poly(ADP-ribose) Polymerase-1 Depletion Research Articles

Role of poly(ADP-ribose) polymerase-1 in regulating human islet cell differentiation

Poly(ADP-ribose) polymerase-1 (PARP1), a fundamental DNA repair enzyme, is known to regulate β cell death, replication, and insulin secretion. PARP1 knockout (KO) mice are resistant to diabetes, while PARP1 overactivation contributes to β cell death. Additionally, PARP1 inhibition (PARPi) improves diabetes complications in patients with type-2 diabetes. Despite these beneficial effects, the use of PARP1 modulating agents in diabetes treatment is largely neglected, primarily due to the poorly studied mechanistic action of PARP1 catalytic function in human β cell development. In the present study, we evaluated PARP1 regulatory action in human β cell differentiation using the human pancreatic progenitor cell line, PANC-1. We surveyed islet census and histology from PARP1 wild-type versus KO mice pancreas in a head-to-head comparison with PARP1 regulatory action for in-vitro β cell differentiation following either PARP1 depletion or its pharmacological inhibition in PANC-1-differentiated islet cells. shRNA mediated PARP1 depleted (SiP) and shRNA control (U6) PANC-1 cells were differentiated into islet-like clusters using established protocols. We observed complete abrogation of new β cell formation with absolute PARP1 depletion while its inhibition using the potent inhibitor, PJ34, promoted the endocrine β cell differentiation and maturation. Immunohistochemistry and immunoblotting for key endocrine differentiation players along with β cell maturation markers highlighted the potential regulatory action of PARP1 and augmented β cell differentiation due to direct interaction of unmodified PARP1 protein elicited p38 MAPK phosphorylation and Neurogenin-3 (Ngn3) re-activation. In summary, our study suggests that PARP1 is required for the proper development and differentiation of human islets. Selective inhibition with PARPi can be an advantage in pushing more insulin-producing cells under pathological conditions and delivers a potential for pilot clinical testing for β cell replacement cell therapies for diabetes.

Abstract P370: The Role Of PJ-34 In The Protection Of Burn-induced Cardiomyopathy

Burn injury results in adverse myocardial remodeling and heart failure through circulatingcatecholamines and androgen and cytokine cascades. The DNA binding protein PARP1(poly ADP ribose polymerase 1) catalyzes a post translational modification to generatePARylation proteins,which changes the normal function of the modified proteins. Bothof PARP-1 and SIRT1 (sirtuin1) are NAD + dependent. In this study, we propose that PJ34 (PARP-1 inhibitor) would protect the function of burn-remodeled cardiomyocytes.Commercial rats were obtained and were subject to 60% total surface body area (TSBA)scald burns by immersing the abdomen and back in boiling water. They were immediatelytreated with the PJ34 post injury. Separately, the cardiomyocytes (Ac16) were exposedto burn-serum replaced culture medium with or without treatment of lentivirus-PARP1 KOand/or SIRT-PGC-1α agonists in vitro . The in vivo experiments showed that burn-ratsexhibited cardiac hypertrophy, fibrosis and an increase in the inflammatory markers IL-1β, IFN-γ and TNFα. Burned rats had an increased oxidative stress, concomitant withelevated PARP-1 activity and reduced Sirtuin-1 (SIRT1) expression. PJ34 treatment ledto increased SIRT1 and Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) levels and attenuated oxidative stress, inflammation, and fibrosis. In vitro tests demonstrated that the treatments of PJ34 and SIRT1-PGC-1α axis agonists incardiomyocytes exposed to burn-serum replaced culture medium led to a significantreduction in mit ROS and mitochondrial dysfunction. In Conclusion, PARP1 depletion byPJ34 in vivo and Letivirus-PARP1 KO Ac16 in vitro attenuated cardiomyopathic featuresin burn-rats through the activation of SIRT1 and its downstream antioxidant defensemechanisms. The results of this study suggest a pivotal role of PARP-1 inhibition intreating burn-induced cardiomyopathy. Keywords: Burn injury; PJ34; PARP1; Cardiomyocyte; Lentivirus.

PARP1 and PRC2 double deficiency promotes BRCA-proficient breast cancer growth by modification of the tumor microenvironment.

