Daxx-beta and Daxx-gamma, two novel splice variants of the transcriptional co-repressor Daxx.

Daxx is involved in transcriptional control and apoptosis. It comprises several domains, including a regulatory C terminus that is responsible for the interaction with numerous proteins such as p53, promyelocytic leukemia protein (PML), and Hsp27. Here, we describe the identification and characterization of two novel variants of Daxx termed Daxx-ß and Daxx-γ, which are generated by alternative splicing. Alternative splicing results in a truncated regulatory C terminus in both proteins. As a consequence, Daxx-ß and Daxx-γ show a markedly decreased affinity to PML, which in turn is associated with a different subnuclear localization of these proteins compared with Daxx. Although Daxx is localized mainly in PML-oncogenic domains (PODs) Daxx-ß and Daxx-γ display a distinct distribution pattern. Furthermore, Daxx-ß and Daxx-γ show a decreased affinity to p53 also due to the truncated C terminus. We provide evidence that the p53 recruitment into PODs is Daxx isoform-dependent. The decreased affinity of Daxx-ß/-γ to p53 and PML results in a diffuse localization of p53 throughout the nucleus. In contrast to Daxx, Daxx-ß and Daxx-γ are unable to repress p53-mediated transcription. Therefore, alternative splicing of Daxx might indicate an additional level in the cellular apoptosis network.

Nitric oxide-mediated protection of endothelial cells from hydrogen peroxide is mediated by intracellular zinc and glutathione.

Oxidative stress may cause endothelial dysfunction and vascular disease. It has been shown that NO protects endothelial cells (EC) against H(2)O(2)-induced toxicity. In addition, it is known that NO within cells induces a zinc release from proteins containing zinc-sulfur complexes. The aim of this study was to investigate whether zinc released intracellularly by NO plays a signaling role in the NO-mediated protection against H(2)O(2) in rat aortic EC. Our results show that the NO-mediated protection toward H(2)O(2) depends on the activities of glutathione peroxidase and glutamate cysteine ligase (GCL), the rate-limiting enzyme of glutathione (GSH) de novo biosynthesis. Moreover, NO increases the synthesis of the antioxidant GSH by inducing the expression of the catalytic subunit of GCL (GCLC). Chelating intracellular "free" zinc abrogates the NO-mediated increase of GCLC and of cellular GSH levels. As a consequence, the NO-mediated protection against H(2)O(2)-induced toxicity is impaired. We also show that under proinflammatory conditions, both cellular NO synthesis and intracellular "free" zinc are required to maintain the cellular GSH levels. Using RNA interference and laser scanning microscopy, we found that the NO-induced expression of GCLC depends on the activation of the transcription factor Nrf2 but not on the activity of the "zinc-sensing" transcription factor MTF-1. These findings show that intracellular "free" zinc plays a signaling role in the protective activity of NO and could explain why maintenance of an adequate zinc status in the endothelium is important to protect from oxidative stress and the development of vascular disease.

Zinc protects endothelial cells from hydrogen peroxide via Nrf2-dependent stimulation of glutathione biosynthesis.

Oxidative stress is one of the main causes of vascular disease. This study aims to investigate the antioxidant activity exerted by zinc in primary rat endothelial cells (EC). Using a 24-h treatment with hydrogen peroxide as a model for oxidative stress, we found that zinc supplementation protects from peroxide-induced cell death via increasing the transcription of the catalytic subunit (heavy chain) of glutamate-cysteine ligase (GCLC) and the concentrations of glutathione (GSH). Conversely, zinc depletion significantly decreased the expression of GCLC and the cellular GSH levels, resulting in an increased susceptibility of EC to oxidative stress. Using confocal microscopy and the RNA silencing technique, we found that zinc upregulates the expression of GCLC by activating the transcription factor Nrf2. Surprisingly, the intracellular zinc sensor, metal-responsive transcription factor-1, is not involved in the zinc-induced expression of GCLC. The present study shows that zinc controls the redox state of EC by regulating the de novo synthesis of GSH. This molecular mechanism may contribute to the elaboration of new nutritional and/or pharmaceutical approaches for protecting the endothelium against oxidative stress.

Evidence for a physiological role of intracellularly occurring photolabile nitrogen oxides in human skin fibroblasts.

