Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression

Background  

S100 proteins, a multigenic family of non-ubiquitous cytoplasmic Ca²⁺-binding proteins, have been linked to human pathologies in recent years. Dysregulated expression of S100 proteins, including S100A9, has been reported in the epidermis as a response to stress and in association with neoplastic disorders. Recently, we characterized a regulatory element within the S100A9 promotor, referred to as MRE that drives the S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner (Kerkhoff et al. (2002) J. Biol. Chem. 277, 41879–41887).     

Identification and biochemical characterization of a Werner's syndrome protein complex with Ku70/80 and poly(ADP-ribose) polymerase-1

Werner's syndrome (WS) is an inherited disease characterized by genomic instability and premature aging. The WS gene encodes a protein (WRN) with helicase and exonuclease activities. We have previously reported that WRN interacts with Ku70/80 and this interaction strongly stimulates WRN exonuclease activity. To gain further insight on the function of WRN and its relationship with the Ku heterodimer, we established a cell line expressing tagged WRN(H), a WRN point mutant lacking helicase activity, and used affinity purification, immunoblot analysis and mass spectroscopy to identify WRN-associated proteins. To this end, we identified three proteins that are stably associated with WRN in nuclear extracts. Two of these proteins, Ku70 and Ku80, were identified by immunoblot analysis. The third polypeptide, which was identified by mass spectrometry analysis, is identical to poly(ADP-ribose) polymerase-1(PARP-1), a 113-kDa enzyme that functions as a sensor of DNA damage. Biochemical fractionation studies and immunoprecipitation assays and studies confirmed that endogenous WRN is associated with subpopulations of PARP-1 and Ku70/80 in the cell. Protein interaction assays with purified proteins further indicated that PARP-1 binds directly to WRN and assembles in a complex with WRN and Ku70/80. In the presence of DNA and NAD(+), PARP-1 poly(ADP-ribosyl)ates itself and Ku70/80 but not WRN, and gel-shift assays showed that poly-(ADP-ribosyl)ation of Ku70/80 decreases the DNA-binding affinity of this factor. Significantly, (ADP-ribosyl)ation of Ku70/80 reduces the ability of this factor to stimulate WRN exonuclease, suggesting that covalent modification of Ku70/80 by PARP-1 may play a role in the regulation of the exonucleolytic activity of WRN.     

Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update

Poly(ADP-ribosylation) is a post-translational modification of proteins playing a crucial role in many processes, including DNA repair and cell death. The best known poly(ADP-ribosylating) enzime, PARP-1, is a DNA nick sensor and uses NAD⁺ to form polymers of ADP-ribose which are further bound to nuclear protein acceptors. To strictly regulate poly(ADP-ribose) turnover, its degradation is assured by the enzyme poly(ADP-ribose) glycohydrolase (PARG). During apoptosis, PARP-1 plays two opposite roles: its stimulation leads to poly(ADP-ribose) synthesis, whereas caspases cause PARP-1 cleavage and inactivation. PARP-1 proteolysis produces an 89 kDa C-terminal fragment, with a reduced catalytic activity, and a 24 kDa N-terminal peptide, which retains the DNA binding domains. The fate and the possible role of these fragments during apoptosis will be discussed.     

Poly(ADP-ribose) polymerase-1 mediated caspase-independent cell death after ischemia/reperfusion

In ischemia/reperfusion (I/R) injury increased intracellular Ca(2+) and production of reactive oxygen species (ROS) may cause cell death by intrinsic apoptotic pathways or by necrosis. In this review, an alternative intrinsic cell death pathway, mediated by poly(ADP-ribose) polymerase-1 (PARP-1) and apoptosis-inducing factor (AIF), is described. ROS-induced DNA strand breaks lead to overactivation of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30), causing excessive use of energetic substrates such as NAD(+) and ATP, inducing cell death either by apoptosis or by necrosis. Recently, it was demonstrated that activation of PARP-1 induces translocation of apoptosis-inducing factor from the mitochondria to the nucleus, causing DNA condensation and fragmentation, and subsequent cell death. This pathway seems to be triggered by depletion of NAD(+) and appears to be caspase independent. Several lines of evidence suggest that this pathway plays a role in I/R injury, although some studies indicate that mitochondrial dysfunction may also trigger AIF translocation and cell death. At present, the exact mechanisms linking PARP-1 and AIF in the induction of the ROS-induced cell death are still unclear. Therefore, it appears that further investigations will yield valuable information on underlying mechanisms and potential interventions to reduce caspase-independent cell death during ischemia-reperfusion.     

