Nuclear localization and DNA binding of ecdysone receptor and ultraspiracle.

The Ecdysone receptor (EcR) is distributed between cytoplasm and nucleus in CHO cells. Nuclear localization is increased by the ligand Muristerone A. The most important heterodimerization partner Ultraspiracle (Usp) is localized predominantly in the nucleus. We used the diethylentriamine nitric oxide adduct DETA/NO, which releases NO and destroys the zinc-finger structure of nuclear receptors, to investigate whether nuclear EcR and Usp interact with DNA. If expressed separately, Usp and EcR in the absence of hormone do not interact with DNA. The hormone-induced increase in nuclear EcR is due to enhanced DNA binding. In the presence of Usp, EcR is shifted nearly quantitatively into the nucleus. Only a fraction (approximately 30%) of the heterodimer is sensitive to DETA/NO. Interaction of the heterodimer with DNA is mediated mainly by the C-domain of EcR. Deletion of the DNA-binding domain of Usp only slightly reduces nuclear localization of EcR/Usp, although the nuclear localization signal of Usp is not present anymore. The results indicate that EcR and Usp can enter the nucleus independently, but cotransport of both receptors mediated by dimerization via the ligand binding domains is possible even in the absence of hormone.

Nitric oxide-mediated inhibition of androgen receptor activity: possible implications for prostate cancer progression.

Chronic inflammation increases the risk of cancer and many cancers, including prostate cancer, arise at sites of chronic inflammation. Inducible nitric oxide synthase (iNOS) is an enzyme dominantly expressed during inflammatory reactions. Although synthesis of high amounts of nitric oxide (NO) by iNOS has been demonstrated in pathophysiological processes, such as acute or chronic inflammation, autoimmune diseases or tumorigenesis, the role of iNOS activity in most of these diseases is poorly understood. Analysing prostate cancer biopsies by immunohistochemistry we found iNOS protein expression in tumor cells strongly paralleled by nitrotyrosine suggesting that iNOS is fully active. In vitro, NO inhibits androgen receptor-dependent promoter activity and prostate specific antigen production as well as DNA-binding activity of the androgen receptor (AR) in a concentration-dependent manner. Inhibition of the activity of androgen receptor-dependent reporter constructs is neither owing to diminished AR protein levels nor owing to an inhibition of its nuclear import. In addition, NO inhibits the proliferation of androgen receptor-positive prostate cancer cells significantly more efficiently than proliferation of androgen receptor-negative prostate cancer cells. In summary, our findings suggest that intratumoral iNOS activity favors development of prostate cancer cells that are able to proliferate androgen receptor-independently, thereby promoting prostate tumor progression.

Cysteine-Zn2+ complexes: unique molecular switches for inducible nitric oxide synthase-derived NO.

Nitric oxide (NO) in the low nanomolar range acts as a transcellular messenger molecule to initiate regulatory and physiological responses in nearby target cells via binding to the soluble guanylate cyclase heme moiety. Higher NO concentrations, as synthesized by the inducible NO synthase (iNOS) during inflammatory processes, show additional effects: NO may react with O2, yielding nitrogen oxides like N2O3 that are able to nitrosate thiols. A variety of proteins involved in very different functions of the cell contain cysteine-Zn2+ complexes. Effects of NO on different proteins containing cysteine-Zn2+ domains and playing essential roles during transcription, protein folding, and proteolysis are discussed. It is suggested that iNOS-derived NO acts as a signal molecule targeting cysteine-Zn2+ linkages, thus enabling cells to react toward nitrosative stress.

Zinc finger proteins as molecular targets for nitric oxide-mediated gene regulation.

A multiplicity of biological functions have been ascribed to nitric oxide (NO). It plays a significant role as a signal as well as a cytotoxic effector molecule. NO may, however, also play regulatory and/or modulatory roles in biology. A growing body of evidence suggests that posttranslational modifications of transcription factors serve a regulating role on gene transcription, particularly after changes of the redox state of the cell. Zinc fingers are the most prevalent transcription factor DNA-binding motif. As NO is able to S-nitrosate thiols of zinc-sulfur clusters leading to reversible disruption of zinc finger structures, this provides a molecular mechanism to regulate the transcription of genes. Current knowledge about effects of NO on the cellular zinc homeostasis and on the gene-regulating activity of zinc finger transcription factors is reviewed.

