Christoph V. Suschek

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 inhibitorl-N 5-(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-ium-1,2-diolate at concentrations from 10 to 1000 μm 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.

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.

Regulation of zinc homeostasis by inducible NO synthase-derived NO: Nuclear metallothionein translocation and intranuclear Zn2+ release

Zn2+ is critical for the functional and structural integrity of cells and contributes to a number of important processes including gene expression. It has been shown that NO exogenously applied via NO donors resulting in nitrosative stress leads to cytoplasmic Zn2+ release from the zinc storing protein metallothionein (MT) and probably other proteins that complex Zn2+ via cysteine thiols. We show here that, in cytokine-activated murine aortic endothelial cells, NO derived from the inducible NO synthase (iNOS) induces a transient nuclear release of Zn2+. This nuclear Zn2+ release depends on the presence of MT as shown by the lack of this effect in activated endothelial cells from MT-deficient mice and temporally correlates with nuclear MT translocation. Data also show that NO is an essential but not sufficient signal for MT-mediated Zn2+ trafficking from the cytoplasm into the nucleus. In addition, we found that, endogenously via iNOS, synthesized NO increases the constitutive mRNA expression of both MT-1 and MT-2 genes and that nitrosative stress exogenously applied via an NO donor increases constitutive MT mRNA expression via intracellular Zn2+ release. In conclusion, we here provide evidence for a signaling mechanism based on iNOS-derived NO through the regulation of intracellular Zn2+ trafficking and homeostasis.

Arginase 1 Overexpression in Psoriasis: Limitation of Inducible Nitric Oxide Synthase Activity as a Molecular Mechanism for Keratinocyte Hyperproliferation

Abnormal proliferation of keratinocytes in the skin appears crucial to the pathogenesis of psoriasis, but the underlying mechanisms remain unknown. Nitric oxide (NO), released from keratinocytes at high concentrations, is considered a key inhibitor of cellular proliferation and inducer of differentiation in vitro. Although high-output NO synthesis is suggested by the expression of inducible NO synthase (iNOS) mRNA and protein in psoriasis lesions, the pronounced hyperproliferation of psoriatic keratinocytes may indicate that iNOS activity is too low to effectively deliver anti-proliferative NO concentrations. Here we show that arginase 1 (ARG1), which substantially participates in the regulation of iNOS activity by competing for the common substrate L-arginine, is highly overexpressed in the hyperproliferative psoriatic epidermis and is co-expressed with iNOS. Expression of L-arginine transporter molecules is found to be normal. Treatment of primary cultured keratinocytes with Th1-cytokines, as present in a psoriatic environment, leads to de novo expression of iNOS but concomitantly a significant down-regulation of ARG1. Persistent ARG1 overexpression in psoriasis lesions, therefore, may represent a disease-associated deviation from normal expression patterns. Furthermore, the culturing of activated keratinocytes in the presence of an ARG inhibitor results in a twofold increase in nitrite accumulation providing evidence for an L-arginine substrate competition in human keratinocytes. High-output NO synthesis is indeed associated with a significant decrease in cellular proliferation as shown by down-regulation of Ki67 expression in cultured keratinocytes but also in short-term organ cultures of normal human skin. In summary, our data demonstrate for the first time a link between a human inflammatory skin disease, limited iNOS activity, and ARG1 overexpression. This link may have substantial implications for the pathophysiology of psoriasis and the development of new treatment strategies.

Distinct in vivo expression patterns of survivin splice variants in renal cell carcinomas

Survivin, a novel member of the inhibitor of apoptosis protein (IAP) family, reduces the susceptibility of tumor cells to proapoptotic stimuli, thereby promoting tumor cell survival during tumor progression and treatment with anticancer drugs. Recently, we identified 2 novel alternative splice variants of survivin, survivin‐2B and survivin‐ΔEx3, which differ in their antiapoptotic properties. Survivin‐2B has lost its antiapoptotic potential and may act as a naturally occurring antagonist of antiapoptotic survivin and survivin‐ΔEx3. Because the in vivo expression of these splice variants in human cancer has not been analyzed so far, 57 renal cell carcinomas (RCCs) were explored using quantitative reverse transcriptase polymerase chain reaction. We found that all RCCs express survivin‐ΔEx3, survivin‐2B and survivin, the latter being the dominant transcript. When we compared early and intermediate stages with late stages of clear cell RCCs, no significant changes in the expression levels of survivin and survivin‐ΔEx3 became evident. However, a significant decrease was observed for the mRNA ratio between survivin‐2B and survivin in late tumor stages (p = 0.036). Chromophilic/papillary RCCs, which are known to be less aggressive than clear cell RCCs, did not show significantly lower expression levels of antiapoptotic survivin and survivin‐ΔEx3, compared with stage‐adjusted clear cell RCCs. Our study demonstrates for the first time in vivo expression of functionally different survivin variants and suggests a role of these survivin splice variants in the progression and clinical behavior of human RCCs.