Poly(ADP-ribose) polymerase-1 inhibition protects the brain against systemic inflammation

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair, but its overactivation can induce cell death. Our aim was to investigate the role of PARP-1 in activation of programmed cell death processes in the brain during systemic inflammation.Our data indicated that lipopolysaccharide (1 mg/kg b.w., i.p.)-evoked systemic inflammation enhanced PARP-1 activity in the mouse brain, leading to the lowering of β-NAD⁺ concentration, to translocation of apoptosis inducing factor from mitochondria to the nucleus, and to enhanced lipid peroxidation. Inhibitor of PARP-1, 3-aminobenzamide (30 mg/kg b.w., i.p.), protected the brain against prooxidative and cell death processes, suggesting involvement of PARP-1 in systemic inflammation-related processes in the brain.     

Poly(ADP-ribose) polymerase-1 inhibition protects the brain against systemic inflammation

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair, but its overactivation can induce cell death. Our aim was to investigate the role of PARP-1 in activation of programmed cell death processes in the brain during systemic inflammation.

Our data indicated that lipopolysaccharide (1 mg/kg b.w., i.p.)-evoked systemic inflammation enhanced PARP-1 activity in the mouse brain, leading to the lowering of β-NAD⁺ concentration, to translocation of apoptosis inducing factor from mitochondria to the nucleus, and to enhanced lipid peroxidation. Inhibitor of PARP-1, 3-aminobenzamide (30 mg/kg b.w., i.p.), protected the brain against prooxidative and cell death processes, suggesting involvement of PARP-1 in systemic inflammation-related processes in the brain.     

Poly(ADP-ribose) polymerase-1 (PARP-1) gene deficiency alleviates diabetic kidney disease

Poly(ADP-ribose)polymerase (PARP) inhibitors prevent or alleviate diabetic nephropathy. This study evaluated the role for PARP-1 in diabetic kidney disease using the PARP-1-deficient mouse. PARP-1?/? and the wild-type (129S1/SvImJ) mice were made diabetic with streptozotocin, and were maintained for 12 weeks. Final blood glucose concentrations were increased ～ 3.7-fold in both diabetic groups. PARP-1 protein expression (Western blot analysis) in the renal cortex was similar in non-diabetic and diabetic wild-type mice (100% and 107%) whereas all knockouts were PARP-1-negative. PARP-1 gene deficiency reduced urinary albumin (ELISA) and protein excretion prevented diabetes-induced kidney hypertrophy, and decreased mesangial expansion and collagen deposition (both assessed by histochemistry) as well as fibronectin expression. Renal podocyte loss (immunohistochemistry) and nitrotyrosine and transforming growth factor-β₁ accumulations (both by ELISA) were slightly lower in diabetic PARP-1?/? mice, but the differences with diabetic wild-type group did not achieve statistical significance. In conclusion, PARP-1?/? gene deficiency alleviates although does not completely prevent diabetic kidney disease.     

Poly(ADP-ribose) polymerase-1 signaling to mitochondria in necrotic cell death requires RIP1/TRAF2-mediated JNK1 activation

Poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation-induced necrosis has been implicated in several pathophysiological conditions. Although mitochondrial dysfunction and apoptosis-inducing factor translocation from the mitochondria to the nucleus have been suggested to play very important roles in PARP-1-mediated cell death, the signaling events downstream of PARP-1 activation in initiating mitochondria dysfunction are not clear. Here we used the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, a potent PARP-1 activator, to study PARP-1 activation-mediated cell death. We found, based on genetic knockouts and pharmacological inhibition, that c-Jun N-terminal kinase (JNK), especially JNK1, but not the other groups of mitogen-activated protein kinase, is required for PARP-1-induced mitochondrial dysfunction, apoptosis-inducing factor translocation, and subsequent cell death. We reveal that receptor-interacting protein 1 (RIP1) and tumor necrosis factor receptor-associated factor 2 (TRAF2), are upstream of JNK in PARP-1 hyperactivated cells, because PARP-1-induced JNK activation was attenuated in RIP1-/- and TRAF2-/- mouse embryonic fibroblast cells. Consistently, knockouts of RIP1 and TRAF2 caused a resistance to PARP-1-induced cell death. Therefore, our study uncovers that RIP1, TRAF2, and JNK comprise a pathway to mediate the signaling from PARP-1 overactivation to mitochondrial dysfunction.     

Poly(ADP-ribose) polymerase-1 protects from oxidative stress induced endothelial dysfunction

Background

Generation of reactive oxygen species (ROS) is a key feature of vascular disease. Activation of the nuclear enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1) is a downstream effector of oxidative stress.