Nitric oxide inhibits the cochaperone activity of the RING finger-like protein DnaJ.

As a consequence of bacterial infection and the ensuing inflammation, expression of the inducible NO synthase results in prolonged synthesis of NO in high concentrations, which among other functions, contributes to the innate defense against the infectious agent. Here we show that NO inhibits the ability of the bacterial cochaperone DnaJ containing a RING finger-like domain to cooperate with the Hsp70 chaperone DnaK in mediating correct folding of denatured rhodanese. This inhibition is accompanied by S-nitrosation of DnaJ as well as by Zn2+ release from the protein. In contrast, NO has no effect on the activity of GroEL, a bacterial chaperone without zinc sulfur clusters. Escherichia coli cells lacking the chaperone trigger factor and thus relying on the DnaJ/DnaK system are more susceptible toward NO-mediated cytostasis than are wild-type bacteria. Our studies identify the cochaperone DnaJ as a molecular target for NO. Thus, an encounter of bacterial cells with NO can impair the protein folding activity of the bacterial chaperone system, thereby increasing bacterial susceptibility toward the defensive attack by the host.

Inducible nitric oxide synthase-derived nitric oxide in gene regulation, cell death and cell survival.

Studies from many laboratories have demonstrated the complex role of NO in inflammatory processes. Prolonged exposure to NO shifts the cellular redox potential to a more oxidized state and this is critically regulated by intracellular levels of reduced glutathione. NO-mediated stress will alter gene expression patterns, and the number of genes known to be involved is steadily increasing. Indeed, due to its S-nitrosating activity in the presence of oxygen, NO can modify the activity of transcription factors containing zinc finger motifs or cysteines within the DNA-binding domain. In addition, we are faced with not only NO acting as a powerful inducer of apoptosis or of necrosis in some cells, but also representing an equally powerful protection from cell death in many instances. Some of these apparent discrepancies may be explained by different capacities of cells to cope with the stress of NO exposure. Here, we review our findings on the complex impact of NO on transcriptional regulation of genes, cell death and cell survival. These NO-mediated actions will contribute to a better understanding of the impact of inducible nitric oxide synthase (iNOS) enzyme activity during inflammatory reactions.

Even after UVA-exposure will nitric oxide protect cells from reactive oxygen intermediate-mediated apoptosis and necrosis.

Reactive oxygen species (ROS) play a pivotal role in UVA-induced cell damage. As expression of the inducible nitric oxide synthase (iNOS) is a normal response of human skin to UV radiation we examined the role of nitric oxide (NO) as a protective agent during or even after UVA1- or ROS-exposure against apoptosis or necrosis of rat endothelial cells. When added during or up to 2 h subsequent to UVA1 or ROS exposure the NO-donor S-nitroso-cysteine (SNOC) at concentrations from 100-1000 microM significantly protects from both apoptosis as well as necrosis. The NO-mediated protection strongly correlates with complete inhibition of lipid peroxidation (sixfold increase of malonedialdehyde formation in untreated versus 1.2-fold with 1 mM SNOC). NO-mediated protection of membrane function was also shown by the inhibition of cytochrome c leakage in UVA1 treated cells, a process not accompanied by alterations in Bax and Bcl-2 protein levels. Thus, the experiments presented demonstrate that NO exposure during or even after a ROS-mediated toxic insult fully protects from apoptosis or necrosis by maintaining membrane integrity and function.

Nitric oxide inhibits endothelial IL-1[beta]-induced ICAM-1 gene expression at the transcriptional level decreasing Sp1 and AP-1 activity.

