyte elastase inhibitor (LEI) by post-translational modification. In vitro, the conversion of LEI into L-DNase II can be induced by incubation of LEI at an acidic pH. In this study, we proposed to analyze the effects of intracellular acidification on this transformation. Amiloride derivatives, like hexamethylene amiloride (HMA), are known to provoke a decrease of cytosolic pH by inhibiting the Na(+)/H(+) antiport. In BHK cells, treatment with HMA-induced apoptosis accompanied by an increase in L-DNase II immunoreactivity and L-DNase II enzymatic activity. Overexpression of L-DNase II precursor led to a significant increase of apoptosis in these cells supporting the involvement of L-DNase II in HMA induced apoptosis. As previously shown in other cells, etoposide-induced apoptosis did not activate L-DNase. On the contrary, LEI overexpression significantly increased cell survival in etoposide-induced apoptosis. Together these results suggest differential roles of LEI and L-DNase II in response to different types of apoptotic inducers.     

Interaction of Leukocyte Elastase Inhibitor/L-DNase II with BCL-2 and BAX

Leukocyte Elastase Inhibitor (LEI, also called serpin B1) is a protein involved in apoptosis among other physiological processes. We have previously shown that upon cleavage by its cognate protease, LEI is transformed into L-DNase II, a protein with a pro-apoptotic activity. The caspase independent apoptotic pathway, in which L-DNase II is the final effector, interacts with other pro-apoptotic molecules like Poly-ADP-Ribose polymerase (PARP) or Apoptosis Inducing Factor (AIF). The screening of LEI/L-DNase II interactions showed a possible interaction with several members of the BCL-2 family of proteins which are known to have a central role in the regulation of caspase dependent cell death. In this study, we investigated the regulation of LEI/L-DNase II pathway by two members of this family of proteins: BAX and BCL-2, which have opposite effects on cell survival. We show that, in both BHK and HeLa cells, LEI/L-DNase II can interact with BCL-2 and BAX in apoptotic and non-apoptotic conditions. These proteins which are usually thought to be anti-apoptotic and pro-apoptotic respectively, both inhibit the L-DNase II pro-apoptotic activity. These results give further insight in the regulation of caspase independent pathways and highlight the involvement of the intracellular environment of a given protein in the determinism of its function. They also add a link between caspase-dependent and independent pathways of apoptosis.     

Nuclear translocation of a leukocyte elastase Inhibitor/Elastase complex during staurosporine-induced apoptosis: role in the generation of nuclear L-DNase II activity

Using L1210 murine leukemia cells, we have previously shown that in response to treatment with drugs having different targets, apoptotic cell death occurs through at least two different signaling pathways. Here, we present evidence that nuclear extracts from staurosporine-treated cells elicit DNase II activity that is not detected in nuclear extracts from cisplatin-treated cells. This activity correlates with the accumulation of two nuclear proteins (70 and 30 kDa) which are detected by an anti-L-DNase II antibody. Partial purification of this DNase II activity suggests that the 30-kDa protein could be the nuclease responsible for staurosporine-induced DNA fragmentation. The 70-kDa protein is also recognized by an anti-elastase antibody, suggesting that it carries residues belonging to both L-DNase II and elastase. Since previous findings showed that L-DNase II was generated from the leukocyte inhibitor of elastase, we propose that the 70-kDa protein results from an SDS-stable association between these two proteins and is translocated from the cytoplasm to the nucleus during staurosporine-induced apoptosis.     

Leukocyte Elastase Inhibitor, the precursor of L-DNase II, inhibits apoptosis by interfering with caspase-8 activation

LEI (Leukocyte Elastase Inhibitor), the precursor of the pro-apoptotic molecule L-DNase II, belongs to the ovalbumin subgroup of serpins. Several serpins can inhibit apoptosis: the viral serpin Crm A inhibits Fas or TNFalpha-induced apoptosis, and overexpression of PAI-2 or PI-9 protects cells from TNFalpha or granzyme B induced apoptosis. We have previously shown that LEI overexpression protects cells from etoposide-induced apoptosis. The molecular reason of this anti-apoptotic activity is now investigated. We show that, in BHK-21 and HeLa cells, LEI anti-protease activity is essential for its anti-apoptotic effect. The protease inhibited is cathepsin D, released from the lysosome during etoposide treatment. Cathepsin D enhances caspase activity in the cell by cleaving procaspase-8 and LEI overexpression slows down this cleavage, protecting cells from apoptosis. This let us presume that high expression of LEI in tumor cells may reduce the efficiency of etoposide as a chemotherapeutic agent.     