Poly (ADP-ribose) polymerase 1 (PARP1) and polycomb-repressive complex 2 (PRC2) are each known for their individual roles in cancer, but their cooperative roles have only been studied in the DNA damage repair process in the context of BRCA-mutant cancers. Here, we show that simultaneous inhibition of PARP1 and PRC2 in the MDA-MB-231 BRCA-proficient triple-negative breast cancer (TNBC) cell line leads to a synthetic viability independent of the mechanisms of DNA damage repair. Specifically, we find that either genetic depletion or pharmacological inhibition of both PARP1 and PRC2 can accelerate tumor growth rate. We attribute this to modifications in the tumor microenvironment (TME) that are induced by double-depleted breast cancer cells, such as promoting intratumoral angiogenesis and increasing the proportion of tumor-promoting type 2 (M2) macrophages. These changes subsequently inhibit cell death and promote proliferation. Mechanistically, we find that PARP1 and PRC2 double depletion induces not only a basal activation of the NF-κB pathway but also a maximal activation of NF-κB within the TME in response to external stimuli such as hypoxia and the presence of macrophages. In summary, our study reveals an unprecedented synthetic viable interaction between PARP1 and PRC2 in BRCA-proficient TNBC and identifies NF-κB as the downstream mediator. DATABASE: RNA-seq data are available in the GEO databases under the accession GSE142769.

Poly(ADP-ribose) Polymerase 1 (PARP1) restrains MyoD-dependent gene expression during muscle differentiation

The myogenic factor MyoD regulates skeletal muscle differentiation by interacting with a variety of chromatin-modifying complexes. Although MyoD can induce and maintain chromatin accessibility at its target genes, its binding and trans-activation ability can be limited by some types of not fully characterized epigenetic constraints. In this work we analysed the role of PARP1 in regulating MyoD-dependent gene expression. PARP1 is a chromatin-associated enzyme, playing a well recognized role in DNA repair and that is implicated in transcriptional regulation. PARP1 affects gene expression through multiple mechanisms, often involving the Poly(ADP-ribosyl)ation of chromatin proteins. In line with PARP1 down-regulation during differentiation, we observed that PARP1 depletion boosts the up-regulation of MyoD targets, such as p57, myogenin, Mef2C and p21, while its re-expression reverts this effect. We also found that PARP1 interacts with some MyoD-binding regions and that its presence, independently of the enzymatic activity, interferes with MyoD recruitment and gene induction. We finally suggest a relationship between the binding of PARP1 and the loss of the activating histone modification H3K4me3 at MyoD-binding regions. This work highlights not only a novel player in the epigenetic control of myogenesis, but also a repressive and catalytic-independent mechanisms by which PARP1 regulates transcription.

SAT-030 Role of PARP-1 in Regulating Cytokine-Mediated Pro-Inflammatory Transcriptional Responses in Macrophages

Macrophages (MF) are major components of the innate immune system involved in mounting inflammatory responses against infection or injury in the host. MF activated by microbial agents, cytokines, or interferon-gamma (IFNg) are highly effective at resolving infections. Mice lacking in the enzyme Poly(ADP-ribose) Polymerase-1 (PARP-1) are resistant to septic shock due to an attenuated pro-inflammatory response. Moreover, PARP-1 modulates the activity of NF-κB, a key pro-inflammatory transcription factor. Although these observations implicate PARP-1 in the regulation of inflammation, its role in innate immune signaling is relatively unexplored. We hypothesized that PARP-1 potentiates pro-inflammatory responses by mediating signal-dependent transcriptional changes in MF. To test this hypothesis, we assessed the effect of PARP-1 inhibition on pro-inflammatory signaling in MF. RNA-seq analysis revealed that PARP inhibition induces significant changes in the LPS- and TNFα-stimulated transcriptomes. Moreover, PARP inhibition, as well as PARP-1 depletion, significantly alters the global expression of IFNg-regulated genes, including a number of key pro-inflammatory cytokines and chemokines. In this regard, we observed that PARP-1 associates with nuclear STAT1α and triggers its poly(ADP-ribosyl)ation (PARylation) in an IFNg-dependent manner. Using a mass spectrometric approach, we identified the sites of PARylation on STAT1α, including a number of glutamate and aspartate residues in its DNA binding and transactivation domains. Using ChIP-seq, we observe significant changes in IFNg-induced STAT1α genomic localization upon treatment with PJ34. Furthermore, PARP inhibition attenuates IFNg-dependent phosphorylation of STAT1. These results suggest that PARP-1-driven PARylation affects DNA-binding and transactivation by STAT1, consequently altering IFNg responses in MF. These studies may open new avenues for using PARP inhibitors to treat autoimmune and inflammatory diseases. This work is supported by the American Heart Association postdoctoral fellowship to RG and a grant (DK069710) from the NIH/NIDDK to WLK.

