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

A Knockout of Poly(ADP-Ribose) Polymerase 1 in a Human Cell Line: An Influence on Base Excision Repair Reactions in Cellular Extracts.

Base excision repair (BER) is the predominant pathway for the removal of most forms of hydrolytic, oxidative, and alkylative DNA lesions. The precise functioning of BER is achieved via the regulation of each step by regulatory/accessory proteins, with the most important of them being poly(ADP-ribose) polymerase 1 (PARP1). PARP1's regulatory functions extend to many cellular processes including the regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. Systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells are extremely limited. In this study, a CRISPR/Cas9-based technique was used to knock out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with the putative PARP1 deletion were characterized by several approaches including PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, quantitative PCR analysis of PARP1 mRNA, Western blot analysis of whole-cell-extract (WCE) proteins with anti-PARP1 antibodies, and PAR synthesis in WCEs. A quantitative PCR analysis of mRNAs coding for BER-related proteins-PARP2, uracil DNA glycosylase 2, apurinic/apyrimidinic endonuclease 1, DNA polymerase β, DNA ligase III, and XRCC1-did not reveal a notable influence of the PARP1 knockout. The corresponding WCE catalytic activities evaluated in parallel did not differ significantly between the mutant and parental cell lines. No noticeable effect of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed either.

PARP1 Promotes Heart Regeneration and Cardiomyocyte Proliferation.

Myocardial infarction causes cardiomyocyte loss, and depleted cardiomyocyte proliferative capacity after birth impinges the heart repair process, eventually leading to heart failure. This study aims to investigate the role of Poly(ADP-Ribose) Polymerase 1 (PARP1) in the regulation of cardiomyocyte proliferation and heart regeneration. Our findings demonstrated that PARP1 knockout impaired cardiomyocyte proliferation, cardiac function, and scar formation, while PARP1 overexpression improved heart regeneration in apical resection-operated mice. Mechanistically, we found that PARP1 interacts with and poly(ADP-ribosyl)ates Heat Shock Protein 90 Alpha Family Class B Member 1 (HSP90AB1) and increases binding between HSP90AB1 and Cell Division Cycle 37 (CDC37) and cell cycle kinase activity, thus activating cardiomyocyte cell cycle. Our results reveal that PARP1 promotes heart regeneration and cardiomyocyte proliferation via poly(ADP-ribosyl)ation of HSP90AB1 activating the cardiomyocyte cell cycle, suggesting that PARP1 may be a potential therapeutic target in treating cardiac injury.

Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.

Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.

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.

Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage

BackgroundN-acetyltransferase 10 (NAT10), an abundant nucleolar protein with both lysine and RNA cytidine acetyltransferase activities, has been implicated in Hutchinson-Gilford progeria syndrome and human cancer. We and others recently demonstrated that NAT10 is translocated from the nucleolus to the nucleoplasm after DNA damage, but the underlying mechanism remains unexplored.MethodsThe NAT10 and PARP1 knockout (KO) cell lines were generated using CRISPR-Cas9 technology. Knockdown of PARP1 was performed using specific small interfering RNAs targeting PARP1. Cells were irradiated with γ-rays using a 137Cs Gammacell-40 irradiator and subjected to clonogenic survival assays. Co-localization and interaction between NAT10 and MORC2 were examined by immunofluorescent staining and immunoprecipitation assays, respectively. PARylation of NAT10 and translocation of NAT10 were determined by in vitro PARylation assays and immunofluorescent staining, respectively.ResultsHere, we provide the first evidence that NAT10 underwent covalent PARylation modification following DNA damage, and poly (ADP-ribose) polymerase 1 (PARP1) catalyzed PARylation of NAT10 on three conserved lysine (K) residues (K1016, K1017, and K1020) within its C-terminal nucleolar localization signal motif (residues 983–1025). Notably, mutation of those three PARylation residues on NAT10, pharmacological inhibition of PARP1 activity, or depletion of PARP1 impaired NAT10 nucleoplasmic translocation after DNA damage. Knockdown or inhibition of PARP1 or expression of a PARylation-deficient mutant NAT10 (K3A) attenuated the co-localization and interaction of NAT10 with MORC family CW-type zinc finger 2 (MORC2), a newly identified chromatin-remodeling enzyme involved in DNA damage response, resulting in a decrease in DNA damage-induced MORC2 acetylation at lysine 767. Consequently, expression of a PARylation-defective mutant NAT10 resulted in enhanced cellular sensitivity to DNA damage agents.ConclusionCollectively, these findings indicate that PARP1-mediated PARylation of NAT10 is key for controlling its nucleoplasmic translocation and function in response to DNA damage. Moreover, our findings provide novel mechanistic insights into the sophisticated paradigm of the posttranslational modification-driven cellular response to DNA damage.7RTHzWMYUwTGweyv2z3-hVVideo