Nitric oxide (NO) plays a pivotal role in human skin biology. Cutaneous NO can be produced enzymatically by NO synthases (NOS) as well as enzyme independently via photodecomposition of photolabile nitrogen oxides (PNOs) such as nitrite or nitroso compounds, both found in human skin tissue in comparably high concentrations. Although the physiological role of NOS-produced NO in human skin is well defined, nothing is known about the biological relevance or the chemical origin of intracellularly occurring PNOs. We here, for the first time, give evidence that in human skin fibroblasts (FB) PNOs represent the oxidation products of NOS-produced NO and that in human skin fibroblasts intracellularly occurring PNOs effectively protect against the injurious effects of UVA radiation by a NO-dependent mechanism. In contrast, in PNO-depleted FB cultures an increased susceptibility to UVA-induced lipid peroxidation and cell death is observed, whereas supplementation of PNO-depleted FB cultures with physiological nitrite concentrations (10 microM) or with exogenously applied NO completely restores UVA-increased injuries. Thus, intracellular PNOs are biologically relevant and represent an important initial shield functioning in human skin physiology against UVA radiation. Consequently, nonphysiological low PNO concentrations might promote known UVA-related skin injuries such as premature aging and carcinogenesis.

The impact of nitrite and antioxidants on ultraviolet-A-induced cell death of human skin fibroblasts.

Nitrite (NO(2)(-)) occurs ubiquitously in biological fluids such as blood and sweat. Ultraviolet A-induced nitric oxide formation via decomposition of cutaneous nitrite, accompanied by the production of reactive oxygen (ROS) or nitrogen species (RNS), represents an important source for NO in human skin physiology. Examining the impact of nitrite and the antioxidants glutathione (GSH), Trolox (TRL), and ascorbic acid (ASC) on UVA-induced toxicity of human skin fibroblasts (FB) we found that NO(2)(-) concentration-dependently enhances the susceptibility of FB to the toxic effects of UVA by a mechanism comprising enhanced induction of lipid peroxidation. While ASC completely protects FB cultures from UVA/NO(2)(-)-induced cell damage, GSH or TRL excessively enhances UVA/NO(2)(-)-induced cell death by a mechanism comprising nitrite concentration-dependent TRL radical formation or GSH-derived oxidative stress. Simultaneously, in the presence of GSH or TRL the mode of UVA/NO(2)(-)-induced cell death changes from apoptosis to necrosis. In summary, during photodecomposition of nitrite, ROS or RNS formation may act as strong toxic insults. Although inhibition of oxidative stress by NO and other antioxidants represents a successful strategy for protection from UVA/NO(2)(-)-induced injuries, GSH and TRL may nitrite-dependently aggravate the injurious impact by TRL or GSH radical formation, respectively.

The transforming acidic coiled coil 3 protein is essential for spindle-dependent chromosome alignment and mitotic survival.

Cancer-associated centrosomal transforming acidic coiled coil (TACC) proteins are involved in mitotic spindle function. By employing gene targeting, we have recently described a nonredundant and essential role of TACC3 in regulating cell proliferation. In this study, we used an inducible RNA interference approach to characterize the molecular function of TACC3 and its role in mitotic progression and cell survival. Our data demonstrate that a TACC3 knockdown arrests G(1) checkpoint-compromised HeLa cells prior to anaphase with aberrant spindle morphology and severely misaligned chromosomes. Interestingly, TACC3-depleted cells fail to accumulate the mitotic kinase Aurora B and the checkpoint protein BubR1 to normal levels at kinetochores. Moreover, localization of the structural protein Ndc80 at outer kinetochores is reduced, indicating a defective kinetochore-microtubule attachment in TACC3-deficient cells. As a consequence of prolonged TACC3 depletion, cells undergo caspase-dependent cell death that relies on a spindle checkpoint-dependent mitotic arrest. TACC3 knockdown cells that escape from this arrest by mitotic slippage become highly polyploid and accumulate supernumerary centrosomes. Similarly, deficiency of the post-mitotic cell cycle inhibitor p21(WAF) exacerbates the effects of TACC3 depletion. Our findings therefore point to an essential role of TACC3 in spindle assembly and cellular survival and identify TACC3 as a potential therapeutic target in cancer cells.