BACKGROUND: Nitric oxide (NO) has frequently been shown to inhibit leukocyte adherence to activated endothelium thus displaying anti-adhesive and immunosuppressive activities. A molecular mechanism contributing to this effect is described. MATERIALS AND METHODS: Primary murine aortic endothelial cells were activated with interleukin (IL)-1beta to express intercellular adhesion molecule-1 (ICAM-1) mRNA in the presence or absence of the physiological spontaneous NO-donor S-nitrosocysteine. Subsequently, semiquantitative RT-PCR and gel shift assays with nuclear extracts were performed to analyse the effects of NO on ICAM-1 mRNA expression and on the activity of transcription factors involved in ICAM-1 transcription. In addition, luciferase reporter gene activity of cytokine-activated cells transiently transfected with an ICAM-1 promoter-luciferase construct and cultured in the presence of the slow-releasing NO-donor DETA/NO was determined. RESULTS: NO at subtoxic concentrations decreases IL-1beta-induced endothelial ICAM-1 mRNA expression. This inhibition occurs at the transcriptional level, as NO affects IL-1b-induced ICAM-1 promoter activity in transiently transfected cells. Using gel-shift assays and double-stranded oligonucleotide consensus sequences of the known transcription factor binding sites of the ICAM-1 promoter, Sp1 and AP-1 were identified as transcriptional activators of IL-1beta-driven ICAM-1 expression. The DNA binding of both of these transcription factors to specific binding sites of the ICAM-1 promoter was decreased in MAEC exposed to NO. CONCLUSIONS: Our studies indicate that the anti-adhesive effect of NO concentrations equivalent to high-output NO synthesis is mediated, at least in part, by inhibition of ICAM-1 expression via a concerted action of NO on the redox-sensitive transcriptional activators Sp1 and AP-1. This molecular mechanism may contribute to the anti-inflammatory actions of NO synthesized by the inducible NO synthase.

Inactivation of zinc finger transcription factors provides a mechanism for a gene regulatory role of nitric oxide.

Nitric oxide (NO) is known to induce Zn(2+) release from the zinc-storing protein metallothionein and to induce Zn(2+) release within the nuclei and cytoplasm of cells. This suggests that zinc finger proteins may be primary targets of NO-induced stress. In this study, the specific interaction of the heterodimeric complex of two zinc finger transcription factors, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) receptor (VDR) and retinoid X receptor (RXR) with 1alpha,25(OH)(2)D(3) response elements (VDREs), was used as a model system. NO was applied to this system via the NO donors SNOC and MAMA/NO and caused a dose-dependent inhibition of VDR-RXR-VDRE complex formation (IC(50) values 0.5-0.8 mM). Ligand-bound or preformed complexes displayed less sensitivity to NO-induced stress. These in vitro effects of NO were found to be reversible. Functional assays in transiently transfected cells indicated that NO can also act in vivo as a repressor of 1alpha,25(OH)(2)D(3) signaling (IC(50) value of the slow NO donor DETA/NO, 0.5 mM). These findings suggest that NO has a modulatory role on transcription factors depending on their sensitivity to NO-induced stress, thus providing a mechanism for a gene regulatory function of NO.

Nitric oxide interferes with islet cell zinc homeostasis.

Zinc is crucial for the biosynthesis, storage, and secretion of insulin in pancreatic islet cells. We have previously presented evidence that NO interferes with cellular Zn(2+) homeostasis and we therefore investigated the influence of chronic NO exposure on the labile islet cell Zn(2+) content. A strong fluorescence activity in a large islet cell subpopulation was found after staining with the Zn(2+)-specific fluorophore Zinquin. Culture for 24 h in the presence of nontoxic concentrations of the slow-releasing NO donor DETA/NO resulted in a significantly reduced Zn(2+)-dependent fluorescence. This appears to be islet specific as in endothelial cells DETA/NO exposure enhanced the Zn(2+)-dependent fluorescence activity in a concentration-dependent manner. These results suggest that NO interferes with cellular Zn(2+) homeostasis, which in islet cells is crucial for proper hormone delivery and thus special cell function.

Implications of inducible nitric oxide synthase expression and enzyme activity.

We summarize here our current knowledge about inducible nitric oxide synthase (NOS) activity in human diseases and disorders. As basic research discovers more and more effects of low or high concentrations of NO toward molecular and cellular targets, successful therapies involving inhibition of NO synthesis or application of NO to treat human diseases are still lacking. This is in part due to the fact that the impact of NO on cell function or death are complex and often even appear to be contradictory. NO may be cytotoxic but may also protect cells from a toxic insult; it is apoptosis-inducing but also exhibits prominent anti-apoptotic activity. NO is an antioxidant but may also compromise the cellular redox state via oxidation of thiols like glutathione. NO may activate specific signal transduction pathways but is also reported to inhibit exactly these, and NO may activate or inhibit gene transcription. The situation may even be more complicated, because NO, depending on its concentration, may react with oxygen or the superoxide anion radical to yield reactive species with a much broader chemical reaction spectrum than NO itself. Thus, the action of NO during inflammatory reactions has to be considered in the context of timing and duration of its synthesis as well as stages and specific events in inflammation.