PARP-1 regulates inflammasome activity by poly-ADP-ribosylation of NLRP3 and interaction with TXNIP in primary macrophages

Poly(ADP-ribose) polymerase-1 (PARP-1) plays an essential role in DNA repair by catalyzing the polymerization of ADP-ribose unit to target proteins. Several studies have shown that PARP-1 can regulate inflammatory responses in various disease models. The intracellular Nod-like receptor NLRP3 has emerged as the most crucial innate immune receptor because of its broad specificity in mediating immune response to pathogen invasion and danger signals associated with cellular damage. In our study, we found NLRP3 stimuli-induced caspase-1 maturation and IL-1β production were impaired by PARP-1 knockout or PARP-1 inhibition in bone marrow-derived macrophages (BMDM). The step 1 signal of NLRP3 inflammasome activation was not affected by PARP-1 deficiency. Moreover, ATP-induced cytosolic ROS production was lower in Parp-1−/− BMDM, resulting in the decreased inflammasome complex assembly. PARP-1 can translocate to cytosol upon ATP stimulation and trigger the PARylation modification on NLRP3, leading to NLRP3 inflammasome assembly. PARP-1 was also a bridge between NLRP3 and thioredoxin-interacting protein (TXNIP) and participated in NLRP3/TXNIP complex formation for inflammasome activation. Overall, PARP-1 positively regulates NLRP3 inflammasome activation via increasing ROS production and interaction with TXNIP and NLRP3, leading to PARylation of NLRP3. Our data demonstrate a novel regulatory mechanism for NLRP3 inflammasome activation by PARP-1. Therefore, PARP-1 can serve as a potential target in the treatment of IL-1β associated inflammatory diseases.

Poly(ADP-Ribose) Polymerase 1 Promotes Inflammation and Fibrosis in a Mouse Model of Chronic Pancreatitis.

Chronic pancreatitis (CP) is an inflammatory disease of the pancreas characterized by ductal obstructions, tissue fibrosis, atrophy and exocrine and endocrine pancreatic insufficiency. However, our understanding is very limited concerning the disease’s progression from a single acute inflammation, via recurrent acute pancreatitis (AP) and early CP, to the late stage CP. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor enzyme activated mostly by oxidative DNA damage. As a co-activator of inflammatory transcription factors, PARP1 is a central mediator of the inflammatory response and it has also been implicated in acute pancreatitis. Here, we set out to investigate whether PARP1 contributed to the pathogenesis of CP. We found that the clinically used PARP inhibitor olaparib (OLA) had protective effects in a murine model of CP induced by multiple cerulein injections. OLA reduced pancreas atrophy and expression of the inflammatory mediators TNFα and interleukin-6 (IL-6), both in the pancreas and in the lungs. Moreover, there was significantly less fibrosis (Masson’s trichrome staining) in the pancreatic sections of OLA-treated mice compared to the cerulein-only group. mRNA expression of the fibrosis markers TGFβ, smooth muscle actin (SMA), and collagen-1 were markedly reduced by OLA. CP was also induced in PARP1 knockout (KO) mice and their wild-type (WT) counterparts. Inflammation and fibrosis markers showed lower expression in the KO compared to the WT mice. Moreover, reduced granulocyte infiltration (tissue myeloperoxidase activity) and a lower elevation of serum amylase and lipase activity could also be detected in the KO mice. Furthermore, primary acinar cells isolated from KO mice were also protected from cerulein-induced toxicity compared to WT cells. In summary, our data suggest that PARP inhibitors may be promising candidates for repurposing to treat not only acute but chronic pancreatitis as well.