Characterisation of Deubiquitylating Enzymes in the Cellular Response to High-LET Ionizing Radiation and Complex DNA Damage

PurposeIonizing radiation, particular high-linear energy transfer (LET) radiation, can induce complex DNA damage (CDD) wherein 2 or more DNA lesions are induced in close proximity, which contributes significantly to the cell killing effects. However, knowledge of the enzymes and mechanisms involved in coordinating the recognition and processing of CDD in cellular DNA are currently lacking.Methods and MaterialsA small interfering RNA screen of deubiquitylation enzymes was conducted in HeLa cells irradiated with high-LET α-particles or protons, versus low-LET protons and x-rays, and cell survival was monitored by clonogenic assays. Candidates whose depletion led to decreased cell survival specifically in response to high-LET radiation were validated in both HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells, and the association with CDD repair was confirmed using an enzyme modified neutral comet assay.ResultsDepletion of USP6 decreased cell survival specifically after high-LET α-particles and protons, but not low-LET protons or x-rays. USP6 depletion caused cell cycle arrest and a deficiency in CDD repair mediated through instability of poly(ADP-ribose) polymerase-1 (PARP-1) protein. Increased radiosensitivity of cells to high-LET protons as a consequence of defective CDD repair was furthermore mimicked using the PARP inhibitor olaparib, and through PARP-1 small interfering RNA.ConclusionsUSP6 controls cell survival in response to high-LET radiation by stabilizing PARP-1 protein levels, which is essential for CDD repair. We also describe synergy between CDD induced by high-LET protons and PARP inhibition, or PARP-1 depletion, in effective cancer cell killing.

CHK2-mediated regulation of PARP1 in oxidative DNA damage response.

Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor, which upon activation, recruits downstream proteins by poly(ADP-ribosyl)ation (PARylation). However, it remains largely unclear how PARP1 activity is regulated. Interestingly, the data obtained through this study revealed that PARP1 was co-immunoprecipitated with checkpoint kinase 2 (CHK2), and the interaction was increased after oxidative DNA damage. Moreover, CHK2 depletion resulted in a reduction in overall PARylation. To further explore the functional relationship between PARP1 and CHK2, this study employed H2O2 to induce an oxidative DNA damage response in cells. Here, we showed that CHK2 and PARP1 interact in vitro and in vivo through the CHK2 SCD domain and the PARP1 BRCT domain. Furthermore, CHK2 stimulates the PARylation activity of PARP1 through CHK2-dependent phosphorylation. Consequently, the impaired repair associated with PARP1 depletion could be rescued by re-expression of wild-type PARP1 and the phospho-mimic but not the phospho-deficient mutant. Mechanistically, we showed that CHK2-dependent phosphorylation of PARP1 not only regulates its cellular localization but also promotes its catalytic activity and its interaction with XRCC1. These findings indicate that CHK2 exerts a multifaceted impact on PARP1 in response to oxidative stress to facilitate DNA repair and to maintain cell survival.

PO-113 PARP-1 depletion in combination with either high or low LET reduces the metastatic potential in cultured human cells by suppression of its MMP activity

IntroductionHadron therapy is producing promising clinical results worldwide. The major advantages are its ability to kill radio-resistant tumour and its anti-metastatic activity. In contrast cells irradiated with gamma rays have been shown to increase the metastatic potential of cancer cells. Poly (ADP-ribose) polymerase-1(PARP-1) inhibitors have been widely used as radiosensitizer and many of them are in clinical trial. The purpose of this study was to investigate the effect of PARP-1 depletion in combination with either Carbon Ion Beam (CIB) or gamma irradiation on metastatic potential of cultured cancerous cells.Material and methodsCancer cells were treated with PARP-1 inhibitor and irradiated with CIB or Gamma. The irradiated cells (0–4Gy) for CIB and (0, 2, 4, 6 and 10 Gy) were tested for cell migratory assay and MMP-2 and MMP-9 activity. Alterations of mRNA expression of the MMP-2, MMP-9 genes and in some of the genes those are involved in cell migration (Cadherin, Fibronectin, Vimentin etc.) were studied.Results and discussionsThe activity of MMP-2 and MMP-9 reduces significantly on CIB exposure, which further diminishes when cells were PARP-1 depleted. On Contrary, MMP- 2 and MMP-9 activity significantly increases when cells were irradiated with gamma, whereas, its activity gets reduced upon PARP-1 depletion on a dose dependant manner. The mRNA expression of the MMP genes and the migratory potential was also found to be down-regulated on a dose dependant manner on CIB irradiated and in PARP-1 depleted CIB irradiated cells. However, MMP expression and migratory ability of the gamma irradiated cells were only found to get reduced only in those which were PARP-1 depleted.ConclusionThus, our study clearly demonstrates that PARP-1 inhibition in combination with either high or low LET can be a promising tool in controlling metastatic cancers.

