Hyperactivation of PARP Triggers Nonhomologous End-Joining in Repair-Deficient Mouse Fibroblasts

Regulation of poly(ADP-ribose) (PAR) synthesis and turnover is critical to determining cell fate after genotoxic stress. Hyperactivation of PAR synthesis by poly(ADP-ribose) polymerase-1 (PARP-1) occurs when cells deficient in DNA repair are exposed to genotoxic agents; however, the function of this hyperactivation has not been adequately explained. Here, we examine PAR synthesis in mouse fibroblasts deficient in the base excision repair enzyme DNA polymerase β (pol β). The extent and duration of PARP-1 activation was measured after exposure to either the DNA alkylating agent, methyl methanesulfonate (MMS), or to low energy laser-induced DNA damage. There was strong DNA damage-induced hyperactivation of PARP-1 in pol β nullcells, but not in wild-type cells. In the case of MMS treatment, PAR synthesis did not lead to cell death in the pol β null cells, but instead resulted in increased PARylation of the nonhomologous end-joining (NHEJ) protein Ku70 and increased association of Ku70 with PARP-1. Inhibition of the NHEJ factor DNA-PK, under conditions of MMS-induced PARP-1 hyperactivation, enhanced necrotic cell death. These data suggest that PARP-1 hyperactivation is a protective mechanism triggering the classical-NHEJ DNA repair pathway when the primary alkylated base damage repair pathway is compromised.     

Poly(ADP-ribose): PARadigms and PARadoxes

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational protein modification (PTM) catalyzed by members of the poly(ADP-ribose) polymerase (PARP) enzyme family. PARPs use NAD⁺ as substrate and upon cleaving off nicotinamide they transfer the ADP-ribosyl moiety covalently to suitable acceptor proteins and elongate the chain by adding further ADP-ribose units to create a branched polymer, termed poly(ADP-ribose) (PAR), which is rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). In recent years several key discoveries changed the way we look at the biological roles and mode of operation of PARylation. These paradigm shifts include but are not limited to (1) a single PARP enzyme expanding to a PARP family; (2) DNA-break dependent activation extended to several other DNA dependent and independent PARP-activation mechanisms; (3) one molecular mechanism (covalent PARylation of target proteins) underlying the biological effect of PARPs is now complemented by several other mechanisms such as protein–protein interactions, PAR signaling, modulation of NAD⁺ pools and (4) one principal biological role in DNA damage sensing expanded to numerous, diverse biological functions identifying PARP-1 as a real moonlighting protein. Here we review the most important paradigm shifts in PARylation research and also highlight some of the many controversial issues (or paradoxes) of the field such as (1) the mostly synergistic and not antagonistic biological effects of PARP-1 and PARG; (2) mitochondrial PARylation and PAR decomposition, (3) the cross-talk between PARylation and signaling pathways (protein kinases, phosphatases, calcium) and the (4) divergent roles of PARP/PARylation in longevity and in age-related diseases.     

Herpes simplex virus 1 infection activates poly(ADP-ribose) polymerase and triggers the degradation of poly(ADP-ribose) glycohydrolase

Herpes simplex virus 1 infection triggers multiple changes in the metabolism of host cells, including a dramatic decrease in the levels of NAD(+). In addition to its role as a cofactor in reduction-oxidation reactions, NAD(+) is required for certain posttranslational modifications. Members of the poly(ADP-ribose) polymerase (PARP) family of enzymes are major consumers of NAD(+), which they utilize to form poly(ADP-ribose) (PAR) chains on protein substrates in response to DNA damage. PAR chains can subsequently be removed by the enzyme poly(ADP-ribose) glycohydrolase (PARG). We report here that the HSV-1 infection-induced drop in NAD(+) levels required viral DNA replication, was associated with an increase in protein poly(ADP-ribosyl)ation (PARylation), and was blocked by pharmacological inhibition of PARP-1/PARP-2 (PARP-1/2). Neither virus yield nor the cellular metabolic reprogramming observed during HSV-1 infection was altered by the rescue or further depletion of NAD(+) levels. Expression of the viral protein ICP0, which possesses E3 ubiquitin ligase activity, was both necessary and sufficient for the degradation of the 111-kDa PARG isoform. This work demonstrates that HSV-1 infection results in changes to NAD(+) metabolism by PARP-1/2 and PARG, and as PAR chain accumulation can induce caspase-independent apoptosis, we speculate that the decrease in PARG levels enhances the auto-PARylation-mediated inhibition of PARP, thereby avoiding premature death of the infected cell.     