Poly(ADP-ribose) polymerase-1 depletion enhances the severity of inflammation in an imiquimod-induced model of psoriasis.

Poly(ADP-ribose) polymerase-1 (PARP1) is a pro-inflammatory protein, whose pro-inflammatory properties were demonstrated in human. The pro-inflammatory properties of PARP1 were shown in Th1- and Th2-mediated inflammatory pathologies, but not Th17-mediated inflammation. Thus, we studied the role of PARP1 in the imiquimod-induced model of psoriasis. To our surprise, in imiquimod-induced psoriasis, PARP1 acted as an anti-inflammatory factor and its genetic deletion exacerbated symptoms. We showed that in the absence of PARP1, the epidermis thickened and the number of TUNEL-positive cells decreased in the epidermis. These data indicate programmed cell death is decreased in keratinocytes. Changes in involucrin expression suggest that keratinocyte differentiation is hampered. Furthermore, epidermal expression of IL6 increased in the psoriasiform lesions of PARP1 knockout mice, suggesting that the inflammatory response is also derailed in the absence of PARP1. Finally, we showed that PARP1 expression is reduced in human psoriatic lesions compared with control skin samples. In imiquimod-treated HPV-KER keratinocytes, PARP inhibition recapitulated the in vivo findings, namely keratinocyte hyperproliferation; furthermore, the mRNA expression of psoriasis-associated cytokines (IL6, IL1β, IL8, IL17 and IL23A) was also induced. The inhibition of TRPV1 abrogated the effects of the combined imiquimod+PARP inhibitor treatment.

Poly(ADP-ribose) Polymerase-1 is required for hepatocyte proliferation and liver regeneration in mice

Liver regeneration is an orchestrated cellular response and the mechanisms underlying it have been extensively studied using the partial hepatectomy (PHx) model. Poly(ADP-Ribose) Polymerase-1 (PARP1) is a common enzyme for post-translational modification of proteins. Here, we aimed to determine the role of PARP1 in liver regeneration. We found that PARP1 activity was strongly associated with hepatocyte proliferation after PHx. PARP1 knockout mice showed impaired liver recovery and suppressed hepatocyte proliferation after PHx. Mechanistically, PARP1 knockout repressed YAP activity and inhibited expression of cell cycle-associated proteins in liver tissues. Therefore, our findings highlight specialized roles for PARP1 in regulating hepatocytes proliferation and liver regeneration.

LPS protects macrophages from AIF-independent parthanatos by downregulation of PARP1 expression, induction of SOD2 expression, and a metabolic shift to aerobic glycolysis

In inflamed tissues or during ischemia-reperfusion episodes, activated macrophages produce large amounts of reactive species and are, thus, exposed to the damaging effects of reactive species. Here, our goal was to investigate the mechanism whereby activated macrophages protect themselves from oxidant stress-induced cell death. Hydrogen peroxide-treated mouse bone marrow-derived macrophages (BMDM) and THP-1 human monocyte-derived cells were chosen as models. We found a gradual development of resistance: first in monocyte-to-macrophage differentiation, and subsequently after lipopolysaccharide (LPS) exposure. Investigating the mechanism of the latter, we found that exposure to intense hydrogen peroxide stress causes poly(ADP-ribose) polymerase-1 (PARP-1) dependent programmed necrotic cell death, also known as parthanatos, as indicated by the protected status of PARP-1 knockout BMDMs and the protective effect of the PARP inhibitor PJ-34. In hydrogen peroxide-treated macrophages, however, apoptosis inducing factor (AIF) proved dispensable for parthanatos; nuclear translocation of AIF was not observed. A key event in LPS-mediated protection against the hydrogen peroxide-induced AIF independent parthanatos was downregulation of PARP1 mRNA and protein. The importance of this event was confirmed by overexpression of PARP1 in THP1 cells using a viral promoter, which lead to stable PARP1 levels even after LPS treatment and unresponsiveness to LPS-induced cytoprotection. In BMDMs, LPS-induced PARP1 suppression lead to prevention of NAD+ depletion. Moreover, LPS also induced expression of antioxidant proteins (superoxide dismutase-2, thioredoxin reductase 1 and peroxiredoxin) and triggered a metabolic shift to aerobic glycolysis, also known as the Warburg effect. In summary, we provide evidence that in macrophages intense hydrogen peroxide stress causes AIF-independent parthanatos from which LPS provides protection. The mechanism of LPS-mediated cytoprotection involves downregulation of PARP1, spared NAD+ and ATP pools, upregulation of antioxidant proteins, and a metabolic shift from mitochondrial respiration to aerobic glycolysis.