The activation of the atypical PKC zeta in light‐induced retinal degeneration and its involvement in L‐DNase II control

Light‐induced retinal degeneration is characterized by photoreceptor cell death. Many studies showed that photoreceptor demise is caspase‐independent. In our laboratory we showed that leucocyte elastase inhibitor/LEI‐derived DNase II (LEI/L‐DNase II), a caspase‐independent apoptotic pathway, is responsible for photoreceptor death. In this work, we investigated the activation of a pro‐survival kinase, the protein kinase C (PKC) zeta. We show that light exposure induced PKC zeta activation. PKC zeta interacts with LEI/L‐DNase II and controls its DNase activity by impairing its nuclear translocation. These results highlight the role of PKC zeta in retinal physiology and show that this kinase can control caspase‐independent pathways.     

On the use of an appropriate TdT-mediated dUTP–biotin nick end labeling assay to identify apoptotic cells

Apoptosis is an essential cellular mechanism involved in many processes such as embryogenesis, metamorphosis, and tissue homeostasis. DNA fragmentation is one of the key markers of this form of cell death. DNA fragmentation is executed by endogenous endonucleases such as caspase-activated DNase (CAD) in caspase-dependent apoptosis. The TUNEL (TdT-mediated dUTP–biotin nick end labeling) technique is the most widely used method to identify apoptotic cells in a tissue or culture and to assess drug toxicity. It is based on the detection of 3′-OH termini that are labeled with dUTP by the terminal deoxynucleotidyl transferase. Although the test is very reliable and sensitive in caspase-dependent apoptosis, it is completely useless when cell death is mediated by pathways involving DNA degradation that generates 3′-P ends as in the LEI/L-DNase II pathway. Here, we propose a modification in the TUNEL protocol consisting of a dephosphorylation step prior to the TUNEL labeling. This allows the detection of both types of DNA breaks induced during apoptosis caspase-dependent and independent pathways, avoiding underestimating the cell death induced by the treatment of interest.     

Multiple apoptotic pathways induced by p53-dependent acidification in benzo[a]pyrene-exposed hepatic F258 cells

Polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene (B[a]P), are ubiquitous genotoxic environmental pollutants. Their DNA-damaging effects lead to apoptosis induction, through similar pathways to those identified after exposure to other DNA-damaging stimuli with activation of p53-related genes and the involvement of the intrinsic apoptotic pathway. However, at a low concentration of B[a]P (50 nM), our previous results pointed to the involvement of intracellular pH (pHi) variations during B[a]P-induced apoptosis in a rat liver epithelial cell line (F258). In the present work, we identified the mitochondrial F0F1-ATPase activity reversal as possibly responsible for pHi decrease. This acidification not only promoted executive caspase activation, but also activated leucocyte elastase inhibitor/leucocyte-derived DNase II (LEI/L-DNase II) pathway. p53 appeared to regulate mitochondria homeostasis, by initiating F0F1-ATPase reversal and endonuclease G (Endo G) release. In conclusion, a low dose of B[a]P induced apoptosis by recruiting a large panel of executioners apparently depending on p53 phosphorylation and, for some of them, on acidification.     

Coordinated and sequential activation of neutral and acidic DNases during interdigital cell death in the embryonic limb

