Signaling mechanism of poly(ADP-ribose) polymerase-1 (PARP-1) in inflammatory diseases

Poly(ADP-ribosyl)ation, attaching the ADP-ribose polymer chain to the receptor protein, is a unique posttranslational modification. Poly(ADP-ribose) polymerase-1 (PARP-1) is a well-characterized member of the PARP family. In this review, we provide a general update on molecular structure and structure-based activity of this enzyme. However, we mainly focus on the roles of PARP-1 in inflammatory diseases. Specifically, we discuss the signaling pathway context that PARP-1 is involved in to regulate the pathogenesis of inflammation. PARP-1 facilitates diverse inflammatory responses by promoting inflammation-relevant gene expression, such as cytokines, oxidation-reduction-related enzymes, and adhesion molecules. Excessive activation of PARP-1 induces mitochondria-associated cell death in injured tissues and constitutes another mechanism for exacerbating inflammation.     

Integrin-mediated suppression of endotoxin-induced DNA damage in lung endothelial cells is sensitive to poly(ADP-ribose) polymerase-1 gene deletion

Endotoxin (LPS) is a cause of adult respiratory distress syndrome (ARDS), a disease which is preceded by acute lung injury involving the pulmonary endothelium. Experimentally, LPS causes acute DNA strand breakage in mouse lung endothelial cells (MLEC). Engagement of integrin cell adhesion receptors inhibits acute DNA breakage, which could be of use in reducing lung injury. Because integrins presumably inhibit DNA damage or activate repair, we hypothesized that the DNA-damage response protein, poly(ADP-ribose) polymerase-1 (PARP-1), regulates the protective action of integrins, as well as sensitivity to LPS. Therefore, the effect of LPS on MLEC cultured from wild-type and PARP-1 knockout mice was determined. Fluorescence microscopic measures were used to assess plasma membrane integrity, PARP activity, DNA strand breakage and DNA repair in attached cells. LPS caused a concentration-dependent increase in the permeability of wild-type MLEC. Engagement of beta1 integrins with an antibody protected wild-type MLEC from this LPS-induced injury. Wild-type cells treated with the PARP-inhibitor, 3-aminobenzamide, and PARP-1 knockout MLEC were also resistant. LPS caused acute DNA breakage in both wild-type and knockout MLEC, but PARP was activated only in wild-type cells. LPS-induced DNA breakage was inhibited by 3-aminobenzamide, but not by knockout. Anti-beta1 integrin antibody also inhibited the DNA breakage and PARP activation caused by LPS in wild-type MLEC. However, integrin engagement did not prevent DNA breakage in PARP-1 knockout cells, despite a similar level of beta1 integrin in wild-type and knockout cells. Thus, integrin engagement, 3-aminobenzamide, and PARP-1 deletion protected MLEC from increases in membrane permeability caused by LPS. PARP-1 deletion also impaired the ability of integrin engagement to inhibit LPS-induced DNA breakage, suggesting that knockout may affect nuclear factors necessary for integrin-mediated suppression of LPS-induced DNA breakage.     

Spatial and functional relationship between poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase in the brain

Poly(ADP-ribose) polymerases (PARPs) are members of a family of enzymes that utilize nicotinamide adenine dinucleotide (NAD(+)) as substrate to form large ADP-ribose polymers (PAR) in the nucleus. PAR has a very short half-life due to its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). PARP-1 mediates acute neuronal cell death induced by a variety of insults including cerebral ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism, and CNS trauma. While PARP-1 is localized to the nucleus, PARG resides in both the nucleus and cytoplasm. Surprisingly, there appears to be only one gene encoding PARG activity, which has been characterized in vitro to generate different splice variants, in contrast to the growing family of PARPs. Little is known regarding the spatial and functional relationships of PARG and PARP-1. Here we evaluate PARG expression in the brain and its cellular and subcellular distribution in relation to PARP-1. Anti-PARG (alpha-PARG) antibodies raised in rabbits using a purified 30 kDa C-terminal fragment of murine PARG recognize a single band at 111 kDa in the brain. Western blot analysis also shows that PARG and PARP-1 are evenly distributed throughout the brain. Immunohistochemical studies using alpha-PARG antibodies reveal punctate cytosolic staining, whereas anti-PARP-1 (alpha-PARP-1) antibodies demonstrate nuclear staining. PARG is enriched in the mitochondrial fraction together with manganese superoxide dismutase (MnSOD) and cytochrome C (Cyt C) following whole brain subcellular fractionation and Western blot analysis. Confocal microscopy confirms the co-localization of PARG and Cyt C. Finally, PARG translocation to the nucleus is triggered by NMDA-induced PARP-1 activation. Therefore, the subcellular segregation of PARG in the mitochondria and PARP-1 in the nucleus suggests that PARG translocation is necessary for their functional interaction. This translocation is PARP-1 dependent, further demonstrating a functional interaction of PARP-1 and PARG in the brain.     