P21-PARP-1 Pathway Is Involved in Cigarette Smoke-Induced Lung DNA Damage and Cellular Senescence

Persistent DNA damage triggers cellular senescence, which may play an important role in the pathogenesis of cigarette smoke (CS)-induced lung diseases. Both p21^CDKN1A (p21) and poly(ADP-ribose) polymerase-1 (PARP-1) are involved in DNA damage and repair. However, the role of p21-PARP-1 axis in regulating CS-induced lung DNA damage and cellular senescence remains unknown. We hypothesized that CS causes DNA damage and cellular senescence through a p21-PARP-1 axis. To test this hypothesis, we determined the levels of γH2AX (a marker for DNA double-strand breaks) as well as non-homologous end joining proteins (Ku70 and Ku80) in lungs of mice exposed to CS. We found that the level of γH2AX was increased, whereas the level of Ku70 was reduced in lungs of CS-exposed mice. Furthermore, p21 deletion reduced the level of γH2AX, but augmented the levels of Ku70, Ku80, and PAR in lungs by CS. Administration of PARP-1 inhibitor 3-aminobenzamide increased CS-induced DNA damage, but lowered the levels of Ku70 and Ku80, in lungs of p21 knockout mice. Moreover, 3-aminobenzamide increased senescence-associated β-galactosidase activity, but decreased the expression of proliferating cell nuclear antigen in mouse lungs in response to CS. Interestingly, 3-aminobenzamide treatment had no effect on neutrophil influx into bronchoalveolar lavage fluid by CS. These results demonstrate that the p21-PARP-1 pathway is involved in CS-induced DNA damage and cellular senescence.     

Poly(ADP-ribose) polymerase 1 accelerates single-strand break repair in concert with poly(ADP-ribose) glycohydrolase

Single-strand breaks are the commonest lesions arising in cells, and defects in their repair are implicated in neurodegenerative disease. One of the earliest events during single-strand break repair (SSBR) is the rapid synthesis of poly(ADP-ribose) (PAR) by poly(ADP-ribose) polymerase (PARP), followed by its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). While the synthesis of poly(ADP-ribose) is important for rapid rates of chromosomal SSBR, the relative importance of poly(ADP-ribose) polymerase 1 (PARP-1) and PARP-2 and of the subsequent degradation of PAR by PARG is unclear. Here we have quantified SSBR rates in human A549 cells depleted of PARP-1, PARP-2, and PARG, both separately and in combination. We report that whereas PARP-1 is critical for rapid global rates of SSBR in human A549 cells, depletion of PARP-2 has only a minor impact, even in the presence of depleted levels of PARP-1. Moreover, we identify PARG as a novel and critical component of SSBR that accelerates this process in concert with PARP-1.     

Loss of Epithelial Hypoxia-Inducible Factor Prolyl Hydroxylase 2 Accelerates Skin Wound Healing in Mice

Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β₃-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.     

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.     

Smad4 disruption accelerates keratinocyte reepithelialization in murine cutaneous wound repair

Keratinocyte reepithelialization is a rate-limiting event in cutaneous wound repair, which involves the migration and proliferation of keratinocytes to cover the denuded dermal surface. Transforming growth factor-β1 (TGF-β1) has the ability to induce epithelial cell migration while inhibiting proliferation, and controversial results have been generated regarding the effect of TGF-β signaling on reepithelialization. In this study, full-thickness skin wounds were made in keratinocyte-specific Smad4 knockout and the control mice. The wound closure, reepithelialization, keratinocyte proliferation, myofibroblast numbers and collagen deposition of were assessed. The results showed that the proliferation of keratinocytes increased, which accelerated the reepithelialization, and led to faster wound repair in the epidermis of Smad4 mutant mice. Upregulation of keratin 17, 14-3-3 sigma and phosphorylated AKT in the hyperproliferative epidermis may be correlated with the accelerated reepithelialization. We conclude that Smad4 plays an inhibitory role in the keratinocyte-mediated reepithelialization of wound healing.     

Valproic Acid Induces Cutaneous Wound Healing In Vivo and Enhances Keratinocyte Motility

Background

Cutaneous wound healing is a complex process involving several signaling pathways such as the Wnt and extracellular signal-regulated kinase (ERK) signaling pathways. Valproic acid (VPA) is a commonly used antiepileptic drug that acts on these signaling pathways; however, the effect of VPA on cutaneous wound healing is unknown.

Methods and Findings

We created full-thickness wounds on the backs of C3H mice and then applied VPA. After 7 d, we observed marked healing and reduced wound size in VPA-treated mice. In the neo-epidermis of the wounds, β-catenin and markers for keratinocyte terminal differentiation were increased after VPA treatment. In addition, α-smooth muscle actin (α-SMA), collagen I and collagen III in the wounds were significantly increased. VPA induced proliferation and suppressed apoptosis of cells in the wounds, as determined by Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining analyses, respectively. In vitro, VPA enhanced the motility of HaCaT keratinocytes by activating Wnt/β-catenin, ERK and phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling pathways.

Conclusions

VPA enhances cutaneous wound healing in a murine model and induces migration of HaCaT keratinocytes.     

Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.     

