Theophylline prevents NAD+ depletion via PARP-1 inhibition in human pulmonary epithelial cells

Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD+, resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD+ pool, and of NAD+-dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD+ levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzyme inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies.     

PARP-1-induced cell death through inhibition of the MEK/ERK pathway in MNNG-treated HeLa cells

Poly(ADP-ribose) polymerase-1 (PARP-1) hyper-activation promotes cell death but the signaling events downstream of PARP-1 activation are not fully identified. To gain further information on the implication of PARP-1 activation and PAR synthesis on signaling pathways influencing cell death, we exposed HeLa cells to the DNA alkylating agent N-methyl-N′-methyl-nitro-N-nitrosoguanidine (MNNG). We found that massive PAR synthesis leads to down-regulation of ERK1/2 phosphorylation, Bax translocation to the mitochondria, release of cytochrome c and AIF and subsequently cell death. Inhibition of massive PAR synthesis following MNNG exposure with the PARP inhibitor PJ34 prevented those events leading to cell survival, whereas inhibition of ERK1/2 phosphorylation by inhibiting MEK counteracted the cytoprotective effect of PJ34. Together, our results provide evidence that PARP-1-induced cell death by MNNG exposure in HeLa cells is mediated in part through inhibition of the MEK/ERK signaling pathway and that inhibition of massive PAR synthesis by PJ34, which promotes sustained activation of ERK1/2, leads to cytoprotection.     

Metabolism of NAD+ in Nuclei of Saccharomyces cerevisiae during Stimulation of Its Biosynthesis by Nicotinamide

The activities of nuclear enzymes involved in NAD⁺ metabolism in Saccharomyces cerevisiae strain 913a-1 and its mutant 110 previously selected as an NAD⁺ producer were investigated. The presence of extracellular nicotinamide increased the total NAD⁺ pool in the cells and increased [³H]nicotinic acid incorporation; however, NAD⁺ concentration in isolated nuclei decreased slightly. The stimulating effect of nicotinamide on intracellular synthesis of NAD⁺ correlated with increases in ADP-ribosyl transferase, NAD⁺-pyrophosphorylase, and NAD⁺ ase activities.     

Photocatalyzed Anaerobic Oxidation of Nicotinamide Coenzyme Dimers to NAD+ by Adriamycin

The nicotinamide adenine dinucleotide dimers (NAD)₂ obtained by electrochemical reduction of NAD⁺ are oxidized by adriamycin in anaerobic photocatalyzed reaction yielding NAD⁺ and 7-deoxyadriamyci-none. Under the same conditions NADH is not oxidized.     

Tankyrase-1 overexpression reduces genotoxin-induced cell death by inhibiting PARP1

Poly(ADP-ribose) polymerases or PARPs are a family of NAD⁺-dependent enzymes that modify themselves and other substrate proteins with ADP-ribose polymers. The founding member PARP1 is localized predominantly in the nucleus and is activated by binding to DNA lesions. Excessive PARP1 activation following genotoxin treatment causes NAD⁺ depletion and cell death, whereas pharmacological PARP1 inhibition protects cells from genotoxicity. This study investigates whether cellular viability and NAD⁺ metabolism are regulated by tankyrase-1, a PARP member localized predominantly in the cytosol. Using a tetracycline-sensitive promoter to regulate tankyrase-1 expression in Madin–Darby canine kidney (MDCK) cells, we found that a 40-fold induction of tankyrase-1 (from 1500 to 60,000 copies per cell) lowers steady-state NAD⁺ levels but does not affect basal cellular viability. Moreover, the induction confers protection against the oxidative agent H₂O₂ and the alkylating agent MNNG, genotoxins that kill cells by activating PARP1. The cytoprotective effect of tankyrase-1 is not due to enhanced scavenging of oxidants or altered expression of Mcl-1, an anti-apoptotic molecule previously shown to be down-regulated by tankyrase-1 in CHO cells. Instead, tankyrase-1 appears to protect cells by preventing genotoxins from activating PARP1-mediated reactions such as PARP1 automodification and NAD⁺ consumption. Our findings therefore indicate a cytoprotective function of tankyrase-1 mediated through altered NAD⁺ homeostasis and inhibition of PARP1 function.     

