Edina Bakondi

Detection of Poly(ADP-ribose) Polymerase Activation in Oxidatively Stressed Cells and Tissues Using Biotinylated NAD Substrate

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by DNA damage. Activated PARP cleaves NAD+ into nicotinamide and (ADP-ribose) and polymerizes the latter on nuclear acceptor proteins. Over-activation of PARP by reactive oxygen and nitrogen intermediates represents a pathogenetic factor in various forms of inflammation, shock, and reperfusion injury. Using a novel commercially available substrate, 6-biotin-17-nicotinamide-adenine-dinucleotide (bio-NAD+), we have developed three applications, enzyme cytochemistry, enzyme histochemistry, and cell ELISA, to detect the activation of PARP in oxidatively stressed cells and tissues. With the novel assay we were able to detect basal and hydrogen peroxide-induced PARP activity in J774 macrophages. We also observed that mitotic cells display remarkably elevated PARP activity. Hydrogen peroxide-induced PARP activation could also be detected in wild-type peritoneal macrophages but not in macrophages from PARP-deficient mice. Application of hydrogen peroxide to the skin of mice also induced bio-NAD+ incorporation in the keratinocyte nuclei. Hydrogen peroxide-induced PARP activation and its inhibition by pharmacological PARP inhibitors could be detected in J774 cells with the ELISA assay that showed good correlation with the traditional [3H]-NAD incorporation method. The bio-NAD+ assays represent sensitive, specific, and non-radioactive alternatives for detection of PARP activation.

Nitric oxide‐peroxynitrite‐poly(ADP‐ribose) polymerase pathway in the skin

Abstract: In the last decade it has become well established that in the skin, nitric oxide (NO), a diffusable gas, mediates various physiologic functions ranging from the regulation of cutaneous blood flow to melanogenesis. If produced in excess, NO combines with superoxide anion to form peroxynitrite (ONOO–), a cytotoxic oxidant that has been made responsible for tissue injury during shock, inflammation and ischemia‐reperfusion. The opposite effects of NO and ONOO– on various cellular processes may explain the ‘double‐edged sword’ nature of NO depending on whether or not cellular conditions favour peroxynitrite formation. Peroxynitrite has been shown to activate the nuclear nick sensor enzyme, poly(ADP‐ribose) polymerase (PARP). Overactivation of PARP depletes the cellular stores of NAD+, the substrate of PARP, and the ensuing ‘cellular energetic catastrophy’ results in necrotic cell death. Whereas the role of NO in numerous skin diseases including wound healing, burn injury, psoriasis, irritant and allergic contact dermatitis, ultraviolet (UV) light‐induced sunburn erythema and the control of skin infections has been extensively documented, the intracutaneous role of peroxynitrite and PARP has not been fully explored. We have recently demonstrated peroxynitrite production, DNA breakage and PARP activation in a murine model of contact hypersensitivity, and propose that the peroxynitrite‐PARP route represents a common pathway in the pathomechanism of inflammatory skin diseases. Here we briefly review the role of NO in skin pathology and focus on the possible roles played by peroxynitrite and PARP in various skin diseases.