Asbestos exposure affects poly(ADP-ribose) polymerase-1 activity: role in asbestos-induced carcinogenesis

Asbestos is known to induce malignant mesothelioma (MM) and other asbestos-related diseases. It is directly genotoxic by inducing DNA strand breaks and cytotoxic by promoting apoptosis in lung target cells. Poly(ADP-ribose) polymerase-1 (PARP1) is a nuclear zinc-finger protein with a function as a DNA damage sensor. To determine whether PARP1 is involved in asbestos-induced carcinogenesis, PARP1 expression and activity as well as DNA damage and repair were evaluated in circulating cells of asbestos-exposed subjects, MM patients and age-matched controls. PARP1 expression and activity were also evaluated in pleural biopsies of MM patients and compared with normal tissue. Accumulation of the pre-mutagenic 8-hydroxy-2'-deoxyguanosine and elevated PARP1 expression were found both in asbestos-exposed subjects and MM patients. Although PARP1 was highly expressed, its activity was relatively low. Low DNA repair efficiency was observed in lymphocytes from MM patients. High expression of PARP1 associated with low PARP activity was also found in MM biopsies. To mimic PARP1 dysfunction, PARP1 expression and activity were induced in immortalised mesothelial cells by their exposure to asbestos in the presence of a PARP1 inhibitor, which resulted in transformation of the cells. We propose that exposure to asbestos inhibits the PARP1 activity possibly resulting in higher DNA instability, thus causing malignant transformation.     

Differential contribution of poly(ADP-ribose)polymerase-1 and -2 (PARP-1 and -2) to the poly(ADP-ribosyl)ation reaction in rat primary spermatocytes

Poly(ADP-ribose)polymerases (PARP-1 and -2) are activated by DNA strand breaks to synthesize protein-bound ADP-ribose polymers from NAD+. The two enzymes are overexpressed in rat spermatocytes and are likely to play a role in meiosis. Indeed parp-2-/- mice, but not parp-1 knockouts, show hypofertility. Aside, PARP-1 and PARP-2 are both involved in DNA damage repair and signalling, but their relative contributions to such processes remain as yet unknown, largely because of the lack of PARP isoform-specific inhibitors that has precluded in vivo studies. Here, we used permeabilized rat primary spermatocytes or isolated spermatocyte nuclei and radiolabelled NAD+ to investigate potential isoform-specific effects on basic features of the poly(ADP-ribosyl)ation reaction, including size of ADP-ribose polymers at different NAD+ concentrations, extent of auto- versus etheromodification, and modulation of such reactions by the PARP inhibitor, PJ34. We found that PARP-1 automodification prevailed over PARP-2 modification. In addition, over 50% of cellular poly(ADP-ribose) was covalently bound to histones H1 and H2. The inhibitory effect of PJ34 appeared to be targeted mainly to the elongation step of the reaction. We propose that a different propensity of PARP-1 and PARP-2 to undergo automodification and/or catalyze etheromodification, both in terms of number of enzyme molecules being involved and amount of bound poly(ADP-ribose), may underlie distinct roles in the regulation of spermatocyte functions.     

