Self-regulatory role of 4-hydroxynonenal in signaling for stress-induced programmed cell death

Within the last two decades, 4-hydroxynonenal has emerged as an important second messenger involved in the regulation of various cellular processes. Our recent studies suggest that HNE can induce apoptosis in various cells through the death receptor Fas (CD95)-mediated extrinsic pathway as well as through the p53-dependent intrinsic pathway. Interestingly, through its interaction with the nuclear protein Daxx, HNE can self-limit its apoptotic role by translocating Daxx to cytoplasm where it binds to Fas and inhibits Fas-mediated apoptosis. In this paper, after briefly describing recent studies on various biological activities of HNE, based on its interactions with Fas, Daxx, and p53, we speculate on possible mechanisms through which HNE may affect a multitude of cellular processes and draw a parallel between signaling roles of H(2)O(2) and HNE.     

Daxx is required for stress-induced cell death and JNK activation

Daxx has been implicated in the modulation of apoptosis in response to various stimuli. In the nucleus, Daxx interacts and colocalizes with the promyelocytic leukemia protein (PML) into the PML-nuclear body. Moreover, overexpressed Daxx positively modulates FAS-ligand and TGFbeta-induced apoptosis. However, recent reports indicate that Daxx can also act as an antiapoptotic factor. As most studies on the role of Daxx in cell death have been conducted using tumour cell lines, we analysed the function of Daxx in physiological settings. We found that Daxx is induced upon exposure to ultraviolet (UV) irradiation and hydrogen peroxide treatment. We employed RNA interference to downregulate Daxx in primary fibroblasts. Remarkably, Daxx-depleted cells are resistant to cell death induced by both UV irradiation and oxidative stress. Furthermore, the downregulation of Daxx results in impaired MKK/c-Jun-N-terminal kinase (JNK) activation. This is the first evidence that Daxx promotes cell death and JNK activation in physiological conditions.     

Daxx enhances Fas-mediated apoptosis in a murine pro-B cell line,BAF3

Daxx has been shown to play an essential in type I interferon (IFN-/β)-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and nuclear translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the mechanism of Daxx-mediated apoptosis signaling in B lymphocyte progenitors, here we introduced an efficient suicide switch in a murine pro-B cell line, BAF3, by expressing FK506-binding protein-fused Fas intracellular domain (FKBP-Fas) and Daxx. It allows us to monitor Fas/Daxx-mediated signal by induction of Fas dimerization with the dimerizer drug AP20187. AP20187-mediated Fas dimerization induced not only apoptosis but also Jun N-terminal kinase (JNK) activation. However, AP20187 had no effect on cells expressing either Fas or Daxx only. Furthermore, expression of a JNK inhibitor, the JNK-binding domain of JIP-1, resulted in resistance to AP20187-mediated apoptosis in cells expressing FKBP-Fas and Daxx. These results imply that our novel suicide switch system may provide a powerful tool to delineate or identify the signaling molecules for Daxx-mediated apoptotic machinery in B lymphocyte progenitors through JNK activation.     

A kinase-independent function of Ask1 in caspase-independent cell death

Ask1 (apoptosis signal-regulating kinase 1) is activated as a consequence of cell exposure to a variety of stresses and can then initiate apoptosis. A known pathway of apoptosis downstream of Ask1 involves the activation of the stress-activated protein kinases, the release of cytochrome c from mitochondria, the activation of caspases, and the fragmentation of nuclei. Here, we characterized a novel mechanism of Ask1-mediated cell killing that is triggered by the interaction with Daxx. Co-transfection of Ask1 and Daxx induced a caspase-independent cell-death process characterized at the morphological level by distinctive crumpled nuclei easily distinguishable from the condensed and fragmented nuclei seen during classical caspase-dependent apoptosis. The kinase activity of Ask1 was not involved in this process, because mutants lacking kinase activity were as efficient as wild type Ask1 in mediating Daxx-induced cell death. Ask1N, a deletant that lacks the C-terminal half including the kinase domain of Ask1, was constitutively active in producing crumpled nuclei. In contrast, Ask1DeltaN, the reciprocal deletant that possesses constitutive kinase activity, produced fragmented nuclei typical of caspase-dependent death processes. We conclude that in addition to a caspase-dependent pro-apoptotic function that depends on its kinase activity, Ask1 possesses a caspase-independent killing function that is independent on its kinase activity and is activable by interaction with Daxx. In the physiological situation, such an activity is induced as a consequence of the translocation of Daxx from the nucleus to the cytoplasm, a condition that occurs following activation of the death receptor Fas.     