3-Aminobenzamide Prevents Concanavalin A-Induced Acute Hepatitis by an Anti-inflammatory and Anti-oxidative Mechanism.

Concanavalin A is known to activate T cells and to cause liver injury and hepatitis, mediated in part by secretion of TNFα from macrophages. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have been shown to prevent tissue damage in various animal models of inflammation. The objectives of this study were to evaluate the efficacy and mechanism of the PARP-1 inhibitor 3-aminobenzamide (3-AB) in preventing concanavalin A-induced liver damage. We tested the in vivo effects of 3-AB on concanavalin A-treated mice, its effects on lipopolysaccharide (LPS)-stimulated macrophages in culture, and its ability to act as a scavenger in in vitro assays. 3-AB markedly reduced inflammation, oxidative stress, and liver tissue damage in concanavalin A-treated mice. In LPS-stimulated RAW264.7 macrophages, 3-AB inhibited NFκB transcriptional activity and subsequent expression of TNFα and iNOS and blocked NO production. In vitro, 3-AB acted as a hydrogen peroxide scavenger. The ROS scavenger N-acetylcysteine (NAC) and the ROS formation inhibitor diphenyleneiodonium (DPI) also inhibited TNFα expression in stimulated macrophages, but unlike 3-AB, NAC and DPI were unable to abolish NFκB activity. PARP-1 knockout failed to affect NFκB and TNFα suppression by 3-AB in stimulated macrophages. Our results suggest that 3-AB has a therapeutic effect on concanavalin A-induced liver injury by inhibiting expression of the key pro-inflammatory cytokine TNFα, via PARP-1-independent NFκB suppression and via an NFκB-independent anti-oxidative mechanism.

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.

Abstract 68: Functional interactions of HPV and PARP-1 in head and neck cancer

Abstract Though the overall incidence of head and neck cancer has been declining over the last several decades, there has been a concomitant and worrisome rise in human-papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC). HPV infection is a necessary but not sufficient condition for cancer development. Unlike in cervical cancer, where several co-factors including smoking and co-infection with other agents are well established, how HPV drives malignant transformation in head and neck cancer and which cellular factors are required are not well characterized. Recent evidence from our group has shown that poly (ADP-ribose) polymerase-1 (PARP-1) is overexpressed and overactive in HPV-positive (HPV(+)), but not HPV-negative (HPV(-)), oropharyngeal squamous cell carcinoma (OPSCC). The significance of PARP-1 overexpression and hyperactivation in cancer is only beginning to emerge. High levels of PARP-1 are found in a fraction of hepatocellular and breast cancers and correlate with poor prognosis. Recently, PARP-1 was implicated in prostate cancer progression. This study explores the role of PARP-1 in HPV infection and HNSCC carcinogenesis. Biochemical methods were used to determine PARP activity in vitro, in head and neck tumors, and during infection with HPV16 pseudovirus (PsV) or overexpression of HPV16 proteins in epithelial cells. The response to PARP inhibition was evaluated with clonogenic survival assays. HPV L1 mRNA in HNSCC was detected using quantitative real-time PCR and HPV L1 protein levels were determined by immunohistochemistry. Here, we show that the HPV16 major capsid protein L1 directly binds PARP-1 and activates poly(ADP-ribosyl)ation in the absence of DNA damage in vitro and in HPV(-) cells. Using PARP-1 knockout cells, we found that PARP-1 expression and enzymatic activity supports initial HPV infection. Furthermore, HPV(-) cells were sensitized to PARP inhibition both after L1 overexpression and after infection with HPV16 PsV particles. In conclusion, we report functional interactions between the viral capsid and cellular protein, signifying a vital role played by PARP-1 in HPV infection and carcinogenesis. Importantly, our data provide a mechanistic explanation for pharmacological PARP inhibition as an intriguing therapeutic opportunity for HPV(+) head and neck cancers. Citation Format: Cyril S. Gary, Michael Hajek, Asel Biktasova, Andrew Sewell, Yarbrough G. Wendell, Natalia Issaeva. Functional interactions of HPV and PARP-1 in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 68.