Poly(ADP-ribose) signaling in cell death

Poly(ADP-ribosyl)ation (PARylation) is a reversible protein modification carried out by the concerted actions of poly(ADP-ribose) polymerase (PARP) enzymes and poly(ADP-ribose) (PAR) decomposing enzymes such as PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Reversible PARylation is a pleiotropic regulator of various cellular functions but uncontrolled PARP activation may also lead to cell death. The cellular demise pathway mediated by PARylation in oxidatively stressed cells has been described almost thirty years ago. However, the underlying molecular mechanisms have only begun to emerge relatively recently. PARylation has been implicated in necroptosis, autophagic cell death but its role in extrinsic and intrinsic apoptosis appears to be less predominant and depends largely on the cellular model used. Currently, three major pathways have been made responsible for PARP-mediated necroptotic cell death: (1) compromised cellular energetics mainly due to depletion of NAD, the substrate of PARPs; (2) PAR mediated translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus (parthanatos) and (3) a mostly elusive crosstalk between PARylation and cell death/survival kinases and phosphatases. Here we review how these PARP-mediated necroptotic pathways are intertwined, how PARylation may contribute to extrinsic and intrinsic apoptosis and discuss recent developments on the role of PARylation in autophagy and autophagic cell death.     

TNF regulates cellular NAD+ metabolism in primary macrophages

The inflammatory cytokine TNF is known to affect glucose and lipid metabolism, where its action leads to a cachexic state. Despite a well-established connection of TNF to metabolism, the relationship between TNF and NAD(+) metabolism remains unclear. In this report, we evaluated the effects of TNF on NAD(+) metabolism in cells that are TNF's primary autocrine target-macrophages. We designed real-time PCR primers to all NAD(+) metabolic enzymes, which we used to examine TNF-induced changes over time. We found that TNF paradoxically up-regulated enzymes that served to increase NAD(+) levels, such as IDO and PBEF, as well as enzymes that decrease NAD(+) levels, such as CD38 and CD157. The significance of these mRNA changes was evaluated by examining TNF-mediated changes in cellular NAD(+) levels. Treatment of macrophages with TNF decreased NAD(+) levels over time, suggesting that increases in NAD(+)-degrading enzymes were dominant. To evaluate whether this was the case, we measured TNF-mediated changes in NAD(+) levels in animals where CD38 was genetically deleted. In CD38-/- macrophages, the effects of TNF were reversed, with TNF increasing NAD(+) levels over time. The significance of our findings is threefold: (1) we establish that TNF affects NAD(+) metabolism by regulating the expression of major NAD(+) metabolic enzymes, (2) TNF-induced decreases in cellular NAD(+) levels were carried out through the up-regulation of extracellularly situated enzymes, and (3) we provide a mechanism for the observed clinical connection of TNF-dependent diseases to tissue reductions in NAD(+) content.     

Dunnione protects against experimental cisplatin-induced nephrotoxicity by modulating NQO1 and NAD+ levels

Despite being an efficacious anticancer agent, the clinical utility of cisplatin is hindered by its cardinal side effects. This investigation aimed to appraise potential protective impact of dunnione, a natural naphthoquinone pigment with established NQO1 stimulatory effects, on cisplatin nephrotoxicity of rats. Dunnione was administered orally at 10 and 20?mg/kg doses for 4 d and a single injection of cisplatin was delivered at the second day. Renal histopathology, inflammatory/oxidative stress/apoptotic markers, kidney function, and urinary markers of renal injury were assessed. Dunnione repressed cisplatin-induced inflammation in the kidneys as indicated by decreased TNF-α/IL-1β levels, and reduced nuclear phosphorylated NF-κB p65. This agent also obviated cisplatin-invoked oxidative stress as elucidated by decreased MDA/GSH levels and increased SOD/CAT activities. Dunnione, furthermore, improved renal histological deteriorations as well as caspase-3 activities and terminal deoxynucleotidyl transferase (TUNEL) positive cells, the indicators of apoptosis. Moreover, it up-regulated nuclear Nrf2 and cytosolic haeme-oxygenase-1 (HO-1) and NQO1 levels; meanwhile, promoted NAD⁺/NADH ratios followed by enhancing the activities of Sirt1 and PARP1; and further attenuated nuclear acetylated NF-κB p65. Dunnione additionally declined cisplatin-evoked retrogression in renal function and upraise in urinary markers of glomerular and tubular injury as demonstrated by decreased serum urea and creatinine with simultaneous reductions in urinary excretions of collagen type IV, podocin, cystatin C, and retinol-binding protein (RBP). Altogether, these findings offer dunnione as a potential protective agent against cisplatin-induced nephrotoxicity in rats.     