Dissociation of Akt1 from its negative regulator JIP1 is mediated through the ASK1-MEK-JNK signal transduction pathway during metabolic oxidative stress: a negative feedback loop

We have previously observed that metabolic oxidative stress-induced death domain-associated protein (Daxx) trafficking is mediated by the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. The relocalized Daxx from the nucleus to the cytoplasm during glucose deprivation participates in a positive regulatory feedback loop by binding to apoptosis signal-regulating kinase (ASK) 1. In this study, we report that Akt1 is involved in a negative regulatory feedback loop during glucose deprivation. Akt1 interacts with c-Jun NH(2)-terminal kinase (JNK)-interacting protein (JIP) 1, and Akt1 catalytic activity is inhibited. The JNK2-mediated phosphorylation of JIP1 results in the dissociation of Akt1 from JIP1 and subsequently restores Akt1 enzyme activity. Concomitantly, Akt1 interacts with stress-activated protein kinase/extracellular signal-regulated kinase (SEK) 1 (also known as MKK4) and inhibits SEK1 activity. Knockdown of SEK1 leads to the inhibition of JNK activation, JIP1-JNK2 binding, and the dissociation of Akt1 from JIP1 during glucose deprivation. Knockdown of JIP1 also leads to the inhibition of JNK activation, whereas the knockdown of Akt1 promotes JNK activation during glucose deprivation. Altogether, our data demonstrate that Akt1 participates in a negative regulatory feedback loop by interacting with the JIP1 scaffold protein.     

TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has a principal role in growth control through both its cytostatic effect on many different epithelial cell types and its ability to induce programmed cell death in a variety of other cell types. Here we have used a screen for proteins that interact physically with the cytoplasmic domain of the type II TGF-beta receptor to isolate the gene encoding Daxx - a protein associated with the Fas receptor that mediates activation of Jun amino-terminal kinase (JNK) and programmed cell death induced by Fas. The carboxy-terminal portion of Daxx functions as a dominant-negative inhibitor of TGF-beta-induced apoptosis in B-cell lymphomas, and antisense oligonucleotides to Daxx inhibit TGF-beta-induced apoptosis in mouse hepatocytes. Furthermore, Daxx is involved in mediating JNK activation by TGF-beta. Our findings associate Daxx directly with the TGF-beta apoptotic-signalling pathway, and make a biochemical connection between the receptors for TGF-beta and the apoptotic machinery.     

Pin1-dependent prolyl isomerization modulates the stress-induced phosphorylation of high molecular weight neurofilament protein

Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis, but the underlying mechanisms are still unclear. Previous studies have shown that Pin1, a peptidylprolyl cis/trans-isomerase, may be actively involved in the regulation of Tau hyperphosphorylation in AD. Here, we show that Pin1 modulates oxidative stress-induced NF-H phosphorylation. In an in vitro kinase assay, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline-directed kinases, Erk1/2, Cdk5/p35, and JNK3 in a concentration-dependent manner. In vivo, dominant-negative (DN) Pin1 and Pin1 small interfering RNA inhibited epidermal growth factor-induced NF-H phosphorylation. Because oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed cortical neurons and HEK293 cells. Both hydrogen peroxide (H(2)O(2)) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 short interference RNA and DN-Pin1 rescues the effect of stress-induced NF-H phosphorylation. The H(2)O(2) and heat shock induced perikaryal phospho-NF-H accumulations, and neuronal apoptosis was rescued by inhibition of Pin1 in cortical neurons. JNK3, a brain-specific JNK isoform, is activated under oxidative and heat stresses, and inhibition of Pin1 by Pin1 short interference RNA and DN-Pin1 inhibits this pathway. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and amyotrophic lateral sclerosis. Thus, Pin1 may be a potential therapeutic target for these diseases.     