Neer Award 2016: reduced muscle degeneration and decreased fatty infiltration after rotator cuff tear in a poly(ADP-ribose) polymerase 1 (PARP-1) knock-out mouse model

Disturbed muscular architecture, atrophy, and fatty infiltration remain irreversible in chronic rotator cuff tears even after repair. Poly (adenosine 5'-diphosphate-ribose) polymerase 1 (PARP-1) is a key regulator of inflammation, apoptosis, muscle atrophy, muscle regeneration, and adipocyte development. We hypothesized that the absence of PARP-1 would lead to a reduction in damage to the muscle subsequent to combined tenotomy and neurectomy in a PARP-1 knockout (KO) mouse model. PARP-1 KO and wild-type C57BL/6 (WT group) mice were analyzed at 1, 6, and 12 weeks (total n = 84). In all mice, the supraspinatus and infraspinatus muscles of the left shoulder were detached and denervated. Macroscopic analysis, magnetic resonance imaging, gene expression analysis, immunohistochemistry, and histology were used to assess the differences in PARP-1 KO and WT mice. The muscles in the PARP-1 KO group had significantly less retraction, atrophy, and fatty infiltration after 12 weeks than in the WT group. Gene expression of inflammatory, apoptotic, adipogenic, and muscular atrophy genes was significantly decreased in PARP-1 KO mice in the first 6 weeks. Absence of PARP-1 leads to a reduction in muscular architectural damage, early inflammation, apoptosis, atrophy, and fatty infiltration after combined tenotomy and neurectomy of the rotator cuff muscle. Although the macroscopic reaction to injury is similar in the first 6 weeks, the ability of the muscles to regenerate was much greater in the PARP-1 KO group, leading to a near-normalization of the muscle after 12 weeks.

Androgen and PARP-1 Regulation of TRPM2 Channels after Ischemic Injury

The calcium-permeable transient receptor potential M2 (TRPM2) ion channel was recently demonstrated to have a sexually dimorphic contribution to ischemic brain injury, with inhibition or knockdown of the channel protecting male brain preferentially. We tested the hypothesis that androgen signaling is required for this male-specific cell-death pathway. Additionally, we tested the hypothesis that differential activation of the enzyme poly (ADP-ribose) polymerase-1 (PARP-1) is responsible for male-specific TRPM2 channel activation and neuronal injury. We observed that administration of the TRPM2 inhibitor clotrimazole (CTZ) 2 hours after onset of ischemia reduced infarct volume in male mice and that protection from ischemic damage by CTZ was abolished by removal of testicular androgens (castration; CAST) and rescued by androgen replacement. Male PARP-1 knockout mice had reduced ischemic damage compared with WT mice and inhibition of TRPM2 with CTZ failed to reduce infarct size. Lastly, we observed that ischemia increased PARP activity in the peri-infarct region of male mice to a greater extent than female mice and the difference was abolished in CAST male mice. Data presented in the current study indicate that TRPM2-mediated neuronal death in the male brain requires intact androgen signaling and PARP-1 activity.

YY1-binding sites provide central switch functions in the PARP-1 gene expression network.