The effects of organic solvents on poly(ADP-ribose) polymerase-1 activity: implications for neurotoxicity

Poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30), also termed as poly(ADP-ribose) synthetase, is a key enzyme in the recognition and repair of damaged DNA. Several conditions (e.g., ischemia-reperfusion or chemical-induced injury) have been shown to overactivate PARP-1, causing neurodegeneration and necrotic or apoptotic cell death from NAD+ and ATP depletion. In contrast, inhibitors of PARP-1 have been shown to have a neuroprotective effect by ameliorating this response. The purpose of this study was to determine the effects of three routinely used organic solvents (ethanol, methanol, and dimethyl sulfoxide (DMSO)) on the activity of purified PARP-1. A dose-response was examined with each of these solvents. A 112% and 82% increase in PARP-1 activity was observed with 15% ethanol and 20% methanol, respectively. In contrast, a near 20% decrease in the activity was observed with 4% DMSO. Kinetic analysis revealed that the maximal velocity remained unchanged with increasing concentrations of DMSO up to 20%, indicating that DMSO is a competitive inhibitor of PARP-1. Thus, PARP-1 inhibition by DMSO depends on NAD+ concentration and in some pathological processes might be significant even at low DMSO concentrations. Our findings suggest that the interpretation of data from dose-response studies obtained when using common organic solvents may be dramatically skewed, either exaggerating the inherent toxicity of the compound or masking its potential for damage.     

Quantitative correlation between cellular proliferation and nuclear poly (ADP-ribose) polymerase (PARP-1)

Treatment of cells with lysophosphatidyl choline and centrifugal extraction can separate poly (ADP-ribose) synthetase (PARP-1) and DNA synthetase activities, permitting the experimental analysis and comparison of both multienzyme systems. Only PARP-1 is being assayed by our system. Ca(2+) and Mg(2+) have minor activating effects, and added histones are without activating action. Short end-blocked dsDNAs at nM concentrations and spermine at mM concentrations are maximally activating coenzymes of poly (ADP-ribose) synthesis. Comparison of non-proliferating non-malignant cells with rapidly growing cancer cells demonstrates that rates of poly (ADP-ribose) synthesis and DNA synthesis are highest in pre-confluent non-malignant cells and in proliferating cancer cells, and lowest in contact-inhibited non-malignant cells. Rates of poly (ADP-ribose) synthesis correlate with the number of enzymatically activable PARP-1 molecules per cell, determined under Vmax conditions where activity is linearly proportional to enzyme protein. Contact-inhibited non-malignant cells exhibit only trans-ADP-ribosylation that is not affected by ATP, while rapid growth, especially in cancer cells, demonstrates extensive auto-poly (ADP)-ribosylation that is strongly inhibited by ATP at concentrations present in cells exhibiting normal bioenergetics. Rates of mRNA synthesis in non-proliferating non-malignant cells and in cancer cells were indistinguishable, indicating that the differences observed between cellular phenotypes are most likely due to reassembly of PARP-1 molecules in nuclei to homo-dimers (in cancer cells) and hetero-dimers (in non-cancer cells). A specific inhibitor and an inactivator of PARP-1 each inhibit DNA synthesis when intact cancer cells are pretreated with these drugs. Direct addition of these drugs to permeabilized cells performing DNA synthesis has no effect on DNA synthesis. The most striking diagnostic signal for cancer cells is activation of PARP-1 and of DNA synthesis.     

Genetic cooperation between the Werner syndrome protein and poly(ADP-ribose) polymerase-1 in preventing chromatid breaks,complex chromosomal rearrangements,and cancer in mice

Werner syndrome is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for Werner syndrome encodes a DNA helicase/exonuclease protein. Participation in a replication complex is among the several functions postulated for the WRN protein. The poly(ADP-ribose) polymerase-1 (PARP-1) enzyme, which is known to bind to DNA strand breaks, is also associated with the DNA replication complex. To determine whether Wrn and PARP-1 enzymes act in concert during cell growth, mice with a mutation in the helicase domain of the Wrn gene (Wrn(Deltahel/Deltahel) mice) were crossed to PARP-1-null mice. Both Wrn(Deltahel/Deltahel) and PARP-1-null/Wrn(Deltahel/Deltahel) cohorts developed more neoplasms than wild-type animals. The tumor spectrum was the same between PARP-1-null/Wrn(Deltahel/Deltahel) mice and Wrn mutants. However, PARP-1-null/Wrn(Deltahel/Deltahel) mice developed neoplasms at a younger age. Mouse embryonic fibroblasts derived from such PARP-1-null/Wrn(Deltahel/Deltahel) mice stop dividing abruptly unlike Wrn(Deltahel/Deltahel) or PARP-1-null cells. PARP-1-null/Wrn(Deltahel/Deltahel) fibroblasts were distinguished by an increased frequency of chromatid breaks, complex chromosomal rearrangements, and fragmentation. Finally, experiments have indicated that the PARP-1 enzyme co-immunoprecipitates with the WRN protein in human 293 embryonic kidney cells. These results suggest that Wrn and PARP-1 enzymes may be part of a complex involved in the processing of DNA breaks.     