Methods

PARP-1(−/−) and PARP-1(+/+) mice were injected with paraquat (PQ; 10 mg/kg i.p.) to induce intracellular oxidative stress. Aortic rings were suspended in organ chambers for isometric tension recording to analyze vascular function.

Results

PQ treatment markedly impaired endothelium-dependent relaxations to acetylcholine in PARP-1(−/−), but not PARP-1(+/+) mice (p < 0.0001). Maximal relaxation was 45% in PQ treated PARP-1(−/−) mice compared to 79% in PARP-1(+/+) mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (SNP) were not altered. After PQ treatment, l-NAME enhanced contractions to norepinephrine by 2.0-fold in PARP-1(−/−) mice, and those to acetylcholine by 3.3-fold, respectively, as compared to PARP-1(+/+) mice. PEG-superoxide dismutase (SOD) and PEG-catalase prevented the effect of PQ on endothelium-dependent relaxations to acetylcholine in PARP-1(−/−) mice (p < 0.001 vs. PQ treated PARP-1(+/+) mice. Indomethacin restored endothelium-dependent relaxations to acetylcholine in PQ treated PARP-1(−/−) mice (p < 0.05 vs. PQ treated PARP-1(+/+).

Conclusion

PARP-1 protects from acute intracellular oxidative stress induced endothelial dysfunction by inhibiting ROS induced production of vasoconstrictor prostanoids.     

Poly(ADP-ribose) polymerase-1 protects neurons against apoptosis induced by oxidative stress

In neurons, DNA is prone to free radical damage, although repair mechanisms preserve the genomic integrity. However, activation of the DNA repair system, poly(ADP-ribose) polymerase (PARP-1), is thought to cause neuronal death through NAD+ depletion and mitochondrial membrane potential (delta psi(m)) depolarization. Here, we show that abolishing PARP-1 activity in primary cortical neurons can either enhance or prevent apoptotic death, depending on the intensity of an oxidative stress. Only in severe oxidative stress does PARP-1 activation result in NAD+ and ATP depletion and neuronal death. To investigate the role of PARP-1 in an endogenous model of oxidative stress, we used an RNA interference (RNAi) strategy to specifically knock down glutamate-cysteine ligase (GCL), the rate-limiting enzyme of glutathione biosynthesis. GCL RNAi spontaneously elicited a mild type of oxidative stress that was enough to stimulate PARP-1 in a Ca2+-calmodulin kinase II-dependent manner. GCL RNAi-mediated PARP-1 activation facilitated DNA repair, although neurons underwent delta psi(m) loss followed by some apoptotic death. PARP-1 inhibition did not prevent delta psi(m) loss, but enhanced the vulnerability of neurons to apoptosis upon GCL silencing. Conversely, mild expression of PARP-1 partially prevented to GCL RNAi-dependent apoptosis. Thus, in the mild progressive damage likely occur in neurodegenerative diseases, PARP-1 activation plays a neuroprotective role that should be taken into account when considering therapeutic strategies.     

Poly(ADP-ribose) polymerase-1 inhibition: preclinical and clinical development of synthetic lethality

The hereditary forms of breast cancer identified by BRCA1 and BRCA2 genes have a defect in homologous DNA repair and demonstrate a dependence on alternate DNA repair processes by base excision repair, which requires poly(ADP-ribose) polymerase 1 (PARP-1). siRNA and deletion mutations demonstrate that interference with PARP-1 function results in enhanced cell death when the malignancy has a defect in homologous recombination. These findings resulted in a plethora of agents in clinical trials that interfere with DNA repair, and these agents offer the potential of being more selective in their effects than classic chemotherapeutic drugs. An electronic search of the National Library of Medicine for published articles written in English used the terms "PARP inhibitors" and "breast cancer" to find prospective, retrospective and review articles. Additional searches were done for articles dealing with mechanism of action. A total of 152 articles dealing with breast cancer and PARP inhibition were identified. PARP inhibition not only affects nonhomologous repair, but also has several other nongenomic functions. Mutational resistance to these agents was seen in preclinical studies. To date, PARP-1 inhibitors were shown to enhance cytotoxic effects of some chemotherapy agents. This new class of agents may offer more therapeutic specificity by exploiting a DNA repair defect seen in some human tumors with initial clinical trials demonstrating antitumor activity. Although PARP inhibitors may offer a therapeutic option for selected malignancies, the long-term effects of these agents have not yet been defined.     

Poly(ADP-ribose) polymerase-1 regulates the stability of the wild-type p53 protein.