Interdigital tissue regression during embryonic development is one of the most representative model systems of morphogenetic cell death, but the degenerative cascade accounting for this process awaits clarification. Although the canonical apoptotic caspase pathway appears to be activated in the interdigital mesenchyme committed to die, neither genetic nor chemical blockage of caspases or their downstream effectors, is sufficient to prevent cell death. Hence, alternative and/or complementary dying pathways must also be responsible for this degenerative process. In this work we have chosen to study the endonucleases during the regression of the interdigital tissue of avian embryos to gain insights into the molecular mechanisms accounting for programmed cell death in this system. We show that caspase activated DNase, which is a neutral DNase associated with the caspase apoptotic pathway, appears to be the main endonuclease only at an initial phase of interdigit regression. However at peak stages of the degenerative process, the acidic DNases L-DNase II and lysosomal DNase IIB become predominant in the system and markers for cell autophagy become moderately up-regulated. Consistent with the activation of acidic endonucleases we observed that microenvironmental pH value in the interdigits decreased to levels only appropriate for acidic enzymes. Furthermore, we found that overexpression of lysosomal DNase IIB in embryonic limb mesoderm promoted cell death, which was also accompanied by up-regulation and activation of L-DNase II. Up-regulation of acidic DNases was maintained in interdigits explanted to culture dishes, where the participation of exogenous professional phagocytes of hematopoietic origin is avoided. Finally, and consistent with all our findings, up-regulation of acidic DNases was much reduced in the webbed interdigits of duck embryos, characterized by a rudimentary interdigital degenerative process. We conclude that the regression of the interdigital tissue involves a coordinated and sequential activation of the caspase and lysosomal degenerative molecular cascades.     

Mutations on the hinge region of leukocyte elastase inhibitor determine the loss of inhibitory function

Leukocyte elastase inhibitor (LEI) is a cytosolic component of lung macrophages and blood leukocytes that inhibits neutrophil elastase. LEI is a member of the serpin superfamily, these proteins, mostly protease inhibitors, are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the hinge region of the reactive centre loop into a beta-sheet of the inhibitor. In this work three mutations were produced in the hinge region of elastase inhibitor that abolish the inhibition activity of LEI and transform the protein in a substrate of the elastase. This result demonstrates that the inhibitory mechanism of serpin is common to LEI.     

Differential involvement of DNases in HeLa cell apoptosis induced by etoposide and long term-culture

We have applied to human HeLa cells two different stimuli of apoptosis: the antitumoral drug etoposide, and a more 'physiological' death condition, obtained by growing cells in the same medium for long time periods, for up to 10 days. Analysis of different parameters demonstrated that in both experimental systems the same apoptotic features are visible. However, the DNA degradation pattern appeared to be different, suggesting the involvement of different DNases. In this view, we have analyzed the activity and expression of Ca2+-Mg2+-dependent and acid DNases. We have observed that DNase I is not modulated during apoptosis. In contrast, the acid L-DNase II (derived from Leukocyte Elastase Inhibitor by post-translational modification), recently identified in our laboratory, is mainly active in the apoptotic pathway induced by long term-culture. Furthermore, we have provided evidence that while caspase 3 is activated by both inducers, caspase 1 is essential only for the etoposide-induced apoptosis.     

DNases and apoptosis.

Here we review the different apoptotic DNases. From a functional point of view, DNases implicated in apoptosis may be classified into three groups: the Ca2+/Mg2+ endonucleases, the Mg2+-endonucleases, and the cation-independent endonucleases. The first group includes DNase I which has no specificity for the linker region, DNase gamma which has some homology with DNase I, and other DNases which cleave DNA in the linker region. Both DNase I and DNase gamma have been cloned. The other nucleases of this category have dispersed molecular weights. Their sequences are unknown and it is difficult to determine their role(s) in apoptosis. It seems that different pathways are present and that these nucleases may be activated either by caspases or serine proteases. The caspase 3 activated DNase (CAD, CPAN, or DFF40) belongs to the Mg2+-dependent endonucleases. DNase II belongs to the third group of acid endonucleases or cation-independent DNases. We have shown the involvement of DNase II in lens cell differentiation. Recently, the molecular structure of two different enzymes has been elucidated, one of which has a signal peptide and appears to be secreted. The other, called L-DNase II, is an intracellular protein having two enzymatic activities; in its native form, it is an anti-protease, and after posttranslational modification, it becomes a nuclease.     

Apoptosis-inducing factor (AIF) and leukocyte elastase inhibitor/l-DNase II (LEI/LDNaseII), can interact to conduct caspase-independent cell death

Programmed cell death is an important factor in tissue homeostasis. Lot of work has been performed to characterize the caspase-dependent cell death. Caspase-independent cell death, although important in many physiological situations, is less investigated. In this work we show that two caspase-independent effectors of cell death, namely apoptosis-inducing factor and leukocyte elastase inhibitor derived DNase II interact and can cooperate to induce cell death. These results contribute to the knowledge of molecular pathways of cell death, an important issue in the development of new therapeutic strategies for the treatment of cancer or neurodegenerative diseases.     

Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death).

Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.     

Multiple effects of the Na+/H+ antiporter inhibitor HMA on cancer cells

Amiloride derivatives are a class of new promising chemotherapeutic agents. A representative member of this family is the sodium–hydrogen antiporter inhibitor HMA (5-(N,N-hexamethylene amiloride), which has been demonstrated to induce cellular intracytosolic acidification and cell death through the apoptotic pathway(s). This work aims at characterizing drug response of human cancer cell lines to HMA. After a first screening revealing that HMA interferes with cancer cell survival, we focused our attention on SW613-B3 colon carcinoma cells, which are intrinsically resistant to a panel of drugs. Searching for the activation of canonical apoptosis, we found that this process was abortive, given that the final steps of this process, i.e. PARP-1 cleavage and DNA ladder, were not detectable. Thus, we addressed caspase-independent paradigms of cell death and we observed that HMA promotes the induction of the LEI/L-DNase II pathway as well as of parthanatos. Finally, we explored the possible impact of autophagy of cell response to HMA, providing the evidence that autophagy is activated in our experimental system. On the whole, our results defined the biochemical reactions triggered by HMA, and elucidated its multiple effects, thus adding further complexity to the intricate network leading to drug resistance.     

ZEN1 is a key enzyme in the degradation of nuclear DNA during programmed cell death of tracheary elements

Tracheary elements (TEs) have a unique cell death program in which the rapid collapse of the vacuole triggers the beginning of nuclear degradation. Although various nucleases are known to function in nuclear DNA degradation in animal apoptosis, it is unclear what hydrolase is involved in nuclear degradation in plants. In this study, we demonstrated that an S1-type nuclease, Zinnia endonuclease 1 (ZEN1), functions directly in nuclear DNA degradation during programmed cell death (PCD) of TEs. In-gel DNase assay demonstrated the presence of a 24-kD Ca(2+)/Mg(2+)-dependent nuclease and a 40-kD Zn(2+)-dependent nuclease as well as ZEN1 in 60-h-cultured cells that included differentiating TEs. Such cell extracts possessed the ability to degrade the nuclear DNA isolated from Zinnia elegans cells in the presence of Zn(2+), and its activity was suppressed by an anti-ZEN1 antibody, indicating that ZEN1 is a central DNase responsible for nuclear DNA degradation. The introduction of the antisense ZEN1 gene into Zinnia cells cultured for 40 h specifically suppressed the degradation of nuclear DNA in TEs undergoing PCD but did not affect vacuole collapse. Based on these results, a common mechanism between animal and plant PCD is discussed.     

A novel paraptosis pathway involving LEI/L-DNaseII for EGF-induced cell death in somato-lactotrope pituitary cells

We have recently reported that EGF triggers an original form of cell death in pituitary cell line (GH4C1) with a phenotype sharing some characteristics of both apoptosis (internucleosomal DNA fragmentation) and paraptosis (caspase-independence and cytoplasmic vacuolization). However, the endonuclease involved in EGF-induced DNA fragmentation has not been assessed so far. In the present work we therefore further explored the putative paraptosis involvement in EGF-induced cell death and asked whether L-DNaseII might be involved. Indeed, this endonuclease is known to mediate internucleosomal DNA fragmentation in caspase independent manner. Our Western blot, immunocytochemistry and enzymatic measurement assays show that EGF triggers a cleavage of Leukocyte Elastase Inhibitor (LEI) precursor into L-DNaseII, its subsequent enzymatic activation and nuclear translocation thus pointing to the involvement of this endonuclease pathway in caspase-independent DNA fragmentation. In addition, EGF-induced cell death can be blocked by paraptosis inhibitor AIP-1/Alix, but not with its anti-apoptotic C-terminal fragment (Alix-CT). Altogether these data suggest that EGF-induced cell death defines a novel, L-DNaseII-mediated form of paraptosis. This work was supported by fellowships from Fondation pour la Recherche medicale (FRM: FDT20030627283), from Association pour la Recherche contre le Cancer (ARC: ML/MLD/CM-A04/4) to JF and Retina France to AT and LP. These two authors equally contributed to this work.