Abstract LB-333: PARP-1 supports HPV infection and HPV-associated head and neck cancer

Abstract Background: As the incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) continues to rise, mechanistic knowledge of HPV16-driven HNSCC remains incomplete. HPV(+) and HPV(-) HNSCCs are molecularly distinct, with altered signaling pathways that have been characterized by proteomic technologies. We recently reported that poly(ADP-ribose)polymerase-1 (PARP-1) is overexpressed in HPV(+) compared to HPV(-) HNSCC. PARP-1 is a multi-functional protein with the ability to produce polymers of ADP-ribose attached to proteins and with major known roles in maintaining genomic integrity, DNA damage repair, transcription, DNA replication, and cell cycle regulation. However, PARP-1 functions in HPV infection and in head and neck cancer have not been fully uncovered. Here, we examine the role of PARP-1 in HPV infection, the interactions of HPV with cellular proteins, and the therapeutic implications of PARP-1 inhibition in HPV(+) HNSCC. Methods: PARP activity was assessed by immunoblotting for levels of poly(ADP-ribose) and by enzymatic PARP-1 ELISA. Expression of HPV genes was measured by RT-PCR. Murine kidney epithelial cells were established from PARP-1 wild-type and knockout mice, and the absence of PARP-1 expression was confirmed by RT-PCR and immunoblotting. Infection with HPV pseudovirus (PsV) containing a GFP expression plasmid was detected by L1 immunofluorescence staining and GFP detection. Survival studies were done in vitro and in vivo with PARP inhibitors veliparib and olaparib. Results: In addition to being overexpressed, we found that PARP-1 was highly enzymatically active in HPV(+) HNSCC as compared to HPV(-) tumors. HPV16 PsV and major capsid protein L1 activated PARP-1 in vitro and in cells. Depletion of PARP-1 and chemical PARP inhibition repressed HPV16 cellular entry, and infection of cells with HPV PsV increased sensitivity of HPV(-) cells to PARP inhibition. Conclusions: We identified a functional role of PARP-1 in initial HPV infection and in HPV-associated cancer. PARP-1 is activated by HPV major capsid protein L1, and HPV(+) HNSCCs maintain PARP-1 enzymatic activity. On top of its role in initial HPV infection, PARP-1 also appears to be important for survival of HPV-infected cells, as inhibition of PARP-1 sensitized cells to HPV PsV infection. These findings suggest that PARP inhibition may hold significant therapeutic potential in the treatment of HPV-associated HNSCC, with our work resulting in the initiation of a window clinical trial ongoing at the Yale Cancer Center. Citation Format: Cassie Pan, Asel Biktasova, Michael Hajek, Andrew Sewell, Tejas Sathe, Gary Bellinger, Wendell Yarbrough, Natalia Issaeva. PARP-1 supports HPV infection and HPV-associated head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-333.

PARP-1 depletion in combination with carbon ion exposure significantly reduces MMPs activity and overall increases TIMPs expression in cultured HeLa cells.