Influence of nitric oxide on the intracellular reduced glutathione pool: different cellular capacities and strategies to encounter nitric oxide-mediated stress.

Different cell types exhibit huge differences towards the cytotoxic action of NO. In search for an explanation, we used subtoxic concentrations of the NO-donors S-nitrosocysteine (SNOC) for short-term challenge and of (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (DETA/NO) for longer periods of exposure, respectively, and subtoxic concentrations of the oxidant H2O2 to determine the impact on intracellular reduced glutathione (GSH) concentrations. We find that GSH concentrations are always decreased, but that different cell types show different responses. Incubation of the relatively NO-sensitive murine lymphocytes with both NO-donors, but not with H2O2, resulted in a nearly complete loss of intracellular GSH. Short-term NO-treatment of P815 mastocytoma cells, also sensitive to NO-mediated cell death, decreased GSH to a similar extent only if either glutathione reductase (GSHR) activity or y-glutamylcysteine synthetase (gammaGCS) activity were inhibited concomitantly by specific inhibitors. Long-term NO-treatment of P815 cells, however, resulted in a significant decrease of GSH that could be further enhanced by inhibiting gammaGCS activity. In contrast, neither short-term nor long-term NO-exposure nor H2O2-treatment affected intracellular GSH levels of L929 fibroblasts, which were previously shown to be extremely resistant towards NO, whereas concomitant gammaGCS inhibition, but not GSHR inhibition, completely decreased GSH concentrations. These results show that different cell types use different pathways trying to maintain glutathione concentrations to cope with nitrosative stress, and the overall capability to maintain a critical amount of GSH correlates with susceptibility to NO-induced cell death.

Nitric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation.

A variety of toxic and modulating events induced by UVA exposure are described to cause cell death via apoptosis. Recently, we found that UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and endothelial cells (ECs). We have now searched for the role of iNOS expression and nitric oxide (NO) synthesis in UVA-induced apoptosis as detected by DNA-specific fluorochrome labeling and in DNA fragmentation visualized by in situ nick translation in ECs. Activation with proinflammatory cytokines 24 h before UVA exposure leading to iNOS expression and endogenous NO synthesis fully protects ECs from the onset of apoptosis. This protection was completely abolished in the presence of the iNOS inhibitor L-N5-(1-iminoethyl)-ornithine (0.25 mM). Additionally, preincubation of cells with the NO donor (Z)-1-[N(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-i um-1, 2-diolate at concentrations from 10 to 1000 microM as an exogenous NO-generating source before UVA irradiation led to a dose-dependent inhibition of both DNA strand breaks and apoptosis. In search of the molecular mechanism responsible for the protective effect, we find that protection from UVA-induced apoptosis is tightly correlated with NO-mediated increases in Bcl-2 expression and a concomitant inhibition of UVA-induced overexpression of Bax protein. In conclusion, we present evidence for a protective role of iNOS-derived NO in skin biology, because NO either endogenously produced or exogenously applied fully protects against UVA-induced cell damage and death. We also show that the NO-mediated expression modulation of proteins of the Bcl-2 family, an event upstream of caspase activation, appears to be the molecular mechanism underlying this protection.

Zinc finger transcription factors as molecular targets for nitric oxide-mediated immunosuppression: inhibition of IL-2 gene expression in murine lymphocytes.

BACKGROUND: Nitric oxide (NO) has frequently been shown to display immunosuppressive activities. We describe here a molecular mechanism contributing to this effect. MATERIALS AND METHODS: Murine T cell lymphoma EL4-6.1 cells were activated with the physiological stimulus interleukin (IL)-1beta to express IL-2 mRNA in the presence or absence of subtoxic concentrations of the physiological spontaneous NO donor S-nitrosocysteine (SNOC). Subsequently, semiquantitative RT-PCR and gel shift assays with nuclear extracts were performed to analyze the effects of NO on IL-2 mRNA expression and on the activity of the dominant regulating transcription factors Sp1, EGR-1, and NFATc. RESULTS: NO inhibits IL-1beta-induced IL-2 mRNA expression in EL4-6.1 cells. The suppressive activity of NO was concentration dependent and found to be completely reversible. Importantly, NO at the concentrations used induced neither apoptosis nor necrosis. Dominant regulation of IL-2 gene expression is known to reside in the zinc finger transcription factors Sp1 or EGR-1 and in the non-zinc finger protein NFAT. NO abrogates the DNA binding activities of recombinant Sp1 and EGR-1. More importantly, gel shift assays also showed a lack of DNA binding of native Sp1 derived from NO-treated nuclear extracts and that from NO-treated viable lymphocytes. This effect is selective, as the DNA binding activity of recombinant NFATc was not affected by NO. CONCLUSION: Inactivation of zinc finger transcription factors by NO appears to be a molecular mechanism in the immunosuppressive activity of NO in mammals, thus contributing to NO-mediated inhibition of IL-2 gene expression after physiological stimuli. The exact understanding of the molecular mechanism leading to NO-mediated, fully reversible suppression of immune reactions may lead to use of this naturally occurring tool as an aid in inflammatory diseases.