Isolation and characterization of an NAD+-degrading bacterium PTX1 and its role in chromium biogeochemical cycle

Microorganisms can reduce toxic chromate to less toxic trivalent chromium [Cr(III)]. Besides Cr(OH)₃ precipitates, some soluble organo-Cr(III) complexes are readily formed upon microbial, enzymatic, and chemical reduction of chromate. However, the biotransformation of the organo-Cr(III) complexes has not been characterized. We have previously reported the formation of a nicotinamide adenine dinucleotide (NAD⁺)-Cr(III) complex after enzymatic reduction of chromate. Although the NAD⁺-Cr(III) complex was stable under sterile conditions, microbial cells were identified as precipitates in a non-sterile NAD⁺-Cr(III) solution after extended incubation. The most dominant bacterium PTX1 was isolated and assigned to Leifsonia genus by phylogenetic analysis of 16S rRNA gene sequence. PTX1 grew slowly on NAD⁺ with a doubling time of 17 h, and even more slowly on the NAD⁺-Cr(III) complex with an estimated doubling time of 35 days. The slow growth suggests that PTX1 passively grew on trace NAD⁺ dissociated from the NAD⁺-Cr(III) complex, facilitating further dissociation of the complex and formation of Cr(III) precipitates. Thus, organo-Cr(III) complexes might be an intrinsic link of the chromium biogeochemical cycle; they can be produced during chromate reduction and then further mineralized by microorganisms.     

Decreased cADPR and increased NAD+ in the Cd38-/- mouse

CD38 is a type II glycoprotein that catalyzes the formation of cyclic ADP-ribose (cADPR), an intracellular calcium signalling molecule, from nicotinamide adenine dinucleotide (NAD⁺). Using a modified version of the fluorimetric cycling assay for cADPR which reduces between-subject variability, we report significant decreases in brain and lung cADPR, which although similar to previously published values, showed much less individual variation. The reduced variation within each group suggests that the range of cADPR is narrower than previously thought, and that the regulatory mechanisms controlling these levels are more finely tuned. We also report significant increases in brain, lung, and kidney NAD⁺ in the Cd38^−/− mouse, and provide the first experimental demonstration of the proximate relationship between CD38 and NAD⁺.     

PARP-1 inhibition does not restore oxidant-mediated reduction in SIRT1 activity

Sirtuin1 (SIRT1) deacetylase and poly(ADP-ribose)-polymerase-1 (PARP-1) respond to environmental cues, and both require NAD⁺ cofactor for their enzymatic activities. However, the functional link between environmental/oxidative stress-mediated activation of PARP-1 and SIRT1 through NAD⁺ cofactor availability is not known. We investigated whether NAD⁺ depletion by PARP-1 activation plays a role in environmental stimuli/oxidant-induced reduction in SIRT1 activity. Both H₂O₂ and cigarette smoke (CS) decreased intracellular NAD⁺ levels in vitro in lung epithelial cells and in vivo in lungs of mice exposed to CS. Pharmacological PARP-1 inhibition prevented oxidant-induced NAD⁺ loss and attenuated loss of SIRT1 activity. Oxidants decreased SIRT1 activity in lung epithelial cells; however increasing cellular NAD⁺ cofactor levels by PARP-1 inhibition or NAD⁺ precursors was unable to restore SIRT1 activity. SIRT1 was found to be carbonylated by CS, which was not reversed by PARP-1 inhibition or selective SIRT1 activator. Overall, these data suggest that environmental/oxidant stress-induced SIRT1 down-regulation and PARP-1 activation are independent events despite both enzymes sharing the same cofactor.     