Evidence is presented for the involvement of the interplay between transcription factor Yin Yang 1 (YY1) and poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of mouse PARP-1 gene (muPARP-1) promoter activity. We identified potential YY1 binding motifs (BM) at seven positions in the muPARP-1 core-promoter (−574/+200). Binding of YY1 was observed by the electrophoretic supershift assay using anti-YY1 antibody and linearized or supercoiled forms of plasmids bearing the core promoter, as well as with 30 bp oligonucleotide probes containing the individual YY1 binding motifs and four muPARP-1 promoter fragments. We detected YY1 binding to BM1 (−587/−558), BM4 (−348/−319) and a very prominent association with BM7 (+86/+115). Inspection of BM7 reveals overlap of the muPARP-1 translation start site with the Kozak sequence and YY1 and PARP-1 recognition sites. Site-directed mutagenesis of the YY1 and PARP-1 core motifs eliminated protein binding and showed that YY1 mediates PARP-1 binding next to the Kozak sequence. Transfection experiments with a reporter gene under the control of the muPARP-1 promoter revealed that YY1 binding to BM1 and BM4 independently repressed the promoter. Mutations at these sites prevented YY1 binding, allowing for increased reporter gene activity. In PARP-1 knockout cells subjected to PARP-1 overexpression, effects similar to YY1 became apparent; over expression of YY1 and PARP-1 revealed their synergistic action. Together with our previous findings these results expand the PARP-1 autoregulatory loop principle by YY1 actions, implying rigid limitation of muPARP-1 expression. The joint actions of PARP-1 and YY1 emerge as important contributions to cell homeostasis.

Cutting Edge: Proteolytic Inactivation of Poly(ADP-Ribose) Polymerase 1 by the Nlrp3 and Nlrc4 Inflammasomes

Caspase-mediated cleavage of the DNA damage sensor poly(ADP-ribose) polymerase 1 (PARP1) is a hallmark of apoptosis. However, it remains unclear whether PARP1 is processed during pyroptosis, a specialized cell-death program that occurs upon activation of caspase-1 in inflammasome complexes. In this article, we show that activation of the Nlrp3 and Nlrc4 inflammasomes induces processing of full-length PARP1 into a fragment of 89 kDa in a stimulus-dependent manner. Macrophages deficient for caspase-1 and those lacking the inflammasome adaptors Nlrp3, Nlrc4, and ASC were highly resistant to cleavage, whereas macrophages lacking the downstream inflammasome effector caspase-7 were partially protected. A modest, but statistically significant, reduction in Nlrp3 inflammasome-induced pyroptosis was observed in PARP1 knockout macrophages. Thus, protease-mediated inactivation of PARP1 is a shared feature of apoptotic, necrotic, and pyroptotic cells.

Increased poly(ADP-ribose) polymerase (PARP)-1 expression and activity are associated with inflammation but not goblet cell metaplasia in murine models of allergen-induced airway inflammation

ABSTRACTInflammation plays a key role in lung injury and in the pathogenesis of asthma. Two murine models of allergic airway inflammation—sensitization and challenge to ovalbumin (OVA) and intratracheal exposure to interleukin-13 (IL13)—were used to evaluate the expression of poly(ADP-ribose) polymerase-1 (PARP-1) in allergic airway inflammation. Inflammation is prominent in OVA-induced allergic asthma, but this inflammation is greatly reduced by a PARP-1 inhibitor and almost eliminated when PARP-1 knockout mice are subjected to the OVA model. The present study temporally evaluated PARP-1 protein expression, localization, and activity, as well as inflammation and goblet cell metaplasia (GCM), in murine lungs following a single OVA challenge or IL13 exposure. Following OVA challenge PARP-1 protein expression and activity were greatly increased, being maximal at 3 to 5 days following OVA exposure and beginning to decrease by day 8. These changes correlated with the timing and degree of inflammation and GCM. In contrast, PARP-1 protein or activity did not change following single IL13 exposure, though GCM was manifested without inflammation. This study demonstrates that both PARP-1 protein and activity are increased by allergen-activated inflammatory mediators, excluding IL13, and that PARP-1 increase does not appear necessary for GCM, one of the characteristic markers of allergic airway inflammation in murine models.