Poly(ADP-ribose) polymerase-1 inhibitor 3-aminobenzamide enhances apoptosis induction by platinum complexes in cisplatin-resistant tumor cells

Cisplatin is one of the most widely used antitumor drugs. However, as all the anticancer drugs currently used in clinic, cisplatin shows the phenomenon of drug resistance (intrinsic or acquired) against a wide variety of tumors. Poly (ADP-ribose) polymerase-1 is an enzyme involved in DNA repair and apoptotic cell death, which may be inhibited to increase cisplatin chemosensitivity of tumor cells so that cisplatin resistance may be circumvented. In the present study we report that PARP-1 inhibitor 3-aminobenzamide (3-AB) increases the cytotoxic activity of the platinum compounds cisplatin, trans-[PtCl(2)(4-picoline)(piperazine)] and transplatin against CH1cisR cisplatin-resistant ovarian tumor cells. In fact, a concentration of 3-AB of 1 mM not only increases the cytotoxic activity of these platinum complexes but also switches the mode of cell death from necrosis to apoptosis. Altogether, these data suggest that pharmacological modulation of PARP-1 by inhibitors may be a suitable strategy to fight against tumor resistance to platinum drugs.     

Local DNA damage by proton microbeam irradiation induces poly(ADP-ribose) synthesis in mammalian cells

Cellular recovery from ionizing radiation (IR)-induced damage involves poly(ADP-ribose) polymerase (PARP-1 and PARP-2) activity, resulting in the induction of a signalling network responsible for the maintenance of genomic integrity. In the present work, a charged particle microbeam delivering 3.2 MeV protons from a Van de Graaff accelerator has been used to locally irradiate mammalian cells. We show the immediate response of PARPs to local irradiation, concomitant with the recruitment of ATM and Rad51 at sites of DNA damage, both proteins being involved in DNA strand break repair. We found a co-localization but no connection between two DNA damage-dependent post-translational modifications, namely poly(ADP-ribosyl)ation of nuclear proteins and phosphorylation of histone H2AX. Both of them, however, should be considered and used as bona fide immediate sensitive markers of IR damage in living cells. This technique thus provides a powerful approach aimed at understanding the interactions between the signals originating from sites of DNA damage and the subsequent activation of DNA strand break repair mechanisms     

Increased frequency of multiradial chromosome structures in mouse embryonic fibroblasts lacking functional Werner syndrome protein and poly(ADP-ribose) polymerase-1

To determine whether the mouse Werner syndrome homologue (Wrn) and the poly (ADP-ribose) polymerase-1 (PARP-1) enzymes act in concert to prevent specific chromosomal rearrangements, mice with a mutation in the helicase domain of the Wrn gene (Wrn(Deltahel/Deltahel) mice) were crossed to PARP-1 null mice. Spectral karyotyping of the mouse metaphases was used in correlation with conventional G-banded karyotype analysis to precisely define the chromosomal aberrations in cells. Although there was no recurrent clonal chromosome aberration, PARP-1 null/Wrn(Deltahel/Deltahel) fibroblasts were distinguished by an increased frequency of chromatid breaks. Interestingly, multiradial structures were the only type of DNA rearrangement that was significantly higher in such PARP-1 null/Wrn(Deltahel/Deltahel) cells. These results indicate that Wrn and PARP-1 enzymes may be part of a protein complex involved in the processing of DNA breaks that can ultimately lead to multiradial structures when both enzymes are nonfunctional. Finally, regions of chromosomes known to be fragile sites in the mouse genome are not more prone to DNA rearrangements in the absence of both PARP-1 and functional Wrn proteins. Moreover, the low number of recurrent rearranged chromosome at any given site suggest a random mutagenesis process in PARP-1 null/Wrn(Deltahel/Deltahel) fibroblasts.     