Biphasic effect of exogenous nitric oxide on proliferation and differentiation in skin derived keratinocytes but not fibroblasts.

Nitric oxide (NO) is known to exert cytotoxic and cytostatic effects in various cells and tissues. Although NO formation in human skin has been convincingly demonstrated, little is known about the NO-mediated effects in skin physiology and pathology. Here, we investigate the influence of NO on proliferation, differentiation, and apoptosis of primary cultures of normal human keratinocytes and fibroblasts. Four different NO donors at concentrations ranging from 0.01 to 5 mM were added every 12 h or 24 h to primary cultures of human keratinocytes and fibroblasts, and cells cultured for up to 3 d in the presence of these compounds. Cultures were examined for necrosis or apoptosis using trypan blue exclusion and in situ nick-translation. Cultures were also screened for the expression of the proliferation marker Ki67 and for an increase in cell numbers using neutral red staining. In addition, keratinocytes were stained for cytokeratin 6 expression to assess differentiation. We find that both keratinocytes and fibroblasts are highly resistant towards necrosis- or apoptosis-inducing effects of NO. In both cell types NO modulates cell growth, albeit in a cell-type specific pattern: cytostasis becomes significant in fibroblasts at concentrations of > or = 0.25 mM of the NO donor. In keratinocytes a biphasic effect is found with increased proliferation at low concentrations ranging from 0.01 to 0.25 mM and cytostasis at concentrations of > or = 0.5 mM. Conversely, expression of cytokeratin 6 is decreased at the lower NO donor concentrations and increased at higher concentrations as an indication of induction of differentiation at higher NO concentrations. Collectively, our results demonstrate that NO modulates proliferation and differentiation in human skin cells, a finding that will help to explain the pathophysiology of human skin diseases. Moreover, these findings suggest that NO generation in human skin diseases is not directly associated with local cell destruction, in contrast to findings in several other human diseases.

Nitric oxide: cytotoxicity versus cytoprotection--how, why, when, and where?

Nitric oxide (NO) has been found to play an important role as a signal molecule in many parts of the organism as well as a cytotoxic effector molecule of the nonspecific immune response. It appears paradoxical that NO on one side acts as a physiological intercellular messenger and on the other side may display cytotoxic activity in vivo. To make things even more complicated, cytoprotective properties of NO are also described. We here review the current understanding of cytotoxic versus cytoprotective effects of NO in mammalian cells and try to highlight the janus-faced properties of this important small molecule.

Nitric oxide mediates intracytoplasmic and intranuclear zinc release.

We previously described that NO. leads to destruction of ZnS clusters and release of Zn2+ from various proteins including zinc finger transcription factors. To assess the relevance in living cells, we investigated, whether exogenous NO. leads to an increase of cytoplasmic and nuclear free Zn2+. L929 cells, mouse splenocytes, or rat aorta endothelial cells were labeled with Zinquin-E, a Zn2+-specific fluorophore, and were treated with two different spontaneous NO donors, S-nitrosocysteine or DETA/NO. Both NO donors strongly increased the Zn2+-dependent fluorescence in the cellular cytosol and also in nuclei as compared to controls. NO-dependent Zn2+ release in splenocytes was quantitated by flow cytometry. These results show for the first time, that nitrosative stress mediates intracellular and intranuclear Zn2+ release which may be relevant in altering gene expression patterns.

Streptozotocin is not a spontaneous NO donor.

The reaction of streptozotocin with oxymyoglobin was analyzed and compared with results using various compounds that spontaneously generate nitric oxide in solution.