BackgroundHadron therapy is an innovative technique where cancer cells are precisely killed leaving surrounding healthy cells least affected by high linear energy transfer (LET) radiation like carbon ion beam. Anti-metastatic effect of carbon ion exposure attracts investigators into the field of hadron biology, although details remain poor. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors are well-known radiosensitizer and several PARP-1 inhibitors are in clinical trial. Our previous studies showed that PARP-1 depletion makes the cells more radiosensitive towards carbon ion than gamma. The purpose of the present study was to investigate combining effects of PARP-1 inhibition with carbon ion exposure to control metastatic properties in HeLa cells.MethodsActivities of matrix metalloproteinases-2, 9 (MMP-2, MMP-9) were measured using the gelatin zymography after 85 MeV carbon ion exposure or gamma irradiation (0- 4 Gy) to compare metastatic potential between PARP-1 knock down (HsiI) and control cells (H-vector - HeLa transfected with vector without shRNA construct). Expression of MMP-2, MMP-9, tissue inhibitor of MMPs such as TIMP-1, TIMP-2 and TIMP-3 were checked by immunofluorescence and western blot. Cell death by trypan blue, apoptosis and autophagy induction were studied after carbon ion exposure in each cell-type. The data was analyzed using one way ANOVA and 2-tailed paired-samples T-test.ResultsPARP-1 silencing significantly reduced MMP-2 and MMP-9 activities and carbon ion exposure further diminished their activities to less than 3 % of control H-vector. On the contrary, gamma radiation enhanced both MMP-2 and MMP-9 activities in H-vector but not in HsiI cells. The expression of MMP-2 and MMP-9 in H-vector and HsiI showed different pattern after carbon ion exposure. All three TIMPs were increased in HsiI, whereas only TIMP-1 was up-regulated in H-vector after irradiation. Notably, the expressions of all TIMPs were significantly higher in HsiI than H-vector at 4 Gy. Apoptosis was the predominant mode of cell death and no autophagic death was observed.ConclusionsOur study demonstrates for the first time that PARP-1 inhibition in combination with carbon ion synergistically decreases MMPs activity along with overall increase of TIMPs. These data open up the possibilities of improvement of carbon ion therapy with PARP-1 inhibition to control highly metastatic cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-016-0703-x) contains supplementary material, which is available to authorized users.

Poly(ADP-ribose) Polymerase 1 Represses Liver X Receptor-mediated ABCA1 Expression and Cholesterol Efflux in Macrophages

Liver X receptors (LXR) are oxysterol-activated nuclear receptors that play a central role in reverse cholesterol transport through up-regulation of ATP-binding cassette transporters (ABCA1 and ABCG1) that mediate cellular cholesterol efflux. Mouse models of atherosclerosis exhibit reduced atherosclerosis and enhanced regression of established plaques upon LXR activation. However, the coregulatory factors that affect LXR-dependent gene activation in macrophages remain to be elucidated. To identify novel regulators of LXR that modulate its activity, we used affinity purification and mass spectrometry to analyze nuclear LXRα complexes and identified poly(ADP-ribose) polymerase-1 (PARP-1) as an LXR-associated factor. In fact, PARP-1 interacted with both LXRα and LXRβ. Both depletion of PARP-1 and inhibition of PARP-1 activity augmented LXR ligand-induced ABCA1 expression in the RAW 264.7 macrophage line and primary bone marrow-derived macrophages but did not affect LXR-dependent expression of other target genes, ABCG1 and SREBP-1c. Chromatin immunoprecipitation experiments confirmed PARP-1 recruitment at the LXR response element in the promoter of the ABCA1 gene. Further, we demonstrated that LXR is poly(ADP-ribosyl)ated by PARP-1, a potential mechanism by which PARP-1 influences LXR function. Importantly, the PARP inhibitor 3-aminobenzamide enhanced macrophage ABCA1-mediated cholesterol efflux to the lipid-poor apolipoprotein AI. These findings shed light on the important role of PARP-1 on LXR-regulated lipid homeostasis. Understanding the interplay between PARP-1 and LXR may provide insights into developing novel therapeutics for treating atherosclerosis.

Down-regulation of poly (ADP-ribose) polymerase 1 leads to change of hydroquinone cytotoxicity in TK6 cells