PARTIAL PROTECTION BY POLY(ADP-RIBOSE) POLYMERASE INHIBITORS FROM NITROXYL-INDUCED CYTOTOXICITY IN THYMOCYTES

Nitroxyl (NO(-)/HNO), has been proposed to be one of the NO(*)-derived cytotoxic species. Although the biological effect of nitroxyl is largely unknown, it has been reported to cause DNA breakage and cytotoxicity. We have therefore investigated whether NO(-)/HNO-induced DNA single-strand breakage activates the nuclear nick sensor enzyme poly(ADP-ribose) polymerase (PARP) and whether PARP activation affects the mode of NO(-)/HNO- induced cell death. NO(-)/HNO generated from Angelis salt (AS, sodium trioxodinitrate) (0-300 microM) induced DNA single-strand breakage, PARP activation, and a concentration-dependent cytotoxicity in murine thymocytes. AS-induced cell death was also accompanied by decreased mitochondrial membrane potential and increased secondary superoxide production. The cytotoxicity of AS, as measured by propidium iodide uptake, was abolished by electron acceptors potassium ferricyanide, TEMPOL, the intracellular calcium chelator BAPTA-AM, and by PARP inhibitors 3-aminobenzamide (3-AB) and PJ-34. The cytoprotective effect of 3-AB was paralleled by increased output of AS-induced apoptotic parameters such as phosphatidylserine exposure, caspase activation, and DNA fragmentation. No significant increase in tyrosine nitration could be observed in AS-treated thymocytes as opposed to peroxynitrite-treated cells, indicating that tyrosine nitration is not likely to contribute to NO(-)/HNO-induced cytotoxicity. Our results demonstrate that NO(-)/HNO-induced PARP activation shifts the default apoptotic cell death toward necrosis in thymocytes. However, as total PARP inhibition resulted only in 30% cytoprotection, PARP-independent mechanisms dominate NO(-)/HNO-induced cytotoxicity in thymocytes.

Cytoprotective effect of gallotannin in oxidatively stressed HaCaT keratinocytes: The role of poly(ADP-ribose) metabolism

Abstract:  Oxidative stress‐induced cytotoxicity is mediated in part by accelerated poly‐ADP ribosylation. Peroxynitrite and hydrogen peroxide cause DNA breakage triggering the activation of the DNA nick sensor enzyme poly(ADP‐ribose) polymerase‐1 (PARP‐1). Overactivation of PARP‐1 leads to cell dysfunction and cell death mainly due to depletion of NAD+ (the substrate of PARP‐1) and ATP. PARP‐1 attaches most ADP‐ribose residues onto itself, leading to downregulation of enzyme activity. Here, we have investigated the role of poly(ADP‐ribose) glycohydrolase (PARG), the poly(ADP‐ribose)‐catabolyzing enzyme in oxidative stress‐induced cytotoxicity in HaCaT cells. We have found that inhibition of PARG by gallotannin (GT) (50 µM) provided significant cytoprotection to peroxynitrite‐ or hydrogen peroxide‐treated HaCaT cells, as assessed by lactate dehydrogenase release and propidium iodide uptake (parameters of necrotic cell death) as well as caspase activation (apoptotic parameter). GT pretreatment has also inhibited the depletion of cellular NAD+ pools in hydrogen peroxide‐ or peroxynitrite‐treated HaCaT cells. GT caused the accumulation of poly(ADP‐ribose) and concomitant inhibition in cellular PARP activity in oxidatively stressed cells. Therefore, PARG is likely to contribute to maintaining the active state of PARP‐1 by continuously removing inhibitory ADP‐ribose residues from PARP‐1.

Age-related loss of stress-induced nuclear proteasome activation is due to low PARP-1 activity

Changes in protein turnover are among the dominant metabolic changes during aging. Of special importance is the maintenance of nuclear protein homeostasis to ensure a coordinated cellular metabolism. Therefore, in the nucleus a special PARP-1-mediated mechanism of proteasomal activation exists to ensure a rapid degradation of oxidized nuclear proteins. It was already demonstrated earlier that the cytosolic proteasomal system declines dramatically with aging, whereas the nuclear proteasome remains less affected. We demonstrate here that the stress-mediated proteasomal activation in the nucleus declines during replicative senescence of human fibroblasts. Furthermore, we clearly show that this decline in the PARP-1-mediated proteasomal activation is due to a decline in the expression and activity of PARP-1 in senescent fibroblasts. In a final study we show that this process also happens in vivo, because the protein expression level of PARP-1 is significantly lower in the skin of aged donors compared to that of young ones. Therefore, we conclude that the rate-limiting factor in poly(ADP-ribose)-mediated proteasomal activation in oxidative stress is PARP-1 and not the nuclear proteasome itself.