Whole Body UVA Irradiation Lowers Systemic Blood Pressure by Release of Nitric Oxide From Intracutaneous Photolabile Nitric Oxide Derivates

Rationale: Human skin contains photolabile nitric oxide derivates like nitrite and S-nitroso thiols, which after UVA irradiation, decompose and lead to the formation of vasoactive NO. Objective: Here, we investigated whether whole body UVA irradiation influences the blood pressure of healthy volunteers because of cutaneous nonenzymatic NO formation. Methods and Results: As detected by chemoluminescence detection or by electron paramagnetic resonance spectroscopy in vitro with human skin specimens, UVA illumination (25 J/cm2) significantly increased the intradermal levels of free NO. In addition, UVA enhanced dermal S-nitrosothiols 2.3-fold, and the subfraction of dermal S-nitrosoalbumin 2.9-fold. In vivo, in healthy volunteers creamed with a skin cream containing isotopically labeled 15N-nitrite, whole body UVA irradiation (20 J/cm2) induced significant levels of 15N-labeled S-nitrosothiols in the blood plasma of light exposed subjects, as detected by cavity leak out spectroscopy. Furthermore, whole body UVA irradiation caused a rapid, significant decrease, lasting up to 60 minutes, in systolic and diastolic blood pressure of healthy volunteers by 11±2% at 30 minutes after UVA exposure. The decrease in blood pressure strongly correlated (R2=0.74) with enhanced plasma concentration of nitrosated species, as detected by a chemiluminescence assay, with increased forearm blood flow (+26±7%), with increased flow mediated vasodilation of the brachial artery (+68±22%), and with decreased forearm vascular resistance (−28±7%). Conclusions: UVA irradiation of human skin caused a significant drop in blood pressure even at moderate UVA doses. The effects were attributed to UVA induced release of NO from cutaneous photolabile NO derivates.

The Role of iNOS in Chronic Inflammatory Processes In Vivo: Is it Damage-Promoting, Protective, or Active at all?

The expression of the inducible nitric oxide synthase (iNOS) is one of the direct consequences of an inflammatory process. Early studies have focused on the potential toxicity of the ensuing high-output NO-synthesis serving as a means to eliminate pathogens or tumor cells but also contributing to local tissue destruction during chronic inflammation. More recently, however, data are accumulating on a protective effect of high-output NO synthesis and - equally important - on a gene-regulatory function that helps to mount a protective stress response and simultaneously aids in down-regulating the proinflammatory response. These findings appear to contrast to the often observed sustained iNOS-expression during chronic inflammatory diseases, as for instance in Psoriasis vulgaris and other conditions with a chronic Th1-like reactivity. We here pose the question as to whether the iNOS is really active in these diseases. We review the data accumulated on iNOS expression in chronic diseases. We also report on the various factors that potentially interfere with proper NO formation by the expressed enzyme. We also highlight the recent findings of how, why and where evidences emerge that impeded NO formation contributes to chronic disease processes and finally present details on our current understanding of such abnormally low NO synthesis and its contribution to the pathophysiological processes of the human proinflammatory skin disease Psoriasis vulgaris.

Cytotoxic action of IL-1β against pancreatic islets is mediated via nitric oxide formation and is inhibited by N G-monomethyl-l-arginine

IL‐1β has been previously shown to act as a cytotoxic agent in islets. Here we show by electron microscopy of alginate encapsulated islets, that islet cell lysis is induced by culturing islets for 24 or 48 h in the presence of IL‐1β. The extent of lysis depends on the IL‐1β concentration and is slightly enhanced by the addition of TNF‐α. Cells can be protected from lysis by N G‐monomethyl‐l‐arginine. Lysis is paralleled by an increase in nitrite concentration in culture supernatants of whole islets but not in supernatants of isolated endocrine cells. The results indicate that IL‐1β toxicity occurs via inducing in non‐endocrine islet cells the synthesis and release of nitric oxide, which has been shown earlier to be highly toxic for islet cells.

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.

Specific age-associated DNA methylation changes in human dermal fibroblasts

Epigenetic modifications of cytosine residues in the DNA play a critical role for cellular differentiation and potentially also for aging. In mesenchymal stromal cells (MSC) from human bone marrow we have previously demonstrated age-associated methylation changes at specific CpG-sites of developmental genes. In continuation of this work, we have now isolated human dermal fibroblasts from young (<23 years) and elderly donors (>60 years) for comparison of their DNA methylation profiles using the Infinium HumanMethylation27 assay. In contrast to MSC, fibroblasts could not be induced towards adipogenic, osteogenic and chondrogenic lineage and this is reflected by highly significant differences between the two cell types: 766 CpG sites were hyper-methylated and 752 CpG sites were hypo-methylated in fibroblasts in comparison to MSC. Strikingly, global DNA methylation profiles of fibroblasts from the same dermal region clustered closely together indicating that fibroblasts maintain positional memory even after in vitro culture. 75 CpG sites were more than 15% differentially methylated in fibroblasts upon aging. Very high hyper-methylation was observed in the aged group within the INK4A/ARF/INK4b locus and this was validated by pyrosequencing. Age-associated DNA methylation changes were related in fibroblasts and MSC but they were often regulated in opposite directions between the two cell types. In contrast, long-term culture associated changes were very consistent in fibroblasts and MSC. Epigenetic modifications at specific CpG sites support the notion that aging represents a coordinated developmental mechanism that seems to be regulated in a cell type specific manner.