Hydroquinone (HQ), one of the most important metabolites derived from benzene, is known to be associated with acute myelogenous leukemia risk; however, its carcinogenic mechanism remains unclear. In a previous study, we found that low-level of benzene exposure down-regulated the expression of poly(ADP-ribose)polymerase 1 (PARP1). Here, we employed RNA interference to knock down PARP1 expression in TK6 cells and explored the potential role of PARP1 in HQ-induced cytotoxicity. The results showed that stable PARP1-knockdown cells were successfully constructed and more than 80% inhibition of PARP1 expression was confirmed. We found that HQ treatment of TK6 cells decreased cell viability, increased cell apoptosis, and caspase3/7 activity. Knockdown of PARP1 in HQ-treated TK6 cells prevented caspase3 activation, and increased apoptosis than that in the wild-type TK6 cells, with fully functional PARP1. The results also showed that down-regulation of PARP1 led to a decrease in cell proliferation and an enhanced susceptibility to HQ-induced cytotoxicity with concentration less than 40 μM than that in normal TK6 cells. Moreover, PARP1-knockdown TK6 cells treated with HQ displayed an increased level of DNA double-strand breaks as measured by olive tail moment. No evidence was obtained of an effect of PARP1 depletion on H2AX phosphorylation induction. Under the experimental conditions, PARP1 has a role in HQ-induced DNA damage repair rather than in long-term chromatin modifications signaled by phosphorylated H2AX.

PARP-1 mediates LPS-induced HMGB1 release by macrophages through regulation of HMGB1 acetylation.

The high-mobility group box protein 1 (HMGB1) is increasingly recognized as an important inflammatory mediator. In some cases, the release of HMGB1 is regulated by poly(ADP-ribose) polymerase-1 (PARP-1), but the mechanism is still unclear. In this study, we report that PARP-1 activation contributes to LPS-induced PARylation of HMGB1, but the PARylation of HMGB1 is insufficient to direct its migration from the nucleus to the cytoplasm; PARP-1 regulates the translocation of HMGB1 to the cytoplasm through upregulating the acetylation of HMGB1. In mouse bone marrow-derived macrophages, genetic and pharmacological inhibition of PARP-1 suppressed LPS-induced translocation and release of HMGB1. Increased PARylation was accompanied with the nucleus-to-cytoplasm translocation and release of HMGB1 upon LPS exposure, but PARylated HMGB1 was located at the nucleus, unlike acetylated HMGB1 localized at the cytoplasm in an import assay. PARP inhibitor and PARP-1 depletion decreased the activity ratio of histone acetyltransferases to histone deacetylases that elevated after LPS stimulation and impaired LPS-induced acetylation of HMGB1. In addition, PARylation of HMGB1 facilitates its acetylation in an in vitro enzymatic reaction. Furthermore, reactive oxygen species scavenger (N-acetyl-l-cysteine) and the ERK inhibitor (FR180204) impaired LPS-induced PARP activation and HMGB1 release. Our findings suggest that PARP-1 regulates LPS-induced acetylation of HMGB1 in two ways: PARylating HMGB1 to facilitate the latter acetylation and increasing the activity ratio of histone acetyltransferases to histone deacetylases. These studies revealed a new mechanism of PARP-1 in regulating the inflammatory response to endotoxin.

Fine-tuning of Smad protein function by poly(ADP-ribose) polymerases and poly(ADP-ribose) glycohydrolase during transforming growth factor β signaling.

BackgroundInitiation, amplitude, duration and termination of transforming growth factor β (TGFβ) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFβ signaling.MethodsA robust cell model of TGFβ signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFβ stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference.ResultsTGFβ signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFβ target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFβ-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1.ConclusionNuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFβ signaling.

Modulation of poly(ADP-ribose) polymerase-1 (PARP-1)-mediated oxidative cell injury by ring finger protein 146 (RNF146) in cardiac myocytes.

Poly(ADP-ribose) polymerase-1 (PARP-1) activation is a hallmark of oxidative stress-induced cellular injury that can lead to energetic failure and necrotic cell death via depleting the cellular nicotinamide adenine dinucleotide (NAD(+)) and ATP pools. Pharmacological PARP-1 inhibition or genetic PARP-1 deficiency exert protective effects in multiple models of cardiomyocyte injury. However, the connection between nuclear PARP-1 activation and depletion of the cytoplasmic and mitochondrial energy pools is poorly understood. By using cultured rat cardiomyocytes, here we report that ring finger protein 146 (RNF146), a cytoplasmic E3-ubiquitin ligase, acts as a direct interactor of PARP-1. Overexpression of RNF146 exerts protection against oxidant-induced cell death, whereas PARP-1-mediated cellular injury is augmented after RNF146 silencing. RNF146 translocates to the nucleus upon PARP-1 activation, triggering the exit of PARP-1 from the nucleus, followed by rapid degradation of both proteins. PARP-1 and RNF146 degradation occurs in the early phase of myocardial ischemia-reperfusion injury; it precedes the induction of heat shock protein expression. Taken together, PARP-1 release from the nucleus and its rapid degradation represent newly identified steps of the necrotic cell death program induced by oxidative stress. These steps are controlled by the ubiquitin-proteasome pathway protein RNF146. The current results shed new light on the mechanism of necrotic cell death. RNF146 may represent a distinct target for experimental therapeutic intervention of oxidant-mediated cardiac injury.