Positive regulation of apoptosis signal-regulating kinase 1 by hD53L1

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase family member that plays a central role in cytokine- and stress-induced apoptosis by activating c-Jun N-terminal kinase and p38 signaling cascades. ASK1-induced apoptotic activity is up-regulated by two cellular factors, Daxx and TRAF2, through direct protein-protein interactions. Daxx and TRAF2 are death receptor-associated proteins in Fas and tumor necrosis factor-alpha pathways, respectively. Recent studies suggest that calcium signaling may regulate ASK1 pathway. Here we report that human D53L1, a member of the tumor protein D52 family involved in cell proliferation and calcium signaling, up-regulates the ASK1-induced apoptosis. The human D53L1 physically interacts with the C-terminal regulatory domain of ASK1 and promotes ASK1-induced apoptotic activity by activating caspase signaling in mammalian cells. In luciferase reporter assays, hD53L1 activates c-Jun N-terminal kinase-mediated transactivation in the presence of ASK1. Expression of hD53L1 enhances autophosphorylation and kinase activity of ASK1 but has no effect on ASK1 oligomerization that is necessary for kinase activity and on binding of ASK1 to MKK6, a downstream factor of ASK1. Taken together, these results suggest that activation of ASK1 by hD53L1 may provide a novel mechanism for ASK1 regulation.     

C-terminus of heat shock protein 70-interacting protein facilitates degradation of apoptosis signal-regulating kinase 1 and inhibits apoptosis signal-regulating kinase 1-dependent apoptosis

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that is regulated under conditions of cellular stress. ASK1 phosphorylates c-Jun N-terminal kinase (JNK) and elicits an apoptotic response. ASK1 activity is regulated at multiple levels, 1 of which is through inhibition by cytosolic chaperones of the heat shock protein (Hsp) 70 family. Among the proteins that determine Hsp70 function, CHIP (C-terminus of Hsp70-interacting protein) is a cochaperone and ubiquitin ligase that interacts with Hsp70 through an amino-terminal tetratricopeptide repeat (TPR) domain. Prominent among the cellular functions mediated by CHIP is protection against physiologic stress. Because ASK1 is known to contain a TPR-acceptor site, we examined the role of CHIP in regulating ASK1 function. CHIP interacted with ASK1 in a TPR-dependent fashion and induced ubiquitylation and proteasome-dependent degradation of ASK1. Targeting of ASK1 by CHIP inhibited JNK activation in response to oxidative challenge and reduced ASK1-dependent apoptosis, whereas short interfering RNA (siRNA)-dependent depletion of CHIP enhanced JNK activation. Consistent with its ability to reduce cytoplasmic ASK1 levels, CHIP triggered the translocation of ASK1 partner protein death-associated protein (Daxx) into the nucleus, where it is known to activate an antiapoptotic response. These results indicate that CHIP regulates ASK1 activity by inducing its ubiquitylation and degradation, which, together with its effects on Daxx localization, provides a mechanism for the antiapoptotic effects of CHIP observed in the face of cellular and physiologic stress.     

Negative control of p53 by Sir2alpha promotes cell survival under stress.

The NAD-dependent histone deacetylation of Sir2 connects cellular metabolism with gene silencing as well as aging in yeast. Here, we show that mammalian Sir2alpha physically interacts with p53 and attenuates p53-mediated functions. Nicotinamide (Vitamin B3) inhibits an NAD-dependent p53 deacetylation induced by Sir2alpha, and also enhances the p53 acetylation levels in vivo. Furthermore, Sir2alpha represses p53-dependent apoptosis in response to DNA damage and oxidative stress, whereas expression of a Sir2alpha point mutant increases the sensitivity of cells in the stress response. Thus, our findings implicate a p53 regulatory pathway mediated by mammalian Sir2alpha. These results have significant implications regarding an important role for Sir2alpha in modulating the sensitivity of cells in p53-dependent apoptotic response and the possible effect in cancer therapy.     