Ethanol-induced downregulation of the angiotensin AT2 receptor in murine fibroblasts is mediated by PARP-1

Molecular mechanisms accompanying ethanol-induced cytotoxicity remain to be defined. The renin–angiotensin system with its respective receptors, the angiotensin AT1 and AT2 receptor (AT1R and AT2R), has been implicated in these processes. The AT2R seems to counteract the pro-inflammatory, pro-hypertrophic, and pro-fibrotic actions of the AT1R and is involved in cellular differentiation and tissue repair. Recently, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel negative transcriptional regulator of the AT2R. However, the complex interactions between ethanol, PARP-1, and the AT2R are largely unknown. In this in vitro study, we aimed to clarify whether acute ethanol treatment modifies AT2R promoter activity or AT2R mRNA and protein levels and whether PARP-1 is involved in ethanol-mediated regulation of the AT2R. Murine fibroblasts of the R3T3 and MEF line (murine embryonic fibroblasts) were exposed to ethanol for 24 h. AT2R promoter activity, mRNA and protein levels were analyzed with and without PARP-1 inhibition and in PARP-1 knockout MEF cells. Expression of PARP-1 was analyzed over course of time, and cell viability and DNA fragmentation were measured on single-cell level by flow cytometry. Ethanol exposition induced substantial downregulation of the AT2R on promoter, mRNA and protein levels in a dose-dependent manner. Pharmacological inhibition or ablation of PARP-1 completely abolished this effect. Ethanol treatment did not have any effect on AT1R mRNA and protein levels in MEF cells. Further, acute ethanol treatment promoted DNA fragmentation and caused transcriptional induction of PARP-1. Our findings reveal that PARP-1 is an upstream transcriptional regulator of the AT2 receptor in the context of ethanol exposure and represses the AT2R gene in fibroblasts in vitro. Variations in expression of the potentially tissue-protective AT2R might contribute to ethanol-mediated pathology.

Abstract 3947: PARP-1 as a therapeutic target in gliomas

Abstract Gliomas are the most common primary tumors in the brain. We use this tumor type as a paradigm of oncogenic transformation. Deregulation of signal transduction pathways are commonly found both in low- and high-grade gliomas. Moreover, all WHO grade III and IV (GBM) show a disruption of cell cycle due to alterations in at least one out of three families of proteins involved in G1/S transition, such as: CDKs, INK4a-ARF or Rb. Oncogenic HRas (HRasV12) expression recapitulates gain-of-function mutations in several growth factor receptors in glial cells, resulting in the activation of different downstream effectors such as Raf/MEK/ERK and PI3K/AKT signaling pathways. The hyperactivation of growth signaling pathways, phenocopied by HRasV12, increases cell proliferation rate in the absence of senescence, facilitating their transformation into tumoral cells. Furthermore, our data indicate that HRasV12 is able to cooperate with Rb loss in the tumor malignization. Poly(ADP-ribose) polymerase-1 (PARP-1) facilitates DNA repair by binding to DNA breaks and attracting DNA repair proteins to the site of DNA damage. Despite its important role in the cellular response to genotoxic stress, PARP-1 is not required for survival and Parp-1−/− mice are viable and fertile. These mice do not develop early onset tumors and tumor latency is increased in PARP-1 knockout mice that are deficient for p53. Accordingly, here we observed that Parp-1−/− astrocytes display a marked proliferative decrease when compared with Parp-1+/+ astrocytes which express HRasV12, either in a Rb+/+ or in a Rb−/− context. Given that loss of PARP-1 are less susceptible to HRasV12 induced transformation, the inactivation of PARP-1 with chemical inhibitors is a potential therapeutic approach to restrain the tumor progression. Interestingly, in our model of gliomagenesis PARP-1 chemical inhibition (PJ34) leads to a significant fall in the proliferative rate in HRasV12 astrocytes comparable to Parp-1−/− astrocytes, and is also able to reduce tumor progression even when HRasV12 cooperates with Rb loss. These results indicate that treatment with PARP-1 inhibitors might be a potential therapy for this type of tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3947.