Abstract C297: PARP1 and BIN1 cooperate to act as a novel co-repressor for E2F1.

Abstract Background: Bridging integrator 1 (BIN1) is a transcriptional co-repressor and inhibits oncogenic activities of c-MYC and E1A oncoproteins1. BIN1 inhibits c-MYC through direct interaction, whereas the mechanism by which BIN1 attenuates the oncogenic transformation mediated by adenovirus E1A remains elusive. Since BIN1 does not physically interact with E1A, we hypothesized that BIN1 diminishes a cellular effector, which can be activated by E1A and is essential for E1A transformation. One of the well-recognized cellular effectors for E1A transformation is the E2F1 transcription factor. Although retinoblastoma (RB) protein is an authentic E2F1 inhibitor, loss of RB does not immediately result in cancer development, suggesting that an anti-E2F1 function, which is independent of RB, compensates for RB deficiency. BIN1 was recently shown to interact with poly (ADP-ribose) polymerase1 (PARP1), a component of transcriptional complex2. Given that PARP1 also interacts with E2F1, we hypothesized functional cross talk between BIN1 and PARP1 to curb E2F1 activity. Experimental Procedures: E2F1 activity was determined using an adenovirus-driven E2F1-sensitive luciferase reporter vector, E2A-Luc. Protein-protein interaction was studied by co-immunoprecipitation followed by Western blot analysis. Chromatin immunoprecipitation assay was used to demonstrate protein-DNA interaction. Colony formation and foci formation assays were performed to investigate BIN1-mediated tumor suppression in the presence and absence of PARP1. Results: We discovered that BIN1 physically interacts with E2F1 and inhibits its transactivation. Interestingly, depletion of PARP1 released endogenous E2F1 activity, regardless of the status of RB expression. Furthermore, BIN1 failed to inhibit E2F1 activity in the absence of PARP1, but RB does not need PARP1 to inhibit E2F1 activity. Chromatin immunoprecipitation assays suggested that BIN1 and PARP1 co-existed on an E2F-responsive promoter. Moreover, in the absence of PARP1, the binding affinities of both BIN1 and E2F1 to the promoter were significantly reduced. In addition, only in the presence of PARP1, ectopically expressed BIN1 inhibited tumor colony formation and HPV16 E7 oncoprotein-dependent cellular transformation. Our results suggest that BIN1 and PARP1 cooperate to inhibit E2F1 activity and suppress oncogenic transformation. Conclusion: We conclude that BIN1 is a novel E2F1 corepressor in cooperation with PARP1. BIN1-PARP1 interaction may serve as a safety device to control deregulated E2F1 activity due to RB loss. This study was supported by NIH R01CA140379 (to D.S.). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C297. Citation Format: Alpana Kumari, Slovénie Pyndiah, Erica K. Cassimere, Daitoku Sakamuro. PARP1 and BIN1 cooperate to act as a novel co-repressor for E2F1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C297.

X-Ray Induced DNA Damage and Repair in Germ Cells of PARP1−/− Male Mice

Poly(ADP-ribose)polymerase-1 (PARP1) is a nuclear protein implicated in DNA repair, recombination, replication, and chromatin remodeling. The aim of this study was to evaluate possible differences between PARP1−/− and wild-type mice regarding induction and repair of DNA lesions in irradiated male germ cells. Comet assay was applied to detect DNA damage in testicular cells immediately, and two hours after 4 Gy X-ray irradiation. A similar level of spontaneous and radiation-induced DNA damage was observed in PARP1−/− and wild-type mice. Conversely, two hours after irradiation, a significant level of residual damage was observed in PARP1−/− cells only. This finding was particularly evident in round spermatids. To evaluate if PARP1 had also a role in the dynamics of H2AX phosphorylation in round spermatids, in which γ-H2AX foci had been shown to persist after completion of DNA repair, we carried out a parallel analysis of γ-H2AX foci at 0.5, 2, and 48 h after irradiation in wild-type and PARP1−/− mice. No evidence was obtained of an effect of PARP1 depletion on H2AX phosphorylation induction and removal. Our results suggest that, in round spermatids, under the tested experimental conditions, PARP1 has a role in radiation-induced DNA damage repair rather than in long-term chromatin modifications signaled by phosphorylated H2AX.