AMPK phosphorylates NAMPT to regulate NAD+ homeostasis under ionizing radiation

Radiation-induced oral mucositis is the most common complication for patients who receive head/neck radiotherapy. Nicotinamide adenine dinucleotide (NAD⁺) is vital for DNA damage repair under ionizing radiation, through functioning as either the substrate for protein poly(ADP-ribosyl)ation at DNA break sites or the cofactor for multiple DNA repair-related enzymes, which therefore can result in a significant consumption of cellular NAD⁺ during DNA repair. Mammalian cells produce NAD⁺ mainly by recycling nicotinamide via the salvage pathway, in which the rate-limiting step is governed by nicotinamide phosphoribosyltransferase (NAMPT). However, whether NAMPT is co-opted under ionizing radiation to timely fine-tune NAD⁺ homeostasis remains elusive. Here we show that ionizing radiation evokes NAMPT activation within 30 min without apparent changes in its protein expression. AMPK rapidly phosphorylates NAMPT at S314 under ionizing radiation, which reinforces the enzymatic activity of NAMPT by increasing NAMPT binding with its substrate phosphoribosyl pyrophosphate (PRPP). AMPK-mediated NAMPT S314 phosphorylation substantially restores NAD⁺ level in the irradiated cells and facilitates DNA repair and cell viability. Our findings demonstrate a new post-translational modification-based signalling route, by which cells can rapidly orchestrate NAD⁺ metabolism to support DNA repair, thereby highlighting NAMPT as a potential target for the prevention of ionizing radiation-induced injuries.     

Conserved water-mediated recognition and dynamics of NAD+ (carboxamide group) to hIMPDH enzyme: water mimic approach toward the design of isoform-selective inhibitor

Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in GMP biosynthesis pathway. Type I hIMPDH is expressed at lower levels in all cells, whereas type II is especially observed in acute myelogenous leukemia, chronic myelogenous leukemia cancer cells, and 10?ns simulation of the IMP–NAD⁺ complex structures (PDB ID. 1B3O and 1JCN) have revealed the presence of a few conserved hydrophilic centers near carboxamide group of NAD⁺. Three conserved water molecules (W1, W, and W1′) in di-nucleotide binding pocket of enzyme have played a significant role in the recognition of carboxamide group (of NAD⁺) to D274 and H93 residues. Based on H-bonding interaction of conserved hydrophilic (water molecular) centers within IMP–NAD⁺-enzyme complexes and their recognition to NAD⁺, some covalent modification at carboxamide group of di-nucleotide (NAD⁺) has been made by substituting the –CONH₂group by –CONHNH₂ (carboxyl hydrazide group) using water mimic inhibitor design protocol. The modeled structure of modified ligand may, though, be useful for the development of antileukemic agent or it could be act as better inhibitor for hIMPDH-II.     

Extracellular NAD+: a novel autocrine/paracrine signal in osteoblast physiology

Intercellular communication allows co-ordination of cell metabolism and sensitivity to extracellular stimuli. In bone cells, paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex intercellular networks, which favours the intercellular exchange of nutrients and second messengers, ultimately controlling the process of bone remodelling. The importance of local factors in bone remodelling is known since many years. Bone cells secrete and respond to a variety signals, among which include prostaglandins, cytokines, growth factors, and ATP. We here report evidence that extracellular NAD(+) is a novel extracellular signal stimulating osteoblast differentiation. We found that HOBIT human osteoblastic cells, which are known to express ADP-ribosyl cyclase/CD38 activity, respond to micromolar concentrations of extracellular NAD(+) with oscillatory increases of the cytosolic Ca(2+) concentration. The initial Ca(2+) response was followed by a time-dependent inhibition of cell growth, the appearance of an epithelial morphology, and by an increase of alkaline phosphatase and osteocalcin expression. Under resting condition HOBIT cells release NAD(+) in the extracellular medium and the release is significantly potentiated by mechanical stimulation. Taken together these results point to NAD(+) as a novel autocrine/paracrine factor involved in stimulation and maintenance of the osteoblast differentiated phenotype.     

Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes

In the present study, we show that the extracellular addition of nicotinamide adenine dinucleotide (NAD⁺) induces a transient rise in [Ca²⁺]_i in human monocytes caused by an influx of extracellular calcium. The NAD⁺-induced Ca²⁺ response was prevented by adenosine triphosphate (ATP), suggesting the involvement of ATP receptors. Of the two subtypes of ATP receptors (P2X and P2Y), the P2X receptors were considered the most likely candidates. By the use of subtype preferential agonists and antagonists, we identified P2X₁, P2X₄, and P2X₇ receptors being engaged in the NAD⁺-induced rise in [Ca²⁺]_i. Among the P2X receptor subtypes, the P2X₇ receptor is unique in facilitating the induction of nonselective pores that allow entry of ethidium upon stimulation with ATP. In monocytes, opening of P2X₇ receptor-dependent pores strongly depends on specific ionic conditions. Measuring pore formation in response to NAD⁺, we found that NAD⁺ unlike ATP lacks the ability to induce this pore-forming response. Whereas as little as 100 μM ATP was sufficient to activate the nonselective pore, NAD⁺ at concentrations up to 2 mM had no effect. Taken together, these data indicate that despite similarities in the action of extracellular NAD⁺ and ATP there are nucleotide-specific variations. So far, common and distinct features of the two nucleotides are only beginning to be understood.     