Daxx deletion mutant (amino acids 501-625)-induced apoptosis occurs through the JNK/p38-Bax-dependent mitochondrial pathway

Death-associated protein (Daxx) deletion mutant (aa 501-625) has been known to be an inducer of apoptosis. In this study, we observed that the Bax-dependent mitochondrial death signaling pathway plays an important role in Daxx501-625-induced apoptosis. Daxx fragment-induced activation of caspase-9 and -3 was mediated through the apoptosis signal-regulating kinase 1 (ASK1)-MEK-c-Jun-N-terminal kinase (JNK)/p38-Bax pathway. By overexpressing JNK-binding domain (JBD) of JIP1, a JNK-inhibitory protein, and treatment with SB203580, a specific p38 inhibitor, DU-145 cells were made resistant to Daxx501-625-induced apoptosis. Capase-3 deficiency, Bax deficiency, or overexpression of a dominant-negative caspase-9 mutant prevented apoptosis, even though the Daxx501-625 fragment still activated the ASK1-MEK-MAPK pathway. Interestingly, Daxx501-625-induced Bcl-2 interacting domain (Bid) cleavage was suppressed in the dominant-negative caspase-9 mutant cells, whereas Bim was still phosphorylated in these cells. These results suggest that cleavage of Bid occurs downstream of caspase-9 activation. In contrast, phosphorylation of Bim is upstream of caspase-9 activation. Taken together, our results suggest that Daxx501-625-induced apoptosis is mediated through the ASK1-MEK-JNK/p38-Bim-Bax-dependent caspase pathway.     

Daxx Upregulation within the Cytoplasm of Reovirus-Infected Cells Is Mediated by Interferon and Contributes to Apoptosis

Reovirus infection is a well-characterized experimental system for the study of viral pathogenesis and antiviral immunity within the central nervous system (CNS). We have previously shown that c-Jun N-terminal kinase (JNK) and the Fas death receptor each play a role in neuronal apoptosis occurring in reovirus-infected brains. Death-associated protein 6 (Daxx) is a cellular protein that mechanistically links Fas signaling to JNK signaling in several models of apoptosis. In the present study, we demonstrate that Daxx is upregulated in reovirus-infected brain tissue through a type I interferon-mediated mechanism. Daxx upregulation is limited to brain regions that undergo reovirus-induced apoptosis and occurs in the cytoplasm and nucleus of neurons. Cytoplasmic Daxx is present in Fas-expressing cells during reovirus encephalitis, suggesting a role for Daxx in Fas-mediated apoptosis following reovirus infection. Further, in vitro expression of a dominant negative form of Daxx (DN-Daxx), which binds to Fas but which does not transmit downstream signaling, inhibits apoptosis of reovirus-infected cells. In contrast, in vitro depletion of Daxx results in increased expression of caspase 3 and apoptosis, suggesting that Daxx plays an antiapoptotic role in the nucleus. Overall, these data imply a regulatory role for Daxx in reovirus-induced apoptosis, depending on its location in the nucleus or cytoplasm.     

Epstein-Barr virus-encoded poly(A)- RNA confers resistance to apoptosis mediated through Fas by blocking the PKR pathway in human epithelial intestine 407 cells

Our recent findings demonstrated that the Epstein-Barr virus-encoding small nonpolyadenylated RNA (EBER) confers resistance to various apoptotic stimuli and contributes to the maintenance of malignant phenotypes in Burkitt's lymphoma. In this study we investigated the role of EBER in the human epithelial Intestine 407 cell line, which is known to be susceptible to Fas (Apo1/CD95)-mediated apoptosis. Fas, a member of the tumor necrosis factor receptor family, transduces extracellular signals to the apoptotic cellular machinery, leading to cell death. Transfection of the EBER gene into Intestine 407 cells significantly protected the cells from Fas-mediated apoptosis, whereas EBER-negative cell lines underwent apoptosis after Fas treatment. EBER bound double-stranded RNA-dependent protein kinase R (PKR), an interferon-inducible serine/threonine kinase, and abrogated its kinase activity. Moreover, expression of the catalytically inactive dominant-negative PKR provided resistance to Fas-induced apoptosis. Expression of EBER or dominant-negative PKR also inhibited the cleavage of poly(ADP-ribose) polymerase, a mediator of the cellular response to DNA damage, downstream of the Fas-mediated apoptotic pathway. These results in combination indicate that EBER confers resistance to Fas-mediated apoptosis by blocking PKR activity in Intestine 407 cells, consistent with the idea that EBER contributes to the maintenance of epithelioid malignancies.