Poly(ADP-ribose) protects vascular smooth muscle cells from oxidative DNA damage

Vascular smooth muscle cells (VSMCs) undergo death during atherosclerosis, a widespread cardiovascular disease. Recent studies suggest that oxidative damage occurs in VSMCs and induces atherosclerosis. Here, we analyzed oxidative damage repair in VSMCs and found that VSMCs are hypersensitive to oxidative damage. Further analysis showed that oxidative damage repair in VSMCs is suppressed by a low level of poly (ADP-ribosyl)ation (PARylation), a key post-translational modification in oxidative damage repair. The low level of PARylation is not caused by the lack of PARP-1, the major poly(ADP-ribose) polymerase activated by oxidative damage. Instead, the expression of poly(ADP-ribose) glycohydrolase, PARG, the enzyme hydrolyzing poly(ADP-ribose), is significantly higher in VSMCs than that in the control cells. Using PARG inhibitor to suppress PARG activity facilitates oxidative damage-induced PARylation as well as DNA damage repair. Thus, our study demonstrates a novel molecular mechanism for oxidative damage-induced VSMCs death. This study also identifies the use of PARG inhibitors as a potential treatment for atherosclerosis. [BMB Reports 2015; 48(6): 354-359]     

p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates

The cell cycle inhibitor p21^CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates containing single-strand breaks, we found that full-length recombinant GST-tagged p21 but not a C-terminal domain truncated form of p21 was able to stimulate the PARP-1 binding to BER intermediates with no significant influence on the catalytic activity of PARP-1. In addition, we investigate whether the activation of PARP-1 through poly(ADP-ribose) (PAR) synthesis, is required for its interaction with p21. We have found that in human fibroblasts and in HeLa cells treated with the DNA alkylating agent N-methyl-N''-nitro-N-nitrosoguanidine (MNNG), the interaction of p21 with PARP-1 was greatly dependent on PAR synthesis. In fact, an anti-PAR antibody was able to co-immunoprecipitate p21 and PARP-1 from extracts of MNNG-treated cells, while blocking PAR synthesis with the PARP-1 inhibitor Olaparib, drastically reduced the amount of p21 co-immunoprecipitated by a PARP-1 antibody. Our results provide the first evidence that p21 can stimulate the binding of PARP-1 to DNA repair intermediates, and that this cooperation requires PAR synthesis.     

Poly(ADP-Ribose) polymerase 1 as a key regulator of DNA repair

Poly(ADP-ribosyl)ation (PARylation) of proteins is one of the immediate cell responses to DNA damage and is catalyzed by poly(ADP-ribose) polymerases (PARPs). When bound to damaged DNA, some members of the PARP family are activated and use NAD⁺ as a source of ADP to catalyze synthesis of poly(ADP-ribose) (PAR) covalently attached to a target protein. PAR synthesis is considered as a mechanism that provides a local signal of DNA damage and modulates protein functions in response to genotoxic agents. PARP1 is the best-studied protein of the PARP family and is widely known аs a regulator of repair of damaged bases and single-strand nicks. Data are accumulating that PARP1 is additionally involved in double-strand break repair and nucleotide excision repair. The review summarizes the literature data on the role that PARP1 and PARylation play in DNA repair and particularly in base excision repair; original data obtained in our lab are considered in more detail.     

Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities

Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response.     

Search for Cellular Stress Biomarkers in Lymphocytes from Patients with Multiple Sclerosis: A Pilot Study

Multiple Sclerosis (MS) is a chronic disease of the central nervous system, the etiology of which, although not completely known, involves inflammation and autoimmunity. In the present study we aimed at identifying molecular markers of apoptosis, cellular stress and DNA damage in isolated peripheral blood mononuclear cells (PBMCs) of MS patients. The analysis was carried on 19 relapsing-remitting untreated MS patients and 13 healthy individuals. We investigated the emergency-driven synthesis of poly(ADP-ribose) (PAR), the expression level of the constitutive enzyme poly(ADP-ribose) polymerase-1 (PARP-1) and the DNA damage-induced phosphorylation of histone H2AX. PAR accumulation, PARP-1 and phosphorylated H2AX (γH2AX) were detected by immunofluorescence experiments on PBMCs isolated from 19 patients and 13 healthy volunteers. Our results show for the first time a net increased amount in PAR and γH2AX in MS patients compared to healthy individuals. Patients were further subdivided in three groups, according to the neuroimaging (MRI)-based classification of disease phase. Remarkably, we found a positive correlation between the level of γH2AX and MS aggressiveness. In addition, apoptosis in PBMCs was monitored by flow cytometry of both phosphatidylserine exposure (revealed by Annexin V-FITC labeling) and membrane permeability to propidium iodide. Our observations provide the evidence that the number of apoptotic cells was significantly higher in patients compared to healthy individuals, thus suggesting that apoptosis could affect MS lymphocyte function.