Poly(ADP-Ribose) Polymerase Mediates Inflammation in a Mouse Model of Contact Hypersensitivity

Mrowietz U, Christophers E, Altmeyer P (1999) Treatment of severe psoriasis with fumaric acid esters: scientific background and guidelines for therapeutic use. The German Fumaric Acid Ester Consensus Conference. Br J Dermatol 141:424–9 Nelson KC, Carlson JL, Newman ML, Sternberg P Jr, Jones DP, Kavanagh TJ et al. (1999) Effect of dietary inducer dimethylfumarate on glutathione in cultured human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 40:1927–35

The role of p38 signaling and poly(ADP-ribosyl)ation-induced metabolic collapse in the osteogenic differentiation-coupled cell death pathway

Osteogenic differentiation is a multistep process regulated by a diverse set of morphogenic and transcription factors. Previously we identified endogenous hydrogen peroxide-induced poly(ADP-ribose) polymerase-1 (PARP1) activation as a mediator of osteodifferentiation and associated cell death. Here we set out to investigate whether or not activation of PARP1 is dependent on DNA breaks and how PARP1 mediates cell death during osteodifferentiation of mesenchymal stem cells and SAOS-2 cells. Here we show that the MAP kinases p38, JNK, and ERK1/2 become activated during the differentiation process. However, only p38 activation depended both on hydrogen peroxide production and on PARP1 activation as the hydrogen peroxide decomposing enzyme catalase, the PARP inhibitor PJ34, and the silencing of PARP1 suppressed p38 activation. Inhibition of p38 suppressed cell death and inhibited osteogenic differentiation (calcium deposition, alkaline phosphatase activity, and marker gene expression) providing further support for the close coupling of osteodifferentiation and cell death. Metabolic collapse appears to be central in the hydrogen peroxide-PARP1-p38 pathway as silencing PARP1 or inhibition of p38 prevented differentiation-associated loss of cellular NAD, inhibition of mitochondrial respiration, and glycolytic activity. We also provide evidence that endogenous hydrogen peroxide produced by the differentiating cells is sufficient to cause detectable DNA breakage. Moreover, p38 translocates from the cytoplasm to the nucleus where it interacts and colocalizes with PARP1 as detected by immunoprecipitation and immunofluorescence, respectively. In summary, hydrogen peroxide-induced PARP1 activation leads to p38 activation and this pathway is required both for the successful completion of the differentiation process and for the associated cell death.

Diabetes-induced oxidative stress in the vitreous humor

Purpose Diabetes is accompanied by fundamental rearrangements in redox homeostasis. Hyperglycemia triggers the production of reactive oxygen and nitrogen species which contributes to tissue damage in various target organs. Proliferative diabetic retinopathy (PDR) is a common manifestation of diabetic complications but information on the possible role of reactive intermediates in this condition with special regard to the involvement of the vitreous in PDR-associated redox alterations is scarce. The aim of the study was to determine key parameters of redox homeostasis [advanced glycation endproducts (AGE); protein carbonyl and glutathione (GSH)] content in the vitreous in PDR patients. Methods The study population involved 10 diabetic patients undergoing surgery for complications of proliferative diabetic retinopathy and 8 control (non-diabetic) patients who were undergoing surgery for epiretinal membranes. Vitreal fluids were assayed for the above biochemical parameters. Results We found elevated levels of AGE in the vitreous of PDR patients (812.10 vs 491.69 ng AGE/mg protein). Extent of protein carbonylation was also higher in the samples of diabetic patients (2.08 vs 0.67 A/100 μg protein). The GSH content also increased in the vitreous of PDR patients as compared to the control group (4.54 vs 2.35 μmol/μg protein), respectively. Conclusion The study demonstrates that diabetes-associated redox alterations also reach the vitreous with the most prominent changes being increased protein carbonylation and increased antioxidant levels.