Role of poly(ADP-ribose) polymerase-1 in the removal of UV-induced DNA lesions by nucleotide excision repair

Among the earliest responses of mammalian cells to DNA damage is catalytic activation of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Activated PARP-1 forms the polymers of ADP-ribose (pADPr or PAR) that posttranslationally modify its target proteins, such as PARP-1 and DNA repair-related proteins. Although this metabolism is known to be implicated in other repair pathways, here we show its role in the versatile nucleotide excision repair pathway (NER) that removes a variety of DNA damages including those induced by UV. We show that PARP inhibition or specific depletion of PARP-1 decreases the efficiency of removal of UV-induced DNA damage from human skin fibroblasts or mouse epidermis. Using NER-proficient and -deficient cells and in vitro PARP-1 assays, we show that damaged DNA-binding protein 2 (DDB2), a key lesion recognition protein of the global genomic subpathway of NER (GG-NER), associates with PARP-1 in the vicinity of UV-damaged chromatin, stimulates its catalytic activity, and is modified by pADPr. PARP inhibition abolishes UV-induced interaction of DDB2 with PARP-1 or xeroderma pigmentosum group C (XPC) and also decreases localization of XPC to UV-damaged DNA, which is a key step that leads to downstream events in GG-NER. Thus, PARP-1 collaborates with DDB2 to increase the efficiency of the lesion recognition step of GG-NER.

Effect of poly(ADP‐ribose) polymerase 1 on integration of the adeno‐associated viral vector genome

Adeno-associated virus type 2 (AAV) has the ability to target integration of its DNA into a specific locus of the human genome. Site-specific AAV integration is mediated by viral Rep proteins, although the role of cellular factors involved in this process is largely unknown. Recent studies provide evidence showing that cellular DNA repair proteins are involved in targeted integration of AAV, although their specific roles are not well defined. In the present study, we investigated the interaction between Rep and proteins of the back-up nonhomologous end-joining pathway (B-NHEJ). We then analyzed the effect of one of these proteins, poly(ADP-ribose) polymerase 1 (PARP1) on AAV integration. We show that AAV Rep interacts with B-NHEJ members DNA ligase III and PARP1 but does not associate with the scaffolding factor XRCC1. Moreover, PARP1 and Rep bind directly and not via DNA-protein interactions. We also found that Rep increases the enzymatic activity of PARP1 potentially through the endonuclease activity of Rep. Finally, we demonstrate that both chemical inhibition of PARP1 and PARP1 depletion using small hairpin RNA enhance integration of the AAV genome in HeLa cells. The findings of the present study indicate that manipulation of PARP1 activity could be used as a tool for developing new, effective AAV-based therapies for the treatment of genetic diseases and cancer.

Small interfering RNA mediated Poly (ADP-ribose) Polymerase-1 inhibition upregulates the heat shock response in a murine fibroblast cell line

Poly (ADP-ribose) polymerase-1 (PARP-1) is a highly conserved multifunctional enzyme, and its catalytic activity is stimulated by DNA breaks. The activation of PARP-1 and subsequent depletion of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP) contributes to significant cytotoxicity in inflammation of various etiologies. On the contrary, induction of heat shock response and production of heat shock protein 70 (HSP-70) is a cytoprotective defense mechanism in inflammation. Recent data suggests that PARP-1 modulates the expression of a number of cellular proteins at the transcriptional level. In this study, small interfering RNA (siRNA) mediated PARP-1 knockdown in murine wild-type fibroblasts augmented heat shock response as compared to untreated cells (as evaluated by quantitative analysis of HSP-70 mRNA and HSP-70 protein expression). These events were associated with increased DNA binding of the heat shock factor-1 (HSF-1), the major transcription factor of the heat shock response. Co-immunoprecipitation experiments in nuclear extracts of the wild type cells demonstrated that PARP-1directly interacted with HSF-1. These data demonstrate that, in wild type fibroblasts, PARP-1 plays a pivotal role in modulating the heat shock response both through direct interaction with HSF-1 and poly (ADP-ribosylation).