Determination of the transglycosidation activity of NAD+ glycohydrolases with 4-(2'-alkyl-sulfanyl-vinyl)-pyridine derivatives generating chromophoric NAD+ analogs

The base exchange of nicotinamide with pyridine derivatives 1a-5a, catalyzed by pig brain NAD(+) glycohydrolase and ADP-ribosyl cyclase from Aplysia californica, generated the corresponding NAD(+) analogs 1b-5b. These analogs exhibited a high absorbance band in the visible region. The transglycosidation rate was determined by monitoring the absorbance increase. Among the tested derivatives, (E)-4-[2-(methylsulfanyl)-vinyl]-pyridine 1a was the most suitable substrate for pig brain NAD(+) glycohydrolase while 4-[1,3]-dithiolan-2-ylidenemethyl-pyridine 3a was the most efficient for ADP-ribosyl cyclase from A. californica.     

NAD+ metabolism and oxidative stress: the golden nucleotide on a crown of thorns

Abstract

In the twentieth century, NAD⁺ research generated multiple discoveries. Identification of the important role of NAD⁺ as a cofactor in cellular respiration and energy production was followed by discoveries of numerous NAD⁺ biosynthesis pathways. In recent years, NAD⁺ has been shown to play a unique role in DNA repair and protein deacetylation. As discussed in this review, there are close interactions between oxidative stress and immune activation, energy metabolism, and cell viability in neurodegenerative disorders and ageing. Profound interactions with regard to oxidative stress and NAD⁺ have been highlighted in the present work. This review emphasizes the pivotal role of NAD⁺ in the regulation of DNA repair, stress resistance, and cell death, suggesting that NAD⁺ synthesis through the kynurenine pathway and/or salvage pathway is an attractive target for therapeutic intervention in age-associated degenerative disorders. NAD⁺ precursors have been shown to slow down ageing and extend lifespan in yeasts, and protect severed axons from degeneration in animal models neurodegenerative diseases.     

Key NAD+-binding residues in human 15-hydroxyprostaglandin dehydrogenase

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins, and is believed to be the key enzyme responsible for the biological inactivation of these biologically potent eicosanoids. The enzyme utilizes NAD(+) specifically as a coenzyme. Potential amino acid residues involved in binding NAD(+) and facilitating enzyme catalysis have been partially identified. In this report, we propose that three more residues in 15-PGDH, Ile-17, Asn-91, and Val-186, are also involved in the interaction with NAD(+). Site-directed mutagenesis was used to examine their roles in binding NAD(+). Several mutants (I17A, I17V, I17L, I17E, I17K, N91A, N91D, N91K, V186A, V186I, V186D, and V186K) were prepared, expressed as glutathione S-transferase (GST) fusion enzymes in Escherichia coli, and purified by GSH-agarose affinity chromatography. Mutants I17E, I17K, N91L, N91K, and V186D were found to be inactive. Mutants N91A, N91D, V186A, and V186K exhibited comparable activities to the wild type enzyme. However, mutants I17A, I17V, I17L, and V186I had higher activity than the wild type. Especially, the activities of I17L and V186I were increased nearly 4- and 5-fold, respectively. The k(cat)/K(m) ratios of all active mutants for PGE(2) were similar to that of the wild type enzyme. However, the k(cat)/K(m) ratios of mutants I17A and N91A for NAD(+) were decreased 5- and 10-fold, respectively, whereas the k(cat)/K(m) ratios of mutants I17V, N91D, V186I, and V186K for NAD(+) were comparable to that of the wild type enzyme. The k(cat)/K(m) ratios of mutants I17L and V186A for NAD(+) were increased over nearly 2-fold. These results suggest that Ile-17, Asn-91, and Val-186 are involved in the interaction with NAD(+) and contribute to the full catalytic activity of 15-PGDH.