3-aminobenzamide,one of poly(ADP-ribose)polymerase-1 inhibitors,rescuesapoptosisin rat models of spinal cord injury

Poly(ADP-ribose)polymerase-1 (PARP-1) is anubiquitous, DNA repair-associated enzyme, which participates in gene expression, cell death, central nerve system (CNS) disorders and oxidative stress. According to the previous studies, PARP-1 over-activation may lead to over-consumption of ATP and even cell apoptosis. Spinal cord injury (SCI) is an inducement towards PARP-1 over-activation due to its massive damage to DNA. 3-aminobenzamide (3-AB) is a kind of PARP-1 inhibitors. The relationship among PARP-1, 3-AB, SCI and apoptosis has not been fully understood. Hence, in the present study, we focused on the effects of 3-AB on cell apoptosis after SCI. Accordingly, SCI model was constructed artificially, and 3-AB was injected intrathecally into the Sprague-Dawley (SD) rats. The results demonstrated an increase in cell apoptosis after SCI. Furthermore, PARP-1 was over-activated after SCI but inhibited by 3-AB injection. In addition, apoptosis-inducing factor (AIF) was inhibited but B-cell lymphoma-2 (Bcl-2) was up-regulated by 3-AB. Interestingly, caspase-3 was not significantly altered with or without 3-AB. In conclusion, our experiments showed that 3-AB, as a PARP-1 inhibitor, could inhibit cell apoptosis after SCI in caspase-independent way, which could provide a better therapeutic target for the treatment of SCI.     

Role of poly(ADP-ribose) polymerase-1 activation in the pathogenesis of diabetic complications: endothelial dysfunction, as a common underlying theme

Hyperglycemia-induced overproduction of superoxide by mitochondrial electron-transport chain triggers several pathways of injury involved in the pathogenesis of diabetic complications [protein kinase C (PKC), hexosamine and polyol pathway fluxes, advanced glycation end product (AGE) formation] by inhibiting glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) activity. Increased oxidative and nitrosative stress activates the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP). PARP activation, on the one hand, depletes its substrate, NAD+, slowing the rate of glycolysis, electron transport, and ATP formation. On the other hand, it inhibits GAPDH by poly(ADP-ribosy)lation. These processes result in acute endothelial dysfunction in diabetic blood vessels, which importantly contributes to the development of various diabetic complications. Accordingly, hyperglycemia-induced activation of PKC isoforms, hexosaminase pathway flux, and AGE formation is prevented by blocking PARP activity. Furthermore, inhibition of PARP protects against diabetic cardiovascular dysfunction in preclinical models. PARP activation is present in microvasculature of human diabetic subjects. The oxidative/nitrosative stress-PARP pathway leads to diabetes-induced endothelial dysfunction, which may be an important underlying mechanism for the pathogenesis of other diabetic complications (cardiomyopathy, nephropathy, neuropathy, and retinopathy). This review focuses on the role of PARP in diabetic complications and the unique therapeutic potential of PARP inhibition in the prevention or reversal of diabetic complications.     

SCF(beta-TrCP1) controls Smad4 protein stability in pancreatic cancer cells

Smad4, also known as deleted in pancreatic carcinoma locus 4 (DPC4), is a critical co-factor in signal transduction pathways activated by transforming growth factor (TGF)-beta-related ligands that regulate cell growth and differentiation. Mutations in Smad4/DPC4 have been identified in approximately 50% of pancreatic adenocarcinomas. Here we report that SCF(beta-TrCP1), a ubiquitin (E3) ligase, is a critical determinant for Smad4 protein degradation in pancreatic cancer cells. We found that F-box protein beta-TrCP1 in this E3 ligase interacted with Smad4 and that SCF(beta-TrCP1) inhibited TGF-beta biological activity in pancreatic cancer cells by decreasing Smad4 stability. Very low Smad4 protein levels in human pancreatic ductal adenocarcinoma cells were observed by immunohistochemistry. By analyzing pancreatic tumor-derived Smad4 mutants, we found that most point-mutated Smad4 proteins, except those within or very close to a mutation cluster region, exhibited higher interaction affinity with beta-TrCP1 and significantly elevated protein ubiquitination by SCF(beta-TrCP1). Furthermore, AsPC-1 and Caco-2, two cancer cell lines harboring Smad4 point mutations, exhibited rapid Smad4 protein degradation due to the effect of SCF(beta-TrCP1). Both Smad4 levels and TGF-beta signaling were elevated by retrovirus-delivered beta-TrCP1 siRNA in pancreatic cancer cells. Therefore, inhibition of Smad4-specific E3 ligase might be a target for therapeutic intervention in pancreatic cancer.