We investigated the interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the product of the tumor suppressor gene p53 using two different approaches. In the first approach, we used primary and immortalized cells derived from wt and PARP-1 -/- mice. We examined whether PARP-1 deficiency would affect the expression of the wild-type (wt) p53 protein. The inactivation of the PARP-1 gene markedly affected the constitutive expression of the wt p53 protein. Interestingly, only the regularly spliced form of wt p53 was reduced to a barely detectable level in consequence to an approximately 8-fold shortening of its half-life, whereas the level of alternatively spliced p53 remained unchanged. Moreover, reconstitution of cells lacking the PARP-1 gene with the human counterpart restored the normal stability of the regularly spliced p53 protein. In the second approach, we performed experiments with c-Ha-ras transformed primary rat cells overexpressing the p53135val mutant alone or in combination with PARP-1. The advantage of this temperature sensitive p53135val mutant is its oncogenic character at 37 degrees C, connected with cytoplasmic localization of p53, and its tumor suppressor activity at 32 degrees C, accompanied by p53 translocation into the nucleus. No noticeable differences in proliferation and G1 accumulationwere observed between cells expressing p53135val with or without PARP-1. On the other hand, a comparison of the recovery of G1 arrested cells after a shift up to 37 degrees C for both cell lines showed dramatic differences in the kinetics. While cells expressing p53135val rapidly reached the characteristic S-phase level after a shift up to basal temperature, cells additionally expressing PARP-1 rested in G1 despite the temperature elevation. This coincided with exclusively cytoplasmic p53 protein in cells expressing p53135val and predominantly nuclear localization of p53 in p53135val +PARP-1 cells, as evidenced by immunostaining. Determination of the p53 level during the maintenance of cells at 32 degrees C revealed a marked decrease in the level of p53 in cells expressing p53135val alone, whereas in cells coexpressing PARP-1, the level of p53 remained largely unaffected. This indicates that the stability of wild-type p53 greatly differed between both cell lines. Furthermore, the inhibition of PARP-1 activity in G1 arrested cells by 3-aminobenzamide abolished its stabilizing effect on the wild-type p53 protein. Taken together, our results indicate that PARP-1 regulates the stability of the wt p53 protein and that its enzymatic activity is necessary for this stabilizing action.     

Poly(ADP-ribose) polymerase-2 is a lipid-modulated modulator of muscular lipid homeostasis

There is a growing body of evidence that poly(ADP-ribose) polymerase-2 (PARP2), although originally described as a DNA repair protein, has a widespread role as a metabolic regulator. We show that the ablation of PARP2 induced characteristic changes in the lipidome. The silencing of PARP2 induced the expression of sterol regulatory element-binding protein-1 and -2 and initiated de novo cholesterol biosynthesis in skeletal muscle. Increased muscular cholesterol was shunted to muscular biosynthesis of dihydrotestosterone, an anabolic steroid. Thus, skeletal muscle fibers in PARP2^?/? mice were stronger compared to those of their wild-type littermates. In addition, we detected changes in the dynamics of the cell membrane, suggesting that lipidome changes also affect the biophysical characteristics of the cell membrane. In in silico and wet chemistry studies, we identified lipid species that can decrease the expression of PARP2 and potentially phenocopy the genetic abruption of PARP2, including artificial steroids. In view of these observations, we propose a new role for PARP2 as a lipid-modulated regulator of lipid metabolism.     

Poly(ADP-ribose) polymerase-1 activity facilitates the dissociation of nuclear proteins from platinum-modified DNA

The affinity of the poly(ADP-ribose) polymerase-1 (PARP-1) for platinum-damaged DNA was first discovered during photo-cross-linking experiments using the photoactive compound Pt-BP6 [J. Am. Chem. Soc.2004, 126, 6536-6537], an analogue of the anticancer drug cis-diamminedichloroplatinum(II), cisplatin. Although PARP inhibitors sensitize cancer cells to cisplatin, there are conflicting reports in the literature about their efficacy. In order to improve our understanding of the mechanism by which PARP inhibition might potentiate the cell-killing ability of cisplatin, and to shed light on the source of the discrepancy among different laboratories, we have in the present study probed the influence of three PARP inhibitors in four types of cancer cells, cervical (HeLa), testicular (NTera2), pancreatic (BxPC3), and osteosarcoma (U2OS), on the results of Pt-BP6 photo-cross-linking experiments and cytotoxicity assays. We find that the activity of PARP proteins following exposure to platinum-modified DNA results in the dissociation of DNA-bound proteins. PARP inhibitors were able to sensitize some, but not all, of the cell lines to cisplatin. This cell line-dependence and the potential consequences of PARP-initiated protein removal from platinum-DNA lesions are discussed. Control experiments revealed that NTera2 cells are especially sensitive to PARP inhibition.